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									     ABC TRANSPORTER MODULATION
       IN ACUTE MYELOID LEUKEMIA
STUDIES FROM THE LEUKEMIC STEM CELL PERSPECTIVE




              MARC HGP RAAIJMAKERS
         ABC TRANSPORTER MODULATION
            IN ACUTE MYELOID LEUKEMIA
STUDIES FROM THE LEUKEMIC STEM CELL PERSPECTIVE



                Een wetenschappelijke proeve op het gebied
                     van de Medische Wetenschappen


                                Proefschrift

Ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen,
           op gezag van de Rector Magnificus prof. dr. C.W.P.M. Blom,
volgens besluit van het College van Decanen in het openbaar te verdedigen op
       vrijdag 17 februari 2006 des namiddags om 1.30 uur precies door




               Marc Hermanus Gerardus Petrus Raaijmakers
                    Geboren op 7 juni 1968 te Veghel
Promotor:
Prof. dr. T.J.M. de Witte

Copromotores:
Dr. R.A.P. Raymakers
Dr. J.H. Jansen

Manuscriptcommissie:
Prof. dr. P.H.M. de Mulder
Prof. dr. R.J. Scheper (Vrije Universiteit, Amsterdam)
Prof. dr. F. G.M. Russel




ISBN-10: 90-9020306-0
ISBN-13: 978-90-9020306-5
© 2006 M.H.G.P. Raaijmakers, Nijmegen

Print: Print Partners Ipskamp, Enschede
Lay-out: Dia Hopmans Scriptura, Nijmegen
Cover photography: M.H.G.P.R., with gratefulness to P.Groenen and D. van de Vendel,
bee-keepers.

The studies described in this thesis were supported financially by grants from the Dutch
Cancer Society and the Stichting Vanderes.

Publication of this thesis was financially supported by the Dutch Cancer Society, Glaxo-
SmithKline, Sigma-Tau, Novartis Oncology, Ortho Biotech, Pfizer, Amgen, Beckman
Coulter.
Sit down before fact as a little child, be prepared to give up every
       preconceived notion, follow humbly wherever or whatever
                  abysses nature leads, or you will learn nothing
                                                   Thomas H. Huxley




                                                  Voor mijn ouders
                                         Ter herinnering aan Twan
6
TABLE OF CONTENTS


Chapter 1   General introduction                                                      9

Chapter 2   Quantitative assessment of gene expression in highly purified            31
            hematopoietic cells using real-time reverse transcriptase polymerase
            chain reaction
            Experimental Hematology 30: 481-487; 2002

Chapter 3   Single cell image analysis to assess ABC-transporter mediated efflux     47
            in highly purified hematopoietic progenitors
            Cytometry;49:135-42, 2002

Chapter 4   ABCB1 mediated drug efflux is reduced in leukemic progenitor cells in    65
            comparison to their normal counterparts in acute myeloid leukemia
            Clinical Cancer Research. Accepted for publication

Chapter 5   The Breast Cancer Resistance Protein in drug resistance of primitive     89
            CD34+38- cells in acute myeloid leukemia
            Clinical Cancer Research. 11: 2436-2444, 2005

Chapter 6   Leukemic CD34+38- progenitor cells display conserved differential       109
            expression of many ATP-binding cassette transporter genes
            Leukemia. Accepted for publication

Chapter 7   Lung-resistance-related protein expression is a negative predictive     129
            factor for response to conventional low but not to intensified dose
            alkylating chemotherapy in multiple myeloma
            Blood; 91:1029-36, 1998

Chapter 8   Impaired Breast Cancer Resistance Protein mediated drug transport       145
            in plasma cells in multiple myeloma
            Leukemia Research. 29:1455-58, 2005

Chapter 9   Summary and perspectives                                                155

Nederlandse samenvatting                                                            165

Dankwoord                                                                           173

Curriculum Vitae                                                                    179

Epilogue                                                                            183

Appendix: color illustrations                                                       189


                                                                                      7
   CHAPTER 1
GENERAL INTRODUCTION
_____   CHAPTER 1 _____________________________________________________________________________________________________




10
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



Hematopoiesis and the CD34+38- hematopoietic stem cell

Hematopoiesis and hematopoietic stem cells
Hematopoiesis is the continuous process of proliferation and terminal differentiation of
hematopoietic stem cells (HSC) into the different blood cell lineages (myeloid, erythroid
and lymphoid). Hematopoiesis is supported by pluripotent HSC defined as cells with the
capacity to engraft, selfrenew and support multilineage differentiation into committed
progenitors and subsequently into all types of blood cells. Characteristically, HSC, like all
other stem cells have the ability to divide assymetrically. Through this process, the division
of a stem cells results in the formation of two daughter cells, one of which is another stem
cell and the other is a committed progenitor cell that is capable of further differentiation and
proliferation but lacks selfrenewal capacity. HSC reside predominantly in the bone marrow
although low numbers are also found in the peripheral blood. Although they were previously
thought to be resting cells recent evidence from mouse experiments show that 8-10 % of
HSC randomly enter the cell cycle every day with all HSCs entering the cell cycle in 1-3
months(1). HSC respond to a variety of conditions by entering the cell cycle, expanding
their numbers by self-renewing proliferation and mobilizing into the bloodstream. These
conditions include myelosuppressive treatment with agents such as cyclophosphamide
and cytokines such as granulocyte colony-stimulating factor (G-CSF) and granulocyte
macrophage colony stimulating factor (GM-CSF).

CD34+38- hematopoietic pluripotent stem cells
HSC and committed progenitor cells in human bone marrow as well as umbilical cord
blood can be identified by their expression of the CD34 membrane phosphoglycoprotein(2).
CD34+ cells account for approximately 1.5% of normal adult mononuclear cells and
contain erythroid, granulomonocytic, megakaryocytic, B and T lymphoid and dendritic cell
precursors. The observation that CD34+ cells provide sustained multilineage hematopoiesis
after allogeneic and autologous transplantation(3), a characteristic of hematopoietic stem
cells, showed that CD34+ cells also contain HSC. These hematopoietic stem cells in
human have subsequently been shown to have a CD34+ CD38- phenotype(4,5), comprising
only 1-10% of CD34+ cells. CD38 is a 45 kDa transmembrane glycoprotein that appears
to play a role as a differentiation molecule(6) and inductior of apoptosis(7,8); CD34+38+ cells
contain the committed progenitor cell types. Additionally CD38 is expressed on most
mature blood cells (majority of B and T-lymphocytes, NK cells and subsets of monocytes,
granulocyes, erythroid cells and platelets)(9-11). CD34+38- cells have a lymphocyte-like
morphology(12), weakly express Thy1(13) and the tyrosine kinase receptors c-kit(14) and Flt-
3(15) but lack antigens displayed on mature hematopoietic cells(16). It appears that CD38 is
a better marker for HSC purification than a cocktail of so-called “lineage markers” which
are antigens expressed on mature blood cells because CD34+ cells undergoing early
differentiation first express CD38 and than lineage markers(17). Evidence that CD34+38-


                                                                                                                    11
_____   CHAPTER 1 _____________________________________________________________________________________________________



hematopoietic cells contain pluripotent hematopoietic stem cells has come from both in
vitro and in vivo assays; the CD34+38- cell population is enriched for long-term culture
initiating cells (LTC-IC)(18-20) and extended LTC-IC(21), which proliferate later and have a
greater regenerative potential. No such cells were found in the CD34+38+ population.
Definite assessment of HSC function(22,23) has come from in vivo assays demonstrating
that CD34+38- cells from adult bone marrow were able to engraft and generate sustained
(re) transplantable multilineage human hematopoiesis in fetal sheep(24) and NOD/SCID
mice(25,26) illustrating the characteristics of stem cells i.e. engraftment, self-renewal and
multilineage cell generation.



Acute myeloid leukemia as a hematopoietic stem cell disease

Acute myeloid leukemia originates from hematopoietic stem cells
Acute myeloid leukemia is characterized by the accumulation of large numbers of blast
cells that fail to differentiate into functional granulocytes or monocytes. The leukemic blasts
have limited proliferative potential suggesting that a small subpopulation of leukemic cells
with extensive proliferation and the capacity for self-renewal must maintain the leukemia(27-
29)
    . This concept of tumorigenic leukemic stem cells has emerged from findings that only a
small subset of leukemic cells is capable of extensive proliferation in vitro and in vivo.
In recent years it has become evident that leukemic stem cells exist and reside within the
CD34+38- cell population in AML. The involvement of CD34+38- cells in leukemogenesis
is demonstrated by the presence of cytogenetically aberrant cells in the CD34+CD38-
compartment(30,31). Additionally, leukemic cells with long-term proliferative ability have
been identified as CD34+CD38-(32). Concluding evidence for the existence for a leukemic
stem cell in acute myeloid leukemia has come from studies using the nonobese diabetic/
severe combined immunodeficient mice (NOD/SCID) mouse model showing that cells with
leukemic stem cell characteristics, defined as capacity for self-renewal and engraftment
potential are present in AML(33,34). These cells were referred to as SCID leukemia initiating
cells or SL-IC. The SL-IC were found exclusively in the primitive CD34+38- fraction of all
patient samples regardless of the lineage markers expressed by the leukemic blasts, the
percentage of CD34+ blast cells or the FAB subtype (with exception of AML-M3). SL-IC
were not found in more committed CD34+38+ progenitors or CD34- cells. These studies
stress that leukemia initiating transformation and progression associated genetic events
occur at the level of these primitive CD34+38- cells. This parallels the hierarchy in normal
bone marrow in which a rare population of CD34+38- cells have stem cell characteristics(35),
supporting the hypothesis that malignant transformation takes place in normal HSC and that
the nature of the genetic event itself determines the differentiation program of the leukemic
clone (Figure 1). A similar role for the CD34+CD38- compartment in leukemogenesis has
been suggested for chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL)
and the myelodysplastic syndrome(36-38)
12
__________________________________________________________________________________________   GENERAL INTRODUCTION _____




                                                                                                  Self-rewal
                                Leukemogenic
                                    event
                      SL-IC                           HSC                          SRC
                                                   CD34+CD38-




                                                                                                 Multipotent
                                                                                                 progenitors
      Clonogenic
        leukemic
      progenitors


                                                                                                  Committed
                                                                                                  progenitors
                                                                                                  CD34+CD38+




                     Leukemia


                                                Lymphocytes         Granulocytes       Erythrocytes       Platelets

Figure 1: Acute myeloid leukemia originates from the normal hematopoietic stem cell. In human normal
hematopoiesis a CD34+CD38- hematopoietic stem cell (HSC) gives rise to the SCID repopulating cell (SRC)
which is capable of self renewal and the production of all forms off mature blood cells through the subsequent
differentiation into multipotential progenitors and committed CD34+38+ hematopoietic progenitors. In AML
leukemic transformation of the HSC leads to the occurrence of a SCID leukemia initiating cell (SL-IC) that is
capable of self renewal and produces both the clonogenic and non-clonogenic blast cells that form the bulk of the
tumor, similar to the hierarchy in normal bone marrow. Adapted from reference 32 © (1997) Macmillan Magazines
Ltd.


Cancer as a stem cell disease
It is increasingly recognized that other forms of cancer, including solid tumors, may arise
from normal stem cells as well(39-42). A subset of cancer cells may have the properties of
cancer stem cells, with self renewal capacity to generate progeny cancer cells with limited
proliferative potential. Normal stem cells have the machinery for self renewal, which is
a hallmark of cancer, already activated and therefore may require fewer mutations for
malignant transformation in comparison to mature cells. In addition, normal stem cells persist
throughout life and undergo an increased number of cell divisions and therefore have greater
chance for the acquisition of mutations required for malignant transformation than short-lived
cells. Cancer stem cells have been characterized using NOD/SCID engraftment and serial
transplantation models in breast cancer(40) and neuronal tumours(43-45). In these diseases
surface markers have been identified that recognize cells which are highly enriched for the
ability to form tumors following transplantation relative to bulk tumor cells.


                                                                                                                      13
_____   CHAPTER 1 _____________________________________________________________________________________________________



Committed progenitors as leukemic stem cell in leukemia
Although HSC are now increasingly recognized as the target of genetic events leading
to malignant transformation, committed progenitors or even differentiated cells may also
become leukemia-initiating cells through the activation of oncogenic pathways that re-
establish stem cell (self-renewal) capacities. Proof of principle for this concept has been
established in mouse models of leukemia(46,47). Additionally transgenic mice models using
promoter regions of myeloid specific genes have shown that myeloid leukemias can also
arise from committed progenitors, since leukemic fusion proteins such as BCR/ABL,
PML-RARa and MLL-ENL(48-50) can be transforming at the level of myeloid progenitors
and are able to give rise to leukemia without HSC involvement. This is likely the case in
promyelocytic leukemia in which the specific t (15,17) PML/RARa reciprocal translocation
is found in CD34+38+ committed progenitors but not in CD34+38- cells(48). Similarly,
recent evidence indicates that the CML stem cell compartment might be dynamic when
it progresses to blast crisis(51); self renewal properties were observed in vitro within the
granulocyte-monocyte progenitor population, a compartment that normally lacks the ability
for self renewal. These results indicate that this progenitor compartment may attain self
renewal properties upon further transformation that are responsible for the expansion of
acute leukemic blasts.

Therapeutical implications of AML as a stem cell disease
The concept that AML arises from the malignant transformation of normal HSC has
important implications for the treatment of the disease; Therapies directed against mature
leukemic cells may produce clinical improvement and responses but are unlikely to be
curative if leukemic stem cells that are responsible for maintenance of the disease are not
targeted or are resistant to therapy (Figure 2). Incomplete chemotherapeutical eradication
of these cells will ultimately result in disease relapse, as is frequently observed in AML.
Cellular antigens or signaling pathways expressed by leukemic cells may not be optimal
therapeutic targets if immature leukemic cells do not express these targets. The leukemic
stem cell is thus the most crucial target in the treatment of AML and therefore elucidation
of the mechanisms conferring resistance against chemotherapy to these cells is of great
importance. Additionally, further characterization of cancer stem cells may lead to improved
diagnostics and therapies by allowing us to better identify and target these crucial cells
within the tumor hierarchy.




14
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



                                       Conventional chemotherapy




                                              remission induction but
                                                   LSC survives
                                                                                                 Disease relapse




                                  Leukemic stem cell targeted therapy




                                                 LSC eradicated: loss of                       Disease cure
                                           ability to generate leukemic cells


              Leukemic stem cell (LSC) with innate resistance



               Drug sensitive bulk of leukemia cells


Figure 2: Stem cell model of drug resistance in AML. Leukemic stem cells (LSC) might be naturally resistant
to chemotherapy through conserved ABC transporter expression, quiescence or other mechanisms such as
DNA repair and anti-apoptotic mechanisms. Incomplete chemotherapeutical eradication of these tumor initiating
stem cells through this innate resistance will eventually lead to recurrence of disease after an initial response to
chemotherapy (which is induced through eradication of the sensitive bulk of tumor cells) as is frequently observed
in AML.




ATP-binding cassette (ABC) transporters

ABC Protein and Gene Organization
The ATP-binding cassette(ABC) transporters represent the largest family of transmembrane
proteins involved in the transmembrane transport of a huge variety of substrates including
sugars, peptides, inorganic anions, amino-acids, oligopeptides, polysaccharides and
proteins vitamins and metallic ions(52,53). ABC transporters use the energy of ATP binding to
drive the transport of these molecules across all cell membranes(54). Proteins are classified
as ABC transporters based on the sequence and organization of their ATP-binding domains,
also known as nucleotide-binding folds NBFs. The NBFs contain characteristic motifs,
Walker A and B, separated by ~90-120 amino acids, found in all ATP-binding proteins.
ABC transporters also contain an additional element, the signature C motif, located just
upstream of the Walker B site(55). The functional protein typically contains two NBFs and
two transmembrane (TM) domains (Figure 3). The TM domains contain 6-11 membrane-


                                                                                                                    15
_____     CHAPTER 1 _____________________________________________________________________________________________________



spanning α-helices and provide the specificity for the substrate. The NBFs are located in
the cytoplasm and transfer the energy to transport the substrate across the membrane. In
eukaryotes, most ABC transporters transport compounds from the cytoplasm to the outside
of the cell or into an extracellular compartment (endoplasmic reticulum, mitochondria,
peroxisome). The eukaryotic ABC transporters are organized either as full transporters
containing two TMs and two NBFs, or as half transporters(55). The latter must form either
homodimers or heterodimers to constitute a functional transporter. ABC genes are dispersed
widely in eukaryotic genomes and are highly conserved between species, indicating that
most of these genes have existed since the beginning of eukaryotic evolution. The human
superfamily of ABC transporters currently consists of 48 members. The genes can be
divided into subfamilies based on similarity in gene structure (half vs. full transporters),
order of the domains, and on sequence homology in the NBF and TM domains, resulting in
seven mammalian ABC gene subfamilies (ABCA, B, C, D, E, F and G family).

                                                     TM domains
 out


     in
                                                                                                             COOH

 NH2                                                      NBF                                              NBF
Figure 3: Schematic representation of a typical ABC transporter protein (ABCB1). ABC transporters consist
a varying number of transmembrane domains. ATP binding that drives the transmembrane transport takes place
in the nuclear binding folds (NBF). The picture represents the motifs found in a full transporter such as ABCB1
(N-TM-NBF-TM-NBF-C). Half transporters with TM-NBF or NBF-TM configuration also exist (e.g. ABCG2).




Multidrug Resistance (MDR)
MDR is defined as resistance to many different drugs with different chemical structures
and different mechanisms of action. Many different mechanisms for the arisal of MDR have
been elucidated including failure of apoptotic mechanisms, alterations in cell cycle check-
points, repair of damaged cellular targets and reduced drug accumulation. Some members
of the ABC transporter family have the ability to export a wide variety of structurally
unrelated chemotherapeutical compounds from cancer cells, thereby conferring MDR
to these cells(56). The best studied ABC transporters that have been implicated in drug
transport are ABCB1, ABCC1 and ABCG2.




16
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



ABCB1
The best characterized ABC transporter is ABCB1, formerly known as MDR1 or P-
glycoprotein(57). The ABCB1 gene maps to chromosome 7q21.1. ABCB1 was the first
human ABC transporter cloned and characterized through its ability to confer a multidrug
resistance phenotype to cancer cells that had developed resistance to chemotherapy
drugs(58). ABCB1 has been demonstrated to be a promiscuous transporter of hydrophobic
substrates, hydrophobic drugs including drugs like colchicine, VP16, adriamycin and
vinblastine as well as lipids, steroids, xenobiotics, and peptides(59).
ABCB1 is a classical ABC transporter composed of two homologous halves, each
containing six transmembrane (TM) domains and an ATP-binding domain, separated by
a flexible linker region (Figure 3). The two halves interact to form a single transporter(60,61).
The major drug binding domains reside in the transmembrane domains. However, the
mutations in mammalian P-glycoproteins that affect substrate specificity are found
throughout the molecule including the TM regions, soluble intra – and extracellular loops
and the ATP-binding domains(60,62). Mutational analysis further showed that the conserved
NBF (walker A and B and signature regions) and the linker region is critical for the
transport function of the protein(63-65). Glycosylation appears to be required for the proper
trafficking of the transporter to the cell surface but is not required for transport function of
P-glycoprotein(66,67). P-gp has phosphorylation-sites but mutants lacking these sites exhibit
normal transport function(68). Recently, polymorphisms in the gene have been described
that are associated with both altered substrate specificity and decreased transport function
of the protein(69). ABCB1 is expressed in tissues with a barrier function such as epithelium of
the liver, kidney, small and large intestine and capillary endothelial cells in brain, ovary and
testis. Data from ABCB1 knockout transgenic mice strongly support a physiological role for
ABCB1 in drug absorption, elimination and detoxification pathways(70,71).

ABCC1
The ABCC1/MRP1 gene, located on chromosome 16p13, was identified in the small-
cell lung carcinoma cell line NCI-H69, a multidrug resistant cell that did not overexpress
ABCB1(72). The ABCC1 transporter confers resistance to doxorubicin, daunorubicin,
vincristine, colchicine and several other compounds, a very similar profile to that of ABCB1.
Unlike ABCB1, however, ABCC1 transports drugs that are conjugated to glutathione by the
glutathione reductase pathway(73). Studies in ABCC1 -/- mice demonstrated hypersensitivity
to etoposide and ABCC1 protected the oropharyngeal mucosa and testicular tubes
indicating that it plays an important role in drug detoxification(74,75). ABCC1 can also
transport leukotrienes, such as leukotriene C4 (LTC4). LTC4 is an important signaling
molecule for the migration of dendritic cells. Migration of dendritic cells from the epidermis
to lymphatic vessels is defective in ABCC1 -/- mice(76).




                                                                                                                    17
_____   CHAPTER 1 _____________________________________________________________________________________________________



ABCG2
ABCG2, or Breast Cancer Resistance Protein (BCRP), is a 655-aa member of the ABCG
subfamily of ABC- membrane transporters first described in drug resistant cell lines that do
not overexpress ABCB1 or ABCC1(77 78,79). BCRP is a half transporter, likely functioning as a
homodimer and confers multidrug resistance to topotecan, mitoxantrone, doxorubicin and
related compounds by ATP dependent drug extrusion(80,81). BCRP is expressed in placental
syncytiotrophoblasts, intestinal epithelium, liver canicular membrane(82) suggesting a
physiological role in detoxification. This is substantiated by studies in bcrp1 -/- mice
which are normally viable without pronounced abnormalities but display abnormalities in
pharmacokinetics and disposal of carcinogens(83-85)

Other ABC transporters involved in extrusion of chemotherapeutical
compounds and drug resistance
The highly conserved homology between different ABC transporters predicts that additional
members may be involved in the extrusion of structurally unrelated chemotherapeutical
compounds. Indeed, an increasing number of ABC transporters have been demonstrated
to cause resistance to cancer chemotherapeutical agents(86,87) (Table 1). Additionally,
recent studies examining ABC transporter gene expression in drug-selected cancer cell
lines demonstrated overexpression of a number of transporters not previously recognized
as associated with drug resistance (ABCA4, ABCA7, ABCB2, ABCB6, ABCB8, ABCB9
and ABCG1)(88). Together, these data outline the emerging picture that ABC transporter
mediated multiple drug resistance may be conferred by simultaneous activity of many
redundant family-members in addition to ABCB1, ABCC1 and ABCG2.

Table 1: ABC transporters involved in the extrusion of chemotherapeutical compounds and drug
resistance.

Transporter            Alternative name            Drug substrates
ABCA2                                              estramustine
ABCB1                  MDR1/ p-glycoprotein        anthracyclins, etoposide, imatinib, taxanes, mitoxantrone, vinca
                                                   alkaloids
ABCB4                  MDR2                        paclitaxel, vinblastine
ABCB11                 BSEP                        paclitaxel
ABCC1                  MRP1                        anthracyclins, etoposide, methotrexate,
ABCC2                  MRP2/cMOAT                  cisplatin, doxorubicin, , etoposide, methotrexate, mitoxantrone,
                                                   vinca alkaloids
ABCC3                  MRP3                        cisplatin, doxorubicin, etoposide, methotrexate, vinca alkaloids
ABCC4                  MRP4                        methotrexate, thiopurines
ABCC5                  MRP5                        6-mercaptopurine, 6-thioguanine
ABCC6                  MRP6                        anthracyclins, etoposide, teniposide
ABCC10                 MRP7                        docetaxel, paclitaxel, vinca alkaloids
ABCC11                 MRP8                        purine and pyrimidine nucleotide analogs
ABCG2                  BCRP/MXR                    mitoxantrone, methotrexate, topetocan, SN-38, imatinib,
                                                   flavopiridol, anthracyclins

18
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



ATP-binding cassette transporters and stem cells

ABC transporters as markers of stem cells
Human (hematopoietic) stem cells are characterized by their ability to efflux fluorescent
dyes. Isolation of stem cells based on this efflux is an efficient method to purify stem cells
from different sources. It has been demonstrated that rhodamine 123 retention is low in the
most primitive hematopoietic cells(89,90). While the rhodamine 123-dull fraction provides long-
term reconstitution following injection into lethally irradiated mice, the rhodamine 123-bright
fraction provides only short term repopulation(91,92). Hoechst 33342 is another fluorescent
dye used for isolation of stem cell fractions, which is called the side-population(93,94). In
combination with rhodamine123 staining, Hoechst 33342-dull rhodamine 123-dull cells are
highly enriched for stem cell function(95). The molecular basis of this phenotype is has long
been unclear but recent studies indicate that ATP-binding cassette (ABC) transmembrane
transporters that efflux the fluorescent dyes are responsible for the dull phenotype of
hematopoietic stem cells. ABCB1 is highly expressed on CD34+ hematopoietic cells
suggesting that efflux pump activity could be responsible for the low retention of rhodamine
123 in primitive cells(96). More recent studies in knock-out mice have indeed shown that
ABCB1 attributes to the rhodamine dull phenotype of hematopoietic stem cells(97-99).
Recently, BCRP has been shown to be highly expressed in a wide variety of stem cells(100)
including immature human hematopoietic progenitors(101) and confer the side population
(SP) phenotype.

Physiological function of ABC transporters in stem cells
The physiological function of ABC transporters in human stem cell biology is currently
unknown. A role for ABC-transporters in the protection from genetic damage by naturally
occurring xenobiotics that are substrates for the transporter have been suggested for
HSC(102,103) and has recently been substantiated by the observation of increased cytotoxicity
to normal bone marrow in ABCB1(104) and ABCG2/BCRP knock-out mice(105).
In addition to protection of these long-lived cells to naturally occurring xenobiotics, a
fundamental role in stem cell biology has been suggested based on studies in Dictyostelium
demonstrating that a rhodamine-123 cellular efflux pump (RhT) with the properties of
a ABC-transporter prevents differentiation of prespore cells(106), possibly by exporting
differentiating factors from the cell interior. This led to the hypothesis that a cell’s response
to a signal that can diffuse across the plasma membrane depends, in part, on the cell’s
ability to remove it from the cytoplasm. According to this hypothesis, HSC might escape the
effect of differentiation factors present in the bone marrow. The idea that ABC transporters
regulate the cellular concentration of signaling molecules may prove to be a general way for
developing organisms to control their response to signals and regulate cell fate decisions.
However, no such a role has been demonstrated for the ABC-transporters identified on
human (hematopoietic) stem cells; Retroviral-induced overexpression of ABCB1 resulted


                                                                                                                    19
_____   CHAPTER 1 _____________________________________________________________________________________________________



in increased hematopoietic repopulating activity and myeloproliferative disease in mice(107-
109)
     . However, when extrapolating these experiments to a primate model, no detectable
adverse consequences have been observed(110) and MDR1 induction in hematopoietic
cells in clinical trials, in an attempt to protect these cells for chemotherapeutical damage,
have not resulted in abnormal hematopoiesis(111,112). Furthermore liver and marrow of adult
mdr1a/1b -/- mice show normal generation, function and multi-tissue trafficking of primitive
hematopoietic cells(113).
In contrast to ABCB1 overexpression studies, overexpression of BCRP/ABCG2 in a mouse
model significantly blocked hematopoietic development and resulted in less progeny in the
bone marrow and peripheral blood(114). However, similarly to ABCB1, ABCG2 -/- null mice
display no defects in steady state hematopoiesis and that, though ABCG2 expression is
required for the SP phenotype, with the loss of ABCG2 repopulating hematopoietic stem
cells are maintained outside the SP-region(115). Recently experiments using ABCB1/ABCG2
double knock-out mice confirmed a lack of effect on hematopoiesis, excluding possible
redundant activity of these transporters(116). The combined data do not support a role for
ABCG2 or ABCB1 in hematopoiesis but support the idea that they protect stem cells
from genetic damage of naturally occurring xenobiotics that are the substrate for these
transporters(117). This seems particularly important for HSC given their life-long presence
in the bone marrow and high proliferative activity during this period. In this context, it
is noteworthy that an anti-apoptotic role for ABCB1 has been demonstrated recently
promoting cell survival by inhibiting caspase activation(118-121).



ABC transporter modulation in the treatment of AML

Expression of ABC transporters ABCB1, ABCC1 and ABCG2 has been widely studied
in leukemic blasts in AML. Targeting of these molecules in clinical trials in an attempt to
enhance chemosensitivity of leukemic blast, however, has been limited to ABCB1.
ABCB1 is frequently expressed in elderly patients with AML. It is detected in 17% of
adults younger than 35 years, 39% in patients between 35 and 50 years and in 71 % of
patients older than 65 years(122). This is accompanied by a rising prevalence of the CD34+
surface phenotype with advancing age(123). AML specimens that express ABCB1 display
reduced cellular accumulation and relative in vitro resistance to antineoplastics such as
anthracyclins(124). Additionally, in the majority of reports investigating de novo AML, ABCB1
is a prognostic variable associated with reduced probability for remission induction and in
some reports, although debatable, inferior leukemia-free and overall survival(125-127). These
observations have led to the introduction of ABCB1 modulators in the treatment of AML
in an attempt to circumvent drug resistance by targeting ABCB1 mediated drug efflux
in leukemic blasts. Initial trials using agents with pleiotropic effects on drug resistance
(Quinine and cyclosporin) showed promising results with increased CR and OS in some


20
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



trials(128). Although concern existed about the effect of ABCB1 modulation on pharmokinetics
as a confounder in these studies, these results fostered the development of second
generation ABCB1 modulators such as PSC 833. PSC 833, a cyclosporin D analog, is a
second generation modulator with increased specificity and potency (10-fold greater than
cyclosporin). Recent phase III trials using PSC 833 (Valsopar) in 2 clinical trials adjusting
the dosing of chemotherapeutical agents are disappointing; An ECOG trial investigating
the addition of PSC to mitoxantrone, etoposide and cytarabine in patients with relapsed
or refractory AML(129) was suspended prematurely after an interim-analysis revealed no
improvement in rate of CR in the experimental arm. In the CALBG study investigating PSC
833 in combination with cytarabin, etoposide and daunorubicin in previously untreated
patients of 60 years and older(130) demonstrated excessive induction mortality with PSC
833, necessitating closure of the study. Both studies showed increased non-hematological
toxicity in the PSC 833 arm and failed to demonstrate beneficial effects on remission
induction in ABCB1 expressing subsets of patients. The outcome of these trials suggest
the presence of other, yet unidentified mechanisms of drug resistance in leukemic cells in
AML but despite these negative results, clinical trials using third generation modulators
(including tariquidar, zosuquidar and laniquidar, all characterized by high-affinity for the
ABCB1 protein and minimal activity against other members of the ABC transporter family)
are in development for clinical use arguing that the disappointing results of earlier trials
may be due to chemotherapy underdosing or schedule disparity(131).



Goal and outline of current thesis

The recognition of AML and other forms of cancer as diseases originating from normal stem
cells dictates a paradigm shift in the treatment of leukemia away from targeting the blast
cells and towards targeting the LSC. The high expression of ABC-transporters involved in
the extrusion of chemotherapeutical compounds on normal (hematopoietic) stem cells could
have major consequences for the treatment of leukemia. If ABC transporter expression and
function is conserved after malignant transformation of stem cells, this would be a major
mechanism of drug resistance to these cells and prevent chemotherapeutical eradication
of this tumor-initiating cell population. In the paradigm shift for the treatment of leukemia,
ABC drug transport inhibitors might thus be thought of as “leukemia stem cell sensitizing
agents” that allow the most crucial and drug-resistant cells in the leukemia-hierarchy
to be destroyed. Whether ABC expression and function is conserved after malignant
transformation of hematopoietic stem cells, however, is currently unknown. Though
expression and function of ABCB1 and ABCG2 have been widely studied in AML, there is
a remarkable lack of studies addressing expression and function in the critical CD34+38-
hematopoietic stem cell population. Additionally, no studies exist aimed at comparing ABC
transporter function between normal and malignant hematopoietic cells.


                                                                                                                    21
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The goal of the research performed and described in this thesis was to gain insight into
the expression, function and role in drug resistance of ABC transporters in CD34+38-
hematopoietic stem cells in AML in comparison to their normal counterparts. We
emphasised on studying ABCB1 and ABCG2 since these transporters are highly expressed
on a variety of human stem cells and their wide substrate specificity includes drugs used
in the treatment of AML (i.e. anthracyclins, mitoxantrone, vinca alkaloids). Additionally we
aimed to study the effect of modulation of these transporters on LSC eradication in AML
and identify additional ABC transporters involved in drug extrusion and chemoresistance of
leukemic CD34+38- stem cells.

Outline of this thesis
Hematopoietic CD34+38- stem cells have a very low frequency in the human adult bone
marrow (approximately 0.1 % of mononuclear cells). The study of these rare hematopietic
stem cells requires the development of adapted assays to study ABC transporter gene
expression and biological activity of these molecules. The first two chapters of this thesis
are dedicated to the development of such stem cell assays.
Chapter 2 describes the development of a novel method to accurately and quantitatively
assess gene expression in highly purified, low cell number samples of CD34+38- cells
from human normal bone marrow and AML. Quantitative assessment of gene expression
in the CD34+CD38- cell population is essential for understanding the molecular events
underlying normal and malignant hematopoiesis. Though real-time -PCR is a sensitive
and flexible mRNA quantitation method, the small number of cells available for analysis
precludes the use of standard methodologies, necessitating alternative strategies like
global cDNA amplification or nested RT-PCR.
A novel real-time quantitative RT-PCR approach using adapted RNA isolation is described.
This assay is validated and the relevance for appropriate reference genes for the study
of gene expression in hematopoietic stem cells is discussed. Using this method the
expression of drug resistance related genes is assessed in these cells in human normal
bone marrow. Chapter 3 describes the development of a method to enable assessment of
the biological activity of ABC transporters in highly purified CD34+38- cells on the single
cell level using fluorescence microscopy and image analysis. Using this method ABCB1
mediated transport is quantitatively assessed in normal CD34+38- hematopoietic cells
in comparison to CD34+38+ committed progenitors. The next two chapters investigate
the expression, biological activity and role in drug resistance of the known ABC stem cell
transporters in CD34+38- cells in AML. Chapter 4 investigates ABCB1 expression and
function in the CD34+38- cell population in AML using monoclonal antibodies and inhibition
assays among which the assay described in chapter 3. Additionally, ABCB1 mediated
transport is studied in hematopoietic stem cells in the pre-leukemic syndrome refractory
anemia.



22
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



In Chapter 5 the role of ABCG2 in drug accumulation and chemoresistance of leukemic
CD34+38- cells is addressed. ABCG2 expression and function is studied in CD34+38- stem
cells in AML in comparison to normal bone marrow and the effect of in vitro modulation of
ABCG2 on chemoresistance of leukemic CD34+38- stem cells is tested. The finding that
drug transport in leukemic CD34+38- cells occurs in the presence of blockage of ABCB1
and ABCG2 as described in chapters 4 and 5 suggested the presence of yet unidentified
drug transporters in leukemic CD34+38- cells. To address this question Chapter 6 describes
the results of profiling of the entire family of ABC transporters in CD34+38- hematopoietic
cells using real-time quantitative RT-PCR on micro-fluidic cards. This technique is validated
corroborating on the results described in chapter 2 and ABC transporter profiles of normal
(G-CSF mobilized and bone marrow) and leukemic CD34+38- cells are described in
comparison to committed CD34+38+ progenitors.
Finally, flow-cytometric analysis of CD34+38- cells as described in the previous chapters
yielded additional data in another CD34 and CD38 defined cell type: the CD34-CD38++
plasma cells. In the second part of this thesis (Chapter 7 and 8) results are described on
drug resistance proteins in these plasma cells in normal bone marrow and the plasma
cell malignancy multiple myeloma. Chapter 8 investigates the role expression and clinical
significance of the lung resistance related protein (LRP) in multiple myeloma. Chapter 8
investigated the role of ABCG2 in primary drug resistance of malignant plasma cells



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                                                                                                                    27
_____   CHAPTER 1 _____________________________________________________________________________________________________



88.     Gillet, J. P., Efferth, T., Steinbach, D., Hamels, J., de Longueville, F., Bertholet, V., and Remacle, J.
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28
__________________________________________________________________________________________   GENERAL INTRODUCTION _____



105. Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. D., and Sorrentino, B. P. Bcrp1 gene expression
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114. Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. M., Sampath, J., Morris, J. J., Lagutina, I., Grosveld,
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115. Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. D., and Sorrentino, B. P. Bcrp1 gene expression
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     mitoxantrone in hematopoietic cells in vivo. Proc.Natl.Acad.Sci.U.S.A, 99: 12339-12344, 2002.
116. Zhou, S., Zong, Y., Lu, T., and Sorrentino, B. P. Hematopoietic cells from mice that are deficient in both
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117. Smeets, M., Raymakers, R., Vierwinden, G., Pennings, A., van de, L. L., Wessels, H., Boezeman, J., and De
     Witte, T. A low but functionally significant MDR1 expression protects primitive haemopoietic progenitor cells
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118. Johnstone, R. W., Cretney, E., and Smyth, M. J. P-glycoprotein protects leukemia cells against caspase-
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120. Pallis, M., Turzanski, J., Higashi, Y., and Russell, N. P-glycoprotein in acute myeloid leukaemia: therapeutic
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     The cyclosporin PSC 833 increases survival and delays engraftment of human multidrug-resistant leukemia
     cells in xenotransplanted NOD-SCID mice. Leukemia, 16: 2388-2394, 2002.

                                                                                                                    29
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122. Leith, C. P., Kopecky, K. J., Chen, I. M., Eijdems, L., Slovak, M. L., McConnell, T. S., Head, D. R., Weick, J.,
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123. te Boekhorst, P. A., de Leeuw, K., Schoester, M., Wittebol, S., Nooter, K., Hagemeijer, A., Lowenberg, B.,
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     and therapeutic strategies. Blood, 104: 1940-1951, 2004.




30
                                             CHAPTER 2
    QUANTITATIVE ASSESSMENT OF GENE EXPRESSION IN
HIGHLY PURIFIED HEMATOPOIETIC CELLS USING REAL-TIME
REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION




                                                      MARC H.G.P. RAAIJMAKERS
                                                            LIESBETH VAN EMST
                                                                THEO DE WITTE
                                                               EWALD MENSINK
                                                       REINIER A.P. RAYMAKERS

                  DEPARTMENT OF HEMATOLOGY AND CENTRAL HEMATOLOGY LABORATORY,
                           UNIVERSITY MEDICAL CENTER NIJMEGEN, THE NETHERLANDS




                                    EXPERIMENTAL HEMATOLOGY 30: 481-487; 2002
_____   CHAPTER 2 _____________________________________________________________________________________________________




32
___________________________________________________________________   GENE EXPRESSION IN HEMATOPOIETIC STEM CELLS _____



Abstract

Objective
Quantitative assessment of gene expression in stem cells is essential for understanding
the molecular events underlying normal and malignant hematopoiesis. The aim of the
present study was to develop a method for the precise quantitating of gene expression in
small subsets of highly purified CD34+CD38- stem cell populations.

Methods
Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was
used to quantitate housekeeping and drug resistance gene expression in cDNA obtained
from 300 CD34+CD38- cells without cDNA amplification or nested PCR techniques.

Results
Validation experiments in cellines showed efficient, representative and reproducible gene
amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in
dilutional experiments and by the detection of the low-copy gene PBGD. GAPDH was found
a useful reference gene in normal and leukemic CD34+CD38- cells but 18S rRNA content,
in contrast, varied 100-1000-fold in these populations. Moreover, expression of 18S rRNA
was significantly lower in leukemic CD34+CD38+ cells compared to normal CD34+CD38+
cells (p=0.002). Expression of MDR-1(18-fold, p<0.0005), MRP-1 (3.8-fold, p<0.05) and
LRP (1.8-fold, NS) was higher in normal CD34+CD38- compared to CD34+CD38+ cells.

Conclusion
Real-time quantitative RT-PCR is a valuable tool for precise quantitating of gene expression
in small subsets of hematopoietic cells. Using this method we show the inappropriateness
of 18S as a reference gene in these progenitors and the downregulation of drug-resistance
related genes early in hematopoiesis.



Introduction

In human normal hematopoiesis, a small subpopulation of immunophenotypically defined
CD34+CD38- cells have stem cell characteristics as shown by in vitro proliferation
characteristics, self-renewal and engraftment potential(1,2). Additionally to its established role
in normal hematopoiesis, accumulating evidence documents a role for the CD34+CD38-
compartment in malignant hematopoiesis. Cells with leukemic stem cell characteristics in
AML are found in the CD34+CD38- subpopulation(3,4), similar to the hierarchy observed
in human normal bone marrow. The CD34+CD38- compartment plays a similar role in
the leukemogenesis of CML, APL and ALL(5-7). This implicates that the leukemia-initiating


                                                                                                                    33
_____   CHAPTER 2 _____________________________________________________________________________________________________



transformation and progression associated genetic events occur in these primitive cells
and not in committed progenitors.
Cellular decisions concerning differentiation are reflected in altered patterns of gene
expression(8). Quantitative assessment of gene expression in the CD34+CD38- cell
population is essential for understanding the molecular events underlying normal and
malignant hematopoiesis. Though RT-PCR is a sensitive and flexible mRNA quantitation
method, the small number of cells available for analysis precludes the use of standard
methodologies, necessitating alternative strategies like global cDNA amplification or nested
RT-PCR(9-12). Quantitation of mRNA transcripts using these strategies in hematopoietic
stem cells thus far have been performed by end-point amplicon measurement quantitating
mRNA signals after blotting.
Real-time PCR has been established as a method for rapid and precise quantitation of nucleic
acids using an integrated system for thermal cycling, real-time fluorescence detection and
subsequent analysis(13,14). Real-time PCR quantitates PCR products in the exponential phase
of amplification by the detection of fluorescent hybridisation probes which are degraded
by the 5’nuclease activity of Taq polymerase. This technique has shown to be superior to
end-point quantitation both in accuracy and reproducibility(15). We report the use of real-time
quantitative RT-PCR for molecular analysis of rare CD34+CD38- stem cells without the need
for prior cDNA amplification or nested PCR. Using this method we examined the expression
of two of the most commonly used RNA’s to normalise patterns of gene expression, the
housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and 18S
ribosomal RNA (18S rRNA) in both normal and leukemic bone marrow. Furthermore, we
investigated the expression of multidrug resistance related genes MDR-1 and MRP-1 and the
LRP (lung-resistance related protein) gene in normal CD34+CD38- cells.



Methods

Human normal and leukemic CD34+ bone marrow cells
Bone marrow was obtained from six healthy bone marrow donors and six patients with
AML at diagnosis after informed consent. Mononuclear cells were isolated by Ficoll
1.077 g/mL (Pharmacy Biotech, Uppsala, Sweden). From normal bone marrow samples,
CD34-positive cells were selected using directly conjugated CD34 antibody- coupled
immunomagnetic beads (M-450 coated with ‘561’ a class III epitope anti-CD34, Dynal,
Oslo, Norway). Isolation, cryopreservation and thawing procedures of the CD34+ cells
have been described previously(16). Purity of CD34 cells was controlled by labelling with
CD34-Cy5 monoclonal antibody (Beckton Dickinson, BV, Etten- Leur, The Netherlands)
using flow cytometry. CD34+ bead selected cells were stained with CD34- CY5 and
CD38-phycoerythrin (PE) monoclonal antibodies (Beckton Dickinson, BV, Etten-Leur, The
Netherlands) for 30 minutes at 4ºC, and washed in HBSS with 10% FCS.


34
___________________________________________________________________   GENE EXPRESSION IN HEMATOPOIETIC STEM CELLS _____



Definition and sorting of CD34+CD38- and CD34+CD38+ cells
A Coulter Epics Elite Flow cytometer was used to sort cell populations. Gating on forward
and right angle scatter was used to exclude dead cells and debris. In AML samples CD34+
cells from the mononuclear fraction were pre-sorted in HBSS 10%FCS. CD34+CD38-
cells of both normal bone marrow and AML were defined as the 1% of CD34+ cells
with the lowest CD38 density. Normal CD34+CD38- cells appeared in a consistently
restricted light-scattering region confirming the lymphoid appearance of these primitive
progenitors(17). The CD34+CD38+ cells were sorted from a gate positioned in the bulk of
CD34+ cells (containing approximately 25% of CD34+ cells) and showed heterogeneous
light-scattering properties, reflecting the morphologically different lineages present in this
progenitor population. The primitive character of both normal and leukemic sorted cells was
confirmed by their rhodamine dull phenotype(18) in rhodamine 123 accumulation and efflux
studies in comparison to the rhodamine-bright appearance of CD34+CD38+ cells (data not
shown).

