- Email: [email protected]
cancer bioassays: cell cycle control, cell proliferation and apoptosis as modifiers of outcome1
Mutation Research 398 Ž1998. 189–195
Series: Current Issues in Mutagenesis and Carcinogenesis, No. 85
1
Transgenic rodent mutationrcancer bioassays: cell cycle control, cell proliferation and apoptosis as modifiers of outcome Ruth A. Roberts
)
Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, UK
Keywords: Cell proliferation; Apoptosis; Genotoxicity; Cell cycle; p53
1. Introduction The advent of transgenic rodent mutation bioassays w1–3x has focused attention on certain practical considerations, such as the requirement for sufficient time between dosing and mutation frequency assessment to allow for the expression of DNA lesions and mutations Žreviewed in w4,5x.. Also, there has been much debate about the consequences of modifying oncogenes and tumour suppressor genes implicated in DNA surveillance and in the expression of mutations in rodent cancer bioassay models w6–8x. Current interest in the use of transgenic rodent assays for regulatory testing has focused on establishing validated and practical test protocols Žreviewed in w4,5x.. However, there has been a growing appreciation of the need also to understand the underlying cell biology, such as the molecular regulation of cell cycle, cell proliferation and apoptosis and the inter-relationship between these key modifiers of cell fate. These factors require consideration in the evaluation and validation process in order to facilitate accurate interpretation and extrapolation of rodent data to hu-
) Tel.: q44 Ž1625. 516-413; fax: q44 Ž1625. 587-964; e-mail: [email protected] 1 No. 84 was published in Mutation Research – Genetic Toxicology and Environmental Mutagenesis.
mans w9x. This article considers key recent findings in the control of cell proliferation and apoptosis in the context of DNA damage. These topics are suggested to be of general relevance to the deployment of transgenic rodent assays and the interpretation of derived test data.
2. The cell cycle There are several key issues to be considered when addressing the effects of cell proliferation and apoptosis on the expression of genotoxicity. Firstly, an understanding of cell cycle regulation is required. Building on this, how do cell proliferation and apoptosis work together to regulate cell fate? The mammalian cell cycle is divided approximately into 4 stages; synthetic ŽS.-phase where the cell replicates DNA and mitotic ŽM.-phase where the cells undergo nuclear and cytoplasmic division Žsee Fig. 1. Žreviewed in w10x.. S-phase and mitosis are divided by two gaps: gap 1 ŽG1 .; and gap 2 ŽG2 .. There are a number of key regulatory points in the cell cycle known as checkpoints where cells pause in their progress through the cycle until they receive the signals necessary to progress. The most crucial checkpoints for genome integrity are the G1 checkpoint where a cell will pause to check sequence
0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 2 3 9 - X
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R.A. Robertsr Mutation Research 398 (1998) 189–195
Fig. 1. The mammalian cell cycle. For details, see text.
integrity before replicating the DNA and the G2rM checkpoint where a cell will pause to check if DNA replication has been completed before chromosomes are divided. The G1 checkpoint is referred to also as the restriction or ‘R’ point since this forms a ‘barrier’; once a cell has traversed this restriction it normally completes the cell cycle.
