The human erythroleukemia K562 cell culture system for identification by pgb16892

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									                                                                                                                MINERVA BIOTEC 2003;15:123-8




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                   The human erythroleukemia K562 cell culture system
                       for identification of inducers of fetal hemoglobin




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                                                          O
                                                                                                                         R. GAMBARI 1, 2




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                                                                                 ED
The human leukemic K562 cell line has been proposed                                 1Department  of Biochemistry and Molecular Biology,
as a useful in vitro model to study the molecular mech-                                              University of Ferrara, Ferrara, Italy,
                                      O
anism(s) regulating the expression of the human                                     2Laboratory for the Development of Pharmacological
embryonic and fetal globin genes, as well as to deter-                                  and Pharmacogenomic Therapy of Thalassemia,
mine the therapeutic potential of new differentiation-                                                              Biotechnology Center,
inducing compounds. This cell line exhibits a low propor-                                             University of Ferrara, Ferrara, Italy
                                                                           M
tion of hemoglobin (Hb) synthesizing cells under stan-
dard cell growth conditions, but it is capable of undergo-
ing erythroid differentiation when treated with a varie-
ty of compounds, such as hemin, cytosine arabinoside                       of various agents able to upregulate the expression
(ara-C), butyric acid, 5-azacytidine, chromomycin and                      of the human γ-globin genes, such as hormones,
                                                   VA


mithramycin, tallimustine, cisplatin and cisplatin analogs.                cytotoxic drugs, hemopoietic cytokines and short fat-
                  R



Following erythroid induction of K562 cells, Hb Portland
(ζ2γ2) and Hb Gower 1 (ζ2ε2) accumulate, due to increase                   ty acids as molecules capable of augmenting HbF
in the expression of human ζ-, ε- and γ-globin genes.                      levels in humans.4-13
K562 cells are suitable for identifying inducers of fetal                     In this respect, the availability of a cellular model
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hemoglobin (HbF). However, after a preliminary screen-                     system for the rapid screening of potential HbF induc-
ing, the molecules able to induce erythroid differentia-                   ers could be of great interest.10, 14, 15 Among cellular
tion of K562 cells should be tested in the 2-phases culture                systems available for the screening of drugs able to
systems of human erythroid precursors from peripher-                       induce erythroid differentiation and possibly involved
al blood, to obtain more informative results on the effects
                                                                           in reactivation of HbF production, the human eryth-
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of inducers on HbF production.
                                                                           roleukemia K562 cell line has been largely exploited.
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KEY WORDS: K562 cells - β-thalassemia - Fetal hemoglobin -
Ara-C - Cytosine arabinoside.
                                                                              This cell line has been indeed proposed as a use-
                                                                           ful in vitro model to study the molecular mecha-
                                                                           nism(s) regulating the expression of embryonic and
I ncrease of fetal hemoglobin (HbF) leads to a signif-                     fetal human globin genes,16-20 as well as to determine
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  icant improvement of the clinical status of patients                     the therapeutic potential of new differentiation-induc-
affected by these hematological disorders, including                       ing compounds.21-28
β-thalassemia and sickle cell anemia.1-3 Therefore,                           The K562 cell line has been isolated and character-
several studies have been focused on the screening                         ized by Lozzio and Lozzio 29 from a patient with chron-
  This work was supported by Associazione Veneta per la Lotta alla
                                                                           ic myelogenous leukemia (CML) in blast crisis. This cell
Thalassemia (Rovigo).                                                      line exhibits a low proportion of Hb-synthesizing cells
                                                                           under standard cell growth conditions, but it is capable
                                                                           of undergoing erythroid differentiation when treated
   Address reprint requests to: Prof. R. Gambari, Department of            with a variety of compounds, as demonstrated by
Biochemistry and Molecular Biology, Via Luigi Borsari 46, 44100 Ferrara,
Italy. E-mail: gam@unife.it                                                Rutherford et al. in 1979,16 studying the effects of



Vol. 15 - No. 2                                             MINERVA BIOTECNOLOGICA                                                     123
GAMBARI                                                                                                      THE HUMAN ERYTHROLEUKEMIA K562 CELL SYSTEM