Cell-lines
The MDR-1 negative RPMI 8226 myeloma parental cell-line and the doxorubicin resistant
subclone RPMI 8226 Dox 40 expressing the MDR-1 phenotype(19) were maintained as
a suspension in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 1%
penicillin (100U/mL), 1% streptomycin (100ug/mL) and 1% L-glutamine at 37ºC in a
humidified atmosphere with 5% CO2 in the presence of 400nM doxorubicin. Experiments
were performed on cells in an exponential growth phase and cultured for 1-2 weeks without
doxorubicin. Heterogeneity in the expression of P-glycoprotein in RPMI 8226 cells used for
experiments was excluded by flow cytometric assessment of P-glycoprotein expression
using the monoclonal antibody MRK 16.

Cell-lysis based RNA preparation and RT-PCR
Using the Elite flow cytometer with an Autoclone Cell Deposition Unit (Coulter, Hiale,
FL), 300 cells were sorted using phosphate buffered saline (PBS) as a sheath solution
into PCR tubes containing 7 µl of lysis solution ( I) (Superscript first-strand buffer, Life
Technologies, Gaithersburg, Md, USA) NP-40 (0.90% vol/vol), 10 mM dithiothreitol (DTT,
Life Technologies), 10 U RNAse inhibitor ( Promega, Madison, Wisc., U.S.A.) and distilled
water. Directly after cell sorting an RT-solution (II) containing 5 µM random hexamer primers
(dN6, Pharmacia, Uppsala, Sweden), 1.25 mM dNTP’s (Pharmacia, Uppsala, Sweden)
10 U RNAse inhibitor and 100 U Mo-MuLV reverse transcriptase (Life Technologies,
Gaithersburg, Md., U.S.A.) was added to a final volume of 10 µl. Lysis solution I and RT-
solution II were kept on ice during the entire procedure. Tubes were centrifuged for 15 s
before entering the cDNA synthesis reaction, performed for 10 minutes at 20°C, followed
by 42°C for 45 minutes and a 10 minute incubation step at 95°C. The total cDNA volume of
10 µl was centrifuged and frozen at –80°C until real-time quantitative PCR was performed.


                                                                                                                    35
_____   CHAPTER 2 _____________________________________________________________________________________________________



After thawing for PCR experiments the cDNA was diluted in distilled water to a final volume
of 50 µl. 2.5 µl of diluted cDNA was used for each PCR reaction.

Real-time quantitative PCR and PCR conditions
The use of real-time PCR has previously been described by our group(20,21). In brief, PCR
reactions were performed in the ABI PRISM 7700 sequence detector in a 50 µl final volume.
The PCR mixture contained buffer (Perkin Elmer/ Applied Biosystems, Foster City, CA,
U.S.A.)) with 5 mM MgCl2, 250 µM dNTP (Pharmacia, Uppsala, Sweden), 1.25 U Amplitaq
Gold-TM (Perkin Elmer/ Applied Biosystems), primers, probe and passive reference dye
Rox. For GAPDH 4.5 mM MgCl2 and 2.5 U Amplitaq Gold- TM was used. The primer and
probe sequences for gene amplification were:

PBGD:
FP 5’-GGCAATGCGGCTGCAA-3’
RP 3’-GGGTACCCACGCGAATCAC-5’
Probe: 5’-CTCATCTTTGGGCTGTTTTCTTCCGCC-3’
18S: Applied biosystems; part number 4308329
GAPDH: Applied biosystems; part number 402869
MDR-1: Applied Biosystems
MRP-1
FP: 5’-GCCCTGTTTGCGGTGATC-3’
RP: 3’-GATGTTGGTTTCCATTTCAGATGA-5’
Probe: 5’-CCGAACCAGCCAGTTCAAGTACGTGG-3’
LRP:
FP: 5’-GGCAGGACAATGAGAGGGTACT-3’
RP: 3’-CCGAACTTGCCCTGTGACAT-5’
Probe: 5’- TGACCGTCCCCCCACGTCACTACT-3’

The taqman-TM probes carried a 5’TET reporter label and a 3’TAMRA quencher group
and were synthesised by PE-Applied Biosystems. DNA-specificity of primers and probes
was excluded by demonstration of a lack of amplification in samples with human DNA.
Additionally, omission of reverse transcriptase in the RT-PCR protocol led to failure of
target gene amplification in the positive controls. The Amplitaq Gold enzyme was activated
by heating for 10 minutes at 95°C. All genes were amplified by a first step of 15 seconds at
95°C followed by three minutes at 60°C for 45 cycles. The relatively long cycle times were
optimised on the basis of a beneficial impact on the detection frequency of low-copy genes.
Initially 50 cycles were performed but samples never became positive after 40 cycles.




36
___________________________________________________________________   GENE EXPRESSION IN HEMATOPOIETIC STEM CELLS _____



Quantitation of target-gene expression
Normalised drug-resistance related gene expression to the internal standard GAPDH is
given by the following equation(22):

T0/R0= K. (1+E) (CT,r – CT,t)

To: Initial number of target gene copies; Ro: Initial number of standard gene copies; E;
Efficiency of amplification; CT,t: Threshold cycle of target gene; CT, r: Threshold cycle of
standard gene; and K: Constant.
The relation is based on the precondition that efficiency of reference and target gene
amplification is approximately equal. Serial dilution of 50 ng RPMI dox40 GITC-isolated
cDNA indeed showed comparable efficiencies for 18S, GAPDH, MDR-1, MRP-1, and LRP
( slope of log input versus Ct was -3.36, -3.44, -3.39, -3.43, -3.44 respectively). Reference
gene (GAPDH) is shown to be equally expressed in the cellpopulations of interest (see
section results).

Experimental design
All normal bone marrow (NBM) and AML samples were evaluated for a specific target
and control gene expression in a single PCR series obviating the need for comparison of
different runs by calculation of run efficiencies. MDR-1 and 18S reactions were performed
within one tube by the use of a double probe assay. No template controls served as
negative controls. For MDR-1 and LRP the myeloma RPMI 8226 parental celline was used
as an additional negative control.
MDR-1, MRP-1 and LRP expression in the dox 40 subline was used as a positive control.
All samples were tested as duplicates. For quantitation, the average value of both
duplicates was used. In the cases that only one of the duplicates showed a positive signal
the expression of the gene was designated as ‘borderline’ since the binary answer of the
PCR will reflect a matter of probability in the case of low density of target molecules(23).

Conventional GITC- RNA isolation and cDNA synthesis
RNA from the RPMI 8226 parental and dox 40 celline was extracted from approximately 106
cells using the guanidinium-thiocyanate acid-phenol-chloroform procedure(24) with minor
modifications as previously described(21). 50 ng of RNA was used for cDNA synthesis. The
cDNA synthesis reaction was performed as previously described(21).

Statistical analysis
Differences in normalised target gene expression between different hematopoietic
subpopulations were calculated using the student t test with a level of significance of p<
0.05.



                                                                                                                    37
_____   CHAPTER 2 _____________________________________________________________________________________________________



Results

Performance of 300-cell real time quantitative RT-PCR: experiments in the
8226 RPMI myeloma cellines
The number of 300 cells was carefully optimised on the basis of preliminary experiments.
Lower cell numbers decreased sensitivity of gene detection whereas increase of the cell
input decreased gene amplification efficiency, possibly caused by PCR inhibition by cell
lysis products (data not shown).

Efficiency of gene amplification
Gene amplification in 2.5 ul of cDNA obtained from 300 cells was both linear and efficient
as assessed by serial dilution of the initial cDNA (figure 1A) and dilution of RPMI dox40
cells (expressing MDR-1 target) to a background of RPMI parental cells (figure 1B) to
exclude possible dilution of inhibitory components present in the initial cDNA. Relative
expression (∆Ct) of target genes to 18S was similar compared to standard (50 ng GITC
isolated RNA) RT- PCR showing that target gene amplification in 300-cell isolated cDNA is
representative (table 1).
                              A

                                                  40,00



                                                  35,00                                             y = -3,282x + 32,909
                           Threshold cycle (Ct)




                                                                                                         R2 = 0,9976

                                                  30,00



                                                  25,00            GAPDH
                                                                                                   y = -3,2462x + 30,922
                                                                   MDR1
                                                                                                        R2 = 0,9987

                                                  20,00
                                                           -1      -0,5         0          0,5              1               1,5
                                                                               log cell amount


                            B

                                                  4 0,00
                        Threshold cycle (Ct)




                                                  3 5,00                                         y = -3,2485x + 26,72
                                                                                                      R2 = 0,9571

                                                  3 0,00


                                                  2 5,00        MDR-1

                                                  2 0,00
                                                           -2           -1,5         -1              -0,5               0
                                                                               log dox40 cells

Figure 1: Efficiency of gene amplification in 300-cell real-time quantitative RT-PCR. A. Serial dilution
of 300-cel cDNA (2.5 µl) obtained from myeloma RPMI dox 40 celline in lysis/Rt-solution showing high

38
___________________________________________________________________   GENE EXPRESSION IN HEMATOPOIETIC STEM CELLS _____



efficiency of PCR reactions over a 1:125 dilution. Efficiency of reference (GAPDH) and target gene (MDR-
1), indicated by the slope of the log dilution curve, are equal (-3.25 and –3.28 respectively). B. Dilutional
experiment of RPMI dox40 cells (expressing MDR-1 target) to a background of RPMI parental cells with a
constant 300-cell number input ; 300, 95, 30, 9 and 3 cells RPMI 8226 dox40 were sorted in 0, 205, 270,
291 and 297 RPMI 8226 parental cells. Efficiency of MDR-1 amplification was –3.25 (R2 0.96).

Table 1: Quantitation of gene expression in 300-cell real-time quantitative RT-PCR: comparison with
standard RT-PCR.

Target          50 ng                            ∆Ct                       300 cell                ∆Ct
18S             10.27                                                      17.87
GAPDH           18.65                            8.38                      26.29                   8.42
MDR-1           19.20                            8.93                      26.64                   8.77
MRP-1           25.65                            15.38                     33.61                   15.74
LRP             23.40                            13.07                     31.58                   13.71

Threshold cycle (Ct) of different target genes in 2.5 µl 300-cell isolated myeloma RPMI dox 40 cDNA versus
50 ng GITC-isolated cDNA using real-time quantitative PCR. Ct value is mean of in duplicate assessment.
Comparable delta Ct ( Ct target genes minus Ct 18S) indicates representative gene amplification in 2.5 µl 300-
cell isolated cDNA compared to standard 50 ng GITC isolated cDNA.



Sensitivity and reproducibility of gene quantitation
The low-copy gene porphobilinogen dehydrogenase (PBGD) was invariably detected in
duplicate in 300 cell cDNA (2.5 µl) at a Ct of 33.0 (mean of four experiments).
GAPDH could still be detected in duplicate (Ct 35.63) in a 1000-fold dilution of the initial
2.5 µl cDNA. Cell sorting and RNA-processing caused little variation in quantitative gene
expression results; the average Ct of independently sorted samples (n=10) was 28.252
(SD 0.57) for MDR-1 and 19.96 (SD 0.81) for 18S. The mean variation between duplicates
was 0.21 Ct (SD 0.05) and 0.40 Ct (SD 0.19) respectively. In two completely independent
experiments the average Ct was 17.56 and 18.21 for 18S, 27.35 and 27.04 for MDR-1 and
33.21 and 33.23 for PBGD indicating a high interexperimental reproducibility.

Quantitative gene expression in CD34CD38 subpopulations in human
normal and leukemic bone marrow

Expression of 18S rRNA and the housekeeping gene GAPDH
The constant input of 300 cells in the samples allows an estimation of the level of
housekeeping gene expression in different hematopoietic subpopulations by comparison
of the threshold cycle (Ct). Figure 2 shows the 18S and GAPDH gene expression in human
CD34+CD38- and CD34+CD38+ cells in human normal (n=6) and leukemic bone marrow
(n=6).




                                                                                                                    39
_____   CHAPTER 2 _____________________________________________________________________________________________________


                     40



                     30
                Ct


                     20

                                                                                                 NBM
                     10
                                                                                                 AML


                     0
                               18S             18S            GAPDH           GAPDH
                                38-             38+             38-             38+
Figure 2: Expression of housekeeping genes in normal and leukemic CD34+ subpopulations as
determined by 300-cell real-time quantitative RT-PCR. Mean levels and standard deviation of 18S rRNA and
GAPDH expression in 300-cell samples of normal and leukemic CD34+CD38- and CD34+CD38+ cellpopulations
as indicated by the threshold cycle (Ct).



No significant differences exist in GAPDH expression between the different CD34+CD38-
and CD34+CD38+ populations in NBM (mean Ct 31.41 ± 1.37 and 29.54 ± 0.63) and AML
(Ct 31.37 ± 1.23 and 31.65 ± 0.84). The relatively low Ct in the normal CD34+CD38+
subpopulation is paralleled by low Ct’s for PBGD and 18S (mean delta Ct’s comparable
between subpopulations) indicating that this reflects initial amounts or quality of RNA in
these samples rather than endogenous GAPDH expression. 18S expression varied widely
within the primitive hematopoietic CD34+CD38- samples in both normal bone marrow (Ct
18.94-25.63; SD 2.60) and AML (Ct 17.63-27.32; SD 3.36) with a 100-fold and 1000-fold
variation respectively calculated with the equation shown in figure 1. A significantly higher
Ct was found for 18S in CD34+CD38+ cells in AML ( mean Ct 23.55; SD 1.91) compared to
normal bone marrow (mean Ct 19.04; SD1.45, p=0.04). This significantly lower expression
of 18S in the leukemic CD34+CD38+ cells was confirmed when the expression was
normalised for GAPDH according to the formula described in the section methods and
depicted as Xn= k. copies target gene per copy GAPDH (figure 3);
In AML samples, 18S expression in the CD34+CD38+ population (mean Xn 376 ± 410) was
lower than in the CD34+CD38+ in normal bone marrow population ( mean Xn 1625 ± 635;
p= 0.002) and the leukemic CD34+CD38- subpopulation (mean Xn 2263 ± 2731; p=0.07).




40
___________________________________________________________________                            GENE EXPRESSION IN HEMATOPOIETIC STEM CELLS _____


                                                                      8000




                 Relative 18S expression: K.copies
                                                                      6000

                                                     18S/copy GAPDH


                                                                      4000




                                                                      2000




                                                                         0
                                                                             38-         38+                  38-         38+
                                                                                   NBM                              AML

Figure 3: Normalised 18S rRNA content in CD34+ subpopulations in normal bone marrow and AML as
determined by real-time quantitative RT-PCR. Expression in CD34+CD38- and CD34+CD38+ cells from paired
samples in normal bone marrow and AML. Expression (Xn) is normalised for the expression of the standard gene
GAPDH.


Expression of drug resistance related genes
We studied the expression of the ABC transporter genes MDR-1 and MRP-1 and the
lung-resistance related protein (LRP) gene in the normal hematopoietic CD34+CD38- and
CD34+CD38+ cells using GAPDH as endogenous reference (table 2). MDR-1 was detected
in all (6/6) CD34+C38- samples but expression was approximately 18-fold lower in the more
differentiated fraction (1/6 CD34+CD38+ samples positive) Three CD34+CD38+ samples
showed borderline expression. MRP-1 was detected in all samples (n=4) but expression
was also invariably lower in CD34+CD38+ cells compared to the CD34+CD38- population
indicating a approximately 3.8-fold lower expression in the more differentiated fraction.
Expression of LRP was detected in all (4/4) CD34+CD38- and CD34+CD38+ samples
without significant differences in mean expression between subpopulations However, a
lower expression in the CD34+CD38+ cells was noted in 3/4 individuals (Xn CD38- vs
CD38+ 0.10 vs 0.018, 0.07 vs 0.01 and 0.06 vs 0.009 respectively).




                                                                                                                                             41
_____   CHAPTER 2 _____________________________________________________________________________________________________



Table 2: Quantitative expression of drug resistance related genes in the CD34+CD38- and CD34+CD38+
cell populations in human normal bone marrow as determined by 300-cell real-time quantitative RT-PCR.

                                        CD38-                   CD38+                  Difference (P-value)
MDR-1
Samples positive                        6/6                     1/6
Mean Ct ( ± SD)                         34.73 ± 1.10            37.09
Mean Xn ( ± SD)                         0.09 ± 0.03             0.005                  18-fold (p<0.0005)
MRP-1
Samples positive                        4/4                     4/4
Mean Ct ( ± SD)                         32.38 ± 0.93            33.30 ± 1.73
Mean Xn ( ± SD)                         0.38 ± 0.18             0.10 ± 0.06            3.8-fold (p<0.05)
LRP
Samples positive                        4/4                     4/4
Mean Ct ( ± SD)                         34.82 ± 0.70            35.28 ± 2.23
Mean Xn ( ± SD)                         0.09 ± 0.03             0.05 ± 0.08            1.8-fold (NS)

Relative quantitation (Xn) is defined as the number of target gene copies relative to the number of the standard
gene (GAPDH) copies. Ct is threshold cycle in 2.5 µl cDNA of 300-cell sample.




Discussion

We present the use of a real-time quantitative RT-PCR method for the precise quantitation
of gene expression in small subsets of human hematopoietic cells.
Combining modified RNA isolation and adjusted PCR-protocols with the sensitivity and
accuracy of real-time quantitative PCR we were able to detect and to quantitate low-copy
gene expression without the need of prior cDNA amplification or nested-PCR strategies.
cDNA amplification is a valuable tool for stem cell gene expression assessment though
interpretation of quantitative results is critically dependent on representative cDNA
amplification(9). Furthermore, the hybridisation signal is usually represented by a smear due
to the presence of truncated cDNA strands of different lengths, compromising sensitivity
and reliability of gene quantitation by end-point evaluation(12). cDNA amplification is also
limited to the detection of transcripts with unique 3’ terminal sequences(10).
Though real-time quantitative RT-PCR has been reported to be applicable to quantitate
gene expression in single cells(22), in our hands the limit of 300 cells was carefully optimised
and lowering the input cell number decreased the detection frequency of the low-copy gene
PBGD. Therefore the method described in this paper cannot be applied to single cells
without prior cDNA amplification strategies.
For quantitative analysis of gene expression the expression of housekeeping genes are
often used as internal standards. The ideal internal standard should be expressed at a
constant level among different cell populations and individuals and should be unaffected
by the experimental conditions(25). The stable input of mRNA of 300 cells in each sample
in our study allows comparison of endogenous gene expression by direct measurement
of Ct’s. Although GAPDH expression has been reported to vary during the cell cycle(26)


42
___________________________________________________________________   GENE EXPRESSION IN HEMATOPOIETIC STEM CELLS _____



and developmental stage(27) no significant differences in expression were found between
primitive and more committed progenitor populations in normal and leukemic bone
marrow.
In contrast, though rRNA is generally considered a suitable and more reliable internal
control than housekeeping genes(28), we found a striking (100-1000-fold) individual variety
in 18S rRNA levels in both normal and leukemic primitive CD34+CD38- cell samples.
Control of rRNA expression has been correlated with differentiation programs(29,30) and
the metabolic activity in mouse embryonic hematopoietic stem cells(31). Whether the wide
variety in 18S rRNA in human primitive CD34+CD38- hematopoietic cells reflect differences
in differentiation within the compartment between individuals is unclear.
A second important finding is the significantly lower level of 18s rRNA in the leukemic
CD34+CD38+ blasts compared to both the leukemic CD34+CD38- compartment and the
CD34+CD38+ subpopulation in normal bone marrow. This finding seems to be in line
with previous reports on impaired processing of ribosomal precursor RNA leading to a
failure of rRNA synthesis and relatively small amounts of 18S rRNA in blast cells of acute
leukemia(32-34). Our results suggests that the putative defect in rRNA synthesis occurs early
in leukemogenesis on the CD34+ progenitor level.
The variety in 18S expression, precluding its use as an appropriate reference gene, stresses
the importance of validating reference genes on expression in specific subpopulations
when designing quantitative gene expression studies.
We studied the normalised quantitative expression of the drug-resistance related genes
MDR-1, MRP-1 and LRP in human primitive CD34+CD38- cells. The expression of MDR-
1 and its protein product P-glycoprotein in hematopoietic stem cells is well known(35) and
likely responsible for the rhodamine-123 dull phenotype of these cells(18). We found that
expression of the gene is strongly downregulated in more differentiated progenitors, in
line with recent findings studying MDR-1 expression in these cells(36). The downregulation
of MRP-1 and LRP during lineage commitment was less striking compared to MDR-1.
Expression of these genes persisted in more differentiated CD34+CD38+ progenitors.
It is tempting to speculate on the function of these genes in human hematopoietic stem
cells. A role in protection of the hematopoietic stem cell against toxic compounds has
been shown for P-glycoprotein(16). Similar detoxification functions are conceivable for
MRP, a membrane bound drug efflux pump, known to have a broad substrate specificity(37),
and LRP which has been implicated in the transport of diverse substrates from the cell
nucleus to the cytoplasm(38). Additionally, MDR-1 and MRP-1 might play a role in prolonged
stem cell survival by protection against apoptosis(39). Finally, it has been speculated that
ABC transporters extrude molecules required for commitment to differentiation, thereby
maintaining the quiescent stem cell state(36-40). Further studies addressing the significance
of these genes in the hematopoietic stem cell and hematopoiesis are clearly warranted.
The technique described in this study will facilitate these and other experiments addressing
gene expression in these small cell subpopulations.


                                                                                                                    43
_____   CHAPTER 2 _____________________________________________________________________________________________________



Acknowledgments

The authors wish to thank A. Pennings and G. Vierwinden for their technical assistance and
and L. van de Locht for help in designing primer and probes for real-time PCR.



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46
                                              CHAPTER 3
             SINGLE CELL IMAGE ANALYSIS TO ASSESS
ABC-TRANSPORTER MEDIATED EFFLUX IN HIGHLY PURIFIED
                             HEMATOPOIETIC PROGENITORS




                                                       MARC HGP RAAIJMAKERS1
                                                        GERARD VAN DEN BOSCH2
                                                                JAN BOEZEMAN2
                                                           THEO J.M. DE WITTE1
                                                       REINIER A.P. RAYMAKERS1

                DEPARTMENT OF HEMATOLOGY1 AND CENTRAL HEMATOLOGY LABORATORY2,
                          UNIVERSITY MEDICAL CENTER NIJMEGEN, THE NETHERLANDS.




                                                   CYTOMETRY;49:135-42, 2002
_____   CHAPTER 3 _____________________________________________________________________________________________________




48
__________________________________________________   ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____



Abstract

Background
Normal and malignant hematopoietic stem cells are characterised by their capacity to
actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC)
transporters to this phenomenon is largely unknown due to the small stem cell numbers
limiting the use of standard methods to assess functional efflux.

Methods
We used epifluorescence microscopy (EFM) in combination with single cell image analysis
to study ABC-transporter mediated efflux in highly purified, viable, CD34+CD38- cells
sorted on an adhesive biolayer. P-glycoprotein and MRP mediated efflux were quantitated
using fluorescent substrates (rhodamine-123 and calcein AM) and specific inhibitors
(verapamil and probenecid, respectively).

Results
Feasibility, sensitivity and reproducibility of rhodamine-123 efflux quantitation using
single cell EFM was shown in cell lines and compared to standard flow cytometrical
assessment. P-glycoprotein mediated transport was higher in CD34+CD38- cells than in
more differentiated progenitors (mean efflux index 2.24 ± 0.35 and 1.14 ± 0.11 respectively,
p=0.01) P-glycoprotein mediated transport was the main determinant of the rhodamine dull
phenotype of these cells. Additionally, significant MRP-mediated efflux was demonstrated
in CD34+CD38- and CD38+ cells (mean efflux index 1.42 ± 0.19 and 1.28 ± 0.18,
respectively).

Conclusion
The described method is a valuable tool for assessment of ABC-transporter mediated efflux
in highly purified single cells. Both P-glycoprotein and MRP mediated efflux are present in
human CD34+CD38- hematopoietic stem cells.



Introduction

In human normal hematopoiesis a rare subpopulation of bone marrow cells have stem cell
characteristics defined as long-term proliferative capacity, self-renewal and engraftment
potential. Identification and isolation of these cells is based on surface marker expression
(e.g. CD34+CD38-lin-)(1) or the ability to exclude fluorescent dyes such as rhodamine-
123 and hoechst (rhodamine-123/hoechst “dull”-phenotype)(2-6). Similar to the situation in
normal hematopoiesis, leukemic cells with stem cell characteristics have been identified
in acute myeloid leukemia, based on the reduced capacity of these cells to accumulate


                                                                                                                   49
_____   CHAPTER 3 _____________________________________________________________________________________________________



fluorescent dyes(7,8). The dull-phenotype of stem cell populations has been attributed to
dye efflux caused by the ATP-binding cassette transporter P-glycoprotein(9,10). Recent data,
however, on gene expression in mouse and human normal and leukemic CD34+CD38-
stem cell compartments suggest the presence of other ABC-transporters in these primitive
hematopoietic progenitors(8-12). ABC-transporters (e.g. P-glycoprotein, multidrug-resistance
proteins (MRP) and breast cancer related protein (BCRP) are involved in the export of a
broad array of chemotherapeutical agents, a phenomenon referred to as multiple drug
resistance (MDR). Activity of these transporters could promote survival of normal and
leukemic stem cells during chemotherapeutic treatment. Therefore, identification and
characterisation of the ABC-transporter mediated efflux in stem cell populations is of great
clinical importance. The expression of ABC-transporters can be assessed by determining
gene or protein expression but discrepancies with chemoresistance and functional drug
efflux have been reported(13) possibly due to post translational modification of proteins or
the presence of yet unidentified efflux pumps. Therefore, assessment of functional drug
efflux is an important tool in determining the significance of ABC-transporters(14). Functional
tests are based on the accumulation of fluorescent dyes such as rhodamine 123 and
calcein-AM(15,16), which are substrates for the P-glycoprotein and MRP mediated transport.
Fluorescence analysis is generally performed by cellular imaging using flow-cytometry(17).
Limitations of flow-cytometric assessment of drug efflux are the relatively large numbers
of cells needed to perform the assays (the CD34+CD38- compartment comprises
approximately 0.01 % of the mononuclear fraction of human normal bone marrow) and the
problems combining certain fluorescent dye substrates with immunophenotypical markers
in double-labelling techniques. Additionally, these methods are not suited to continuously
study efflux kinetics and the effect of efflux modulators at the level of single cells. In an effort
to overcome these limitations we developed an ABC-transporter-mediated efflux assay
combining highly purified cell sorting on adhesive protein layers and computerised single
cell image analysis to allow quantitative and kinetic study of efflux mechanisms in small
subsets of hematopoietic cells. Using this assay we studied the levels of P-glycoprotein
and MRP related efflux in normal primitive CD34+CD38- hematopoietic cells compared to
more differentiated CD34+CD38+ progenitors.



Material and methods

Cell lines and human CD34+ bone marrow cells
The RPMI 8226 myeloma parental cell line and the doxorubicin resistant subline RPMI
8226 Dox 40 expressing P-glycoprotein(18) were maintained as a suspension in RPMI
1640 medium supplemented with 10% (v/v) fetal calf serum, 1% penicillin (100U/mL), 1%
streptomycin (100µg/mL) and 1% L-glutamine at 37°C in a humidified atmosphere with 5%
CO2 in the presence of 400nM doxorubicin. Experiments were performed on cells in an


50
__________________________________________________   ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____



exponential growth phase and cultured for 1-2 weeks without doxorubicin. The human T-
cell Jurkat cell line was cultured under the same conditions as the RPMI 8226 myeloma cell
line without doxorubicin. Bone marrow was obtained from healthy allogeneic bone marrow
transplantation donors after informed consent. Mononuclear cells were isolated by Ficoll
1.077 g/mL (Pharmacy Biotech, Uppsala, Sweden). CD34-positive cells were isolated using
directly conjugated CD34 antibody- coupled immunomagnetic beads (M-450 coated with
‘561’ a class III epitope anti-CD34, Dynal, Oslo, Norway). Isolation, cryopreservation and
thawing procedures of the CD34+ cells have been described previously(10). Purity of CD34
cells was controlled by labelling with CD34-Cy5 monoclonal antibody (Beckton Dickinson,
BV, Etten- Leur, The Netherlands) using flow cytometry.

Chemicals
Mussel adhesive protein (MAP; bioglue ultra; Swedish BioScience Lab., Floda, Sweden)
is a formulation of polyphenolic proteins which are the key components of the anchoring
glue secreted by the marine mussel Mytilus edulis(19). The usefulness of MAP for
the immobilisation of non-adherent cells for fluorimetric studies has been described
previously(20). MAP was diluted in PBS and stored at a stock concentration of 1 mg/ml
(4°C).
Rhodamine-123 (Sigma chemical Co., St. Louis, MO, U.S.A.) was dissolved in distilled
water and stored in stock concentration 0.1 mg/ml (4°C). Calcein-AM (Molecular probes,
OR, U.S.A.) was dissolved in DMSO and stored in stock solution 1 mg/ml (4°C). Verapamil
(Knoll AG, Ludwigshaven, Germany) was stored at room temperature in stock concentration
2.5mg/ml. Probenecid (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in 1
ml phosphate buffered saline (PBS) with 40 µl 1 M NaOH and stored at –20°C in stock
concentration 5.708 mg/ml.

Flow-cytometric isolation of CD34+CD38- cells and cell sorting
CD34+ bead selected cells were stained with CD34- CY5 and CD38- phycoerythrin (PE)
monoclonal antibodies (Beckton Dickinson, BV, Etten-Leur, The Netherlands) at 4°C for
30 minutes. A Epics Elite flow cytometer ( Coulter, Miami, Florida, U.S.A) was used to sort
cell populations. Gating on forward and right angle scatter was used to exclude dead cells
and debris. Cells were excitated with a single Argon ion laser emitting at 548 nm and 647
nm , running at 15 mW (standard setting). CD34+CD38- cells were defined as the 1% of
CD34+ cells with the lowest CD38-PE density as shown in figure 1. CD34+CD38+ cells
were sorted from a gate positioned as indicated in figure1, containing approximately 25%
of CD34+ cells. CD34+CD38- cells appeared in a consistently restricted light-scattering
region confirming the lymphoid appearance of these primitive progenitors(21)(figure 1).




                                                                                                                   51
_____   CHAPTER 3 _____________________________________________________________________________________________________




                      A                                        B




                       CD34-Cy5C
                                   A 1%

                                                                     A 1.0 %           B 25.7%




                                      CD38-PE                             CD38-PE
                     C                                         D
                       FS




                                                                FS




                                          RAS                                    RAS
Figure 1: Flow-cytometric analysis of bead-selected CD34+ bone marrow cells stained with CD34-Cy5 and
CD38-PE. Scatter and histogram (A and B) based on CD38-PE antigen density level. Sort region A defined as 1%
of the overall CD34+ population with the lowest CD38-PE antigen density. Region B defines the CD34+CD38+
subpopulation. The lymphoid character of the primitive progenitors in region A is confirmed by a restricted light-
scattering region (C) compared the heterogeneous light scattering properties of the CD34+CD38+ population
(D).


Rhodamine-123 and Calceine-AM accumulation and efflux studied using
direct cell sorting and single cell image analysis

Cell immobilisation and manipulation
Five hundred cells of the cell lines or human CD34 progenitor cells were sorted in 8 µl of
washing buffer solution containing NaCl 0.9% supplemented with natriumdifosfate (6.6% v/
v), albumin (1% v/v) and glucose (1% v/v) applied on a glass cover slip coated with mussel
adhesive protein (10 µl/cm2).
Cells were allowed to adhere for 10 minutes in a perfusion chamber constructed to enable
cell environment manipulation (incubation and washing steps) within the microscope
environment (figure 2). The microscope was placed in an incubator to allow all procedures
to run at 37°C which is essential for physiologic efflux studies.




52
__________________________________________________   ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____




Figure 2: Inverted microscope system for analysis of dye efflux placed in a 37°C incubator. Inset: Holder
of adhesive protein coated cover slip and perfusion chamber. The coated cover slip with the cell population of
interest is positioned in the middle of the holder and the perfusion chamber is placed on it. Washing steps and
addition of specific dye efflux modulators are performed through a perfusion system coupled to the perfusion
chamber.


Uptake and efflux of fluorescent probes
Studies on P-glycoprotein mediated efflux were performed with rhodamine-123 (0.30 µgr/
ml) as a substrate for P-glycoprotein and verapamil (20 µgr/ml) as an inhibitor of efflux.
Though rhodamine-123 is a MRP substrate as well, it was shown that in short term (60 min)
drug accumulation assays, rhodamine-123 can be regarded as P-glycoprotein specific(22).
Moreover, in CD34+ cells verapamil does not seem to inhibit MRP(23). For MRP-mediated
efflux calcein acetoxymethyl ester (calcein-AM, 10µgr/ml)) was used as a substrate. Since
P-glycoprotein actively extrudes the calcein-AM but not the fluorescent calcein, which is
selectively extruded by MRP(16), all experiments on MRP function were performed in the
presence of verapamil to block P-glycoprotein-mediated efflux of calcein-AM. Probenecid
(0.20mM), a specific and effective modulator of MRP(24) was used to modulate the calcein
efflux by MRP. After immobilisation, cells were incubated with either rhodamine-123 or
calcein-AM dissolved in Iscove’s containing 0.5% v/v heat inactivated fetal calfs serum
(FCS, Hyclone, Logan, Utah, U.S.A.) with or without efflux modulator during 30 minutes.
Cells were washed with washing buffer at 37°C, again in the presence or absence of efflux
modulators, and allowed to efflux the dye for an additional 15 minutes. The time interval of 15
minutes was based on the observation of efflux kinetics in the CD34+CD38- compartment
showing the majority of rhodamine-123 efflux taking place within 15 minutes.


                                                                                                                   53
_____   CHAPTER 3 _____________________________________________________________________________________________________



Fluorescence microscopy
During efflux, focal planes were adjusted and conventional light microscopy images were
stored from different fields to identify cells. Fluorescence images were acquired after 15
minutes of efflux. The images were acquired on a Zeiss Axiovert 35M inverted microscope
(Thorwood, New York, USA) equipped with a 40x oil (N.A. 1.3) objective, perfusion chamber
with possibility for using disposable glass cover slips and a cooled 756x580 pixel resolution
CDD-camera (Variocam, PCO computer optics, Kellheim, Germany) coupled with the pixel
pipeline in a Macintosh Quadro 800. The microscope was placed in a 37°C controlled
incubator. Cells were exited with a mercury arc lamp using band pass filter (BP) 440-490
nm for rhodamine-123 and calcein or BP 510-560 nm for propidium iodide (PI). Emission
was measured with BP 515-565 and longpass filter 610nm. The perfusion chamber was
positioned in x, y, and z directions with an EK 86 MTP scanning table (Marzhauser, Wetzlar,
Germany) controlled by the serial interface of the Macintosh or manually by the operator.
Calibration of fluorescence signal detection was performed using flowset fluorespheres
(Coulter Corporation, FI, U.S.A.). Finally, propidium iodide (10 µg/ml, Sigma chemical Co.,
St. Louis, MO, U.S.A.) was added to the washing buffer to confirm cell viability. Cell viability
typically exceeded 90% of cells. Dead cells were excluded from analysis.

Image analysis
Fluorescence intensity analysis of individual cells was performed on TCL-Image 4.6
software package (TNO, Delft, The Netherlands). Regions of interest (ROI) were manually
defined by the operator around cells as well as around empty backgrounds in bright field
images. The area (A) and fluorescence (F) of each ROI was determined in fluorescence
images. The fluorescence intensity (FI) of the cell in a ROI was calculated:

FI= Fcell / A cell – F background /A background

This formula corrects for differences in cell size between lymphoid-like CD34+CD38- cells
and the larger CD34+CD38+ cells (fig 1) as well as background illumination. At least 30
cells (range 30-50) were evaluated per sample. Fluorescence intensity of the overall cell
population is expressed as mean fluorescence intensity (MFI) and depicted in arbitrary
fluorescence units (AU). P-glycoprotein and MRP-mediated efflux was quantitated as
the ratio of mean fluorescence intensity in the presence/absence of a specific inhibitor
(verapamil and probenecid) and depicted as “efflux index”.

Rhodamine-123 efflux measurement using flow-cytometry
In order to compare single cell image analysis with conventional flow-cytometry, P-
glycoprotein-mediated efflux studies on the RPMI 8226 cell lines were performed using both
methods. For flow-cytometric efflux assessment, RPMI 8226 parental and Dox 40 cells were
incubated (2.105 cells/ml) in Iscove’s medium with a rhodamine-123 concentration of 0.3


54
__________________________________________________   ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____



µg/ml (30 min, 37°C) in the presence or absence of 20µg/ml verapamil. After centrifugation,
cells were resuspended in dye-free Iscove’s medium again in the presence or absence of
verapamil and incubated at 37°C to allow efflux to take place during 15 minutes. Cellular
dye content after 15 minutes efflux was measured with the Epics Elite Flow cytometer.
Cells were excited with a single Argon ion laser emitting at 488 nm, running at 15 mW
(standard setting). Gating on forward and right angle scatter was used to exclude dead
cells and debris. Fluorescence intensity of cellular rhodamine was assessed using a 525
nm BP filter.

Statistics
For statistical analysis of differences in MFI between subpopulations the t- test and two-
way ANOVA analysis were used as appropriate.



Results

Validation of efflux measurement using single cell image analysis

Sensitivity and linearity of rhodamine-123 fluorescence quantitation
In order to assess the sensitivity and linearity of rhodamine-123 fluorescence detection we
incubated Jurkat cells, which do not express P-glycoprotein, at increasing concentrations
(0.01-0.1 µgr/ml) of rhodamine-123 during 30 minutes and quantitated the fluorescence
intensity (figure 3a). A fluorescence signal was detected in Jurkat cells incubated with the
lowest (0.01 µgr/ml) concentration of rhodamine-123 and cellular fluorescence intensity
showed a linear increase with rhodamine-123 concentration (correlation coefficient
0.99) within a range (2000-25000 AU) similar to that observed in CD34+ hematopoietic
progenitors (data not shown).

Demonstration of P-glycoprotein mediated efflux in the 8226 RPMI myeloma cell lines
Rhodamine-123 efflux in the RPMI 8226 Dox 40 and parental cell line was assessed
using single cell image analysis as well as standard flow cytometry (figure 3b). A lack of P-
glycoprotein mediated efflux was demonstrated by both assays in the RPMI 8226 parental
cell line (efflux index 0.72 ± 0.14 and 0.86 ± 0.09 respectively). The RPMI 8226 Dox 40 cells
showed a significant P-glycoprotein mediated efflux using both FCM (index 36.20 ± 0.32)
and single cell image analysis (index 4.58 ± 1.83). Analysis of P-glycoprotein mediated
efflux in Dox 40 cells on consecutive days in order to assess the reproducibility of efflux
measurement using single cell image analysis demonstrated no significant differences in
fluorescence intensities or efflux indices; The MFI with/without verapamil was 35244 ( ±
2940) / 10356 ( ± 1250) and 41669 ( ± 8334)/ 9104 ( ± 1802) respectively. This resulted in
efflux indices of 3.40 ± 0.69 and 4.58 ± 1.83 respectively (differences not significant).


                                                                                                                   55
_____   CHAPTER 3 _____________________________________________________________________________________________________


                A
                              30000

                              25000

                  MFI (AU)    20000
                                                                                      R2 = 0.9901
                              15000

                              10000

                               5000

                                    0
                                        0.00   0.02       0.04       0.06      0.08         0.10      0.12
                                                      Rhodamine-123 concentration (ug/ml)

                B                35

                                 30

                                 25

                                 20

                                 15

                                 10

                                    5

                                    0
                             FCM                      Parental                               Dox 40


                 C
                              70000

                              60000

                              50000

                              40000

                              30000

                              20000

                              10000

                                    0
                                                      Parental                               Dox 40
                             SCIA
Figure 3: Validation experiments for single cell efflux measurement. A. Confirmation of linearity and
sensitivity of rhodamine-123 fluorescence detection with increasing incubation concentrations of rhodamine-123
in a human T-cell Jurkat cell line. B. Comparison of rhodamine-123 fluorescence with (black bars) and without
(white bars) verapamil in the human myeloma RPMI 8226 parental (sensitive) and doxorubicin resistant (Dox 40)
cell line using single cell image analysis (SCIA) and flow cytometry (FCM).




56
__________________________________________________          ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____



P-glycoprotein and MRP mediated efflux in primitive hematopoietic
progenitors

P- glycoprotein mediated rhodamine-123 efflux
Rhodamine-123 accumulation and efflux in the absence and presence of verapamil was
assessed in the CD34+CD38- and CD34+CD38+ subpopulations in four samples (figure
4). CD34+CD38- progenitors had a rhodamine-123 “dull”phenotype; in the absence
of verapamil the rhodamine-123 fluorescence intensity was invariably lower in the
CD34+CD38- subpopulation compared to the CD34+CD38+ population (average MFI
14.27 vs 25.97 AU respectively).
    A                              CD38-                                                          CD38+

                   - Verapamil                   + Verapamil                    - Verapamil                    + Verapamil




    B
                          CD38-                                                  CD38+

         Sample           MFI ± SEM                    P-value   Efflux          MFI ±SEM                     P-value   Efflux

                          - Verapamil    + Verapamil                             - Verapamil    + Verapamil
         1                13.96±0.82     25.19±1.17    <0.0001   1.80±0.19       21.63±0.86     26.13±1.30    0.036     1.21±0.11
         2                15.05±0.65     26.04±1.85    <0.0001   1.73±0.20       23.37±1.06     20.87±1.07    0.10      0.89±0.09
         3                12.29±1.52     39.79±5.64    <0.0001   3.23±0.86       17.84±0.58     25.05±1.48    0.00003   1.40±0.13
         4                15.79±1.15     34.77±3.23    <0.0001   2.20±0.36       41.02±3.19     42.54±3.38    0.75      1.04±0.16
                                                               #                                                    #
         Average          14.27          31.45         < 0.0001 2.24±0.35        25.97          28.65         0.017     1.14±0.11


    C              40

                   35

                   30
                   25
                                                                                               - Verapamil
        MFI (AU)




                   20                                                                          + Verapamil
                   15

                   10

                    5
                    0
                                        38-                          38+

Figure 4: P-glycoprotein mediated efflux in CD34+ hematopoietic progenitors. A. Typical example of
rhodamine-123 fluorescence images in CD38- and CD38+ subpopulations after accumulation and efflux in the
presence or absence of verapamil. B. Table showing rhodamine-123 fluorescence intensity (MFI) in the presence/
absence of verapamil and P-glycoprotein efflux index in CD34+ subpopulations in four bone marrow samples (# =
as assessed by two-way ANOVA analysis). C. Rhodamine fluorescence intensity in presence/absence verapamil
in CD34+ cell populations: average of four samples.
                                                                                                                                 57
_____   CHAPTER 3 _____________________________________________________________________________________________________



Addition of verapamil increased the accumulation of rhodamine-123 in CD34+CD38- cells
significantly in all (4/4) samples (average MFI without/with verapamil 14.27 and 31.45 AU
respectively, p<0.0001) but only in 2/4 CD34+CD38+ samples (average MFI without/with
verapamil 25.97 and 28.65 respectively, p=0.017). Interestingly, addition of verapamil
completely restored rhodamine-123 accumulation to the level of more differentiated
CD38+ progenitors in all samples, indicating that P-glycoprotein mediated efflux is the
main determinant of the rhodamine “dull” phenotype of these cells. Consequently, the efflux
index (defined as the ratio of MFI presence/absence inhibitor) was invariably higher in the
CD34+CD38- subpopulation compared to CD34+CD38+ cells (mean value 2.24 ± 0.35 vs
1.14 ± 0.11, p=0.01).

MRP mediated calcein-AM efflux
The role of MRP-mediated efflux in CD34+CD38- hematopoietic progenitors using the
MRP substrate calcein-AM and the MRP inhibitor probenecid was assessed in three bone
marrow samples (figure 5).




Figure 5: MRP mediated efflux in CD34+ hematopoietic progenitors. A. Example of calcein fluorescence
images in CD38- and CD38+ subpopulations after accumulation and efflux in the presence or absence of
probenecid. B. Table showing calcein fluorescence intensity (MFI) in the presence/absence of probenecid and
MRP efflux index in CD34+ subpopulations in three bone marrow samples (# = as assessed by two-way ANOVA
analysis).




