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Apoptotic versus genotoxic potential of anti-tumor agents: a concept of duality in unity
Medical Hypotheses (2003) 61(5–6), 643–650 ª 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0306-9877(03)00266-4
Apoptotic versus genotoxic potential of anti-tumor agents: a concept of duality in unity B. Vladan,1 Z. Milic´evic´,2 P. -S. Biljana,3 A. Nedeljkovic-Kurepa4 1
ICN-Galenika Pharmaceuticals, Institute for Biomedical Research, Belgrade, Yugoslavia; 2Laboratory for Molecular Biology and Endocrinology, ‘‘Vinca’’ Institute for Nuclear Sciences, Belgrade, Yugoslavia; 3University of Belgrade, Faculty of Pharmacy, Belgrade, Yugoslavia; 4Institute for Cancer research ‘‘KBC’’, Belgrade, Yugoslavia
Summary Recent advances in anti-tumor therapy have raised a problem of secondary tumors and tumor resistance. Secondary tumors induced by chemotherapeutic agents as a consequence of primary therapy have poor prognostic outcome. Many new insights into molecular controls of cell cycle progression of normal and cancer cells can provide a useful framework in order to identify potential targets for anti-tumor therapies. One of the most promising strategies is the possibility to modulate apoptosis induced by anti-tumor agents. Cancer cell survival after chemotherapy will depend on specific checkpoints and/or repair pathways that have been lost, leading either to greater susceptibility to anti-tumor agents when the repair of damage is most important for survival or to greater resistance when the apoptotic response is more important. We have proposed a hypothesis that views survival and apoptotic processes (duality) in normal and tumor cells as genetically coupled (unity). We introduce, through a theoretical background, a new pathway of apoptotic inhibition. The proposed process of apoptotic inhibition is induced by mutation fixation in which recombination/repair processes (hRAD genes) play an important role. These coupled processes (duality in unity), to our view, underline tumor resistance by apoptotic inhibition and mutation fixation in normal cells exposed to anti-tumor agents. ª 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Pharmaceutical agents in response to there potential mutagenicity and genotoxicity show that in more and less time can induce genomic and chromosomal instability with changes on the genetic (point mutations, Loss of Heterozygosity (LOH) and Microsatellite Instability (MI)) and chromosomal level (structural and numerical aberrations-aneuploidy) with a possible consequence of secondary tumors. Carcinogenesis can be perceived as multistage process (initiation, promotion and progression) in which a dysfunction of reparative mechanisms Received 21 October 2002 Accepted 26 March 2003 Correspondence to: Dr. Bajic Vladan DVM, PhD, ICN-Yu, Pasterova 2 (c/o Prof. Dr. Raki c Ljubisav) Belgarde 11000, Serbia, Yugoslavia. Phone: +381-11-3610314; Fax: +381-11-3610-053; E-mail: [email protected]
and inhibition of apoptosis predominate. Programmed cell death is an orderly and genetically controlled form of cell death. Apoptosis can be viewed as a process that is complementary to mitosis. By the coordination of these two processes there is a balance between proliferation of tissue mass and atrophy. Programmed cell death is also a fundamental mechanism by which damaged cells are recognized and excluded from the organism. Dysfunction of this process has been seen as the basis of pre-neoplastic and neoplastic proliferation, neurodegenerative and autoimmune diseases. Viewing carcinogenesis as a three-stadium process: (1) dysfunction or inhibition of reparative processes, (2) inhibition of apoptosis and (3) inability to terminate cell proliferation we have proposed a hypothesis of a new pathway of apoptosis inhibition induced by anti-tumor agents. Pharmacodynamics of anticancer drugs consists of two distinctive steps. The first step includes the
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mitosis
mitosis Pro-survival processes
Pro-Apoptotic processes
DUALITY
UNITY Stability under genetic control
MUTATION FIXATION
INHIBITION OF APOPTOSIS
Fig. 1 Pro-apoptotic coupled pro-survival processes and genome stability.
interaction with its cellular target which is not lethal per se. The commitment of the cell to undergo apoptosis forms the second step. The efficacy of anticancer drugs is determined by the ability to selectively sensitize tumor cells to apoptosis which depends to a large extent from the expression of various oncogenes, such as bcl-2, p53, bax, ras, c-myc and others, and from endogenous factors. In the proposed hypothesis, we have inclined that hRAD51, 52 and possible other hRAD genes are implicated in proliferative (pro-survival) processes that can lead to mutation fixation and apoptotic inhibition in normal cells. We have to emphasis that pro-survival processes are coupled with pro-apoptotic processes (duality) but have the same genetic background, i.e., they are genetically entwined (unity) (Fig. 1). We also propose that the underlying mechanism of tumor cell resistance in chemotherapy is based on apoptosis inhibition. If inhibition of apoptosis is coupled by mutation fixation, we could finally answer an intriguing question: What are the mechanisms of apoptotic control in normal and cancer cells in which antitumor agents promote genomic instability, genotoxicity and consequently carcinogenesis, when it is well known that anti-tumor agents are the most powerful inductors of apoptosis? GENOTOXICITY OF ANTI-TUMOR AGENTS In genetic toxicology today there is a need for high quality genotoxicity data to be derived and integrated with other relevant toxicological studies on new pharmaceutical agents in order to provide information on the mechanisms of possible carcinogenic action (1–3). Antitumor agents are recognized as being clastogenic, aneugenic and recombinagenic (4). A vast number of chemotherapeutical agents can induce secondary tumors (ANLL type, or acute nonlymphoblastic leukemia) as a single-agent in primary tumor chemotherapy (5). Medical Hypotheses (2003) 61(5–6), 643–650
Secondary tumors are refractory to novel agents and have poor prognostic outcome. One of the main mechanisms of primary/secondary tumor resistance to chemotherapy is apoptotic inhibition and repair dysfunction. Chromosomal alterations, such as deletions, amplifications, inversions, three nucleotide expansions, LOH, microsatellite instability, loss of genetic imprinting and translocations, are commonly observed in transformed cell lines, which has lead to the hypothesis that genetic instability may play an important role in oncogenesis. Chromosome breakage, as it might arise from double-strand breaks (DSB) has been postulated to be the initial event in these processes (6). Current models suggest that genomic instability is crucial in the accumulation of the multiple alterations required for tumorogenesis. However, the nature of the initial damage responsible for the origin of genomic instability remains poorly understood. Defects in recombination/repair pathways that process DNA damage, such as (DSB), have been proposed as a mechanism of genome instability in some human cancers. Both homologous mitotic recombination (HMR), causing loss of heterozygosity (LOH) of a wild type allele, and structural chromosome aberrations (CA) involve the formation of double-strand breaks in DNA. DSB is a strong promotor for initiation of apoptosis (7). DSB can be perceived as the corner stone in which coupled pro-apoptotic and pro-survival processes determine cell fate (apoptosis or survival) (Fig. 2). APOPTOSIS AND SURVIVAL PROCESSES The molecular pathway of apoptosis starts with an initiating phase by a signal from external stimuli, such as ligation to distinct receptors, or to irreversible cellular or nuclear damage. The initiation phase is followed by a decision phase. During this phase transduction of the apoptotic stimuli to nuclear and cytoplasmatic target proteins– enzymes occurs. The transduction phase includes activation of endonucleases and enzymatic alteration of the cytoskeleton (stimuli for survival?). The execution phase is started when the cell has arrived at the stage of no return. In morphological sense, apoptosis differs from necrosis in that there is cellular shrinkage and chromatin condensation, followed by fragmentation of nuclear components with in membrane-bound vesicles, which are cleared by phagocytosis without damage to adjacent tissue. There are numerous proteins, which modulate the apoptotic mechanism in a positive or negative direction (8,9). C-myc oncogene was the first gene to be viewed as an expression of the dual nature of apoptosis promotion and cell proliferation. The mitogenic and apoptotic ª 2003 Elsevier Ltd. All rights reserved.
