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					                         Mitochondria in the Tumourigenic Phenotype



                                       By Bertrand C. Liang

                                 Copyright 2011 Bertrand C. Liang

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Table of Contents

    Acknowledgements
    List of Publications
    List of Figures
    Monograph Abstract
    Introduction: the Tumourigenic Phenotype
    Investigation of copy number alterations of mitochondrial DNA in human gliomas,
    including investigations of mitochondrial DNA as a transposable element
    Evaluation of the biology of mitochondrial alterations in the neoplastic phenotype,
    including the effect of removing mitochondrial DNA (creation of rho- tumor cells) and
    evaluation of the ontogeny of mitochondrial membrane potential changes in a model of
    neoplastic transformation
    Evaluation of the mechanism and potential of inhibition of mitochondrial function in
    effecting tumour cell death as a transition to approaches to the treatment of patients with
    cancer
    Discussion: Mitochondria and Cancer
    A Model of the Tumourigenic Phenotype
    Monograph Bibliography
    About the Author



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Acknowledgments


    As is the usual case, the generous support received for this work cannot be adequately
    conveyed in a short page of gratitude. Mentors, colleagues, and friends, both new and old,
    in the scientific, clinical and industrial worlds, are far too numerous to list individually,
    but of whom I hope understand my significant indebtedness to them, as well as my most
    heartfelt thanks.
    My sincere appreciation to Professor Martin Grootveld, Dr. Richard Iles, Dr. Neil Morgan
    and Dr. Subhash Anand MBE, who took time out of very busy schedules to be part of this
    work; grateful thanks.
    My most significant note of thanks is to my supportive family –Christopher A. Liang,
    Kate M. A. Liang, Bryan A. Liang, Anita C. Liang and Anna R. Winn – and particularly,
    my loving and understanding wife, Diane M. Liang - who appreciate not only the value of
    education, but the rationale of life-long learning, both as example and beneficiary. Finally,
    to my father, Charles C. Liang, PhD, a victim of cancer at too young an age – a mentor,
    and in later life, a dear friend; I would have very much liked for him to both understand
    this work and see this day, for I miss him and his wise scientific insight and counsel very,
    very much.



                                                ***




                                         TO MY FATHER
               “It does not matter how slowly you go, so long as you do not stop.”




                                                ***
List of Publications


     BC Liang. Evidence for association of mitochondrial DNA sequence amplification and
     nuclear localization in human low grade gliomas. Mutation Research, 354:27-33, 1996.
     BC Liang, Hays L. Mitochondrial DNA copy number changes in human gliomas. Cancer
     Letters, 105:167-173, 1996.
     L Cavalli, Varella-Garcia M, Liang BC. Diminshed tumorgenic phenotype achieved by
     depletion of mitochondrial DNA. Cell Growth and Differentiation, 8:1189-1198, 1997.
     BC Liang, Ullyatt E. Chemosensitization of glioblastoma cells to
     bis-dichloroethyl-nitrosourea with tyrphostin AG17. Clinical Cancer Research, 4:773-781,
     1998.
     LR Cavalli, Liang BC. Mutagenesis, Tumorigenicity, and Apoptosis: Are the
     Mitochondria involved? Mutation Research (Fundamental and Molecular Mechanisms of
     Mutagenesis), 398:19-26, 1998.
     BC Liang, Ullyatt E. Increased sensitivity to cis-diamminedichloroplatinum-induced
     apoptosis with mitochondrial DNA depletion. Cell Death and Differentiation, 5:694-701,
     1998.
     E Ullyatt, Liang BC. 2’3’-dideoxycytidine is a potent inducer of apoptosis in glioblastoma
     cells. Anticancer Research, 18:1859-64, 1998.
     BC Liang, Miller L, A Weller. Ethyl-nitrosourea transformed astrocytes exhibit
     mitochondrial membrane hyperpolarization and constrained apoptosis. Apoptosis,
     4:89-97, 1999.



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List of Figures


     Figure 1. Sequence of extracted cDNA clone.
     Figure 2. Results of fluorescent in situ hybridization (FISH) of the rhodamine-labelled
     mitochondrial genome probe to tumour nuclei.
     Figure 3. Ethidium bromide/acridine orange assay for chromatin condensation.
     Figure 4. Mitochondrial membrane potential (mitochondrial membrane
     potential)/Mitochondrial mass (MM) ratio.
     Figure 5. Effects of oligomycin on U937 ATP levels.
     Figure 6. BCNU dose response.
     Figure 7. A model of the development of the tumourigenic phenotype, including
     mitochondrial involvement.
                                                 ***




Monograph Abstract

     Cancer arises from the accumulation of nuclear and cytoplasmic abnormalities, a
     phenomenon which allows for the expression of the tumourigenic phenotype. The studies
     documented herein have evaluated the role of mitochondria in this phenotype. An
     increased copy number of mitochondrial DNA (mtDNA) was observed, along with
     changes in the expression of cancer cell lines and primary tumour tissue. Such
     amplification was associated with mtDNA within the nucleus, and in low grade
     (ontologically-early) tumours. Tumour cell lines were depleted of mtDNA, creating “rho-”
     cells, with minimal mtDNA/functional mitochondria. While rho- cells retained
     immortality, there was a loss of anchorage-independence and xenograft growth; moreover,
     these cells became more sensitive to the effects of cytotoxic agents. When normal
     mitochondria were replaced into the rho- cells, the cells reverted to the parental
     phenotype, including amplification of mtDNA. To further evaluate the early onset of
     mitochondrial alterations, normal human astrocytes were exposed to ethyl-nitrosourea, a
     process which created transformed cells. Hyperpolarization of the mitochondrial
     membrane was noted as an early manifestation of transformation, with corresponding
     decreases in mitochondrial mass. The inhibition of the glycolytic enzyme
     phosphofructokinase diminished the development of the tumourigenic phenotype in the
     transformed cells. Tumour cells were treated with mitochondrial DNA-polymerase gamma
     inhibitor 2’,3’-dideoxycytidine (ddC) and tyrphostin AG17; both compounds
     demonstrated an ability to effectively inhibit mitochondrial function and thereby either
     directly, or in combination with other cytotoxic drugs (1,3-bis-dichloroethyl-nitrosourea,
     BCNU), effect apoptosis. Hence, the mitochondrion serves as an important element in the
     tumourigenic phenotype, and clinical approaches targeting this organelle have potential
     for the development of effective treatment regimens for patients with cancer.



                                                 ***




Introduction : the Tumourigenic Phenotype
      Conceptually, understanding the behavior of tumor cells by analysis of the phenotype is an
      important aspect required to develop preventative measures, in addition to treatment
      modalities which are both effective and safe for patients with cancer. This “tumourigenic
      phenotype” has, in the past, been typically evaluated using approaches primarily
     addressing nuclear encoded genes. However, it is clear that the influence of cytoplasmic
     components of the cell also can play a role in the manifestations of malignancy therein. As
     a component of the cytoplasm, the mitochondrion has, in the past, been minimally
     investigated as a source of influence on the tumourigenic phenotype.
     In order to further investigate the role of the mitochondria in this specific phenotype, an
     evaluation of copy number and fidelity of mitochondrial DNA was performed, as well as a
     determination of the role of functional mitochondria (manifest by removing mitochondrial
     DNA and evaluation of mitochondrial membrane potential) and phenotypic alterations
     based on sensitivity to programmed (or other mechanisms) cell death.



                                                 ***




Investigation of copy number alterations of mitochondrial DNA in human gliomas, including
      investigations of mitochondrial DNA as a transposable element


     Cancer arises from the accumulation of DNA and cytoplasmic abnormalities, which
     allows for the expression of a resultant phenotype. This phenotype – the tumourigenic
     phenotype – is manifested by certain elements, such as lack of terminal differentiation,
     immortality, anchorage-independent growth and resistance to cellular death from
     cytotoxic and further inducing stimuli (Cavalli and Liang, 1998). Gene amplification (of
     nuclear genes) is an important aspect of this multistep pathway to the neoplastic
     phenotype, and occurs in most, if not all, tumour types (Liang et. al., 1995). Indeed,
     cytogenetic and molecular cytogenetic data suggest that this genetic finding is an
     intermediate step in the progression of cells in the multistep pathway noted above. Within
     this context, several techniques have been developed to isolate variably expressed and
     amplified genes in human cancer, using brain tumors (gliomas) as a model system
     (correlating with the clinical interest in this tumor type). Using a molecular subtractive
     hybridization approach (Liang, 1995), an amplified cDNA was isolated which revealed
     mitochondrial (cf. nuclear) sequence from a glioblastoma cell line (PFAT-MT), an
     unexpected finding. Whilst mtDNA had been observed to be structurally abnormal in the
     past (Firkin and Clark-Walker, 1979; Gianni et. al., 1980), no studies had noted significant
     changes in copy number. Consequently, further studies were focused on attempts to
     understand the role of mitochondria, and mitochondrial DNA (mtDNA), in the
     tumourigenic phenotype, since the differential expression screen suggested a level of
     amplification and/or expression which differed from that of corresponding normal tissues.
     Of especial note is that the mitochondrial genome is the only known extranuclear genome
     in mammalian cells, and consists of 16,569 base pairs arranged in a circular array, with
     each strand expressed as a single polycistronic message. There are effectively no introns
     in this genome, and all mRNA derived from this genome are transcribed and translated
     within the mitochondria (Scheffler, 1999).
     The first study conducted after the identification of this cDNA fragment (Genbank
Accession MDL U25123, or “U25123”) (Figure 1) involved an evaluation of further
tumour cell lines in addition to primary tumours, in order to determine the frequency of
differential expression and/or amplification.