58
__________________________________________________   ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____



MRP mediated efflux, as indicated by a significant increase of calceine fluorescence after
addition of probenecid, was found in 2/3 CD34+CD38- samples (efflux index 1.44 ± 0.12,
p<0.0001 and 1.73 ± 0.15, p<0.0001, respectively). The lack of MRP related efflux in the
remaining sample occurred both in the CD34+CD38- and CD34+CD38+ cell population
and was accompanied by a discordant high calceine fluorescence in these cells compared
to other samples. Interestingly, this sample displayed the highest P-glycoprotein mediated
efflux in CD34+CD38- cells. In contrast to P-glycoprotein mediated efflux, no significant
difference in MRP-mediated efflux was found between CD38- and CD38+ subpopulations
(average efflux index 1.42 ± 0.19 and 1.28 ± 0.18 respectively).



Discussion

We used epifluorescence microscopy (EFM) in combination with image analysis to study
ABC-transporter mediated efflux in highly purified cells from hematopoietic stem cell
populations. This experimental approach enabled the study of fluorescent dye efflux in
small numbers of immunophenotypically defined cells.
Epifluorescence microscopy has previously been shown to allow accurate fluorescence
measurements in the assessment of efflux and intracellular redistribution properties of
ABC-transporters in human cells and yeast(25-27).
However, instrumental and procedure optimisation is required to circumvent limitations
commonly encountered in EFM(28,29). In our experimental approach, the problem of out of
focus plain image artefacts was limited since cells were adhered on a bioadhesive layer
before analysis, reducing out of focus information. To further reduce artefacts, focal planes
were adjusted for each field before fluorescence images were taken. Background noise and
shading was corrected for by spatially matched references for each evaluated target cell.
Finally, photobleaching(30) was prevented by short light exposure times and the selection
of different fields for consecutive images. No significant differences in mean fluorescence
intensity between consecutive images were observed (data not shown).
An important issue is the sensitivity of efflux detection using EFM. In general, fluorescence
detection is more sensitive using FCM compared to EFM due to higher and more
directed excitation intensities of argon lasers compared to mercury lamps and the lack of
fluorescence background in FCM. We compared the sensitivity of efflux detection in EFM to
flow cytometric analysis. The higher efflux ratio in the RPMI 8226 Dox 40 cell line measured
by FCM probably reflects the inherently higher sensitivity of FCM. Experimental bias
induced by the necessary centrifuge washing step, prolonging the duration of efflux in the
FCM experiment, might play an additional role in these rapidly pumping cells. The studies
in the Jurkat cell line, however, showed that detection of rhodamine-123 using EFM was
both sensitive and linear in the fluorescence intensity range observed in the efflux studies
in hematopoietic cells. In order to further optimise sensitivity of efflux detection in our


                                                                                                                   59
_____   CHAPTER 3 _____________________________________________________________________________________________________



experimental approach we combined fluorescent dye retention and efflux in the presence
and absence of inhibitors in a single assay. Combination improves sensitivity compared to
seperate uptake or efflux studies(17). The relatively short duration of uptake and efflux was
used for practical (time-limiting) reasons and optimised for CD34+CD38- cells based on our
own and others(2) observations of rapid efflux in these primitive hematopoietic progenitors.
Extension of efflux time, however, is likely to further increase sensitivity of efflux detection
in cells with lower pump activities.
Using the described method we showed that P-glycoprotein strongly mediates rhodamine-
123 efflux in the primitive CD34+CD38- hematopoietic progenitors compared to more
differentiated CD34+CD38+ progenitor cells. These findings are in line with previous
reports on the role of P-glycoprotein in primitive hematopoietic subsets. Chaudhary et al(31)
found that the population of lymphoid bone marrow cells within the antigenic phenotype
of pluripotent stem cells (CD34++, HLA-DR low, CD33-) shows the highest level of P-
glycoprotein mediated dye efflux among CD34+ progenitor cells. In these cells a rhodamine
efflux ratio, comparable (3-4-fold) to our results, was found in a 10 hours efflux assay as
assessed by FCM.
Similar results have been reported by Uchida et al(2) demonstrating that within the CD34+
subpopulation rhodamine-123 efflux was most rapid in the more primitive Thy-1 subset.
These studies used relatively large subpopulations within the CD34+ population due to flow-
cytometric limitations compared to the current single cell image analysis method, enabling
us to study a cell population comprising only 1% of the overall CD34+ cell population. In the
majority of these highly purified cells the blockade of efflux by the P-glycoprotein inhibitor
verapamil reconstituted the level of rhodamine-123 fluorescence intensity to the level of
more differentiated CD34+CD38+ cells. This suggest that P-glycoprotein mediated efflux
is the main determinator of rhodamine-123 accumulation in the majority of these primitive
progenitor cells rather than cycle status(32,33) or number of mitochondria(34). Down regulation
of P-glycoprotein mediated efflux observed in CD34+CD38+ cells could be related to cell
differentiation or proliferation as has been reported previously by our group(35). However, a
small subpopulation of CD34+CD38- cells (3-4%; data not shown) preserved a rhodamine
dull phenotype in the presence of verapamil, suggesting the presence of other factors
responsible for the rhodamine dull phenotype in these cells. This finding seems to
concur with the observation by Zijlmans et al(36) who reported the presence of a small
subpopulations of rhodamine dull cells (3-6%) in which rhodamine accumulation was not
increased after addition of verapamil (Rho-/Rho (VP)- in mouse bone marrow, containing
cells with long term repopulating ability. The mechanisms underlying the rhodamine dull
phenotype ( including the possible presence of other rhodamine transporters) and the
biological significance in terms of stem cell characteristics of this population in human
normal bone marrow remain to be determined.




60
__________________________________________________   ABC-TRANSPORTER MEDIATED EFFLUX IN HEMATOPOIETIC STEM CELLS _____



In addition to P-glycoprotein mediated efflux we established the presence of MRP mediated
efflux in human CD34+CD38- hematopoietic cells. The presence of MRP-mediated efflux
has been reported previously for normal CD34+ hematopoietic cells(23,37) but to our
knowledge no studies have been performed to investigate this in the CD34+CD38- stem
cell compartment. In contrast to P-glycoprotein mediated efflux, MRP mediated efflux seem
to be rather stable during early hematopoietic differentiation.
Given the role of these efflux mechanisms in protecting cells against cytotoxic agents(38,39),
these data underscore the possible deleterious effect of the combination of P-glycoprotein
and MRP blocking agents with chemotherapeutical regimens, used to enhance
chemosensitivity of cancer cells, on the CD34+CD38- stem cell compartment in normal
bone marrow.
In conclusion, we developed a method to study ABC-transporter mediated efflux in highly
purified cells at the single cell level and demonstrated both P-glycoprotein and MRP-
mediated efflux in primitive CD34+CD38- hematopoietic cells. This assay provides a tool to
study ABC-transporter mediated efflux in (malignant) stem cell populations obtained from a
variety of sources, for example in clinical samples of AML(40) and other leukemias.



Acknowledgements

The authors like to thank A. Pennings and G. Vierwinden for technical assistance, H.
Broxterman (Free University Hospital Amsterdam, the Netherlands) for providing the
myeloma 8226 cell lines and H. Waite (University of California, Marine biotechnology
Center, USA) for providing MAP. RHGP is a recipient of a grant from the Dutch Cancer
Society. This work was further supported by a grant from the Vanderes Foundation.



References
1.   Bhatia M, Wang JC, Kapp U, Bonnet D, Dick JE. Purification of primitive human hematopoietic cells capable
     of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997; 94: 5320-5.
2.   Uchida N, Combs J, Chen S, Zanjani E, Hoffman R, Tsukamoto A. Primitive himan hematopoietic cells
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                                                                                                                   61
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      primitive hematopoietic cell. Nat Med 1997; 3: 730-7.




                                                                                                                   63
                                                                 CHAPTER 4
ABCB1 MEDIATED DRUG EFFLUX IS REDUCED IN LEUKEMIC
    PROGENITOR CELLS IN COMPARISON TO THEIR NORMAL
                   COUNTERPARTS IN ACUTE MYELOID LEUKEMIA




                                                                           MARC H.G.P. RAAIJMAKERS
                                                                             ELKE P.L.M. DE GROUW
                                                                                    JOOP H. JANSEN
                                                                            BERT A. VAN DER REIJDEN
                                                                                LEONIE H.H. HEUVER
                                                                                THEO J.M. DE WITTE
                                                                            REINIER A.P. RAYMAKERS

                                 DEPARTMENT OF HEMATOLOGY AND CENTRAL HEMATOLOGY LABORATORY
                                                           UNIVERSITY MEDICAL CENTER NIJMEGEN




IN PART ADAPTED FROM: ABCB1 MODULATION IS NOT AN EFFECTIVE STRATEGY TO CIRCUMVENT DRUG EXTRUSION
    FROM PRIMITIVE LEUKEMIC PROGENITORS AND MAY PREFERENTIALLY TARGET RESIDUAL NORMAL PROGENITORS
                   IN ACUTE MYELOID LEUKEMIA.   CLINICAL CANCER RESEARCH. ACCEPTED FOR PUBLICATION.
                                                                  IN PART: SUBMITTED FOR PUBLICATION
_____   CHAPTER 4 _____________________________________________________________________________________________________




66
_______________________________________________   REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



Abstract

Acute myeloid leukemia (AML) originates from hematopoietic CD34+38- stem cells.
Unraveling the mechanisms that confer chemoresistance to these cells is essential to
provide molecular targets for leukemic stem cell eradication. The multidrug ATP-binding-
cassette transporter ABCB1 is widely studied in AML but its expression and function in
leukemic CD34+38- cells in comparison to their normal counterparts is unknown.
We demonstrate that ABCB1 is the major drug transporter in CD34+38- cells in bone marrow
from healthy controls (n=16) as demonstrated by the abrogation of mitoxantrone extrusion
by ABCB1 modulators (verapamil and PSC833; efflux index 1.99 ± 0.08). Surprisingly,
ABCB1 mediated drug extrusion was invariably reduced in CD34+38- cells in AML (n=15;
efflux index 1.21 ± 0.05) which resulted in increased intracellular mitoxantrone accumulation
in these cells. Active drug extrusion from these cells occurred in the presence of ABCB1
modulators in the majority of samples, pointing in the direction of redundant drug efflux
mechanisms. Reduced ABCB1 activity was not observed in CD34+38- cells constituting
normal hematopoiesis from autologous transplanted patients in long-term remission but
preceded the development of overt leukemia in refractory anemia, suggesting that it is
an early leukemic characteristic rather than a characteristic of normal hematopoietic cells
preceding malignant transformation. In line with this, residual normal CD34+38- cells were
identified in an AML patient by their conserved ABCB1 mediated mitoxantrone extrusion
capacity. Together these results indicate that ABCB1 modulation is not an effective strategy
to circumvent mitoxantrone extrusion from leukemic CD34+38- cells and may preferentially
target residual normal progenitors in AML.



Introduction

Cancer is increasingly recognized as a disease originating from the transformation of
normal stem cells, retaining the capacity for self-renewal(1). This concept for the origin of
tumorigenesis was confirmed by the postulation of acute myeloid leukemia as a stem cell
disease(2). Evidence for this concept has come from studies using the NOD-SCID mouse
model showing that cells with leukemic engraftment and selfrenewal potential in AML are
found in the CD34+CD38- subpopulation(3). This CD34+CD38- phenotype of leukemia
initiating cells was observed regardless of the lineage markers expressed by the leukemic
blasts, percentage of cells expressing the CD34 surface antigen or the FAB subtype(3). A
similar role for the CD34+CD38- compartment in leukemogenesis has been suggested
for CML and ALL(4,5). This parallels the hierarchy in normal bone marrow in which a rare
population of CD34+38- cells have stem cell characteristics(6). These studies stress that
leukemia-initiating transformation and progression associated genetic events occur at the
level of these primitive CD34+38- cells. Consequently, incomplete chemotherapeutical


                                                                                                                  67
_____   CHAPTER 4 _____________________________________________________________________________________________________



eradication of these cells may ultimately result in disease relapse. Therefore elucidation
of the mechanisms conferring resistance against chemotherapy to these cells is of crucial
importance(7).
The ABCB1 encoded P-glycoprotein is a highly conserved membrane-bound ATP
binding cassette transporter which extrudes a wide variety of structurally unrelated
chemotherapeutical compounds across the cell membrane8), conferring the multidrug
resistance phenotype in cancer cells. ABCB1, together with another member of the ABC-
transporter family ABCG2 (BCRP), is highly expressed in a wide variety of stem cells
including normal hematopoietic CD34+38- stem cells(9-11). Its physiological function in these
cells is believed to be the protection against genetic damage caused by both environmental
and naturally occurring xenobiotics that are substrates for these transporters. Importantly,
conserved ABCB1 mediated transport after malignant transformation of normal stem cells
would represent a formidable obstacle for chemotherapeutical eradication of cancer stem
cells.
ABCB1 expression and function has been extensively studied in AML. The observation
that it is frequently expressed and that expression is associated with adverse treatment
outcome(12) has led to the introduction of ABCB1 modulators to enhance chemotoxicity of
various chemotherapeutical agents in clinical trials. Trials using PSC-833 (valspodar) as a
highly potent and specific inhibitor of ABCB1 function have, however, failed to reach their
intended endpoints of improved complete remission rates and have been complicated by
increased toxicity(13).
In interpreting these negative results it is important to know whether the leukemia-initiating
CD34+38- hematopoietic subpopulation is targeted by ABCB1 modulation. The vast amount
of studies on ABCB1 expression and function in AML have been performed on general
blast populations. Its expression and function in leukemic CD34+38- cells and the effect
of ABCB1 modulators on drug accumulation in these cells is unknown. AML is generally
considered to be an ABCB1 expressing malignancy reflecting a conserved physiological
function of CD34+ progenitor cells(14) . This assumption is based on the observation that
ABCB1 is preferentially expressed in CD34+ blasts in AML(15) and in CD34+ cells in normal
bone marrow(16), but a direct comparison of ABCB1 expression and function between
normal and malignant progenitor cells has never been made before.
The aim of the current study was to investigate ABCB1 expression and the effect of
ABCB1 modulation on drug accumulation in CD34+38- cells in AML in comparison to their
counterparts in normal bone marrow. We report that ABCB1 mediated transport is impaired
in leukemic CD34+38- cells in comparison to their normal counterparts and that additional
drug transport mechanisms, not inhibited by ABCB1 modulators, confer drug extrusion
from these cells.




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_______________________________________________   REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



Methods

Bone marrow samples
Bone marrow was obtained after informed consent from healthy bone marrow donors
and patients with AML at diagnosis. Mononuclear cells were isolated by Ficoll 1.077
g/ml (Pharmacy Biotech, Uppsala, Sweden). Isolation, cryopreservation and thawing
procedures of cells have been described previously(17) and were identical for normal and
leukemic bone marrow samples.

CD34+CD38- hematopoietic cells
Cells were stained with CD34- CY5 or fluorescein isothiocyanate (FITC) and CD38-
phycoerythrin (PE) monoclonal antibodies (Becton Dickinson, BV, Etten-Leur, The
Netherlands) for 30 minutes at 4ºC and washed in Hank’s balanced salt solution (HBSS)
with 1% v/v heat inactivated fetal calf serum (FCS, Hyclone, Logan, Utah, U.S.A.). A
Coulter Epics Elite Flow cytometer was used to define cell populations. Gating on forward
and right angle scatter was used to exclude dead cells and debris. CD34+CD38- cells of
both normal bone marrow and AML were defined as the cells with CD38-PE fluorescence
within the first decade of emission as shown in figure 1. CD34+CD38- cells appeared in a
consistently restricted light-scattering region confirming the lymphoid appearance of these
primitive progenitors(18). The CD34+CD38+ cells were sorted from a gate positioned in the
bulk of CD34+ cells separated by a decade from CD34+38- cells as indicated in figure1 and
showed more heterogeneous light-scattering properties.

Flow cytometric assessment of ABCB1/P-glycoprotein expression
ABCB1 expression was analyzed using the ABCB1 specific antibody MRK16, recognizing an
external epitope of the protein, in three-color flow cytometric assays as previously described(12).
Briefly, cells were blocked with pooled human serum and stained with MRK16 (mouse IgG2a;
1.0 ug/ 106 cells) and subsequently with biotin goat- anti mouse IgG2a antibody (1.0ug/
106 cells; Southern biotech, Birmingham, AB). A 10-minute incubation with goat serum to
block non-specific binding was followed by staining with fluorescein isothiocyanate (FITC)-
conjugated CD34 and phycoerythrin (PE)-conjugated CD38. MRK16 staining was detected
with streptavidin conjugated to Texas Red (Red613; Becton Dickinson). Appropriate IgG2a
isotype controls at the same protein concentration as the relevant antibodies were used as
controls. ABCB1 protein expression was quantitated as the median fluorescence channel shift
(MRK16 /Isotype control) in CD34+38-, CD34+38+ and CD34- cell populations. To analyze
samples, the histogram of MRK16 expression is overlaid on that of the control. Differences in
fluorescence are assessed using the Kolmogorov-Smirnov (KS) statistic, denoted D, which
measures the difference between two distribution functions and generates a value ranging
from -1.0 to 1.0. This method accurately identifies small differences in fluorescence and is
useful in detection of low level MDR1 expression(12). MRK16 staining intensity is categorized


                                                                                                                  69
_____   CHAPTER 4 _____________________________________________________________________________________________________



as follows: negative (D < 0.10), dim (0.10 < D < 0.15), moderate (0.15 < D > 0.25) and bright
(D > 0.25).

Flow cytometric assessment of ABCB1 mediated mitoxantrone transport
Cells were stained with CD34-FITC and CD38-PE membrane markers, washed in HBSS
1% FCS and pre-incubated with or without verapamil (Knoll AG, Ludwigshaven, Germany
(20 ug/ml) or PSC-833 (Novartis Pharma AG, Basel; 2 uM) as inhibitors for ABCB1 mediated
transport for 20 minutes in Iscove’s modified Dulbecco’s medium supplemented with 1%
FCS. Mitoxantrone (Novantrone, Lederle, Netherlands;10 uM) was added and cells were
incubated for 2 hrs at 37°C, 5% CO2, with or without verapamil or PSC-833. Subsequently,
cells were allowed an additional 1-hr efflux in drug free medium with/without inhibitor.
Cellular mitoxantrone fluorescence was measured on a flow-cytometer (Coulter Elite)
equipped with an argon laser. Fluorescence was assessed at an excitation wavelength of
635 nm through a 670-nm band-pass filter in a three-color protocol with CD34-FITC and
CD38-PE. At least 200 CD34+38- cells were analyzed in each sample.
ABCB1 mediated transport was assessed by quantitating the effect of ABCB1 inhibition
on mitoxantrone accumulation and depicted as the ratio of intracellular mitoxantrone
fluorescence (MFI) in the presence/absence of verapamil or PSC833.
To assess interexperimental reproducibility, ABCB1 mediated transport in CD34+38- cells
was assessed in a normal bone marrow sample in completely independent experiments
performed on separate days (n=7). ABCB1 mediated efflux index was 2.10 ± 0.09 (mean ±
SEM), indicating good reproducibility of the assay. This sample was subsequently included
in other experiments as a control. Preliminary experiments demonstrated similar ABCB1
mediated efflux in cryopreserved samples when compared with fresh cells in normal bone
marrow and AML, in line with a previous report addressing this issue(19).

Single cell image analysis for assessment of ABCB1 mediated transport of
rhodamine-123 in CD34+38- cells
We developed an assay to study ABCB1 mediated transport of the xenobiotic compound
rhodamine- 123 in immunophenotypically defined stem cells, sorted on adhesive biolayers.
This assay has been comprehensively described elsewhere(20).
The mean rhodamine-123 fluorescence intensity (MFI) of CD34+38- and CD34+38+ cells
was calculated using TCL-image 4.6 software package (TNO, Delft, the Netherlands) and
depicted in arbitrary fluorescence units (AU). ABCB1 mediated transport was quantitated
as the ratio of mean fluorescence intensity in the presence/absence of verapamil and
depicted as “efflux index”. Propidium iodide (10 µg/ml, Sigma chemical Co., St. Louis, MO,
U.S.A.) was added to confirm cell viability which typically exceeded 90% of cells. Dead
cells were excluded from analysis.




70
_______________________________________________   REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



Fluorescence in situ hybridization (FISH)
CD34+38- cells were sorted and lysed in 15 ul KCl (75 uM) on a glass cover slip. Cells were
fixed in methanol:acetic acid and stored at 4°C until analysis. FISH was performed using
the LSI AML1/ETO Dual Color, Dual Fusion t(8;21) Probe or the LSI EGR1 (5q31) Dual
Color Probe (Vysis, Downers Grove, IL, U.S.A.) according to manufacturer’s instructions.
One hundred cells were analyzed microscopically for t(8;21) or chromosome 5 (5q31)
cytogenetic abnormalities, as appropriate.

Adapted real-time quantitative RT-PCR for assessment of CBFB-MYH11
gene expression in CD34+38- cells
A linear real-time quantitative RT-PCR approach has been developed for quantitation
of gene expression in low-frequency hematopoietic stem cells and was described
comprehensively elsewhere(11) .

Primer and probe sequences were:
GAPDH:
GAPDH control reagents (Applied Biosystems; part number 402869)
CBFB-MYH11 (Type D) fusion gene:
Forward primer: 5’-CATTAGCACAACAGGCCTTTGA-3’
Reverse primer: 5’- CCTCGTTAAGCATCCCTGTGA-3’
Probe: TET-ATAGAGACAGGTCTCATCG

Normalized gene expression to the internal standard GAPDH is given by the following
equation:

T0/R0 (Xn) = K. (1+E) (CT,r – CT,t)

To: Initial number of target gene copies; Ro: Initial number of standard gene copies; E;
Efficiency of amplification; CT,t: Threshold cycle of target gene; CT, r: Threshold cycle of
standard gene; and K: Constant.

Statistical analysis
Differences in ABCB1 gene expression and function between different normal and leukemic
cell populations were calculated using the students t-test with a level of significance of p<
0.05.




                                                                                                                  71
_____   CHAPTER 4 _____________________________________________________________________________________________________



Results

ABCB1 is the major determinant of mitoxantrone extrusion in CD34+38-
cells in normal bone marrow
CD34+38- hematopoietic cells were flow-cytometrically defined as described in the section
methods (figure 1).

                                          NBM




                                                                   CD34
                                                                          SSC
                                 CD34




                                        CD38




                                          AML
                                                                   CD34




                                                                          SSC
                                 CD34




                                        CD38

Figure 1: Definition of CD34+38- hematopoietic cells in normal bone marrow and acute myeloid leukemia
(for color reproduction: see appendix). Representative example of normal bone marrow (NBM) and acute
myeloid leukemia (AML). CD34+38- cells were defined flow-cytrometrically as the CD34-FITC+ cells (indicated in
blue) with CD38-PE expression within the first decade of fluorescence emission (indicated in red) and compared with
CD34+38+ cells (indicated as blue gated) with exclusion of a decade between CD38- and CD38+ cells. CD34- cells
are shown in gray. CD34+38- cells exhibited restricted light-scattering characteristics (SSC) confirming the lymphoid
appearance of these cells (inset). The median frequency of CD34+38- cells was 0.1 % of mononuclear cells (MNC)
(range 0.1%-0.3%) in NBM and 0.2 % of MNC (range 0.1%- 10%) in AML. No difference existed in average CD38
density between normal and leukemic CD34+38- cells (MFI 0.57 ± 0.05 SD, range 0.40-0.70 and MFI 0.53 ± 0.12
SD, range 0.30-0.70).


ABCB1 protein expression was assessed in hematopoietic cell populations using the
monoclonal antibody MRK-16. ABCB1 was differentially expressed in CD34+38- cells in
comparison to more differentiated cell populations (figure 2). A decrease in ABCB1 protein
expression in CD34+38+ and CD34- cells was observed in all samples examined.




72
_______________________________________________               REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



                                             0.4
                                                                                                           NBM
                                            0.35

                 ABCB1 protein expression
                                                                                                           AML
                                             0.3
                    MRK16 (D-Value)
                                            0.25

                                             0.2

                                            0.15

                                             0.1

                                            0.05

                                              0
                                                   CD34+38-             CD34+38+                   CD34-

Figure 2: ABCB1 is differentially expressed in CD34+38- cells in both normal bone marrow and acute
myeloid leukemia. P-glycoprotein expression was assessed flow-cytometrically using the MRK16 monoclonal
antibody and valued using the Kolmogorov-Smirnov (KS) statistic, denoted D, as described in the section
methods. MRK16 staining intensity is categorized as follows: negative (d < 0.10), dim (0.10 < D < 0.15), moderate
(0.15 < D > 0.25) and bright (D > 0.25). P-glycoprotein was preferentially expressed in CD34+38- cells in
comparison to more differentiated CD34+38+ progenitors in all normal (n=10; black dots) and leukemic (n=10;
white dots) samples examined. No difference existed in levels of expression in CD34+38- cells between NBM and
AML (Average D-value 0.16 ± 0.03 and 0.16 ± 0.04 respectively).


We next studied ABCB1 mediated transport using mitoxantrone as a substrate.
Mitoxantrone was used as a substrate because of its favorable fluorescence emission
characteristics in multi-parameter flowcytometry and its importance as a drug frequently
used for remission induction and consolidation in AML.
After two hours exposure to mitoxantrone followed by an hour efflux in drug free medium,
CD34+38- cells in normal bone marrow were mitoxantrone “dull” compared to CD34+38+
cells (MFI 3.08 ± 0.23 vs. 7.29 ± 0.50, p<0.001; n=16). Inhibition of ABCB1 mediated
transport by verapamil significantly increased mitoxantrone fluorescence in CD34+38- cells
(figure 3A). In line with expression profiles, ABCB1 mediated efflux was invariably higher
in CD34+38- cells (mean efflux index 1.99 ± 0.08, range 1.64-2.92) compared to more
differentiated CD34+38+ (1.45 ± 0.06) cells.
The phenomenon of verapamil-sensitive drug efflux has also been attributed to ABCG2/
BCRP mediated transport, though at higher concentrations of verapamil than used in our
experiments(21). To demonstrate that the observed mitoxantrone efflux in CD34+38- cells is
mediated by ABCB1 rather than other transporters, additional experiments were performed.
First, similar results were found when the ABCB1 specific inhibitor PSC-833 was used in a
subgroup of samples (n=4); (mean EI ± SEM 2.03 ± 0.12 and 1.38 ± 0.09 for CD34+38- and
CD34+38+ cells, respectively; Figure 3A).
Secondly, using single cell image analysis, we assessed ABCB1 mediated transport in
single CD34+38- cells using rhodamine-123 as a substrate which is not a substrate for
BCRP(22) (figure 3B); Rhodamine-123 accumulation was significantly lower in CD34+CD38-
cells compared to CD34+CD38+ cells in normal bone marrow (p<0.001), similar to the


                                                                                                                              73
_____   CHAPTER 4 _____________________________________________________________________________________________________



mitoxantrone “dull” phenotype of CD34+38- cells. Blocking of ABCB1 mediated transport
by verapamil increased rhodamine-123 fluorescence significantly. ABCB1 mediated efflux
was found in all examined (n=5) CD34+CD38- samples (mean efflux index 3.74 ± 1.81).




                                         MITOXANTRONE
                                                                                                        12.00
                                                                                                                No inhibitor




                                                                                        MITOXANTRONE
                                                                         No inhibitor                           Verapamil
                                                                                                         8.00
                                                                         Verapamil



                                                        EVENTS
                                                                                                         4.00
                                                                         PSC833
                                                                                                         0.00
                                                                 MITOXANTRONE             A2                      34+38-       34+38+
                                                         A1

                                                          NO INHIBITOR   VERAPAMIL                      12000
 CD34




                                                                                        RHODAMINE-123
                                                                                                         9000

        CD38                                                                                             6000

                                                                                                         3000

                                                                                                            0
                                                        B1       RHODAMINE-123           B2                     34+38-         34+38+


Figure 3: ABCB1 is the major mitoxantrone and rhodamine-123 transporter in hematopoietic CD34+38-
cells in human normal bone marrow (for color reproduction: see appendix). A1 Representative example of
flow-cytometric assessment of ABCB1 mediated mitoxantrone transport in CD34+38- cells in NBM. CD34+38-
cells display low intracellular mitoxantrone fluorescence in comparison to CD34+38+ cells (inset) which can be
increased significantly by blockage of ABCB1 mediated transport by verapamil/PSC833. A2 Average mitoxantrone
fluorescence (n=16) (depicted as mean fluorescence intensity (MFI) in CD34+ cells in the absence (shaded bars)
or presence (black bars) of verapamil. B1 Representative example of the effect of ABCB1 inhibition by verapamil
on rhodamine-123 fluorescence in CD34+38- cells assessed by single cell image analysis. B2. Average values ±
S.D. of rhodamine-123 fluorescence (n=5)(depicted as arbitrary units) in the absence (shaded bars) or presence
(black bars) of verapamil. Rhodamine-123 accumulation was significantly lower in CD34+CD38- cells compared
to CD34+CD38+ cells (rhodamine-123 fluorescence 2820 ± 501 AU vs. 9369 ± 2367 AU). Blockage of ABCB1
mediated transport significantly increases rhodamine-123 accumulation in CD34+38- cells.



Thirdly, experiments with the BCRP/ABCG2 specific inhibitor KO143 in these CD34+38-
hematopoietic cells demonstrated only a minor contribution of these transporters to the
mitoxantrone dull phenotype of these cells, as we described recently(23).
To obtain further insight in the relative contribution of ABCB1 mediated mitoxantrone
extrusion to the mitoxantrone “dull” appearance ot these cells, serial measurements of
mitoxantrone fluorescence were performed in a subpanel of samples (n=7) both after 2
hours exposure with mitoxantrone and after an additional hour of efflux in drug free medium
in the presence and absence of verapamil (figure 4A). Inhibition of ABCB1 by verapamil
completely abrogated mitoxantrone efflux during an hour efflux in drug-free medium in
CD34+38- cells (MFI 6.74 ± 0.34 vs. 6.74 ± 0.49 respectively).




74
_______________________________________________                               REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



                                                          10
                                                                A                   NBM

                                                           8




                        Mitoxantrone fluorescence (MFI)
                                                          6


                                                           4


                                                           2
                                                                     P=0.0001                             ns
                                                          10
                                                                B                     AML

                                                           8


                                                           6

                                                           4

                                                           2
                                                                     P=0.0001                            P=0.006

                                                               2 hrs MITO     + 1 hr DFM        2 hrs MITO     + 1 hr DFM
                                                                     - VERA                               +VERA

Figure 4: ABCB1 modulation abrogates mitoxantrone extrusion from normal but not leukemic CD34+38-
cells. To assess whether mitoxantrone efflux occurs in CD34+38- cells in the presence of ABCB1 modulation
(verapamil=VERA), serial measurements were performed of mitoxantrone fluorescence after 2 hrs of exposure
to mitoxantrone (MITO) and after an additional hour of efflux in drug free medium (DFM), both in the presence
and absence of ABCB1 modulation. Inhibition of ABCB1 by verapamil completely abrogates mitoxantrone efflux
during the additional hour of efflux in DFM in CD34+38- cells from normal bone marrow (MFI 6.74 ± 0.34 vs. 6.74
± 0.49 respectively; figure 4A) but not in AML (MFI 6.90 ± 0.55 vs. 6.26 ± 0.62 respectively, difference using T-
test for paired samples p=0.006; Figure 4B) (average value mitoxantrone ± SEM represented by bar, individual
samples represented by symbols). When cells were put on ice during the additional hour of efflux, no decrease in
mitoxantrone fluorescence was observed.


Together, these results demonstrate that ABCB1 mediated transport is the major
determinant of the mitoxantrone dull phenotype of CD34+CD38- cells in human normal
bone marrow.

Impaired ABCB1 mediated transport is a biological commonality in
CD34+38- hematopoietic cells in AML
Conserved ABCB1 mediated drug extrusion from CD34+38- cells after malignant
transformation would be an important obstacle for chemotherapeutical eradication of
these cells in AML. We therefore investigated ABCB1 expression and function of this cell
population in AML in comparison to their counterparts in normal bone marrow.
Investigations were performed on a panel of bone marrow samples of 20 untreated AML
patients (table 1).




                                                                                                                                              75
_____   CHAPTER 4 _____________________________________________________________________________________________________



Table 1: Patient characteristics.

N                                            20
Sex
M                                            9
F                                            11
Age (median/range)                           50(15-71)
% CD34                                       31 (1-91)
FAB
M1                                           6
M2                                           8
M4                                           3
M5                                           3
Cytogenetics
Normal                                       9
t(8;21)                                      4 (+2 complex)
(6;11)                                       2
45XO                                         1
Complex                                      4



The FAB M3 subtype was not included in this study because controversy exist about the
origin of leukemic stem cells in this subtype, which may not be CD34+38- cells(3). The
panel consisted predominantly of CD34+ leukemias with a median percentage of CD34+
cells of 31% (range 1-91). CD34+38- cells in AML were immunophenotypically defined as
indicated in figure 1. These cells showed comparable scatter characteristics and CD38
expression in comparison to their counterparts in normal bone marrow. FISH analyses in
AML patients who carried a cytogenetic abnormality and from whom sufficient cells were
available for analysis demonstrated a predominantly leukemic character of CD34+38- cells
in these samples (n=5; table 2).

Table 2: CD34+38- cells in AML are predominately of leukemic origin.

             Cytogenetic             % cytogenetic aberrant
Pat. nr.     aberrancy               CD34+38- cells                ABCB1 efflux-index
                                                                   Rhodamine-123                   Mitoxantrone
                                                                   3.74 (1.76-5.66)A               1.99 (1.64-2.94)A
1.           -5,-7,-15,-17,t(2:3)    56%                           0.84                            1.32
2.           t(8;21)                 90%                           NA                              0.92
3.           t(8;21)                 95%                           0.81                            1.15
4.           t(8;21)                 90%                           1.39                            NA
5.           t(8;21)                 100%                          NA                              1.28
The incidence of cytogenetic aberant cells in the CD34+38- cell population as assessed by FISH and ABCB1
mediated transport in these cells. NA= material not available for analysis.
A
  Values normal bone marrow: mean (range).




76
_______________________________________________               REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



ABCB1/Pglycoprotein expression in CD34+CD38- cells in AML as assessed by the MRK-
16 monoclonal antibody was comparable to expression of their counterparts in normal
bone marrow (figure 2). Similar to the expression in normal bone marrow, decreased
protein expression in CD34+38+ and CD34- subsets was observed in all samples.
Next, we studied the effect of ABCB1 modulation on drug accumulation in CD34+38- cells
in AML (figure 5);

                                                                                                             12.00

                                      MITOXANTRONE
                                                                                                                     No inhibitor




                                                                                             MITOXANTRONE
                                                                                                                     Verapamil
                                                                             No inhibitor
                                                                                                              8.00

                                                                             Verapamil
                                                     EVENTS
                                                                                                              4.00
                                                                             PSC833
                                                                                                              0.00
                                                                                              A2                     34+38-         34+38+
                                                      A1        MITOXANTRONE
                                                                                                         12000
                                                       NO INHIBITOR          VERAPAMIL




                                                                                             RHODAMINE-123
CD34




                                                                                                             9000

                                                                                                             6000
       CD38
                                                                                                             3000

                                                                                                                0
                                                                                                                     34+38-         34+38+
                                                     B1           RHODAMINE-123               B2


Figure 5: ABCB1 transport is impaired in hematopoietic CD34+38- cells in acute myeloid leukemia
(for color reproduction: see appendix). A1. Representative example demonstrating increased intracellular
mitoxantrone accumulation in comparison to CD34+38- cells from normal bone marrow due to impaired
ABCB1 mediated transport in CD34+38- cells illustrated by a lack of effect of verapamil/PSC833 on intracellular
mitoxantrone fluorescence. A2. Average mitoxantrone fluorescence (depicted as mean fluorescence intensity
(MFI) in CD34+ cells in the absence (shaded bars) or presence (black bars) of verapamil. B1. Representative
example of impaired ABCB1 mediated rhodamine-123 extrusion from CD34+38- cells, illustrated by the lack
of effect of ABCB1 inhibition by verapamil on intracellular rhodamine-123 fluorescence assessed by single cell
image analysis. B2. Average values ± S.D. Rhodamine-123 fluorescence was significantly lower in CD34+CD38-
cells compared to CD34+CD38+ cells (3106 ± 267 AU vs. 4594 ± 373 AU) but blockage of ABCB1 by verapamil
has no effect on rhodamine-123 fluorescence.


Intracellular mitoxantrone accumulation in CD34+38- cells in AML upon exposure and
subsequent efflux in drug free medium was significantly higher in comparison to their
normal counterparts (MFI 4.54 ± 0.44 vs. 3.08 ± 0.23 ; p<0.001; figure 5A). This increased
mitoxantrone accumulation was due to reduced ABCB1 mediated extrusion from these
cells; blockage of ABCB1 mediated transport by verapamil increased mitoxantrone
fluorescence in CD34+38- cells to a much lower extent than in normal bone marrow (MFI
4.54 ± 0.44 vs. 5.42 ± 0.52 in the absence vs. presence of verapamil respectively; p=0.009;
figure 5A).
ABCB1 mediated efflux, defined as the fluorescence ratio with modulator divided by drug
fluorescence without modulator, was invariably impaired in CD34+38- cells in AML (n=15)
compared to these cells in normal bone marrow (mean efflux index 1.21 ± 0.05 (0.93-1.51);


                                                                                                                                             77
_____   CHAPTER 4 _____________________________________________________________________________________________________



p<0.0001; figure 6, panel A). Impairment of ABCB1 function was not restricted to CD34+38-
cells in AML; impaired ABCB1 function in comparison to normal counterparts was also
found for CD34+38+ cells (mean EI 1.17 ± 0.03, range 1.05-1.31; p<0.001).

                                                                    1.99    1.21    2.18          1.94      1.53    1.48        2.02    1.36
                                                                   ±0.08   ±0.05   ±0.08         ±0.06     ±0.02   ±0.05       ±0.08   ±0.09
                                                        3.00
           ABCB1 mediated mitoxantrone transport (EI)




                                                               A                           B                               C

                                                        2.50
                      in CD34+38- cells




                                                        2.00



                                                        1.50



                                                        1.00
                                                                                               AML
                                                                                                 AML     AML
                                                                                                          AML      MM
                                                                                                                   MM
                                                                   NBM
                                                                   NBM     AML
                                                                           AML     MM           allo
                                                                                                 allo    auto
                                                                                                          auto     auto
                                                                                                                   auto        RA
                                                                                                                               RA      RA - AML
                                                                                                                                       RA-AML
                                                        0.50

Figure 6: Impaired ABCB1 mediated drug transport in CD34+38- cells in AML is a biological commonality
that is the result of early leukemic alterations rather than a characteristic of normal CD34+38- cells
predisposing to malignant transformation. A. ABCB1 mediated mitoxantrone transport in hematopoietic
CD34+38- cells from normal bone marrow (NBM) and AML samples depicted as an efflux index (EI) of
mitoxantrone fluorescence in the presence/absence of verapamil. Each sample is represented by a black dot.
Average ABCB1 efflux index ± SEM is depicted on top. MM = CD34+38- cells from multiple myeloma.
B. ABCB1 mediated transport in autologous transplanted cells constituting normal hematopoiesis in AML
(AUT-AML) is significantly higher than in AML at diagnosis and similar to ABCB1 transport after autologous
transplantation in MM (AUTO-MM) indicating that impaired ABCB1 function is not a characteristic of normal
CD34+38- cells predisposing to malignant transformation in AML. ALLO-AML= long-term remission after
allogeneic transplantation. C. Patients suffering from refractory anemia (RA) that have normal ABCB1 mediated
mitoxantrone transport show no malignant progression whereas patients that developed AML after RA (RA-AML)
have impaired ABCB1 mediated transport in CD34+38- cells at the moment of RA diagnosis.



To confirm that impaired verapamil-inhibitable transport in CD34+38- cells in AML is indeed
due to impaired ABCB1 mediated transport, additional experiments were performed. First,
similar results were found when PSC-833 was used as an inhibitor (n=4, average efflux
index in CD34+38- cells 1.23 ± 0.16; figure 5A).
Additionally, impaired ABCB1 mediated transport in CD34+38- cells in AML was confirmed
using rhodamine-123 as a substrate in single cells (figure 5B). CD34+CD38- cells in AML
(n=8) were rhodamine-123 dull compared to the more differentiated CD34+CD38+ cells.
Addition of verapamil did not increase rhodamine-123 fluorescence in CD34+38- cells. The
mean efflux index was 0.99 ± 0.07 (figure 7).




78
_______________________________________________                                 REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____




                        ABCB1 transport (EI) in CD34+38-cells
                                                                5.50


                                                                4.50


                                                                3.50


                                                                2.50


                                                                1.50

                                                                       RHODAMINE 123
                                                                0.50
                                                                                        NBM                     AML

Figure 7: Impaired ABCB1 mediated rhodamine-123 transport in CD34+38- cells is a biological
commonality in AML. ABCB1 mediated rhodamine-123 transport in hematopoietic CD34+38- cells from normal
bone marrow (NBM) and AML samples depicted as an efflux index (EI) of rhodamine-123 fluorescence in the
presence/absence of verapamil. Each sample is represented by a black dot.


Finally, analysis of mitoxantrone efflux mediated by the ATP-binding cassette transporters
ABCG2 (BCRP), performed in a series of normal and leukemic bone marrow samples
including those described in this paper, using the ABCG2-specific fumitremorgin C analog
KO143 as inhibitor, demonstrated no difference between normal and leukemic CD34+38-
cells(23).
To unravel whether additional mechanisms confer drug transport from these cells, serial
measurements of mitoxantrone fluorescence were performed in a subpanel of AML
samples (n=10) both after 2 hours exposure with mitoxantrone and after an additional
hour of efflux in drug free medium in the presence and absence of ABCB1 blockage by
verapamil (figure 4B). A significant decrease in mitoxantrone fluorescence was observed
during an hour efflux in drug free medium in the presence of verapamil (MFI 6.90 ± 0.55 vs.
6.26 ± 0.62 respectively, difference using T-test for paired samples p=0.006) in the majority
of patients, pointing in the direction of additional drug extrusion mechanisms. No decrease
in mitoxantrone fluorescence was observed when cells were put on ice during the efflux in
drug free medium.
These results indicate that ABCB1 mediated mitoxantrone transport is reduced in CD34+38-
cells in AML in comparison to their counterparts in normal bone marrow.
Modulation of ABCB1 alone does not abrogate drug efflux from these cells due to the
activity of promiscuous, non verapamil- inhibitable transport mechanisms.

Impairment of ABCB1 mediated transport is an early leukemic characteristic
of CD34+38- hematopoietic cells in AML
The observation that impaired ABCB1 mediated transport is a biological commonality in
AML and not observed in normal bone marrow opened the possibility that impaired function
of the major xenobiotic transporter in normal hematopietic stem cells predisposes to the


                                                                                                                                                79
_____   CHAPTER 4 _____________________________________________________________________________________________________



malignant transformation of these cells. Alternatively, impaired ABCB1 function could be
an early characteristic induced by leukemia-associated genetic events in hematopoietic
CD34+38- cells. To address this question we studied ABCB1 mediated transport in AML
patients in which normal hematopoiesis was restored after bone marrow transplantation.
Long term survivors following autologous bone marrow transplantation were compared
with long term survivors following allogeneic transplantation. Patient and CD34+38- cell
characteristics are shown in table 3.

Table 3: Autologous vs allogeneic transplanted patients: Patient and CD34+38- cell characteristics.

                                                                 Autologous BMT (n=11)         Allogeneic BMT (n=9)
Age                                                              38 (19-55)                    34 (26-56)
Sex                                      M                       6                             2
                                         F                       5                             7

FAB                                      M1                      1                             0
                                         M2                      4                             6
                                         M4                      3                             1
                                         M5                      2                             2
                                         M6                      1                             0

Cytogenetics                             Normal                  6                             4
                                         T (8;21)                2                             3
                                         T (9;11)                1                             0
                                         T (16;16)               1                             0
                                         Complex                 1                             2

CD34+38- cells                           CD38 (MFI)              5.72 ± 0.84(SD)               4.83 ± 0.42(SD)
                                         Incidence (%MNC)        0.21 ± 0.27(SD)               0.40 ± 0.15(SD)
ABCB1 transport assessment                                       38 (12-80)                    37 (12-140)
(months after BMT)
Survival (months after BMT)                                      74 (38-139)                   158 (59-254)

BMT=Bone marrow transplantation.