levels do not fluctuate significantly; it is the level of their cognate cyclins that vary as the cell oscillates through S to M and back to G1. Fig. 2 depicts progression through the cell cycle, indicating the peak of cyclin D 1 which occurs early in G1 and of cyclin E which is required to traverse the G1 to S boundary Žreviewed in w10x.. As cyclin D 1 peaks in early G1 , it complexes with and activates cyclin-dependent kinase 4 ŽCDK4.. CDK4 has as its major target substrate the retinoblastoma protein ŽRb. Žreviewed in w12x.. Similarly, as cyclin E peaks in the G1 to S transition, it is able to complex with CDK2, augmenting the phosphorylation of Rb. Hyperphosphorylation of Rb permits dissociation from DNA where it is bound to and complexed with the elongation factors such as E2F-1 and DP1 Žreviewed in w13x.. When complexed with Rb, E2F-1 is unable to transcribe the genes associated with, and essential for, cell proliferation, such as the immediate early genes c-myc, c-fos, c-jun and genes involved in DNA synthesis, such as dihydrofolate reductase and thymidylate synthetase. However, the phosphorylation of the Rb protein by CDK2 and CDK4 permits dissociation of Rb from E2F-1rDP1 permitting the commencement of proliferation Žreviewed in w12x.. The cyclin-CDK network forms a very elegant control mechanism for DNA replication and cell
3. Cell cycle regulation: CDKs, CDIs and Rb Progression of the cells through the cell cycle is regulated by two distinct families of proteins known as cyclins and cyclin-dependent kinases ŽCDKs; Fig. 1. Žreviewed in w10,11x.. Each cyclin has a CDK binding partner. For example, cyclin D 1 forms a heterodimer with CDK4 and is one of the major signals regulating the early G1 checkpoint. Cyclins are so called because their levels peak and fall during progression through the cell cycle Žsee Fig. 2.. Thus, as the level of cyclin rises, it becomes available to heterodimerise with its appropriate CDK. This, in turn, permits activation of the CDK and, as the name implies, these activated CDKs can phosphorylate their substrate or target protein. CDKs are crucial for the regulation of the cell cycle but their
Fig. 2. Cell cycle control. For details, see text.
R.A. Robertsr Mutation Research 398 (1998) 189–195
division. However, there is a further level of complexity in the regulation of the cell cycle; each CDK has a cyclin-dependent kinase inhibitor or CDI Žreviewed in w14x.. Several of these have been characterised and fall broadly into two families. The family of greatest interest when considering cell cycle regulation and DNA damage is the p21 family of CDIs Žreviewed in w15x.. p21 itself is also known as WAF-1 Žand is not to be confused with p21ras .. p21WA F-1 is a CDI that acts on cyclins D and D 1 to prevent the activation of the CDKs that control the G1 checkpoint w16x. Thus, activation of p21WA F-1 causes a cell cycle arrest in early G1 w16x. What relevance does this have to the expression of genotoxicity? Data emerging more recently have shown that the major mechanism for activation of p21WA F-1 is via p53 w17x and the major activator of p53 is DNA damage Žreviewed in w18x..
4. DNA damage and p53 The p53 protein was described originally as an oncogene by two separate groups, both of whom noted a prominent polypeptide in cells transformed by the DNA tumour virus, SV40 w19,20x. However, it became apparent over the next few years that p53 is not an oncogene, but belongs in the opposing camp of tumour suppressor genes. Retrospectively, many of the early experiments demonstrating oncogenic activity of p53 unknowingly used mutant p53. This realisation coincided with a key paper on human colorectal cancer concluding that p53 had all the classical attributes of a tumour suppressor gene w21x. One of the unanimously accepted concepts regarding p53 is that it acts as a molecular stress response device. DNA damage elevates levels of active p53 by two mechanisms Žreviewed in w22x.. Firstly, there is increased transcription and translation. However, DNA damage also causes a stabilisation of the p53 protein which normally has a short half-life Žreviewed in w18,22x.. Thus, p53 is a cellular brake that arrests the cell cycle in early G1 and is required when the integrity of the genome is challenged. Also, p53 is a sensor of DNA damage which earned it the title of ‘guardian of the genome’ w23x. Thus, DNA damage causes elevation of p53; p53 elevates p21WA F-1 and p21WA F-1 causes a G1 arrest. Clues to
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the mechanism by which p53 elevates p21 were offered when it was discovered that another function of the multi-faceted p53 protein is as a transcription factor w22x. Subsequently, it was shown that p53 binds to the p21WA F-1 promoter and thus is responsible for transcriptional upregulation of p21WA F-1, the CDI crucial to the regulation of cell cycle progression. Thus, we have a complete pathway through from DNA damage to cell cycle arrest in early G1. Thus far, we have considered how DNA damage caused by genotoxic insult causes a p53-mediated G1 arrest. The relevance of this to the expression of genotoxicity becomes apparent when considering subsequent events. This p53-mediated G1 arrest permits a choice between three paths; escape into Sphase and cell proliferation, protracted G1 arrest pending DNA repair or cell deletion via apoptosis. Apoptosis is a mechanism whereby cells with potentially hazardous lesions are deleted from the organ and the organism and thus will not give rise to cancer prone progeny Žreviewed in w24x.. This G1 decision point is crucial to the expression of genotoxicity and will be discussed in more detail later.