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Chemotherapeutical Agents Ch emotherapeutical Ag ents
Double DoubleStrand StrandBreak Br eak DSB DSB
Apoptotic potential/apoptosis
Genotoxic potential/survival
RAD-52 epistatic group RAD-52 epistatic gr oup Homolgous HomolgousDNA DNArecombination recombinationrepair re pa ir
APOPTOSIS AND SURVIVAL PROCESSES Fig. 2 Double-strand break (DSB) as a common denominator of the genotoxic and apoptotic potential of anti-tumor agents.
pathways are genetically inseparable (10). Cell proliferation/pro-survival and apoptotic pathways are coupled/ duality in unity! One of the key mediator for growth factor-dependent cell survival is a small guanosin triphosphatase-protein Ras. Of various pathways that are activated by Ras, the pathway that comprises a series of sequentially activated protein kinases Raf, MEK (MAPK kinase) and MAPK have been found to promote cell survival and inhibit apoptosis (10). The Ras oncoproteins provide a particularly interesting example of how proliferative and apoptotic pathways have been entwined (genetically inseparable). Ras proteins are key transducers of mitogenic signals through their activation of Raf-MAP kinase pathways, a fact attested by the high frequency of Ras activating mutations in human cancer. Ras proteins are also involved in transducing survival signals from a receptor such as IGFI to downstream factors such as PI3-kinase, Akt and Bad (11). Survival-promoting cytokines suppress the activity of the protein BAD, a pro-apoptotic member of the bcl-2 family, by induction of phosphorylation of BAD at two critical sites, serine 112 and serine 136. Phosphorylation of BAD leads to the dissociation of BAD from pro-survival Bcl-2 and the association with the members of the 14-3-3 family of proteins. The regulation of BAD by ª 2003 Elsevier Ltd. All rights reserved.
these phophorylation events suggests that BAD is a point of convergence for multiple signaling pathways that cooperate in order to promote cell survival. Therefore oncogenic mutation of Ras appears able to simultaneously activate cell proliferation and suppress the concomitant apoptosis – a potentially catastrophic combination. Paradoxically, however when activated Ras is expressed in untransformed cells, it triggers precisely a opposite response – a profound p53 growth arrest, frequently accompanied by apoptosis both of which are mediated by Ras kinase raf (11). The p53 mitogen activated protein kinase (MAPK) plays a key role in the activation of p53 by genotoxic stress when induced by anti-tumor agents (12). Ras can activate cell proliferation and suppress apoptosis, but when expressed in untransformed cells, it triggers an apoptotic response. Again, we conclude that these processes are coupled. Therefore, survival signaling pathways maybe directly/ indirectly responsible for mutation fixation. APOPTOSIS, CELL CYCLE CHECKPOINT CONTROL AND ADAPTATION Gene products that regulate the progression of the cell cycle affect apoptosis. Inhibition of apoptosis is consequence of G1–S checkpoint loss. This results in genomic Medical Hypotheses (2003) 61(5–6), 643–650
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instability and survival of damaged cells. Signals for cell cycle checkpoint control are DSB, UV-dimmers and centromere alterations (13). A number of checkpoint genes that are thought to be responsible for monitoring chromosome integrity and preventing cell cycle progression in the presence of damaged DNA-DSB. Abrogation of these damage-monitoring checkpoints may contribute to chromosome rearrangements and therefore can lead to genetic instability. Molecular mechanisms of genetic control of DSB repair are not well understood in humans (13). In Saccharomyces cerevisiae DSB repair is well understood, it generally involves RAD52-dependent recombination. Disrepair of DSB can be the result of illegitimate recombination between non-homologous DNA sequences. Gupat et al. (14) showed that mitotic recombination and, too much lesser extent deletion may be the primary mechanism for the relative high frequency of in vivo LOH observed in normal human T cells. Because LOH leads to the expression of recessive tumor suppresser genes in many cancers, there is significant implication that LOH produced by alteration of RAD52 recombination repair can be the initial event leading to genetic instability (15) and mutation fixation. Signal-tranduction pathways systems have the ability for adaptation. Cells of S. cerevisiae can fixate non-repaired DSB and progress into mitosis after a delay in the G2 phase. Transactivation of p21waf=Cip1 represents absolute requirement for p53-dependent G1 arrest, but alterations in expression of p53 binding protein mdm-2 can result in adaptation of the G2 control point. A major part of p53-mediated growth arrest proceeds through induction of the cyclin-dependent kinase (CdK) inhibitor p21. Raderschall et al. (16) have shown that Rad 51 expression, Rad 51 foci formation and p21 expression are interrelated, suggesting a functional link between mammalian Rad 51 protein and p21-mediated cell cycle regulation (16). Restoration of defective control points would restore the inhibition of the apoptotic answer of cancerous cells to anti-tumor agents (17). REPAIR SYSTEMS AND TUMOR RESISTANCE Mismatch repair system promotes mutational frequency (in tumor cells) in cases of deficiency or defect, but also forms resistance to mutagenic/genotoxic agents in various tumors. It has been observed that cells with a functional mismatch repair (MMR) system respond to treatment with alkylating agents by arresting cell cycle progression in G1 and/or G2 phase. Cells with a deficient MMR system can only arrest the cell cycle in the G1 phase. This indicates that the MMR system integrates with the G2 checkpoint, and the arrest in the G1 Medical Hypotheses (2003) 61(5–6), 643–650
phase is seemingly independent of the MMR system (18,19). The observation of apoptosis in testicular and ovarian cancers with cis-platin sensitivity of these cancers is explained by an increased expression of the hMSH2 protein and a lost ability of MMR system to repair cis-platin DNA lesions. Defects in mismatch repair are associated with cis-platin resistance (19). The MMR system also detects cis-platin DNA adducts and loss of MMR results in resistance to cis-platin (20). Observations support the hypothesis that mismatch repair defects in hMUtL alpha and hMUtS alpha, but not in hMUtS beta, contribute to increased replicative bypass in cells exposed to cis-platin. Replicative bypass introduces drug resistance by preventing futile cycles of translesion synthesis and mismatch correction (21). Anti-cancer activity of intra and interstrand alkylating agents arises from its ability to damage DNA, with the major adducts formed being intrastrand (GpG) and d(ApG) crosslinks. These crosslinks bend and unwind the DNA duplex. The altered structure attracts high mobility group (HMG) domain proteins and other proteins (21). Nucleotide excision repair (NER) is also responsible for the repair of platinum–DNA lesions. The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of DNA damage sites during NER. Recombination pathways act independently of NER and are of equal importance to NER as genoprotective systems against cis-platin cytotoxicity. Taken together these results shed new light on cell survival processes and chemotherapy resistance (22). Previous studies have shown that high-mobility group (HMG) domain proteins such as HMG1 sensitize cells to cis-platin by shielding the major DNA adducts from nucleotide excision repair. It is still not known if these genes are turned on by DNA damage (formation of DNA adducts) or by a downstream of biological consequences induced by the damage of DNA (22). HMG proteins constitute a class of important architectural proteins involved in transcription regulation of genes, participate in gene rearrangements which are linked to the emergence of benign tumors and have the ability to recognize DNA–cis-platin adducts selectively. HMG sensitize cells to cis-platin by affecting DNA replication. The implication of HMG proteins to shield DNA adducts from NER repair have dual consequences: one is sensitizing tumor cells to cis-platin, but the other opens a question: would shielding DNA adducts in normal cells bring apoptosis inhibition and consequently possible induction of secondary tumors by cis-platin? cis-platin is known to induce secondary tumors of the ANLL type after primary treatment. Also, it should be considered that some HMG proteins, acting as architectural proteins ª 2003 Elsevier Ltd. All rights reserved.