ABCB1 mediated efflux was assessed when hematopoiesis was morphologically normal.
Patients following allogeneic transplantation had complete donor hematopoiesis as
assessed by XY chromosome karyotyping or single nucleotide polymorphism analysis at
the time of evaluation. Polyclonality of hematopoiesis in autologous transplanted female
patients was confirmed using the HUMARA-assay (data not shown). All patients are still
alive without recurrence of disease after a median follow-up of 74 and 158 months in
autologous and allogeneic transplanted patients respectively.
ABCB1 mediated mitoxantrone transport in allogeneic transplanted CD34+38- cells was
similar to that observed in normal bone marrow (mean efflux index 1.94 ± 0.06, 1.76-2.29;
figure 6, panel B and figure 8). ABCB1 mediated transport in autologous transplanted
CD34+38- cells was significantly higher than in CD34+38- cells in AML at diagnosis (EI 1.53
± 0.02 vs. 1.21 ± 0.05, p<0.001), although lower than in allogeneic transplanted cells.
80
_______________________________________________   REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



                           A      ALLOGENEIC                                   EI=2.30

                                                                              No inhibitor
                                                                              Verapamil


                                  AUTOLOGOUS                                   EI=1.48

                          CD34
                                                                              No inhibitor
                                                                              Verapamil


                                  CD38                              MITOXANTRONE


                           B
                                  RA                                            EI=2.23

                                                                              No inhibitor
                                                                               Verapamil



                                  RA-AML                                        EI=1.24
                           CD34




                                                                              No inhibitor
                                                                              Verapamil


                                                                   MITOXANTRONE
                                   CD38

Figure 8: Representative examples of ABCB1 mediated mitoxantrone efflux in CD34+38- cells from
autologous and allogeneic transplanted cells constituting normal hematopoiesis in long-term remission,
refractory anemia (RA), refractory anemia progressing towards AML (RA-AML) (for color reproduction:
see appendix).



To address whether the lower ABCB1 activity in CD34+38- cells in autologous transplanted
patients in comparison to normal CD34+38- cells was transplantation-related or a
preleukemic characteristic of these cells, as a control, ABCB1 mediated transport was
assessed in CD34+38- hematopoietic stem cells in multiple myeloma (MM). MM is a
plasma cell malignancy that is derived from differentiated clonogenic B-cells rather than
multipotential CD34+38- cells and is therefore regarded as a malignancy not originating
from hematopoietic stem cells. ABCB1 mediated transport in CD34+38- cells from
bone marrow obtained from patients with MM (n=5) was similar to that in normal bone
marrow (Efflux index 2.18 ± 0.08; figure 6, panel A) and significantly higher than in AML
(p<0.001). Autologous transplanted cells from 2 patients with multiple myeloma (MM) that
demonstrated normal ABCB1 mediated transport in their CD34+38- cells at diagnosis
displayed reduced ABCB1 activity similar to the activity observed in CD34+38- cells from
autologous transplanted AML patients (figure 6, panel B), indicating that the reduction in
function in autologous transplants in AML in comparison to allogeneic transplanted cells
is treatment-related rather than a pre-leukemic characteristic. Together, these findings
argue against the possibility that impaired ABCB1 mediated transport is a characteristic of
normal, polyclonal, CD34+38- cells predisposing to malignant transformation in AML.



                                                                                                                  81
_____   CHAPTER 4 _____________________________________________________________________________________________________



Impaired ABCB1 mediated transport is an early characteristic of hematopoietic
CD34+38- cells in patients with refractory anemia developing acute myeloid leukemia
To gain further insight into the timing and significance of impaired ABCB1 mediated
transport in leukemogenesis, we studied bone marrow of patients suffering from refractory
anemia (RA) and patients with refractory anemia with ring sideroblasts (RARS). RA
and RARS are preleukemic syndromes representing subgroups of the myelodysplastic
syndrome (MDS) characterized by the absence of leukemic blasts in the bone marrow
(<5%) and peripheral blood (<1%)(24). Malignant transformation to AML occurs in 10-20% of
patients with RA/RARS(25)). We selected RA/RARS patients who received supportive care
only (no chemotherapy or bone marrow transplantation) with a follow-up of at least 2 years.
Eleven patients were identified who matched these criteria and from whom bone marrow at
diagnosis was available for analysis (table 4).

Table 4: ABCB1 mediated mitoxantrone transport in CD34+38- cells at diagnosis related to malignant
transformation in RA/RARS patients.

                                         ABCB1 mediated
Nr.      Sex      Age     Diagnosis      transport (EI)         AML      Latency period       Survival       Status
1.       F        68      RARS           1.88                   no                            87             -
2.       M        66      RA             1.86                   no                            89             -
3.       M        59      RA             2.42                   no                            39             -
4.       M        56      RARS           2.23                   no                            79             Alive
5.       F        50      RA             1.86                   no                            46             Alive
6.       M        54      RA             2.09                   no                            31             Alive
7.       F        68      RA             2.00                   no                            58             -
8.       M        25      RA             1.80                   no                            172            Alive
                                         2.02 ± 0.08A
9.       M        44      RA             1.24                   yes      32                   34             -
10.      M        76      RA             1.30                   yes      24                   26             -
11.      F        55      RA             1.53                   yes      48                   56             -
                                         1.36 ± 0.07A
RA=refractory anemia. RARS=refractory anemia with ringed sideroblasts. A average ± S.E.M.


ABCB1 mediated mitoxantrone transport in CD34+38- cells was assessed in all patients
(table 4; figure 6; panel C and figure 8). CD34+38- cells in 8/11 patients displayed normal
(defined as efflux index values within the range found in normal bone marrow) ABCB1
mediated transport (mean efflux index 2.02 ± 0.08, range 1.80-2.42). None (0/8) of these
patients developed AML with a median follow-up of 58 months. Impaired ABCB1 mediated
efflux at diagnosis was found in CD34+38- cells in 3/11 patients (1.36 ± 0.09, range 1.24-
1.53). These 3 patients developed AML after a latency period of 2-4 years. Together these
data demonstrate that impaired ABCB1 mediated transport in hematopoietic progenitors
is an early leukemic event, preceding the development of clinically overt AML in patients
suffering from refractory anemia.
82
 _______________________________________________    REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



ABCB1 mediated drug transport identifies a subpopulation of residual normal CD34+38-
hematopoietic stem cells in a patient with CBFB-MYH11 positive AML
The finding that impairment of ABCB1 function is not a characteristic of normal
hematopoietic stem cells preceding malignant transformation but rather an early leukemic
event implies that in AML residual normal CD34+38- hematopoietic cells may be identified
by their conserved ABCB1 mediated transport capacity.
Indeed, during additional monitoring, one patient was identified with a clearly discernable
CD34+38- mitoxantrone dull population caused by ABCB1 mediated drug efflux within the
range observed in normal bone marrow (figure 9).


                                                    No inhibitor

                                                    Verapamil
                 CD34+38-
                 ABCB1 efflux ±
                                                        ABCB1
                                                        EI=1.48

                                                                           GAPDH                  CBFB-MYH11
                                                                           Ct 30.13                 Ct 38.27

                                                                      Xn 4.3 10 -3 Copies CBFB-MYH11/copy GAPDH
                              MITOXANTRONE
CD34




                                                                               CBFB-MYH11 not detectable

                                                                           GAPDH                   CBFB-MYH11
       CD38                                  CD34                          Ct 29.74                  Ct >45

                                                     No inhibitor

                                                     Verapamil
                CD34+38-
                ABCB1 efflux ++

                                                         ABCB1
                                                         EI=2.50


Figure 9: ABCB1 mediated mitoxantrone transport identifies a subpopulation that harbors residual
normal CD34+38- cells in a patient with CBFB-MYH11 AML (for color reproduction: see appendix). A patient
was identified in which mitoxantrone discriminated a population of CD34+38- mitoxantrone dull cells. The dull
phenotype was determined by strong ABCB1 mediated efflux (CD34+38- ABCB1 efflux ++), within the range
observed in CD34+38- cells observed in normal bone marrow (EI=2.50). The CBFB-MYH11 (type D) transcript
could be detected at the level of the positive control ( blast population from CBFB-MYH11 positive patient; Xn
= 5.1. 10-3 copies CBFB-MYH11/Copy GAPDH) in the CD34+38- mitoxantrone bright population. CBFB-MYH11
transcripts were not detected in the CD34+38- ABCB1 efflux ++ population.


Because the intracellular presence of mitoxantrone precluded the use of FISH, we used
real-time quantitative RT-PCR to quantify the number of CBFB-MYH11 transcripts in these
CD34+38- subpopulations. The number of transcripts in the CD34+38- cell population was


                                                                                                                    83
_____   CHAPTER 4 _____________________________________________________________________________________________________



similar to that in the positive control (blast population from CBFB-MYH11, type D positive
patient) (Xn 4.3 .10-3 vs. 3.5 .10-3 copies CBFB-MYH11/copy GAPDH, respectively),
whereas no transcripts were detected in the CD34+38- ABCB1 efflux ++ subpopulation.
This sample provides proof of principle that ABCB1 mediated drug efflux may indeed
identify a subpopulation of CD34+38- cells that harbor residual normal cells at least in a
subgroup of AML patients.



Discussion

In this report we demonstrate that ABCB1 mediated drug transport is impaired in leukemic
CD34+38- progenitor cells in comparison to their normal counterparts in AML. This
finding was initially unanticipated since AML is generally considered to be an ABCB1
over-expressing malignancy with levels of expression reflecting the expression in normal
bone marrow(14). This assumption, however, is based on the observation that ABCB1 is
preferentially expressed in CD34+ blasts in AML(15) similar to preferential expression of
ABCB1 in these cells in normal bone marrow(16), thus reflecting patterns of expression
rather than direct comparison of ABCB1 expression and function of normal and malignant
cell subpopulations. Expression data in our study confirm the preferential expression of
ABCB1 in CD34+ cells in AML(15) at levels comparable to those observed in normal bone
marrow. ABCB1 transport capacity, however, was markedly reduced both in CD34+38- and
in CD34+38+ cells in AML.
The molecular determinants behind impaired ABCB1 mediated transport in hematopoietic
cells in AML remain to be elucidated. The finding in this study that reduced ABCB1 function
in CD34+38- hematopoietic cells is an early leukemic event preceding the development
of clinically overt leukemia in patients suffering from refractory anemia, rather than a
preleukemic characteristic of normal hematopoietic cells argues against polymorphisms
in the ABCB1 gene(26) as an explanation. ABCB1 downregulation has been reported as
a downstream event of oncoproteins central in leukemogenesis such as AML1-ETO(27)
and TEL-AML1(28). Additionally, down-regulation of ABCB1 by the PML-RARa leukemic
oncoprotein in the preleukemic phase of the disease(29) has recently been described in
transgenic mice. Although ABCB1 protein expression in this study was similar in CD34+38-
cells from normal bone marrow and AML, this does not always reflect gene expression,
possibly through lack of sensitivity of protein expression detection. Consequently,
downregulation of ABCB1 expression by oncoproteins cannot be excluded as a possible
mechanism behind the reduced transport capacity. To address this issue we are currently
profiling gene expression in normal and leukemic CD34+38- hematopoietic cells.
Alternatively, factors governed by the cellular environment such as disruption of cellular
ATP- metabolism, membrane integrity or cell cycle regulation(30) could be involved in
reduced ABCB1 function in leukemic progenitor cells.


84
_______________________________________________   REDUCED ABCB1 MEDIATED TRANSPORT IN LEUKEMIC PROGENITOR CELLS _____



The finding in this study that reduced ABCB1 mediated transport in CD34+38- cells is
a biological commonality and an early leukemic event preceding the development of
clinically overt AML in the pre-leukemic disorder refractory anemia, raises questions about
its biological significance in leukemogenesis. It is tempting to hypothesize that impaired
xenobiotic transport in hematopoietic cells that are the target for leukemic transformation
attributes to increased susceptibility for progression-associated genetic events induced by
naturally occurring genotoxic xenobiotics required for malignant transformation. Interestingly,
a decrease in ABCB1 function in CD34+ hematopoietic cells has been described in aplastic
anemia (AA), another hematopoietic disorder associated with xenobiotic exposure(31). AA
is associated with an increased risk for the development of clonal hematopoietic disorders
with about 25% of patients developing acute leukemia(32). Additionally, polymorphisms in
the ABCB1 gene, associated with reduced expression and function of ABCB1, have been
related to the occurrence of acute leukemia(33) and other tumors(34).
It has to be noted however that leukemia initiating capacity using NOD-SCID models
is found in only about 1/1000 CD34+38- cells in AML and therefore our results do not
necessarily apply to these repopulating cells. Studies investigating the leukemia engrafting
capacity of CD34+38- ABCB1 efflux++ and efflux- subpopulations are warranted to
definitely address whether reduced ABCB1 function is required to confer the leukemic stem
cell phenotype to hematopoietic CD34+38- hematopoietic cells. The low frequency of cells
in the cell samples we examined and the use of mitoxantrone precluded these experiments
in the current study.
Regardless the underlying mechanism and biological significance, the finding that ABCB1
mediated transport is impaired in CD34+38- cells in AML has important clinical implications;
The recognition of AML as a disease originating from these cells dictates a paradigm
shift in the treatment towards the eradication of this crucial stem cell population in AML.
Unraveling the mechanisms underlying chemoresistance in these cells is therefore of great
importance. Our results indicate that ABCB1 plays a limited role in mitoxantrone extrusion
from leukemic CD34+38- cells. In this study we used mitoxantrone as a drug because of
its favorable emission characteristics in multiparameter flow cytometry. It remains to be
determined whether the ABCB1 extrusion capacity for other drugs, such as daunorubicin
and idarubicin is reduced in leukemic CD34+38- cells as well. The observation that
rhodamin-123 extrusion capacity is reduced, however, suggests that reduced function may
comprise multiple (drug) substrates. The relatively mitoxantrone and rhodamine-123 dull
phenotype of CD34+38- cells in AML in comparison to more differentiated CD34+38+ cells
in the absence of ABCB1 mediated transport in our experiments, points to the direction
of additional redundant mitoxantrone transporters in these cells. We have recently
documented ABCG2/BCRP as one of these additional drug transporters in leukemic
CD34+38- cells(23). Modulation of additional transporters is likely to be required to increase
mitoxantrone accumulation and to enhance mitoxantrone induced eradication of leukemic
stem cells in AML.


                                                                                                                  85
_____   CHAPTER 4 _____________________________________________________________________________________________________




Additionally, the current study demonstrates that residual normal CD34+38- cells are
likely to harbor within the ABCB1 efflux ++ subpopulation. This seems congruent with
the report(35) that the capacity to efflux hoechst 33342 (side population) identifies a
subpopulation of CD34+38- residual normal cells in AML, whereas CD34+38- hoechst
bright cells were leukemic. Although these authors could not relate hoechst 33342
efflux with ABCB1 protein expression in the overall side population of cells, this was not
investigated in the CD34+38- subset. The current report demonstrates that differences
in ABCB1 mediated efflux discriminates normal from leukemic CD34+38- cells. We can
however not completely exclude the possibility that yet unidentified transporters that efflux
rhodamine-123 and mitoxantrone and are blocked by verapamil and PSC 833 are involved.
The hoechst transporter ABCG2 is not involved because we recently demonstrated that its
transport capacity is conserved in CD34+38- cells in AML in comparison to their normal
counterparts(23).
Translated to the use of ABCB1 modulation in AML, the finding that residual normal
CD34+38- cells in AML exhibit high ABCB1 mediated drug efflux, suggests that this
approach may preferentially target residual normal hematopoietic cells whereas leukemic
CD34+38- cells escape its effect through the action of redundant mechanisms. These
laboratory findings could provide an alternative explanation for the poor results of ABCB1
modulation by PSC 833 on long-term disease outcome in clinical trials(13,36). The early
reports of improved clinical outcome in a clinical trial using cyclosporin as a ABCB1
modulator(37) may actually reflect this assumption since cyclosporin is a more promiscuous
ABC-transporter inhibitor with effects on both ABCB1 and ABCG2(12).
These data prompt further research to elucidate the transport mechanisms involved in the
extrusion of drugs from leukemic CD34+38- cells to provide novel targets for the eradication
of this crucial cell population within the AML hierarchy.



Acknowledgments

The authors like to thank A. Pennings and G. Vierwinden for technical assistance. The
Hematology Data Base Center Nijmegen is thanked for providing material and clinical data.
We are grateful to F. Russel for critically reviewing the manuscript.



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88
                                          CHAPTER 5
THE BREAST CANCER RESISTANCE PROTEIN IN DRUG
        RESISTANCE OF PRIMITIVE CD34+38- CELLS
                           IN ACUTE MYELOID LEUKEMIA




                                                  MARC H.G.P. RAAIJMAKERS1
                                                    ELKE P.L.M. DE GROUW2
                                                       LEONIE H.H. HEUVER2
                                                   BERT A. VAN DER REIJDEN2
                                                           JOOP H. JANSEN2
                                                            RIK J. SCHEPER3
                                                         GEORGE SCHEFFER3
                                                       THEO J.M. DE WITTE1
                                                   REINIER A.P. RAYMAKERS1

           DEPARTMENT OF HEMATOLOGY1 AND CENTRAL HEMATOLOGY LABORATORY2
          UNIVERSITY MEDICAL CENTER NIJMEGEN, AND DEPARTMENT OF PATHOLOGY3,
                                FREE UNIVERSITY AMSTERDAM, THE NETHERLANDS




                            CLINICAL CANCER RESEARCH. 11: 2436-2444, 2005
_____   CHAPTER 5 _____________________________________________________________________________________________________




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Abstract

Purpose
Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete
chemotherapeutical eradication of leukemic CD34+38- stem cells is likely to result in
disease relapse. The purpose of this study was to investigate the role of the Breast Cancer
resistance Protein (BCRP/ABCG2) in drug resistance of leukemic stem cells and the effect
of its modulation on stem cell eradication in AML.

Experimental design
BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody)
and the effect of its modulation (using the novel fumitremorgin C analog KO143) on
intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in
leukemic CD34+38- cells.

Results
BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP
mediated drug extrusion by the novel fumitremorgin C analog KO143 resulted in increased
intracellular mitoxantrone accumulation in these cells in the majority of patients. This
increase, however, was much lower than in the mitoxantrone resistant breast cancer cell
line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage
due to the presence of additional drug transport mechanisms, among which P-glycoprotein
and Multiple Drug Resistance Protein (MRP). In line with these findings, selective blockage
of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells.

Conclusions
These results demonstrate that drug extrusion from leukemic stem cells is mediated by the
promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather
than modulation of single mechanisms, is therefore likely to be required to circumvent drug
resistance and eradicate leukemic stem cells in AML.



Introduction

Cancer is increasingly recognized as a disease originating from the transformation of
normal stem cells, retaining the capacity for self-renewal(1). This emerging concept for
the origin of tumorigenesis was stimulated by the postulation of acute myeloid leukemia
as a stem cell disease. Cells with leukemic stem cell characteristics, defined as leukemic
engraftment potential and self renewal capacity, in acute myeloid leukemia are found in
the CD34+CD38- cell population. The involvement of CD34+38- cells in leukemogenesis


                                                                                                                    91
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is suggested by the presence of cytogenetically aberrant cells in the CD34+CD38-
compartment as demonstrated by FISH and PCR for leukemia specific translocations(2,3).
Additionally, leukemic cells with long-term proliferative ability both in vitro and in vivo have
been identified as CD34+CD38-(4). Studies using the NOD-SCID mouse model have shown
that cells with leukemic engraftment and self renewal potential in AML are found in the
CD34+CD38- subpopulation(5,6) and not in more differentiated CD34+CD38+ cells. These
studies strongly argue that leukemia initiating transformation and progression associated
genetic events occur at the level of these primitive cell populations. Importantly, incomplete
chemotherapeutical eradication of cancer stem cells is likely to result in disease relapse.
Elucidation of the mechanisms conferring resistance to these cells is therefore essential to
provide novel targets for stem cell eradication in AML.
The ATP-binding cassette protein family is a large family of highly conserved membrane
proteins transporting a wide variety of substrates across the cell membrane(7) . Several
members, among which MDR1 (ABCB1) and BCRP (ABCG2) extrude a variety of
structurally unrelated chemotherapeutical compounds, thereby conferring a multi-drug
resistance phenotype to cancer cells.
BCRP (ABCG2) is a 655-aa member of the ABCG subfamily of ABC- membrane transporters
first described in drug resistant cell lines(8-10). BCRP is a half transporter functioning as a
dimer and confers multidrug resistance to topotecan, mitoxantrone, doxorubicin and
related compounds by ATP dependent drug extrusion(9,11). BCRP is expressed in placental
syncytiotrophoblasts, intestinal epithelium, liver canicular membrane(12) suggesting
a physiological role in detoxification. Recently, BCRP has been shown to be highly
expressed in a wide variety of stem cells(13) including immature human hematopoietic
progenitors(14). BCRP is proposed to protect these long-lived cells from naturally occurring
toxic substrates(15).
KO143 is a novel fumitrimorgin C (FTC) analogue which has recently been shown to be
an extremely potent BCRP inhibitor(16) . KO143 is a specific inhibitor of BCRP and much
more potent than currently known inhibitors of BCRP such as FTC(17) and GF120918(18).
Importantly, KO 143 is non toxic at effective in vitro and in vivo concentrations which makes
it one of the most promising compounds for development of clinical modulators of BCRP
mediated efflux. The purpose of the current study was to investigate the role of BCRP in
drug resistance of leukemic CD34+38- stem cells and the effect of its modulation on stem
cell eradication in AML.



Methods

Bone marrow samples and CD34+38- hematopoietic cells
Bone marrow was obtained after informed consent from healthy allogeneic bone marrow
donors and patients with AML at diagnosis. Patient characteristics are shown in table 1.


92
_____________________________________________________________________________   BCRP IN LEUKEMIC PROGENITOR CELLS _____



Table 1: Patient characteristics.

N                                                     22
Sex
M                                                     12
F                                                     10
Age (median/range)                                    52 (20-68)
% CD34                                                40 (3-88)
FAB
M1                                                    5
M2                                                    12
M4                                                    1
M5                                                    4
Cytogenetics
Normal                                                12
t(8;21)                                               4
t(11;19)                                              1
+8                                                    2
Complex                                               3



Mononuclear cells were isolated by density gradient centrifugation using Ficoll 1.077
g/mL (Pharmacia Biotech, Uppsala, Sweden). Isolation, cryopreservation and thawing
procedures of cells have been described previously(19) and were identical for normal
and leukemic bone marrow samples. After thawing, cells were stained with fluoresceïne
isothiocyanate (FITC) or CY5 labeled CD34 and CD38- phycoerythrin (PE) monoclonal
antibodies (Becton Dickinson, BV, Etten-Leur, The Netherlands) for 30 minutes at 4ºC and
washed in HBSS with 1% v/v heat inactivated fetal calf’s serum (FCS, Hyclone, Logan,
Utah, U.S.A.). A Coulter Epics Elite Flow cytometer was used to define cell populations.
Gating on forward and right angle scatter was used to exclude dead cells and debris.
CD34+CD38- cells of both normal bone marrow and AML were defined as the cells with
CD38-PE fluorescence within the first decade of emission. CD34+CD38- cells appeared
in a consistently restricted light-scattering region confirming the lymphoid appearance of
these primitive progenitors(20). The CD34+CD38+ cells were sorted from a gate positioned
in the bulk of CD34+ cells and showed more heterogeneous light-scattering properties.

BCRP cell line
The human breast cancer cell line MCF7-MR (gift from R.Scheper, Free University
Hospital, Amsterdam, the Netherlands) is resistant to mitoxantrone and overexpresses
BCRP but mot MRP and MDR1 (P-glycoprotein)(21). MCF7-MR cells were cultured in RPMI
1640 medium supplemented with 10 % FCS in the presence of 80 nM mitoxantrone.

BCRP expression
BCRP was detected with the BXP21 monoclonal antibody (gift from R. Scheper, Free
University Hospital, Amsterdam, the Netherlands) which recognizes an internal epitope


                                                                                                                    93
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of the protein(12) and has been shown to specifically bind BCRP, displaying no cross
reactivity with other proteins(12). In AML patient samples BXP21 was more sensitive than
another BCRP specific moab BXP 34(22). BXP21 was used in a three color flow cytometric
assay, which, in preliminary experiments prooved to be much more sensitive than
immunocytochemical assessment using immunofluorescence-microscopy, as previously
recognized for other ATP-binding cassette transporters(23). First cells (1×106) were fixed
and permeabilised (“Fix & Perm”, Caltag Lab., Burlingame, CA, U.S.A., according to
manufacturer’s instructions) and subsequently washed in HBSS 1% FCS. Following
fixation, cells were blocked with pooled human serum for 10 minutes and incubated with
BXP21 (2.5 ug/ml) or isotype control IgG2a in the same concentration for 60 min at 4C.
BXP21 was detected by fluorescein (FITC)-conjugated goat anti mouse F(ab)2 fragments
(5.0 ug/ml) for 30 minutes. A 10 minute incubation with goat serum to block non-specific
binding was followed by staining with CY5-conjugated CD34 and phycoerythrin (PE)-
conjugated CD38. BCRP appropriate isotype control at the same protein concentration as
the relevant antibody was used as control. BCRP protein expression was quantitated as
the median fluorescence channel shift (BXP21/Isotype control).

ABCG2 gene expression
RNA was isolated from subfractions (105 cells) of G-CSF mobilzed CD34+ hematopoietic
cells using cDNA synthesis and real time quantitative RT-PCR was performed as described
previously(24) . ÁBCG2 target gene expression was quantitated relatively to housekeeping
genes glyceraldehyde-3-phosphate- dehydrogenase (GAPDH) and hydroxymethylbilane
synthase (HMBS) as described previously(24). We earlier showed that GAPDH is an
appropriate gene for standardisation of target gene expression in human CD34+38- and
CD34+38+ hematopoietic cell populations(24).

Primer and probe sequences for gene amplification:
ABCG2: Taqman gene expression assay (Apllied Biosystems) no HS 00184979 GAPDH:
Taqman gene expression assay (Apllied Biosystems) no HS 99999905
HMBS: Taqman gene expression assay (Apllied Biosystems) no HS00609297

BCRP mediated mitoxantrone efflux
BCRP function was tested using mitoxantrone as a substrate and the fumitremorgin C
analog KO143 as an inhibitor for BCRP; Mitoxantrone, rather than an anthracyclin was
used as a substrate because mitoxantrone appears to be a more sensitive substrate to
detect BCRP mediated efflux as suggested by resistance profiles in cell line models(8,11).
The novel fumitremorgin C analog KO143 (0.1 uM) was used as a specific inhibitor for
BCRP mediated efflux. KO143 was shown to be the most potent inhibitor currently available
and, importantly, is BCRP-specific (without inhibition of MDR1 or MRP) at the concentration
used in our assay(16). Flow cytometric measurement of FTC-sensitive mitoxantrone efflux


94
_____________________________________________________________________________   BCRP IN LEUKEMIC PROGENITOR CELLS _____



has been shown to be a sensitive and specific method for measuring the function of BCRP
in both selected and unselected cell lines(25)
Cells were stained with CD34-FITC and CD38-PE membrane markers, washed in HBSS 1%
FCS and pre-incubated with or without BCRP inhibitor for 20 minutes in Iscove’s modified
Dulbecco’s medium supplemented with 1% FCS. Mitoxantrone (10 uM) was added and
cells were incubated for 2 hrs at 37C, 5% CO2, with or without inhibitor. Subsequently,
cells were washed in ice cold HBSS 1% FCS and kept on ice until flow-cytometric analysis
or allowed an additional 1 hr efflux in drug free medium with or without inhibitor to further
enhance sensitivity of the assay. Cellular mitoxantrone fluorescence was measured on a
FACS-elite cytometer equipped with an argon laser. Fluorescence was assessed at an
excitation wavelength of 635 nm through a 670 nm band-pass filter in a three color protocol
with CD34-FITC and CD38-PE (figure 1B). At least 200 CD34+38- cells were analyzed in
each sample.
BCRP mediated efflux was quantitated as the ratio of mitoxantrone fluorescence (MFI)
in the presence/absence of KO143 and assessed in CD34+CD38-, CD34+CD38+ and
CD34- cells as defined. Interexperimental variability was assessed by performing triplicate,
independently performed, experiments in a panel of four different normal bone marrow
samples. BCRP mediated efflux, assessed in 200 CD34+38- cells and indicated as an
efflux index was 1.05 ± 0.03 (mean ± SEM), 0.88 ± 0.06, 1.11 ± 0.02 and 1.09 ± 0.03
in these samples. Based on the average SEM (0.036) observed in these preliminary
experiments, a cut-off level of EI 1.05 was used to define significant BCRP mediated efflux
in individual samples.

P-glycoprotein and multiple drug resistance protein (MRP) mediated drug
efflux
Flow cytometric assessment of P-glycoprotein and MRP mediated mitoxantrone efflux was
performed as described above using verapamil (Knoll AG, Ludwigshaven, Germany (20
ugr/ml) and probenecid (0.5 mM) as inhibitors for P-glycoprotein and MRP, respectively.

Fluorescence in situ hybridization (FISH)
CD34+38- cells were sorted and lysed in 15 ul KCL (75 uM) on a glass cover slip. Cells were
fixed in methanol:acetic acid and stored at 4°C until analysis. FISH was performed using
the LSI AML1/ETO Dual Color, Dual Fusion t (8;21) Probe or the LSI EGR1 (5q31) Dual
Color Probe (Vysis, Downers Grove, IL, U.S.A.) according to manufacturer’s instructions.
One hundred cells were analyzed microscopically for t(8;21) or chromosome 5 (5q31)
cytogenetic abnormalities, as appropriate.

96-well chemosensitivity assay
CD34+38- cells were sorted in 96-well, round bottom, plates (200 cells/well) containing
Iscove’s medium supplemented with 10% FCS with/without mitoxantrone (1.0 uM) and


                                                                                                                    95
_____   CHAPTER 5 _____________________________________________________________________________________________________



with/without KO 143 in different concentrations (0.1, 1.0 and 10.0 uM). Plates were
incubated at 37°C, 5% CO2 for 48 hours. All experiments were performed in quadruplicate.
Cell apoptosis was assessed at 24 hrs and 48 hrs using propidium iodide (PI) as a cell
death marker. Cell images were acquired on a Zeiss Axiovert 35M inverted microscope
(Thorwood, New York, USA) equipped with a 40x oil (N.A. 1.3) objective and a cooled
756x580 pixel resolution CDD-camera (Variocam, PCO computer optics, Kellheim,
Germany) coupled with the pixel pipeline in a Macintosh Quadro 800. Cells were excited
with a mercury arc lamp using band pass filter 510-560 nm for propidium iodide (PI).
Emission was measured with a longpass filter 610nm.

Statistical analysis
The Student’s T-test was used to calculate significant differences. Correlations were
calculated using correlation coefficients. Data are presented as mean ± standard error of
the mean (SEM). P values < 0.05 were considered significant.



Results

BCRP expression and BCRP mediated mitoxantrone efflux in MCF7-MR
cells
BCRP expression in the MCF7-MR cell line, indicated as BXP21/IgG2a isotype control,
using the current protocol was 44.7 ± 4.5 (mean ± SD of three experiments; figure 1a).
BCRP-mediated efflux was examined using KO143 as a specific inhibitor of BCRP. KO143
has been shown to be specific for BCRP at concentrations of 0.1-1.0 uM, not inhibiting
MDR1 (P-glycoprotein) or MRP1 at these concentrations(16). The effect of KO143 on
cellular mitoxantrone (10 uM) fluorescence in MCF7-MR cells, measured after 2 hours
accumulation with/without inhibitor, is shown in figure 1b.
Mitoxantrone fluorescence (MFI) of MF7-MR cells after 2 hrs accumulation without
mitoxantrone and in the presence of mitoxantrone without/with KO143 were 0.4 ± 0.1 (SD),
9.1 ± 0.3 and 28.5 ± 6.7, respectively (n=3). The mean BCRP mediated mitoxantrone
efflux, indicated as a fluorescence shift index was 3.13 ± 0.62. Mitoxantrone efflux during
an additional hour in drug-free medium was completely blocked by KO143, illustrating
the potency of KO143 as a BCRP inhibitor and increasing sensitivity of BCRP efflux
assessment in the MCF7-cell line (mean efflux index 5.70 ± 0.35, figure 1B2). Importantly,
these data confirm that KO143 at the concentration used in our assays (0.1 uM) completely
blocks BCRP mediated mitoxantrone efflux, as demonstrated earlier(16).




96
_____________________________________________________________________________   BCRP IN LEUKEMIC PROGENITOR CELLS _____



                A
                              IgG2a                               BXP-21                                       IgG2a
                                                                                                               BXP-21




                B1
                             -KO143                                 + KO143                                    - KO143
                                                                                                               + KO143




                B2
 MITOXANTRONE




                              -KO143                               + KO143                                     - KO143
                                                                                                               + KO143




Figure 1: BCRP expression and BCRP mediated mitoxantrone efflux in MCF7-MR cells. A. Flow cytometric
assessment of BCRP expression using secondary FITC labeled F(ab)2 fragments against monoclonal antibody
BXP21 (dotted line) or isotype control. B. Mitoxantrone fluorescence (on y-axis) in the presence (dotted line) or
absence of the BCRP specific inhibitor KO143 (0.1 uM) after 2 hr accumulation (B1) and an additional hour of
efflux in drug free medium (B2).


BCRP is preferentially expressed and functionally active in primitive
CD34+38- hematopoietic cells in human normal bone marrow
In order to compare BCRP expression and function in normal and malignant hematopoietic
stem cells, first, BCRP expression was assessed in hematopoietic subpopulations in seven
normal bone marrow samples (figures 2 and 3).
BCRP expression was higher in CD34+38- cells (mean BXP21/IgG2a index 7.95 ± 1.32
compared to more differentiated CD34+38+ progenitors (6.81 ± 0.98; p= 0.05, using
student’s T-test for paired samples) and sharply decreased during further differentiation in
CD34- cells ( 3.36 ± 0.62; p<0.001)(figure 3A).




                                                                                                                        97
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                                                                                 IgG2a                   No inhibitor
                                                                                 BXP21                   KO143 (0.1uM)

                            A1                             B1                            C1                              D1




                                  MITOXANTRONE




                                                                 EVENTS
CD34




       CD38                                      CD34                     FITC                    MITOXANTRONE

                            A2                             B2                            C2                              D2




                            A3                             B3                            C3                              D3




Figure 2: Flow-cytometric assessment of BCRP expression and BCRP mediated mitoxantrone efflux
in CD34+38- hematopoietic in normal bone marrow and AML (for color reproduction: see appendix).
A.Definition of CD34+38- hematopoietic cells in human normal bone marrow (NBM) and AML. CD34+38- cells
were defined flow-cytrometrically as the CD34 expressing cells (indicated in blue) with CD38 expression within
the first decade of fluorescence emission (indicated in red) and compared with CD34+38+ cells (indicated as
blue gated) with an exclusion of a decade between CD38- and CD38+. CD34- cells are shown in grey. CD34+38-
cells were found invariably in both normal bone marrow (A1) and both CD34+ (> 10% CD34+ cells in bone
marrow by definition, A2) and CD34- leukemias (A3). The median frequency of CD34+38- cells was 0.1 % of
mononuclear cells in normal bone marrow (range 0.1%-0.3%) and 0.2 % in acute myeloid leukemia (range 0.1%-
10%). B. Cellular mitoxantrone fluorescence in different cell populations after 2 hrs incubation with mitoxantrone
(10 uM) in the absence of BCRP inhibitor. CD34+38- cells have a mitoxantrone “dull” phenotype. C. BCRP
protein expression in CD34+38- cells as determined by the BXP-21 antibody (dotted line) vs isotype control.
D. Mitoxantrone fluorescence in CD34+38- cells in the presence (dotted line) or absence of the BCRP specific
inhibitor KO143 (0.1 uM)




98
_____________________________________________________________________________                              BCRP IN LEUKEMIC PROGENITOR CELLS _____



       A                                                                B
                                  15.00
                                                                                                    1.60
                                                                  N=7                                                                     N=5/7
                                                                                                    1.50
  BCRP expression (BXP21/IgG2a)




                                                                        BCRP mediated efflux (EI)
                                                                                                    1.40
                                  10.00


                                                                                                    1.30


                                                                                                    1.20

                                   5.00
                                                                                                    1.10


                                                                                                    1.00


                                   0.00                                                             0.90
                                          34+38-   34+38+   34-                                              34+38-      34+38+          34-

Figure 3: BCRP is preferentially expressed and functionally active in CD34+38- hematopoietic cells in
human normal bone marrow. A. BCRP expression indicated as BXP21/IgG2a isotype control. Mean values
are indicated as bars and individual samples are represented by lines. BCRP expression is higher in CD34+38-
cells compared to more differentiated CD34+38+ progenitors and CD34- cells (7.95 ± 1.32, 6.81 ± 0.98; p= 0.05
and 3.36 ± 0.62; p<0.001, respectively). B. BCRP mediated mitoxantrone efflux indicated as efflux index (EI) in
different hematopoietic subpopulations.



To provide additional evidence that BCRP is preferentially expressed in CD34+38-
hematopoietic cells in comparison to committed progenitors and differentiated cells, ABCG2
gene expression was assessed in these cell populations using real-time quantitative RT-
PCR. Since bone marrow samples yielded insufficient cell numbers to reliably perform
real-time quantitative RT-PCR on the relatively low-copy transcript ABCG2, we used
GCS-F mobilized peripheral blood CD34+ cells obtained from two normal controls. ABCG2
expression was assessed relatively to house keeping genes as described previously(24) in
CD34+38-, CD34+38+ and differentiated cells (monocytes) (figure 4).




                                                                                                                                               99
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                                             0.3
          ABCG2 expression (ABCG2/HMBS)




                                             0.2




                                             0.1




                                               0




                                          0.0015
        ABCG2 expression (ABCG2/GAPDH)




                                           0.001




                                          0.0005




                                               0
                                                   34+38-      34+38+                     monocytes


Figure 4: ABCG2 is highly expressed in normal hematopoietic stem cells. ABCG2 expression was assessed
in different subfractions from GCSF-mobilized peripheral blood in two normal donors and quantitated relative to
the housekeeping genes HMBS and GAPDH (indicated on the y-axis as copy’s ABCG2/ copy HMBS/GAPDH)


These results confirm, in an independent assay, that primitive hematopoietic stem cells
express high levels of ABCG2, in line with previous reports(14,26).
Next, we investigated BCRP mediated efflux in CD34+38-, CD34+38+ and CD34-
hematopoietic subpopulations, using mitoxantrone as a substrate; cellular mitoxantrone
fluorescence (MFI) after 2 hrs mitoxantrone accumulation was low in CD34+38- cells,
compared to more differentiated CD34+38+ progenitors and CD34- cells (4.4 ± 0.29, 9.29
± 0.83 and 7.63 ± 0.55 respectively). BCRP mediated mitoxantrone efflux contributed to
the mitoxantrone “dull” appearance of primitive CD34+38- cells as evidenced by the ability
of KO143 to increase fluorescence intensity; Significant BCRP mediated efflux, defined
as a mitoxantrone fluorescence (MFI) index with/without KO143 (0.1 uM) > 1.05 (see
section “methods”), was found in CD34+38- cells in 5/7 (71%) samples. In 2/7 samples no
BCRP mediated efflux was found in either hematopoietic subpopulation. In those samples
displaying BCRP mediated efflux, this was invariably and significantly higher in CD34+38-


100
_____________________________________________________________________________                               BCRP IN LEUKEMIC PROGENITOR CELLS _____



cells compared to CD34+38+ and CD34- cells (mean efflux index 1.22 ± 0.08, 1.13 ± 0.04;
p=0.03 and 1.11 ± 0.03; p=0.03, respectively; figures 2 and 3B).
These data show that BCRP is preferentially expressed and functionally active in primitive
CD34+38- hematopoietic cells compared to more differentiated cells in human normal
bone marrow.

BCRP expression and function is conserved in primitive leukemic CD34+38-
hematopoietic cells in AML

BCRP is preferentially expressed and functionally active in CD34+38- hematopoietic
cells in AML
BCRP expression was assessed in different hematopoietic subpopulations in 22 de novo
AML samples (figure 2 and 5).

             A                                                                    B
                                                                                                     1.4
                                                                 N=22                                                                      N=14/22
                                 20.00

                                                                                                    1.35
 BCRP expression (BXP21/IgG2a)




                                                                                                     1.3

                                 15.00
                                                                        BCRP mediated efflux (EI)




                                                                                                    1.25




                                                                                                     1.2
                                 10.00


                                                                                                    1.15




                                                                                                     1.1
                                  5.00




                                                                                                    1.05




                                  0.00                                                                 1
                                         34+38-   34+38+   34-                                             34+38-        34+38           34-


Figure 5: BCRP is preferentially expressed and mediates mitoxantrone efflux in CD34+38- hematopoietic
cells in AML. BCRP expression A. indicated as BXP21/IgG2a isotype control and BCRP mediated mitoxantrone
efflux B. indicated as efflux index (EI) in different hematopoietic subpopulations. Mean values are indicated as
bars and individual samples represented by lines.


Expression was higher in CD34+38- cells compared to more differentiated CD34+38+ and
CD34- cell populations in 19/22 samples investigated. Mean BCRP expression was 9.00 ±
1.01 (range 1.59-21.4), 6.53 ± 0.68 (range 2.84-14.89); p<0.001 and 5.75 ± 0.58 (range 1.66-
10.13); p<0.001, respectively. BCRP expression in CD34+38- cells in AML was not significantly


                                                                                                                                                101
_____   CHAPTER 5 _____________________________________________________________________________________________________



different from the expression in these cells in normal bone marrow (p=0.30). These results
indicate that BCRP is preferentially expressed in primitive CD34+38- cells in the majority of
AML samples, similar to the expression profile in normal bone marrow. We next investigated
whether the preferential expression of BCRP in leukemic CD34+38- cells was reflected by
increased BCRP mediated transport in these cells; In AML, similar to the situation in normal
bone marrow, CD34+38- hematopoietic cells accumulated the lowest amount of mitoxantrone
relative to more differentiated CD34+38+ and CD34- cell populations (MFI 6.6 ± 0.49, 10.0 ±
0.66, 10.34 ± 0.74, respectively). To determine the contribution of BCRP mediated efflux to
the mitoxantrone “dull” phenotype of leukemic CD34+38- cells, BCRP mediated mitoxantrone
efflux was assessed in 22 leukemic bone marrow samples using KO143 (0.1 uM) as an
inhibitor; In the majority of samples (14/22=64%), BCRP mediated efflux was detected in
CD34+38- cells. The remaining 8 samples displayed no BCRP mediated efflux in either cell
population. In the samples displaying BCRP mediated efflux, efflux was significantly higher
in CD34+38- cells compared to CD34+38+ and CD34- cells (Efflux index 1.20 ± 0.03, 1.08 ±
0.02; p=0.003 and 1.06 ± 0.002; p=0.001, respectively, Figure 5B). The differences remain
significant when the overall population (n=22) is considered (Efflux index 1.11 ± 0.04, 1.03 ±
0.02; p=0.005 and 1.02 ± 0.02; p=0.003, respectively). No significant differences were found
when BCRP mediated efflux in different subpopulations in AML was compared with these
populations in normal bone marrow. No correlation existed between BCRP expression or
BCRP-mediated mitoxantrone efflux and mitoxantrone accumulation in leukemic CD34+38-
cells (r –0.22 and –0.27, respectively).


BCRP is expressed and mediates mitoxantrone efflux in leukemic CD34+38-
hematopoietic cells
The CD34+38- cell population in AML harbors residual normal CD34+38- cells(27). To
exclude the possibility that the expression and function of BCRP is present in residual
normal rather than leukemic cells within the CD34+38- cell compartment in AML, we
performed FISH analysis on leukemia associated cytogenetic aberrations in these cells
(n=5); Table 2 shows that either BCRP expression or efflux is present in CD34+38- cells
with predominantly or exclusively leukemic cells within this compartment (sample 2-5).

Table 2: BCRP is expressed and mediates mitoxantrone efflux in leukemic CD34+38- cells.

Patient          Cytogenetics           % 34+38- aberrant cells          BCRP            BCRP-mediated transport
1.               -5q31                  56                               4.95            0.94
2.               t(8;21)                90                               1.59            1.22
3.               t(8;21)                90                               NA              1.10
4.               t(8;21)                100                              3.19            1.03
5.               t(8;21)                95                               8.52            1.18
BCRP expression (BXP21/IgG2a) and BCRP mediated mitoxantrone efflux (indicated as efflux index) related to
percentage of cytogenetically aberrant cells in the CD34+38- cell population in AML.
NA= no material available for analysis.


102
_____________________________________________________________________________   BCRP IN LEUKEMIC PROGENITOR CELLS _____



Together, these results demonstrate that BCRP is preferentially expressed and functionally
active in primitive leukemic hematopoietic CD34+38- cells in the majority of patients. This
phenotype reflects a conserved physiological function, similar to the situation in normal
bone marrow.