5. DNA damage and apoptosis One clue to how a G1 arrest via p53 signals apoptosis was discovered when it was found that Bax, a ‘killer’ member of the Bcl2 gene family, has a binding site for p53 in its promoter region. Thus, like p21WA F-1 , p53 directly upregulates transcription of Bax w25x. Members of the Bcl2rBax family of protein molecules are key to the regulation of cellular commitment to apoptosis with some members of the family protecting from cell death ŽBcl-2, Bcl-X L . and others associated with cell killing ŽBax, Bad, Bak. Žreviewed in w24,26x.. Initially, it was proposed that Bcl2–Bcl2 protein homodimers provided a survival signal whereas Bax–Bax protein homodimers caused cell killing w27,28x. However, it has become apparent more recently that when Bax levels exceed levels of Bcl2, free Bax may undergo a conformational change, exposing the so-called Bcl2 homology domain 3 ŽBH3. w26,29x which initiates the apoptosis pathway via activation of the caspase ŽICE-like protease. cascade w30,31x. Recently, it was demonstrated that the release of cytochrome c from mitochondria
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plays a role in this pathway to apoptotic cell death w32x, although the mechanism of is not yet understood. A recent paper suggests that p53 induces apoptosis through a three-step process involving the transcriptional activation of redox genes, the formation of reactive oxygen species and the oxidative degeneration of mitochondrial components, culminating in cell death w33x. In addition to p53-dependent mechanisms, under certain conditions, DNA damage can initiate apoptosis through a p53-independent route involving direct activation of caspases via the FasL–Fas receptor pathway and activation of FADD ŽFas-activated death domain. w34,35x. Whether and how this p53-independent pathway to apoptosis acts alongside the more extensively characterised p53-dependent pathway remains to be determined. The recent discovery of a p53-related protein that can induce apoptosis, p73 w36x, may provide further insight into the relationship between DNA damage and apoptosis.
6. p53-mediated G 1 arrest: a decision point? After DNA damage has caused a p53-mediated G1 arrest, the DNA damaged cell can progress down one of three possible pathways ŽFig. 3.. The cell could remain in a G1 arrest pending repair. Alternatively, the cell could undergo apoptosis and would be deleted from the organ or from the body. If apoptosis is the fate of that particular cell then the DNA damage sustained would not be expressed in assays of genotoxicity. Alternatively, the cell could undergo S-phase and proceed through the G1rS checkpoint. If this were to occur before the DNA was fully repaired then any remaining DNA damage would become incorporated into the genome by mis-sense mismatch replication during S-phase w37x. This would yield a positive result in in vitro and in vivo mutation assays w3x. These three possible outcomes are not mutually exclusive at either the cell or the tissue level since the cell could enter S-phase before completion of repair. In addition to the fate of this individual cell, a variety of routes may be taken within a tissue where some cells will undergo apoptosis, some cells will repair and others will replicate. What parameters impinge on the decision between apoptosis, repair and S-phase? Of fundamental im-
Fig. 3. Events downstream of DNA damage. For details, see text.