Both cell lines and primary tumours were evaluated to determine whether this was an in
vitro phenomenon only, or whether this was pathologically important and therefore
detectable at the primary tissue level; moreover, the investigation of different stages of
glioma (low grade – pilocytic tumors, astrocytomas; intermediate grade – anaplastic
astrocytomas; high grade – glioblastomas) was performed to determine the relative
ontogeny of any findings. Indeed, evaluations of the genomic DNA of these tissues
revealed frequent amplification of not only the cell lines, but also the primary tumours; of
much significance was the finding that lower grade tumours also harboured the
amplification of this U25123 sequence, an observation suggesting that this was an early
event in the multistep pathway towards malignancy, in distinct contrast to nuclear DNA
amplification in cancer, which is generally more considered to be an intermediate step.
Correlated with this was expression data in a subset of tumours in which RNA was
available; these revealed an increased level of expression of the cDNA in both lower grade
and cell line samples.
To further investigate the amplification of mtDNA in the glioma tumors and cell lines,
probes from each segment of the mitochondrial genome were created. Interestingly, most
of the mtDNA in the tumours and cell lines revealed an amplification process, although a
segment which included components of the electron transport chain (including ND1 and
ND2, viz. amino acid positions 4121 - 5275) had minimal increases (if any) in copy
number. Indeed, in some cases the minimal hybridization on Southern blot suggested a
deletion of that area of the mitochondrial genome.
These findings – i.e. amplification of the mitochondrial sequence in gliomas, the
ontologically-early identification of amplification and heterogeneous areas of mtDNA
amplification (suggesting deletions in the mitochondrial genome) – extended early
findings of abnormal mtDNA structure in cancer (Clayton and Smith, 1975; Clayton and
Vinograd, 1967) which were previously unreported in the literature. Moreover, the
association with petit mutants in yeast (Ragnini and Fukuhara, 1989; Traven et. al., 2001),
where truncated mtDNA is associated with a dysfunction of the mitochondrion and high
degrees of amplification of the remaining DNA, was a unique insight, as was the
hypothesis that a miscommunication between the nuclear and mitochondrial genomes was
occurring, observations which would have implications in the later studies conducted (see
below).
As a result of the discovery of mtDNA amplification, and the hypothesis that the
pathobiology could recapitulate findings in yeast, the next set of experiments evaluated
tumours with regard to “mtDNA escape”. In yeast, mtDNA escape occurs as a
manifestation of the communication between the nuclear and mitochondrial genome
(Campbell and Thorsness, 1998; Thorsness and Weber, 1996), and has been postulated to
be related to both genetic and environmental control. mtDNA has been found transposed
to the nucleus in cultured cells (HeLa) as well as carcinogen-induced hepatic carcinoma
cells (Shay and Werbin, 1992; Corral et. al., 1989). Indeed, it has been hypothesized that
high copy numbers of mtDNA may be required to transfer mitochondrial sequences from
mitochondrial sources to the nucleus in cancer, a phenomenon similar to that in yeast
(Netter and Robineau, 1989; Liang, 1996).
Given the above finding of mtDNA amplification, an evaluation of the presence of
mtDNA in the nucleus of tumour cells was performed, using interphase nuclei with
fluorescent DNA probes (interphase fluorescent in situ hybridization, or “interphase
FISH”) (Figure 2). Moreover, lower grade tumours were employed in order to evaluate the
early stage findings noted in previous experiments.
Utilizing the U25123 probe, mtDNA showed increases in copy number in all the lower
grade tumours studied, but not the normal brain specimens. When evaluated by the
interphase FISH technique using fluorescent probes of the entire mitochondrial genome, at
least 69% of the nuclei revealed mitochondrial sequence hybridization in the tumour
specimens. In comparison, when evaluating two normal brain samples, as well as a
glioblastoma specimen (all of which did not reveal increases in mtDNA copy number), <
8% of nuclei revealed hybridization with the mtDNA fluorescent probes. This observation
suggested that there was an association with the amplification of mtDNA and the finding
of “promiscuous” mtDNA sequences in the nuclear compartment. Indeed, this was the
first description of nuclear localization of mtDNA found to be associated with mtDNA
amplification, and as an ontologically early event; furthermore, it was the first description
of such an event found in primary tumour tissues, rather than cell culture experiments.
Hence, in this specific aim, the evaluation of copy number, sequence, promiscuity and
diversity of tumour grade was performed, and the experiments involved revealed
previously unreported results which suggested the importance of mitochondria and its
DNA in the tumourigenic phenotype.
Data noted in alterations of the mitochondrial genome were followed by investigations by
others, i.e. those regarding its importance in various other tumour types. Penta et. al.
(2001) noted that in addition to gliomas, breast, liver and colon cancers showed increases
in mtDNA/expression, with deletions in other tumours including those of the colon,
stomach, bladder, head/neck and lung. Carew and Huang (2002) also identified mtDNA as
being altered in these tumour types, and additionally, described findings in ovarian,
esophageal, pancreatic, renal cell and prostate cancer, as well as thyroid cancers and
hematologic malignancies. In particular, these authors noted gliomas to be the
best-characterised tumours with respect to mtDNA alterations, noting the studies
documented in this dissertation, as well as the study by Kirches et. al. (1999). Beheshti et.
al. (2003) found mtDNA amplification in neuroblastoma, and Haugen et. al. (2003) found
an increased expression of mtDNA encoded genes in papillary thyroid carcinomas. Jia et.
al. (1999) showed increases in mtDNA levels with the mdr-1 genotype, and Tarantul et. al.
(2000) found in SIV-infected primate non-Hodgkin lymphoma (NHL) evidence for
amplified mitochondrial sequences; similarly, Nikolaev et. al. (2001) showed mtDNA
increases in simian NHL, and Nenasheva et. al. (2004) found such sequences increased in
SV40-transformed fibroblasts, observations suggesting that these modifications serve as
early markers for the development and/or pathogenesis of malignancy. However, Dani et.
al. (2004) evaluated breast, colorectal, gastric and head/neck tumours for the mtDNA4977
deletion (“common deletion”), and concluded that such tumour cells are essentially free of
this mutation; Kiebish and Seyfried (2005) also did not find significant mtDNA mutations
in mouse brain tumours, and concluded these did not play a role in either
chemically-induced or spontaneous mouse brain tumours. However, this has not been
confirmed by others; indeed, primary tumour cells in these tumour types did show
alterations in the mtDNA4977 region, amongst other mutations (Kirches et. al., 2001;
Kamalidehghan et. al., 2006; Poetsch et. al., 2004; Chung et. al., 2000; Polyak et. al.,
1998; Dai et. al., 2006). Furthermore, the D-loop region (where mtDNA initiates
replication, and is non-coding) has been frequently associated with alterations in copy
number as well as mutation in osteosarcoma, uterine serous carcinoma, gastric carcinoma,
and ovarian, endometrial and breast cancer (Zhu et. al., 2005; Guo and Guo, 2006; Pejovic
et. al., 2004; Maximo et. al., 2001; Liu et. al., 2001; Wang et. al., 2005; Tan et. al., 2002;
Lievre et. al., 2006); indeed, Duncan et. al. (2000) identified a novel human mitochondrial
D-loop RNA species which was up-regulated subsequent to cellular immortalization,
supporting data herein that mtDNA alterations represent an early modification in
generation of the tumourigenic phenotype. Moreover, associations of D-loop mutations
have been noted in the background of mutation of p53, associated with changes in
expression of mitochondrial proteins (Zhou et. al., 2003; Gochhait et. al., 2008). Of note,
others have found a distribution of mutations throughout the mitochondrial genome
(Jakupciak et. al. 2005; Ivanova et. al., 1998). These studies support a role for mtDNA in
the tumourigenic phenotype, and directly support the findings first reported herein of
alterations in copy number (particularly increases) as an ontologically-early aspect of the
pathway to malignant behaviour.
Czarnecka et. al. (2007) describe cancer as a “mitochondriopathy”, and note the alterations
of mtDNA in various tumors, as well as insertion of mtDNA sequences into the nuclear
genome, with findings in hepatoma and meningioma (Bianchi et. al., 2001; Yamauchi,
2005; Sorenson and Fleisher, 1996). Interestingly, in both human myeloid leukemia HL60
and colorectal carcinoma COLO 320DM cells, amplification of mtDNA was found to be
associated with mitochondrial sequences identified in the nucleus; in normal fibroblasts
and peripheral blood T-cells, no promiscuous mtDNA sequence was noted (Hirano, 1999).
Similarly, in mouse and rat model systems, mtDNA-like inserts were found at higher
frequencies in tumour tissue nuclei when expressed relative to those of normal cells
(Hadler et. al., 1998). Finally, Ricchetti et. al. (2004) found mtDNA sequences
interrupting intronic regions of MADH2, a tumour suppressor gene in colorectal cancer
(Eppert et. al., 1996), and Borensztajn et. al. (2002) and Turner et. al. (2003) showed
promiscuous mtDNA in the nuclear genome in both the intron and exon regions, which
were associated with clinical phenotypes of disease (Factor VII deficiency and
Pallister-Hall Syndrome, respectively). These studies directly confirm results documented
in this dissertation regarding human gliomas; such results suggest that this may represent
an important mechanism in the generation of the tumourigenic phenotype. These mtDNA
changes could have significant implications regarding both the developmental
mechanisms and detection of malignant cells in patients with cancer. Indeed, Copeland,
(2002), Modica-Napolitano and Singh, (2002), Saffroy et. al. (2004), Chatterjee et. al.
(2006), and Paul and Mukhopadhyay, (2007), have noted the relevance of mtDNA
mutations and their potential consequences regarding both the diagnosis and treatment of
patients with malignancy; in fact, both diversity of changes throughout the mitochondrial
genome (Ray et. al., 2000; Cai et. al., 2004) in addition to specific polymorphisms
     (G10398A: Darvishi et. al., 2007; Canter et. al., 2005) have been noted to be associated
     with risk for cancer. Jiang et. al. (2005) found that there was an increased mtDNA content
     in saliva associated with head/neck cancer patients; treatment with apoptosis-inducing
     therapy (but not surgery alone) was associated with a decline in mtDNA levels (Jiang et.
     al., 2006). Hence, alterations in mtDNA may serve as a diagnostic measure reflecting
     response to therapeutic intervention with modalities such as radiation therapy. Indeed,
     Polyak et. al. (2002) have filed a patent to utilize the presence or absence of mtDNA
     mutations to detect tumour cells as a diagnostic approach in cancer patients.



                                                 ***




Evaluation of the biology of mitochondrial alterations in the neoplastic phenotype, including
     the effect of removing mitochondrial DNA (creation of rho- tumor cells) and evaluation
     of the ontogeny of mitochondrial membrane potential changes in a model of neoplastic
     transformation


     As an extension of the findings noted of the importance of mtDNA in neoplastic tissue
     from Specific Aim 1, a further understanding of the pathobiology involved was evaluated
     at the level of the phenotypic effects of decreasing the amplification and mitochondrial
     function, which employed techniques to decrease the levels of mtDNA (“rho-“ cells). In
     this manner, the direct effect of mtDNA, and mitochondrially-encoded biomolecules,
     would be evaluable, and the concepts potentially translated into the treatment of malignant
     disease.
     Neoplastic cells (MCF-7, a well-known breast cancer cell line, and DBTRG-O5MG,
     derived from a glioblastoma), were treated with low concentrations of ethidium bromide
     (EtBr). EtBr is an inhibitor of mtDNA replication (Nass, 1972); hence, during cell growth
     and passage, the initial number of mtDNA molecules is decreased by a factor of two on
     their treatment with this agent. These cells were passaged for at least 30 doublings, and
     thus, < 1% of the original number of mtDNA molecules (viz. 1/230) were present. With
     such treatment, the cells became auxotrophic on uridine and pyruvate. The requirement for
     uridine relates to that for dihydrooratate dehydrogenase (an enzyme of pyrimidine
     biosynthesis, localized to the inner mitochondrial membrane) to have an active electron
     transport chain for normal function. The auxotrophic dependence of rho- cells on pyruvate
     is not definitively known or well described, but most likely relates to the inability of the
     cell to oxidize cytoplasmically-produced NADH. Cells were also noted to have minimal or
     no hybridization on dot blot to mtDNA probes, a monitoring system indicative of
     phenotypic rho- cells.
     Further characterization was performed of the rho- vs. rho+ (parent) cells. Evaluations of
     the genes bcl-2, bax, mdr1, mrp and O6-alkyltransferase were conducted by reverse
     northern blots. These genes have been associated with changes in responsiveness to
     cytotoxic chemotherapy, and believed to be important in maintenance of the tumourigenic
     phenotype (Hickman, 1996; Susin et. al., 1996). Moreover, both BCL2 and BAX can be
located at the inner mitochondrial membrane (Susin et. al., 1996) after being translated
cytoplasmically. In all cases (bcl-2, bax, mdr1, mrp and O6-alkyltransferase), using
reverse northern blotting, although each gene was expressed, there were no changes in
expression observed with these genes in the rho- cells when compared to their rho+
parents.
A key aspect of the tumourigenic phenotype is the ability of such cells to grow in an
anchorage-independent fashion (Nikiforov et. al., 1996). Both rho+ parent and rho- cells
were assessed with regard to their abilities to form colonies in soft agar, an indication of
anchorage-independence. Whilst both types of cells were able to attach to plastic tissue
culture flasks, formation of colonies only occurred in the rho+ parent cells that harboured
mtDNA, with minimal colony formation found in either MCF-7 or DBTRG-O5MG rho-
cells. Indeed, even prolonged incubation of the rho- cells (over double the length of time
of that for rho+ cells) in soft agar did not result in additional colonies being formed.
Employment of a cell-conditioned medium from the rho+ parent cells into the rho- cell
culture experiments did not result in any additional colonies being formed; furthermore,
cell-conditioned medium from the rho- cells did not inhibit colony formation in the rho+
cells. When evaluated in xenograft experiments, only rho+ cells showed growth in the
flanks of nude mice; no growth was found with the rho- cells. It has been noted that
altered expression of bcl-2 and mutant p53 are potential mediators of anchorage
independence (Nikiforov et. al., 1996). In these neoplastic cells, the p53 gene was wild
type (Kruse et. al., 1992; Engel and Young, 1978), and no changes between rho+ and rho-
cells in the expression of bcl-2 was noted. These data strongly suggested that changes in
mtDNA, and mitochondria, were playing a role in anchorage independence, a
manifestation of the tumourigenic phenotype.
Further studies were performed on the rho+ parents, and their derived rho- cells, with
respect to sensitivity to apoptosis effected by cytotoxic chemotherapeutic agents.
DBTRG-O5MG cells were treated with 1,3-bis-dichloroethyl-nitrosourea (BCNU), a
common drug utilised in the therapy of patients with glioblastoma; MCF-7 cells were
exposed to cis-diamino-dichloro platinum(II) (CDDP), one of the most commonly
employed chemotherapy drugs in cancer treatment. In both cases, rho- cells were
distinctly and significantly more sensitive to the respective agents than their parent rho+
cells, a previously unreported observation. Interestingly, despite a lack of mtDNA, both
rho+ and rho- cells underwent apoptosis as a mechanism of cell death, as noted by
morphologic criteria and chromatin condensation after exposure to these cytotoxic agents.
This was observed at all doses of BCNU and CDDP investigated and in both MCF-7 and
DBTRG-O5MG cells, i.e. an increased dosing level did not revert cells to a necrotic
mechanism of cell death. Whilst it had been hypothesized that the putative drug
resistance/apoptosis resistance genes bcl-2, bax, mdr1, mrp and O6-alkyltransferase would
be involved in the phenotype changes, no changes were noted in expression. Hence,
mitochondria must play additional role(s) in the drug resistance phenotype, at least in the
manifestation of resistance to apoptosis. Of note, however, is that removal of functional
mitochondria via depletion of mtDNA failed to prevent apoptotic cell death, an
observation indicating that actively-translated, mitochodrially-encoded molecules are not,
per se, required for apoptosis induction by cytotoxic chemotherapeutic agents, a clearly
novel finding.
In order to further evaluate the potential function of the mitochondria, “cybrids” of the
rho- cells were created. Cybrids are fusion cells of the rho- ones and normal cells which
harbour mitochondria, in order to replace this organelle in order to further study
mitochondrial function, and is a technique utilised significantly in the evaluation of
mitochondrial encephalomyelopathies. In this case, normal platelets were employed,
rather than either DBTRG-O5MG or MCF-7 de novo enucleated cells and cytoplast
transformation, in order to avoid nuclear and cytoplasmic component influences of the
tumour cells on the cybrid phenotype, i.e. entirely normal mitochondria with a minimal
level of influence of nuclear-encoded genes; platelets are, by definition, fragmented
aspects of cells, and thus anuclear. Subsequent to fusion, the cells were selected in uridine-
and pyruvate-free medium, and assessed to determine mtDNA identity of the donor, copy
number, anchorage independence, and sensitivity to apoptosis via cytotoxic agents.
Interestingly, a novel finding was that cybrids created from the rho- cells and platelet
mitochondria showed mtDNA levels which were similar to that of the rho+ ones, viz.
amplified; of particular note is that this level was higher than that of peripheral blood
platelets and lymphocytes, an observation suggesting that a cellular influence to maintain
increased mtDNA levels is still acting through the rho- state, and subsequent to platelet
fusion. Moreover, the morphology of the cybrids differed from the rho- cells, and more
resembled the rho+ cells, a potential reflection of the role of mitochondria on cell
morphology (Felty and Roy, 2005).
When evaluating the cybrids for anchorage-independent growth, both MCF-7 and
DBTRG-O5MG showed a return of this aspect of the tumourigenic phenotype. Indeed,
both neoplastic cybrids essentially demonstrated a return to the rho+ cell levels of colonies
formed in soft agar, with colony sizes and densities almost if not identical in nature. The
growth as xenografts in nude mice also returned in both cell lines after mitochondrial
transfer. Furthermore, the relative sensitivity of cells to the effects of cytotoxic
chemotherapy – viz. induction of apoptosis – also changed from rho- levels to those of the
rho+ cells. MCF-7 and DBTRG-O5MG cells returned to their levels of sensitivity to
CDDP and BCNU, respectively, which was observed prior to removal of mtDNA
amplification. This phenotypic reversion toward a more apoptosis-resistant state (at least
with respect to chemotherapeutic agents) showed no difference with regard to the overall
mechanism of cell death when compared to those of rho+ and rho- cells (Figure 3).
Hence, these data were the first to suggest that the nuclear influences controlling mtDNA
copy numbers are maintained through the rho- state, and that mitochondria are at least
participating in the maintenance of the tumourigenic phenotype, manifested as
anchorage-independence and relative resistance to the cytotoxic effects of
apoptosis-inducing agents CDDP and BCNU. Because cybrids were constructed from
normal mitochondria, tumour mitochondria/mtDNA per se is not responsible for the
observed phenotypic change of the immortal but diminished tumourigenic rho- cells,
which revert to the de novo phenotype. Thus, mitochondria play a direct role in neoplastic
maintenance (with nuclear encoded genes, which support mtDNA amplification), and yet
may not be strictly required for apoptotic cell death.
To further understand the pathological significance of the role of mitochondria in
malignancy, the mitochondrial membrane potential was evaluated in a model system of
normal cell transformation, again to determine the relative ontogeny of mitochondrial
modifications associated with progression to the tumourigenic phenotype. This is of
particular interest, since further studies have shown, in chemotherapy resistant cell lines,
that mitochondrial dysfunction is manifested by changes in mitochondrial membrane
potential, dissociated from the energy-generating capacity of mitochondria (Fantin et. al.,
2002). Moreover, evaluation of shunting cells towards oxidative phosphorylation by the
inhibition of glycolysis was also evaluated to determine the potential value of using the
aforementioned data in a therapeutic manner.
Normal human astrocytes (NHA) were treated with ethyl-nitrosourea (ENU) in order to
derive transformed astrocytes. Cells were initially observed to become immortal,
continuing to divide after untreated NHA reached terminal division; experiments were
performed subsequent to the achievement of immortality (anchorage independence, low
density growth). In addition, phosphofructokinase (PFK) is the major regulatory enzyme
in glycolysis, and is allosterically-inhibited by citrate; cells were also treated with this
glycolysis inhibitor to determine the effects of metabolic manipulation on the expressed
phenotype.
NHA cells were unable to divide past approximately 20 doublings, and, moreover, could
not grow in an anchorage-independent manner or under low density conditions. In
contrast, cells exposed to ENU became immortal, and at approximately 26 doublings,
achieved the ability to grow in soft agar and in low density. Interestingly, when NHA cells
were treated with ENU and pre- and co-treated with the PFK inhibitor citrate, they still
became immortal, dividing well past terminal differentiation. However, despite achieving
the ability to avoid replicative senescence, these dually-exposed cells did not gain
anchorage independence, nor the ability to grow in a low density. When ENU-transformed
clones were subsequently treated with citrate (cf. pre- and co-treatment) and evaluated for
growth under low density conditions, at and above an added level of 100 M of this
agent, there was a decrease in colony formation. Hence, although ENU treatment resulted
in immortal and anchorage independent cells, pre- and co-treatment of cells with citrate
with subsequent ENU exposure diminished the morphological changes and transformed
characteristics of the cells; use of citrate to “treat” malignant cells also decreased the
transformed phenotype, albeit at higher concentrations. This was the first report of
changes in cellular phenotype by the modification of glycolytic enzymes in
chemically-transformed cells.
When evaluating mitochondrial membrane potential, additional alterations were found. In
NHA cells, mitochondrial membrane potential was found to progressively decrease with
increasing doublings, until terminal division. Citrate did not exert an effect on the NHA
cells with respect to mitochondrial membrane potential. In contrast, when ENU was
administered, after cells underwent 3 – 5 doublings, the ENU cells exhibited a
significantly higher polarized mitochondrial membrane potential, when compared to that
of NHA cells. With subsequent doublings, the ENU cells did decrease in mitochondrial
membrane potential, although values were, in general, higher than NHA (or citrate
exposed cells), with maintenance of an elevated membrane potential after the development
of immortality, an observation which was previously unreported. Interestingly, in parallel
with the lack of anchorage-independence and growth in low density, pre- and co-exposure
of cells to citrate did not result in the increased mitochondrial membrane potential
observed treated with ENU alone. No difference was noted between the
ENU/citrate-exposed cells, and the NHA or NHA citrate-exposed cells with respect to
mitochondrial membrane potential.
Furthermore, mitochondrial mass was also influenced by exposure to ENU and the
development of a transformed phenotype. The NHA and NHA/citrate cells showed a very
gradual, modest decline of mitochondrial mass, with successive doublings. With ENU
administration, however, after ca. 10 doublings, there was a substantial drop in
mitochondrial mass, to about 25% of the starting mass measured. Such mass changes
stabilized after about 16 doublings, and were maintained after the achievement of
immortality, and also during the achievement of anchorage independence. Citrate
prevented the dramatic drop in mitochondrial mass; even with immortality achieved, the
mitochondrial mass of ENU/citrate-treated cells was not different from NHA cells which
were terminally differentiated. When calculating the mitochondrial membrane
potential/mitochondrial mass ratios, such data revealed a hyperpolarized state of the
mitochondrial membrane of the ENU-transformed cells, which was prevented by citrate
exposure both prior to and concomitantly (Figure 4). These data suggested that
hyperpolarization of the mitochondrial membrane represents is an early manifestation of
the transformed/tumourigenic phenotype, associated with anchorage independence, and
may be mitigated by use of the PFK glycolytic inhibitor citrate.