Additional transporters are involved in drug efflux in leukemic CD34+38- hematopoietic cells
The relatively small effect of BCRP blockage by KO143 on cellular mitoxantrone
concentration and the lack of correlation between BCRP expression or BCRP mediated
drug efflux and mitoxantrone fluorescence in leukemic CD34+38- cells suggest that BCRP
is not the major determinant of the intracellular drug concentration in these cells. To assess
the contribution of other transporters to mitoxantrone efflux in leukemic CD34+38- cells,
we investigated whether mitoxantrone efflux took place in CD34+38- cells in the presence
of BCRP blockage. For this purpose cells were incubated with mitoxantrone (10 uM) with
KO143 (0.1 uM) for 2 hrs and subsequently allowed to efflux mitoxantrone in drug free
medium for an additional hour in the presence of KO143 (0.1 uM). In all AML samples
(n=10) mitoxantrone fluorescence (MFI) in CD34+38- cells decreased during the additional
hour of efflux with a MFI decrease of 20% ± 12% (mean ± SD). When cells were put
on ice after mitoxantrone accumulation to block active transport, no decrease in cellular
mitoxantrone was observed. The observation that KO143 in BCRP blocking concentration
can not completely abrogate drug efflux in CD34+38- cells indicates that additional
transporters are mediating mitoxantrone efflux in these cells.
To assess the role of other ATP-binding cassette transporters in drug extrusion from
leukemic stem cells, P-glycoprotein (ABCB1/MDR1) and MRP (ABCC2) mediated
mitoxantrone efflux was assessed in these 10 samples displaying drug efflux in the
presence of BCRP inhibition (table 3).

Table 3: Additional drug efflux mechanisms are involved in mitoxantrone efflux from leukemic stem cells.

Pat. Nr.               BCRP (ABCG2)                    P-glycoprotein (ABCB1)                     MRP (ABCC2)
1                      1.27                            1.25                                       1.51
2                      ns                              ns                                         ns
3                      ns                              1.15                                       ns
4                      1.22                            ns                                         ns
5                      1.10                            1.13                                       ns
6                      1.10                            ns                                         1.12
7                      ns                              1.15                                       na
8                      1.08                            ns                                         1.10
9                      1.08                            1.19                                       1.28
10                     ns                              1.12                                       ns
mean ± SEM             1.14 ± 0.03                     1.17 ± 0.02                                1.25 ± 0.09
P-glycoprotein and MRP mediated mitoxantrone efflux, indicated as efflux index, in leukemic CD34+38- cells.
ns= not significant (defined as efflux index < 1.05). na= no material available for analysis



                                                                                                                   103
_____   CHAPTER 5 _____________________________________________________________________________________________________



CD34+38- cells in 8/10 samples displayed either significant P-glycoprotein or MRP
mediated transport or both. The combined effect of P-glycoprotein and MRP inhibition on
mitoxantrone accumulation exceeded the effect of BCRP blockage in all these samples.
Additionally, the observation that drug efflux occurs in the presence of BCRP blockage
in a sample with only BCRP mediated efflux (sample 4) and a sample in which no ABC-
transporter activity could be demonstrated (sample 2) suggests the presence of yet
unidentified drug extrusion mechanisms.
Together these results indicate that BCRP is not the predominant drug efflux mechanism
in leukemic CD34+38- stem cells and that other mechanisms, among which P-glycoprotein
and MRP, play a role in drug efflux from leukemic stem cells.


Modulation of BCRP by the fumitremorgin C analog KO143 does not enhance drug
induced apoptosis in leukemic CD34+38- cells
Because inhibition of BCRP with KO143 (0.1 uM) increases mitoxantrone concentration
only moderately in primitive CD34+38- leukemic cells compared to the drug resistant MCF-
7 cell line and other drug transporters are active in these leukemic cells, we raised the
question whether selective modulation of BCRP could enhance chemosensitivity of these
cells. To investigate this, a 96-well cytotoxicity assay was employed on sorted CD34+38-
cells in 5 AML samples that displayed BCRP-mediated drug efflux (median efflux index
1.25 (1.21-1.37)(figure 6).
Mitoxantrone (1.0 uM) significantly induced apoptosis in leukemic CD34+38- cells at 48
hrs of incubation in all samples (relative increase in apoptotic cells compared to drug free
medium 1.66 ± 0.23; p=0.007)(Figure 6). KO143 at concentrations that completely block
BCRP mediated efflux (0.1-1.0 uM; as demonstrated in the MCF7-MR cell line) did not
significantly increase drug induced apoptosis (relative increase 1.81 ± 0.32 and 1.87 ± 0.37
respectively). Enhanced apoptosis when KO143 was used at high concentration (10 uM)
(average increase 2.57 ± 0.58, (figure 6), likely reflected intrinsic toxicity of the compound
rather than modulation of drug efflux mechanisms as demonstrated by increased apoptosis
in controls lacking mitoxantrone and the observation that KO143 at 10 uM did not further
increase intracellular mitoxantrone concentration in drug accumulation assays (data not
shown).
These results argue that specific modulation of BCRP, though functionally active in these
samples, does not significantly enhance drug induced apoptosis in leukemic CD34+38-
cells.




104
_____________________________________________________________________________   BCRP IN LEUKEMIC PROGENITOR CELLS _____




 B                                     3.5
Relative increase in apoptotic cells




                                        3


                                       2.5


                                        2


                                       1.5


                                         1


                                       0.5


                                        0
                                             MITO    + KO143              +KO143                      + KO143
                                             1.0uM    0.1uM                1.0uM                        10uM

  Figure 6: Specific inhibition of BCRP does not increase mitoxantrone induced apoptosis in primitive
  leukemic CD34+38- hematopoietic cells. Effect of KO143 on mitoxantrone induced apoptosis in leukemic
  CD34+38- cells. A. Typical example of microscope images of leukemic CD34+38- cells of a patient in the
  presence/absence of mitoxantrone and KO143. Apoptotic (PI-positive) cells are depicted in black (negative
  images). DFM=drug free medium, MITO= mitoxantrone alone, + KO143= mitoxantrone in the presence of
  KO143 at different concentrations. B. Mitoxantrone induced cell death depicted as relative increase in apoptotic
  cells in sorted CD34+38- cells in the presence/ absence of KO143. Values represent the mean ± S.E.M. of five
  patients. The white bars represent control experiments with KO143 at different concentrations in the absence
  of mitoxantrone. No significant increase in mitoxantrone induced apoptosis is seen when KO143 is added in
  BCRP specific concentrations (0.1-1.0 uM). Enhanced apoptosis when KO143 was used at high concentration
  (10 uM) reflects intrinsic toxicity of the compound as demonstrated by increased apoptosis in controls lacking
  mitoxantrone.



  Discussion

  Incomplete eradication of cancer stem cells is likely to result in disease relapse. Elucidation
  of the mechanisms conferring chemoresistance to these cells is therefore of major
  importance. In this report we demonstrate that the ATP-binding cassette transporter BCRP/
  ABCG2 is preferentially expressed in the leukemic CD34+38- stem cell subpopulation and
  contributes to mitoxantrone efflux in these cells.
  Recently, several studies have addressed the expression of BCRP in leukemic blasts in
  AML(22,26). Abott et al(26) found relatively low levels of BCRP gene expression compared


                                                                                                                   105
_____   CHAPTER 5 _____________________________________________________________________________________________________



to drug resistant BCRP clones in newly diagnosed adult AML patients. Flow cytometry
revealed very small subpopulations of BCRP expressing cells, suggesting that BCRP
expression may be limited to a small cell subpopulation. Similar findings have been
reported by Sargent et al(28), who demonstrated, using imunocytochemistry with the BXP-
34 antibody, that BCRP is expressed in a minority of leukemic cells in AML. Although the
results of these studies suggested the possibility of high BCRP expression in a small
subpopulation of cells, until now the subpopulations of BCRP expressing cells in AML
had not been identified. We here demonstrate that BCRP is preferentially expressed in a
subpopulation of primitive CD34+38- leukemic cells, which have previously been shown
to comprise leukemic stem cells. BCRP expression and efflux in leukemic CD34+38- cells
was not different from expression and function in normal CD34+38- cells. This shows that
the high BCRP expression in leukemic CD34+38- cells reflects a conserved physiological
function of BCRP in primitive hematopoietic cells rather than being a leukemia- associated
phenotype; BCRP has previously been shown to be highly expressed in a wide variety
of human normal stem cells(13), including hematopoietic stem cell populations(14) and is
believed to protect these long-lived cells against genetic damage induced by naturally
occurring xenobiotic toxins(15).
Incomplete chemotherapeutical eradication of leukemia-initiating CD34+38- cells due to
intrinsic chemoresistance, is likely to result in disease relapse in acute myeloid leukemia.
The preferential expression in the majority of newly diagnosed AML patients suggested
that BCRP might be a target for stem cell eradication in AML. To test this hypothesis, we
assessed the effect of BCRP modulation on the chemosensitivity of leukemic CD34+38-
cells. Importantly, selective blockage of BCRP by KO143, the most potent BCRP inhibitor
currently available(16) and capable of completely blocking BCRP as confirmed in the MCF7-
MR cell line, did not increase mitoxantrone induced apoptosis to leukemic CD34+38- cells.
This lack of a chemosensitizing effect of BCRP modulation by KO143 was anticipated by
several observations in this study; First BCRP expression and BCRP mediated efflux were
not correlated with intracellular mitoxantrone accumulation in CD34+38- cells, suggesting
that other determinants are involved in the mitoxantrone “dull” phenotype. Secondly,
BCRP expression and the effect of KO143 on mitoxantrone accumulation was moderate
in leukemic CD34+38- cells compared to the drug resistant MCF7-MR cell line in which
BCRP confers resistance to mitoxantrone. Finally, we observed substantial drug efflux in
leukemic CD34+38- cells in the presence of BCRP inhibition, demonstrating that other
drug transporters, among which P-glycoprotein and MRP, are active in these cells. The
presence of these promiscuous drug extrusion mechanisms might provide an explanation
for the poor correlation between BCRP expression and function reported in literature(22, 29)
and the observation in this study that KO143 increased mitoxantrone accumulation in only
64% of samples expressing BCRP.
Together, these findings argue that selective modulation of BCRP is not sufficient to
circumvent resistance of leukemic CD34+38- cells. Alternatively, we cannot completely


106
_____________________________________________________________________________   BCRP IN LEUKEMIC PROGENITOR CELLS _____



exclude the possibility that KO143 does not completely inhibit BCRP in leukemic cells. The
report that KO143 is the most potent BCRP inhibitor known thus far, tested in a panel of
cell lines and our finding that it completely blocks efflux in the MCF7-MR cell line argues
against this possibility.
Simultaneous modulation of several transporters, among which BCRP, could be required
to sufficiently increase drug accumulation and eradicate CD34+38- stem cells in AML. The
recent report of improved clinical outcome in a trial using cyclosporin as a P-glycoprotein
inhibitor in AML(30), in contrast to the earlier, consistently poor, results of selective
P-glycoprotein modulation on long-term disease outcome, could be in line with this
assumption since cyclosporin is a more promiscuous ABC-transporter inhibitor with effects
on both P-glycoprotein and BCRP(31). Together, these data warrant further research into
the usefulness and feasibility of broad-spectrum ABC-transporter inhibitors to eradicate
leukemic stem cells in AML.



Acknowledgments

The authors like to thank A. Pennings and G. Vierwinden for technical assistance. J.Allen
(Department of Experimental Therapy, Netherlands Cancer Institute, Amsterdam, the
Netherlands) is thanked for providing KO143.



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      primitive hematopoietic cell. Nat Med 1997;3:730-737.
7.    Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily. Genome
      Res 2001;11:1156-1166.
8.    Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer
      cells. Proc Natl Acad Sci USA 1998;95:15665-15670.
9.    Ross DD, Yang W, Abruzzo LV, et al. Atypical multidrug resistance: breast cancer resistance protein
      messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 1999;91:429-433.



                                                                                                                   107
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10.     Maliepaard M, van Gastelen MA, de Jong LA, et al. Overexpression of the BCRP/MXR/ABCP gene in a
        topotecan-selected ovarian tumor cell line. Cancer Res 1999;59:4559-4563.
11.     Allen JD, Brinkhuis RF, Wijnholds J, Schinkel AH. The mouse Bcrp1/Mxr/Abcp gene: amplification and
        overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res
        1999;59:4237-4241.
12.     Maliepaard M, Scheffer GL, Faneyte IF, et al. Subcellular localization and distribution of the breast cancer
        resistance protein transporter in normal human tissues. Cancer Res 2001;61:3458-3464.
13.     Zhou S, Schuetz JD, Bunting KD, et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of
        stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028-1034.
14.     Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump
        and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507-512.
15.     Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP. Bcrp1 gene expression is required for
        normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in
        hematopoietic cells in vivo. Proc Natl.Acad Sci USA 2002;99:12339-12344.
16.     Allen JD, van Loevezijn A, Lakha, JM, et al. Potent and specific inhibition of the breast cancer resistance
        protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol
        Cancer Ther 2002;1:417-425.
17.     Rabindran SK, Ross DD, Doyle LA, Yang W, Greenberger LM. Fumitremorgin C reverses multidrug
        resistance in cells transfected with the breast cancer resistance protein. Cancer Res 2000;60:47-50.
18.     de Bruin M, Miyake K, Litman T, Robey R, Bates SE. Reversal of resistance by GF120918 in cell lines
        expressing the ABC half-transporter, MXR. Cancer Lett 1999;46:117-126.
19.     Smeets M, Raymakers R, Vierwinden G, et al. A low but functionally significant MDR1 expression protects
        primitive haemopoietic progenitor cells from anthracycline toxicity. Br J.Haematol 1997;96:346-355.
20.     Terstappen LW, Huang S, Safford M, Lansdorp PM, Loken MR. Sequential generations of hematopoietic
        colonies derived from single nonlineage-committed CD34+. Blood 1991;77:1218-1227.
21.     Taylor CW, Dalton WS, Parrish PR, et al. Different mechanisms of decreased drug accumulation in
        doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line. Br J Cancer
        1991;63:923-929.
22.     van der Kolk DM, Vellenga E, Scheffer GL, et al. Expression and activity of breast cancer resistance protein
        (BCRP) in de novo and relapsed acute myeloid leukemia. Blood 2002;99:3763-3770.
23.     Broxterman HJ, Lankelma J, Pinedo HM, et al. Theoretical and practical considerations for the measurement
        of P-glycoprotein function in acute myeloid leukemia. Leukemia 1997;11:1110-1118.
24.     Raaijmakers MH, van Emst L, de Witte T, Mensink E, Raymakers RA. Quantitative assessment of gene
        expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain
        reaction. Exp.Hematol 2002;30:481-487.
25.     Robey RW, Honjo Y, van de LA., et al. A functional assay for detection of the mitoxantrone resistance
        protein, MXR (ABCG2). Biochim Biophys Acta 2001;1512: 171-182.
26.     Abbott B L, Colapietro AM, Barnes Y, Marini F, Andreeff M, Sorrentino BP. Low levels of ABCG2 expression
        in adult AML blast samples. Blood 2002;100:4594-4601.
27.     Feuring-Buske M, Haase D, Buske C, Hiddemann W, Wormann B. Clonal chromosomal abnormalities in
        the stem cell compartment of patients with acute myeloid leukemia in morphological complete remission.
        Leukemia 1999;13:386-392.
28.     Sargent JM, Williamson CJ, Maliepaard M, Elgie AW, Scheper RJ, Taylor CG. Breast cancer resistance
        protein expression and resistance to daunorubicin in blast cells from patients with acute myeloid leukaemia.
        Br J Haematol 2001;115:257-262.
29.     Suvannasankha A, Minderman H, O’loughlin KL, et al. Breast cancer resistance protein: Discordance between
        expression and function in acute myeloid leukemia. Blood 2003;100(11, supplement):Abstract nr 244.
30.     List AF, Kopecky KJ, Willman CL, et al. Benefit of cyclosporine modulation of drug resistance in patients with
        poor-risk acute myeloid leukemia: a Southwest Oncology Group study. Blood 2001;98:3212-3220.
31.     Leith CP, Chen IM, Kopecky KJ, et al. Correlation of multidrug resistance (MDR1) protein expression with
        functional dye/drug efflux in acute myeloid leukemia by multiparameter flow cytometry: identification of
        discordant MDR-/efflux+ and MDR1+/efflux- cases. Blood 1995;86:2329-2342.

108
                                     CHAPTER 6
LEUKEMIC CD34+38- PROGENITOR CELLS DISPLAY
CONSERVED DIFFERENTIAL EXPRESSION OF MANY
   ATP-BINDING CASSETTE TRANSPORTER GENES




                                                   MARC HGP RAAIJMAKERS*
                                                           ELKE DE GROUW*
                                                           JOOP H. JANSEN
                                                   BERT A. VAN DER REIJDEN
                                                       THEO J.M. DE WITTE
                                                    REINIER AP RAYMAKERS
                                     * THESE AUTHORS CONTRIBUTED EQUALLY TO THIS ARTICLE



         DEPARTMENT OF HEMATOLOGY AND CENTRAL HEMATOLOGY LABORATORY,
                  UNIVERSITY MEDICAL CENTER NIJMEGEN, THE NETHERLANDS




                                     LEUKEMIA. ACCEPTED FOR PUBLICATION
_____   CHAPTER 6 _____________________________________________________________________________________________________




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__________________________________________________   ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____



Abstract

Introduction
Acute myeloid leukemia (AML) is considered a disease originating from hematopoietic
CD34+38- stem cells. Incomplete chemotherapeutical eradication of leukemic CD34+38-
stem cells is likely to result in disease relapse. Elucidation of the mechanisms conferring
resistance to these cells is therefore essential to provide novel targets for stem cell
eradication in AML.

Methods
We studied gene expression of 45 ABC-transporters (the complete ABCA, B, C, D and G
family) in human hematopoietic CD34+38- cells in comparison to committed CD34+38+
progenitors in normal G-CSF mobilized or bone marrow cells (n=11) and their malignant
counterparts in AML (n=11). Gene expression was assessed with a novel real-time
quantitative RT-PCR approach using microfluidic low-density arrays.

Results
In normal CD34+38- cells 36 transporters were expressed of which 24 displayed significant
differential expression in comparison to committed progenitors. In addition to known stem
cell transporters (ABCB1, ABCG2 and ABCC1) these differentially expressed genes
included many members not previously associated with stem cell biology. ABC transporter
gene expression profile of CD34+38- cells was largely conserved in AML and included
expression of all 13 members currently associated with drug extrusion and resistance.

Conclusions
These data suggest an important role for previously unrecognized ABC-transporters
in hematopoietic stem cell biology. Additionally, the identification of many, previously
unidentified, drug transporters in leukemic stem cells prompts further research to test their
role in drug resistance and value as potential new targets to enhance chemotherapeutical
eradication of leukemic stem cells in AML



Introduction

AML is considered a disease originating from hematopoietic CD34+38- stem cells. In AML,
cells with leukemic stem cell characteristics, defined as leukemic engraftment potential and
self-renewal capacity, are found in the CD34+CD38- cell population, similar to the hierarchy
in normal bone marrow. The involvement of CD34+38- cells in leukemogenesis is suggested
by the presence of cytogenetically aberrant cells in the CD34+CD38- compartment as
demonstrated by FISH and PCR for leukemia specific translocations(1 2). Additionally,


                                                                                                                  111
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leukemic cells with long-term proliferative ability both in vitro and in vivo have been identified
as CD34+CD38-(3). Studies using the NOD-SCID mouse model have shown that cells with
leukemic engraftment and self renewal potential in AML are found in the CD34+CD38-
subpopulation (4,5) and not in more differentiated CD34+CD38+ cells. These studies strongly
argue that leukemia initiating transformation and progression associated genetic events
occur at the level of these primitive cell populations. Incomplete chemotherapeutical
eradication of leukemic stem cells is likely to result in disease relapse. Elucidation of the
mechanisms conferring resistance to these cells is therefore essential to provide novel
targets for stem cell eradication in AML.
Normal hematopoietic (CD34+38-) stem cells (HSC) are characterized by their ability to
extrude a variety of fluorescent compounds such as Hoechst and rhodamine123, resulting
in a fluorescent-dye “dull” phenotype of these cells. The molecular basis of this phenotype
is the expression of the ATP-binding cassette (ABC) transmembrane transporters
ABCB1(6-9) and ABCG2(10,11), that efflux the fluorescent dyes. ABC-transporters represent
the largest family of transmembrane proteins involved in the transmembrane transport of
a huge variety of substrates including sugars, peptides, inorganic anions, amino-acids,
oligopeptides, polysaccharides and proteins vitamins and metallic ions(12,13) The human
superfamily of ABC transporters currently consists of 48 members. The genes can be
divided into subfamilies based on similarity in gene structure resulting in seven mammalian
ABC gene subfamilies (ABCA, B, C, D, E, F and G family).
The physiological function of ABC transporters in human stem cell biology is largely
unknown. A role for ABC-transporters in the protection from genetic damage by naturally
occurring xenobiotics that are substrates for the transporter have been suggested for
hematopoietic stem cells(14,15) and has recently been substantiated by the observation of
increased cytotoxicity to normal bone marrow in ABCB1(7) and ABCG2/BCRP knock-out
mice(16).
Recently, an increasing number of ABC transporters has been associated with extrusion
of xenobiotic compounds and chemotherapeutical agents(17) and thus may be important
for stem cell protection. The expression of the vast majority of ABC transporters on
hematopoietic CD34+38- stem cells, however, is currently unknown.
Identification of such drug transporters may be particularly relevant for the treatment of
hematological malignancies originating from hematopoietic CD34+38- stem cells such
as AML since conserved expression of ABC transporters involved in drug resistance
after malignant transformation of CD34+38- hematopoietic stem cells would represent
an important mechanism of drug resistance to these cells(18). We recently demonstrated
conserved expression and function of ABCG2 in CD34+38- cells in AML but observed
significant mitoxantrone extrusion from these cells in the presence of ABCG2 inhibition(19),
suggesting the presence of additional drug transporters in these cells.
In the current study we profiled the expression of 45 ABC transporters (the complete
subfamilies A, B, C, D and G) in CD34+38- hematopoietic stem cells and committed


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__________________________________________________   ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____



CD34+38+ progenitors from normal bone marrow and AML using real-time RT-PCR.
Using this approach we demonstrate the differential expression of a large number of
ABC transporters in hematopoietic stem cells suggesting an important biological function
of these molecules in hematopoietic stem cell biology. These transporters include many
transporters associated with drug transport of which the expression is conserved in the
CD34+38- leukemic stem cell population in AML.



Methods

Isolation of CD34+38- and CD34+38+ hematopoietic cells
Bone marrow or granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood
was obtained after informed consent from healthy bone marrow donors and patients with
AML at diagnosis. Mononuclear cells were isolated by Ficoll 1.077 g/ml (Pharmacy Biotech,
Uppsala, Sweden). Isolation, cryopreservation and thawing procedures of cells have
been described previously(15) and were identical for normal and leukemic bone marrow
samples.
Cells were stained with CD34- CY5 or fluorescein isothiocyanate (FITC) and CD38-
phycoerythrin (PE) monoclonal antibodies (Becton Dickinson, BV, Etten-Leur, The
Netherlands) for 30 minutes at 4ºC and washed in Hank’s balanced salt solution (HBSS)
with 1% v/v heat inactivated fetal calf serum (FCS, Hyclone, Logan, Utah, U.S.A.). A
Coulter Epics Elite Flow cytometer was used to define cell populations. Gating on forward
and right angle scatter was used to exclude dead cells and debris. CD34+CD38- cells of
both normal bone marrow and AML were defined as the cells with CD38-PE fluorescence
within the first decade of emission as shown in figure 1. CD34+CD38- cells appeared in a
consistently restricted light-scattering region confirming the lymphoid appearance of these
primitive progenitors(20). The CD34+CD38+ cells were sorted from a gate positioned in the
bulk of CD34+ cells separated by a decade from CD34+38- cells as indicated in figure1 and
showed more heterogeneous light-scattering properties.

RNA isolation
Sorted cells were centrifuged at 1500 rpm for 5 minutes, and supernatant was removed
completely. Total RNA was extracted using a Zymo research Mini RNA isolation II kit (Zymo
research, USA) according to the manufacturer’s protocol. Reverse transcriptase PCR was
performed using 5x 1st strand buffer (Life Technologies), DTT (10mM, Life Technologies), dN6
(5mg/ml, Amersham Pharmacia Biotech), dNTP (0.625 mM, Amersham Pharmacia Biotech),
Rnasin (20 U, Promega) and M-MLV reverse transcriptase (200 U, Life Technologies) in a
total volume of 20 µl. The reaction mixture was incubated at 20°C for 10 minutes, at 42°C
for 60 minutes followed by heat inactivation of the enzyme at 95°C for 10 minutes. Before
storage at -80°C the cDNA was diluted in distilled water to a final volume of 100µl.


                                                                                                                  113
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Real-time RT-PCR

Design low-density array-microfluidic card
By using pre-configured Taqman low-density array-micro fluidic cards (Applied Biosystems,
USA), 45 ABC transporters in combination with 3 housekeeping genes were quantified
simultaneously by real time RT-PCR in single samples. Primer sets for real rime RT-PCR
were designed by Applied Biosystems (http://myscience.appliedbiosystems.com/index.jsp)
and spotted on the micro fluidic card. For quantification of each sample a 2x Taqman
Universal mastermix and 50µl cDNA template was added to a total volume of 100 µl. PCR
was performed in an Applied Biosystems 7900 HT Fast Real-time PCR sequence detector
by 40 cycles of 30 seconds at 95°C and 1 minute at 60°C.

Quantitation of gene expression
Normalized drug-resistance related gene expression to the internal standard glyceraldehyde-
3-phosphate- dehydrogenase (GAPDH) is given by the following equation(21,22):

T0/R0= K. (1+E) (CT,r – CT,t)

To: Initial number of target gene copies; Ro: Initial number of standard gene copies; E;
Efficiency of amplification; CT,t: Threshold cycle of target gene; CT, r: Threshold cycle of
standard gene; and K: Constant.
ABC transporter gene expression was quantitated relatively to housekeeping genes
GAPDH, hydroxymethylbilane synthase (HMBS) and porphobilinogen dehydrogenase
(PBGD). We earlier showed that GAPDH is an appropriate gene for standardization of
target gene expression in human CD34+38- and CD34+38+ hematopoietic cell populations
from normal bone marrow and AML(21)

Statistical analysis
Differences in normalized target gene expression between different hematopoietic
subpopulations were calculated using the Student’s t-test with a level of significance op
p<0.05.




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Results

ATP-binding cassette transporter gene expression in normal hematopoietic
CD34+38- cells

Normalized levels of ABC transporter gene expression in CD34+38- cells
We assessed gene expression of 45 ABC-transporters by real-time RT-PCR using low-
density array microfluidic cards in normal CD34+38- cells of G-CSF-mobilized peripheral
blood (n=8) or bone marrow (n=3) from allogeneic stem cell donors. CD34+38- cells were
purified as described in the section “methods”. CD34+38- cDNA input varied between
samples due to variability in number of cells available for sorting (average threshold
cycle (Ct) GAPDH 24.18 ± 0.56 SEM; range 20.97-26.30). We have previously shown
that GAPDH is an appropriate reference gene for normalizing target gene expression in
CD34+38- hematopoietic cells with similar levels of expression in committed CD34+38+
progenitors in both normal bone marrow and AML(21). Normalized CD38 gene expression
was 0.13 ± 0.02 copy CD38/copy GAPDH (mean ± SEM) and 0.39± 0.09 copy CD38/copy
GAPDH for CD34+38- and CD34+38+ populations respectively, indicating a median 2.88 ±
0.37-fold higher expression in CD34+38+ cells.
Normalized levels of ABC transporter gene expression in comparison to GAPDH are shown
in figure 1. Similar results were found when HMBS or PBGD were used as reference genes
(data not shown). The expression of 36 ABC-transporters was detected in CD34+38-
cells (figure 1). 24 ABC transporters were detected in all (11/11) samples. The lower
frequency of expression in the other 12 ABC transporters is likely to reflect the relatively
low expression level of these genes in addition to the lower amount of cDNA input in some
samples, precluding the detection of these low-copy genes, rather than biological binary of
expression. This is supported by the finding that genes with the highest level of expression
were detected invariably in all samples and that low frequency ABC transporters are the
genes with relative low expression levels (figure 1). This is further corroborated by the
finding that in the sample with the highest input (Ct GAPDH 20.97) 34 transporters were
detected (exceptions are ABCD2 and ABCC8).
Levels of ABC transporter gene expression were generally low in comparison to GAPDH
expression (mean expression level 0.059 ± 0.009 copies/copy GAPDH - 6.19.10-(5) copies/
copy GAPDH for highest (ABCC1) and lowest (ABCA8) expressed genes respectively).
Highest levels of expression were found for ABCC1, ABCA2, ABCB2, ABCB7, ABCD4
and ABCB1 respectively, confirming the relatively high expression of known stem cell
transporters ABCB1/ MDR1 and ABCC1/MRP1.
ABC-transporter gene expression levels were not significantly different in G-CSF mobilized
CD34+38- cells in comparison to their counterparts in normal bone marrow (data not
shown) with the exception of ABCB8 (copies ABCB8/copy GAPDH 0.014 ± 0.002 SEM
vs. 0.003± 0.0004 respectively; p< 0.001) and ABCB5 that was detected in all G-CSF
mobilized samples (8/8) but not in normal bone marrow (0/3).
                                                                                                                  115
_____                            CHAPTER 6 _____________________________________________________________________________________________________

 Copies target gene/copy GAPDH


                                 10-1



                                 10-2


                                 10-3



                                 10-4
                                        ABCA10
                                        ABCA12
                                        ABCA13




                                        ABCB10
                                        ABCB11




                                        ABCC10
                                        ABCC11
                                        ABCC12
                                        ABCC13




                                        ABCG1
                                        ABCG2
                                        ABCG4
                                        ABCG5
                                        ABCG8
                                        ABCA1
                                        ABCA2
                                        ABCA3
                                        ABCA4
                                        ABCA5
                                        ABCA6
                                        ABCA7
                                        ABCA8
                                        ABCA9



                                        ABCB1
                                        ABCB2
                                        ABCB3
                                        ABCB4
                                        ABCB5
                                        ABCB6
                                        ABCB7
                                        ABCB8
                                        ABCB9


                                        ABCC1
                                        ABCC2
                                        ABCC3
                                        ABCC4
                                        ABCC5
                                        ABCC6
                                        ABCC7
                                        ABCC8
                                        ABCC9




                                        ABCD1
                                        ABCD2
                                        ABCD3
                                        ABCD4
                                        11
                                        11
                                        11

                                        11

                                        11




                                        10
                                        11
                                        11
                                        11


                                        11
                                        11
                                        10
                                        11
                                        11

                                        11


                                        11
                                        11
                                        10



                                        11



                                        11

                                        11
                                        11
                                        11
                                        0

                                        6

                                        1
                                        9
                                        7
                                        0




                                        4
                                        8




                                        4

                                        7
                                        4



                                        0
                                        2
                                        0

                                        0
                                        0
                                        2

                                        4



                                        8
                                        0
                                        0
                                        0
Figure 1: ABC transporter gene expression in normal hematopoietic CD34+38- cells. Gene expression of 45
ABC transporters was assessed in CD34+38- hematopoietic cells from G-CSF-mobilized peripheral blood or bone
marrow (n=11) from allogeneic stem cell donors. Levels of gene expression are depicted as natural logarithmic
values normalized for the expression of the housekeeping gene GAPDH. Mean values of gene expression of
positive samples (± SEM) are represented by bars. On the X-axis frequency of expression (number of samples in
which gene expression was detectable) is shown.



ABC transporter gene expression in CD34+38- cells in comparison to committed
CD34+38+ progenitors
Cellular decisions concerning differentiation are reflected in altered patterns of gene
expression and therefore differential expression of genes in hematopoietic stem cells
in comparison to committed progenitors may be more relevant for understanding the
molecular events underlying normal and malignant hematopoiesis than absolute levels
of gene expression. For this reason we compared ABC transporter gene expression in
CD34+38- hematopoietic cells to the expression in committed CD34+38+ progenitors
(figure 2).
All 36 ABC transporter genes that were detected in CD34+38- cells were preferentially
expressed in CD34+38- cells in comparison to CD34+38+ committed progenitors, although
expression of none of these genes was restricted to hematopoietic CD34+38- stem cells.
This differential expression reached statistical significance, using student t-test for paired
samples; p≤0.05) in 24 ABC transporter genes (figure 2). Among these significantly
differential expressed genes were the anticipated transporters previously associated with
hematopoietic stem cells ABCB1 (median 5.0-fold difference; p<0.001), ABCC1 (1.66-
fold; p=0.003) and ABCG2 (1.97- fold; p=0.005). Additionally, many ABC transporters not


116
__________________________________________________                                   ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____



previously associated with hematopoietic stem cell biology were identified that displayed
significant differential expression in CD34+38- cells. In ranking order of differential
expression: 1. ABCA13 (33.4-fold; p=0.05) 2. ABCA9 (30.1-fold; p=0.04), 3. ABCB5 (21.36-
fold; p=0.02), 4. ABCC6 (16.6-fold ; p=0.05), 5. ABCA10 (13.2-fold ; p=0.01), 6. ABCA6
(9.0-fold ; p=0.004), 7.ABCA5 (5.4-fold ; p< 0.001) 8. ABCB4 (5.0-fold ; p=0.03), 9. ABCB1
(5.0-fold p<0.01), 10. ABCG1 (4.5-fold ; p=0.01).
In conclusion these results demonstrate that hematopoietic stem cells display differential
expression of a large number of ABC-transporters, including many members not previously
associated with stem cell biology.




                                    1000
 Differential expression (X-fold)




                                                            *

                                    100
                                                    *               *                *
                                                                                                              *
                                                                *
                                     10                                 *                              *                                *
                                                *                                *
                                           **                                                                                   *
                                                        *
                                                                                             *     *                   *
                                                                            **           *                                          ** *
                                          ABCA10
                                          ABCA12
                                          ABCA13




                                          ABCB10
                                          ABCB11




                                          ABCC10
                                          ABCC11
                                          ABCC12
                                          ABCC13




                                          ABCG1
                                          ABCG2
                                          ABCG4
                                          ABCG5
                                          ABCG8
                                          ABCA1
                                          ABCA2
                                          ABCA3
                                          ABCA4
                                          ABCA5
                                          ABCA6
                                          ABCA7
                                          ABCA8
                                          ABCA9



                                          ABCB1
                                          ABCB2
                                          ABCB3
                                          ABCB4
                                          ABCB5
                                          ABCB6
                                          ABCB7
                                          ABCB8
                                          ABCB9


                                          ABCC1
                                          ABCC2
                                          ABCC3
                                          ABCC4
                                          ABCC5
                                          ABCC6
                                          ABCC7
                                          ABCC8
                                          ABCC9




                                          ABCD1
                                          ABCD2
                                          ABCD3
                                          ABCD4




Figure 2: Differential expression of ABC-transporters in hematopoietic CD34+38- stem cells in
comparison to committed CD34+38+ progenitors. Difference in levels of gene expression between CD34+38-
and CD34+38+ cells is depicted at the y-axis as median X-fold difference in samples that expressed the ABC
transporter in CD34+38- cells. Transporters for which differential expression reached statistical significance
(paired analysis using student t-test for all samples; p<0.05) are indicated with an asterix.


ABC transporter gene expression in CD34+38- hematopoietic progenitors in
AML
The differential expression of many ABC transporter genes among which many genes
associated with the extrusion of chemotherapeutical compounds and drug resistance
prompted us to investigate the expression of these transporters in the leukemia- initiating
CD34+38- cell population in AML. We and others have previously shown that these cells
are predominantly of leukemic origin in the majority of samples(19,23).
ABC transporter gene expression was assessed by real-time PCR in CD34+38- cells from
11 AML patients at diagnosis. Patient characteristics are listed in table 1. FAB M3 was not


                                                                                                                                                  117
_____   CHAPTER 6 _____________________________________________________________________________________________________



included in this study because this subtype is not considered a disease originating from
CD34+38- stem cells as evidenced by the failure of CD34+38- cells to engraft leukemia
in the NOD-SCID model(5) . Samples were selected with a relatively high percentage of
CD34+ cells in order to enable the sorting of sufficient CD34+38- cells for the quantitation
of low-copy genes.

Table 1: Patient characteristics.

Patients                             11
Age (median; range)                  55 (10-72)
Sex
M                                    4
F                                    7
FAB subtype
Mo                                   2
M1                                   3
M2                                   2
M4                                   2
M5                                   2
CD34 % (median, range)               82 (19-90)


CD34+38- cells isolated from AML patients were comparable to normal CD34+38- cell
samples regarding CD38 expression both on protein level (average MFI 9.6 ±2.24 and 8.4
± 1.34 respectively, difference not significant) and CD38 gene expression (median 0.11
± 0.06 copy CD38/copy GAPDH and 0.13 ± 0.02 copy CD38/copy GAPDH respectively;
difference not significant). CD34+38- cDNA input was similar for AML and normal samples
(average Ct GAPDH 23.47 ± 0.69 and 24.18 ±0.56 respectively).
In CD34+38- cells in AML 40 ABC-transporter genes were expressed (figure 3). All genes
expressed in normal CD34+38- cells were detected in CD34+38- cells in AML with the
exception of ABCA8 that was detected in CD34+38- cells of 1/11 normal samples but not in
CD34+38- cells in AML. Additionally 5 ABC transporters were detected with low frequency
in AML but not in normal CD34+38- cells (ABCA4, ABCC7, ABCC9, ABCC11, and ABCG4).
A significant difference in frequency of expression between normal and leukemic CD34+38-
cells was found for ABCA13 (10/11 vs. 3/11 respectively; p< 0.001) and ABCB5 (8/11 vs.
1/11 respectively p<0.001).
Levels of expression of ABC transporters did not differ significantly between CD34+38-
cells in AML and their normal counterparts for the majority of ABC transporters that
were expressed in both normal and leukemic samples (31/35). Significant lower levels
of expression in CD34+38- cells in AML were found for ABCA3 (mean 0.002 ± 0.001
copy/copy GAPDH vs. 0.012 ±0.003; p=0.004), ABCA5 (0.005 ± 0.002 vs., 0.012 ± 0.003;
p<0.001),ABCB1 (0.01 ± 0.002 vs. 0.03 ± 0.005; p<0.001) and ABCB7 (0.03 ± 0.006 vs.
0.04 ± 0.006; p=0.04).
ABC transporter gene expression profiles for individual AML samples are included in the in
supplementary data.
118
__________________________________________________      ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____




                                                            *
                                                    *
 Copies target gene/copy GAPDH


                                 10-1
                                            *
                                        *

                                 10-2



                                 10-3



                                 10-4
                                        ABCA10
                                        ABCA12
                                        ABCA13




                                        ABCB10
                                        ABCB11




                                        ABCC10
                                        ABCC11
                                        ABCC12
                                        ABCC13




                                        ABCG1
                                        ABCG2
                                        ABCG4
                                        ABCG5
                                        ABCG8
                                        ABCA1
                                        ABCA2
                                        ABCA3
                                        ABCA4
                                        ABCA5
                                        ABCA6
                                        ABCA7
                                        ABCA8
                                        ABCA9



                                        ABCB1
                                        ABCB2
                                        ABCB3
                                        ABCB4
                                        ABCB5
                                        ABCB6
                                        ABCB7
                                        ABCB8
                                        ABCB9


                                        ABCC1
                                        ABCC2
                                        ABCC3
                                        ABCC4
                                        ABCC5
                                        ABCC6
                                        ABCC7
                                        ABCC8
                                        ABCC9




                                        ABCD1
                                        ABCD2
                                        ABCD3
                                        ABCD4
                                        10
                                        11




                                        11




                                        11
                                        11
                                        11


                                        11
                                        11
                                        11
                                        10
                                        11

                                        11
                                        10

                                        11
                                        11


                                        22

                                        11



                                        11

                                        11
                                        11
                                        11
                                        9
                                        2
                                        9
                                        9

                                        0
                                        7
                                        5
                                        0
                                        3



                                        6
                                        1




                                        1


                                        3


                                        8
                                        1

                                        4

                                        2
                                        0
                                        5

                                        3



                                        6
                                        1
                                        0
                                        0
                                                *       *
Figure 3: ABC transporter gene expression in CD34+38- cells in AML. Gene expression of 45 ABC
transporters was assessed in CD34+38- hematopoietic cells from AML patients (n=11) at diagnosis. Levels of
gene expression are depicted as natural logarithmic values normalized for the expression of the housekeeping
gene GAPDH. Mean values of gene expression of positive samples (± SEM) are represented by bars. On the
X-axis frequency of expression (number of samples in which gene expression was detectable) is shown. ABC
transporters with significant different frequency or level of expression in comparison to normal CD34+38- cells are
indicated with an asterix.



Similar to the expression pattern in normal CD34+38- cells, significant differential
expression in comparison to CD34+38+ cells was found for most (27/40) ABC transporter
genes detected in CD34+38- cells in AML. (figure 4) Together these results demonstrate that
ABC transporter gene expression levels in hematopoietic stem cells and their differential
expression in comparison to CD34+38+ cells is largely conserved in AML.




                                                                                                                     119
_____                              CHAPTER 6 _____________________________________________________________________________________________________



                                   1000
Differential expression (X-fold)



                                                            *                      *                                       *
                                    100                                        *                               *
                                                                                                       *
                                                                *                                                                           *
                                                                                                                   *
                                                                      *
                                     10            *                                               *
                                            **                                                             *           *                *
                                                                          **           ***     *                               *   **
                                          ABCA10
                                          ABCA12
                                          ABCA13




                                          ABCB10
                                          ABCB11




                                          ABCC10
                                          ABCC11
                                          ABCC12
                                          ABCC13




                                          ABCG1
                                          ABCG2
                                          ABCG4
                                          ABCG5
                                          ABCG8
                                          ABCA1
                                          ABCA2
                                          ABCA3
                                          ABCA4
                                          ABCA5
                                          ABCA6
                                          ABCA7
                                          ABCA8
                                          ABCA9



                                          ABCB1
                                          ABCB2
                                          ABCB3
                                          ABCB4
                                          ABCB5
                                          ABCB6
                                          ABCB7
                                          ABCB8
                                          ABCB9


                                          ABCC1
                                          ABCC2
                                          ABCC3
                                          ABCC4
                                          ABCC5
                                          ABCC6
                                          ABCC7
                                          ABCC8
                                          ABCC9




                                          ABCD1
                                          ABCD2
                                          ABCD3
                                          ABCD4
Figure 4: Differential expression of ABC-transporters in CD34+38- cells in comparison to CD34+38+
progenitors in AML Difference in levels of gene expression between CD34+38- and CD34+38+ cells is depicted
at the y-axis as median X-fold difference in samples that expressed the ABC transporter in CD34+38- cells.
Transporters for which differential expression reached statistical significance (paired analysis using student t-test
for all samples; p<0.05) are indicated with an asterix.


Expression of ABC transporter genes involved in drug resistance in CD34+38
hematopoietic cells in AML
Failure to eradicate CD34+38- leukemic stem cells in AML is likely to result in disease
relapse. Therefore elucidation of the mechanisms that confer chemoresistance to these
cells is of crucial importance. Currently, 13 members of the ABC transporter family are
associated with extrusion of chemotherapeutical compounds and drug resistance(24).
Table 2 shows the expression of these genes in CD34+38- cells in AML. In addition to
expected expression of ABCB1, ABCC1 and ABCG2, all other drug transport related ABC
transporter genes were expressed in CD34+38- cells in AML at varying frequency. Six
drug transport related genes were found in CD34+38- cells of all patients examined and a
significant differential expression in CD34+38- cells in comparison to CD34+38+ cells was
documented in 10/13 ABC transporters detected.
These results demonstrate that the ABC transporter profile of leukemic CD34+38- cells
includes many members implicated in drug extrusion and resistance in addition to the
known stem cell ABC transporters ABCB1, ABCG2 and ABCC1.




