ARTICLE IN PRESS

Journal of Theoretical Biology 227 (2004) 253–264

Model of the developing tumorigenic phenotype in mammalian cells and the roles of sustained stress and replicative Tatiana V. Karpinets*, Brent D. Foy Department of Physics, Wright State University, 3640 Colonel Glenn Hwy, Dayton, OH, 45435, USA Received 7 August 2003; received in revised form24 October 2003; accepted 4 November2003

Abstract

The molecular mechanisms that drive mammalian cells to the development of cancer are the subject of intense biochemical, genetic and medical studies. But for the present, there is no comprehensive model that might serve as a general framework for the interpretation of experimental data. This paper is an attempt to create a conceptual model of the mechanism of the developing tumorigenic phenotype in mammalian cells, defined as having high genomic instability and proliferative activity. The basic statement in the model is that acquired by tumor cells are not caused directly by external DNA damaging agents, but instead are produced by the cell itself as an output of a Mutator Response similar to the bacterial ‘‘SOS response’’ and characterized by the initiation of error-prone progression and an elevated rate of . This response may be induced in arrested mammalian cells by intracellular and extracellular proliferative signals combined with blocked apoptosis. The mutant cells originated by this response are subjected to via apoptosis and turnover. This selection process favors the survival of cells with high proliferative activity and the suppression of apoptosis resulting in the long run in the appearance of immortalized cells with high proliferative activity. Either a sustained stressful environment accompanied by continuing apoptotic cell death, or replicative senescence, provides conditions suitable for activation of the Mutator Response, namely the emergence of arrested cells with blocked apoptosis and the induction of proliferative signal. It also accelerates the selection process by providing continuing cell turnover. The proposed mechanism is described at the level of involved metabolic pathways and proteins and substantiated by the related experimental data available in the literature. r 2003 Elsevier Ltd. All rights reserved.

Keywords: Cancer; Tumorigenesis; Genomic instability; Apoptosis; Cellular senescence; Stress response

1. Introduction mutations and associates this nature to the mechanism of natural selection arising fromenvironmentalpres- In spite of different manifestations, etiology, and sures accompanied with cell death. In tumorigenesis clinical features of different cancers, they share several natural selection may pick cells with the traits which principal common characteristics both at the level of are beneficial to cell survival, and genomic instability their development and progression and at the level of may supply a wide repertoire of mutant cells for this function of an individual cancer cell. In almost all selection. instances deregulated cell proliferation and suppressed Several papers draw attention to consideration of cell death together provide a minimal underlying plat- tumorigenesis as a process of natural selection via formnecessary for neoplastic progression ( Evan and apoptosis to provide a growth advantage for cancer cells Vousden, 2001). These changes are provided by muta- (Cairns et al., 1988; Evan and Vousden, 2001; Khong tions residing in cancer cells that lead to a gain of and Restifo, 2002). The experimental studies of so-called function for protooncogenes and loss of function for ‘‘adaptive’’ mutations in bacteria and yeast arising in tumor suppressor genes. The fact that similar mutations cells after their exposure to a selective stressful environ- are often observed reveals the adaptive nature of ment, such as medium containing lactose as the only carbon source in experiments with lac mutants of *Corresponding author. Tel.: +937-775-2760; fax: +937-775-2222. , reveal possible mechanisms, called E-mail address: [email protected] (T.V. Karpinets). ‘‘SOS response’’, of this at the molecular