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that bring many of the transcription factors into precise three-dimensional shapes may have a critical role in neoplastic transformation (as to that of some transcriptional factors may have themselves) (21). In addition to its classical role in the cellular stress response, heat shock proteins (HSP) play a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival (23). A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitions and essential protein chaperones HSP 90 and HSP 70. Chaperones play a role within the regulatory network of the cell cycle and within signal cascades. HSP 70 and HSP 90 associate with proteins of the mitogen- activated signal cascade, particularly with the Src-kinase, tyrosine receptor kinases, Raf and the MAPkinase activating kinase (MEK) (24). This supports a notion of a protective function of chaperones and stress proteins in the maintenance of the cell cycle and the signaling processes. HSP 70 is identified recently as a potent anti-apoptotic factor (25–29). HSP 90 is involved in negative regulation of apoptosis (30). The complexicity of tumor resistance through apoptotic inhibition shows us that repair, transcriptional and replicative processes are not separated in pro-survival and/or pro-apoptotic pathways but highly entwined through genetically controlled processes. HOMOLOGOUS RECOMBINATIONAL REPAIR AND DSB Homologue recombination is defined as a process in which genetic information is exchanged between two DNA molecules that share sequence identity. This process is exemplified by genetic recombination during meiosis, helps to increase genetic diversity as well as to ensure proper chromosome segregation (31). Major function of HR in mitotic cells is DNA repair and is mediated through out the RAD52 epistasis group of genes. Molecular genetics of HR in mammals are poorly understood. In yeast genes RAD52 epistasis group including RAD50, RAD51, 52, 54, 55, 57, MRE11 and XRS2, are involved in both homology-dependent DSB break repair and meiotic HR. DSB is also a place of potential recombination (31,32). Persistent DSB could be the initial event leading to loss of heterozygosity by defective recombinational repair and consequently to the expression of recessive oncogenes seen in malignant cells. DSB also affects the checkpoint control mechanism of the cell cycle. DNA damage (DSB) results in activation of p53 through an incompletely understood mechanism, that involves possible activation of ATM (ataxia-telangiectasia mutated gene) and chk 2 kinase (33,34). Chen et al. (35) have provided evidence that ATM and c-Abl ª 2003 Elsevier Ltd. All rights reserved.
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kinase (c-abl, an oncogene is also a downstream effector in the ATM DNA damage response pathway and has been shown to phosphorylate hRAD51 and alter to bind to DNA in vitro) are required for correct post-translational modification and assembly of Rad 51 protein complexes (35). Wyllie et al. (10) have reported studies that have demonstrated that aneuploid cells in colorectal cancer have sustained obligatory recombinational elements that must have involved DSB (10). Other studies have shown that p53 activation following DSB can initiate apoptosis (Fig. 3). These descriptive human data are compatible with the idea that aneuploid cells of colon rectal cancer may have arisen from cells that sustained DSB but failed to enter apoptosis! Powerful mechanisms are expressed in order to activate apoptosis when cells that replicate are comforted with DSB. But, apoptosis can be overridden by deregulated recombinational processes through the RAD epitasis group (39). Rad52 interacts with RAD51 through its C-end, and is attached to DNA by its N-tail. RAD52 homologue recombination is the main rout for DNA (DSB) repair in S. cerevisiae. Rad52 has an increased expression in S and G2 phase of the cell cycle, possible in connection with cell cycle checkpoints. On the 3’ end and of the nontranslational part of mouse RAD52-cDNA there is ATTTA motif that is explained as a controller for iRNA degradation of some genes like c-fos or c-myc. C-myc is a oncogene implicated in proliferative and apoptotic (coupled) processes in tumor cells. Expression of c-myc, if increased, has a better chance to lead cells to apoptosis by anti-tumor agents. hRAD51 is major tumor suppressor gene. RAD51 is a member of the RecA/RAD51 family of proteins that have been implicated in recombination/reparative processes. RAD51 proteins are directly/or indirectly associated to p53, BRCA 1 and BRCA 2. Dysfunction of RAD51 can promote cancer and instability, i.e., hRAD promotes chromosome stability and protects DNA against DNAcrosslinks and other damages (36,37). hRAD deficient cells accumulate chromosome breakages prior to cell death. p53 is directly linked to homologue recombination processes via RAD51/RecA protein interaction. Interaction of RAD51 and RAD52 genes show that mutation in one of the genes can change the expression of the others (22,31). Increased expression of RAD51 genes in humans are related to DSB repair, in meiosis and V/D/J recombination during he time of lymphocyte maturation (31). The hRad51 appears to be regulated in at least two ways: (a) transcriptionally, by genes that confer a proliferative potential, as well as by checkpoint signaling pathways that regulate DNA damage responses (b) at the protein level, where interactions with other molecules lead to distinct cellular localization of hRad51 in nuclear foci (39). Medical Hypotheses (2003) 61(5–6), 643–650
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RAD RAD51, RAD invovlved RAD52, 52,RAD50, RAD50,RAD51, RAD 54, 54, RAD55, RAD55, MRE11 MR E11and and XRS2 XRS2 are are invovlved ininhomology depedent double strand break repair homology depedent doub le stra nd break repair
Survival Survival signaling signaling pathways pa thways IGF-1 IGF- 1 interleucin interl eucin3 3 ras/raf-1 ras/raf-1 kinase kinase toto bcl-2 bcl-2 defect defectininp53 p53 mdm-2 mdm-2
inhibition inhibition of of PCD/apoptosis PCD/apoptosi s
Proapoptotic Proapoptotic pathways pathways bcl-2 bcl-2fosphorilation fosphorilatio n p53 p53 myc expression myc expression high highmobility mobility group grou p proteins(HMG) proteins(HMG ) BRCA1 BRCA1and an d2 2 hRAD51 hR AD51
Proliferative Prol iferati veand an dapoptotic apopt oticpathways pathw aysare are coupled coup led
Mu tation fixation fixationby byadatation adatationofofcell cellcycle cyclecheck checkpoint point controll Mutation contro Genom Genom instability instabilit y Fig. 3 A schematic representation of pro-survival and pro-apoptotic (coupled) processes that is based on complex interaction of homologous recombination repair, abrogation of cell cycle checkpoint control and replicative ‘bypass’ which can lead to inhibition of apoptosis by mutation fixation.