120
__________________________________________________   ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____


Table 2: Expression of ABC transporter genes associated with extrusion of chemotherapeutical
compounds in CD34+38- cells in AML

                                                            Differential expression
                                                            In comparison to
             Frequency of        Level of expression        CD34+38+cells                 Chemotherapeutical
Gene         expression (%)      (copy/copy GAPDH)          (median X-fold; p-value)      compounds
ABCA2        11/11               0.044± 0.014               3.51-fold; p=0.001            estramustine
ABCB1        11/11               0.011±0.002                4.19-fold; p< 0.001           anthracyclins, etoposide,
                                                                                          imatinib, taxanes,
                                                                                          mitoxantrone, vinca
                                                                                          alkaloids
ABCB4        6/11                0.0004±0.0001              5.14-fold; p=0.022            paclitaxel, vinblastine
ABCB11       1/11                6.4.10-6                   0.34-fold                     paclitaxel
ABCC1        11/11               0.027 ± 0.017              2.45-fold; p=0.014            anthracyclins, etoposide,
                                                                                          methotrexate,
ABCC2        10/11               0.0017 ±0.0012             5.01-fold; p=0.027            cisplatin, doxorubicin,
                                                                                          etoposide, methotrexate,
                                                                                          mitoxantrone, vinca
                                                                                          alkaloids
ABCC3        3/11                0.0015±0.0006              56.47-fold; p=0.079           cisplatin, doxorubicin,
                                                                                          etoposide, methotrexate,
                                                                                          vinca alkaloids
ABCC4        11/11               0.0058 ± 0.0057            3.26-fold; p=0.046            methotrexate, thiopurines
ABCC5        11/11               0.011 ± 0.003              3.36-fold; p=0.007            6-mercaptopurine,
                                                                                          6-thioguanine
ABCC6        8/11                0.0005 ± 0.0003            3.71-fold; p=0.048            anthracyclins, etoposide,
                                                                                          teniposide
ABCC10       11/11               0.0041 ± 0.0018            3.35-fold; p=0.005            docetaxel, paclitaxel, vinca
                                                                                          alkaloids
ABCC11       1/11                0.0002 ± 0.0002            751.94-fold                   purine and pyrimidine
                                                                                          nucleotide analogs
ABCG2        6/11                0.0004 ± 0.0003            10.75-fold; p= 0.051          mitoxantrone,
                                                                                          methotrexate, topetocan,
                                                                                          SN-38, imatinib,
                                                                                          flavopiridol, anthracyclins

Differential expression is depicted as the median value of the X-fold difference between CD34+38- and
CD34+38+ cells in patients in which the gene is expressed in CD34+38- cells




                                                                                                                    121
_____   CHAPTER 6 _____________________________________________________________________________________________________



Discussion

In this report we describe the ABC transporter gene expression profiling of normal
CD34+38- hematopoietic cells using real-time RT-PCR on low-density micro-arrays. Using
this approach we describe the differential expression of many, previously unrecognized
ABC transporters on normal hematopoietic cells and conservation of expression pattern
and levels after malignant transformation in AML.
Besides the known stem cell transporters ABCB1(6,25) and ABCG2(10,26), a number of
other differentially expressed transcripts were anticipated based on reports from mouse
hematopoietic stem cell gene profiling using high density affymetrix arrays. Ramalho et
al(27), investigating mouse side population cells in comparison to the bone marrow main
population using Affymetrix U74Av2 Showed that ABCG1, ABCG2, ABCB1, ABCB2,
ABCB3, ABCB8, ABCD3, ABCF3 were differentially expressed in this hematopoietic
stem cell population. All these genes that displayed differential expression in mouse SP
cells were significantly differential expressed in normal CD34+38- cells in comparison to
committed progenitors in our study. Similarly, gene profiling in fetal Sca+AA4.1+Kit+Lin-
liver hematopoietic cells versus stem cell-depleted AA4.1- cells(28) identified 14 differentially
expressed ABC transporters (ABCA1, ABCA2, ABCA5, ABCA7, ABCA13, ABCB1, ABCB2,
ABCB9, ABCC1, ABCC4, ABCF3, ABCG1, ABCG3, ABCF2), all of which, when included,
are differentially expressed in human CD34+38- cells in our study. (ABCG3 does not have
a human ortholog).
Finally, Langmann et all(30) investigated ABC transporter gene expression using real time
RT-PCR in a panel of human tissues including whole bone marrow (mixed RNA sample
83 donors). Most ABC transporters were detected in bone marrow with exception of
ABCA4, ABCB5, ABCB11, ABCC7, ABCC8, ABCC11, ABCC12, ABCG4, ABCG5 and
ABCG8. Interestingly, of the 10 members not detected in whole bone marrow, in 7
transporters we were not able to detect expression in CD34+38- cells indicating high
concordance between these data sets. Two transporters in which no expression could
be detected in the Langmann study (ABCB11 and ABCC8), showed expression in only
the minority of samples in our study. The only exception was ABCB5 that was detected in
8/11 samples in our study. Interestingly, however, ABCB5 was detected only in all G-CSF
mobilized CD34+38- samples but not in NBM suggesting that expression of ABCB5 is
G-CSF mobilization-related or a specific characteristic of peripheral blood hematopoietic
stem cells. Interestingly, ABCB5 has been implicated in cell fusion of progenitor cells(31),
opening the possibility that it is a specific characteristic of mobilized cells enabling them to
participate in fusion events in peripheral tissues.
Together these comparisons with reported expression of ABC transporter in hematopoietic
stem cell populations show high concordance with the results from our study indicating
great reliability of the “gold standard” real-time RT-PCR gene quantitation method used.
In addition to the anticipated differentially expressed genes however, we identified a large


122
__________________________________________________   ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____



number of previously unrecognized ABC transporter genes that displayed differential
expression in hematopoietic stem cells.
The sensitivity of real-time PCR in comparison to cDNA and oligonucleotide arrays(32),
especially in quantifying low-copy genes, likely explains the identification of these genes in
the current study. It is noteworthy that many of the differentially expressed ABC transporter
genes, not previously recognized, in CD34+38- cells are indeed low-copy genes when
compared to the expression of the housekeeping gene GAPDH and are not detected above
background in cDNA arrays. Additionally, the lack of necessity for mRNA amplification, the
limited number of ABC transporters represented in some cDNA arrays and differences in
cell populations studied (CD34+38- versus the more heterogeneous side-population and
CD34+38+ versus whole bone marrow in other studies) could explain the identification of
novel differentially expressed transcripts in hematopoietic cells in this study.
The identification of many previously unrecognized ABC transporters with differential
expression in hematopoietic stem cells raises questions about the physiological function
in these cells. In addition to protection of these long-lived cells to naturally occurring
xenobiotics, a role in stem cell biology for ABC transporters has been suggested based
on studies in Dictyostelium demonstrating that a rhodamine-123 cellular efflux pump with
the properties of a ABC-transporter prevents differentiation of prespore cells(33) through
efflux of a differentiation inducing factor.The results presented in this study suggested
that hematopoietic stem cells may escape the effect of differentiation factors present in
the bone marrow through extrusion of these factors by ABC transporters. However, no
such role has been demonstrated for the ABC-transporters currently identified on human
(hematopoietic) stem cells. It remains to be determined whether novel transporters identified
in this study serve such function in human hematopoietic cells. Interestingly, among the
ranking order 10 most differentially expressed ABC transporters 5 members of the ABCA
family are listed (ABCA5, ABCA6, ABCA9, ABCA10 and ABCA13). Interestingly, four of
these genes (ABCA5, A6, A9 and A10) are arranged in a cluster on chromosome 17q24
representing a phylogenetically distinct subgroup within the ABCA gene subfamily(34,35).
Though the substrates and function of these ABCA transporters are currently unknown,
recent studies have led to the concept that some of these transporters may serve critical
physiological functions in the transmembrane transport of endogenous lipid substrates
such as phospholipids and essential fatty-acids, substrates involved in the regulation of
differentiation of hematopoietic cells. (reviewed in(36)). Additionally, ABCA13 has recently
been shown to have a putative promoter sequence containing multiple binding sites for
hematopoietic stem cell transcription factors suggesting a key role in hematopoiesis(37).
Clearly, further research is warranted to elucidate the role of these ABCA transporters in
stem cell biology and is currently ongoing at our laboratory.
Regardless their role in stem cell biology, conserved expression of ABC transporters
involved in drug extrusion after malignant transformation of hematopoietic stem cell in
AML, as demonstrated in this study, could greatly impede sensitivity of these cells to


                                                                                                                  123
_____   CHAPTER 6 _____________________________________________________________________________________________________



chemotherapeutical eradication. In this study we demonstrated expression of all 13 ABC
transporter members currently associated with drug resistance(24). However, given the
high structure similarity of ABC transporters it is conceivable that additional members
will be associated with drug resistance. A recent study examining ABC-transporter gene
expression in drug-selected cancer cell lines demonstrated over expression of a number of
transporters thus far not recognized as associated with drug resistance (ABCA4, ABCA7,
ABCB2, ABCB3, ABCB6, ABCB8, ABCB9, ABCC6, ABCG1)(17) suggesting that these
transporters are involved in drug resistance as well. Interestingly, all these additional
transporters are expressed in a differential fashion in CD34+38- hematopoietic stem cells
in AML as well. Obviously, care has to be taken in extrapolating these gene expression
data to clinical drug resistance since gene expression will not necessarily reflect protein
expression and function but our data suggest that drug efflux mediated drug resistance
of leukemic stem cells is conferred by many members of the ABC family of membrane
transporters with possibly promiscuous action on different chemotherapeutical compounds.
Modulation of additional transporters is therefore likely to be required to increase drug
accumulation and induce chemotherapeutical eradication of leukemic stem cells in AML.
These data have too be taken into account when interpreting the largely poor results of
ABCB1 modulation on long-term disease outcome in clinical trials(38,39).
Additionally, the similar patterns of ABC-transporter expression in normal and leukemic
hematopoietic stem cells predict that modulation of these transporters in AML as a
therapeutic strategy to target leukemic stem cells will target residual normal hematopoietic
stem cells as well and is therefore likely to encounter increased bone marrow toxicity.
Nevertheless, the identification of previously unrecognized ABC transporters in leukemic
stem cells offer the basis for future studies investigating the role of these ABC transporters
in resistance of leukemic stem cells and their usefulness as therapeutical targets to
eradicate this crucial cell population in AML.




124
                                                                                                                             10
                                                                                                                                                                                    10
                                                                                                                                                                                                                                           10




                                                                                                                                        1000

                                                                                                                                  100
                                                                                                                                                                                         100
                                                                                                                                                                                               1000




                                                                                                                                               10-4
                                                                                                                                                      10-3
                                                                                                                                                             10-2
                                                                                                                                                                    10-1
                                                                                                                                                                                                      10-4
                                                                                                                                                                                                             10-3
                                                                                                                                                                                                                    10-2
                                                                                                                                                                                                                           10-1
                                                                                                                                                                                                                                                100
                                                                                                                                                                                                                                                      1000
                                                                                                                                                                                                                                                                                                       100

                                                                                                                                                                                                                                                                                                  10
                                                                                                                                                                                                                                                                                                             1000




                                                                                                                                                                                                                                                             10-4
                                                                                                                                                                                                                                                                    10-3
                                                                                                                                                                                                                                                                           10-2
                                                                                                                                                                                                                                                                                  10-1
                                                                                                                                                                                                                                                                                                                    10-4
                                                                                                                                                                                                                                                                                                                           10-3
                                                                                                                                                                                                                                                                                                                                  10-2
                                                                                                                                                                                                                                                                                                                                         10-1
                                                                                                                    ABCA1                                                  ABCA1                                                  ABCA1                                                  ABCA1
                                                                                                                    ABCA2                                                  ABCA2                                                  ABCA2                                                  ABCA2
                                                                                                                    ABCA3                                                  ABCA3                                                  ABCA3                                                  ABCA3
                                                                                                                    ABCA4                                                  ABCA4                                                  ABCA4                                                  ABCA4
                                                                                                                    ABCA5                                                  ABCA5                                                  ABCA5                                                  ABCA5
                                                                                                                    ABCA6                                                  ABCA6                                                  ABCA6                                                  ABCA6
                                                                                                                    ABCA7                                                  ABCA7                                                  ABCA7                                                  ABCA7
                                                                                                                    ABCA8                                                  ABCA8                                                  ABCA8                                                  ABCA8
                                                                                                                    ABCA9                                                  ABCA9                                                  ABCA9                                                  ABCA9
                                                                                                                    ABCA10                                                 ABCA10                                                 ABCA10                                                 ABCA10
                                                                                                                    ABCA12                                                 ABCA12                                                 ABCA12                                                 ABCA12
                                                                                                                    ABCA13                                                 ABCA13                                                 ABCA13                                                 ABCA13
                                                                                                                    ABCB1                                                  ABCB1                                                  ABCB1                                                  ABCB1
                                                                                                                    ABCB2                                                  ABCB2                                                  ABCB2                                                  ABCB2
                                                                                                                    ABCB3                                                  ABCB3                                                  ABCB3                                                  ABCB3
                                                                                                                    ABCB4                                                  ABCB4                                                  ABCB4                                                  ABCB4
                                                                                                                    ABCB5                                                  ABCB5                                                  ABCB5                                                  ABCB5
                                                                                                                    ABCB6                                                  ABCB6                                                  ABCB6                                                  ABCB6
                                                                                                                    ABCB7                                                  ABCB7                                                  ABCB7                                                  ABCB7
                                                                                                                    ABCB8                                                  ABCB8                                                  ABCB8                                                  ABCB8
                                                                                                                    ABCB9                                                  ABCB9                                                  ABCB9                                                  ABCB9
                                                                                                                    ABCB10                                                 ABCB10                                                 ABCB10                                                 ABCB10
                                                                                                                    ABCB11                                                 ABCB11                                                 ABCB11                                                 ABCB11
                                                                                                                    ABCC1                                                  ABCC1                                                  ABCC1                                                  ABCC1
                                                                                                                    ABCC2                                                  ABCC2                                                  ABCC2                                                  ABCC2
                                                                                                                    ABCC3                                                  ABCC3                                                  ABCC3                                                  ABCC3
                                                                                                                    ABCC4                                                  ABCC4                                                  ABCC4                                                  ABCC4
                                                                                                                    ABCC5                                                  ABCC5                                                  ABCC5                                                  ABCC5
                                                                                                                    ABCC6                                                  ABCC6                                                  ABCC6                                                  ABCC6
                                                                                                                    ABCC7                                                  ABCC7                                                  ABCC7                                                  ABCC7
                                                                                                                    ABCC8                                                  ABCC8                                                  ABCC8                                                  ABCC8
                                                                                                                    ABCC9                                                  ABCC9                                                  ABCC9                                                  ABCC9
                                                                                                                    ABCC10                                                 ABCC10                                                 ABCC10                                                 ABCC10
                                                                                                                    ABCC11                                                 ABCC11                                                 ABCC11                                                 ABCC11
                                                                                                                    ABCC12                                                 ABCC12                                                 ABCC12                                                 ABCC12
                                                                                                                    ABCC13                                                 ABCC13                                                 ABCC13                                                 ABCC13
                                                                                                                    ABCD1                                                  ABCD1                                                  ABCD1                                                  ABCD1
                                                                                                                    ABCD2                                                  ABCD2                                                  ABCD2                                                  ABCD2
                                                                                                                    ABCD3                                                  ABCD3                                                  ABCD3                                                  ABCD3
                                                                                                                    ABCD4                                                  ABCD4                                                  ABCD4                                                  ABCD4
                                                                                                                    ABCG1                                                  ABCG1                                                  ABCG1                                                  ABCG1
                                                                                                                    ABCG2                                                  ABCG2                                                  ABCG2                                                  ABCG2
                                                                                                                    ABCG4                                                  ABCG4                                                  ABCG4                                                  ABCG4
                                                                                                                    ABCG5                                                  ABCG5                                                  ABCG5                                                  ABCG5
                                                                                                                    ABCG8                                                  ABCG8                                                  ABCG8                                                  ABCG8
                                                                                                                                                                                                                                                                                                                                           MO




                                                                                                                                                                      M5
                                                                                                                                                                                                                             M2
                                                                                                                                                                                                                                                                                    M1
                                                                                                                    ABCA1                                                  ABCA1
                                                                                                                                                                           ABCA2                                                  ABCA1                                                  ABCA1
                                                                                                                    ABCA2                                                                                                         ABCA2                                                  ABCA2
                                                                                                                    ABCA3                                                  ABCA3
                                                                                                                                                                           ABCA4                                                  ABCA3                                                  ABCA3
                                                                                                                    ABCA4                                                                                                         ABCA4                                                  ABCA4
                                                                                                                    ABCA5                                                  ABCA5
                                                                                                                                                                           ABCA6                                                  ABCA5                                                  ABCA5
                                                                                                                    ABCA6                                                                                                         ABCA6                                                  ABCA6
                                                                                                                    ABCA7                                                  ABCA7
                                                                                                                                                                                                                                  ABCA7                                                  ABCA7
                                                                                                                                                                                                                                                                                                                                                                   __________________________________________________




                                                                                                                    ABCA8                                                  ABCA8
                                                                                                                                                                           ABCA9                                                  ABCA8                                                  ABCA8
                                                                                                                    ABCA9                                                                                                         ABCA9                                                  ABCA9
                                                                                                                    ABCA10                                                 ABCA10
                                                                                                                                                                           ABCA12                                                 ABCA10                                                 ABCA10
                                                                                                                    ABCA12                                                                                                        ABCA12                                                 ABCA12
                                                                                                                    ABCA13                                                 ABCA13
                                                                                                                                                                           ABCB1                                                  ABCA13                                                 ABCA13
                                                                                                                    ABCB1                                                                                                         ABCB1                                                  ABCB1
                                                                                                                    ABCB2                                                  ABCB2
                                                                                                                                                                           ABCB3                                                  ABCB2                                                  ABCB2
                                                                                                                    ABCB3                                                                                                         ABCB3                                                  ABCB3
                                                                                                                    ABCB4                                                  ABCB4
                                                                                                                                                                           ABCB5                                                  ABCB4                                                  ABCB4
                                                                                                                    ABCB5                                                                                                         ABCB5                                                  ABCB5
                                                                                                                    ABCB6                                                  ABCB6
                                                                                                                                                                           ABCB7                                                  ABCB6                                                  ABCB6
                                                                                                                    ABCB7                                                                                                         ABCB7                                                  ABCB7
                                                                                                                    ABCB8                                                  ABCB8
                                                                                                                                                                           ABCB9                                                  ABCB8                                                  ABCB8
                                                                                                                    ABCB9                                                                                                         ABCB9                                                  ABCB9
                                                                                                                    ABCB10                                                 ABCB10




      committed CD34+38+ cells (fold-difference; lower graph).
                                                                                                                                                                           ABCB11                                                 ABCB10                                                 ABCB10
                                                                                                                    ABCB11                                                                                                        ABCB11                                                 ABCB11
                                                                                                                    ABCC1                                                  ABCC1
                                                                                                                                                                           ABCC2                                                  ABCC1                                                  ABCC1
                                                                                                                    ABCC2                                                                                                         ABCC2                                                  ABCC2
                                                                                                                    ABCC3                                                  ABCC3
                                                                                                                                                                           ABCC4                                                  ABCC3                                                  ABCC3
                                                                                                                    ABCC4                                                                                                         ABCC4                                                  ABCC4
                                                                                                                    ABCC5                                                  ABCC5
                                                                                                                                                                           ABCC6                                                  ABCC5                                                  ABCC5
                                                                                                                    ABCC6                                                                                                         ABCC6                                                  ABCC6
                                                                                                                    ABCC7                                                  ABCC7
                                                                                                                                                                           ABCC8                                                  ABCC7                                                  ABCC7
                                                                                                                    ABCC8                                                                                                         ABCC8                                                  ABCC8
                                                                                                                    ABCC9                                                  ABCC9
                                                                                                                                                                           ABCC10                                                 ABCC9                                                  ABCC9
                                                                                                                    ABCC10                                                                                                        ABCC10                                                 ABCC10
                                                                                                                    ABCC11                                                 ABCC11
                                                                                                                                                                           ABCC12                                                 ABCC11                                                 ABCC11
                                                                                                                    ABCC12                                                                                                        ABCC12                                                 ABCC12
                                                                                                                    ABCC13                                                 ABCC13
                                                                                                                                                                           ABCD1                                                  ABCC13                                                 ABCC13
                                                                                                                    ABCD1                                                                                                         ABCD1                                                  ABCD1
                                                                                                                    ABCD2                                                  ABCD2
                                                                                                                                                                           ABCD3                                                  ABCD2                                                  ABCD2
                                                                                                                    ABCD3                                                                                                         ABCD3                                                  ABCD3
                                                                                                                    ABCD4                                                  ABCD4
                                                                                                                                                                           ABCG1                                                  ABCD4                                                  ABCD4
                                                                                                                    ABCG1                                                                                                         ABCG1                                                  ABCG1
                                                                                                                    ABCG2                                                  ABCG2
                                                                                                                                                                           ABCG4                                                  ABCG2                                                  ABCG2
                                                                                                                    ABCG4                                                                                                         ABCG4                                                  ABCG4
                                                                                                                    ABCG5                                                  ABCG5
                                                                                                                                                                           ABCG8                                                  ABCG5                                                  ABCG5
                                                                                                                    ABCG8




                                                                                                                                                                                                                             M2
                                                                                                                                                                                                                                  ABCG8                                                  ABCG8




                                                                                                                                                                      M5
                                                                                                                                                                                                                                                                                    M4
                                                                                                                                                                                                                                                                                                                                           M1




                                                                                                                                                                           ABCA1                                                  ABCA1                                                  ABCA1
                                                                                                                                                                           ABCA2                                                  ABCA2                                                  ABCA2
                                                                                                                                                                           ABCA3                                                  ABCA3                                                  ABCA3
                                                                                                                                                                           ABCA4                                                  ABCA4                                                  ABCA4
                                                                                                                                                                           ABCA5                                                  ABCA5                                                  ABCA5
                                                                                                                                                                           ABCA6                                                  ABCA6                                                  ABCA6
                                                                                                                                                                           ABCA7                                                  ABCA7                                                  ABCA7
                                                                                                                                                                           ABCA8                                                  ABCA8                                                  ABCA8
                                                                                                                                                                           ABCA9                                                  ABCA9                                                  ABCA9
                                                                                                                                                                           ABCA10                                                 ABCA10                                                 ABCA10
                                                                                                                                                                           ABCA12                                                 ABCA12                                                 ABCA12
                                                                                                                                                                           ABCA13                                                 ABCA13                                                 ABCA13
                                                                                                                                                                           ABCB1                                                  ABCB1                                                  ABCB1
                                                                                                                                                                           ABCB2                                                  ABCB2                                                  ABCB2
                                                                                                                                                                           ABCB3                                                  ABCB3                                                  ABCB3
                                                                                                                                                                           ABCB4                                                  ABCB4                                                  ABCB4
                                                                                                                                                                           ABCB5                                                  ABCB5                                                  ABCB5
                                                                                                                                                                           ABCB6                                                  ABCB6                                                  ABCB6
                                                                                                                                                                           ABCB7                                                  ABCB7                                                  ABCB7
                                                                                                                                                                           ABCB8                                                  ABCB8                                                  ABCB8
                                                                                                                                                                           ABCB9                                                  ABCB9                                                  ABCB9
                                                                                                                                                                           ABCB10                                                 ABCB10                                                 ABCB10
                                                                                                                                                                           ABCB11                                                 ABCB11                                                 ABCB11
                                                                                                                                                                           ABCC1                                                  ABCC1                                                  ABCC1
                                                                                                                                                                           ABCC2                                                  ABCC2                                                  ABCC2
                                                                                                                                                                           ABCC3                                                  ABCC3                                                  ABCC3
                                                                                                                                                                           ABCC4                                                  ABCC4                                                  ABCC4
                                                                                                                                                                           ABCC5                                                  ABCC5                                                  ABCC5
                                                                                                                                                                           ABCC6                                                  ABCC6                                                  ABCC6
                                                                                                                                                                           ABCC7                                                  ABCC7                                                  ABCC7
                                                                                                                                                                           ABCC8                                                  ABCC8                                                  ABCC8
                                                                                                                                                                           ABCC9                                                  ABCC9                                                  ABCC9
                                                                                                                                                                           ABCC10                                                 ABCC10                                                 ABCC10
                                                                                                                                                                           ABCC11                                                 ABCC11                                                 ABCC11
                                                                                                                                                                           ABCC12                                                 ABCC12                                                 ABCC12
                                                                                                                                                                           ABCC13                                                 ABCC13                                                 ABCC13
                                                                                                                                                                           ABCD1                                                  ABCD1                                                  ABCD1
                                                                                                                                                                           ABCD2                                                  ABCD2                                                  ABCD2
                                                                                                                                                                           ABCD3                                                  ABCD3                                                  ABCD3
                                                                                                                                                                           ABCD4                                                  ABCD4                                                  ABCD4
                                                                                                                                                                           ABCG1                                                  ABCG1                                                  ABCG1
                                                                                                                                                                           ABCG2                                                  ABCG2                                                  ABCG2
                                                                                                                                                                           ABCG4                                                  ABCG4                                                  ABCG4
                                                                                                                                                                           ABCG5                                                  ABCG5                                                  ABCG5
                                                                                                                                                                           ABCG8                                                  ABCG8                                                  ABCG8
                                                                                                                                                                                                                             M4
                                                                                                                                                                                                                                                                                    MO
                                                                                                                                                                                                                                                                                                                                           M1




      represent relative expression (copies target gene/copy GAPDH; upper graphs) and expression in comparison to


125
      Appendix: ABC transporter profiles of CD34+38- cells from 11 patients with AML at diagnosis. Graphs
                                                                                                                                                                                                                                                                                                                                                ABC-TRANSPORTER GENE PROFILING IN LEUKEMIC PROGENITOR CELLS _____
_____   CHAPTER 6 _____________________________________________________________________________________________________



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38.     Baer, M. R., George, S. L., Dodge, R. K., O’Loughlin, K. L., Minderman, H., Caligiuri, M. A., Anastasi, J.,
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128
                                                                CHAPTER 7
        LUNG-RESISTANCE-RELATED PROTEIN EXPRESSION IS
           A NEGATIVE PREDICTIVE FACTOR FOR RESPONSE TO
           CONVENTIONAL LOW BUT NOT TO INTENSIFIED DOSE
          ALKYLATING CHEMOTHERAPY IN MULTIPLE MYELOMA



                                                                               H.G.P. RAAIJMAKERS
                                                                                  M.A.I. IZQUIERDO
                                                                                   H.M. LOKHORST
                                                                                      C. DE LEEUW
                                                                                    J.A.M. BELIEN
                                                                                      A.C. BLOEM
                                                                                     A.W. DEKKER
                                                                                    R.J. SCHEPER
                                                                                    P. SONNEVELD

  DEPARTMENTS OF HAEMATOLOGY AND IMMUNOLOGY, THE UNIVERSITY HOSPITAL UTRECHT; THE DEPARTMENT OF
HAEMATOLOGY, UNIVERSITY HOSPITAL ROTTERDAM DIJKZIGT, ROTTERDAM; AND THE DEPARTMENT OF PATHOLOGY,
                                                UNIVERSITY HOSPITAL VRIJE UNIVERSITEIT, AMSTERDAM,
                                                                                 THE NETHERLANDS




                                                                          BLOOD; 91:1029-36, 1998
_____   CHAPTER 7 _____________________________________________________________________________________________________




130
________________________________________________________________________________________   LRP IN MULTIPLE MYELOMA _____



Abstract

This study was undertaken to assess the significance of lung-resistance related protein
(LRP) expression in plasma cells from untreated multiple myeloma (MM) patients and
to determine whether LRP was associated with a poor response and survival in patients
treated with different dose regimens of melphalan. Seventy untreated patients received
conventional oral dose melphalan (0.25 mg/kg, day 1 to 4) combined with prednisone (MP)
or intravenous intermediate IDM; 70 mg/m2) or high- (140 mg/m2) dose Melphalan (HDM).
LRP expression was assessed with immunocytochemistry using the LRP-56 monoclonal
antibody. LRP expression was found in 47% of patients. In the MP treated patients, LRP
expression was a significant prognostic factor regarding response induction (P < .05), event
free survival (P < .003), and overall survival (P < .001). In the intensified dose melphalan
treated patients LRP did not have a prognostic value. The response rates of LRP-positive
patients to MP and IDM/HDM were 18% versus 81%, respectively (P < .0001). We conclude
that LRP is frequently expressed in untreated MM patients and is an independent predictor
for response and survival in patients treated with MP. Pretreatment assessment of LRP
identifies a subpopulation of patients with a poor probability of response to conventional
dose melphalan. Dose intensification of melphalan is likely to overcome LRP-mediated
resistance.



Introduction

Alkylating agents and corticosteroids are still the mainstay of therapy for patients with
multiple myeloma (MM).(1,2) However, only approximately 50% to 60% of patients respond
to oral melphalan and prednisone in conventional dose resulting in a median response
duration of 1 to 2 years and a median survival of about 3 to 4 years. Dose-intensification
studies have shown a dose-response relationship for melphalan(3-5) in MM; therefore, high-
dose melphalan has been used to improve response rates and survival.
Melphalan, like other alkylating agents, exerts its cytotoxic effect through the covalent
linkage of alkyl groups to DNA. Resistance against alkylating agents includes both cellular
and extracellular factors. In cell line studies resistance to melphalan has been attributed
to a decreased drug uptake caused by alterations in either the number or the affinity of
membrane-bound proteins.(6) An alternative explanation may be an increased cellular
detoxifcation by glutathione S-transferases.(7) So far, studies in hematological malignancies
such as MM have failed to show a role of these laboratory findings in clinical specimens.(8)
Multiple drug resistance (MDR) has been identifed as an important path of drug resistance
in MM. MDR is the phenomenon of cancer cells developing cross-resistance to a
variety of structurally unrelated chemotherapeutic compounds such as vinca-alkaloids,
anthracyclines, and epipodophyllotoxins.(9) MDR is associated with the expression of


                                                                                                                    131
_____   CHAPTER 7 _____________________________________________________________________________________________________



the drug transport mediating proteins P-glycoprotein (PgP) and the multidrug resistance-
related protein (MRP).(10) The increasing evidence of additional mechanisms of MDR led
recently to the identifcation of a novel protein associated with MDR, originally termed the
lung-resistance protein (LRP).
The LRP gene has recently been cloned and identifed as the human p110 major vault
protein.(11) Vaults are novel cellular organelles first described by Kedersha and Rome in
1986,(12) which are thought to mediate intracellular transport of a wide variety of substrates.
LRP has been found to be widely distributed in human normal tissues and in tumors,
closely reflecting the susceptibility to chemotherapy of different tumor types.(13) Importantly,
recent studies in myeloma and other human cancer cell lines relate LRP expression to
resistance against the alkylating agent melphalan(14,15) (and W.S. Dalton et al, personal
communication, July 1997).
In the current study we have assessed LRP expression in myeloma patients, and based
on these results we introduce this MDR-related protein as a putative important marker of
clinical resistance to the alkylating agent melphalan resulting in an adverse prognosis.
Moreover, we describe the overcoming of LRP-related resistance against melphalan by
dose intensification.



Patients and methods

The study was performed on nonselected, sequentially stored frozen cytocentrifuge
slides prepared from Ficoll-Hypaque-purifed bone marrow aspirates obtained from all MM
patients with newly diagnosed disease who were treated with melphalan-based regimens
between January 1987 and November 1995.
Additionally, bone marrow aspirates of three normal donors for allogeneic bone marrow
transplantation (BMT) and the aspirates of five patients with monoclonal gammopathy of
undetermined significance (MGUS) were studies for LRP expression.

Patients
Seventy unselected patients treated at the Departments of Haematology of the University
Hospital Utrecht or the University Hospital Rotterdam Dijkzigt were studied. Clinical staging
was defined according to the criteria proposed by Salmon and Durie.(16) Median age of
patients treated with conventional-dose oral melphalan and prednisone was 67 years.
Patients treated with intravenous (IV) intermediate or high-dose melphalan were median
52 years. The performance status was determined according to the criteria of the Eastern
Cooperative Oncology Group (ECOG): 0, normal; 1, ambulant with symptoms; 2, bedrest
less than 50% of the day; 3, bedrest greater than 50% of the day; 4, bedrest all day. The
patient characteristics are summarized in Table 1.



132
________________________________________________________________________________________   LRP IN MULTIPLE MYELOMA _____


Table 1: Patient Characteristics.

                                                  ALL                    IDM/HDM*                       MP†
No. patients                                      70                     32                             38
Age
 Median                                           61                     52                             67
 Range                                            34-86                  35-65                          35-85
Sex (male:female)                                 44:26                  18:14                          26:12
M-component
 IgG                                              44                     22                             22
 IgA                                              16                      7                              9
 IgD                                              1                                                      1
 LCD                                              9                       3                              6
k:λ                                               41:29                  19:13                          22:16
Stage:
 II-A                                             13                      5                              8
 III-A                                            50                     25                             25
 III-B                                             7                      2                              5
Performance status (ECOG)
 0                                                 9                      4                              5
 1                                                22                      9                             13
 2                                                21                      9                             12
 3                                                13                      7                              6
 4                                                 5                      3                              2
Abbreviation: LCD, light chain disease.
*Patients treated with 2 courses of intravenous intermediate dose melphalan, 70 mg/m2 with an interval of 6
weeks (n = 20) or a single IV high-dose melphalan, 140 mg/m2 (n = 12).
†Patients treated with intermittent oral melphalan 0.25 mg/kg/d and prednisone 2 mg/kg/d.



Chemotherapy regimens and response evaluation
Patients received melphalan as first-line treatment, either in combination with prednisone
(MP, 38 patients) or as monotherapy in intermediate dose (IDM, 20 patients) or high dose
(HDM, 12 patients). Patients under 65 years of age were candidates for IDM or HDM,
unless they refused intensive treatment. Patients refusing intensive treatment and patients
over 65 years received MP. Performance status was no selection criterium for treatment
modality. The intermittent MP regimen consisted of oral melphalan 0.25 mg/kg/d and
prednisone 2 mg/kg/d administered for 4 days. Courses were repeated every 6 weeks.
IDM (melphalan 70 mg/m2) was administered by rapid IV infusion. Two courses of IDM
were given with an interval of 6 weeks.(17) The HDM regimen (140 mg/m2) consisted of a
single dose. Response was determined by standard criteria for myeloma response.(18) A
partial response was defined as a reduction of at least 50% in serum M protein or urinary
light chain concentration with no progression of lytic bone lesions, without increase of bone
pain or anemia. A complete response (CR) was defined as complete disappearance of
myeloma proteins from serum and urine and normalization of the bone marrow. Response
in patients treated with MP was determined after 4 courses, or earlier when progression
was obvious. When a partial response (≥50% reduction in M protein) was achieved,


                                                                                                                    133
_____   CHAPTER 7 _____________________________________________________________________________________________________



therapy was continued for at least 1 year. Patients with a minimal response (between 25%
and 50% reduction in M protein) received another four courses. Patients unresponsive
after four courses (less than 25% reduction in M-protein concentration) and patients with
a minimal response after four courses but no further improvement of response after eight
courses, continued with secondline chemotherapy, usually a combination of vincristine,
adriamycin, and dexamethasone (VAD). Patients treated with IDM or HDM were evaluated
2 months after the second IDM or single-dose HDM, respectively. Nonresponding patients
were also treated with VAD.

Immunocytochemical staining of LRP
LRP expression was determined by an alkaline phosphatase immunocytochemical
detection method(19) using the specific murine monoclonal antibody (MoAb) LRP-56 (IgG2b)
that was obtained after immunization of mice with the non-Pgp multidrug-resistant human
nonsmall lung cancer cell line SW-1573/2R120.(14) Bone marrow cells were separated
by Ficoll-Hypaque, washed twice with minimal essential medium (MEM; GIBCO, Grand
Island, NY) and stored at -20°C until use. Cytocentrifuged slides were airdried overnight
and fixed in acetone for 10 minutes.
After preincubation for 20 minutes with 10% rabbit serum in phosphate-buffered
saline plus 1% bovine serum albumin (PBS/BSA; Sigma Chemical Co, St Louis, MO),
cytospins were incubated with LRP-56 (diluted 1:500 in 1% BSA) or with idiotype
matched control (nonspecific mouse IgG-1; Cappel: Organon Teknica 50327/36345)
for 1.5 hours. Next, rabbit anti-mouse immunoglobulin (RAM; Dakopatts Z 259, DAKO
Corp, Glastrup, Denmark) diluted 1:25 for 1 hour was added followed by incubation with
alkaline phosphatase substrate (APAAP; Dakopatts D 651, DAKO), diluted 1:50 for 1
hour. Incubations with RAM and APAAP were repeated for 0.5 hour. The color reaction
was produced using a Neufuchsin (Merck 4041; Merck, Darmstadt, Germany) substrate
incubating for 40 minutes. All incubations were performed at room temperature. Between
incubation steps, slides were washed thoroughly in PBS for 10 minutes. Finally, cytospins
were counterstained in diluted hematoxilin and washed with tap water. Simultaneously, the
LRP-positive fibrosarcoma HT1080 DR4 control cell line(20) was stained as control for the
immunocytochemical assay.
All slides were examined and scored independently by two observers, blinded to the
clinical data. Plasma cells were identified on morphological criteria. At least 250 plasma
cells were evaluated. A sample was considered to be LRP-positive if ≥10% of the plasma
cells stained with the LRP-56 antibody and the idiotype matched controls were indeed
negative. These criteria were based on previous experience with LRP-56 staining in 155
cancer specimens, which indicated that a 10% cut-off value may distinguish two groups of
LRP-expressing tumors.(13,21)




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________________________________________________________________________________________   LRP IN MULTIPLE MYELOMA _____



Determination of prognostic factors
The serum B2-microglobulin level was determined by means of a competitive enzyme
immunoassay (Phadezym; Pharmacia, Uppsala, Sweden). The plasma cell labeling index
(LI) was measured by the incorporation of bromodeoxyuridine as described previously.(22)
Serum levels of lactate dehydrogenase (LDH) were measured according to standard
methods.

Statistical analysis
Data analysis was performed using the SPSS statistical software package (SPSS Inc,
Chicago, IL).
Prognostic parameters such as age were determined at diagnosis and were retrospectively
assessed for their relationship with LRP expression. The response rates were compared
between LRP and prognostic factors expression groups. Qualitative variables were
analyzed using the chi-squared test. Multivariate analysis was performed using step-
wise discriminant analysis. Overall survival was measured in months from the moment of
diagnosis, providing 95% confidence intervals. Actuarial survival curves were estimated
using the Kaplan-Meier method,(23) and differences in survival between subgroups were
compared with the log-rank test (Mantel-Cox).(24) Also, the hazard rates for each variable
were calculated with the Cox-regression model using enter and remove limits of 0.05 and
0.1. Hypotheses were evaluated at a significance level of 0.05. Two-sided statistical tests
were used in all analyses.



Results

Frequency and pattern of LRP expression
LRP was expressed in 47% (33/70) of bone marrow samples of patients with newly
diagnosed myeloma. In the MP-treated population 47% (17/38) of patients were LRP
positive as compared with 50% (16/32) in the IDM/HDM-treated population. The staining
of the LRP-56 MoAb in the LRP-positive myeloma cells was invariably cytoplasmatic
in the perinuclear region, in a granular fashion (Figure 1). The intensity of the staining
was generally strong, but variance in staining intensity was too small to justify objective
classification between aspirates. LRP expression in positive bone marrow samples was
heterogeneous, typically showing LRP-56 immunoreactivity in the majority of myeloma
cells (median 50%, range 10 to 90). There were no samples with LRP expression in less
than 10% of the plasma cells. LRP was not expressed in plasma cells of normal donors (0/
3) or patients with MGUS (0/5). In the majority of MM patients, LRP expression was found
in granulocytic marrow components, irrespective of expression on plasma cells.




                                                                                                                    135
_____   CHAPTER 7 _____________________________________________________________________________________________________




Figure 1: Alkaline phosphatase immunocytohistochemical staining using the MoAb LRP-56 of
cytocentrifuged bone marrow cells containing >95% plasma cells of a patient with multiple myeloma.
Cytospins were counterstained in diluted hemotoxiline. LRP immunoreactivity in a granular fashion in the
cytoplasm is present in almost all plasma cells (A). Isotype control is negative (B).


Correlation with established prognostic factors
Using previously defined cut-off levels we studied the distribution of the plasma cell LI%,
serum B2-microglobulin level, and serum LDH in relation to plasma cell LRP expression.
The cut-off level for LI was ≥2%,(25) for B2-microglobulin ≥4 µg/mL,(26) and for LDH ≥300
U/L.(26) LRP expression was associated with high LDH levels at diagnosis (X2, P = .05).
LRP did not correlate with serum B2-microglobulin (P = .1), plasma cell LI% (P = .07), or
age (P = .9).

LRP expression and response to melphalan chemotherapy
The response to chemotherapy consisting of MP or IDM/HDM is summarized in Table
2; in 38 patients treated with standard MP the overall response rate was 37% (14/38).
There were no complete remissions obtained with MP. In this group, LRP expression was
associated with a poor response to induction treatment. Fifty-two percent (11/21) of the
LRP-negative patients achieved a remission as compared with 18% (3/17) of the patients
with LRP-positive myeloma at diagnosis (X2, P = .027). By univariate analysis, bone
marrow plasma cell LI, serum B2-microglobulin, and serum LDH did not have a significant
prognostic value regarding the response to MP therapy (Table 2).


136
                                                                                           Table 2: LRP Expression, Prognostic Factors, and Clinical Outcome.
LRP IN MULTIPLE MYELOMA _____




                                                                                                                                                                                                                                                                     137
                                                                                                                           ALL Patients                                       MP Treated Patients                               IDM/HDM Treated Patients
                                                                                                             Response                Median                        Response               Median                        Response            Median
                                                                                           Parameter    No     (%)      P-Value*   Survival (mo)   P-Value   No.     (%)    P-Value*    Survival (mo)   P-Value   No.     (%)    P-Value* Survival (mo)    P-Value
                                                                                           Total        70      59                                           38       37                                          32       84
                                                                                           LRP (%)
                                                                                            <10         37      68        .06            54        <.002     21       52      <.05           40         <.001     16       88         .29        69          .36
                                                                                            ≥10         33      48                       28                  17       18                     22                   16       81                    nr
                                                                                           B2M
                                                                                            <4 mg/L     41      63        .49            46                  20       35       .8            38         <.17      21       90         .48        69          .82
________________________________________________________________________________________




                                                                                            ≥4 mg/L     29      55                       33         .22      18       39       .3            28                   11       82                    43
                                                                                           LI%†
                                                                                            <2          41      63        .68            28        <.04      18       39                     28          .1       23       83         .31        69          .87
                                                                                            ≥2          19      58                       nr                  10       20                     nr                    9      100                    76
                                                                                           LDH†
                                                                                            <300 µ/L    42      55        .66            40        <.01      24       33       .8            38          .0001    19       83         .79        69          .97
                                                                                            ≥300 µ/L    15      66                       30                   7       29                     11                    8       88                    46
                                                                                           Abbreviation: nr, not reached.
                                                                                           *By chi-squared test.
                                                                                           †Data were not available from all patients.
_____   CHAPTER 7 _____________________________________________________________________________________________________



A remission was achieved in 84% (27/32) of the patients treated with intensified dose
melphalan, including 8 patients who achieved a CR (25%). No significant difference
was found between the response rate in the LRP-negative patients (88%, 14/16) and
the LRP positive population (81%, 13/16; P = .285). The subgroups of LRP-positive and
LRP-negative IDM/HDMtreated patients showed no statistically significant differences in
distribution of LDH, LI%, B2-microglobulin, or age. A comparison of responses between
the two regimens in LRP-positive patients showed a significant higher response rate with
IDM/ HDM as compared with MP (81% v 18%, P <.0001). In LRP-negative patients a better
response rate with IDM/HDM (88% v 52%, P = .006) was also observed.