Finally, cells were evaluated to determine their levels of spontaneous apoptosis. NHA
cells progressively underwent a higher level of apoptosis with each successive doubling,
as did NHA cells treated with citrate. However, ENU cells only maintained a low level of
apoptosis, which did not attain the same levels as the NHA- or NHA/citrate-treated cells at
any cell culture doubling. In contrast, cells with ENU, both pre- and co-treated with
citrate, had an increased degree of spontaneous apoptosis, which was, however, lower,
than that of the NHA- and NHA/citrate-treated cells at each doubling. Hence, the
treatment of NHA cells with ENU was associated with a decreased extent of programmed
cell death and the achievement of immortality; citrate increased the level of spontaneous
apoptosis in ENU cells (although not to the same degree as that of the NHA cells).
Thus, data derived from this specific aim indicate that mitochondria and/or its genome
play a significant role in human cancer, and that alterations of this organelle can effect
basic changes in the phenotypic representation of malignancy (anchorage independence;
xenograft growth; relative resistance to apoptosis via cytotoxic chemotherapeutic agents).
Furthermore, these modifications are found early in the ontogeny of the tumourigenic
phenotype, since the development of transformed astrocytes was found to involve early
changes in mitochondrial membrane potential, hyperpolarization of the mitochondrial
membrane, and constraints on apoptosis, all of which could be modified by inhibition of
the glycolytic enzyme PFK.
These data directly implicated the mitochondrion in the tumourigenic phenotype; these
observations were subsequently confirmed and extended to further systems. Karthikeyan
and Resnick (2005) noted the consequences of mitochondrial dysfunction on genome
stability, which included an identification of the importance of heteroplasmy (differences
in mtDNA types within the same cell and/or organ), as well as effects of diminished
mitochondrial gene expression which influence both the invasive as well as the
transformative phenotype. Moreover, Xue et. al. (2001) using
Boc.Aspartyl(O-methyl)CH2F, removed mitochondria from neurons and HeLa leukemia
cells; such removal did not damage other cellular components, but essentially irreversibly
committed these cells to death via an apoptotic mechanism. Hence, in cells with
mitochondria inhibited or removed, apoptosis is more readily effected when expressed
relative to that occurring in control cells. Dong et. al. (2002) showed that mitochondrial
transcription factor A (mTfam) (a key regulator of DNA transcription) in Morris (mouse)
hepatoma cells was significantly over-expressed (over 10-fold when compared to normal
liver), with augmentation of downstream mitochondrial gene expression. Moreover,
mTfam protein was also found in the hepatoma cell nucleus, suggesting a role in
nuclear–mitochondrial communication in this neoplastic lesion. Indeed, these findings
indicated that the mitochondrial influence on the observed tumourigenic phenotype is
complex, and involves a myriad of different proteins interacting on both upstream and
downstream pathways. Grandemange et. al. (2005) performed an elegant study, in which
human dermal fibroblasts acquired transformed properties (morphologic changes,
anchorage-independent growth, growth in xenografts) with stimulation of mitochondrial
activity, using p43. Moreover, such transformation was associated with an increase in
c-Jun and c-Fos expression, and virtual extinction of tumour suppressor genes p53,
p21WAF1, and Rb. When mitochondria were then inhibited using chloramphenicol, a loss
of the tumourigenic phenotype was observed (although restoration of the tumour
suppressor genes did not occur). This study thus identified p43, at least in dermal
fibroblasts, as a potential target mechanism by which the early involvement of
mitochondria in the tumourigenic phenotype may be manifested. Similarly, additional
studies in differing model systems have identified proteins which may be responsible for
observation of the importance of mitochondria in the malignant phenotype. In a
clinically-based study, Ambrosone et. al. (1999) found an association of manganese
superoxide dismutase (a protein transported to mitochondria) genotypes (with limitations
on transport into mitochondria) with breast cancer risk; Roy et. al. (2007) noted
estrogen-related processes in the nuclear–mitochondrial communication, via
estrogen-induced mitochondrial oxidants. Ohnami et. al. (1999) showed that K-ras impacts
the expression of mitochondrial genes; when utilizing an anti-sense approach to pancreatic
cancer cells, they identified 11 over-expressed genes, which were all mitochondrial in
origin. This continues to suggest the importance of the nuclear–mitochondrial
communication pathway, where K-ras represents an important mediator of mitochondrial
gene expression and changes in phenotype. Mills et. al. (1999) identified CD14 as a
potential mediator of apoptosis in HL60 cells; they documented that inhibition of
mitochondria using rotenone, antimycin A, and oligomycin all resulted in an increased
expression of CD14 in these leukemic cells, followed by loss of viability and apoptosis.
Magda et. al. (2008) studied A549 rho- cells (lung cancer) induced by exposure to EtBr
(although auxotrophy was not reported in the study). This group showed a less
proliferative ability in the rho- cells, but a greater level of expression of intermediate
filaments, suggesting a more invasive phenotype. This observation is interesting in light of
the hypothesis by Felty and Roy (2005) that malignant cell spreading may be ascribable,
in part, to the contraction and expansion of mitochondria, rather than only the maintenance
of tension on the cytoskeleton via binding to the extracellular matrix. Moreover, these
A549 rho- cells, whilst able to grow in mouse xenografts, required longer induction
periods with smaller tumours than their rho+ parents. This suggests a definite difference in
phenotype, perhaps manifest by differences in nuclear gene expression (Li et. al., 2008;
Ismail and Bateman, 2009). Interestingly, rho- cells increased the expression of MHC-1 in
addition to glucuronidation genes, relevant in immune surveillance and detection function;
this indicates the relevance of mitochondria to the extrinsic manifestations of apoptosis
induction, an observation similar to that made by Mills et. al. (1999) with the expression
of CD14, and our studies with CD95 (Fas) (Liang, Depletion of Mitochondrial DNA from
Glioblastoma Cells results in Increased Fas Expression with Increased Sensitivity to
Apoptosis, manuscript in preparation).
As noted, the communication between the nucleus and mitchondria as hypothesized from
these studies (maintenance of amplified mtDNA/increased expression and reversion of
phenotype of cybrids; alterations in mitochondrial membrane potential during the
transformation of NHA cells) represents an important aspect of the maintenance of the
tumourigenic phenotype. In particular, gradual reductions of mtDNA levels in
rhabdomyoblasts (transformed muscle cells) using EtBr effected changes in mitochondrial
membrane potential and elevated free Ca2+ concentrations in the cytosol (Biswas et. al.,
2005, Amuthan et. al., 2001); these changes resulted in alterations of genes related to
Ca2+ transport and storage, which could be reverted once cells were allowed to grow
without EtBr present. Interestingly, as mtDNA levels were declining (but not absent),
there was an increase in the ability of the rhabdomyoblasts to move through a
reconstituted Matrigel membrane, an observation suggesting a migratory phenotype which
was lost once mtDNA levels were allowed to return to their control values. These data
suggested that mtDNA levels are affected not only early in the tumourigenic phenotype,
but also in ontologically-subsequent manifestations of malignancy, and could serve as a
marker which identifies a pathway by which the migratory phenotype is effected. Hence,
using feedback to the mitochondria and mtDNA levels, the nuclear–mitochondrial
communication could effect the expression of nuclear genes in response to the
microenvironment milieu; this occurs in yeast at a variety of rho- settings (Butow and
Avadhani, 2002; Giudice et. al., 2005).
The maintenance of the tumourigenic phenotype was also found to be related to
mitochondrial membrane potential, the work contained within this dissertation being the
earliest study conducted with regard to the identification of the early stages of chemical
transformation in mitochondrial membrane potential. Interestingly, in neu-initiated
mammary epithelial cell tumours with increased mitochondrial membrane potential
values, inhibition of glycolytic pathways using short hairpin RNAs of LDH-A decreased
mitochondrial membrane potential and reduced the ability of these cells to proliferate in
both de novo and hypoxic environments (Fantin et. al., 2006), supporting this researcher’s
data acquired with the use of citrate as a glycolytic inhibitor of PFK, with associated
changes in membrane potential reflective of the overall tumorigenic phenotype (i.e.
resistance to apoptosis and growth in xenografts). Other researchers have hypothesized
similar approaches (Bucay, 2007). Matarrese et. al. (2001) showed in a drug resistant
human T-lymphoblastoid CEM cell line VBL100 variant (with amplified mtDNA), which
was sensitive to TNF- , so that the induction of apoptosis was attributable to the
depolarization of mitochondrial membrane potential per se. Indeed, these authors showed
a direct correlation between susceptibility to apoptosis (by various agents) and the state of
the mitochondria (mitochondrial membrane potential); prevention of the decrease in the
membrane potential, and thus mitochondrial function, using bongokreic acid (which
inhibits the decrease in mitochondrial membrane potential by antagonising the
adenine-nucleoside transporter, which, in turn, is responsible for allowing
mitochondrial-membrane permeability transition and thus a run down of the potential), the
apoptosis-inducing effects could be mitigated by the various agents which effected the
intrinsic (viz. mitochondrial) pathway of apoptosis (but not the extrinsic pathway).
                                                 ***




Evaluation of the mechanism and potential of inhibition of mitochondrial function in
     effecting tumour cell death as a transition to approaches to the treatment of patients
     with cancer