0022-5193/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtbi.2003.11.005 ARTICLE IN PRESS 254 T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 level. It involves a transient limitation of mismatch (1) An initial assault on a mammalian cell’s DNA such repair function, point mutations (akin to microsatellite as a chemical carcinogen or radiation, or replicative instability in humans) with the participation of senescence, will lead to cell cycle arrest. Once in cell special error-prone DNA polymerase, DinB (Pol IV), cycle arrest, the induction of intracellular and and gene amplification (similar to larger-scale chromo- extracellular proliferative signal in combination somal instability) (Cairns et al., 1988; Bridges, 1994; with the blockade of apoptosis abrogates cell cycle Hendrickson et al., 2002). The increased rate of arrest and activates error-prone cell cycle progres- spontaneous mutations provided by these mechanisms sion thereby generating spontaneous mutations in and stimulated by various kinds of stress allows the of emerged cells. We will refer to the cells to adapt to it (McKenzie and Rosenberg, 2001). emergence of mutant cells as a result of error-prone Evidence exists that these ‘‘directed’’ adaptive mutations division as the Mutator Response (MR). A critical actually result froma standard Darwinian process, in event in developing the MR is to overcome cell cycle which natural selection acts in several sequential steps to arrest under these conditions. direct mutations to adaptation (Hendrickson et al., (2) The mutant cells originated by the MR are 2002). The stressful environment acts to favor a subjected to natural selection via apoptosis. Typi- succession of cell types with progressively adjusted cally the assault on cellular DNA described in (1) phenotype. above will not only lead to cell cycle arrest for some The crucial point in the increased mutation rate in cells, but will also produce conditions of continuing E. coli under stress is the promotion of mutations by cell turnover in the tissue due to cell death and special mutator error-prone DNA polymerases able to subsequent renewal. Under these conditions of elevate substitution and frameshift mutations in un- continuing cell turnover, the selection process damaged DNA (McKenzie and Rosenberg, 2001). The favors the survival of cells with mutations that recent discovery of error-prone DNA polymerases in allow themto avoid apoptosis, overcomecell cycle humans with functions similar to those in yeast and arrest, and promote proliferation. In the long run it bacteria implies that similar molecular mechanism of results in the appearance of transformed cells adaptive may exist in humans (Woodgate, (immortalized cells with high proliferative activity). 1999; Lawrence and Maher, 2001; Goodman, 2002). This suggests that mammalian cells may retain their The above model involves the simultaneous presence rudimentary ability from bacteria to achieve an elevated of several factors, both intracellular and extracellular, in mutation rate, and this ability may be triggered by order to give rise to transformed cells. These factors are certain environmental conditions and realized at the proliferative signals, cell cycle arrest, blockade of level of individual somatic cells at the molecular level. apoptosis and a cellular environment with continuing The parallel between the ‘‘adaptive’’ mutations and cell turnover providing rapid selection. One condition in oncogene-induced transformations strongly supports which these factors can coincide is when cells or tissue this idea (Hall, 1995). are exposed to sustained stress. Sustained stress here is The goal of this paper is (i) to present a model of the defined as situation in which cells undergo the continu- mechanism of the developing tumorigenic phenotype, ing apoptotic cell death and renewal. taking into account the adaptive features of mammalian The model remains a hypothesis at this point due to cells; (ii) to provide support fromthe published the challenge of experimentally confirming large-scale literature for this mechanism at the level of metabolic pathway interactions. Nevertheless the information pathways and proteins; and (iii) to discuss the role of a available in the literature about the pathways and sustained stressful environment and cellular aging in individual proteins involved in tumorigenic transforma- providing conditions required for the establishment of tions supports the presented scheme of cellular pro- the tumorigenic phenotype. cesses. The proposed mechanism at the level of known cellular processes and pathways is given below.

2. Model description and support from the 2.1. The activation of mutator response (step 1) published literature The combinations of cellular events that correspond The proposed model reconstructs a sequence of to normal cell division and MR are presented in Fig. 2. cellular events in the developing tumorigenic phenotype According to this scheme MR may be induced in cells by in mammalian cells (Fig. 1) that follows fromthe superposition of proliferative signal and stress-related generalization of cancer research at the level of cell survival signal, if these cells are arrested as a result of (i) culture or model organisms. It is based on the following irreparable damages or (ii) telomere shortening (repli- statements that represent the steps in the cellular cative senescence). Fig. 3 gives details to the super- mechanism of tumorigenesis. position of signals depicted in Fig. 2 for MR. It also ARTICLE IN PRESS T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 255

Assault on STEP 1 (1) Stressfull conditions leading to continuing cellular DNA apoptotic cell death and renewal, and/or (2) or replicative growth stimulating agents senescence

Extracellular and Cell cycle arrest + intracellular + Blocked apoptosis proliferative signals

Mutator response MUTATOR RESPONSE

Mutant cells

STEP 2 Natural selection via apoptosis

NATURAL SELECTION Transformed cells VIA APOPTOSIS

Fig. 1. The model of cellular events in the developing tumorigenic phenotype in mammalian cells.