                                                                                             roid-induced K562 cells originate a red pellet, indicat-
      K562 cells                                                    100
                                                                                             ing accumulation of large amounts of Hb. Great advan-




                                                                                                          A
      Left: control
      Right: erythroid induced                                                               tages of this system are reproducibility of the induc-
                                                                                             tion process and availability of easy detection proto-
                                                                                             cols. Human erythroleukemia K562 cells are usually




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                                                                     75
                                                                                             cultured in humidified atmosphere of 5% CO2 in RPMI
                                     Benzidine-positive cells (%)



                                                                                             medium supplemented with 10% fetal bovine serum,
                                                                                             50 units/ml penicillin and 50 µg/ml streptomycin.33
                                                                                             Weekly medium changes are required to sustain in
                                                                     50                      vitro cell growth. Treatment of K562 cells with induc-




              D
                                                                                             ers is carried out by adding the appropriate drug con-
                                                                                             centrations at the onset of the cell cultures (cells are
  Erythroid induced K562 cells                                                               seeded at 30 000 cells/ml). The medium, usually, is not
                                                                     25                      changed during the induction period.31-34
         GH E                                                                                   Figure 2 shows the most used approach to follow
                                                                                             erythroid induction of K562 cells. The first is the ben-



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                                                                                             zidine-assay. Briefly, a benzidine-H2O2 solution is
            M
                                                                      0                      freshly prepared and used to identify benzidine pos-
                                                                                             itive (Hb containing) K562 cells.27 To this aim, 5 µl of
                                                                          2    4     6   8
            T
Figure 1.—Pellets of uninduced and erythroid induced K562 cells. Only
                                                                              Days
                                                                                             a K562 cell suspension is diluted 1:1 with ice-cooled
                                                                                             benzidine solution and K562 cells assuming a blue-
                                                                                             stain are counted under a microscope. This assay is
pellets from induced cells are red, indicating Hb production. In the
          A

right side of the panel, the kinetic of the increase of benzidine-positive,                  performed daily. Figure 2A shows a comparison
hemoglobin-containing cells is shown in K562 cells treated with ara-C.                       between a control K562 cell population (up) and
                                                                                             erythroid-induced K562 cells (down). Figure 1 (right
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                                                                                             side of the panel) shows a typical kinetic of appear-
hemin. In this and closely related papers,16-18 it was
                                                                                             ance of benzidine positive cells. Monitoring cell num-
demonstrated that hemin causes no major changes
                                                                                             ber/ml is at this stage important, in order to deter-
in the rate of cell growth of K562 cells but induces a
                                                                                             mine activation of terminal stages of differentiation,
reversible accumulation of embryonic and fetal hemo-
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                                                                                             involving block of cell growth.27
globins. The cells can be indefinitely subcultured in the                                       Following erythroid induction of K562 cells, Hb
presence of the inducer. Thus, the control of hemo-                                          Portland (ζ2γ2) and Hb Gower 1 (ζ2ε2) accumulate,
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globins production in K562 cells does not depend on                                          due to increase in the expression of human ζ-, ε- and
irreversible differentiation.16-18 Since then, a large num-                                  γ-globin genes. This is usually monitored by cellu-
ber of papers were published describing inducers of                                          lose-acetate gel electrophoresis (Figure 2B).15-27
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erythroid differentiation, such as cytosine arabino-                                            Northern blotting (Figure 2C) and RT-PCR (Figures
side (ara-C),30 butyrates,31 5-azacytidine,32 chromo-                                        2D, E) are used to determine the effects of erythroid
mycin and mithramycin,15 tallimustine,27, 33 cisplatin                                       inducers on the expression of globin genes (examples
and cisplatin analogs.34 Unlike hemin, most of these                                         of the results obtained are shown in Figure 2). These
inducers inhibit cell growth and activate terminal cell                                      experiments conclusively demonstrated that K562 do
division of K562 cells, as demonstrated by culturing                                         not express the adult β- and δ-globin genes and, with-
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K562 cells in semi-solid medium. In these experimen-                                         in α-like globin genes, the ζ-globin genes are the
tal conditions, large-sized colonies are not differentiat-                                   most actively transcribed genes.
ed, while small-sized colonies contain large amounts                                            In conclusion, this cell line appears to be commit-
of Hb.                                                                                       ted to differentiate along the embryo-fetal erythroid dif-
                                                                                             ferentiation pathway.
  Features of erythroid induction of K562 cells                                                 However, K562 cells express also HbF, particularly
                                                                                             when induced to differentiation by certain molecules,
  Figure 1 shows pellets from uninduced and eryth-                                           including butyrates,28 apicidin 25 and valproate.26 This
roid induced K562 cells. As clearly evident, only eryth-                                     feature could be of interest for the development of