     With data obtained as noted, interest focused on a translational evaluation of
     understandings of the mechanisms of programmed cell death using chemotherapeutic
     agents, combined with the mitochondrion as a target for treatment.
     Further mechanistic work was performed with CDDP. Indeed, this is one of the most
     commonly utilised chemotherapeutic agents, and recent data has suggested that nucelotide
     excision repair (NER) genes may play a significant role in the resistance of cancer cells to
     this drug. A further goal was to determine in further tumour cell types (e.g. hematologic
     malignancies) whether the findings of the impact of mitochondria on the tumourigenic
     phenotype could be extended. Using the U937 cell line (derived from a histiocytic
     lymphoma), rho- cells were created using EtBr, as were cybrids, using normal human
     platelet mitochondria in order to avoid potential nuclear-encoded cytoplasmic components
     which could exert an impact on results. Expression of the NER genes ercc1 and ercc2 was
     evaluated, since these NER genes have been found to be important in platinum(II)
     drug-resistance in ovarian carcinoma patients; moreover, the BCL-2 family genes bcl-2
     and bax, as well as the drug resistance genes mdr and mrp, were also evaluated.
     Rho- U937 cells were found to have an increased susceptibility to cell death induced by
     CDDP when compared to the rho+ parent, that being observed ca. twice as sensitive to the
     drug. When U937 cybrids were assessed for relative drug resistance, no difference was
     noted between the cybrids and the rho+ cells at any CDDP dose level tested, an
     observation similar to that of previous results obtained with MCF-7 breast cancer and
     DBTRG-O5MG glioma cultured cells. Moreover, an increased copy number was again
     noted in the U937 cybrids, similar to the rho+ cells, and this observation is similar to the
     findings previously noted in breast and glioma cancer cybrids. mRNA expression levels
     were evaluated in the NER, BCL-2 family member, and drug resistance genes; whilst
     there were relative differences in expression of the different genes evaluated, no changes
     were noted between the rho+ and rho- cells, nor in the cybrids. Hence, depletion of
     mtDNA from parental cells did not alter the expression of putative genes potentially
     responsible for overall CDDP resistance. Moreover, as previously noted, cells continued
     to die an apoptotic death, irrespective of whether they were rho+, rho- or cybrid in nature.
     Furthermore, in order to determine whether changes (i.e. decreases) in
     mitochondrially-derived ATP levels accounted for the increases in sensitivity to
     CDDP-induced apoptosis in the rho- cells, the mitochondrial F0F1-ATPase was inhibited
     with oligomycin in the rho+, rho- and cybrid cells. Interestingly, for all cell types, no
     evidence for differences in ATP levels between all samples collected before and after
     treatment with oligomycin were found (Figure 5).
Moreover, no differences were noted between the ATP concentrations of each cell type.
This observation suggests the generation of ATP by these cells is less dependent on
oxidative phosphorylation, but rather glycolysis, in accordance with further earlier studies
(Warburg, 1926); hence, mitochondrial energy production per se (or lack thereof) is not
responsible for the increased sensitivity of rho- cells to CDDP-induced cell death, a
previously unreported finding.
Consequently, the mitochondria may be an appropriate mechanistic target to sensitize cells
to apoptosis using cytotoxic agents. Therefore, the penultimate goal is to determine an
approach which would allow for mitigation of the tumourigenic phenotype manifested by
an increased sensitivity to programmed cell death by cytotoxic chemotherapeutic agents,
but one which would have minimal (if any) toxicity toward normal tissues.
Hence, further studies evaluated agents which were noted to have anti-mitochondrial
activity. 2’,3’-dideoxycytidine (ddC) is an anti-retroviral agent, and is a potent inhibitor of
DNA polymerase gamma, and consequently a distinct anti-mitochondrial activity
(Bouayadi et. al., 1997). It is currently approved for patients with acquired immune
deficiency syndrome (AIDS) as part of the multi-agent cocktail treatment regimen. Two
tumour cell lines (glioma), DBTRG-O5MG and DK-MG, were exposed to ddC at doses
ranging from 10 nM to 100 M. At all doses, ddC effected morphological changes in
cells, consisting of larger cell bodies with more prominent processes and a ground glass
appearance. Such changes occurred at relatively early stages, i.e. within 2-3 days
following the commencement of ddC treatment, regardless of dose. Indeed, at the onset of
the morphologic changes noted, alterations of mitochondrial membrane potential were
also noted, with a decrease in polarized mitochondria at all doses evaluated; up to 31%
diminished in DTBRG-O5MG and 84% in DK-MG cells treated with ddC. At all doses of
ddC studied, cell death ensued eventually in both glioma cell lines, which was apoplectic
in nature; cells became non-viable in < 6 hours after first being noted to detach from the
tissue culture flask. A threshold between 100 nM and 1 M was noted in both
DBTRG-O5MG and DK-MG cell lines, where the time between exposure to ddC and
subsequent cell death decreased from approximately 30 days to <10 days. This
observation suggested that the mechanism of ddC-induced programmed cell death may be
a saturable phenomenon (e.g. enzyme- or receptor-mediated). Interestingly, at some doses
of ddC (between 10 nM and 15 M), a delay (but not total rescue) in the death of both
cell lines could be obtained with supplementation of the medium with glucose, uridine and
pyruvate, suggesting the cells lacked a fully functional respiratory chain; however, cells
treated with higher doses of ddC were unresponsive to such supplementation. This
suggests that mitochondria (in a functional rho- state) are involved in at least one
mechanism of the observed ddC-induced cellular death, although since rescue was only
partially possible with glucose, uridine and pyruvate, and the cells eventually died, that the
overall mechanisms effecting apoptosis are likely to be more complex and multifactorial.
Again, in all cases the mechanism of death was apoptosis. These data are suggestive that
mitochondria may per se serve as a target for therapy, but the therapeutic index may
represent an issue (particularly as most regimens contain multiple drugs). Whilst the
toxicity of ddC clinically can be managed (peripheral neuropathy, neutropenia), the type
of dosing that might be required (chronic dosing, with other cytotoxic agents) could result
in some not insignificant levels of organ toxicity. Hence, agents which have a wider
therapeutic index and more selective towards mitochondria, particularly within
combination regimens, might be more appropriate for patient therapy.
Hence, a search for compounds which could target the mitochondrion ensued; in
particular, compounds which had lipophillic (i.e. diffusible into the tumour cell and past
mitochondrial membranes), and cationic (allowing attraction and retention in the anionic
environment of the mitochondrion) characteristics were sought. A compound which fit
these criteria was tyrphostin AG17 [NSC 242557,
(3,5-di-tert-butyl-4-hydroxybenzylidene) malononitrile] (Burger et. al., 1995). This agent
also has tyrosine kinase inhibitory activity, which is manifest as a growth retardation
mechanism in other studies (Ara and Teicher, 1996). AG17 was used at non-growth
inhibitory concentrations (0.25 M) in order to avoid normal cell toxicity, and to evaluate
the potential anti-mitochondrial contribution to any cytotoxicity noted in combination
treatments.
Treatment of tumour cell lines DBTRG-O5MG and DK-MG, as well as NHA cells, with
AG17 in culture at 0.25 M did not inhibit growth. In order to determine whether the
agent was affecting mitochondria in either tumour or normal cells, evaluations of
auxotrophy on uridine and pyruvate was assessed after 30 days of exposure. On removal
of such supplements, glioma cells stopped proliferating and gradually became detached
from the tissue culture flasks over periods of 7 to 15 days; staining with EtBr and acridine
orange showed chromatin condensation, indicative of apoptotic cell death. In contrast, in
the NHA cells, removal of uridine and pyruvate supplementation did not result in either
detachment nor increased apoptosis as noted in the tumour cell lines. Of note was the
observation that NHA cells did undergo replicative senesence after about 20 doublings,
but this was not affected by the treatment of cells with AG17. Consequently, the AG17
cell treatment effected a auxotrophic phenotype in tumor (but not normal) cells, indicative
of diminished respiratory chain function, which did not occur in NHA. This suggested a
higher selectivity of AG17 towards tumour mitochondria over those of normal cells at
these lower doses, and the avoidance of non-targeted and potential toxic effects might be
possible with this agent.
In order to further define the putative mitochondrial mechanism, mitochondrial membrane
potential was determined with the treatment of cells with AG17. In tumour cells, treatment
with the agent was associated with a decline in the prevalence of polarized mitochondria,
from 32 – 65% (as noted by cytofluorimetric analysis), with the highest level of
depolarization observed with the lengthiest exposure to this agent. Of note was the
observation that mitochondrial mass was found to be diminished in these glioma cells,
reflecting a higher overall relative polarization of the mitochondria, and thus an even more
impressive decrease in overall degree of depolarization of mitochondrial membrane
potential. Moreover, the mitochondrial mass was also found to decrease with prolonged
exposure to AG17, suggesting that the decrease in mitochondrial membrane potential was
at least in part ascribable to reductions of the overall mitochondrial mass in the tumour
cells. In contrast, NHA cells displayed only slight reductions in mitochondrial mass, and
approximately similar mitochondrial membrane potential values over the study period.
With a more prolonged evaluation (> 45 – 50 days), reductions in mitochondrial
membrane potential were noted, both in the AG17-treated and untreated cells (data not
shown).
The evaluations of AG17 treated cells were then focused on their susceptibilities to
cytotoxic agent-induced apoptosis (Figure 6). In both tumour cell lines, treatment with
BCNU (at the highest dose level,144 g/ml) was associated with significant increases in
apoptotic cell death after AG17 exposure of 2 days in DBTRG-O5MG, and 3 days in
DK-MG glioma cell lines. Indeed, exposure to AG17 after 2 or 3 days was associated with
increased sensitivity to lower doses of BCNU as well. Maximally, after 30 days of
exposure to AG17, treatment with BCNU reduced the number of viable cells by over 99%.
A linear relationship was noted between survival at a BCNU dose of 144 g/ml and
mitochondrial membrane potential in the glioma cell lines. In contrast, NHA cells
sensitivity to BCNU was not modified (LD50 of 40 g/ml for both AG17 treated and
untreated cells). In all cases, the mechanism of BCNU-induced cell death was not altered
when mitochondrial membrane potential and mitochondrial mass are diminished by AG17
treatment, and thus decreases in the prevalence of both polarized and total mitochondria,
with increases in sensitivity to cytotoxic agents, still gives rise to an apoptotic death.