Fig. 2. Possible cellular events induced by proliferative signal in normal mammalian cells (a), cells with damaged DNA (b), and senescent cells (c). Cell cycle progression initiated by the proliferative signal is accomplished in normal cells and cells with repairable damage by the emergence of healthy progeny (outputs 1 and 2). If damages cannot be repaired the normal cell cycle progression cannot be accomplished, and the damaged cell undergoes apoptosis (output 3) or initiates the Mutator Response (output 4), when apoptosis is blocked by the stress-related survival signal. The same output takes place in the senescent cell with the arrested growth if the activated proliferative and survival signals coincide in this cell. ARTICLE IN PRESS 256 T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264

Fig. 3. Cellular processes and pathways involved in the Mutator Response of mammalian cells. indicates known pathways and genes that may be The blockade of apoptosis is activated by extracellular engaged in the mechanism of MR in mammalian cells. survival signals, like basic fibroblast growth factor, According to this mechanism the coincidence of vascular endothelial growth factor, insulin-like growth intracellular proliferative signal with the cell cycle arrest factor I, hepatocyte growth factor, interleukin-4, inter- and the blockade of apoptosis is the initiating event for leukin 2 and others. They may be secreted by cells in abrogation of cell cycle arrest at G1 and G2 check- stressful situations accompanied by their death. Ras/ points. This coincidence is a rare situation in mamma- Raf/MEK/ERK pathway is a well known MAP kinase lian cells, though each of these factors taken separately cascade that is activated as a result of this signaling is typical for cells. inducing proliferation and suppressing cellular apopto- Cell cycle arrest is a regular cellular response to tic machinery (Shields et al., 2000). This pathway DNA damage. The involvement of p53 pathway and promotes survival by activation of transcription factors TGF/Smad signal transduction in cell cycle arrest including c-myc (Khosravi-Far et al., 1998) and by at G1 and G2 checkpoints is common for different suppression of apoptosis targeting mdm2 (a major cell lines (Hellin et al., 2000; Zhang et al., 2002; Lutz regulator of p53) or PI3K/PTEN/Akt pathways (Bonni and Knaus, 2002). It is believed that these pathways et al., 1999; Ries et al., 2000). Recently the role of basic induce cell cycle arrest either to allow cell repair fibroblast growth factor was shown in the protection of damages or to activate cell apoptotic machinery if endothelial cells fromthe intrinsic pathway of apoptosis damage are not reparable or repair mechanisms are by mitochondrial translocation of Raf-1 kinase, i.e. by failed. Cell cycle arrest is also imposed in senescent cells mechanism that is independent of MEK kinase (Alavi as a result of telomere shortening. These cells remain et al., 2003). viable, but arrest growth at G1 phase via activation of the p53 and pRb/p16 pathways (Shay and Wright, 2.1.1. Abrogation of cell cycle arrest at G1 checkpoint 2001). The cell’s ability to overcome cell cycle arrest at G1 Any cell activates intracellular proliferative signal to checkpoint with the induction of MR and the activation enter division. In mammals transcriptional factor c-myc of DNA synthesis mechanism, which is parallel to the is a crucial mediator of signals that controls cell number well-known error-free Rb pathway, is demonstrated in and therefore cell decision to divide or not to divide several recent studies (Alevizopoulos et al., 1997; Lukas (Trumpp et al., 2001). C-myc is commonly activated in et al., 1997; Santoni-Rugiu et al., 2000). This alternative cells induced to proliferate by a wide range of growth mechanism initiates aberrant DNA synthesis and factors, cytokines and mitogens, whether the cells destabilizes the cellular genome. Several lines of belong to rapidly dividing tumor or to regenerating evidence support the involvement of c-myc, and Ras/ liver (Grandori et al., 2000; Ohri et al., 2002). Raf/MEK/ERK pathway in the activation of this ARTICLE IN PRESS T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 257 mechanism and in the establishment of the transformed and had many rounds of replication before the establish- cellular state (Land et al., 1986; Schwartz et al., 1986; ment of the transformed state. Several genomic studies Santoni-Rugiu et al., 2000; Bazarov et al., 2001) support the idea that telomere dysfunction typical for including the development of neovasculature (Watnick senescent cells provokes chromosomal instability, in- et al., 2003). cluding regional amplifications and deletions, that drives Though the exact molecular mechanisms of over- carcinogenesis (Counter et al., 1992; de Lange, 1995; coming the G1 checkpoint and the interaction between O’Hagan et al., 2002). c-myc and Ras in the induction of mutant cellular It was also found that cell cycle arrest induced in the response are not clear, several experimental findings senescent cells by p53 pathway may be overcome by present evidence that c-myc and Ras coexpression may inactivation of p53, and it results in further telomere lead to cyclin E-dependent kinase activity and S phase erosion and massive cell death (Shay and Wright, 2001). induction without pRb phosphorylation via a dramatic But if inactivation of p53 is accompanied by