portance is genetic background, defined as the profile of normal or mutated genes that are transcriptionally active or silent in a given cell at a given time Žw38x; reviewed in w39,40x.. Secondly, the severity and the nature of the damage sustained can affect the cellular outcome w41x. Thus, different types of genotoxic damage may be perceived very differently by the cell and, as such, the downstream responses of the cell will differ w41x. Thirdly, and most importantly, is the trophic environment in which the cell finds itself Žw42,43x; reviewed in w40x.. The trophic environment comprises many factors, such as the natural growth factors endogenous in that tissue. For example, in the liver, hepatocyte growth factor ŽHGF., epidermal growth factor ŽEGF. and transforming growth factor-a ŽTGFa . play key roles in cell survival and proliferation reviewed in w44x. Similarly, in the haemopoietic system, growth factors, such as granulocyte macrophage-colony stimulating factor ŽGM-CSF. would affect the fate of cells. Also, there are several cytokines including the interleukins and tumour necrosis factor-a ŽTNFa . that affect
R.A. Robertsr Mutation Research 398 (1998) 189–195
cellular fate in particular tissues w45x. Cytokines assume particular importance since consideration must be given to both the endogenous background levels of these molecules and their increased production in response to high doses of genotoxic chemicals. In addition to natural molecules, such as growth factors and cytokines, there are other modifiers of S-phase and apoptosis, such as non-genotoxic carcinogens, which can profoundly affect the expression of genotoxicity. These tumour promoters frequently induce S-phase, thereby enhancing fixation of DNA damage and also suppress apoptosis preventing the deletion of DNA damaged cells from the tissue Žreviewed in w46x..
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ered p53 analogue, p73 w36x. Further levels of complexity may be in operation also, as illustrated by recent data showing increased cell survival over controls at low levels of damage w47x. At low levels of the non-genotoxic chemical, dimethylformamide ŽDMF., cellular stress and damage were associated with upregulation of survival signals allowing increased survival of the damaged cells over and above control. Further investigation revealed that the cells were upregulating platelet derived growth factor ŽPDGF. and insulin-like growth factor ŽIGF. receptors to enhance cellular survival.
8. Conclusions 7. Thresholds Considering the molecular mechanisms that control the expression of genotoxicity, it is probable that different cellular defence processes operate in response to different levels of damage. This would argue against a linear model for risk assessment at low doses. For example, at high doses of a genotoxic chemical, cells may engage the apoptotic pathway. At intermediate doses, the DNA damage may be below the level required for apoptosis, but might favour, instead, a G1 arrest pending repair, coupled with some G1rS leakage giving rise to mutation. At low doses, the G1 arrest may be absolute until repair is complete. Even an acceptance of the existence of such thresholds does not permit an accurate prediction of outcome; with decreasing dose, cells with reduced levels of damage may survive rather than be deleted, enhancing the perceived rate of mutation. Conversely, a decreased dose could fall below the threshold of cytotoxicity, removing stress cytokines and thus the stimulus for replication in the remaining viable, but DNA-damaged cells. This would be associated with a decreased rate of mutation. This variable outcome with decreasing dose is, of course, speculative and will remain so until the precise threshold for p53 activation of arrest, executor or repair genes is defined. Also, p53-independent pathways that may have different thresholds for activation may be in operation, such as apoptosis triggered by the Fas pathway w34x or by the recently discov-
This paper has reviewed current understanding of the control of cell cycle with the aim of suggesting that the expression of genotoxicity can be altered profoundly by cell proliferation and apoptosis. There are several undeniable truths that need consideration in the use of transgenic rodents in mutation and cancer bioassays. First, certain types of DNA damage, such as single-stranded DNA breaks are more prone to triggering apoptosis than others w41x. Second, transgenic models for mutagenesis require both survival and proliferation of the damaged cells to give rise to progeny with a mutated marker gene. Third, the ultimate expression of genotoxicity is the development of altered foci and preneoplastic lesions and that requires the survival, proliferation and expansion of the DNA-damaged cells. These are dependent on many parameters, such as the p53 status of the target cells and cell cycle regulation in the target tissue.
Acknowledgements The author is grateful to John Ashby for invaluable discussion and for critical appraisal of the manuscript.