These data suggest that the use of AG17 as a chemosensitizer acts, at least in part, at a
mitochondrial level. Since this compound is largely a lipophillic cationic species, it and
related compounds should be valuable for treatment of tumours where an increased
anionic mitochondrial membrane potential is present, as noted in these glioma cell lines as
well as those of other cancers (Modica-Napolitano et. al., 1996). Indeed, in view of the
ability to diffuse into and be retained within the mitochondrial space, this may serve as the
mechanism involved in studies which revealed cellular chemosensitisation by AG17.
Within this specific aim, the finding that targeting mitochondria to enhance the potential
sensitivity of tumour cells to cytotoxic agents is of particular importance with respect to
the design of new chemotherapeutic and other approaches with available as well as
newly-developed novel agents. Indeed, such increases in sensitivity of tumours to
chemotherapy could allow the disease process to be more accessible to therapeutic
intervention, with limited toxic effects to normal tissues.
With the findings of changes in the tumourigenic phenotype related to mitochondria, a
great deal of interest has been focused on targeting organelle-related functions for the
treatment of human malignancy (Ngyuyen and Hussain, 2007). Yen et. al. (2005)
confirmed data noted previously in U937 cells, in rho- osteosarcoma (143b cell line)
treated with CDDP; that particular group showed increases in sensitivity to CDDP, which
were related to significantly increased caspase 3 activation. Indeed, these 143b rho- cells
were also found to have an increased release of cytochrome c into cytoplasm with
exposure to CDDP, an observation suggesting that caspase activation, in addition to
increases in the magnitude of response to CDDP, play a role in rho- apoptotic sensitivity.
Moreover, Cummings and Schnellmann (2002) also showed at least partial (50%) reliance
of CDDP-induced apoptosis on caspase 3 activation in normal renal proximal tubular
cells, without changes in ATP concentrations; further potential mechanisms included those
speculated to involve either BAX or BID proteins, but most likely are not ascribable to
caspase 8 or 9, which were inhibited in these studies. Similarly, Schwerdt et. al. (2005),
studying normal collecting duct-derived cells, showed a similar increased sensitivity to
CDDP when incubated with mitochondrial inhibitors; again, no apparent modifications in
ATP levels were observed. In U937 lymphoma cells, Troyano et. al. (2001) also showed
that ATP levels were maintained in cells where mitochondria were inhibited with
oligomycin, a result further emphasising that data contained herein demonstrated that
these cells are not dependent on mitochondria for energy production. However, of much
interest was the additional finding that depletion of glutathione (GSH)(a reactive oxygen
species scavenger, transported into the mitochondria) increased the sensitivity of cells to
CDDP-induced apoptosis when mitochondria were inhibited, an observation similar to
those of Kharbangar et. al. (2000) and our studies conducted on rho- U937 cells.
Moreover, with increases in duration of CDDP exposure, a necrotic phenotype was noted,
along with a decline in mitochondrial membrane potential. This suggested GSH may serve
as a candidate for moderating sensitivity, as well as being involved in the mechanism of
mitochondrially-mediated cell death, and that mitochondria, at the extreme, may also play
a role in necrotic cell death. In contrast, Yang et. al. (2006) found that in head and neck
cancer cell lines which were enucleated, there was no difference in sensitivity to CDDP
when compared to parental cells harbouring nuclei; however, rho- cells were more
resistant to the effects of CDDP, i.e., up to five times more resistant to the effects of the
drug. Similarly, Lee et. al. (2004) found, when evaluating TRAIL-induced apoptosis in
SK-Hep1 hepatoma cells, that rho- cells were more resistant than parental cells to the
effects of this cytokine; a different model (HeLa cells treated with photodynamic therapy
or adriamycin) also showed resistance to apoptosis in rho- cells (Singh et. al., 1999).
However, these data were in contrast to the observations by Mizutani et. al. (2009), which
revealed cybrids derived from tumour mitochondria were more resistant to CDDP than
rho- cells, an observation which agreed with previously cited results and results herein,
and Mizumachi et. al. (2008) who documented that head/neck cancer cells which were
anthracycline-resistant had significantly higher mtDNA content. Furthermore, in
alternative model systems, photodynamic approaches have also been shown to reveal
increases in cell death with mtDNA depletion (Kessel and Sun, 1999; Morgan et. al.,
2000); indeed, in L1210 murine leukemia cells, there was a significant increase in their
sensitivity to mitochondrially-related photodamage in rho- cells, a process giving rise to
an apoptotic death; when made resistant to ddC, CEM lymphoblast cells showed increases
in their mtDNA content and expression (Bjerke et. al., 2008). These differences may arise
from the various cell sensitivities to apoptosis (based on grade and stage, and thus nuclear
gene expression, e.g. bax expression), the effects of CDDP directly on the mitochondria,
and/or alterations in mitochondria during the development of CDDP-resistance, and hence
its susceptibility to the platinum(II) ion-based chemotherapy. This hypothesis has been
proposed by a number of research groups (Liang and Ullyatt, 1998; Lee et. al., 2004c;
Yang et. al., 2006; Hirama et. al., 2006; Cullen et. al., 2007). Nonetheless, sensitivity to
apoptotic-inducing stimuli, such as CDDP, can be manifested by the state of the
mitochondria, which has direct therapeutic implications with regard to both targeting and
combination therapies.
In order to translate these mitochondrial findings towards clinical practice, investigations
with ddC were performed. Interestingly, since ddC is an approved agent, both mechanistic
studies and a limited body of clinical evaluations have been completed. Lund et. al. (2007)
were able to demonstrate the direct effects of ddC on mtDNA copy number; indeed, there
was a significant depletion of mtDNA with ddC treatment in HepG2 hepatocelluar
carcinoma cells, without this agent exerting an effect on the metabolism of such cells.
These results confirm our data acquired on glioma cells, which became
auxotrophically-dependent on uridine and pyruvate when treated with ddC. Similarly,
Bouayadi et. al. (1997) showed that ddC could effect apoptosis in B16 melanoma cells,
and related this finding not only to DNA polymerase gamma inhibition (that found in
mitochondria), but also to DNA polymerase beta (which is typically found to be
down-regulated in normal cells, but overexpressed in some tumour cells). This
phenomenon was also observed by Louat et. al. (2001), and suggests that ddC has a
multitude of functions by which its anti-neoplastic activities are manifested. However, it is
clear that a significant, and probably dominant, effect of this agent is through the
mitochondrion, since the sensitizing effects of ddC to the apoptotic phenotype require a
decrease in mtDNA levels/transcripts to a critical threshold level (Hentosh and Tibudan,
1997). Indeed, the relative resistance to ddC has been explored in CEM T-lymphoblast
cells (created by their incubation at a low concentration of the anti-nucleoside agent)
(Bjerke et. al., 2008); ddC-resistant cells showed an amplification of mtDNA relative to
the corresponding de novo cells, with significant increases in the expression of mTfam.
These data suggest that mTfam may be a mitochondrially-based mechanism of drug
resistance to compounds such as nucleoside inhibitors as a manifestation of the
tumourigenic phenotype. Previous work has also shown the importance of mTfam in
cancer cells (Dong et. al., 2002). Furthermore, ddC has also been noted to have sensitizing
effects with ionizing radiation; indeed, Coucke et. al. (1997) showed a dose response to
sensitivity of ddC treatment with radiation. This is of particular interest since radiotherapy
generates reactive oxygen species (ROS) in vivo, including aggressively-reactive hydroxy
radical (.OH); inhibition of mitochondrial processes could potentially diminish this effect.
Moreover, Humer et. al. (2008) showed a synergistic effect to azidothymidine (which has
a similar anti-mitochondrial effect to ddC), with CDDP in melanoma cells. However,
these results are confounded by the possible complexation of azidothymidine by the
platinum(II) [Pt(II)] metal ion centre in CDDP. Indeed, in experiments focused on
evaluations of their synergistic inhibitory effects on melanoma cell growth, these agents
     were co-administered in the same medium, and Pt(II) readily forms such complexes with
     thymidine (De Napoli et. al.,1999). Moreover, a further complicating factor is that CDDP
     can also undergo ligand-substitution reactions with selected amino acids (either 'free' or
     protein-incorporated), or further biomolecules, present in the culture medium employed,
     particularly methionine and protein residues of this amino acid for which Pt(II) has a high
     affinity for its side-chain thioether group sulphur donor atom. Mitochondria must
     therefore harbour further complex mechanisms outside of changes in expression which
     alter the phenotype of therapeutic resistance as a manifestation of the tumourigenic
     phenotype.
     Clinically, the use of ddC in combination chemotherapy has shown evidence of efficacy.
     In adult patients with T-cell lymphoma, Besson et. al. (2002) treated patients with CHOP
     induction (cyclophosphamide, hydroxyurea, vincristine and prednisone); in a subset of
     patients, this was followed by consolidation with etoposide and an anti-nucleoside
     (typically ddC, but in some cases azidothymidine). Patients treated with the consolidation
     regimen showed a significantly increased survival when compared to those with induction
     only with CHOP (17 months vs. 3 months, p = 0.004); no major toxicity was noted,
     although there were issues with some cases of peripheral neuropathy. Although this study
     was small (n = 29), it showed the potential benefit of addressing multiple targets,
     including mitochondria, in neoplastic diseases. In addition, patients with AIDS-related
     lymphoma were also treated with low dose methotrexate, bleomycin, doxorubicin,
     cyclophosphamide, vincristine and dexamethasone, in addition to ddC, in a single arm
     study (Levine et. al., 1996). Complete response was noted in 56% of patients, including
     those with poor prognosis factors (AIDS diagnosis prior to lymphoma; < 200/mm3 CD4
     lymphocytes). Interestingly, no peripheral neuropathy was noted, and improvement in
     AIDS-related markers was observed. These observations suggest that ddC may indeed be
     valuable in the treatment of certain cancers, as noted herein; the key will be to ensure that
     the therapeutic index, particularly with the anticipated longer time of therapy and when
     administered in combination with other agents, is wide enough to avoid toxicity. In this
     regard, compounds such as tyrphostin AG17 could be of interest, since this agent exerts
     little or no effect on normal cells (astrocytes); Wallace and Starkov (2000) noted that the
     selective delivery of compounds to the mitochondria, as speculated with AG17, could be a
     key opportunity for therapeutic intervention. This compound continues to be of interest as
     a tyrosine kinase and mitochondrial inhibitor in cancer (Levitt and Koty, 1999) as well as
     in gliomas (Amirlak and Couldwell, 2004).




                                                 ***




Discussion : Mitochondria and Cancer
     The key goal of the above reported studies was to determine the importance of the
     mitochondria in the tumourigenic phenotype. In particular, it was a primary objective to
     determine if the findings regarding the phenotype could be translated into relevant
     approaches to the treatment of cancer patients. These studies have shown the importance
     of mitochondria/mtDNA in genomic as well as cellular biologic phenomena involved in
     human cancer, both in cultured cell models as well as primary tumour tissue, especially
     with regard to the manifestations of tumourigenicity. Moreover, these studies provided the
     first observations of a myriad of different findings, including:


     mtDNA amplification in cancer (primarily gliomas)
     Heterogenous areas of mtDNA amplified (associated with deletions)
     Low-grade tumours and cell lines
     Promiscuous mtDNA into the nuclear genome, associated as an ontologically-early event,
     and found in primary tumour tissues
     Anchorage-independent growth reliant upon mitochondrial/mtDNA-encoded genes
     Increased sensitivity to cytotoxic chemotherapeutic drugs reliant upon
     mitochondrial/mtDNA-encoded genes
     Apoptotic cell death via cytotoxic agents not dependent on mtDNA-encoded molecules
     Nuclear influences responsible for increased copy number of mtDNA in cybrid
     experiments
     Hyperpolarization of mitochondrial membrane potential as an early change in the
     development of the tumourigenic phenotype induced chemically
     Modification of the tumourigenic phenotype at an early stage of neoplastic development
     by inhibitors of glycolytic enzymes
     A decline of ATP levels not responsible for CDDP-induced cell death
     The targeting mitochondria specifically to chemosensitize tumours to further
     apoptosis-inducing agents is an approach to be translated to clinical practice

     These findings have been confirmed with subsequent studies performed using various
     malignancy models. Finally, and of most relevance, the targeting of mitochondria as a
     therapy for cancer has become a design mechanism for both clinical and translational
     scientists, intent on improving the outcome of patients suffering from this set of critical
     and terminal diseases (Fulda and Kroemer, 2009; Gogvadze et. al., 2009).



                                                  ***




A Model of the Tumourigenic Phenotype


     The investigations reported herein have investigated the role that mitochondria play in the
     tumourigenic phenotype, and the potential of these phenomena for use as a target in the
     treatment of cancer. Mitochondrial alterations are ontologically-early processes and
     manifest throughout the multistep pathway to malignancy – with both nuclear genetic and
     genomic alterations, as well as those at the level of the mitochondria and its genome.
     These changes evolve over the lifecycle of the malignant cell and milieu. As noted,
     Nenasheva et. al. (2004) demonstrated that SV40-transformed fibroblasts showed
     evidence of mtDNA increases, an observation which was in accordance with this
dissertation’s findings of early changes noted in lower grade glial tumors. In contrast, Lee
et. al. (2004b, 2005) found varying results in hepatocellular, lung, colorectal and gastric
cancers, where some but not all tumour types showed an amplification of mtDNA. Of
particular interest was association of the higher levels of mtDNA with less somatic
mutation at the D-loop, and that mtDNA reductions occurred (with increases in D-loop
mutations) in later stage cancers (Lee et. al., 2004b). Combined with the data presented in
this dissertation, these findings support a model where mtDNA amplification represents an
early aspect of the generation and support of the tumourigenic phenotype, potentially
ascribable to oxidative stress and nuclear-mitochondrial altered communication (as in petit
mutants in yeast), followed by additional manifestations of malignancy, where glycolytic
pathways predominate, and nuclear mutations support tumourigenicity. While this would
not obviate maintenance of high mtDNA levels, the external and genomic milieu would
determine whether the tumour cell is required to have amplification (particularly, for
example, in the context of certain types of drug resistance) (Jia et. al., 1999; Troyano et.
al., 2001; Bjerke et. al., 2008; Dong et. al., 2002). It has recently been noted that
promiscuous mtDNA anomalies are of particular relevance at regulatory sequences in
gene-rich locations of the genome, within both introns and exons, including, for example,
transcriptional modulators and expressed genes responsible for diverse functions such as
thiamine transporters, serine proteases and T-cell receptor beta-chain (Richetti et. al.,
2004; Liang, unpublished results). Hence, alterations in mtDNA copy number and
expression, as well as promiscuous mtDNA, are clearly an early event in the tumourigenic
phenotype, and this observation has been validated in a variety of neoplasms subsequent
to the findings noted in gliomas herein. Moreover, epigenetic findings with mtDNA
depletion have been observed, with aberrant methylation at promoter regions of genes
putatively important in cancer (e.g. endothelin B receptor, 06-methylguanine-DNA
methyltransferase, and E-cadherin) (Xie et. al., 2007). Hence, this area of cancer biology,
as well as the mechanisms up- and downstream of mtDNA amplification, continues to be
evaluated to improve our understanding of the mechanisms involved as well as the
ramifications in human cancer cells.
Data from studies reported in Specific Aim 2, as well as those of Zhu et. al. (2002) suggest
that the initiating event, at least for transformation towards immortality, would be nuclear
in nature. Indeed, evidence from mouse epidermal growth factor-transformed cells
suggests the initial influence on mtDNA copy number may be attributable to regulation of
nuclear encoded proteases (e.g. mitochondrially-located LON protease); similarly,
heregulin, which activates the erb-b2 pathway, can also increase expression of mtDNA
genes, such as COXII (Sun et. al., 2002). On the phenotypic level, in colorectal cancer, a
lower mtDNA content was associated with later clinical stage, which in turn was
associated with a reduced five year disease free survival (Lin et. al., 2008). In
radiation-induced preleukemic lesions, increases in cytochrome c oxidase activity was
noted; with maturation of the thymic lymphomas, there was a reduction of expression of
the COXI/II/III genes, data suggesting that the mitochondrial changes are early in this
model (Verlaet et. al., 2002). Furthermore, in cells which harbour HMGA1
over-expression (associated with advanced malignancy and increased metastatic
potential), mtDNA levels (as well as mitochondrial mass) were found to be reduced,
although, interestingly, and consistent with the findings herein, susceptibility to apoptosis
was increased (particularly to inhibitors of glycolysis) (Dement et. al., 2007). Indeed, Wu
et. al. (2005) showed in gastric cancers that mtDNA was depleted in the most infiltrating
class of tumours. Collectively, these data suggest that mtDNA alterations, in particular
increases in copy number and/or expression, are early in the manifestation of the
tumourigenic phenotype, both with respect to ontogeny and stage of disease; with an
infiltrating or migratory phenotype, levels of mtDNA appear to become normalized in
some circumstances, suggesting a distinct difference in phenotype. This has been observed
in a variety of other studies evaluating the biology of the migratory vs. growth neoplastic
phenotypes (Hehlgans et. al., 2007; Harradine and Akhurst, 2006). It could thus be
postulated that reversion back to a growth phenotype would effect the specific alterations
noted previously, including changes in mtDNA levels and resultant phenotype. In one
sense, this would suggest that using anti-mitochondrial approaches to treat cancers may
require specific adaptations to target early or clinically established lesions, and additional
approaches for metastatic cells, which may be more sensitive to certain types of apoptotic
stimuli (but not expected to be similarly responsive to the anti-mitochondrial approaches,
per se). Figure 1 shows a model of the development of malignancy, highlighting the
potential mitochondrial involvement as supported by data presented herein and further
published literature.




Figure 7. A model of the development of the tumourigenic phenotype, including mitochondrial
involvement.