activation decrease in the level of p27 (a well-known inhibitor of of Ras and human telomerase reverse transcriptase, then cyclin E/cdk2 complexes) (Lukas et al., 1997; Donnellan such cells remain viable and may be readily transformed and Chetty, 1999; Bazarov et al., 2001). Taking into (Hahn et al., 1999). This phenomenon is similar to account that overexpression of cyclin E closely relates transformations considered above, which take place as a to malignant transformations and the establishment result of abrogation of cell cycle arrest imposed by of a mutator phenotype with chromosomal instability severe DNA damage. According to the presented facts, (Donnellan and Chetty, 1999), these results support the we hypothesize that replicative senescence may be idea that the cooperation of c-myc and Ras activates overcome in human cells by activation of stress-induced MR in the arrested cells. survival signals, which would then promote the induc- tion of MR in the senescent cells. As the expression of 2.1.2. Abrogation of cell cycle arrest at G2 checkpoint telomerase and Ras and inactivation of p53 are not The abrogation of p53 mediated cell cycle arrest at G2 features of all tumors, a deeper understanding of the checkpoint by H-ras was also demonstrated in vitro. It molecular mechanisms for induction of MR in senescent was shown that expressed H-ras in HeLa cells regulates cells will remain a future challenge. the G2/M checkpoint in a p53-independent manner and can induce a premature release from G2 arrest by 2.1.4. The involvement of error-prone polymerases and modifying cyclin B1 (Santana et al., 2002; Concin et al., stress-induced rearrangements in error-prone cell cycle 2003). Recent studies in human mammary epithelial progression cells demonstrates that overexpressed c-myc in these There is no direct evidence in the published literature cells significantly attenuates the G2/M arrest following for human cells that the increased rate of mutations treatment with ionizing radiation, allowing the cells to induced by coexpression of c-myc and Ras in the enter mitosis. At the molecular level, the ectopic arrested cells involves the mechanisms of error-prone overexpression of c-myc after gamma-irradiation leads cell-cycle progression. Indirect support for such an idea to an unusually high sustained level of expression of is based on biochemical studies in cell-culture cyclin B1 (Sheen et al., 2003). The failure of G2 mitotic model systems that on the one hand demonstrate the checkpoint generates tetraploid cells, which is often participation of error-prone polymerases and transla- observed as an early step in tumorigenesis and precedes tional stress-induced mutagenesis in the stress response the formation of aneuploid cells (Andreassen et al., of prokaryotes, and on the other hand reveal similarity 1996). The overexpressed Akt also can overcome a G(2)/ in the enzymes and mechanisms in prokaryotes and M cell cycle checkpoint induced by DNA damage. A eukaryotes involved in the establishment of genomic recent study with mouse embryo fibroblasts shows that instability. cells expressing activated Akt continue to divide, with- Broad lines of evidences indicate the involvement of out being eliminated by apoptosis, in the presence of error-prone polymerases (pol II, pol IV and pol V) in continuous exposure to mutagen and accumulate muta- stress-induced mutagenesis in E. coli (SOS-response). tions (Kandel et al., 2002). This Mutator Response performs translesion synthesis and generates spontaneous mutations when normal 2.1.3. Abrogation of cell cycle arrest in senescent cells replication is stalled (Fijalkowska et al., 1997; Good- A crucial feature of many human tumor cells in man, 2002). It is believed that if the cell cannot repair all comparison to normal ones is their ability to maintain lesions on the genome, then to minimize cell death from telomere length by expressing telomerase or by special the blockage of DNA synthesis, it switches on the mechanism termed ‘‘alternative lengthening of telo- mechanism of translesion synthesis with the involvement meres’’ (Oulton and Harrington, 2000; Chang et al., of mutagenic polymerases. These enzymes are evolutio- 2003). Nevertheless telomeres are usually shorter in narily designed to generate mutations by copying lesions tumors, and it implies that tumor cells are not young with high genetic fidelity and undamaged DNA with ARTICLE IN PRESS 258 T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 very low fidelity (Zhang et al., 2002; Woodgate, 1999; mental stress in and yeast are similar to Kunkel et al., 2003). those usually found in tumors (Dykhuizen, 1993; In past few years error-prone DNA polymerases Dunhamet al., 2002 ). (Y family DNA polymerases) with functions similar to The Mutator Response activated in sustained stress is those in prokaryotes were identified in numerous human not the only example in which mammalian cells switch tissues and other eukaryotes (Friedberg, 2001; Lawrence on the mechanism of increased mutation rate with the and Maher, 2001; Goodman, 2002). They are commonly involvement of error-prone polymerases. The diversity involved in lesion bypass of different types of DNA of high-affinity antibodies in the mammalian immune lesions and the mechanism of translesion DNA synthesis responses is provided by generation of DNA mutations is conserved from E. coli and S. cerevisiae to humans in dividing lymphocytes (Nossal, 2003). In this case (Woodgate, 1999; Wang, 2001; Friedberg et al., 2001). somatic hypermutation, gene conversion and class- There are several reports on human error-prone poly- switch recombination provide an efficient mutator merases that demonstrate their error-prone abilities mechanism to diversify functional antigen receptors (Matsuda et al., 2000; Zhang et al., 2002; Kunkel et al., and in this way to adapt themto a wide range of 2003) and the participation in DNA replication at DNA different antigens. damaged sites in S phase, when replication fork is Summarizing the information about error-prone blocked (Limoli et al., 2000; Kannouche et al., 2001). polymerases and stress-induced mutagenesis, we may Specifically Limoli et al. (2000) demonstrated that DNA conclude that the engagement of error-prone poly- replication arrest after UV damage recruits an alter- merases in DNA replication and translational stress- native pathway involving error-prone polymerase eta to induced mutagenesis provide universal mechanisms for resolve an arrested replication fork. It is associated with the increase of the mutation rate in prokaryotic and increased genomic rearrangements that result from eukaryotic cells when such genome instability are critical double-strand breakage and rejoining in cells, when for their survival. p53 is inactivated. Mutagenesis experiments indicate that another human error-prone DNA polymerase 2.2. Natural selection of mutant cells by apoptosis (step 2) kappa (an error-prone enzyme that is frequently over- expressed in human tumors), may be recruited when One of the fundamental statements in the proposed replicative complexes are stalled, and its involvement model is that in the second step in the developing affects the accuracy of DNA replication (Bergoglio et al., tumorigenic phenotype mutant cells emerging as a result 2002). Overexpression of human polbeta, indicated in of MR are subjected to natural selection via apoptosis several types of cancer, may induce high rates of (Fig. 1). Apoptotic cell death is also involved in the first frameshift mutations and in this way contribute to step of tumorigenesis, the induction of MR (Fig. 2), in carcinogenesis (Yamada and Farber, 2002). which apoptosis is activated by checkpoint pathways in In addition to SOS-response, a number of other cells with DNA damage if the damage cannot be mutator responses have been described in E. coli repaired and therefore the cells must be eliminated from (Humayun, 1998; Balashov and Humayun, 2002). the tissue. At the step of natural selection apoptotic cell Translational stress-induced mutagenesis (TSM) repre- death is part of the cellular mechanism that controls the sents an especially intriguing mutagenic pathway that total number of cells in the tissue. It is activated in all demonstrates links between translation, DNA replica- progeny cells by default if they don’t obtain a survival tion and recombination. The expression of TSM signal fromother cells to deactivate their apoptotic requires a functional homologous recombination sys- machinery. The acquisition of survival signal by a cell is tem, an important pathway for generating genetic a matter of chance, but this chance is greater in the diversity, as well as for repairing damages resulting environment with continuing production of survival fromDNA breaks, especially during S and G 2 phases. signals. The following findings support the involvement The mechanism of TSM pathway involves DNA strand of apoptosis in the selection of beneficial mutations exchange enzymes to bring together two homologous during tumorigenesis and suggest a possible mechanism DNA molecules and catalyse the exchange of their DNA of its initiation and implementation. strands in homologous recombination (Bianco et al., 1998). The same mechanism of homologous recombina- 2.2.1. Apoptosis is a universal cellular mechanism to get tion and nonhomologous end joining is also described rid of unnecessary or potentially dangerous cells for mammalian cells (Lundin et al., 2002). Moreover The ability to ablate cells is an essential and enzymes catalyzing the strand exchange reaction appear constructive process that is involved in ontogeny, to be conserved frombacteria to human( Seitz and quality-control, repair mechanisms and the regulation Kowalczykowski, 2000), and genome rearrangements of cell number (Hall et al., 1994; Meier et al., 2000; (amplification, loss of heterozygosity, breakpoints, Bergmann et al., 2002). During the ontogeny of many aneuploidy) observed as responses to selective environ- organs, cells are over-produced to generate and to ARTICLE IN PRESS T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 259 maintain the complex structures of functional tissues. survival signal after division are subjected to apoptosis. The dependency of animals on cell death for their The importance of apoptosis and extrinsic signals in greater complexity, plasticity and longevity is reflected tight regulation of total cell number was demonstrated in a far more explicit redundancy in mechanisms that for neural stemcells, for epithelial cells and associated regulate cell death (Meier et al., 2000). Though the exact fibroblasts of the mammalian gastrointestinal tract (Hall mechanism by which apoptosis is initiated in different et al., 1994; Sommer and Rao, 2002). situations and cell types in vivo remains unclear, two distinct pathways were identified