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R.A. Robertsr Mutation Research 398 (1998) 189–195 w36x C.A. Jost, M.C. Marin, W.G. Kaelin Jr., p73 is a human p53-related protein that can induce apoptosis, Nature 389 Ž1997. 191–194. w37x F.C. Richardson, M.C. Dyroff, J.A. Boucheron, J.A. Swenberg, Differential repair of O4-alkylthymidine following exposure to methylating and ethylating hepatocarcinogens, Carcinogenesis 6 Ž1985. 625–629. w38x C.A. Midgley, B. Owens, C.V. Briscoe, D.B. Thomas, D.P. Lane, P.A. Hall, Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo, J. Cell Sci. 108 Ž1995. 1843–1848. w39x R.A. Roberts, J.A. Hickman, D.W. Nebert, J.H. Richburg, T.L. Goldsworthy, Perturbation of the mitosisrapoptosis balance: a fundamental mechanism in toxicology, Symp. Overview, Fundam. Appl. Toxicol. 38 Ž1997. 107–115. w40x A.H. Wyllie, Clues in the p53 murder mystery, Nature 389 Ž1997. 237–238. w41x A.C. Wood, P. Elvin, J.A. Hickman, Induction of apoptosis by anti-cancer drugs with disparate modes of action: kinetics of cell death and changes in c-myc expression, Br. J. Cancer 71 Ž1995. 937–941. w42x M.K. Collins, G.R. Perkins, A. Lopez-Rivas, Growth factors
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Series: Current Issues in Mutagenesis and Carcinogenesis, No. 85
1
Transgenic rodent mutationrcancer bioassays: cell cycle control, cell proliferation and apoptosis as modifiers of outcome Ruth A. Roberts
)
Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, UK
Keywords: Cell proliferation; Apoptosis; Genotoxicity; Cell cycle; p53
1. Introduction The advent of transgenic rodent mutation bioassays w1–3x has focused attention on certain practical considerations, such as the requirement for sufficient time between dosing and mutation frequency assessment to allow for the expression of DNA lesions and mutations Žreviewed in w4,5x.. Also, there has been much debate about the consequences of modifying oncogenes and tumour suppressor genes implicated in DNA surveillance and in the expression of mutations in rodent cancer bioassay models w6–8x. Current interest in the use of transgenic rodent assays for regulatory testing has focused on establishing validated and practical test protocols Žreviewed in w4,5x.. However, there has been a growing appreciation of the need also to understand the underlying cell biology, such as the molecular regulation of cell cycle, cell proliferation and apoptosis and the inter-relationship between these key modifiers of cell fate. These factors require consideration in the evaluation and validation process in order to facilitate accurate interpretation and extrapolation of rodent data to hu-
) Tel.: q44 Ž1625. 516-413; fax: q44 Ž1625. 587-964; e-mail: [email protected] 1 No. 84 was published in Mutation Research – Genetic Toxicology and Environmental Mutagenesis.
mans w9x. This article considers key recent findings in the control of cell proliferation and apoptosis in the context of DNA damage. These topics are suggested to be of general relevance to the deployment of transgenic rodent assays and the interpretation of derived test data.
2. The cell cycle There are several key issues to be considered when addressing the effects of cell proliferation and apoptosis on the expression of genotoxicity. Firstly, an understanding of cell cycle regulation is required. Building on this, how do cell proliferation and apoptosis work together to regulate cell fate? The mammalian cell cycle is divided approximately into 4 stages; synthetic ŽS.-phase where the cell replicates DNA and mitotic ŽM.-phase where the cells undergo nuclear and cytoplasmic division Žsee Fig. 1. Žreviewed in w10x.. S-phase and mitosis are divided by two gaps: gap 1 ŽG1 .; and gap 2 ŽG2 .. There are a number of key regulatory points in the cell cycle known as checkpoints where cells pause in their progress through the cycle until they receive the signals necessary to progress. The most crucial checkpoints for genome integrity are the G1 checkpoint where a cell will pause to check sequence
0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 2 3 9 - X
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R.A. Robertsr Mutation Research 398 (1998) 189–195
Fig. 1. The mammalian cell cycle. For details, see text.
integrity before replicating the DNA and the G2rM checkpoint where a cell will pause to check if DNA replication has been completed before chromosomes are divided. The G1 checkpoint is referred to also as the restriction or ‘R’ point since this forms a ‘barrier’; once a cell has traversed this restriction it normally completes the cell cycle.