BRCA 1 and BRCA 2 (Breast Cancer Susceptibility gene 1 & 2) participate in the cell cycle progression, apoptosis and DNA repair pathways mediated through interaction with RAD51 recombinase in response to genotoxic stress. The role of BRCA1 protein in apoptotic pathway as well as an important role in the embriogenesis has also been suggested (37,38). BRCA central region harbors a binding site for RAD51 protein, a eukaryotic homologue of RecA protein of E. Coli. RAD51 is involved in repairing DNA double-strand breaks. One of the function of BRCA1 protein with the respect to its cell cycle-dependent manner of its expression and hyperphosphorylation might be in the cell cycle regulation of the G1–S transition and/or G2/M phase (38). Loss of BRCA 2 causes misrepair of chromosomal DSB occurring between repeated sequences by stimulating use of an error-prone homologous recombinational pathway. Furthermore, loss of BRCA 2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C induced DNA cross-links at the expense of error-free repair by sister chromatid recombination (43). RAD53 codes serine/threonine kinases. RAD54 codes a family of proteins that are ATP-dependent and stimulated in the presence of DNA. Mutant RAD52 cells are defective for NER and also, there is a defect in the repMedical Hypotheses (2003) 61(5–6), 643–650
lication processes after UV-irradiation. Mutant RAD52 does not affect sister chromatide exchange (SCE) and other intrachromosomal events (31). Little is known about the correlation of recombination repair and tumor resistance. We propose that recombination repair is strongly associated with a new pathway of apoptotic inhibition. Apoptotic processes are overwhelmed by prosurvival and adaptation processes inducing mutation fixation. In accordance to our given hypothesis recent results have shown that RAD51 recombinational pathway can be suppressed by Bcl-2 family of proteins (42). The Bcl-2 gene therefore combines two separable cancer-prone phenotypes: apoptosis repression and a genetic instability/mutator phenotype. The dual phenotype could represent a mammalian version of the bacterial SOS repair system (42). RecA proteins are involved in SOS error-prone repair mechanisms in Escherichia coli. The SOS error-prone repair system is RecA and LEXA-dependent process. DSB can induce a signal for the activation of RecA protein activation. RecA protein is activated by LexA protein degradation. SOS mechanism is a survival process in prokaryotic cells with an increased mutation rate. Mutants that don’t have RecA protein are easily killed from UV-radiation because of deficient repair mechanisms. From cells that survive no mutants could be detected. RecA gene is highly ª 2003 Elsevier Ltd. All rights reserved.
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conserved through evolution. Mutator phenotype in tumor cells show mismatch repair deficiency, but not always recombination repair deficiency. This proposed mechanism inserts mutation fixation in tumor cells and consequently apoptotic inhibition. Using analogy for the SOS repair mechanism in prokaryotes to eukaryotic cells, we can hypothesize that ‘SOS’ repair mechanism has evolved to be integrated in the complex system of apoptosis, i.e., inhibitors of apoptosis by mutation fixation. Homologous recombination repair systems are implicated in both systems, and are therefore crucial for genome stability. Example: Altered polymerases expressed through the SOS error-prone repair system in prokaryotes has an analogy in the activation of polymerase beta that can bypass DNA adducts during DNA replication. This bypass process is responsible for the resistance of some tumor cell lines against cis-platin (40,41). Recombination repair coupled with checkpoint control abrogation may be analogue for SOS error-prone survival pathways. We propose a new apoptotic inhibition pathway in which the hypothesized anti-apoptotic protein (MFP or mutation fixation protein) may have a major role in chemotherapy resistance. This SOS error-prone analogue mechanism (?) may be also induced by persistent DSB in normal cells exposed to genotoxic agents, and therefore be responsible for mutation fixation and genomic instability.
FUTURE PERSPECTIVES Results of studies of HR coupled with processes that inhibit apoptotic response in tumor cells can also have fundamental implications of understanding causes and possible mechanisms of DSB fixation and other types of chromosome damage that consequently can lead to genomic instability. We have clearly seen that genomic stability is maintained by dual processes (proliferative versus apoptotic) in which oncogenes play a crucial role. Understanding molecular events that lead to genomic instability, including components involved in damage susceptibility, repair, cell cycle control and apoptosis will be required for development of new chemotherapeutical strategies. We have proposed that hRad 51, 52 and etc., proteins are implicated in coupled pro-apoptoic, pro-survival processes by adaptation. These processes may lead to mutation fixation by apoptotic inhibition in tumor cells, thus resulting in resistance to chemotherapy. The proposed mutation fixation process in normal cells may also be the initial event leading to genome instability and consequently to promotion of oncogenesis. Restoration of apoptosis by modulating the extrinsic and intrinsic regulators of apoptosis in resistant tumor cells can vastly improve the effectiveness of anti-tumor therapy.
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19. Vaisman A., Varchenko M., Umar A., Chaney S. G. The role of hMLH1, hMSH3, and hMSH6 defects in cis-platin and oxaplatin resistance: correlation with replicative bypass of platinum–DNA adducts. Canc Res 1998; 16: 3579–3585. 20. He Q., Liang C. H., Lippard S. J. Steroid hormones induce HMG1 over expression and sensitize breast cancer cells to cis-platin and carboplatin. Proc Natl Acad Sci USA 2000; 97(11): 5768–5772. 21. Wunderlich V., Botter M. High-mobility-group proteins and cancer – a emerging link. J Cancer Res Clin Oncol 1997; 123: 133–140. 22. Zdraveski, Melloow J. A., Marinus M. G. et al. Multiple pathways of recombination define cellular responses to cis-platin. Mut Res 1999; 431: 135–140. 23. Jolly C., Morimoto R. I. Role of heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst 2000; 92: 1564–1572. 24. Helmbrecht K., Zeise E., Rensing L. Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Prolif 2000; 33: 341–365. 25. Jaattela M., Wissing D., Kokholm K., Kallunki T., Egeblad M. HSP 70 exerts its anti-apoptotic function downstream of caspase-3 like proteases. EMBO J 1998; 17: 6124–6134. 26. Beere H. M., Wolf B. B., Cain K., Mosser D. D., Majboubi A., Kuwana T., Tailor P., Morimoto R. I., Cohen G. M., Gren D. B. Heat shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2000;(2): 469–475. 27. Li C. Y., Lee J. S., Ko Y. G., Kim Ji, Seo J. S. Heat shock protein 70 inhibits apoptosis downstream to citochrome c release and upstream caspase-3 activation. J Biol Chem 2000; 275: 25665–25671. 28. Mosser D. D., Caron A. W., Bourget L., Meriin A. B., Sherman M. Y., Morimoto R. I., Massie B. The chaperone function of HSP-70 is required for protection against stressinduced apoptosis. Mol Cell Biol 2000; 20: 7146–7159. 29. Nylandsted J., Rahde M., Brand K., Bastholm L., Elling F., Jaattela M. Selective depletion of Heat shock protein 70 (HSP 70) activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2. Proc Natl Acad Sci USA 2000; 97: 7871–7876. 30. Pandey P., Saleh A., Nakazawa A., Kumar S., Srinivasula S. M., Kumar V., Weichselbaum R., Nalin C., Alnemri E. S., Kufe D., Kharbanda S. Negative regulation of cytochrome C-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat-shock protein 90. EMBO J 2000; 19(16): 4310–4322.