in this signal transduc- 2.2.3. Possible mechanism of natural selection via tion that were reviewed elsewhere (Zimmerman et al., apoptosis and apoptotic crises 2002). Extrinsic receptor-linked apoptosis requires the Taking into account that (i) social control of cell ligation of death receptors, like tumor necrosis factor death implies the competition between cells for the receptor-1 and Fas receptor, leading to the activation of limited quantity of survival signal secreted by neighbor- procaspase-8. Intrinsic mitochondria-mediated apopto- ing cells and that (ii) MR gives randommutationsand sis is regulated by the pro- and anti-apoptotic members therefore implies a lot of deleterious and lethal muta- of the Bcl-2 family. This pathway is induced by cellular tions in the genome of the emerging cells, many stress such as DNA damage and is mediated by the produced mutant cells will undergo mitochondria- mitochondrial release of cytochrome c. mediated apoptosis initiated in them after their emer- gence. Cells with loss-of-function mutations in tumor- 2.2.2. The regulation of cell number via extracellular suppressor genes avoid the apoptotic death and will be control of mitochondria-mediated apoptosis accumulated in the cell population. Gain-of-function It is found that for some mammalian cells pro- mutations in protooncogenes will be also beneficial for grammed death occurs by default unless suppressed by cells, because in this way they acquire high proliferative signals fromother cells ( Raff, 1998). This is known as activity independent of the quantity of survival signal in social control of cell death. Such dependence on specific the environment. These cells have a greater chance to survival signals provides a simple way to eliminate survive and have a sustained turnover. As a result of this misplaced cells, to regulate cell number, and to select selection process the number of transformed cells will cells with beneficial mutations. For example dead cells increase with time compared to normal ones. But some are a prominent feature of the thymic landscape as only time is needed for the required set of tumorigenic 5% of developing thymocytes are exported as mature mutations to appear. Until then most emerged mutant T cells. Most thymocytes die because they are not cells undergo apoptosis. If this mechanism is correct, the positively selected and do not receive a survival signal establishment of transformed state would be accompa- (Starr et al., 2003). Recent studies show the role of nied by extensive cell apoptosis at an early stage of endothelial cells in the transducing of the proliferative tumorigenesis. In highly developed organisms this signal to restore number of hepatocytes after liver phenomenon does not necessarily result in macroscopic injury. In response to activation of vascular endothelial changes that may be easily identified both in vitro and growth factor receptor-1 these cells secrete several in vivo, because the mechanism of programmed cell proteins, including hepatocyte growth factor, that death is quickly implemented at the level of an stimulate hepatocyte proliferation and reduce tissue individual cell without any consequences for other cells. damage in a liver injury model (LeCouter et al., 2003). Nevertheless the phenomenon of apoptotic crises in the Examples of survival signals that may be involved in early stage of transformation is well-established in vitro. the social control of cell death are soluble cytokines and During this stage the majority of cells die before hormones, synaptic connections, and direct physical permanent malignancy will be established (Radfar interactions with neighboring cells and extracellular et al., 1998; Unnikrishnan et al., 1999). Indirect evidence matrix. Different cell types require differing combina- of extensive apoptotic cell death at tumorigenesis has tions of survival signals, which are only available within also been demonstrated in vivo. It was shown that discrete somatic environments (Meier et al., 2000). The cancer patients have an increased level of DNA main reported mechanism of the survival signals is the fragments in blood plasma originating from apoptotic suppression of intrinsic apoptotic pathway. Specifically, and necrotic cells (Jahr et al., 2001). the survival of a subset of midline glia cells in Drosophila depends upon direct suppression of apoptosis via the 2.3. Role of sustained stress in tumorigenic EGF receptor/RAS/MAPK pathway (Bergmann et al., transformations 2002). In some cases the apoptosis suppression may be activated via Bcl-2 or Bcl-X (Raff, 1998; Opferman and Continuing apoptotic cell death may arise as a result Korsmeyer, 2003). By means of this regulation neigh- of long-termsevere cellular injuries induced by various boring cells mutually control their death or survival by forms of environmental stress. It is known that the re- secreting extracellular factors. Cells that fail to receive a generation process follows injury of various mammalian ARTICLE IN PRESS 260 T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 tissues, and that it is triggered by changes in cytokine short-termcell injury and apoptotic death), the damaged expression, leading to activation of specific transcription cells are quickly subjected to apoptosis or survive after factors (Abe et al., 2000; Ohri et al., 2002). This signal repairing, and only undamaged cells are exposed to the transduction relates