levels do not fluctuate significantly; it is the level of their cognate cyclins that vary as the cell oscillates through S to M and back to G1. Fig. 2 depicts progression through the cell cycle, indicating the peak of cyclin D 1 which occurs early in G1 and of cyclin E which is required to traverse the G1 to S boundary Žreviewed in w10x.. As cyclin D 1 peaks in early G1 , it complexes with and activates cyclin-dependent kinase 4 ŽCDK4.. CDK4 has as its major target substrate the retinoblastoma protein ŽRb. Žreviewed in w12x.. Similarly, as cyclin E peaks in the G1 to S transition, it is able to complex with CDK2, augmenting the phosphorylation of Rb. Hyperphosphorylation of Rb permits dissociation from DNA where it is bound to and complexed with the elongation factors such as E2F-1 and DP1 Žreviewed in w13x.. When complexed with Rb, E2F-1 is unable to transcribe the genes associated with, and essential for, cell proliferation, such as the immediate early genes c-myc, c-fos, c-jun and genes involved in DNA synthesis, such as dihydrofolate reductase and thymidylate synthetase. However, the phosphorylation of the Rb protein by CDK2 and CDK4 permits dissociation of Rb from E2F-1rDP1 permitting the commencement of proliferation Žreviewed in w12x.. The cyclin-CDK network forms a very elegant control mechanism for DNA replication and cell
3. Cell cycle regulation: CDKs, CDIs and Rb Progression of the cells through the cell cycle is regulated by two distinct families of proteins known as cyclins and cyclin-dependent kinases ŽCDKs; Fig. 1. Žreviewed in w10,11x.. Each cyclin has a CDK binding partner. For example, cyclin D 1 forms a heterodimer with CDK4 and is one of the major signals regulating the early G1 checkpoint. Cyclins are so called because their levels peak and fall during progression through the cell cycle Žsee Fig. 2.. Thus, as the level of cyclin rises, it becomes available to heterodimerise with its appropriate CDK. This, in turn, permits activation of the CDK and, as the name implies, these activated CDKs can phosphorylate their substrate or target protein. CDKs are crucial for the regulation of the cell cycle but their
Fig. 2. Cell cycle control. For details, see text.
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division. However, there is a further level of complexity in the regulation of the cell cycle; each CDK has a cyclin-dependent kinase inhibitor or CDI Žreviewed in w14x.. Several of these have been characterised and fall broadly into two families. The family of greatest interest when considering cell cycle regulation and DNA damage is the p21 family of CDIs Žreviewed in w15x.. p21 itself is also known as WAF-1 Žand is not to be confused with p21ras .. p21WA F-1 is a CDI that acts on cyclins D and D 1 to prevent the activation of the CDKs that control the G1 checkpoint w16x. Thus, activation of p21WA F-1 causes a cell cycle arrest in early G1 w16x. What relevance does this have to the expression of genotoxicity? Data emerging more recently have shown that the major mechanism for activation of p21WA F-1 is via p53 w17x and the major activator of p53 is DNA damage Žreviewed in w18x..
4. DNA damage and p53 The p53 protein was described originally as an oncogene by two separate groups, both of whom noted a prominent polypeptide in cells transformed by the DNA tumour virus, SV40 w19,20x. However, it became apparent over the next few years that p53 is not an oncogene, but belongs in the opposing camp of tumour suppressor genes. Retrospectively, many of the early experiments demonstrating oncogenic activity of p53 unknowingly used mutant p53. This realisation coincided with a key paper on human colorectal cancer concluding that p53 had all the classical attributes of a tumour suppressor gene w21x. One of the unanimously accepted concepts regarding p53 is that it acts as a molecular stress response device. DNA damage elevates levels of active p53 by two mechanisms Žreviewed in w22x.. Firstly, there is increased transcription and translation. However, DNA damage also causes a stabilisation of the p53 protein which normally has a short half-life Žreviewed in w18,22x.. Thus, p53 is a cellular brake that arrests the cell cycle in early G1 and is required when the integrity of the genome is challenged. Also, p53 is a sensor of DNA damage which earned it the title of ‘guardian of the genome’ w23x. Thus, DNA damage causes elevation of p53; p53 elevates p21WA F-1 and p21WA F-1 causes a G1 arrest. Clues to
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the mechanism by which p53 elevates p21 were offered when it was discovered that another function of the multi-faceted p53 protein is as a transcription factor w22x. Subsequently, it was shown that p53 binds to the p21WA F-1 promoter and thus is responsible for transcriptional upregulation of p21WA F-1, the CDI crucial to the regulation of cell cycle progression. Thus, we have a complete pathway through from DNA damage to cell cycle arrest in early G1. Thus far, we have considered how DNA damage caused by genotoxic insult causes a p53-mediated G1 arrest. The relevance of this to the expression of genotoxicity becomes apparent when considering subsequent events. This p53-mediated G1 arrest permits a choice between three paths; escape into Sphase and cell proliferation, protracted G1 arrest pending DNA repair or cell deletion via apoptosis. Apoptosis is a mechanism whereby cells with potentially hazardous lesions are deleted from the organ and the organism and thus will not give rise to cancer prone progeny Žreviewed in w24x.. This G1 decision point is crucial to the expression of genotoxicity and will be discussed in more detail later.