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31. Ivana Sunjarevic. Isolation and characterization of RAD 52 gene locus in mice. Doctoral Thesis. Faculty of Biological Science. University of Belgrade, 1997. 32. Scully R., Chen J., Weaver D. et al. Association of BRCA 1 with Rad 51 in meiotic and meiotic cells. Cell 1997; 88: 265–275. 33. Bauman P., West S. C. Role of the human RAD51 protein in homologous recombination and double-strand break repair. Trends Biochem Sci 1998; 23: 247–251. 34. Papazisis K. T. Cell cycle and cancer. Ann Acad Stud 2000; 3: 62–65. 35. Chen G., Yuan S. F., Liu W., Xu Y. et al. Radiation-induced assembly of Rad 51 and Rad 52 Recombination complex requires ATM and c-Abl. J Biol Chem 1999; 274(18): 12748–12752. 36. Zhang H. B., Tombline G., Weber B. L. BRCA1 andBRCA 2 and damage response: collision and collsion. Cell 1998; 92: 433–436. 37. Yamamoto A., Taki T., Yagi H., Yoshida K., Yoshimmura Y., Nishimune Y., Morita T. Molecular genetic cell cycle-depedent expression of the mouse Rad 51 gene in proliferating cells. Genetics 1996; 251: 1–12. 38. Raicevic L., Radulovic S. Breast cancer susceptibility gene 1-BRCA 1. Arch Oncol 2000; 8(1): 21–23. 39. Schmutte C., Tombline G., Rheim K., Sadoff M. M., Fishel R. Characterization of human Rad51 genomic locus and examination of tumors with 15q14-15 loss of heterozygosity. Cancer Res 1999; 59: 4564–4569. 40. Judson P. L., Watson J. M., Fowler W. C., Haskill J. S. cis-platin inhibits pacitaxel- induced apoptosis in cis-platin-resistant ovarian cancer cell lines: possible explanation for failure of combination therapy. Cancer Res 1999; 59: 2425–2432. 41. Hoffmann J. S., Locker D., Villani G., Leng M. HMG1 protein inhibits the translesion synthesis of the major DNA cis-platin adduct by cell extracts. J Mol Biol 1997; 270(4): 539–543. 42. Saintigny Y., Dumay A., Lambert S., Lopez B. S. A novel role for the Bcl-2 protein family: specific suppression of the Rad 51 recombination pathway. EMBO J 2001; 20(10): 2596–2607. 43. Tutt A., Bertwistle, Valentine J. et al. Mutation in Brca 2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences. EMBO J 2001; 20(17): 4704–4716.
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Apoptotic versus genotoxic potential of anti-tumor agents: a concept of duality in unity B. Vladan,1 Z. Milic´evic´,2 P. -S. Biljana,3 A. Nedeljkovic-Kurepa4 1
ICN-Galenika Pharmaceuticals, Institute for Biomedical Research, Belgrade, Yugoslavia; 2Laboratory for Molecular Biology and Endocrinology, ‘‘Vinca’’ Institute for Nuclear Sciences, Belgrade, Yugoslavia; 3University of Belgrade, Faculty of Pharmacy, Belgrade, Yugoslavia; 4Institute for Cancer research ‘‘KBC’’, Belgrade, Yugoslavia
Summary Recent advances in anti-tumor therapy have raised a problem of secondary tumors and tumor resistance. Secondary tumors induced by chemotherapeutic agents as a consequence of primary therapy have poor prognostic outcome. Many new insights into molecular controls of cell cycle progression of normal and cancer cells can provide a useful framework in order to identify potential targets for anti-tumor therapies. One of the most promising strategies is the possibility to modulate apoptosis induced by anti-tumor agents. Cancer cell survival after chemotherapy will depend on specific checkpoints and/or repair pathways that have been lost, leading either to greater susceptibility to anti-tumor agents when the repair of damage is most important for survival or to greater resistance when the apoptotic response is more important. We have proposed a hypothesis that views survival and apoptotic processes (duality) in normal and tumor cells as genetically coupled (unity). We introduce, through a theoretical background, a new pathway of apoptotic inhibition. The proposed process of apoptotic inhibition is induced by mutation fixation in which recombination/repair processes (hRAD genes) play an important role. These coupled processes (duality in unity), to our view, underline tumor resistance by apoptotic inhibition and mutation fixation in normal cells exposed to anti-tumor agents. ª 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Pharmaceutical agents in response to there potential mutagenicity and genotoxicity show that in more and less time can induce genomic and chromosomal instability with changes on the genetic (point mutations, Loss of Heterozygosity (LOH) and Microsatellite Instability (MI)) and chromosomal level (structural and numerical aberrations-aneuploidy) with a possible consequence of secondary tumors. Carcinogenesis can be perceived as multistage process (initiation, promotion and progression) in which a dysfunction of reparative mechanisms Received 21 October 2002 Accepted 26 March 2003 Correspondence to: Dr. Bajic Vladan DVM, PhD, ICN-Yu, Pasterova 2 (c/o Prof. Dr. Raki c Ljubisav) Belgarde 11000, Serbia, Yugoslavia. Phone: +381-11-3610314; Fax: +381-11-3610-053; E-mail: [email protected]
and inhibition of apoptosis predominate. Programmed cell death is an orderly and genetically controlled form of cell death. Apoptosis can be viewed as a process that is complementary to mitosis. By the coordination of these two processes there is a balance between proliferation of tissue mass and atrophy. Programmed cell death is also a fundamental mechanism by which damaged cells are recognized and excluded from the organism. Dysfunction of this process has been seen as the basis of pre-neoplastic and neoplastic proliferation, neurodegenerative and autoimmune diseases. Viewing carcinogenesis as a three-stadium process: (1) dysfunction or inhibition of reparative processes, (2) inhibition of apoptosis and (3) inability to terminate cell proliferation we have proposed a hypothesis of a new pathway of apoptosis inhibition induced by anti-tumor agents. Pharmacodynamics of anticancer drugs consists of two distinctive steps. The first step includes the
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mitosis
mitosis Pro-survival processes
Pro-Apoptotic processes
DUALITY
UNITY Stability under genetic control
MUTATION FIXATION
INHIBITION OF APOPTOSIS
Fig. 1 Pro-apoptotic coupled pro-survival processes and genome stability.