to generation of the mitogenic synthesized growth factors. As the proliferative signal signal and to the prevention cell apoptosis. Therefore is not sustained, cellular senescence is not promoted as in an environment with continuing apoptotic cell death, well. In the absence of arrested cells, proliferation severely injured cells with arrested growth have a chance induced by growth factors is accompanied by regular to acquire both proliferative and stress-related survival cell cycle progression and MR is not activated. In signals. As stated above, the activation of proliferative addition, transient stress doesn’t provide sufficient time signal in the arrested cells with blocked apoptosis is and continuing cell turnover for natural selection via crucial for the activation of MR. In addition arrested apoptosis. cells in sustained stress may appear as a result of an From the comparison of activated cellular mechan- increased rate of cellular senescence stimulated by isms in sustained and transient stresses, if an agent continued apoptotic death and proliferation. Each induces cellular injury over a long time period leading round of cell division is accompanied by shortening of to continuing cell death, there is higher chance that telomeres and therefore the emerged cells approach this agent will cause carcinogenic transformations in replicative senescence (Chang et al., 2003). There is mass the exposed cells and in the long run will incite the of evidence in literature on the induction of DNA development of a malignancy. This inference of the damage, cell cycle arrest and cellular apoptosis with model has support from numerous studies on nonmuta- activation of p53 and TGF/Smad pathways and also genic chemical carcinogens (Ames and Gold, 1997). proliferative signals in response to exposure of many They show that (i) more than half the chemicals, toxic agents (Pietenpol and Stewart, 2002). The activa- whether synthetic or natural, are classified as carcino- tion of MAP kinase signaling by various forms of gens when tested at the maximum tolerated dose in environmental stress, including oxidative stress and the standard rodent cancer bioassays, (ii) there is a perfect exposure of diverse xenobiotics, is also well-established correlation between cancer causation and cell division (Aikawa et al., 1997; Laskin et al., 2002). It was also in the target tissue. Even in the case of tobacco smoke shown that cells respond very differently to transient physiological stresses (not necessarily genotoxic) aggra- and prolonged activation of MAP kinase signaling vated by smoking are the leading risk factor in the p53- (Murphy et al., 2002). Sustained ERK activation associated etiology of lung cancer (Rodin and Rodin, induces stable activation of c-Fos and the stabilization 2000). The proposed model explains at the molecular of the activity of AP-1 transcription factors to which c- level why chronic cell killing and consequent cell myc also belongs. Therefore prolonged activation of replacement is a risk factor for cancer. MAP kinase signaling in cells subjected to sustained stress may result in the persistent activation of 3.2. The model and etiology of human cancers intracellular proliferative signal necessary for the induc- tion of MR. In addition, continuing apoptotic cell death The most important inference of the model is that and renewal in sustained stress provide sufficient time mutations acquired by cells in sustained stress are not for cell turnover and therefore accelerate the process of caused directly by external DNA damaging agents, but natural selection of the mutant cells emerging as a result instead are produced by the cell itself as an output of the of MR. The requirement of a long-term influence of MR. The numerous known risk factors for adult cancer, stressful condition for the establishment of tumorigenic such as exposure to smoking, radiation, and cancer- phenotype and its quick subsequent progression are causing chemicals and viruses, function in the model by well-known features of most cancers, a characteristic promoting the establishment of the MR. First, these that may be explained by the proposed model. factors induce a sustained stressful environment in cells resulting in their continuing death and renewal, which indicates the presence of proliferative signals and can 3. Discussion lead to blocked apoptosis. Second, direct DNA damage fromthese external agents induces cell-cycle arrest, 3.1. Transient stress does not provide the necessary which is another necessary condition for the MR. conditions for the establishment of the transformed In some tumors, though, mutations are not necessa- cellular state rily produced by the MR. It is believed that in hereditary cancer the first mutation in the carcinogenic process is According to the model the initiation of error-prone inherited and just transmitted through the germline. The cell-cycle progression in the arrested cell requires a inherited mutation usually occurs in a tumor-suppressor coincidence of intracellular proliferative signal with the gene, protooncogene or DNA repair gene (Boman et al., blockade of apoptosis. If stress is transient (there is only 2002). The steps between these mutations and full blown ARTICLE IN PRESS T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264 261 cancer are not clear at present. One possibility is that Though cancer incidence increases exponentially these particular