Expression of LRP and survival
Kaplan-Meier survival curves of LRP-positive and LRP-negative patients are presented in
Figures 2 through 4. An inverse correlation was found between LRP expression and survival
duration. In the complete group, the median survival of LRP-positive patients was 28
months (95%-Cl: 23 to 33), whereas the median survival duration of LRP negative patients
was 54 months (Cl: 26 to 82; P <.002; hazard ratio [HR] = 2.9 (1.4-5.7); Table 2, Fig 2). This
difference was likely caused by the 38 MP-treated patients who had a median survival 40
months in LRP-negative (95%-Cl: 41 to 69) and 22 months in LRP-positive patients (95%-
Cl: 18 to 26; P = .0006, HR = 4.1 (1.7-9.8); Table 2, Fig 3). In the IDM/HDM-treated patients
no significant difference in survival between LRP-positive and LRP-negative patients was
observed (median survival 69 months [95%-Cl 14 to 124]) in LRP-negative patients and not
reached in LRP-positive patients (P = .365, HR = 1.7 [0.5-5.9; Table 2, Fig 4]).
By univariate analysis LRP (P <.002) was the strongest adverse prognostic marker
for survival in the whole population followed by bone marrow plasma LI, serum B2-
microglobulin, and serum LDH (Table 2). In MP-treated patients high serum LDH (P =
.0001) and LRP expression (P <.0006) both had an adverse effect on survival (Table 2).
Multivariate analysis showed that in the whole population (50 cases available for analysis)
only LRP expression was an independent prognostic factor (P = .03). In the subgroup
of MP-treated patients (25 cases available) LRP (P = .03) and serum LDH (P = .0001)
remained statistically significant for survival. In the patients treated with IDM/HDM none of
the prognostic factors affected survival. Age had no prognostic significance for survival by
either univariate or multivariate analysis.
In the subgroup of responding patients we performed statistical analysis regarding event-
free survival (EFS). Within the responding MP-treated patients, the EFS was remarkable
shorter in LRP-positive patients. Three LRP-positive patients treated with MP relapsed
after 6, 7, and 15 months, respectively (median 7 months, 95%-Cl: 5 to 9), whereas the
EFS of LRP-negative MP-treated patients was median 24 months (n = 11, 95%-Cl; 16 to
32; P <.003). EFS of IDM/HDMtreated LRP-positive patients was median 22 months (n
= 14, 95%-Cl: 14 to 30) versus 24 months (n = 13, 95%-Cl: 15 to 33; P = .182) for LRP-
negative patients.


138
________________________________________________________________________________________                                       LRP IN MULTIPLE MYELOMA _____


                                                    1,0

                                                     ,9

                                                     ,8




                              Cumulative survival
                                                     ,7

                                                     ,6
                                                                                                LRP neg. (N=37)
                                                     ,5

                                                     ,4

                                                     ,3
                                                                              LRP pos. (N=33)
                                                     ,2

                                                     ,1
                                                              P=0.0016
                                                    0,0
                                                          0     12       24   36     48      60      72     84    96   108   120

                                                                                          Time (months)

Figure 2: Patients treated with melphalan, either at conventional dose and combined with prednisone or
administered as an intensified (70/140 mg/m2 IV). Probability of survival from the start of treatment.

                                                    1,0

                                                     ,9

                                                     ,8
                              Cumulative survival




                                                     ,7

                                                     ,6

                                                     ,5
                                                                                              LRP neg. (N=21)
                                                     ,4

                                                     ,3

                                                     ,2                         LRP pos. (N=17)

                                                     ,1       P=0.0006

                                                    0,0
                                                          0     12       24   36     48      60      72     84    96   108   120

                                                                                          Time (months)

Figure 3: Patients treated with conventional melphalan and prednisone. Probability of survival from the
start of treatment.
                                                    1,0

                                                     ,9

                                                     ,8
                                                                                       LRP neg. (N=16)
                              Cumulative survival




                                                     ,7

                                                     ,6

                                                     ,5          LRP pos. (N=16)

                                                     ,4

                                                     ,3

                                                     ,2

                                                     ,1
                                                              P=0.3645
                                                    0,0
                                                          0     12       24   36     48      60      72     84    96   108   120

                                                                                          Time (months)

Figure 4: Patients treated with IV intensified melphalan (70/140 mg/m2 IV). Probability of survival from the
start of treatment.



                                                                                                                                                        139
_____   CHAPTER 7 _____________________________________________________________________________________________________



Discussion

Our findings indicate that LRP is widely expressed in untreated MM and that it is
associated with a low probability of response and a shorter survival in patients treated with
a conventional MP regimen. LRP positivity was found in 47% of the patients with newly
diagnosed MM. This figure is consistent with data indicating the widespread expression
of LRP in untreated human malignancies.(13) Multivariate analysis showed that LRP was
an adverse prognostic marker that was independent for serum B2-microglobulin, bone
marrow plasma cell LI, serum LDH, and age. Interestingly, LRP-related resistance to
MP may initially be overcome by dose intensification of melphalan as suggested by the
outcome of patients treated with IDM and HDM.
These observations add proof to the recent in vitro and clinical studies identifying LRP
as an independent predictor for chemoresistance against melphalan. Studies undertaken
to assess the in vitro sensitivity of the RPMI, 8226 human myeloma cell line to several
cytotoxic drugs showed that by exposure to melphalan an MDR subline emerged, termed
8226 LR5, which is resistant to melphalan and highly upregulates LRP expression in
absence of other MDR proteins (showing a drug accumulation defect). Also exposure to
mitoxantrone resulted in a highly LRP-positive cell population (8226 MR40), which showed
additional resistance to melphalan, again in absence of other MDR-related proteins (W.S.
Dalton et al, personal communication, July 1997).
Moreover, in human cancer cell lines derived from 8 cancer types, using
immunocytohistochemical detection methods, a significant correlation between LRP
expression in these cancer types and in vitro sensitivity to melphalan was found. No
correlation was found between the expression of other MDRrelated proteins and melphalan
sensitivity.(15) These in vitro results are in line with our clinical finding of an association
between LRP expression on myeloma cells in untreated patients and lack of response to
oral melphalan chemotherapy in these patients.
Further evidence for the relationship of LRP with chemoresistance to both classical and
MDR-related drugs is provided by several recent clinical studies. In patients with adult
myeloid leukemia(27) and patients with FIGO stage III/IV ovarian cancer,(21) LRP expression
of malignant cells was significantly correlated with inferior response to chemotherapy,
including cisplatin and alkylating agents, and with shorter overall survival.
The precise mechanism of LRP-related chemoresistance, however, is still unsolved. To
date the biological function of LRP as a major constituent of the human vault protein is
unknown. A small fraction of vaults are localized to the nuclear membrane and nuclear pore
complexes, raising the possibility that vaults mediate the bidirectional transport of a variety
of substrates between the nucleus and the cytoplasm.(28) In support of this view, entrapment
of drugs in exocytotic vesicles and decreased nuclear to cytoplasmic drug ratios were
reported in LRP-overexpressing multidrug resistant cells.(29,30) Interestingly, melphalan and
cisplatin exert their main cytotoxic effect in the cell nucleus having very similar modes of


140
________________________________________________________________________________________   LRP IN MULTIPLE MYELOMA _____



action on nucleic acids. This makes it tempting to hypothesize that vaults are involved in
the nucleo-cytoplasmic exchange of these drugs.
Our findings cannot exclude that LRP is a pleiotropic marker of resistance coexpressed
with other (MDR-related) drug resistance genes. In this study we did not assess the
expression of other MDR-related proteins, ie, Pgp and MRP. Therefore, no conclusion can
be drawn on the individual roles of each resistance protein in these MM samples. However,
we and others found that PgP is expressed in very low frequency (<5%) in untreated MM
and has no prognostic value at that stage.(31,32) In contrast, PgP is highly expressed in
VAD-refractory MM,(31,33) whereas MRP is not expressed above background values in the
majority of MM samples.(34) Moreover, in in vitro experiments and clinical studies MDR and
MRP did not confer resistance to melphalan.(15,21)
Obviously other nonclassical MDR transport mechanisms of resistance must be involved in
MM, which may explain why resistance occurs in LRP-negative patients.
An alternate conclusion from the adverse prognostic value of LRP in patients treated with
the MP regimen could be an inverse relation between LRP expression and the sensitivity of
myeloma cells to corticosteroids. Previous studies have shown that steroid dose intensity
is one of the most important predictors of treatment outcome. Considering this, the use
of prednisone in patients receiving oral melphalan is an important difference between the
treatment groups. However, recent in vitro studies in acute lymphocytic leukemia using
flow cytometry and a methylthiotetrazole (MTT) assay have shown a lack of relation
between LRP expression and resistance to prednisolone (M.L. den Boer et al, Department
of Paediatrics, Free University Hospital, Amsterdam, The Netherlands, personal
communication, January 1997). In addition to a potential role in chemoresistance LRP may
be associated with a biologically more aggressive state of MM. This is not only suggested
by the fact that EFS of LRP-positive patients tended to be shorter but also by the relation
between LRP and elevated LDH levels in untreated disease. LRP did not correlate with
serum B2-microglobulin, LI, or age. High LDH levels in untreated MM have been related to
high tumor mass, unusual clinical features like extraosseous masses, and hypodiploidy or
low RNA content of plasma cells, possibly reflecting a late stage of myeloma transformation
and a poor clinical outcome.(26) Also in our study MP-treated patients with elevated LDH
levels survived significantly shorter. More detailed studies on the relation of LRP with
clinipathological and cytogenetic parameters in MM are therefore warranted.
Our findings are clinically important because LRP-related resistance to the MP regimen
may be circumvented by dose intensification of melphalan. LRP-positive patients had a
significant better response to IDM/HDM as compared with MP. The dose-response relation
for melphalan has been widely documented, and dose escalation has been clinically applied
in recent years.(3-5) In general, previously untreated patients have superior response rates
to intensive regimens as compared with MP, and remissions are of good quality.(5) Our
results indicate that assessment of the LRP status might identify a patient population that
can initially benefit from dose intensification and in which this regimen could be considered
as a first-line treatment above the MP regimen.
                                                                                                                    141
_____   CHAPTER 7 _____________________________________________________________________________________________________



In conclusion, in this study we assessed the expression of LRP in untreated myeloma and
introduce this novel drug resistance-related protein as a prognostic factor for response
to the MP regimen and survival. Moreover, we report that LRP-associated resistance to
melphalan may be circumvented by dose intensification, creating the possibility to select
patients who benefit from these regimens. Further studies on the expression of LRP and
other mechanisms of drug resistance seem warranted to confirm these results and to clarify
the functional characterization and the biological role of LRP in myeloma and other tumor
types.



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                                                                                                                    143
                                                      CHAPTER 8
IMPAIRED BREAST CANCER RESISTANCE PROTEIN MEDIATED
                            DRUG TRANSPORT IN PLASMA CELLS
                                                  IN MULTIPLE MYELOMA




                                                              MARC H.G.P. RAAIJMAKERS1
                                                                ELKE P.L.M. DE GROUW2
                                                                   LEONIE H.H. HEUVER2
                                                               BERT A. VAN DER REIJDEN2
                                                                       JOOP H. JANSEN2
                                                                     GEORGE SCHEFFER3
                                                                        RIK J. SCHEPER3
                                                                   THEO J.M. DE WITTE1
                                                               REINIER A.P. RAYMAKERS1

                        DEPARTMENT OF HEMATOLOGY1 AND CENTRAL HEMATOLOGY LABORATORY2,
                     UNIVERSITY MEDICAL CENTER NIJMEGEN “ST. RADBOUD”, THE NETHERLANDS,
         DEPARTMENT OF PATHOLOGY3, FREE UNIVERSITY HOSPITAL, AMSTERDAM, THE NETHERLANDS




                                                   LEUKEMIA RESEARCH. 29:1455-58, 2005
_____   CHAPTER 8 _____________________________________________________________________________________________________




146
______________________________________________________________________________________   BCRP IN MULTIPLE MYELOMA _____



Abstract

The Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP-binding-cassette
transporter involved in the transport of drugs used in the treatment of multiple myeloma
(MM). Its expression, function and clinical significance in MM, however, are unknown. We
report that BCRP is preferentially expressed and functionally active in normal plasma cells
but that its function is significantly impaired in plasma cells in newly diagnosed MM. The
data presented argue against a role for BCRP in primary drug resistance in MM and the
utilisation as a molecular target as such but warrant research into its (patho)physiological
role in normal and malignant plasma cells.



Introduction

Multiple drug resistance (MDR) is a major obstacle for response to chemotherapy in patients
with multiple myeloma (MM). MDR in MM has been associated with drug efflux by the ATP-
binding cassette (ABC) transporter P-glycoprotein (ABCB1) and expression of the lung
resistance-related protein (LRP)(1). Studies in the multidrug resistant 8226 myeloma cell line
have demonstrated additional mechanisms involved in the MDR phenotype of malignant
plasma cells including an additional energy-dependent drug efflux pump(2), identified as
the breast cancer resistance protein (BCRP/ABCG2)(3). BCRP is a 655-aa member of the
ABCG subfamily of ABC- membrane transporters encoded by the BCRP gene and confers
multidrug resistance to topotecan, mitoxantrone, doxorubicin and other anthracyclins
by ATP dependent drug extrusion. Recent findings suggest that BCRP may also play a
role in the transport of steroids(4), one of the main drugs in anti-myeloma treatment. The
expression of BCRP in myeloma cell lines and its involvement in transporting anthracyclins
steroids suggest that BCRP could be involved in drug resistance against these agents in
MM. This prompted us to investigate BCRP expression, function and role in resistance to
vincristine, adriamycin and dexamethasone (VAD) chemotherapy in de novo MM.



Methods

Bone marrow samples and plasma cells
Bone marrow was obtained after informed consent from healthy allogeneic bone marrow
donors and MM patients at diagnosis. Patient characteristics are shown in table 1.




                                                                                                                   147
_____   CHAPTER 8 _____________________________________________________________________________________________________


Table 1: Patient characteristics, BCRP expression, BCRP mediated mitoxantrone transport and response
to VAD treatment.

                                            BCRP             BCRP efflux MDR1 efflux Initial
Nr      Age     Sex     Stage     Ig        (BXP21/IgG)      (EI)        (EI)        treatment             Response
1.      59      M       III-a     IgG-k     3.68             1.13             0.97          VAD            CR
2.      50      M       III-a     IgA-l     7.79             0.98             1.35          VAD            PR
3.      64      F       II-a      IgA-l     7.29             0.92             1.01          VAD            PR
4.      59      M       III-a     IgG-k     7.31             1.03             1.51          VAD            PD
5.      58      M       III-a     IgA-k     7.71             1.09             1.24          VAD            CR
6.      61      F       III-a     BJ-k      4.58             1.29             1.25          VAD            PR
7.      43      F       III-a     IgA-l     2.85             1.06             1.12          VAD            CR
8.      39      M       II-a      IgG-k     3.88             1.07             1.25          VAD            SD
9.      58      M       III-b     IgG-k     2.40             1.01             1.13          VAD            PR
10      63      M       II-a      IgA-k     3.79             1.01             1.05          VAD            SD
CR=complete remission; PR=partial remission; SD=stable disease; PD=progressive disease


All patients received vincristine, adriamycin and dexamethasone (VAD) chemotherapy as
initial treatment. Response was evaluated after 3 courses according to the criteria of the
Eastern Cooperative Oncology Group (ECOG). Isolation, cryopreservation and thawing
procedures of cells have been described previously(5). Plasma cells were identified as
CD38hi, side scatterhi cells (figure1).


                      NBM                                                     NI
                                                                              KO143


                      CD34+
                                                                EVENTS




                                              PC
              CD34




                     CD38                                                MITOXANTRONE

                      MM                                                      NI
                                                                              KO143
                       CD34+


                                                PC
                                                                EVENTS
              CD34




                     CD38                                                MITOX ANTRONE
Figure 1: BCRP mediated mitoxantrone efflux in normal and malignant plasma cells. Typical examples of
CD34-FITC and CD38-PE labeled bone marrow mononuclear cells in normal bone marrow (NBM) and multiple
myeloma (MM). Plasma cells (PC) were defined as CD38hi, side scatterhi cells. Inhibition of BCRP by the
fumitremorgin C analog KO143 significantly increases mitoxantrone fluorescence in normal but not in malignant
plasma cells, indicating deficient drug efflux in the latter. (NI=no inhibitor)


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______________________________________________________________________________________   BCRP IN MULTIPLE MYELOMA _____



Plasma cell nature was confirmed with the specific monoclonal antibody VS38c (data
not shown) and the number was correlated with the number of plasma cells identified
morphologically on cytospins in MM patients.

BCRP expression and function
BCRP was detected as described previously(5) using the BXP21 monoclonal antibody (2.5
ug/ml) in a three-color flowcytometric assay using fluorescein (FITC)-conjugated goat
anti mouse F(ab)2 fragments (5.0 ug/ml), CY5-conjugated CD34 and phycoerythrin (PE)-
conjugated CD38 and quantified as the median fluorescence channel shift (BXP21/Isotype
control). CD34 was included in all flowcytometric analyses to allow comparison of BCRP
expression and function in plasma cells with CD34 cell populations and to allow CD34+ and
CD34- cells to serve as internal controls.
BCRP function was tested flow-cytometrically using mitoxantrone as a substrate and the
fumitremorgin C (FTC) analog KO143 as an inhibitor for BCRP as described previously (5) ;
KO143 (0.1 uM) is the most potent inhibitor currently available and, importantly, is BCRP-
specific (without inhibition of MDR1 or MRP). BCRP mediated efflux was quantified as
the mitoxantrone fluorescence (MFI) shift in the presence/absence of KO143 (depicted as
efflux index) and assessed in CD38hi/ SSchi, CD34+ and CD34- cells as defined (figure 1).
At least 2000 events were evaluated in each cell population.

P-glycoprotein (MDR1/ABCB1) mediated drug efflux
Flow cytometric assessment of P-glycoprotein mediated mitoxantrone efflux was performed
as described above using verapamil (Knoll AG, Ludwigshaven, Germany (20 ug/ml) as
inhibitor for P-glycoprotein mediated efflux instead of KO143.

Statistical analysis
Differences in expression and function between cell populations and normal and malignant
plasma cells were calculated using the student t-test with a level of significance of p ≤ 0.05.



Results

BCRP is preferentially expressed and functionally active in normal plasma
cells
BCRP was expressed in plasma cells in all normal bone marrow samples examined (n=10)
(figure 2A). The average BCRP expression (BXP21/ Isotype control) was 5.58 ± 0.70
(S.E.M) (range 3.10 -10.24). Expression was significantly higher in plasma cells compared
to other CD34- cells (mean BXP21/Isotype control 2.92 ± 0.52, p=0.002; range 1.60-
6.08) in all samples examined and was similar to the expression in CD34+ hematopoietic
progenitors (mean 6.18 ± 0.77, p=0.13; range 4.04-10.93).


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BCRP mediated efflux was significantly higher in plasma cells (mean efflux index 1.20 ±
0.06 SEM, range 0.9-1.53) than in both CD34- cells (mean efflux index 1.03 ± 0.04, range
0.75-1.11; p< 0.001) and CD34+ hematopoietic cells (mean efflux index 1.07 ± 0.04, range
0.89-1.37; p< 0.001) in all samples examined (figure 2B).
The contribution of BCRP to mitoxantrone efflux in plasma cells was similar to P-glycoprotein
mediated efflux (efflux index 1.22 ± 0.05, range 1.01-1.56; NS) (figure 2C). Together these
results indicate that BCRP is preferentially expressed and functionally active in human
normal plasma cells and mediates mitoxantrone efflux in these cells in cooperation with
P-glycoprotein mediated transport.

BCRP mediated drug transport is impaired in plasma cells in multiple
myeloma
The preferential expression and activity of BCRP in normal plasma cells and its possible
implications for primary drug resistance in MM, prompted us to investigate BCRP
expression and function in in malignant plasma cells in MM at diagnosis. BCRP was
detected on plasma cells in all MM samples examined (n=10) (figure 2A).
Expression was similar to the expression in normal plasma cells (mean BXP-21/ isotype
control 5.13 ± 0.68, range 2.40-7.79; NS). BCRP mediated mitoxantrone efflux, however,
was significantly impaired in malignant plasma cells compared to their normal counterparts
(mean efflux index 1.06 ± 0.03, range 0.92-1.29; p=0.02) (figure 1 and 2B). BCRP mediated
efflux in CD34- and CD34+ cells in MM did not significantly differ from normal bone marrow
and therefore provided a suitable internal control for BCRP efflux assessment in MM
samples. Together these results indicate that BCRP mediated drug efflux, which is an
intrinsic characteristic of normal plasma cells, is impaired in malignant plasma cells in the
majority of MM patients.
P-glycoprotein mediated efflux, in contrast, was unaltered in malignant plasma cells
compared to plasma cells in normal bone marrow (mean efflux index 1.19 ± 0.05, range
0.97-1.51; difference ns) (figure 2C), indicating that intrinsic P-glycoprotein mediated efflux
is the more important determinant of mitoxantrone accumulation in malignant plasma
cells.
In line with the deficient function of the protein, no correlation was found between BCRP
expression and response to initial VAD treatment (table1); 3/4 patients with high BCRP
expression (arbitrarily defined as BXP-21/Isotype ≥ 5.0) responded to treatment compared
to 4/6 patients with low BCRP expression. Additionally, responses were achieved in the
two patients that displayed significant BCRP mediated efflux in their plasma cells, arguing
against an important role for BCRP in clinical resistance to VAD treatment in MM patients.




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                                                 A
                                                     11.00            NBM

                                                                      MM
                                                      9.00

                     BCRP expression
                       (BXP21/IgG)
                                                      7.00


                                                      5.00


                                                      3.00


                                                      1.00
                                                             PC             *   34-           34+
                                                                                      *

                                                 B   1.60
                                                                      NBM
                                                     1.50
                     mitoxantrone efflux (EI)




                                                                      MM

                                                     1.40
                         BCRP mediated




                                                     1.30

                                                     1.20


                                                     1.10

                                                     1.00
                                                             PC                 34-           34+
                                                                  *         *         *   *         *

                                                 C
                                                     2.20             NBM
                      mitoxantrone efflux (EI)
                      P-glycoprotein mediated




                                                     2.00             MM

                                                     1.80

                                                     1.60

                                                     1.40

                                                     1.20

                                                     1.00
                                                             PC                 34-           34+
                                                                                          *         *
Figure 2: BCRP mediated drug efflux is impaired in malignant plasma cells. BCRP expression (A), BCRP
mediated drug efflux (B) and P-glycoprotein mediated drug efflux (C) in human normal and malignant plasma
cells. Average values (±SEM) are given for cell populations in normal bone marrow (grey bars) and multiple
myeloma (open bars). Individual samples are represented by dots. Efflux indices (EI) ≤ 1.00 are indicated on
the x-axis. Significant differences (p<0.05) in comparison to normal plasma cells (grey bars in far left corner) are
indicated with an asterix.




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Discussion

In this report we demonstrate that the ATP-binding cassette transporter ABCG2/BCRP
is preferentially expressed and functionally active in human normal plasma cells. BCRP
expression in bone marrow cells is thought to be restricted to a small population of
hematopoietic stem cells(6). Strikingly, BCRP mediated efflux in plasma cells exceeded the
rate of transport observed in highly purified CD34+38- stem cells(5) , raising questions about
the physiological function of the protein in these highly specialized cells. Intriguingly, cells
with constitutive BCRP expression such as epithelial cells of the digestive tract, biliary ducts
and the mammary gland, are all sites at which apico-luminal transport of immunoglobulins
takes place. Together these data warrant further research into the role of BCRP in plasma
cells and its possible involvement in immunoglobulin secretion.
BCRP mediated drug transport was found to be impaired in plasma cells in MM. Interestingly,
deficient transport function in malignant plasma cells has been described before and been
attributed to dysfunction of P-glycoprotein(7). These authors investigated P-glycoprotein
function using cyclosporin A as an inhibitor. Cyclosporin A has later been shown to be a
more promiscuous inhibitor with effects both on P-glycoprotein and BCRP. The findings
described in this paper suggest that impaired function of BCRP likely accounts at least
partly for the deficient transporter function in plasma cells observed by these authors.
The mechanisms behind impaired BCRP mediated drug efflux in malignant plasma cells
remain to be elucidated; MM is characterised by genetic instability in plasma cells and it
is conceivable that mutations in the BCRP gene, that have been shown to alter substrate
specificity, are responsible for impaired mitoxantrone transport. Alternatively, impaired
BCRP mediated drug transport in malignant plasma cells could reflect competitive
engagement of the protein in other processes in these cells. Involvement in secretion of
autocrine factors such as interleukins, which is frequently observed in MM, and has been
described for ATP-binding-cassette transporters(8), could explain impaired drug efflux by
BCRP. The possibility of alternate functions of BCRP in malignant plasma cells, related to
disease pathogenesis, prompts further research.
Regardless the underlying mechanism, impaired BCRP mediated drug transport is likely
to undermine its role in drug resistance in MM; This is supported by the lack of relation
between BCRP expression and response to initial VAD chemotherapy in this study. Though
the number of patients was small, the combined in vitro and clinical data argue against a
role for BCRP in drug resistance in de novo MM and the utilisation of BCRP modulators to
circumvent it.




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Acknowledgments

The authors like to thank G. Vierwinden and A. Pennings for excellent technical assistance.
A. Schinkel and J.D. Allen (Netherlands Cancer Institute, Amsterdam) are thanked for
providing KO-143. MHGPR is a recipient from a grant from the Dutch Cancer Society
(Nederlandse Kanker Bestrijding-KWF). This work was further supported by a grant from
the Vanderes Cancer Foundation.



References
1.    Raaijmakers HG, Izquierdo MA, Lokhorst HM, de Leeuw C, Belien JA, Bloem AC, Dekker AW, Scheper
      RJ, Sonneveld P. Lung-resistance-related protein expression is a negative predictive factor for response
      to conventional low but not to intensified dose alkylating chemotherapy in multiple myeloma. Blood 1998;
      91(3):1029-1036.
2.    Hazlehurst LA, Foley NE, Gleason-Guzman MC, Hacker MP, Cress AE, Greenberger LW, De Jong MC,
      Dalton WS. Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell
      line. Cancer Res 1999; 59(5):1021-1028.
3.    Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage H, Dietel M, Greenberger L, Cole SP,
      Doyle LA. Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in
      mitoxantrone-selected cell lines. J Natl Cancer Inst 1999; 91(5):429-433.
4.    Janvilisri T, Venter H, Shahi S, Reuter G, Balakrishnan L, van Veen HW. Sterol transport by the human
      breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis. J Biol Chem 2003; 278(23):
      20645-20651.
5.    Raaijmakers MH, de Grouw EP, Heuver LH, van der Reijden BA, Jansen JH, Scheper RJ, Scheffer GL, de
      Witte TJ, Raymakers RA. Breast cancer resistance protein in drug resistance of primitive CD34+38- cells in
      acute myeloid leukemia. Clin Cancer Res 2005; 11(6):2436-2444.
6.    Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M,
      Nakauchi H, Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells
      and is a molecular determinant of the side-population phenotype. Nat Med 2001; 7(9):1028-1034.
7.    Pilarski LM, Szczepek AJ, Belch AR. Deficient drug transporter function of bone marrow-localized and
      leukemic plasma cells in multiple myeloma. Blood 1997; 90(9):3751-3759.
8.    Drach J, Gsur A, Hamilton G, Zhao S, Angerler J, Fiegl M, Zojer N, Raderer M, Haberl I, Andreeff M, Huber
      H. Involvement of P-glycoprotein in the transmembrane transport of interleukin-2 (IL-2), IL-4, and interferon-
      gamma in normal human T lymphocytes. Blood 1996; 88(5):1747-1754.




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        CHAPTER 9
SUMMARY AND PERSPECTIVES
_____   CHAPTER 9 _____________________________________________________________________________________________________




156
______________________________________________________________________________________   SUMMARY AND PERSPECTIVES _____



Summary

Acute myeloid leukemia (AML) is considered a disease originating from hematopoietic
CD34+38- stem cells. The recognition of AML and other forms of cancer as stem cell
diseases propagates a paradigm shift in the treatment of leukemia away from targeting the
blast cells and towards targeting the LSC.
The high expression of ABC-transporters involved in the extrusion of chemotherapeutical
compounds on normal (hematopoietic) stem cells could have major consequences for
the treatment of leukemia. If ABC transporter expression and function is conserved
after malignant transformation of stem cells, this would be a major mechanism of drug
resistance to these cells and prevent chemotherapeutical eradication of this tumor-initiating
cell population. In the paradigm shift for the treatment of leukemia, ABC drug transport
inhibitors might be thought of as “leukemia stem cell sensitizing agent” that allow the most
crucial and drug-resistant cells in the leukemia-hierarchy to be destroyed. Whether ABC
expression and function is conserved, however, is currently unknown. Though expression
and function of ABCB1 and ABCG2 have been widely studied in AML, there is a remarkable
lack of studies addressing expression and function in the critical CD34+38- hematopoietic
stem cell population. Additionally, no studies exist aimed at comparing ABC transporter
function between normal and malignant hematopoietic cells. The goal of the research
performed and described in this thesis was to gain insight into the expression, function and
role in drug resistance of ABC transporters in CD34+38- hematopoietic stem cells in AML
in comparison to their normal counterparts.
The first two chapters of this thesis are dedicated to the development and validation
of essentials tools required to study ABC transporter expression and function in highly
purified hematopoietic cells, precluding the use of standard protocols because of the small
numbers of cells available for study;
In chapter 2 we present the use of a real-time quantitative RT-PCR method for the precise
quantitation of gene expression in small subsets of human hematopoietic cells. Combining
modified RNA isolation and adjusted PCR-protocols with the sensitivity and accuracy of real-
time quantitative PCR we were able to detect and to quantitate low-copy gene expression
without the need of prior cDNA amplification or nested-PCR strategies in as little as 300
highly purified CD34+38- hematopoietic cells from both normal bone marrow and AML.
Validation experiments in cell lines showed efficient, representative and reproducible gene
amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in
dilution experiments and by the detection of the low-copy gene PBGD. GAPDH was found
a useful reference gene in normal and leukemic CD34+CD38- cells. 18S rRNA, in contrast,
varied 100-1000-fold in these populations, showing its inappropriateness as a reference
gene in these progenitors. Finally, using this method we showed the preferential expression
of ABCB1, ABCC1 and LRP in normal CD34+38- cells. The technique described in this
study has facilitated other experiments addressing gene expression in hematopoietic stem
cells both in our lab (chapter IV, V and VI) and in other labs(1).
                                                                                                                   157
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Chapter 3 describes the development of a method to study ABC transporter mediated efflux
in highly purified single CD34+38- hematopoietic stem cells. We used epifluorescence
microscopy in combination with single cell image analysis to quantify ABC transporter
mediated efflux in CD34+38- cells sorted on adhesive biolayers. ABCB1 and ABCC1
mediated efflux were quantified using fluorescent substrates (rhodamine 123 and calcein-
AM) and specific inhibitors (verapamil and probenecid). This method proved feasible and
reproducible, although sensitivity is reduced in comparison to standard flow-cytometrical
assessment of functional, ABC transporter conferred, efflux. Using single cell image analysis
we demonstrated that ABCB1 is preferentially active in CD34+38- cells in comparison to
committed CD34+38+ cells (in line with gene expression studies as described in chapter
2) and the major molecular determinant of the rhodamine 123 dull phenotype of these
cells. Additionally, significant ABCC1 mediated efflux was demonstrated in CD34+38-
hematopoietic stem cells.
In Chapter 4 and 5 we investigated expression and functional activity of ABCB1 and
ABCG2 in hematopoietic CD34+38- hematopoietic stem cells in AML in comparison to
their normal counterparts. We emphasized on studying ABCB1 and ABCG2 since these
transporters are highly expressed on a variety of human stem cells and their wide substrate
specificity includes drugs used in the treatment of AML (i.e. anthracyclins, mitoxantrone,
vinca alkaloids).
In chapter 4 we studied ABCB1 expression and function in CD34+38- cells in AML in
comparison to their counterparts in human normal bone marrow. We demonstrate that
ABCB1 is the major molecular determinant of the mitoxantrone and rhodamine123 dull
phenotype of CD34+38- cells in normal bone marrow as evidenced by the blockage of
transport of these compounds by ABCB1 inhibitors verapamil and PSC-833. Surprisingly,
ABCB1 mediated transport was invariably impaired in the CD34+38- hematopoietic cell
population in AML regardless of disease subtype. Impaired ABCB1 mediated transport
preceded the development of leukemia in patients suffering from refractory anemia but was
not found in autologous transplanted cells from patients in long-term remission constituting
normal hematopoiesis, demonstrating that it is an early leukemic rather than a preleukemic
characteristic of normal CD34+38- cells. In line with this, proof of principle is reported that
ABCB1 efflux capacity can identify a subpopulation with residual normal CD34+38- cells
in AML. Importantly, ABCB1 modulation abrogated drug efflux in normal but not leukemic
CD34+38- cells due to activity of additional drug transporters in leukemic cells.
Together these results demonstrate that reduced ABCB1 mediated transport in CD34+38-
hematopoietic cells is an early characteristic and biological commonality of leukemic
CD34+38- cells in AML. ABCB1 mediated efflux may identify a subpopulation of residual
normal CD34+38- cells in this disease. These findings and the observation that redundant
transport mechanisms, not inhibitable by ABCB1 modulators, mediate drug extrusion from
leukemic CD34+38- cells, have to be taken into account when interpreting the overall
disappointing results on ABCB1 modulation in clinical trials in AML.


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______________________________________________________________________________________   SUMMARY AND PERSPECTIVES _____



The purpose of the investigations described in chapter 5 was to investigate the role of the
Breast Cancer resistance Protein (BCRP/ABCG2) in drug resistance of leukemic stem cells
and the effect of its modulation on stem cell eradication in AML.
BCRP was preferentially expressed (measured flow-cytometrically using the BXP21
monoclonal antibody) in leukemic CD34+38- cells which represented a conserved
physiological function after malignant transformation. Blockage of BCRP mediated drug
extrusion by the novel fumitremorgin C analog KO143 resulted in increased intracellular
mitoxantrone accumulation in these cells in the majority of patients. This increase, however,
was much lower than in the mitoxantrone resistant breast cancer cell line MCF7-MR and
significant drug extrusion occurred in the presence of BCRP blockage due to the presence
of additional drug transport mechanisms, among which ABCB1 and ABCC1 (MRP). In
line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro
chemosensitivity of leukemic CD34+38- cells.
These results confirm that drug extrusion from leukemic stem cells is mediated by the
promiscuous action of ABCB1, ABCG2 and additional transporters.
The observation that additional drug efflux mechanisms are active in leukemic CD34+38-
stem cells prompted us to investigate the expression of other ABC transporter in these
cells.
In chapter 4 and 5 we found that in leukemic CD34+38- cells additional drug transport
mechanisms are active to extrude chemotherapeutiacal compounds. To identify these
drug efflux mechanisms, in chapter 6, we studied gene expression of 45 ABC-transporters
(the complete ABCA, B, C, D and G family) in human hematopoietic CD34+38- cells in
comparison to committed CD34+38+ progenitors in normal G-CSF mobilized and bone
marrow cells (n=12) and their malignant counterparts in AML (n=10). Gene expression
was assessed with a novel real-time quantitative RT-PCR approach using microfluidic
low-density arrays. In normal CD34+38- cells 36 transporters were expressed of which
24 displayed significant differential expression in comparison to committed progenitors. In
addition to known stem cell transporters (ABCB1, ABCG2 and ABCC1) these differentially
expressed genes included many members not previously associated with stem cell biology
such as the family of ABCA6-like ABC transporters clustered on chromosome 17. The
ABC transporter gene expression profile of CD34+38- cells was largely conserved in
AML (although exceptions occurred) and included expression of all 13 members currently
associated with drug extrusion and resistance. These data suggest an important role for
previously unrecognized ABC-transporters in hematopoietic stem cell biology. Additionally,
the identification of many, previously unidentified, drug transporters in leukemic stem cells
prompts further research to test their role in drug resistance and value as potential new
targets to enhance chemotherapeutical eradication of leukemic stem cells in AML

In the studies described in chapter 2-6, CD34 and CD38 membrane markers were used to
identify hematopoietic stem cells from bone marrow. CD38 not only identifies populations


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of hematopoietic stem cells but is also used to identify the CD34-38+++ plasma cells in
bone marrow. Chapter 7 and 8 describe the expression and clinical significance of the
drug resistance genes ABCG2 and the Lung Resistance related Protein (LRP) in CD34-
38+++ plasma cells in normal bone marrow and in their malignant counterparts in multiple
myeloma. Chapter 7 describes the expression and clinical significance of LRP, a protein
associated with MDR (multiple drug resistance) in myeloma. It is demonstrated that LRP
is frequently expressed in plasma cells in untreated disease and that it is an important
negative prognostic factor regarding response induction, event free survival and overall
survival in patients treated with convential dose melphalan and prednisone . Interestingly,
dose intensification (intravenous intermediate/high dose melphalan) abrogated the
prognostic significance of LRP likely by overcoming LRP mediated drug resistance of
malignant plasma cells. LRP thus identifies a subpopulation of myeloma patients with a
poor probability of response to standard MP chemotherapy (18%) that could benefit from
dose intensification (response rate 81%). In chapter 8 it is demonstrated that ABCG2 is
preferentially expressed and functionally active in normal plasma cells but that its function is
significantly impaired in plasma cells in newly diagnosed MM. In line with deficient function
of the protein in malignant plasma cells, no relation was found between ABCG2 expression
and response to VAD chemotherapy. The data presented argue against a role for ABCG2
in primary drug resistance in multiple myeloma and the utilization as a molecular target as
such.

Concluding remarks and perspectives

I. ABC transporters and drug resistance of leukemic stem cells: Implications for the
   clinical use of ABC transporter modulation and future research
In this thesis it is demonstrated that in primitive leukemic CD34+38- cells ABCB1 mediated
transport is reduced in comparison to normal hematopoietic stem cells. ABCB1 mediated
drug efflux identifies residual normal hematopoietic stem cells in AML. Additionally it is
shown that drug efflux in leukemic CD34+38- cells is conferred by the simultaneous action
of many, previously unrecognized, redundant ABC transporters. What implications do
these laboratory findings have on the current practice of ABCB1 modulation to enhance
chemosensitivity of leukemic (stem) cells in AML? Based on these laboratory lessons
it seems justified to critically re-assess the use of ABCB1 modulation as a therapeutic
strategy in AML from the standing point of the leukemic stem cells asking three questions.

1. Is ABCB1 modulation an effective strategy to eradicate leukemic stem cells?
   Extrapolating these in vitro data it seems unlikely that modulation of a single drug
   efflux mechanism, such as ABCB1 or ABCG2, will result in sufficient increase in drug
   accumulation, circumvent drug resistance and eventually eradicate this leukemic stem
   cell population. Additionally, in this context, it has to be realized that drug extrusion


160
______________________________________________________________________________________   SUMMARY AND PERSPECTIVES _____



   is only one way of cancer cells to evade chemotoxicity. Many other mechanisms are
   probably active and it is reasonable to assume that these mechanisms, in parallel to
   ABC transporters, preferentially protect the long lived hematopoietic stem cells and their
   malignant counterparts(2).

2. Is ABCB1 modulation a leukemia- specific targeting therapy?
   The data in this thesis demonstrate that ABCB1 mediated drug efflux identifies a
   population of residual normal stem cells in AML. In leukemic CD34+38- cells ABCB1
   efflux is actually reduced in comparison to their normal counterparts. These findings
   demonstrate that ABCB1 modulation is not a leukemia-specific targeted therapy and
   in fact, may preferentially target normal rather than leukemic stem cells. Additionally,
   although not the subject of this thesis, ABCB1 modulation targets its physiological role
   in many other tissues especially those involved in drug detoxification.

   The negative answers to both answers challenge the rationale for the use of ABCB1
   modulation as a strategy to target leukemic stem cells in AML and have to be taken
   into account when interpreting the disappointing results from clinical trials showing no
   benefit of ABCB1 modulation at the cost of increased toxicity. The clinical observation
   that ABCB1 modulation has not resulted in increased overall and relapse free survival in
   AML, as would be anticipated if ABC drug transport inhibitors function as “leukemia stem
   cell sensitizing agent” that kill the leukemia-initiating cell population, seems congruent
   with the laboratory findings described in this thesis.
   On the other hand our data do not definitely exclude the possibility that “true” leukemic
   stem cells harbor in the ABCB1 mediated CD34+38- mitoxantrone dull population that is
   described in chapter 4 of this thesis. This would imply that ABCB1 mediated drug efflux
   (or rather verapamil and PSC inhibitable drug efflux) is indeed the major xenobiotic
   transport mechanism in these cells, despite the expression of other ABC transporters,
   comparable to normal hematopoietic stem cells. To definitively address this question
   NOD-SCID experiments need to be performed transplanting the CD34+38- mitoxantrone
   dull population to see whether it engrafts normal or leukemic hematopoiesis. If true
   leukemic stem cells harbor in the ABCB1 efflux ++ population, ABCB1 modulation is
   likely to augment chemotherapy induced eradication of leukemic stem cells and could
   be related to improved OS but this will be at the cost of severe hematological toxicity.

3. Does challenging the role of ABCB1 in drug resistance of leukemic stem cells dismiss
   the value of ABCB1 as a prognostic marker in AML, one of the hallmark rationales for
   ABCB1 modulation in this disease?
   First it has to be stated that its prognostic significance is demonstrated for induction of
   remission, a prognostic value for DFS and OS is less clear. When extrapolated to the
   hierarchy of AML it is conceivable that ABCB1 modulation can enhance chemosensitivity


                                                                                                                   161
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   of the bulk of leukemic blasts but not the leukemic stem cell population that is protected
   by redundant mechanisms. Indeed, our observations in chapter 4 demonstrated that
   ABCB1 modulation is capable of abrogating mitoxantrone efflux from CD34+38+ cells
   but not the CD34+38- leukemia-initiating cell population. This will result in remission
   induction but eventually relapse will occur from the leukemic stem cell population.
   Alternatively, it is conceivable that expression and activity of ABCB1 is a pleiotropic
   marker of prognosis; it may be a marker of immature (CD34+38-) leukemias as
   suggested by the differential expression of ABCB1 in CD34+38- cells as demonstrated
   in chapter IV. These cells are likely characterized by the conserved function of other
   innate drug resistance and anti-apoptotic mechanisms that protect primitive progenitor
   cells, including many drug-resistance related ABC-transporters as demonstrated in
   chapter VI. The prognostic value of ABCB1 may reflect the innate drug resistance
   conferred by these associated drug resistance and anti-apoptotic mechanisms, rather
   than functional activity of a single drug extrusion mechanism. The observation that
   ABCB1 is not upregulated at relapse and that ABCB1 related clonal selection does not
   occur after failure of standard therapy is congruent with these alternative explanations
   of the prognostic value of ABCB1 in AML.


Future studies investigating the role of ABC transporter in chemoresistance of
malignant stem cells
The observation that drug efflux in leukemic CD34+38- cells is conferred by the
simultaneous action of several redundant transport mechanisms suggests that the use of
broad-spectrum inhibitors for ABC transporter modulation is required for the eradication
leukemic stem cells rather than the modulation of single efflux mechanisms. However, the
strategy of broad spectrum modulation of ABC transporters is likely to encounter enhanced
hematological toxicity since the expression of the majority of these transporters in leukemic
stem cells represents a conserved physiological function of normal hematopoietic stem
cells as demonstrated in this thesis. Additionally non-hematological toxicity is anticipated
since many of these transporters serve important functions in detoxification processes.
Therefore it is essential to first elucidate the role of the novel stem cell transporters identified
in this thesis in drug resistance of leukemic stem cells. Studies comparing ABC transporter
profiles in refractory leukemia and at relapse will be of crucial importance to identify those
transporters involved in clinical drug resistance and relapse and are currently ongoing at
our laboratory. Additionally, in vitro and in vivo studies using ABC transporter-null mice
could give definitive insights into the role of individual ABC transporters in (malignant) stem
cell protection against chemotherapeutical agents.
It will be important to unravel the regulatory pathways of ABC transporter expression
in normal and malignant hematopoietic stem cells to identify specific targets that may
distinguish leukemic stem cells from their normal counterparts and ABC transporter
regulatory pathways in other tissues and their stem cells.


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______________________________________________________________________________________   SUMMARY AND PERSPECTIVES _____



Finally, when performing these investigations it remains important to realize that ABC
transporters are only a player in the wide spectrum of drug resistance in cancer stem
cells. These long-lived cells are likely to be naturally resistant to chemotherapy through
their quiescence, precluding efficacy of drugs that target either the cell cycle or rapidly
dividing cells, their capacity for DNA repair and reluctance to enter apoptosis. Tackling drug
resistance in cancer requires ongoing research into all mechanisms of drug resistance
which should continue focusing on the cancer stem cell rather than (or in addition to) its
progeny to offer future patients an increasing change on cure from this disease.