As a future approach for therapy, there have been a significant number of studies now
evaluating the targeting of the tumourigenic phenotype via the mitochondria both as
treatment (Lee et. al., 2004b; Marin-Hernandez et. al., 2003; Preston and Singh, 2001; Teo
et. al., 2006) as well as in a preventative approach (Sun et. al., 2004; Hail, 2005; Hail and
Lotan, 2009). Petit et. al. (1999) as well as Liminga et. al. (1999) who, noting work
performed herein, postulated that the toxicity of certain anti-mitochondrial compounds,
might be utilised to treat and/or sensitize cancer cells to the effects of other agents. Joshi
et. al. (1999) also targeted mitochondria using a novel approach with fatty acid agents
(most likely to include the apoptosis-inducing agent ceramide) to effect cell death, which
was interestingly dependent on an intact respiratory chain; Fantin et. al. (2002) showed,
using an approach previously described with AG17, that a compound (F16) with a
delocalized positive charge would accumulate in the anionic mitochondria, effecting cell
death in an upstream independent manner, and “may represent a specific, low toxicity
approach in cancer treatment”, a postulate agreeing with the statement from this researcher
    that “circumvention of resistance to drug-induced apoptosis, by targeting mitochondria in
    the laboratory and clinical setting, will be interesting to determine the viability of such an
    approach in cancer therapy”. Indeed, with the data noted with ddC, there is some level of
    proof-of-concept that this approach could have significant viability in the future, and there
    are current efforts ongoing to use this postulated approach to modify the tumourigenic
    phenotype in patients with cancer (Fulda and Kroemer, 2009; Gogvadze et. al., 2009).
    Certainly, given the model hypothesised, targeting mitochondria at both early stages
    subsequent to the development of immortality, as well as at the time of the cell growth
    phenotype, would appear to represent the most prudent time for therapy; in addition, if
    cells which harbour a migratory phenotype have a hyperpolarized mitochondrial
    membrane potential (Fantin et. al., 2002), appropriate use of anti-mitochondrial agents
    such as lipophillic cations (e.g., AG17 or F16) could have significant utility when
    administered alone, or (most likely) in combination treatment regimens.