mutations directly increase the rate of with age, this disease also takes place in children. additional mutations. Combining an elevated rate of Due to the relative rarity of cancer in children, the mutation with the natural selection discussed in the etiology of most childhood cancers is unknown. How- model presented in this paper may then lead to cancer. ever, the model presented here may prove to have However we think that it is more likely that these some application in children. It has been shown that inherited mutations promote the development of the many childhood cancers include risk factors asso- MR in cells, and thus indirectly increase the rate of ciated with heredity, virus infections and exposure additional mutations. The inherited mutations in some to toxic agents (Linet et al., 2003). Such assaults cases may fill roles necessary for the MR. For example a may lead to cell cycle arrest and produce a stressful defective tumor-suppressor gene leads to suppression of tissue environment, thereby promoting tumorigenesis in cellular apoptotic machinery and mutation in a proto- ways similar to adults. Also, the levels of proliferative oncogene leads to induction of sustained proliferative signals will be high at many points in the life cycle of signal. In other cases the inherited mutations may lead children. to the generation of a stressed tissue environment with frequent apoptosis of mutated cells, and then this 3.3. Limitations of the model, its advantages and possible stressed tissue environment will contain the proliferative implementations signals combined with cells in arrest that lead to the MR. Many cellular processes considered in the model, The Mutator Response may be activated in cells such as the initiation of error-prone cell cycle progres- experiencing conditions other than sustained stress. Any sion and the natural selection of cells with beneficial agent that stimulates cell division, and is characterized mutations via apoptosis, cannot be described for the by sustained exposure, may create an environment present at the level of specific genes or involved suitable for the activation of the MR. The reason is molecular functions. The presented model suggests that sustained stimulation of cell proliferation induces a only a general scheme of cellular behavior in sustained continuing cell death in order to keep the total cell stress that follows fromexperimentalstudies of biolo- number in the tissue stable and this apoptotic death in gical processes, pathways and molecular functions turn activates stress related survival signals to block related to cancer, DNA damage, molecular genetics apoptotic machinery of the neighboring cells. In and . The model engages the main addition, over long periods of time, continuing cell pathways that are well known to be implicated in turnover promotes cellular senescence which is also one cancer development, and assigns a task and a mec- of the factors for the induction of the Mutator hanism of its performance to each of them. This Response. Well-known growth stimulating agents are global view of molecular mechanisms involved in the hormones, growth factors, and viruses. The involvement developing tumorigenic phenotype should aid future of these agents in carcinogenesis is shown for many studies by (1) providing a framework to explain contra- types of tumors. The observations include an increased dictory facts about protooncogenes and tumor-suppres- risk of breast and endometrial cancer in postmenopausal sor genes with respect to their influence on cell death women with elevated serum estrogens and androgens or survival, the etiology of different cancer diseases, (Lamar et al., 2003); a stimulation of prostate cancer by the resistance of tumor cells to immune attack, and androgens (Castagnetta et al., 1995); and the pro- the genetic changes in the progression of cancers; motion of colorectal carcinogenesis by hyperinsulinae- (2) enabling the prediction of cellular outcome in mia (Nilsen and Vatten, 2001). All known oncogenic various stressful conditions; and (3) highlighting the viruses, both DNA- and RNA-containing, activate cell role of environmental factors that may induce or growth (McCance, 1999). For example all known accelerate tumorigenic transformations and toxicants Burkitt’s lymphoma tumors carry a reciprocal chromo- with carcinogenic properties. This model may facili- somal translocation in the proximity of the c-myc that tate the design of experiments in biochemical and leads to deregulation of its expression (Ruf et al., 1999). genetic studies and in studies with gene arrays. It also Prolonged exposure to elevated levels of growth opens perspectives for mathematical simulation of stimulating agents thus provides three parts to the tumorigenesis. tumorigenic process as described in the model. They promote earlier senescence via increased cell turnover, and they provide proliferative and survival signals. Of Acknowledgements course normal aging in the presence of normal levels of growth signals also leads eventually to cellular senes- This study was supported by a grant fromthe Air cence, which correlates with the increase in cancer Force Office of Scientific Research. We also thank incidence with age. Duncan Greenwood for comments on the paper. ARTICLE IN PRESS 262 T.V. Karpinets, B.D. Foy / Journal of Theoretical Biology 227 (2004) 253–264

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