interaction with its cellular target which is not lethal per se. The commitment of the cell to undergo apoptosis forms the second step. The efficacy of anticancer drugs is determined by the ability to selectively sensitize tumor cells to apoptosis which depends to a large extent from the expression of various oncogenes, such as bcl-2, p53, bax, ras, c-myc and others, and from endogenous factors. In the proposed hypothesis, we have inclined that hRAD51, 52 and possible other hRAD genes are implicated in proliferative (pro-survival) processes that can lead to mutation fixation and apoptotic inhibition in normal cells. We have to emphasis that pro-survival processes are coupled with pro-apoptotic processes (duality) but have the same genetic background, i.e., they are genetically entwined (unity) (Fig. 1). We also propose that the underlying mechanism of tumor cell resistance in chemotherapy is based on apoptosis inhibition. If inhibition of apoptosis is coupled by mutation fixation, we could finally answer an intriguing question: What are the mechanisms of apoptotic control in normal and cancer cells in which antitumor agents promote genomic instability, genotoxicity and consequently carcinogenesis, when it is well known that anti-tumor agents are the most powerful inductors of apoptosis? GENOTOXICITY OF ANTI-TUMOR AGENTS In genetic toxicology today there is a need for high quality genotoxicity data to be derived and integrated with other relevant toxicological studies on new pharmaceutical agents in order to provide information on the mechanisms of possible carcinogenic action (1–3). Antitumor agents are recognized as being clastogenic, aneugenic and recombinagenic (4). A vast number of chemotherapeutical agents can induce secondary tumors (ANLL type, or acute nonlymphoblastic leukemia) as a single-agent in primary tumor chemotherapy (5). Medical Hypotheses (2003) 61(5–6), 643–650
Secondary tumors are refractory to novel agents and have poor prognostic outcome. One of the main mechanisms of primary/secondary tumor resistance to chemotherapy is apoptotic inhibition and repair dysfunction. Chromosomal alterations, such as deletions, amplifications, inversions, three nucleotide expansions, LOH, microsatellite instability, loss of genetic imprinting and translocations, are commonly observed in transformed cell lines, which has lead to the hypothesis that genetic instability may play an important role in oncogenesis. Chromosome breakage, as it might arise from double-strand breaks (DSB) has been postulated to be the initial event in these processes (6). Current models suggest that genomic instability is crucial in the accumulation of the multiple alterations required for tumorogenesis. However, the nature of the initial damage responsible for the origin of genomic instability remains poorly understood. Defects in recombination/repair pathways that process DNA damage, such as (DSB), have been proposed as a mechanism of genome instability in some human cancers. Both homologous mitotic recombination (HMR), causing loss of heterozygosity (LOH) of a wild type allele, and structural chromosome aberrations (CA) involve the formation of double-strand breaks in DNA. DSB is a strong promotor for initiation of apoptosis (7). DSB can be perceived as the corner stone in which coupled pro-apoptotic and pro-survival processes determine cell fate (apoptosis or survival) (Fig. 2). APOPTOSIS AND SURVIVAL PROCESSES The molecular pathway of apoptosis starts with an initiating phase by a signal from external stimuli, such as ligation to distinct receptors, or to irreversible cellular or nuclear damage. The initiation phase is followed by a decision phase. During this phase transduction of the apoptotic stimuli to nuclear and cytoplasmatic target proteins– enzymes occurs. The transduction phase includes activation of endonucleases and enzymatic alteration of the cytoskeleton (stimuli for survival?). The execution phase is started when the cell has arrived at the stage of no return. In morphological sense, apoptosis differs from necrosis in that there is cellular shrinkage and chromatin condensation, followed by fragmentation of nuclear components with in membrane-bound vesicles, which are cleared by phagocytosis without damage to adjacent tissue. There are numerous proteins, which modulate the apoptotic mechanism in a positive or negative direction (8,9). C-myc oncogene was the first gene to be viewed as an expression of the dual nature of apoptosis promotion and cell proliferation. The mitogenic and apoptotic ª 2003 Elsevier Ltd. All rights reserved.
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Chemotherapeutical Agents Ch emotherapeutical Ag ents
Double DoubleStrand StrandBreak Br eak DSB DSB
Apoptotic potential/apoptosis
Genotoxic potential/survival
RAD-52 epistatic group RAD-52 epistatic gr oup Homolgous HomolgousDNA DNArecombination recombinationrepair re pa ir
APOPTOSIS AND SURVIVAL PROCESSES Fig. 2 Double-strand break (DSB) as a common denominator of the genotoxic and apoptotic potential of anti-tumor agents.
pathways are genetically inseparable (10). Cell proliferation/pro-survival and apoptotic pathways are coupled/ duality in unity! One of the key mediator for growth factor-dependent cell survival is a small guanosin triphosphatase-protein Ras. Of various pathways that are activated by Ras, the pathway that comprises a series of sequentially activated protein kinases Raf, MEK (MAPK kinase) and MAPK have been found to promote cell survival and inhibit apoptosis (10). The Ras oncoproteins provide a particularly interesting example of how proliferative and apoptotic pathways have been entwined (genetically inseparable). Ras proteins are key transducers of mitogenic signals through their activation of Raf-MAP kinase pathways, a fact attested by the high frequency of Ras activating mutations in human cancer. Ras proteins are also involved in transducing survival signals from a receptor such as IGFI to downstream factors such as PI3-kinase, Akt and Bad (11). Survival-promoting cytokines suppress the activity of the protein BAD, a pro-apoptotic member of the bcl-2 family, by induction of phosphorylation of BAD at two critical sites, serine 112 and serine 136. Phosphorylation of BAD leads to the dissociation of BAD from pro-survival Bcl-2 and the association with the members of the 14-3-3 family of proteins. The regulation of BAD by ª 2003 Elsevier Ltd. All rights reserved.
these phophorylation events suggests that BAD is a point of convergence for multiple signaling pathways that cooperate in order to promote cell survival. Therefore oncogenic mutation of Ras appears able to simultaneously activate cell proliferation and suppress the concomitant apoptosis – a potentially catastrophic combination. Paradoxically, however when activated Ras is expressed in untransformed cells, it triggers precisely a opposite response – a profound p53 growth arrest, frequently accompanied by apoptosis both of which are mediated by Ras kinase raf (11). The p53 mitogen activated protein kinase (MAPK) plays a key role in the activation of p53 by genotoxic stress when induced by anti-tumor agents (12). Ras can activate cell proliferation and suppress apoptosis, but when expressed in untransformed cells, it triggers an apoptotic response. Again, we conclude that these processes are coupled. Therefore, survival signaling pathways maybe directly/ indirectly responsible for mutation fixation. APOPTOSIS, CELL CYCLE CHECKPOINT CONTROL AND ADAPTATION Gene products that regulate the progression of the cell cycle affect apoptosis. Inhibition of apoptosis is consequence of G1–S checkpoint loss. This results in genomic Medical Hypotheses (2003) 61(5–6), 643–650