II. ABC transporters and stem cell biology
Besides determining the role of ABC transporters in drug resistance of cancer stem cells,
an even greater challenge for future research will be the elucidation of the physiological
function of these molecules in human normal and malignant stem cells. Characterization
of molecules with tightly controlled expression patterns during differentiation represents an
important approach to understanding regulation of hematopoietic stem cell commitment. In
this thesis we describe the expression of many ABC transporters not previously associated
with stem cell biology in hematopoietic stem cells. The physiological function of these
ABC transporters in human stem cell biology is currently unknown. Important insights
to its potential function has come from studies in Dictyostelium demonstrating that a
rhodamine-123 cellular efflux pump with the properties of an ABC transporter prevents
differentiation of prespore cells(3), possibly by exporting differentiating factors from the
cell interior. This led to the hypothesis that a cell’s response to a signal that can diffuse
across the plasma membrane depends, in part, on the cell’s ability to remove it from the
cytoplasm. According to this hypothesis, hematopoietic stem cells might escape the effect
of cell fate determination and differentiation factors present in the bone marrow. The idea
that ABC transporters regulate the cellular concentration of signaling molecules may prove
to be a general way for developing organisms to control their response to signals and
regulate cell fate decisions. However, no such a role has been demonstrated for the ABC-
transporters identified on human (hematopoietic) stem cells thus far. The ABC transporter
gene expression profile of hematopoietic stem cells as described in chapter 6 of this
thesis has revealed a number of transporter candidates that may serve these functions in
human stem cells. The differential expression of 5 members of the ABCA family (ABCA6-
like subfamily on chromosome 17q24 and ABCA13) within the top ten ranking order of
differentially expressed ABC transporter genes may be of particular importance since
recent studies have led to the concept that transporters of the ABCA subclass serve critical
physiological functions in the transmembrane transport of endogenous lipid substrates
such as phospolipids and essential fatty-acids, substrates involved in the regulation of
differentiation of hematopoietic cells (reviewed in(4)).
It will be important to establish a “stemness” profile for ABC transporters, investigating
their expression not only in hematopoietic stem cells but also stem cells from different


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sources, including mesenchymal and embryonic stem cells. These investigations will
identify ABC transporters that are crucial to stem cell biology. Subsequently the role of
these transporters, including the ABCA6-like family members, in stem cell biology and
hematopoiesis must be characterized functionally using both in vitro RNA inhibition and
vector overexpression studies and in vivo transplantation models. These studies are
currently ongoing at our laboratory. The increasing recognition that cancer originates from
normal stem cells prompts these investigations since the developmental programs that
regulate gene expression profiles are tightly controlled and may lead to cancer if perturbed.
It is conceivable that molecules involved in cell fate decisions and differentiation of these
stem cells have a key role in carcinogenesis, a process characterized by the failure of
differentiation. Consequently, members with aberrant expression in malignant stem cells,
such as ABCA13 as described in chapter 6, are among the first candidates that functional
characterization.
Ultimately, elucidation of the role of ABC transporters in and stem cells biology and
malignant hematopoiesis may provide novel targets for stem cell eradication in leukemia
and other forms of cancer independent from their role in drug resistance.



References
1.      Jamieson, C. H., Ailles, L. E., Dylla, S. J., Muijtjens, M., Jones, C., Zehnder, J. L., Gotlib, J., Li, K., Manz,
        M. G., Keating, A., Sawyers, C. L., and Weissman, I. L. Granulocyte-macrophage progenitors as candidate
        leukemic stem cells in blast-crisis CML. N.Engl.J.Med., 351: 657-667, 2004.
2.      Dean, M., Fojo, T., and Bates, S. Tumour stem cells and drug resistance. Nat.Rev.Cancer, 5: 275-284,
        2005.
3.      Good, J. R. and Kuspa, A. Evidence that a cell-type-specific efflux pump regulates cell differentiation in
        Dictyostelium. Dev.Biol., 220: 53-61, 2000.
4.      Rizzo, M. T. The role of arachidonic acid in normal and malignant hematopoiesis. Prostaglandins
        Leukot.Essent.Fatty Acids, 66: 57-69, 2002.




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Nederlandse samenvatting

Acute myeloide leukemie is een beenmergziekte die onstaat door maligne transformatie van
normale bloedvormende stamcellen. Deze veranderingen in de normale hematopoietische
stamcel leiden uiteindelijk tot leukemie, gekenmerkt door opeenstapeling van grote
hoeveelheden leukocyten in beenmerg en perifeer bloed. De leukemische stamcel kan
gezien worden als de “moedercel” en onderhoudt de leukemische kloon. Incomplete
eradicatie van de leukemische stamcel zal uiteindelijk leiden tot relapse van de ziekte. Dit
wordt frequent waargenomen in patienten met AML waarbij meestal wel een remissie wordt
bereikt maar vaak later een relapse optreedt. Kennelijk zijn de huidige chemotherapeutische
behandelingen derhalve wel in staat tot eradicatie van de bulk van leukemische cellen
maar overleeft de leukemische stamcel. Inzicht in de mechanismen waarmee deze cel
zich verdedigt tegen chemotherapeutische behandeling en de ontwikkeling van strategieen
om deze mechanismen te omzeilen zijn daarom van cruciaal belang in de toekomstige
behandeling van AML en waarschijnlijk vele andere vormen van kanker.
Een van de mechanismen waarmee de leukemische stamcel zich zou kunnen vededigen
tegen chemotherapeutische behandelingen is de expressie van membraaneiwitten die een
groot aantal structureel onverwante stoffen, waaronder vele chemotherapeutica, de cel
uitpompen, de zogenaamde ATP-binding cassette (ABC) transporters.
Doel van dit proefschrift is inzicht te krijgen in de rol van ABC transporters in de resistentie
van leukemische stamcellen tegen chemotherapie en het effect van ABC transporter
modulatie op stamcel-eradicatie in AML.
Onderzoek van resistentie-mechanismen in leukemische stamcellen wordt bemoeilijkt
door het zeer zeldzame karakter van deze cellen in normaal en leukemisch beenmerg.
Ongeveer 1/ 100.000-1000.000 cellen in beenmerg betreft een stamcel. “Standaard”
onderzoeksmethoden zijn niet toepasbaar op zulke kleine aantallen cellen. De eerste
twee hoofdstukken van dit proefschrift beschrijven de ontwikkeling van methodologie om
het onderzoek naar de expressie en functie van ABC-transporters in hematopoietische
stamcellen mogelijk te maken.

In hoofdstuk 2 wordt de ontwikkeling beschreven van een real-time quantitatieve reverse-
transcriptase polymerase chain reaction (real-time RT-PCR) techniek voor het precies
kwantificeren van genexpresie in kleine hematopoietische celpopulaties die sterk verrijkt
zijn voor normale en leukemische stamcellen. In deze techniek worden kleine aantallen
hematopoietische progenitorcellen, gekenmerkt door membraanexpressie van CD34 maar
afwezigheid van CD38 (CD34+38-), flow-cytometrisch gesorteerd. Door een gemodificeerde
RNA isolatie-procedure en PCR-protocol te combineren met de sensitiviteit en precisie van
real-time quantitatieve RT-PCR, kunnen genen die laag tot expressie komen kwantitatief
gedetecteerd worden in slechts 300 normale of leukemische CD34+38- cellen, zonder de
noodzaak tot cDNA amplificatie of “nested” PCR technieken. Validatie experimenten in


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cellijnen laten efficiente, representatieve en reproduceerbare gen-amplificatie zien met
behulp van deze 300-cell RT-PCR methode. De sensitiviteit ervan werd bevestigd in
cDNA dilutie-experimenten en detectie van het low-copy gen PBGD. GAPDH bleek een
geschikt referentie-gen voor de relatieve kwantificering van targetgenen omdat het gelijke
niveaus van expressie heeft in zowel normale als leukemische CD34+38- cellen en hun
meer gedifferentieerde CD34+38+ nakomelingen. De expressie van ribosomaal RNA
(18S), een veelgebruikt referentiegen, daarentegen bleek 100-1000 voudig te varieren
en is daarom ongeschikt als referentiegen in deze celpopulaties. Tot slot tonen we met
behulp van deze nieuwe RT-PCR techniek de preferentiele expressie aan van de ABC-
transporters ABCB1 en ABCC1 in normale hematopoietische stamcellen in vergelijking tot
meer gedifferentieerde CD34+38+ cellen.

Hoofdstuk 3 beschrijft de ontwikkeling van een methode om ABC-transporter gemedieerde
efflux in CD34+38- hemtopoietische progenitorcellen te bestuderen. ABC tranporter
gemedieerde efllux wordt gekwantificeerd in gesorteerde en op biolayers geimmobiliseerde
CD34+38- cellen met behulp van epi-fluorescentie-microscopie en beeld-analyse van
individuele cellen. De activiteit van specifieke ABC transporters ( ABCB1 en ABCC1)
wordt bepaald door specifieke fluorescente substraten ( rhodamine 123 en calceine-AM
respectievelijk) in combinatie met specifieke inhibitoren van deze pompen (verapamil
en probenecid). Deze methode bleek goed uitvoerbaar en reproduceerbaar, hoewel de
sensitiviteit van fluorescentie-microscopie minder was dan van flow-cytometrische bepaling
van efflux van fluorescente substaten. Aangetoond wordt dat ABCB1 is de belangrijkste
rhodamine-123 transporter is in normale CD34+38- cellen en dat, in overeenkomst met de
genexpressieprofielen uit hoofdstuk 1, ook ABCC1 preferentieel actief is in deze cellen.

De resultaten van het onderzoek beschreven in hoofdstuk 2 en 3 postuleren ABCB1,
in overeenkomst met data in de literatuur, als een belangrijke transporter in normale
CD34+38- cellen. De geconserveerde expressie en functie van deze drug-transporter
na maligne transformatie in AML zou een belangrijk reistentiemechanisme vormen
tegen chemotherapeutische eradicatie van de leukemische stamcel in AML. Of ABCB1
inderdaad geconserveerd is en wat het effect is van de modulatie van deze transporter om
de accumulatie van chemotherapie in de leukemische stamcel cel is het onderwerp van
studie in hoofdstuk 4.
In dit hoofdstuk bestudeerden we ABCB1 expressie en functie in leukemische CD34+38-
cellen in vergelijking tot hun normale tegenhangers. De resultaten bevestigen dat
ABCB1 de belangrijkste transporter van rhodamine 123 en het chemotherapeuticum
mitoxantrone is in normale CD34+38- cellen. Inhibitie van ABCB1 met verapamil en PSC-
833 leidde tot blokkade van het transport van deze stoffen uit deze cellen. In tegenstelling
tot onze veronderstellingen bleek dit echter niet het geval in leukemische CD34+38-
cellen. ABCB1 gemedieerde efflux was zonder uitzondering verminderd in deze cellen


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ondanks vergelijkbare membraanexpressie van het eiwit. Inhibitie van ABCB1 leidde
niet tot blokkade van efflux in deze cellen, wijzend op de aanwezigheid van additionele,
redundante, mitoxantrone transporters in leukemische stamcellen. Van belang, residuaal
normale CD34+38- cellen konden in AML beenmerg geidentificeerd worden op basis van
hun geconserveerde ABCB1 gemedieerde mitoxantrone transport.
Deze bevindingen impliceren dat modulatie van ABCB1 niet leidt tot omzeilen van drug
efflux uit leukemische stamcellen en mogelijk juist de residuaal normale stamcelpopulatie
schaadt in deze ziekte. Deze bevindingen vormen een belangrijke verklaring voor de
overwegend teleurstellende resultaten van ABCB1 modulatie op overleving in klinische
trials in deze ziekte en pleiten tegen verder gebruik van deze strategie om stamcellen te
eradiceren in leukemie.

De opvallende bevinding dat ABCB1 gemedieerd transport verminderd is in de cellen van
waaruit leukemie ontstaat leidt tot de hypothese dat constitutief verminderde activiteit
van de belangrijkste xenobiotica-transporter in deze cellen mogelijk betrokken is bij een
verhoogde gevoeligheid voor genotoxische schade door xenobiotica, voorafgaand aan het
ontstaan van leukemie. Om deze hypothese te toetsen werd onderzocht of het verminderd
ABCB1 gemedieerde efflux aanwezig is in normale CD34+38- cellen in patienten met AML.
ABCB1 functie in cytogenetisch normale CD34+38- cellen in autoloog getransplanteerd
beenmerg in langdurige remissie met normale hematopoiese bleek vergelijkbaar met
autoloog getransplanteerde CD34+38- cellen in de niet- stamcelziekte multiple myeloma
hetgeen suggereert dat het defect in ABCB1 transport niet constitutief aanwezig is maar
een leukemische verandering van deze cellen. Deze bevinding is congruent met de
bevinding dat residuaal normale progenitorcellen in AML gekenmerkt worden door een
geconserveerde ABCB1 transportcapaciteit zoals beschreven in het eerste gedeelte van
dit hoofdstuk.
Verminderd ABCB1 transport ging wel vooraf aan het ontstaan van klinisch overte leukemie
in patienten die lijden aan refractaire anemie. In patienten die geen leukemie ontwikkelden
werd zonder uitzondering een normale ABCB1 gemedieerde efflux in CD34+38- celllen
waargenomen op het moment van de diagnose refractaire anemie.
Deze resultaten tonen aan dat verminderd ABCB1 gemedieerd transport in hematopoietische
progenitorcellen een vroeg-leukemische verandering is die voorafgaat aan het ontstaan
van klinisch overte leukemie in patienten met refractaire anemie. Toekomstig onderzoek
zal moeten aantonen of verminderde werkzaamheid van deze belangrijke xenobiotica-
tranporter daadwerkelijk betrokken is bij het onstaan van leukemie of een “bystander”
fenomeen betreft in leukemische transformatie.

De bevinding dat in leukemische CD34+38- progenitorcellen mitoxantrone transport
plaatsvindt in de aanwezigheid van ABCB1 blokkade suggereert de aanwezigheid van
additionele drug transporters in deze cellen.


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In hoofdstuk 5 wordt de rol van ABCG2, een multiple-drug ABC transporter met
gedocumenteerde expressie in humane stamcellen van diverse origine, bestudeerd in
drug resistentie van leukemische CD34+38- progenitorcellen.
ABCG2, gemeten middels flow-cytometrie met het BXP21 monoclonaal antilichaam en
real time RT-PCR, kwam preferentieel tot expressie in zowel normale als leukemische
CD34+38- cellen. Blokkade van ABCG2 gemedieerde efflux met de nieuwe, specifieke
en potente inhibitor KO143 resulteerde in toegenomen accumulatie van mitoxantrone
in leukemische CD34+38- cellen in de meerderheid van AML patienten. Deze toename
was echter gering in vergelijking tot de toename in de ABCG2 positieve cellijn MCF7-MR
en significante efflux van mitoxantrone vond plaats in de aanwezigheid van KO143 in
leukemische CD34+38- cellen. In overeenstemming met deze bevindingen leidde blokkade
va ABCG2 met KO143 niet tot toename van de in vitro chemosensitiviteit van leukemische
CD34+38- cellen.
De resultaten van deze studie bevestigen dat drug extrusie uit leukemische progenitorcellen
tot stand komt door de redundante activiteit van additionele transportmechanismen en
dat modulatie van een enkele transporter waarschijnlijk niet leidt tot voldoende toename
van intracellulaire accumulatie van chemotherapeutica en vervolgens drug-geinduceerde
celdood.

Om deze additionele drug transport- mechanismen te identificeren wordt in hoofdstuk 6
de studie beschreven waarin we de genexpressie van 45 ABC transporters (de complete
ABCA, B, C, D en G familie) in normale en leukemische CD34+38- cellen bepalen
gebruik makend van real-time kwantitatieve RT-PCR op microfluente “low-density” arrays.
Genexpressie werd bestudeerd in CD34+38- progenitorcellen in vergelijking tot meer
gedifferentieerde CD34+38+ cellen in normale, G-CSF gemobiliseerde, CD34+ cellen en
AML. In normale CD34+38- cellen werd expressie gevonden van 36 transporters waarvan
er 24 significant hoger tot expressie kwamen in vergelijking tot CD34+38+ cellen. Naast
ABCB1 en ABCG2 betrof het veel familieleden van ABC-transporter families die niet eerder
in verbinding gebracht zijn met stamcel-biologie, zoals de ABCA6 subfamilie waarvan de
leden geclusterd zijn op chromosoom 17. Het ABC transporter genexpressie profiel van
normale CD34+38- cellen bleek grotendeels geconserveerd in deze cellen in AML inclusief
de expressie van de 13 ABC-transporters die momenteel in verband worden gebracht met
efflux van chemotherapeutische agentia.
Deze data suggeren een belangrijke rol voor veel, tevoren onbekende, ABC transporers
in hematopoietische stamcelbiologie. Aanvullend onderzoek is nodig om de rol van
deze transporters in resistentie van de leukemische stamcel verder te ontleden maar
de bevinding dat vele drug-transport gerelateerde transporters tot expressie komen in
leukemische progenitorcellen bevestigd de stelling dat gelijktijdige modulatie van deze
mechanismen, mogelijk door te richten op gemeenschappelijke pathways, nodig is on drug
efflux uit deze cellen te omzeilen.


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In de studies beschreven in de hoofdstukken 2-6 van dit proefschrift werden CD34 en
CD38 monoclonale antilichamen gebruikt voor de flow-cytometrische identificatie van
hematopoietische stamcellen. CD38 is tevens een belangrijke marker voor de identificatie
van plasmacellen (CD34-CD38++). In hoofdstuk 7 en 8 wordt hiervan gebruik gemaakt
en de expressie en klinische betekenis beschreven van ABCG2 en het Lung-resistance
Related Protein (LRP) in plasmacellen in normaal beenmerg en hun maligne tegenhangers
in patienten met multiple myeloom (MM).
Hoofdstuk 7 beschrijft de expressie en klinische betekenis van LRP, een eiwit geassocieerd
met multiple drug resistentie, in MM. Aangetoond wordt dat LRP frequent tot expressie
komt op plasmacellen in onbehandelde ziekte en dat dit een belangrijke negatieve
prognostische factor is met betrekking tot repons- inductie, event-vrije overleving en
totale overleving in patienten die behandeld worden met conventionele doses melfalan en
prednison. Intensificatie van de dosering (intermediaire of hoge dosis melfalan) deed de
prognostische waarde van LRP verdwijnen, mogelijk door LRP gemedieerde resistentie
van maligne plasmacellen te omzeilen. LRP identificeert dus een populatie MM patienten
die slecht respondeert op conventionele doses melfalen (18% respondeert) maar baat zou
kunnen hebben bij dosis-intensivering (respons 81% in LRP positieve patienten).
In hoofdstuk 8 wordt tenslotte de rol van ABCG2 in primaire resistentie van MM
patienten onderzocht. ABCG2 komt hoog tot expressie en is functioneel actief in normale
plasmacellen maar de functie van het eiwit blijkt significant verminderd in MM op het
moment van diagnose. Er bleek dan ook geen correlatie tussen expressie van ABCG2 op
maligne plasmacellen en de respons op VAD (vincristine, adriamycine en dexamethason)
therapie in deze patienten. De data pleiten tegen een rol van ABCG2 in primaire resistentie
van MM patienten tegen VAD chemotherapie en derhalve het gebruik van dit eiwit als
een target in het omzeilen van resistentie in deze ziekte. De opvallend hoge expressie en
functionele activiteit in normale plasmacellen waarborgt verder onderzoek naar de rol van
dit eiwit in deze gespecialiseerde cellen.




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DANKWOORD
_____   DANKWOORD _____________________________________________________________________________________________________




174
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 Er zijn vele mensen die mij geholpen hebben dit proefschrift tot een goed eind te
 brengen.

 Allereerst mijn promotor,
 Prof. Dr. T.J.M. de Witte, beste Theo
 Jij was diegene die me naar Nijmegen haalde en vertrouwen stelde in mijn kwaliteiten
 als onderzoeker. Je gaf me de ruimte deze kwaliteiten verder te ontplooien en mijn eigen
 wegen te kiezen in het onderzoek. Hierin wist ik me altijd gesteund door jouw belangstelling
 en positivisme. Voor al deze zaken ben ik je dankbaar.

 Mijn co-promotores,
 Dr. R.A.P. Raymakers, beste Reinier
 Van alle mensen in dit dankwoord ben jij zeker diegene die het meest direct betrokken
 geweest is bij de wetenschappelijke vormgeving van dit proefschrift. Je hebt me echter
 ook in een vroeg stadium de vrijheid gegeven om mijn eigen onderzoeksinstincten te
 volgen. Ik heb je leren kennen als een duizendpoot, veelzijdig, ruimhartig, integer, met een
 groot relativeringsvermogen en iemand die in de dagelijkse hektiek van het academische
 wezen nooit de menselijke maat uit het oog verliest. Deze eigenschappen maken je tot een
 bijzonder mens. Ik had me geen betere co-promotor kunnen wensen.

 Dr. J.H. Jansen, beste Joop, sta me toe jouw naam in een adem te noemen met die
 van Bert van der Reijden. Met jullie komst naar Nijmegen is ons onderzoek en mijn
 horizon verruimd met kennis van moleculaire biologie en de introductie van elementaire
 onderzoekstechnieken. Jullie kritische, maar altijd opbouwende, geest heeft geleid tot vele
 inspirerende discussies die ik als hoogtepunten in het doen van onderzoek beschouw en
 elementair zijn voor de progressie ervan. Daarnaast getuigt de manier waarop jullie het
 lab hebben opgezet van het feit dat gedrevenheid en ambitie hand in hand kunnen gaan
 met oog voor menselijke verhoudingen. Mede dankzij jullie werk van de afgelopen jaren
 is de basis geschapen voor verder basaal wetenschappelijk en translationeel onderzoek
 naar “ABC transporters in hematopoietische stamcellen” op het Centraal Hematologisch
 laboratorium in Nijmegen.

 Vanaf mijn eerste stap op het Centraal Hematologisch Laboratorium tot op de dag van
 vandaag is dit een omgeving gebleken waar ik me thuis heb gevoeld. Dit gevoel heeft
 naast de liefde voor het basale onderzoek alles te maken met de mensen met wie ik op het
 lab heb kunnen werken en leven;
 Allereerst de mensen die met analytische ondersteuning en het verrichten van experimenten
 direct bij hebben gedragen aan het onderzoek:
 Gerard van de Bosch: Jij was diegene die me heeft geleerd een pipet vast te houden. Ik
 dank je voor het geduld, de toegewijdheid en het fanatisme waarmee je dit deed en de
 experimenten met de fluorescentie-microscopie volbracht.
                                                                                                                    175
_____   DANKWOORD _____________________________________________________________________________________________________



Liesbeth van Emst en Leonie Heuver, dank voor jullie accurate analytische ondersteuning.
Samenwerking met jullie leverde behalve een grote hoeveelheid wetenschappelijke data
een zo mogelijk nog grotere hoeveelheid “sociale” data op.
Gerty Vierwinden: Dank voor je tomeloze inzet, warme belangstelling en sociale
betrokkenheid. Jouw aanwezigheid op het lab is altijd sfeerbepalend geweest.
Elke de Grouw wil ik bedanken voor het verrichten van de weerbarstige transport-assays
en het voortzetten van andere experimenten. Sterkte met de laatste loodjes van jouw
proefschrift.
Tot slot, Arie Pennings. Meer nog dan een bekwaam flow-cytometrist die de hoeksteen
vormde van de flow-cytometrische bepalingen in dit proefschrift is met jouw overlijden ook
de bezielende kracht verdwenen die zo bepalend was voor de sfeer op het lab.

Het onderzoek beschreven in dit proefschrift integreerde veel verschillende geledingen
binnen het CHL (moleculaire hematologie, flow-cytometrie en beeldverwerking en de
beenmergtransplantatie-unit). Van alle mensen uit deze geledingen heb ik niet anders
dan hartelijkheid, bereidwilligheid en medewerking ervaren. Zonder de mensen die deze
geledingen gezicht geven zou dit proefschrift er niet zijn geweest. De laagdrempeligheid
en uitstekende onderlinge sfeer (zoals tot uiting komend in de jaarlijkse labdag) zijn
bijzonder en moeten gekoesterd worden.
Een aantal mensen wil ik met name noemen. Allereerst de mensen “van het eerste uur” die
me onmiddellijk in hun midden hebben opgenomen: Peter Linssen, Carel Trilsbeek, Gerard
van de Bosch en later ook Paul Brans en Jos Paardekoper. Dank voor het openhouden
van het venster naar de wereld met onze dagelijkse besprekingen van “de toestand in
de wereld” tijdens de lunch wat zo noodzakelijk is in de tijdelijke hypomanie van het
onderzoeksbestaan.
Jan Boezeman. Steun en toeverlaat in het oplossen van problemen op het gebied van de
flow-cytometrie, beeldverwerking en niet in de laatste plaats statistiek.
Louis van de Locht voor technische hulp en discussies met betrekking tot PCR, en
Ewald Mensink voor de waardevolle discussies die belangrijk waren voor hoofdstuk twee
van dit proefschrift.

Verder ben ik dank verschuldigd aan:
De mede-auteurs voor hun bijdrage aan het onderzoek beschreven in dit proefschrift.
De opleiders Interne Geneeskunde (Prof. dr. Jos van der Meer, Prof. dr. Paul Stuyt en dr.
Jacqueline de Graaf). Onderzoek doen naast klinische taken is complex maar waarschijnlijk
beter mogelijk in Nijmegen dan elders in Nederland. Dank voor het organiseren van de
opleiding en het scheppen van de voorwaarden die noodzakelijk zijn voor het combineren
van basaal wetenschappelijk onderzoek met klinische taken.
Frans Russel en Roos Masereeuw (Afdeling Farmacologie en Toxicologie) voor de



176
______________________________________________________________________________________________________   DANKWOORD _____



 voortdurende samenwerking. In de nabije toekomst hoop ik in de gelegenheid te zijn onze
 samenwerking verder gestalte te geven.
 De Staf Hematologie, voor het recruteren van, en de zorg voor de patienten zoals
 beschreven in de studies in dit proefschrift; Datacentrum Hematologie voor het onderhouden
 en beschikbaar stellen van klinische data; Henk Lokhorst voor mijn introductie in de wereld
 van de ABC-transporters.

 Tot slot,
 onderzoek doen is een uitgesproken absorberend proces gebleken dat de neiging heeft je
 het zicht te ontnemen op de dingen die wezenlijker zijn dan wetenschappelijke inzichten
 en verdiensten. Het bewegen in het continue spanningsveld tussen de aantrekkingskracht
 van het onderzoek en de aandacht voor de belangrijkste mensen om je heen is de grootste
 uitdaging gebleken waarmee ik in mijn promotie-onderzoek geconfronteerd ben. De
 meeste dank ben ik verschuldigd aan de mensen die me geholpen hebben hierin de goede
 weg te vinden;

 Allereerst mijn ouders ,
 Dank voor jullie voortdurende en onvoorwaardelijke steun door de jaren heen. Jullie
 zijn erin geslaagd mij de kansen te geven die jullie nooit hebben gehad zonder mij met
 verwachtingen te belasten. Een bezoek aan jullie vormt voor mij altijd een terugkeer naar
 de wortels en het perspectief van alledag. Een warme deken. Mijn zus: lieve Doreen, ook
 jou ben ik hiervoor dankbaar.

 Mijn paranimfen,
 Tijd met jullie doorbrengen is waarschijnlijk hetgeen wat het meest heeft geleden onder de
 onderzoeksperikelen en zeker een van de dingen die ik het meest heb gemist. Gelukkig
 zijn jullie nooit te beroerd gebleken me op subtiele en zonodig minder verfijnde wijze aan
 te geven wanneer ik te lang uit het zicht verdwijn. Deze directheid beschouw ik als het
 belangrijkste teken van de vitaliteit van onze vriendschap.

 Lieve Karin,
 Niemand kent de hoogte- en dieptepunten bij de totstandkoming van dit proefschrift beter
 dan jij. Door deze momenten met jou te delen hebben ze eeuwigheidswaarde verkregen.
 Dank voor je liefde, geduld, begrip, steun, lach en een prachtige dochter.

 Lieve Emma,
 Dit proefschrift zal zeker jouw goedkeuring hebben want het behoort tot je favoriete genre;
 het is namelijk een boek “met letters”. Tegen de tijd dat je het kunt lezen en begrijpen zul je
 het op de juiste waarde weten te schatten: Veel over weinig en dan nog lang niet compleet.
 De meeste dingen zijn relatief. Jij vormt daarop de belangrijkste uitzondering.


                                                                                                                    177
CURRICULUM VITAE
_____   CURRICULUM VITAE _______________________________________________________________________________________________




180
________________________________________________________________________________________________   CURRICULUM VITAE _____



 De auteur van dit proefschrift werd op 7 juni 1968 geboren te Veghel. Na afronding van het
 Gymnasium aan het Mgr. Zwijsencollege te Veghel werd in 1986 aangevangen met de studie
 Geneeskunde aan de Universiteit Utrecht. Gedurende de co-schappen deed hij onderzoek
 naar de rol van ABC- transporters in hematologische maligniteiten (dr. H.M. Lokhorst,
 afdeling Hematologie, Academisch Ziekenhuis Utrecht). In 1995 werd het artsexamen
 behaald waarna hij achtereenvolgens werkzaam was als arts-officier bij de Koninklijke
 Marine, arts-assistent Interne Geneeskunde (AGNIO) in het St. Antoniusziekenhuis te
 Nieuwegein en arts–assistent Klinische Microbiologie (AGIO) aan het Eijkman- Winkler
 Instituut voor Medische Microbiologie (opleider Prof. dr. J. Verhoef).
 In april 1999 werd de opleiding tot internist aangevangen aan het UMCN “St. Radboud”
 (opleider Prof. dr. J.W.M. van der Meer) in combinatie met het onderzoek zoals beschreven
 in dit proefschrift. Het onderzoek werd mede gefinancierd door een persoonlijke beurs
 verkregen van de Nederlandse Kankerbestrijding- Koningin Wilhelmina Fonds.
 In 2005 vond registratie tot internist plaats en werd de opleiding tot internist-hematoloog
 aangevangen (opleider Prof. dr. T.J.M. de Witte). Sinds 2006 is hij verantwoordelijk voor het
 onderzoeksprogramma “ De rol van ABC-transporters in de biologie en chemoresistentie
 van normale en maligne stamcellen” op de afdeling Hematologie van het UMCN “St
 Radboud”. Hij woont samen met Karin van Oijen en is de trotse vader van Emma Felicia.
 Begin 2006 wordt hun tweede kind verwacht.




                                                                                                                     181
EPILOGUE
_____   EPILOGUE ________________________________________________________________________________________________________




184
________________________________________________________________________________________________________   EPILOGUE _____



The cover illustration: on the analogy between the honeybee
queen and the hematopoietic stem cell

“Inter omnia insecta principatus apibus, et jure praecipua admiratio. Pliny. Lib. 11.c.5

The cover illustration of this thesis shows the queen of the Western Honey bee (Apis
mellifera). Apis mellifera, unlike many other insects, has taken remarkable steps along
the evolutionary road from a purely solitary existence to the highly developed colony
organisation exhibited by the honeybees today. The hierarchy of the honeybee society
resembles that of the human bone marrow and interesting parallels exist between the role
of the queen in the honeybee hierarchy and the hematopoietic stem cell in human bone
marrow.

The honey bee queen and “stem cell” characteristics
A typical bee colony consist of about 40.000-50.000 bees. All these bees are the progeny
of a single queen. (which actually resembles the stem cell frequency in human normal bone
marrow that is estimated on 1-10.000-100.000 mononuclear cells). This progeny consists
of differentiated bees, i.e. both worker bees and drones (which sole function is fertilizing the
queen). In this respect the queen resembles one of the key characteristics of hematopoietic
stem cells, i.e. the ability to give rise to progeny capable of multilineage differentiation. In
contrast to the hierarchy in human bone marrow were different functions are performed
by distinct terminally differentiated cells, worker bees perform differentiated activities (and
congruent phenotypic changes) during subsequent stages with increasing age.
In addition to multilineage differentiation, the honeybee queen possesses abilities that
resemble the capacity for self-renewal and engraftment, characteristics that define the
hematopoietic stem cell; the queen is able to a form of “self-renewal” by laying eggs in so-
called queen-cells that are subsequently bred into new queens on a special diet provided
by worker bees. Each year about half a dozen queens are bred in case the queen leaves
the hive to start another colony (“swarming” of the bees). The queen that has left the colony
is able to “engraft” a new colony, although this is only possible when about half the bees of a
colony join her (“accessory” bees) to establish a new nest (“micro-environment) to facilitate
the engraftment. Interestingly, the fate of bee precursors (i.e. larva differentiation towards
worker bees and new queens) essentially relies on micro-environmental circumstances
such as the kind of cell an egg is laid in (worker cell vs. queen cell) and the amount and
quality of food that is presented to the larva. Recent evidence in HSC suggests that the
faith of early hematopoietic progenitors is similarly determined by the stem cell “niche”
i.e. direct environmental factors, in addition to predefined (epi)- genetic differentiation
programs found in hematopoietic precursors.




                                                                                                                     185
_____   EPILOGUE ________________________________________________________________________________________________________



Survival of the honey bee queen
One of the characteristics of a hematopoietic stem cell is that it is long-lived. This is
paralleled in the bee-hierarchy in which the life expectancy of a bee queen is about 4-5
years (the age is marked on their abdomen by bee-keepers with a spot of a particular
colour, in case of the front cover red, indicating a 2-year old queen). This life span strikingly
exceeds the lifespan of its progeny with drones living about 4-5 weeks and workers 4-
weeks to several months depending on the season in which it emerges from the brood-
cell. The mechanisms by which queen bees, and hematopoietic stem cells for that matter,
survive for extended periods of time remain largely to be elucidated but the queen honey
bee has become so highly specialized in creating progeny that it is quite unable to collect
food for herself. She cannot, therefor, survive for very long on her own and is dependent on
its environment (nourishment by worker bees among other factors) for survival. This was
recognized even by the first beekeepers as evidenced by their accounts;

              “A bee, like man cannot live alone, if she be alone she dies. Bees
              conserve community unto their last. They express if not great reverence,
              yet I am sure great love to their Commander”. Samuel Purchas (1657).
              A theatre of Political Flying-insects.

The dependence on external factors for survival resembles the hematopoietic stem cells
that cannot be cultured and maintained without the support of stromal layers and other
bone marrow cells that create the required microenvironment and produce numerous
growth factors required for stem cell survival ex vivo. Mounting evidence suggests that,
similarly, in vivo, hematopoietic stem cells are dependent on the appropriate survival
signalling factors from their microenvironment (stem cell niche). This is of key importance
to realize when examining intrinsic mechanisms of resistance and survival of both normal
and malignant stem cells alike.
So what do we learn from all this? Clearly, the differences outweigh the similarities when
comparing bees with bone marrow cells. The variety in the principle organisation of
complex networks, however, tends to be limited and follow repetitive patterns at molecular,
cellular, multicellular, organism and social levels. It is worth taking notice of our natural
environment and extrapolating knowledge in “simpler” systems to obtain novel insights in
more complex ones. If not for this, the wonderful experiments performed by the zoologist
Karl von Frisch(1) in order to gain insight in the communication of bees (deciphering the
famous “dances of the bees”), for which he was awarded the Nobel Prize in 1973, are a
masterpiece of empirical research and should be considered obligatory literature for any
researcher, especially those trying to unravel the cross-talk between stem cells and other
components of the complicated society called the human bone marrow.




186
________________________________________________________________________________________________________   EPILOGUE _____



Literature
1.    Karl von Frisch. The dancing bees. An account of the live and senses of the honeybee. Methuen & Co. LTD.
      London, 1954
2.    Colin G. Butler. The new naturalist. The world of the honeybee. Collins, London 1954.




                                                                                                                     187
___________________________________________________________________________________   APPENDIX: COLOR ILLUSTRATIONS _____



APPENDIX: COLOR ILLUSTRATIONS
Chapter 4

                              NBM




                                                                    CD34
                                                                            SSC
                     CD34




                            CD38




                              AML
                                                                    CD34




                                                                            SSC
                     CD34




                            CD38

Figure 1: Definition of CD34+38- hematopoietic cells in normal bone marrow and acute myeloid leukemia.
Representative example of normal bone marrow (NBM) and acute myeloid leukemia (AML). CD34+38- cells were
defined flow-cytrometrically as the CD34-FITC+ cells (indicated in blue) with CD38-PE expression within the first
decade of fluorescence emission (indicated in red) and compared with CD34+38+ cells (indicated as blue gated)
with exclusion of a decade between CD38- and CD38+ cells. CD34- cells are shown in gray. CD34+38- cells
exhibited restricted light-scattering characteristics (SSC) confirming the lymphoid appearance of these cells (inset).
The median frequency of CD34+38- cells was 0.1 % of mononuclear cells (MNC) (range 0.1%-0.3%) in NBM and 0.2
% of MNC (range 0.1%- 10%) in AML. No difference existed in average CD38 density between normal and leukemic
CD34+38- cells (MFI 0.57 ± 0.05 SD, range 0.40-0.70 and MFI 0.53 ± 0.12 SD, range 0.30-0.70).




                                                                                                                     189
                                         MITOXANTRONE
                                                                                                             12.00
                                                                                                                         No inhibitor




                                                                                            MITOXANTRONE
                                                                          No inhibitor                                   Verapamil
                                                                                                              8.00
                                                                          Verapamil




                                                        EVENTS
                                                                                                              4.00
                                                                           PSC833
                                                                                                              0.00
                                                                  MITOXANTRONE                  A2                         34+38-       34+38+
                                                         A1

                                                          NO INHIBITOR    VERAPAMIL                          12000
      CD34




                                                                                             RHODAMINE-123
                                                                                                              9000

             CD38                                                                                             6000

                                                                                                              3000

                                                                                                                    0
                                                        B1        RHODAMINE-123               B2                        34+38-           34+38+

Figure 3: ABCB1 is the major mitoxantrone and rhodamine-123 transporter in hematopoietic CD34+38-
cells in human normal bone marrow. A1 Representative example of flow-cytometric assessment of ABCB1
mediated mitoxantrone transport in CD34+38- cells in NBM. CD34+38- cells display low intracellular mitoxantrone
fluorescence in comparison to CD34+38+ cells (inset) which can be increased significantly by blockage of ABCB1
mediated transport by verapamil/PSC833. A2 Average mitoxantrone fluorescence (n=16) (depicted as mean
fluorescence intensity (MFI) in CD34+ cells in the absence (shaded bars) or presence (black bars) of verapamil.
B1 Representative example of the effect of ABCB1 inhibition by verapamil on rhodamine-123 fluorescence in
CD34+38- cells assessed by single cell image analysis. B2. Average values ± S.D. of rhodamine-123 fluorescence
(n=5)(depicted as arbitrary units) in the absence (shaded bars) or presence (black bars) of verapamil. Rhodamine-
123 accumulation was significantly lower in CD34+CD38- cells compared to CD34+CD38+ cells (rhodamine-123
fluorescence 2820 ± 501 AU vs. 9369 ± 2367 AU). Blockage of ABCB1 mediated transport significantly increases
rhodamine-123 accumulation in CD34+38- cells.

                                                                                                          12.00
                                       MITOXANTRONE




                                                                                                                        No inhibitor
                                                                                          MITOXANTRONE




                                                                                                                        Verapamil
                                                                           No inhibitor
                                                                                                             8.00

                                                                           Verapamil
                                                        EVENTS




                                                                                                             4.00
                                                                           PSC833
                                                                                                             0.00
                                                                                           A2                           34+38-          34+38+
                                                          A1     MITOXANTRONE
                                                                                                      12000
                                                           NO INHIBITOR    VERAPAMIL
                                                                                          RHODAMINE-123
      CD34




                                                                                                             9000

                                                                                                             6000
              CD38
                                                                                                             3000

                                                                                                                0
                                                                                                                        34+38-          34+38+
                                                        B1        RHODAMINE-123           B2

Figure 5: ABCB1 transport is impaired in hematopoietic CD34+38- cells in acute myeloid leukemia. A1
Representative example demonstrating increased intracellular mitoxantrone accumulation in comparison to
CD34+38- cells from normal bone marrow due to impaired ABCB1 mediated transport in CD34+38- cells illustrated
by a lack of effect of verapamil/PSC833 on intracellular mitoxantrone fluorescence. A2. Average mitoxantrone
fluorescence (depicted as mean fluorescence intensity (MFI) in CD34+ cells in the absence (shaded bars) or
presence (black bars) of verapamil. B1. Representative example of impaired ABCB1 mediated rhodamine-123
extrusion from CD34+38- cells, illustrated by the lack of effect of ABCB1 inhibition by verapamil on intracellular
rhodamine-123 fluorescence assessed by single cell image analysis. B2. Average values ± S.D. Rhodamine-123
fluorescence was significantly lower in CD34+CD38- cells compared to CD34+CD38+ cells (3106 ± 267 AU vs.
4594 ± 373 AU) but blockage of ABCB1 by verapamil has no effect on rhodamine-123 fluorescence.
                                 A               ALLOGENEIC                           EI=2.30

                                                                                     No inhibitor
                                                                                     Verapamil


                                                 AUTOLOGOUS                           EI=1.48




                                 CD34
                                                                                     No inhibitor
                                                                                     Verapamil


                                                 CD38                         MITOXANTRONE


                                  B
                                                 RA                                    EI=2.23

                                                                                      No inhibitor
                                                                                      Verapamil



                                                 RA-AML                                EI=1.24
                                  CD34




                                                                                      No inhibitor
                                                                                      Verapamil


                                                                             MITOXANTRONE
                                                  CD38

Figure 8: Representative examples of ABCB1 mediated mitoxantrone efflux in CD34+38- cells from
autologous and allogeneic transplanted cells constituting normal hematopoiesis in long-term remission,
refractory anemia (RA), refractory anemia progressing towards AML (RA-AML).


                                                          No inhibitor

                                                          Verapamil
                      CD34+38-
                      ABCB1 efflux ±
                                                                ABCB1
                                                                EI=1.48

                                                                                  GAPDH                CBFB-MYH11
                                                                                  Ct 30.13               Ct 38.27

                                                                             Xn 4.3 10 -3 Copies CBFB-MYH11/copy GAPDH
                                  MITOXANTRONE
       CD34




                                                                                     CBFB-MYH11 not detectable

                                                                                 GAPDH                 CBFB-MYH11
              CD38                                CD34                           Ct 29.74                Ct >45

                                                              No inhibitor

                                                              Verapamil
                     CD34+38-
                     ABCB1 efflux ++

                                                                 ABCB1
                                                                 EI=2.50

Figure 9: ABCB1 mediated mitoxantrone transport identifies a subpopulation that harbors residual
normal CD34+38- cells in a patient with CBFB-MYH11 AML. A patient was identified in which mitoxantrone
discriminated a population of CD34+38- mitoxantrone dull cells. The dull phenotype was determined by strong
ABCB1 mediated efflux (CD34+38- ABCB1 efflux ++), within the range observed in CD34+38- cells observed in
normal bone marrow (EI=2.50). The CBFB-MYH11 (type D) transcript could be detected at the level of the positive
control ( blast population from CBFB-MYH11 positive patient; Xn = 5.1. 10-3 copies CBFB-MYH11/Copy GAPDH)
in the CD34+38- mitoxantrone bright population. CBFB-MYH11 transcripts were not detected in the CD34+38-
ABCB1 efflux ++ population.
Chapter 5
                                                                          IgG2a                     No inhibitor
                                                                          BXP21                     KO143 (0.1uM)

                       A1                           B1                            C1                               D1




                            MITOXANTRONE




                                                          EVENTS
CD34




       CD38                                CD34                    FITC                  MITOXANTRONE

                       A2                           B2                            C2                               D2




                       A3                           B3                            C3                               D3




Figure 2 :Flow-cytometric assessment of BCRP expression and BCRP mediated mitoxantrone efflux in
CD34+38- hematopoietic in normal bone marrow and AML. A.Definition of CD34+38- hematopoietic cells in
human normal bone marrow (NBM) and AML. CD34+38- cells were defined flow-cytrometrically as the CD34
expressing cells (indicated in blue) with CD38 expression within the first decade of fluorescence emission
(indicated in red) and compared with CD34+38+ cells (indicated as blue gated) with an exclusion of a decade
between CD38- and CD38+. CD34- cells are shown in grey. CD34+38- cells were found invariably in both normal
bone marrow (A1) and both CD34+ (> 10% CD34+ cells in bone marrow by definition, A2) and CD34- leukemias
(A3). The median frequency of CD34+38- cells was 0.1 % of mononuclear cells in normal bone marrow (range
0.1%-0.3%) and 0.2 % in acute myeloid leukemia (range 0.1%- 10%). B. Cellular mitoxantrone fluorescence
in different cell populations after 2 hrs incubation with mitoxantrone (10 uM) in the absence of BCRP inhibitor.
CD34+38- cells have a mitoxantrone “dull” phenotype. C. BCRP protein expression in CD34+38- cells as
determined by the BXP-21 antibody (dotted line) vs isotype control. D. Mitoxantrone fluorescence in CD34+38-
cells in the presence (dotted line) or absence of the BCRP specific inhibitor KO143 (0.1 uM)

								
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