124                                                                              MINERVA BIOTECNOLOGICA                                        June 2003
THE HUMAN ERYTHROLEUKEMIA K562 CELL SYSTEM                                                                                        GAMBARI




                                                                                       A
                                         Induced differentiation of
                                                K562 cells




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      A            Benzidine-test                                                        Analysis of Hb by                         B
                                                                                          cellulose acetate




              D
                                                                                         gel electrophoresis

                                                                                                           Hb Portland
         GH E                                                                                              Hb A
                                                                                                           Hb F



                                                                                 ®
                                                                                                           Hb Gower 1
            M
                                                     RNA
            T                                                                                              wells line
          A

                       Northern blotting                                                    Quantitative RT-PCR

     C            γ-globin
                                                                                                                                   E
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                                                                                 10
                   probe
                                                                           ∆Rn
                                                                                  -1
                                                                                 10
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                                                                                  -2
                                                                                 10
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                  actin                                                                 4   8   12 16   20 24 28   32   36   40
                  probe                                                                                 Cycle
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                                                                                 10

                                                                           ∆Rn
                                                                                  -1
                                                                                 10
      D                      RT-PCR
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                                                                                  -2
                                                                                 10




                                                                                        4   8   12 16   20 24 28   32 36     40
                                                                                                        Cycle



Figure 2.—Morphological and molecular biology assays to study erythroid differentiation of K562 cells. The benzidine test (A) and the elec-
trophoresis on cellulose-acetate gels (B) give informations on the proportion of K562 cells induced to erythroid differentiation and on the
type of Hb produced. The increase of signal in Northern blotting (C) and RT-PCR (D) assays indicates an increase of production of γ-globin
mRNA. Minor changes are observed studying actin mRNA. Quantitative RT-PCR analysis (E) gives informations on the fold induction of γ-
globin mRNA.




Vol. 15 - No. 2                                         MINERVA BIOTECNOLOGICA                                                         125
GAMBARI                                                                        THE HUMAN ERYTHROLEUKEMIA K562 CELL SYSTEM




HbF inducers useful for treatment of β-thalassemia             under the control of the human γ-globin gene promot-
and sickle cell disease. For instance, Witt et al.28 recent-   er. In such experiments, K562 cells are very useful. For




                                                                           A
ly reported a study on 14 butyrate derivatives and             instance, Hudgins et al.,22 in order to verify whether
retinoic acid with respect to production of HbF by             stimulation of an element in the human γ-globin pro-
K562 cells. Among these compounds, 4 novel butyrate            moter by phenylbutyrate and its analogues might con-




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derivatives with HbF-inducing activity were identi-            tribute to their mechanism of action, used a transient
fied. Combined treatment with the butyrate deriva-             expression system involving K562 cells transfected
tive tributyrin and retinoic acid in vitro led to a 7-         with a luciferase reporter gene driven by the mini-
fold increase of hemoglobin synthesis, allowing the            mum γ-globin gene promoter. Transcriptional activa-
conclusion that tributyrin and retinoic acid might be          tion in this experimental system correlated well with




              D
promising drugs for clinical trials to treat patients with     the capacity of an aromatic fatty acid to stimulate HbF
β-hemoglobinopathies.28                                        production in erythroid precursors.22
                                                                  In a more recent study, Vadolas et al.24 described the
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        Erythroid induction of K562 cells
      and ability to stimulate HbF production
                                                               development of enhanced green fluorescence pro-
                                                               tein (GFP) reporter systems based on bacterial artifi-
                                                               cial chromosomes to monitor the activity of the