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instability and survival of damaged cells. Signals for cell cycle checkpoint control are DSB, UV-dimmers and centromere alterations (13). A number of checkpoint genes that are thought to be responsible for monitoring chromosome integrity and preventing cell cycle progression in the presence of damaged DNA-DSB. Abrogation of these damage-monitoring checkpoints may contribute to chromosome rearrangements and therefore can lead to genetic instability. Molecular mechanisms of genetic control of DSB repair are not well understood in humans (13). In Saccharomyces cerevisiae DSB repair is well understood, it generally involves RAD52-dependent recombination. Disrepair of DSB can be the result of illegitimate recombination between non-homologous DNA sequences. Gupat et al. (14) showed that mitotic recombination and, too much lesser extent deletion may be the primary mechanism for the relative high frequency of in vivo LOH observed in normal human T cells. Because LOH leads to the expression of recessive tumor suppresser genes in many cancers, there is significant implication that LOH produced by alteration of RAD52 recombination repair can be the initial event leading to genetic instability (15) and mutation fixation. Signal-tranduction pathways systems have the ability for adaptation. Cells of S. cerevisiae can fixate non-repaired DSB and progress into mitosis after a delay in the G2 phase. Transactivation of p21waf=Cip1 represents absolute requirement for p53-dependent G1 arrest, but alterations in expression of p53 binding protein mdm-2 can result in adaptation of the G2 control point. A major part of p53-mediated growth arrest proceeds through induction of the cyclin-dependent kinase (CdK) inhibitor p21. Raderschall et al. (16) have shown that Rad 51 expression, Rad 51 foci formation and p21 expression are interrelated, suggesting a functional link between mammalian Rad 51 protein and p21-mediated cell cycle regulation (16). Restoration of defective control points would restore the inhibition of the apoptotic answer of cancerous cells to anti-tumor agents (17). REPAIR SYSTEMS AND TUMOR RESISTANCE Mismatch repair system promotes mutational frequency (in tumor cells) in cases of deficiency or defect, but also forms resistance to mutagenic/genotoxic agents in various tumors. It has been observed that cells with a functional mismatch repair (MMR) system respond to treatment with alkylating agents by arresting cell cycle progression in G1 and/or G2 phase. Cells with a deficient MMR system can only arrest the cell cycle in the G1 phase. This indicates that the MMR system integrates with the G2 checkpoint, and the arrest in the G1 Medical Hypotheses (2003) 61(5–6), 643–650
phase is seemingly independent of the MMR system (18,19). The observation of apoptosis in testicular and ovarian cancers with cis-platin sensitivity of these cancers is explained by an increased expression of the hMSH2 protein and a lost ability of MMR system to repair cis-platin DNA lesions. Defects in mismatch repair are associated with cis-platin resistance (19). The MMR system also detects cis-platin DNA adducts and loss of MMR results in resistance to cis-platin (20). Observations support the hypothesis that mismatch repair defects in hMUtL alpha and hMUtS alpha, but not in hMUtS beta, contribute to increased replicative bypass in cells exposed to cis-platin. Replicative bypass introduces drug resistance by preventing futile cycles of translesion synthesis and mismatch correction (21). Anti-cancer activity of intra and interstrand alkylating agents arises from its ability to damage DNA, with the major adducts formed being intrastrand (GpG) and d(ApG) crosslinks. These crosslinks bend and unwind the DNA duplex. The altered structure attracts high mobility group (HMG) domain proteins and other proteins (21). Nucleotide excision repair (NER) is also responsible for the repair of platinum–DNA lesions. The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of DNA damage sites during NER. Recombination pathways act independently of NER and are of equal importance to NER as genoprotective systems against cis-platin cytotoxicity. Taken together these results shed new light on cell survival processes and chemotherapy resistance (22). Previous studies have shown that high-mobility group (HMG) domain proteins such as HMG1 sensitize cells to cis-platin by shielding the major DNA adducts from nucleotide excision repair. It is still not known if these genes are turned on by DNA damage (formation of DNA adducts) or by a downstream of biological consequences induced by the damage of DNA (22). HMG proteins constitute a class of important architectural proteins involved in transcription regulation of genes, participate in gene rearrangements which are linked to the emergence of benign tumors and have the ability to recognize DNA–cis-platin adducts selectively. HMG sensitize cells to cis-platin by affecting DNA replication. The implication of HMG proteins to shield DNA adducts from NER repair have dual consequences: one is sensitizing tumor cells to cis-platin, but the other opens a question: would shielding DNA adducts in normal cells bring apoptosis inhibition and consequently possible induction of secondary tumors by cis-platin? cis-platin is known to induce secondary tumors of the ANLL type after primary treatment. Also, it should be considered that some HMG proteins, acting as architectural proteins ª 2003 Elsevier Ltd. All rights reserved.
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that bring many of the transcription factors into precise three-dimensional shapes may have a critical role in neoplastic transformation (as to that of some transcriptional factors may have themselves) (21). In addition to its classical role in the cellular stress response, heat shock proteins (HSP) play a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival (23). A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitions and essential protein chaperones HSP 90 and HSP 70. Chaperones play a role within the regulatory network of the cell cycle and within signal cascades. HSP 70 and HSP 90 associate with proteins of the mitogen- activated signal cascade, particularly with the Src-kinase, tyrosine receptor kinases, Raf and the MAPkinase activating kinase (MEK) (24). This supports a notion of a protective function of chaperones and stress proteins in the maintenance of the cell cycle and the signaling processes. HSP 70 is identified recently as a potent anti-apoptotic factor (25–29). HSP 90 is involved in negative regulation of apoptosis (30). The complexicity of tumor resistance through apoptotic inhibition shows us that repair, transcriptional and replicative processes are not separated in pro-survival and/or pro-apoptotic pathways but highly entwined through genetically controlled processes. HOMOLOGOUS RECOMBINATIONAL REPAIR AND DSB Homologue recombination is defined as a process in which genetic information is exchanged between two DNA molecules that share sequence identity. This process is exemplified by genetic recombination during meiosis, helps to increase genetic diversity as well as to ensure proper chromosome segregation (31). Major function of HR in mitotic cells is DNA repair and is mediated through out the RAD52 epistasis group of genes. Molecular genetics of HR in mammals are poorly understood. In yeast genes RAD52 epistasis group including RAD50, RAD51, 52, 54, 55, 57, MRE11 and XRS2, are involved in both homology-dependent DSB break repair and meiotic HR. DSB is also a place of potential recombination (31,32). Persistent DSB could be the initial event leading to loss of heterozygosity by defective recombinational repair and consequently to the expression of recessive oncogenes seen in malignant cells. DSB also affects the checkpoint control mechanism of the cell cycle. DNA damage (DSB) results in activation of p53 through an incompletely understood mechanism, that involves possible activation of ATM (ataxia-telangiectasia mutated gene) and chk 2 kinase (33,34). Chen et al. (35) have provided evidence that ATM and c-Abl ª 2003 Elsevier Ltd. All rights reserved.