5. DNA damage and apoptosis One clue to how a G1 arrest via p53 signals apoptosis was discovered when it was found that Bax, a ‘killer’ member of the Bcl2 gene family, has a binding site for p53 in its promoter region. Thus, like p21WA F-1 , p53 directly upregulates transcription of Bax w25x. Members of the Bcl2rBax family of protein molecules are key to the regulation of cellular commitment to apoptosis with some members of the family protecting from cell death ŽBcl-2, Bcl-X L . and others associated with cell killing ŽBax, Bad, Bak. Žreviewed in w24,26x.. Initially, it was proposed that Bcl2–Bcl2 protein homodimers provided a survival signal whereas Bax–Bax protein homodimers caused cell killing w27,28x. However, it has become apparent more recently that when Bax levels exceed levels of Bcl2, free Bax may undergo a conformational change, exposing the so-called Bcl2 homology domain 3 ŽBH3. w26,29x which initiates the apoptosis pathway via activation of the caspase ŽICE-like protease. cascade w30,31x. Recently, it was demonstrated that the release of cytochrome c from mitochondria
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plays a role in this pathway to apoptotic cell death w32x, although the mechanism of is not yet understood. A recent paper suggests that p53 induces apoptosis through a three-step process involving the transcriptional activation of redox genes, the formation of reactive oxygen species and the oxidative degeneration of mitochondrial components, culminating in cell death w33x. In addition to p53-dependent mechanisms, under certain conditions, DNA damage can initiate apoptosis through a p53-independent route involving direct activation of caspases via the FasL–Fas receptor pathway and activation of FADD ŽFas-activated death domain. w34,35x. Whether and how this p53-independent pathway to apoptosis acts alongside the more extensively characterised p53-dependent pathway remains to be determined. The recent discovery of a p53-related protein that can induce apoptosis, p73 w36x, may provide further insight into the relationship between DNA damage and apoptosis.
6. p53-mediated G 1 arrest: a decision point? After DNA damage has caused a p53-mediated G1 arrest, the DNA damaged cell can progress down one of three possible pathways ŽFig. 3.. The cell could remain in a G1 arrest pending repair. Alternatively, the cell could undergo apoptosis and would be deleted from the organ or from the body. If apoptosis is the fate of that particular cell then the DNA damage sustained would not be expressed in assays of genotoxicity. Alternatively, the cell could undergo S-phase and proceed through the G1rS checkpoint. If this were to occur before the DNA was fully repaired then any remaining DNA damage would become incorporated into the genome by mis-sense mismatch replication during S-phase w37x. This would yield a positive result in in vitro and in vivo mutation assays w3x. These three possible outcomes are not mutually exclusive at either the cell or the tissue level since the cell could enter S-phase before completion of repair. In addition to the fate of this individual cell, a variety of routes may be taken within a tissue where some cells will undergo apoptosis, some cells will repair and others will replicate. What parameters impinge on the decision between apoptosis, repair and S-phase? Of fundamental im-
Fig. 3. Events downstream of DNA damage. For details, see text.