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                                                               ε-, Gγ-, Aγ-, β- and δ-globin genes in the β-globin locus.
            M
           in erythroid precursor cells
                                                               Their study shows relevant differences in the expres-
   In vitro studies demonstrated that known inducers
            T                                                  sion of each of the globin genes. In agreement with
of erythroid differentiation in K562 cells, such as            data on direct induction of K562 cells to erythroid
hydroxyurea,8, 35 butyrates,31 5-azacytidine,32 mithram-       differentiation, they demonstrated that hemin is a
ycin,36 are also capable of inducing HbF production            potent inducer of GFP expression from GFP-modi-
          A

when administered singularly or in combination to              fied ε-, Gγ-, Aγ-globin constructs.
normal erythroid cells. This feature is shared by most
of the erythroid inducers of K562 cells. One recently
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published example is valproate, a molecule which                          Limits of the K562 cell system
has been found to stimulate HbF synthesis in patients
with sickle cell disease. In agreement with these data,           We have already pointed out that K562 cells are
Witt et al. demontrated HbF induction in K562 cells.26         not suitable to study modulation of the expression of
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   Despite the fact that many inducers of K562 cell            the β- and δ-globin genes. For instance, the use of
differentiation are able to stimulate at high levels HbF       GFP-modified β- and δ-globin gene constructs in trans-
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production in normal erythroid precursor cells, impor-         fection experiments using K562 cells consistently pro-
tant exceptions have been described, such as for               duced very low levels of GFP expression after hem-
instance ara-C, the most potent inducer of erythroid           in induction, demonstrating that this differentiation
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differentiation of K562 cells, which does not stimulate        system mimics only embryo-fetal erythroid differen-
HbF production in normal erythroid precursor cells.            tiation.24 On the other hand, expression of the β-glo-
Of interest is the fact that the K562 cells inducers           bin gene is very important to assess for a true prefe-
butyric acid, hydroxyurea and 5-azacytidine have               rential induction of expression of γ-globin genes by
been already useful for the treatment of experimen-            potential HbF inducers.
tal animal systems and β-thalassemia patients.1, 7, 37-39
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                                                                  As far as the overall gene expression is concerned,
                                                               it should be pointed out that the K562 cell line was
                                                               derived from a patient with CML. Several chromoso-
          Advantages of the K562 cell system                   mal abnormalities are present in the karyotype of
                                                               K562 cells, including the presence of a Philadelphia
  The most important advantages of the K562 cell               chromosome carrying a 9/22 translocation, with an
system are reproducibility of the results obtained and         amplification of the bcr/abl sequences. Therefore,
simplicity of the assays to be performed to study              this system should not be considered as a “normal” dif-
erythroid induction. This system has been also                 ferentiation system, but simply a model for a 1st drug
employed for screening studies using reporter genes            screening.




126                                              MINERVA BIOTECNOLOGICA                                          June 2003
THE HUMAN ERYTHROLEUKEMIA K562 CELL SYSTEM                                                                                                   GAMBARI




                                                                              inducers. Following this preliminary screening, the
      Putative HbF inducer
                                                                              molecules able to induce erythroid differentation of




                                                                                              A
                                                                              K562 cells should be tested in the 2-phases culture
                                    Identification of molecules
                                                                              systems of human erythroid precursors from periph-
                                       inducing erythroid
     First screening on                                                       eral blood.14, 41, 42 Only this system allows to obtain
                                          differentiation




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         K562 cells                  and increasing γ-globin
                                                                              conclusive informative results on the effects of induc-
                                      mRNA accumulation
                                                                              ers on HbF production.
      Cell growth
      Erythroid differentiation
      Hemoglobin production

      mRNA accumulation                                                                                   References




              D
      (Northern blotting)
                                  Second screening on
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                                                                                       ®
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   K562 cells will be employed in the next future to test                         et al. Effect of hydroxyurea on the frequency of painful crises in
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  PY RV


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      RI


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   IN




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Vol. 15 - No. 2                                                   MINERVA BIOTECNOLOGICA                                                          127
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              D
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            M
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          A

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128                                                           MINERVA BIOTECNOLOGICA                                                            June 2003

								
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