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kinase (c-abl, an oncogene is also a downstream effector in the ATM DNA damage response pathway and has been shown to phosphorylate hRAD51 and alter to bind to DNA in vitro) are required for correct post-translational modification and assembly of Rad 51 protein complexes (35). Wyllie et al. (10) have reported studies that have demonstrated that aneuploid cells in colorectal cancer have sustained obligatory recombinational elements that must have involved DSB (10). Other studies have shown that p53 activation following DSB can initiate apoptosis (Fig. 3). These descriptive human data are compatible with the idea that aneuploid cells of colon rectal cancer may have arisen from cells that sustained DSB but failed to enter apoptosis! Powerful mechanisms are expressed in order to activate apoptosis when cells that replicate are comforted with DSB. But, apoptosis can be overridden by deregulated recombinational processes through the RAD epitasis group (39). Rad52 interacts with RAD51 through its C-end, and is attached to DNA by its N-tail. RAD52 homologue recombination is the main rout for DNA (DSB) repair in S. cerevisiae. Rad52 has an increased expression in S and G2 phase of the cell cycle, possible in connection with cell cycle checkpoints. On the 3’ end and of the nontranslational part of mouse RAD52-cDNA there is ATTTA motif that is explained as a controller for iRNA degradation of some genes like c-fos or c-myc. C-myc is a oncogene implicated in proliferative and apoptotic (coupled) processes in tumor cells. Expression of c-myc, if increased, has a better chance to lead cells to apoptosis by anti-tumor agents. hRAD51 is major tumor suppressor gene. RAD51 is a member of the RecA/RAD51 family of proteins that have been implicated in recombination/reparative processes. RAD51 proteins are directly/or indirectly associated to p53, BRCA 1 and BRCA 2. Dysfunction of RAD51 can promote cancer and instability, i.e., hRAD promotes chromosome stability and protects DNA against DNAcrosslinks and other damages (36,37). hRAD deficient cells accumulate chromosome breakages prior to cell death. p53 is directly linked to homologue recombination processes via RAD51/RecA protein interaction. Interaction of RAD51 and RAD52 genes show that mutation in one of the genes can change the expression of the others (22,31). Increased expression of RAD51 genes in humans are related to DSB repair, in meiosis and V/D/J recombination during he time of lymphocyte maturation (31). The hRad51 appears to be regulated in at least two ways: (a) transcriptionally, by genes that confer a proliferative potential, as well as by checkpoint signaling pathways that regulate DNA damage responses (b) at the protein level, where interactions with other molecules lead to distinct cellular localization of hRad51 in nuclear foci (39). Medical Hypotheses (2003) 61(5–6), 643–650
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RAD RAD51, RAD invovlved RAD52, 52,RAD50, RAD50,RAD51, RAD 54, 54, RAD55, RAD55, MRE11 MR E11and and XRS2 XRS2 are are invovlved ininhomology depedent double strand break repair homology depedent doub le stra nd break repair
Survival Survival signaling signaling pathways pa thways IGF-1 IGF- 1 interleucin interl eucin3 3 ras/raf-1 ras/raf-1 kinase kinase toto bcl-2 bcl-2 defect defectininp53 p53 mdm-2 mdm-2
inhibition inhibition of of PCD/apoptosis PCD/apoptosi s
Proapoptotic Proapoptotic pathways pathways bcl-2 bcl-2fosphorilation fosphorilatio n p53 p53 myc expression myc expression high highmobility mobility group grou p proteins(HMG) proteins(HMG ) BRCA1 BRCA1and an d2 2 hRAD51 hR AD51
Proliferative Prol iferati veand an dapoptotic apopt oticpathways pathw aysare are coupled coup led
Mu tation fixation fixationby byadatation adatationofofcell cellcycle cyclecheck checkpoint point controll Mutation contro Genom Genom instability instabilit y Fig. 3 A schematic representation of pro-survival and pro-apoptotic (coupled) processes that is based on complex interaction of homologous recombination repair, abrogation of cell cycle checkpoint control and replicative ‘bypass’ which can lead to inhibition of apoptosis by mutation fixation.
BRCA 1 and BRCA 2 (Breast Cancer Susceptibility gene 1 & 2) participate in the cell cycle progression, apoptosis and DNA repair pathways mediated through interaction with RAD51 recombinase in response to genotoxic stress. The role of BRCA1 protein in apoptotic pathway as well as an important role in the embriogenesis has also been suggested (37,38). BRCA central region harbors a binding site for RAD51 protein, a eukaryotic homologue of RecA protein of E. Coli. RAD51 is involved in repairing DNA double-strand breaks. One of the function of BRCA1 protein with the respect to its cell cycle-dependent manner of its expression and hyperphosphorylation might be in the cell cycle regulation of the G1–S transition and/or G2/M phase (38). Loss of BRCA 2 causes misrepair of chromosomal DSB occurring between repeated sequences by stimulating use of an error-prone homologous recombinational pathway. Furthermore, loss of BRCA 2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C induced DNA cross-links at the expense of error-free repair by sister chromatid recombination (43). RAD53 codes serine/threonine kinases. RAD54 codes a family of proteins that are ATP-dependent and stimulated in the presence of DNA. Mutant RAD52 cells are defective for NER and also, there is a defect in the repMedical Hypotheses (2003) 61(5–6), 643–650
lication processes after UV-irradiation. Mutant RAD52 does not affect sister chromatide exchange (SCE) and other intrachromosomal events (31). Little is known about the correlation of recombination repair and tumor resistance. We propose that recombination repair is strongly associated with a new pathway of apoptotic inhibition. Apoptotic processes are overwhelmed by prosurvival and adaptation processes inducing mutation fixation. In accordance to our given hypothesis recent results have shown that RAD51 recombinational pathway can be suppressed by Bcl-2 family of proteins (42). The Bcl-2 gene therefore combines two separable cancer-prone phenotypes: apoptosis repression and a genetic instability/mutator phenotype. The dual phenotype could represent a mammalian version of the bacterial SOS repair system (42). RecA proteins are involved in SOS error-prone repair mechanisms in Escherichia coli. The SOS error-prone repair system is RecA and LEXA-dependent process. DSB can induce a signal for the activation of RecA protein activation. RecA protein is activated by LexA protein degradation. SOS mechanism is a survival process in prokaryotic cells with an increased mutation rate. Mutants that don’t have RecA protein are easily killed from UV-radiation because of deficient repair mechanisms. From cells that survive no mutants could be detected. RecA gene is highly ª 2003 Elsevier Ltd. All rights reserved.
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conserved through evolution. Mutator phenotype in tumor cells show mismatch repair deficiency, but not always recombination repair deficiency. This proposed mechanism inserts mutation fixation in tumor cells and consequently apoptotic inhibition. Using analogy for the SOS repair mechanism in prokaryotes to eukaryotic cells, we can hypothesize that ‘SOS’ repair mechanism has evolved to be integrated in the complex system of apoptosis, i.e., inhibitors of apoptosis by mutation fixation. Homologous recombination repair systems are implicated in both systems, and are therefore crucial for genome stability. Example: Altered polymerases expressed through the SOS error-prone repair system in prokaryotes has an analogy in the activation of polymerase beta that can bypass DNA adducts during DNA replication. This bypass process is responsible for the resistance of some tumor cell lines against cis-platin (40,41). Recombination repair coupled with checkpoint control abrogation may be analogue for SOS error-prone survival pathways. We propose a new apoptotic inhibition pathway in which the hypothesized anti-apoptotic protein (MFP or mutation fixation protein) may have a major role in chemotherapy resistance. This SOS error-prone analogue mechanism (?) may be also induced by persistent DSB in normal cells exposed to genotoxic agents, and therefore be responsible for mutation fixation and genomic instability.
FUTURE PERSPECTIVES Results of studies of HR coupled with processes that inhibit apoptotic response in tumor cells can also have fundamental implications of understanding causes and possible mechanisms of DSB fixation and other types of chromosome damage that consequently can lead to genomic instability. We have clearly seen that genomic stability is maintained by dual processes (proliferative versus apoptotic) in which oncogenes play a crucial role. Understanding molecular events that lead to genomic instability, including components involved in damage susceptibility, repair, cell cycle control and apoptosis will be required for development of new chemotherapeutical strategies. We have proposed that hRad 51, 52 and etc., proteins are implicated in coupled pro-apoptoic, pro-survival processes by adaptation. These processes may lead to mutation fixation by apoptotic inhibition in tumor cells, thus resulting in resistance to chemotherapy. The proposed mutation fixation process in normal cells may also be the initial event leading to genome instability and consequently to promotion of oncogenesis. Restoration of apoptosis by modulating the extrinsic and intrinsic regulators of apoptosis in resistant tumor cells can vastly improve the effectiveness of anti-tumor therapy.
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