                                                 ***




Monograph Bibliography

    AMBROSONE, C. B., FREUDENHEIM, J. L., THOMPSON, P. A., BOWMAN, E.,
    VENA, J. E., MARSHALL, J. R., GRAHAM, S., LAUGHLIN, R., NEMOTO, T. &
    SHIELDS, P. G. 1999. Manganese superoxide dismutase (MnSOD) genetic
    polymorphisms, dietary antioxidants, and risk of breast cancer. Cancer Res, 59, 602-6.
    AMIRLAK, B. & COULDWELL, W. T. 2003. Apoptosis in glioma cells: review and
    analysis of techniques used for study with focus on the laser scanning cytometer. J
    Neurooncol, 63, 129-45.
    AMUTHAN, G., BISWAS, G., ZHANG, S. Y., KLEIN-SZANTO, A.,
    VIJAYASARATHY, C. & AVADHANI, N. G. 2001. Mitochondria-to-nucleus stress
    signaling induces phenotypic changes, tumor progression and cell invasion. EMBO J, 20,
    1910-20.
    ARA, G. & TEICHER, B. 1996. Relationship of cellular energy parameters to cytotoxicity
    for AG-17, lonidamine and cyclocreatine in four human tumor cell lines International
    Journal of Oncology, 8, 865 - 873.
    BEHESHTI, B., BRAUDE, I., MARRANO, P., THORNER, P., ZIELENSKA, M. &
    SQUIRE, J. A. 2003. Chromosomal localization of DNA amplifications in neuroblastoma
    tumors using cDNA microarray comparative genomic hybridization. Neoplasia, 5, 53-62.
    BESSON, C., PANELATTI, G., DELAUNAY, C., GONIN, C., BREBION, A.,
    HERMINE, O. & PLUMELLE, Y. 2002. Treatment of adult T-cell leukemia-lymphoma
    by CHOP followed by therapy with antinucleosides, alpha interferon and oral etoposide.
    Leuk Lymphoma, 43, 2275-9.
    BIANCHI, N. O., BIANCHI, M. S. & RICHARD, S. M. 2001. Mitochondrial genome
    instability in human cancers. Mutat Res, 488, 9-23.
    BISWAS, G., ANANDATHEERTHAVARADA, H. K. & AVADHANI, N. G. 2005.
    Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial
DNA-depleted C2C12 myocytes. Cell Death Differ, 12, 266-78.
BJERKE, M., FRANCO, M., JOHANSSON, M., BALZARINI, J. & KARLSSON, A.
2008. Increased mitochondrial DNA copy-number in CEM cells resistant to delayed
toxicity of 2',3'-dideoxycytidine. Biochem Pharmacol, 75, 1313-21.
BORENSZTAJN, K., CHAFA, O., ALHENC-GELAS, M., SALHA, S., REGHIS, A.,
FISCHER, A. M. & TAPON-BRETAUDIERE, J. 2002. Characterization of two novel
splice site mutations in human factor VII gene causing severe plasma factor VII deficiency
and bleeding diathesis. Br J Haematol, 117, 168-71.
BOUAYADI, K., HOFFMANN, J. S., FONS, P., TIRABY, M., REYNES, J. P. &
CAZAUX, C. 1997. Overexpression of DNA polymerase beta sensitizes mammalian cells
to 2',3'-deoxycytidine and 3'-azido-3'-deoxythymidine. Cancer Res, 57, 110-6.
BURGER, A. M., KAUR, G., ALLEY, M. C., SUPKO, J. G., MALSPEIS, L., GREVER,
M. R. & SAUSVILLE, E. A. 1995. Tyrphostin AG17,
[(3,5-Di-tert-butyl-4-hydroxybenzylidene)- malononitrile], inhibits cell growth by
disrupting mitochondria. Cancer Res, 55, 2794-9.
BUTOW, R. A. & AVADHANI, N. G. 2004. Mitochondrial signaling: the retrograde
response. Mol Cell, 14, 1-15.
CAI, Q., SHU, X. O., WEN, W., CHENG, J. R., DAI, Q., GAO, Y. T. & ZHENG, W.
2004. Genetic polymorphism in the manganese superoxide dismutase gene, antioxidant
intake, and breast cancer risk: results from the Shanghai Breast Cancer Study. Breast
Cancer Res, 6, R647-55.
CAMPBELL, C. L. & THORSNESS, P. E. 1998. Escape of mitochondrial DNA to the
nucleus in yme1 yeast is mediated by vacuolar-dependent turnover of abnormal
mitochondrial compartments. J Cell Sci, 111 ( Pt 16), 2455-64.
CANTER, J. A., KALLIANPUR, A. R., PARL, F. F. & MILLIKAN, R. C. 2005.
Mitochondrial DNA G10398A polymorphism and invasive breast cancer in
African-American women. Cancer Res, 65, 8028-33.
CAREW, J. S. & HUANG, P. 2002. Mitochondrial defects in cancer. Mol Cancer, 1, 9.
CAVALLI, L. R. & LIANG, B. C. 1998. Mutagenesis, tumorigenicity, and apoptosis: are
the mitochondria involved? Mutat Res, 398, 19-26.
CAVALLI, L. R., VARELLA-GARCIA, M. & LIANG, B. C. 1997. Diminished
tumorigenic phenotype after depletion of mitochondrial DNA. Cell Growth Differ, 8,
1189-98.
CHATTERJEE, A., MAMBO, E. & SIDRANSKY, D. 2006. Mitochondrial DNA
mutations in human cancer. Oncogene, 25, 4663-74.
CHUNG, K., LEE, K., SHIM, S., KIM, J. & SONG, E. 2000. Analysis of mitochondrial
DNA mutation in hepatoma Journal of Biochemistry, 33, 417-421.
CLAYTON, D. A. & SMITH, C. A. 1975. Complex mitochondrial DNA. Int Rev Exp
Pathol, 14, 1-67.
CLAYTON, D. A. & VINOGRAD, J. 1967. Circular dimer and catenate forms of
mitochondrial DNA in human leukaemic leucocytes. Nature, 216, 652-7.
COPELAND, W. C., WACHSMAN, J. T., JOHNSON, F. M. & PENTA, J. S. 2002.
Mitochondrial DNA alterations in cancer. Cancer Invest, 20, 557-69.
CORRAL, M., PARIS, B., BAFFET, G., TICHONICKY, L., GUGUEN-GUILLOUZO,
C., KRUH, J. & DEFER, N. 1989. Increased level of the mitochondrial ND5 transcript in
chemically induced rat hepatomas. Exp Cell Res, 184, 158-66.
COUCKE, P. A., LI, Y. X., COPACEANU, M. L., PASCHOUD, N., COTTIN, E.,
OZSAHIN, M. & MIRIMANOFF, R. O. 1997. Cell line specific radiosensitizing effect of
zalcitabine (2',3'-dideoxycytidine). Acta Oncol, 36, 199-205.
CULLEN, K. J., YANG, Z., SCHUMAKER, L. & GUO, Z. 2007. Mitochondria as a
critical target of the chemotheraputic agent cisplatin in head and neck cancer. J Bioenerg
Biomembr, 39, 43-50.
CUMMINGS, B. S. & SCHNELLMANN, R. G. 2002. Cisplatin-induced renal cell
apoptosis: caspase 3-dependent and -independent pathways. J Pharmacol Exp Ther, 302,
8-17.
CZARNECKA, A. M., GAMMAZZA, A. M., FELICE, V. D., ZUMMO, G. &
CAPPELLO, F. 2007. Cancer as a “Mitochondriopathy”. J. Cancer Mol, 3, 71-79.
DAI, J. G., XIAO, Y. B., MIN, J. X., ZHANG, G. Q., YAO, K. & ZHOU, R. J. 2006.
Mitochondrial DNA 4977 BP deletion mutations in lung carcinoma. Indian J Cancer, 43,
20-5.
De Napoli, L., Lacovino, R., Messere, A., Montesarchio, D., Picciali, G., Romanelli, A.,
Ruffo, F., Saviano, M. 1999. Synthesis of platinum(II) complexes of thymidine and
1-methylthymine (1-MeThy); crystal structure of cis-[PtCl(1-MeThy)(PPh3)2]. J Chem
Soc Dalton Trans 1999,1945-1950
DANI, M. A., DANI, S. U., LIMA, S. P., MARTINEZ, A., ROSSI, B. M., SOARES, F.,
ZAGO, M. A. & SIMPSON, A. J. 2004. Less DeltamtDNA4977 than normal in various
types of tumors suggests that cancer cells are essentially free of this mutation. Genet Mol
Res, 3, 395-409.
DARVISHI, K., SHARMA, S., BHAT, A. K., RAI, E. & BAMEZAI, R. N. 2007.
Mitochondrial DNA G10398A polymorphism imparts maternal Haplogroup N a risk for
breast and esophageal cancer. Cancer Lett, 249, 249-55.
DEL GIUDICE, L., MASSARDO, D. R., PONTIERI, P. & WOLF, K. 2005. Interaction
between yeast mitochondrial and nuclear genomes: null alleles of RTG genes affect
resistance to the alkaloid lycorine in rho0 petites of Saccharomyces cerevisiae. Gene, 354,
9-14.
DEMENT, G. A., MALONEY, S. C. & REEVES, R. 2007. Nuclear HMGA1 nonhistone
chromatin proteins directly influence mitochondrial transcription, maintenance, and
function. Exp Cell Res, 313, 77-87.
DONG, X., GHOSHAL, K., MAJUMDER, S., YADAV, S. P. & JACOB, S. T. 2002.
Mitochondrial transcription factor A and its downstream targets are up-regulated in a rat
hepatoma. J Biol Chem, 277, 43309-18.
DUNCAN, E. L., PERREM, K. & REDDEL, R. R. 2000. Identification of a novel human
mitochondrial D-loop RNA species which exhibits upregulated expression following
cellular immortalization. Biochem Biophys Res Commun, 276, 439-46.
ENGEL, L. W. & YOUNG, N. A. 1978. Human breast carcinoma cells in continuous
culture: a review. Cancer Res, 38, 4327-39.
EPPERT, K., SCHERER, S. W., OZCELIK, H., PIRONE, R., HOODLESS, P., KIM, H.,
TSUI, L. C., BAPAT, B., GALLINGER, S., ANDRULIS, I. L., THOMSEN, G. H.,
WRANA, J. L. & ATTISANO, L. 1996. MADR2 maps to 18q21 and encodes a
TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal
carcinoma. Cell, 86, 543-52.
FANTIN, V. R., BERARDI, M. J., SCORRANO, L., KORSMEYER, S. J. & LEDER, P.
2002. A novel mitochondriotoxic small molecule that selectively inhibits tumor cell
growth. Cancer Cell, 2, 29-42.
FANTIN, V. R., ST-PIERRE, J. & LEDER, P. 2006. Attenuation of LDH-A expression
uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance.
Cancer Cell, 9, 425-34.
FELTY, Q. & ROY, D. 2005. Estrogen, mitochondria, and growth of cancer and
non-cancer cells. J Carcinog, 4, 1.
FIRKIN, F. C. & CLARK-WALKER, G. D. 1979. Abnormal mitochondrial DNA in acute
leukaemia and lymphoma. Br J Haematol, 43, 201-6.
FULDA, S. & KROEMER, G. 2009. Targeting mitochondrial apoptosis by betulinic acid
in human cancers. Drug Discov Today.
GIANNI, A. M., DALLA-FAVERA, R. & POLLI, E. 1980. Restriction enzyme analysis
of human leukemic mitochondrial DNA. Leuk Res, 4, 155-60.
GOCHHAIT, S., BHATT, A., SHARMA, S., SINGH, Y. P., GUPTA, P. & BAMEZAI,
R. N. 2008. Concomitant presence of mutations in mitochondrial genome and p53 in
cancer development - a study in north Indian sporadic breast and esophageal cancer
patients. Int J Cancer, 123, 2580-6.
GOGVADZE, V., ORRENIUS, S. & ZHIVOTOVSKY, B. 2009. Mitochondria as targets
for chemotherapy. Apoptosis, 14, 624-40.
GRANDEMANGE, S., SEYER, P., CARAZO, A., BECUWE, P., PESSEMESSE, L.,
BUSSON, M., MARSAC, C., ROGER, P., CASAS, F., CABELLO, G. &
WRUTNIAK-CABELLO, C. 2005. Stimulation of mitochondrial activity by p43
overexpression induces human dermal fibroblast transformation. Cancer Res, 65, 4282-91.
GUO, X. G. & GUO, Q. N. 2006. Mutations in the mitochondrial DNA D-Loop region
occur frequently in human osteosarcoma. Cancer Lett, 239, 151-5.
HADLER, H. I., DEVADAS, K. & MAHALINGAM, R. 1998. Selected nuclear LINE
elements with mitochondrial-DNA-like inserts are more plentiful and mobile in tumor
than in normal tissue of mouse and rat. J Cell Biochem, 68, 100-9.
HAIL, N., JR. 2005. Mitochondria: A novel target for the chemoprevention of cancer.
Apoptosis, 10, 687-705.
HAIL, N., JR. & LOTAN, R. 2009. Cancer chemoprevention and mitochondria: targeting
apoptosis in transformed cells via the disruption of mitochondrial bioenergetics/redox
state. Mol Nutr Food Res, 53, 49-67.
HALABE BUCAY, A. 2007. The biological significance of cancer: mitochondria as a
cause of cancer and the inhibition of glycolysis with citrate as a cancer treatment. Med
Hypotheses, 69, 826-8.
HARRADINE, K. A. & AKHURST, R. J. 2006. Mutations of TGFbeta signaling
molecules in human disease. Ann Med, 38, 403-14.
HAUGEN, D. R., FLUGE, O., REIGSTAD, L. J., VARHAUG, J. E. & LILLEHAUG, J.
R. 2003. Increased expression of genes encoding mitochondrial proteins in papillary
thyroid carcinomas. Thyroid, 13, 613-20.
HEHLGANS, S., HAASE, M. & CORDES, N. 2007. Signalling via integrins:
implications for cell survival and anticancer strategies. Biochim Biophys Acta, 1775,
163-80.
HENTOSH, P. & TIBUDAN, M. 1997. 2-Chloro-2'-deoxyadenosine, an antileukemic
drug, has an early effect on cellular mitochondrial function. Mol Pharmacol, 51, 613-9.
HICKMAN, J. A. 1996. Apoptosis and chemotherapy resistance. Eur J Cancer, 32A,
921-6.
HIRAMA, M., ISONISHI, S., YASUDA, M. & ISHIKAWA, H. 2006. Characterization of
mitochondria in cisplatin-resistant human ovarian carcinoma cells. Oncol Rep, 16,
997-1002.
HIRANO, T., SHIRAISHI, K., ADACHI, K., MIURA, S., WATANABE, H. &
UTIYAMA, H. 1999. Co-localization of mitochondrial and double minute DNA in the
nuclei of HL-60 cells but not normal cells. Mutat Res, 425, 195-204.
HUMER, J., FERKO, B., WALTENBERGER, A., RAPBERGER, R., PEHAMBERGER,
H. & MUSTER, T. 2008. Azidothymidine inhibits melanoma cell growth in vitro and in
vivo. Melanoma Res, 18, 314-21.
ISMAIL, A. & BATEMAN, A. 2009. Expression of TBX2 promotes
anchorage-independent growth and survival in the p53-negative SW13 adrenocortical
carcinoma. Cancer Lett, 278, 230-40.
IVANOVA, R., LEPAGE, V., LOSTE, M. N., SCHACHTER, F., WIJNEN, E.,
BUSSON, M., CAYUELA, J. M., SIGAUX, F. & CHARRON, D. 1998. Mitochondrial
DNA sequence variation in human leukemic cells. Int J Cancer, 76, 495-8.
JAKUPCIAK, J. P., WANG, W., MARKOWITZ, M. E., ALLY, D., COBLE, M.,
SRIVASTAVA, S., MAITRA, A., BARKER, P. E., SIDRANSKY, D. & O'CONNELL,
C. D. 2005. Mitochondrial DNA as a cancer biomarker. J Mol Diagn, 7, 258-67.
JIA, L., LIU, K. Z., NEWLAND, A. C., MANTSCH, H. H. & KELSEY, S. M. 1999.
Pgp-positive leukaemic cells have increased mtDNA but no increased rate of proliferation.
Br J Haematol, 107, 861-9.
JIANG, W. W., MASAYESVA, B., ZAHURAK, M., CARVALHO, A. L.,
ROSENBAUM, E., MAMBO, E., ZHOU, S., MINHAS, K., BENOIT, N., WESTRA, W.
H., ALBERG, A., SIDRANSKY, D., KOCH, W. & CALIFANO, J. 2005. Increased
mitochondrial DNA content in saliva associated with head and neck cancer. Clin Cancer
Res, 11, 2486-91.
JIANG, W. W., ROSENBAUM, E., MAMBO, E., ZAHURAK, M., MASAYESVA, B.,
CARVALHO, A. L., ZHOU, S., WESTRA, W. H., ALBERG, A. J., SIDRANSKY, D.,
KOCH, W. & CALIFANO, J. A. 2006. Decreased mitochondrial DNA content in
posttreatment salivary rinses from head and neck cancer patients. Clin Cancer Res, 12,
1564-9.
JOSHI, B., LI, L., TAFFE, B. G., ZHU, Z., WAHL, S., TIAN, H., BEN-JOSEF, E.,
TAYLOR, J. D., PORTER, A. T. & TANG, D. G. 1999. Apoptosis induction by a novel
anti-prostate cancer compound, BMD188 (a fatty acid-containing hydroxamic acid),
requires the mitochondrial respiratory chain. Cancer Res, 59, 4343-55.
KAMALIDEHGHAN, B., HOUSHMAND, M., PANAHI, M. S., ABBASZADEGAN, M.
R., ISMAIL, P. & SHIROUDI, M. B. 2006. Tumoral cell mtDNA approximately 8.9 kb
deletion is more common than other deletions in gastric cancer. Arch Med Res, 37,
848-53.
KARTHIKEYAN, G. & RESNICK, M. A. 2005. Impact of mitochondria on nuclear
genome stability. DNA Repair (Amst), 4, 141-8.
KESSEL, D. & SUN, H. H. 1999. Enhanced responsiveness to photodynamic
therapy-induced apoptosis after mitochondrial DNA depletion. Photochem Photobiol, 70,
937-40.
KHARBANGAR, A., KHYNRIAM, D. & PRASAD, S. B. 2000. Effect of cisplatin on
mitochondrial protein, glutathione, and succinate dehydrogenase in Dalton
lymphoma-bearing mice. Cell Biol Toxicol, 16, 363-73.
KIEBISH, M. A. & SEYFRIED, T. N. 2005. Absence of pathogenic mitochondrial DNA
mutations in mouse brain tumors. BMC Cancer, 5, 102.
KIRCHES, E., KRAUSE, G., WARICH-KIRCHES, M., WEIS, S., SCHNEIDER, T.,
MEYER-PUTTLITZ, B., MAWRIN, C. & DIETZMANN, K. 2001. High frequency of
mitochondrial DNA mutations in glioblastoma multiforme identified by direct sequence
comparison to blood samples. Int J Cancer, 93, 534-8.
KRUSE, C. A., MITCHELL, D. H., KLEINSCHMIDT-DEMASTERS, B. K.,
FRANKLIN, W. A., MORSE, H. G., SPECTOR, E. B. & LILLEHEI, K. O. 1992.
Characterization of a continuous human glioma cell line DBTRG-05MG: growth kinetics,
karyotype, receptor expression, and tumor suppressor gene analyses. In Vitro Cell Dev
Biol, 28A, 609-14.
LEE, H. C., LI, S. H., LIN, J. C., WU, C. C., YEH, D. C. & WEI, Y. H. 2004a. Somatic
mutations in the D-loop and decrease in the copy number of mitochondrial DNA in human
hepatocellular carcinoma. Mutat Res, 547, 71-8.
LEE, H. C., YIN, P. H., LIN, J. C., WU, C. C., CHEN, C. Y., WU, C. W., CHI, C. W.,
TAM, T. N. & WEI, Y. H. 2005. Mitochondrial genome instability and mtDNA depletion
in human cancers. Ann N Y Acad Sci, 1042, 109-22.
LEE, M. D., SHE, Y., SOSKIS, M. J., BORELLA, C. P., GARDNER, J. R., HAYES, P.
A., DY, B. M., HEANEY, M. L., PHILIPS, M. R., BORNMANN, W. G., SIROTNAK, F.
M. & SCHEINBERG, D. A. 2004b. Human mitochondrial peptide deformylase, a new
anticancer target of actinonin-based antibiotics. J Clin Invest, 114, 1107-16.
LEE, M. S., KIM, J. Y. & PARK, S. Y. 2004c. Resistance of rho(0) cells against
apoptosis. Ann N Y Acad Sci, 1011, 146-53.
LEVINE, A. M., TULPULE, A., ESPINA, B., BOSWELL, W., BUCKLEY, J.,
RASHEED, S., STAIN, S., PARKER, J., NATHWANI, B. & GILL, P. S. 1996. Low dose
methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone
with zalcitabine in patients with acquired immunodeficiency syndrome-related lymphoma.
Effect on human immunodeficiency virus and serum interleukin-6 levels over time.
Cancer, 78, 517-26.
LEVITT, M. L. & KOTY, P. P. 1999. Tyrosine kinase inhibitors in preclinical
development. Invest New Drugs, 17, 213-26.
LI, M., WANG, J., NG, S. S., CHAN, C. Y., CHEN, A. C., XIA, H. P., YEW, D. T.,
WONG, B. C., CHEN, Z., KUNG, H. F. & LIN, M. C. 2008. The four-and-a-half-LIM
protein 2 (FHL2) is overexpressed in gliomas and associated with oncogenic activities.
Glia, 56, 1328-38.
LIANG B. C. 1995. Direct isolation of variably amplified cDNAs from human tumor cell
lines. International Journal of Oncology, 7, 611-615.
LIANG, B. C. 1996. Evidence for association of mitochondrial DNA sequence
amplification and nuclear localization in human low-grade gliomas. Mutat Res, 354,
27-33.
LIANG, B. C. & HAYS, L. 1996. Mitochondrial DNA copy number changes in human
gliomas. Cancer Lett, 105, 167-73.
LIANG, B. C., MELTZER, P. S., GUAN, X. Y. & TRENT, J. M. 1995. Gene
amplification elucidated by combined chromosomal microdissection and comparative
genomic hybridization. Cancer Genet Cytogenet, 80, 55-9.
LIANG, B. C. & ULLYATT, E. 1998. Chemosensitization of glioblastoma cells to
bis-dichloroethyl-nitrosourea with tyrphostin AG17. Clin Cancer Res, 4, 773-81.
LIEVRE, A., BLONS, H., HOULLIER, A. M., LACCOURREYE, O., BRASNU, D.,
BEAUNE, P. & LAURENT-PUIG, P. 2006. Clinicopathological significance of
mitochondrial D-Loop mutations in head and neck carcinoma. Br J Cancer, 94, 692-7.
LIMINGA, G., JONSSON, B., NYGREN, P. & LARSSON, R. 1999. On the mechanism
underlying calcein-induced cytotoxicity. Eur J Pharmacol, 383, 321-9.
LIN, P. C., LIN, J. K., YANG, S. H., WANG, H. S., LI, A. F. & CHANG, S. C. 2008.
Expression of beta-F1-ATPase and mitochondrial transcription factor A and the change in
mitochondrial DNA content in colorectal cancer: clinical data analysis and evidence from
an in vitro study. Int J Colorectal Dis, 23, 1223-32.
LIU, V. W., SHI, H. H., CHEUNG, A. N., CHIU, P. M., LEUNG, T. W., NAGLEY, P.,
WONG, L. C. & NGAN, H. Y. 2001. High incidence of somatic mitochondrial DNA
mutations in human ovarian carcinomas. Cancer Res, 61, 5998-6001.
LOUAT, T., SERVANT, L., ROLS, M. P., BIETH, A., TEISSIE, J., HOFFMANN, J. S.
& CAZAUX, C. 2001. Antitumor activity of 2',3'-dideoxycytidine nucleotide analog
against tumors up-regulating DNA polymerase beta. Mol Pharmacol, 60, 553-8.
LUND, K. C., PETERSON, L. L. & WALLACE, K. B. 2007. Absence of a universal
mechanism of mitochondrial toxicity by nucleoside analogs. Antimicrob Agents
Chemother, 51, 2531-9.
MAGDA, D., LECANE, P., PRESCOTT, J., THIEMANN, P., MA, X., DRANCHAK, P.
K., TOLENO, D. M., RAMASWAMY, K., SIEGMUND, K. D. & HACIA, J. G. 2008.
mtDNA depletion confers specific gene expression profiles in human cells grown in
culture and in xenograft. BMC Genomics, 9, 521.
MARIN-HERNANDEZ, A., GRACIA-MORA, I., RUIZ-RAMIREZ, L. &
MORENO-SANCHEZ, R. 2003. Toxic effects of copper-based antineoplastic drugs
(Casiopeinas) on mitochondrial functions. Biochem Pharmacol, 65, 1979-89.
MATARRESE, P., TESTA, U., CAUDA, R., VELLA, S., GAMBARDELLA, L. &
MALORNI, W. 2001. Expression of P-170 glycoprotein sensitizes lymphoblastoid CEM
cells to mitochondria-mediated apoptosis. Biochem J, 355, 587-95.
MAXIMO, V., SOARES, P., SERUCA, R., ROCHA, A. S., CASTRO, P. &
SOBRINHO-SIMOES, M. 2001. Microsatellite instability, mitochondrial DNA large
deletions, and mitochondrial DNA mutations in gastric carcinoma. Genes Chromosomes
Cancer, 32, 136-43.
MILLS, K. I., WOODGATE, L. J., GILKES, A. F., WALSH, V., SWEENEY, M. C.,
BROWN, G. & BURNETT, A. K. 1999. Inhibition of mitochondrial function in HL60
cells is associated with an increased apoptosis and expression of CD14. Biochem Biophys
Res Commun, 263, 294-300.
MIZUMACHI, T., SUZUKI, S., NAITO, A., CARCEL-TRULLOLS, J., EVANS, T. T.,
SPRING, P. M., ORIDATE, N., FURUTA, Y., FUKUDA, S. & HIGUCHI, M. 2008.
Increased mitochondrial DNA induces acquired docetaxel resistance in head and neck
cancer cells. Oncogene, 27, 831-8.
MIZUTANI, S., MIYATO, Y., SHIDARA, Y., ASOH, S., TOKUNAGA, A., TAJIRI, T.
& OHTA, S. 2009. Mutations in the mitochondrial genome confer resistance of cancer
cells to anticancer drugs. Cancer Sci.
MODICA-NAPOLITANO, J. S., KOYA, K., WEISBERG, E., BRUNELLI, B. T., LI, Y.
& CHEN, L. B. 1996. Selective damage to carcinoma mitochondria by the rhodacyanine
MKT-077. Cancer Res, 56, 544-50.
MODICA-NAPOLITANO, J. S. & SINGH, K. K. 2002. Mitochondria as targets for
detection and treatment of cancer. Expert Rev Mol Med, 4, 1-19.
MORGAN, J., POTTER, W. R. & OSEROFF, A. R. 2000. Comparison of photodynamic
targets in a carcinoma cell line and its mitochondrial DNA-deficient derivative.
Photochem Photobiol, 71, 747-57.
NASS, M. M. 1972. Differential effects of ethidium bromide on mitochondrial and
nuclear DNA synthesis in vivo in cultured mammalian cells. Exp Cell Res, 72, 211-22.
NENASHEVA, V. V., NIKOLAEV, A. I., DUBOVAIA, V. I., INOZEMTSEVA, L. S.,
MANUILOVA, E. S., MARTYNENKO, A. V. & TARANTUL, V. Z. 2004. [Analysis of
expression of a series of lymphoma-specific genes in human fibroblasts immortalized by
SV40 virus]. Mol Biol (Mosk), 38, 265-75.
NETTER, P. & ROBINEAU, S. 1989. The differential overamplification of short
sequences in the mitochondrial DNA of rho- petites in Saccharomyces cerevisiae
stimulates recombination. Gene, 83, 25-38.
NGUYEN, D. M. & HUSSAIN, M. 2007. The role of the mitochondria in mediating
cytotoxicity of anti-cancer therapies. J Bioenerg Biomembr, 39, 13-21.
NIKIFOROV, M. A., HAGEN, K., OSSOVSKAYA, V. S., CONNOR, T. M., LOWE, S.
W., DEICHMAN, G. I. & GUDKOV, A. V. 1996. p53 modulation of anchorage
independent growth and experimental metastasis. Oncogene, 13, 1709-19.
NIKOLAEV, A. I., MARTYNENKO, A. V., KALMYRZAEV, B. B., INOZEMTSEVA,
L. I., DUBOVAIA, V. I., HUNSMANN, G., BODEMER, V. & TARANDUL, V. Z. 2001.
[Amplification of transcription of separate mitochondrial genes in human and monkey
B-cell non-Hodgkin's lymphoma]. Mol Biol (Mosk), 35, 120-7.
OHNAMI, S., MATSUMOTO, N., NAKANO, M., AOKI, K., NAGASAKI, K.,
SUGIMURA, T., TERADA, M. & YOSHIDA, T. 1999. Identification of genes showing
differential expression in antisense K-ras-transduced pancreatic cancer cells with
suppressed tumorigenicity. Cancer Res, 59, 5565-71.
PAUL, M. K. & MUKHOPADHYAY, A. K. 2007. Cancer — the mitochondrial
connection Biologia, 62, 371-380.
PEJOVIC, T., LADNER, D., INTENGAN, M., ZHENG, K., FAIRCHILD, T., DILLON,
D., EASLEY, S., MARCHETTI, D., SCHWARTZ, P., LELE, S., COSTA, J. & ODUNSI,
K. 2004. Somatic D-loop mitochondrial DNA mutations are frequent in uterine serous
carcinoma. Eur J Cancer, 40, 2519-24.
PENTA, J. S., JOHNSON, F. M., WACHSMAN, J. T. & COPELAND, W. C. 2001.
Mitochondrial DNA in human malignancy. Mutat Res, 488, 119-33.
PETIT, T., IZBICKA, E., LAWRENCE, R. A., NALIN, C., WEITMAN, S. D. & VON
HOFF, D. D. 1999. Activity of MKT 077, a rhodacyanine dye, against human tumor
colony-forming units. Anticancer Drugs, 10, 309-15.
POETSCH, M., DITTBERNER, T., PETERSMANN, A. & WOENCKHAUS, C. 2004.
Mitochondrial DNA instability in malignant melanoma of the skin is mostly restricted to
nodular and metastatic stages. Melanoma Res, 14, 501-8.
POLYAK, K., LI, Y., ZHU, H., LENGAUER, C., WILLSON, J. K., MARKOWITZ, S.
D., TRUSH, M. A., KINZLER, K. W. & VOGELSTEIN, B. 1998. Somatic mutations of
the mitochondrial genome in human colorectal tumours. Nat Genet, 20, 291-3.
POLYAK, K., VOGELSTEIN, B. & KINZLER, K. W. 2004. Subtle mitochondrial
mutations as tumor markers U.S. patent application 10/053,611.
PRESTON, T. & SINGH, G. 2001. Mitochondrial signaling and cancer. Advances in Cell
Aging and Gerontology, 7, 103-130.
PRESTON, T. J., ABADI, A., WILSON, L. & SINGH, G. 2001. Mitochondrial
contributions to cancer cell physiology: potential for drug development. Adv Drug Deliv
Rev, 49, 45-61.
RAGNINI, A. & FUKUHARA, H. 1989. Genetic instability of an oligomycin resistance
mutation in yeast is associated with an amplification of a mitochondrial DNA segment.
Nucleic Acids Res, 17, 6927-37.
RAY, A. J., TURNER, R., NIKAIDO, O., REES, J. L. & BIRCH-MACHIN, M. A. 2000.
The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet
radiation exposure in human skin. J Invest Dermatol, 115, 674-9.
RICCHETTI, M., TEKAIA, F. & DUJON, B. 2004. Continued colonization of the human
genome by mitochondrial DNA. PLoS Biol, 2, E273.
ROY, D., FELTY, Q., NARAYAN, S. & JAYAKAR, P. 2007. Signature of mitochondria
of steroidal hormones-dependent normal and cancer cells: potential molecular targets for
cancer therapy. Front Biosci, 12, 154-73.
SAFFROY, R., CHIAPPINI, F., DEBUIRE, B. & LEMOINE, A. 2004. Emerging
Concepts in the Analysis of Mitochondrial Genome Instability Current Genomics, 5,
309-325.
SCHEFFLER, I. E. 1999. Biogenesis. Mitochondria. New York: Wiley-Liss.
SCHWERDT, G., FREUDINGER, R., SCHUSTER, C., WEBER, F., THEWS, O. &
GEKLE, M. 2005. Cisplatin-induced apoptosis is enhanced by hypoxia and by inhibition
of mitochondria in renal collecting duct cells. Toxicol Sci, 85, 735-42.
SHAY, J. W. & WERBIN, H. 1992. New evidence for the insertion of mitochondrial
DNA into the human genome: significance for cancer and aging. Mutat Res, 275, 227-35.
SINGH, K. K., RUSSELL, J., SIGALA, B., ZHANG, Y., WILLIAMS, J. & KESHAV, K.
F. 1999. Mitochondrial DNA determines the cellular response to cancer therapeutic
agents. Oncogene, 18, 6641-6.
SORENSON, M. D. & FLEISCHER, R. C. 1996. Multiple independent transpositions of
mitochondrial DNA control region sequences to the nucleus. Proc Natl Acad Sci U S A,
93, 15239-43.
SUN, S. Y., HAIL, N., JR. & LOTAN, R. 2004. Apoptosis as a novel target for cancer
chemoprevention. J Natl Cancer Inst, 96, 662-72.
SUN, Y., LIN, H., ZHU, Y., MA, C., YE, J. & LUO, J. 2002. Induction or suppression of
expression of cytochrome C oxidase subunit II by heregulin beta 1 in human mammary
epithelial cells is dependent on the levels of ErbB2 expression. J Cell Physiol, 192,
225-33.
SUSIN, S. A., ZAMZAMI, N., CASTEDO, M., HIRSCH, T., MARCHETTI, P.,
MACHO, A., DAUGAS, E., GEUSKENS, M. & KROEMER, G. 1996. Bcl-2 inhibits the
mitochondrial release of an apoptogenic protease. J Exp Med, 184, 1331-41.
TAN, D. J., BAI, R. K. & WONG, L. J. 2002. Comprehensive scanning of somatic
mitochondrial DNA mutations in breast cancer. Cancer Res, 62, 972-6.
TARANTUL, V. Z., NIKOLAEV, A. I., MARTYNENKO, A., HANNIG, H.,
HUNSMANN, G. & BODEMER, W. 2000. Differential gene expression in B-cell
non-Hodgkin's lymphoma of SIV-infected monkey. AIDS Res Hum Retroviruses, 16,
173-9.
TEO, J. W., THAYALAN, P., BEER, D., YAP, A. S., NANJUNDAPPA, M., NGEW, X.,
DURAISWAMY, J., LIUNG, S., DARTOIS, V., SCHREIBER, M., HASAN, S.,
CYNAMON, M., RYDER, N. S., YANG, X., WEIDMANN, B., BRACKEN, K., DICK,
T. & MUKHERJEE, K. 2006. Peptide deformylase inhibitors as potent antimycobacterial
agents. Antimicrob Agents Chemother, 50, 3665-73.
THORSNESS, P. E. & WEBER, E. R. 1996. Escape and migration of nucleic acids
between chloroplasts, mitochondria, and the nucleus. Int Rev Cytol, 165, 207-34.
TRAVEN, A., WONG, J. M., XU, D., SOPTA, M. & INGLES, C. J. 2001. Interorganellar
communication. Altered nuclear gene expression profiles in a yeast mitochondrial dna
mutant. J Biol Chem, 276, 4020-7.
TROYANO, A., FERNANDEZ, C., SANCHO, P., DE BLAS, E. & ALLER, P. 2001.
Effect of glutathione depletion on antitumor drug toxicity (apoptosis and necrosis) in
U-937 human promonocytic cells. The role of intracellular oxidation. J Biol Chem, 276,
47107-15.
TURNER, C., KILLORAN, C., THOMAS, N. S., ROSENBERG, M., CHUZHANOVA,
N. A., JOHNSTON, J., KEMEL, Y., COOPER, D. N. & BIESECKER, L. G. 2003.
Human genetic disease caused by de novo mitochondrial-nuclear DNA transfer. Hum
Genet, 112, 303-9.
ULLYATT, E. & LIANG, B. C. 1998. 2',3'-Dideoxycytidine is a potent inducer of
apoptosis in glioblastoma cells. Anticancer Res, 18, 1859-63.
VERLAET, M., DUYCKAERTS, C., RAHMOUNI, S., DENIS, G., HUMBLET, C.,
GREIMERS, R., SLUSE, F. E., BONIVER, J. & DEFRESNE, M. P. 2002. Transient
modifications of respiratory capacity in thymic cells during murine radioleukemogenesis.
Free Radic Biol Med, 33, 76-82.
WALLACE, K. B. & STARKOV, A. A. 2000. Mitochondrial targets of drug toxicity.
Annu Rev Pharmacol Toxicol, 40, 353-88.
WANG, Y., LIU, V. W., XUE, W. C., TSANG, P. C., CHEUNG, A. N. & NGAN, H. Y.
2005. The increase of mitochondrial DNA content in endometrial adenocarcinoma cells: a
quantitative study using laser-captured microdissected tissues. Gynecol Oncol, 98, 104-10.
WARBURG, O., WIND, F. & NEGELEIN, E. 1926. Uber den Stoffwechsel von Tumoren
in Korper. Journal of Molecular Medicine, 5, 829-832.
WELTER, C., KOVACS, G., SEITZ, G. & BLIN, N. 1989. Alteration of mitochondrial
DNA in human oncocytomas. Genes Chromosomes Cancer, 1, 79-82.
WU, C. W., YIN, P. H., HUNG, W. Y., LI, A. F., LI, S. H., CHI, C. W., WEI, Y. H. &
LEE, H. C. 2005. Mitochondrial DNA mutations and mitochondrial DNA depletion in
gastric cancer. Genes Chromosomes Cancer, 44, 19-28.
XIE, C. H., NAITO, A., MIZUMACHI, T., EVANS, T. T., DOUGLAS, M. G.,
COONEY, C. A., FAN, C. Y. & HIGUCHI, M. 2007. Mitochondrial regulation of cancer
associated nuclear DNA methylation. Biochem Biophys Res Commun, 364, 656-61.
XUE, L., FLETCHER, G. C. & TOLKOVSKY, A. M. 2001. Mitochondria are selectively
eliminated from eukaryotic cells after blockade of caspases during apoptosis. Curr Biol,
11, 361-5.
YAMAMOTO, H., TANAKA, M., KATAYAMA, M., OBAYASHI, T., NIMURA, Y. &
OZAWA, T. 1992. Significant existence of deleted mitochondrial DNA in cirrhotic liver
surrounding hepatic tumor. Biochem Biophys Res Commun, 182, 913-20.
YAMAUCHI, A. 2005. Rate of gene transfer from mitochondria to nucleus: effects of
cytoplasmic inheritance system and intensity of intracellular competition. Genetics, 171,
1387-96.
YANG, Z., SCHUMAKER, L. M., EGORIN, M. J., ZUHOWSKI, E. G., GUO, Z. &
CULLEN, K. J. 2006. Cisplatin preferentially binds mitochondrial DNA and
voltage-dependent anion channel protein in the mitochondrial membrane of head and neck
squamous cell carcinoma: possible role in apoptosis. Clin Cancer Res, 12, 5817-25.
YEN, H. C., TANG, Y. C., CHEN, F. Y., CHEN, S. W. & MAJIMA, H. J. 2005.
Enhancement of cisplatin-induced apoptosis and caspase 3 activation by depletion of
mitochondrial DNA in a human osteosarcoma cell line. Ann N Y Acad Sci, 1042, 516-22.
ZHOU, S., KACHHAP, S. & SINGH, K. K. 2003. Mitochondrial impairment in
p53-deficient human cancer cells. Mutagenesis, 18, 287-92.
ZHU, W., QIN, W., BRADLEY, P., WESSEL, A., PUCKETT, C. L. & SAUTER, E. R.
2005. Mitochondrial DNA mutations in breast cancer tissue and in matched nipple aspirate
fluid. Carcinogenesis, 26, 145-52.
ZHU, Y., WANG, M., LIN, H., HUANG, C., SHI, X. & LUO, J. 2002. Epidermal growth
factor up-regulates the transcription of mouse lon homology ATP-dependent protease
through extracellular signal-regulated protein kinase- and
phosphatidylinositol-3-kinase-dependent pathways. Exp Cell Res, 280, 97-106.



                                           ***
About the Author
     Bert Liang is a serial entrepreneur, scientist, clinician and author with experience in
     the technology, strategy and venture industries and communities. He has published
     over 50 articles, chapters, monographs and books, in the scientific, business and
     fiction areas. He lives in San Diego, with his wife, two children and golden doodle
     dog, Roxy.

                          Other Titles by Bert Liang at Smashwords.com:


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