portance is genetic background, defined as the profile of normal or mutated genes that are transcriptionally active or silent in a given cell at a given time Žw38x; reviewed in w39,40x.. Secondly, the severity and the nature of the damage sustained can affect the cellular outcome w41x. Thus, different types of genotoxic damage may be perceived very differently by the cell and, as such, the downstream responses of the cell will differ w41x. Thirdly, and most importantly, is the trophic environment in which the cell finds itself Žw42,43x; reviewed in w40x.. The trophic environment comprises many factors, such as the natural growth factors endogenous in that tissue. For example, in the liver, hepatocyte growth factor ŽHGF., epidermal growth factor ŽEGF. and transforming growth factor-a ŽTGFa . play key roles in cell survival and proliferation reviewed in w44x. Similarly, in the haemopoietic system, growth factors, such as granulocyte macrophage-colony stimulating factor ŽGM-CSF. would affect the fate of cells. Also, there are several cytokines including the interleukins and tumour necrosis factor-a ŽTNFa . that affect
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cellular fate in particular tissues w45x. Cytokines assume particular importance since consideration must be given to both the endogenous background levels of these molecules and their increased production in response to high doses of genotoxic chemicals. In addition to natural molecules, such as growth factors and cytokines, there are other modifiers of S-phase and apoptosis, such as non-genotoxic carcinogens, which can profoundly affect the expression of genotoxicity. These tumour promoters frequently induce S-phase, thereby enhancing fixation of DNA damage and also suppress apoptosis preventing the deletion of DNA damaged cells from the tissue Žreviewed in w46x..
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ered p53 analogue, p73 w36x. Further levels of complexity may be in operation also, as illustrated by recent data showing increased cell survival over controls at low levels of damage w47x. At low levels of the non-genotoxic chemical, dimethylformamide ŽDMF., cellular stress and damage were associated with upregulation of survival signals allowing increased survival of the damaged cells over and above control. Further investigation revealed that the cells were upregulating platelet derived growth factor ŽPDGF. and insulin-like growth factor ŽIGF. receptors to enhance cellular survival.
8. Conclusions 7. Thresholds Considering the molecular mechanisms that control the expression of genotoxicity, it is probable that different cellular defence processes operate in response to different levels of damage. This would argue against a linear model for risk assessment at low doses. For example, at high doses of a genotoxic chemical, cells may engage the apoptotic pathway. At intermediate doses, the DNA damage may be below the level required for apoptosis, but might favour, instead, a G1 arrest pending repair, coupled with some G1rS leakage giving rise to mutation. At low doses, the G1 arrest may be absolute until repair is complete. Even an acceptance of the existence of such thresholds does not permit an accurate prediction of outcome; with decreasing dose, cells with reduced levels of damage may survive rather than be deleted, enhancing the perceived rate of mutation. Conversely, a decreased dose could fall below the threshold of cytotoxicity, removing stress cytokines and thus the stimulus for replication in the remaining viable, but DNA-damaged cells. This would be associated with a decreased rate of mutation. This variable outcome with decreasing dose is, of course, speculative and will remain so until the precise threshold for p53 activation of arrest, executor or repair genes is defined. Also, p53-independent pathways that may have different thresholds for activation may be in operation, such as apoptosis triggered by the Fas pathway w34x or by the recently discov-
This paper has reviewed current understanding of the control of cell cycle with the aim of suggesting that the expression of genotoxicity can be altered profoundly by cell proliferation and apoptosis. There are several undeniable truths that need consideration in the use of transgenic rodents in mutation and cancer bioassays. First, certain types of DNA damage, such as single-stranded DNA breaks are more prone to triggering apoptosis than others w41x. Second, transgenic models for mutagenesis require both survival and proliferation of the damaged cells to give rise to progeny with a mutated marker gene. Third, the ultimate expression of genotoxicity is the development of altered foci and preneoplastic lesions and that requires the survival, proliferation and expansion of the DNA-damaged cells. These are dependent on many parameters, such as the p53 status of the target cells and cell cycle regulation in the target tissue.
Acknowledgements The author is grateful to John Ashby for invaluable discussion and for critical appraisal of the manuscript.
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