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Beatson Institute for Cancer Research, Bearsden,1 Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom2
Received 28 January 2004/ Returned for modification 15 March 2004/ Accepted 23 June 2004
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ABSTRACT |
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ionizing radiation and H2O2 persists for longer in c-Jun–/– MEF than in wild-type MEF. Unexpectedly, p53 expression, phosphorylation, and transcriptional activity are largely unaffected by oxygen exposure, indicating that the accumulation of spontaneous DNA damage does not result in chronic activation of p53 as judged by conventional criteria. Finally, we find that c-Jun associates with nuclear foci containing H2AX and ATM following irradiation, suggesting a potential role for c-Jun in DNA repair processes per se. |
INTRODUCTION |
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The senescence phenotype of c-Jun–/– MEF becomes apparent after only a short time in culture (19, 31, 37), suggesting that this growth arrest may be due to environmental stress. The major environmental stress encountered by cells in culture is exposure to hyperoxia. Under normal physiological conditions in the embryo, cells in vivo are exposed to a maximum of 5% oxygen, in contrast to the 21% oxygen that cells experience when placed in conventional atmospheric culture. Further evidence that in vitro culture generates genotoxic stress is the finding that mouse fibroblasts isolated from day 14 gestation embryos express p53, whereas cells in the corresponding day 14 embryos do not show any detectable p53 expression (15).
These observations led to the hypothesis that hyperoxia might limit life span in mouse cells as a consequence of DNA damage accumulation. Recently, this concept has been substantiated in a report demonstrating that hyperoxia and DNA damage induced by oxidative stress are major determinants of proliferative capacity in mouse cells (26). It seems likely that significant background levels of hyperoxia-induced DNA damage are continually offset by active repair processes during atmospheric culture, since MEF that lack DNA damage sensors, such as ATM, sustain high levels of chromosomal breakage (33). Furthermore, MEF deficient for a number genes involved in signaling and/or repairing DNA damage also display premature senescence in vitro, indicating that the effects of culture hypoxia can be exacerbated by defects in DNA repair processes. Examples include cells deficient for ATM, Brca1, Hus1, Ku80, and DNA ligase IV (7, 10, 24, 36, 39).
Given the similarities between the senescence phenotype of c-Jun–/– MEF and MEF with defects in either DNA damage recognition or repair, we hypothesized that irreversible growth arrest in cells lacking c-Jun might be attributable to more rapid accumulation of DNA damage under hyperoxic culture conditions. Here, we show that c-Jun-deficient MEF can proliferate successfully under low oxygen conditions and that the growth arrest observed in conventional culture is associated with spontaneous DNA damage accumulation. We also show that DNA damage induced by ionizing radiation (IR) or hydrogen peroxide (H2O2) persists for longer than normal in cells lacking c-Jun, suggestive of diminished DNA repair capacity. Finally, we found that c-Jun itself can associate with sites of DNA damage, as judged by colocalization with nuclear foci containing H2AX and ATM.
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MATERIALS AND METHODS |
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Retroviral infection. pBabe and pBabe c-Jun vectors were used to produce retrovirus in the Phoenix/Eco packaging cell line. pBabe c-Jun S63/73A and pBabe D284-286 were kind gifts of R. Wisdom, and pBabe Tam67-GFP was a kind gift of R. Hennigan; they have been described elsewhere (18, 37). Proliferating passage 1 MEF were incubated in viral supernatant in the presence of 5 µg of Polybrene/ml overnight to permit infection followed by selection in medium containing hygromycin (50 µg/ml).
Western blotting. Whole-cell extracts were prepared from cultures as described previously (22). Extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting as described previously (6). The antisera used for Western blotting were as follows: anti-p53 (Santa Cruz pAB240), anti-Ser15 phosphorylated p53 (Cell Signaling #9284), anti-p21 (Santa Cruz C-19), anti-c-Jun (Becton Dickinson 610327), anti-Erk (Sigma M5670), anti-cyclin D1 (Santa Cruz sc-200044), anti-cyclin D2 (Neomarkers Ab-3), anti-cyclin D3 (Santa Cruz sc-6283), anti-cyclin E (Santa Cruz sc-481), anti-cyclin A (Santa Cruz sc-596), and anti-Cdk2 (Santa Cruz sc-163).
Reporter assays. The pGL3-Mdm2 or pGL3-p21 firefly luciferase reporter construct was transfected into MEF in combination with a renilla luciferase construct (pRL-SV40) (Promega). Cells were transfected overnight with 5 µg of DNA and harvested 48 h later. Cells were lysed, and firefly and renilla luciferase activities were measured with a luminometer. Firefly luciferase activities were normalized to both renilla activity (transfection efficiency) and protein concentration.
Flow cytometry. For DNA content analysis, trypsinized cultures were fixed in 70% ethanol prior to staining with propidium iodide and analysis using a Becton-Dickinson FACScan flow cytometer.
Comet assays. A Trevigen comet assay kit was used to perform both neutral and alkali denaturing comet assays following the manufacturer's instructions. Where cells were cultured under low oxygen conditions, buffers and cells were flushed with nitrogen until cells were immobilized in agarose and lysed to prevent oxidative damage during manipulation following transfer from low oxygen. Samples were stained with SYBR green prior to analysis by fluorescence microscopy. One hundred cells were scored per sample.
Immunocytochemistry. Cells were cultured on glass coverslips at 3% O2 for 24 h prior to fixation in 3.7% formaldehyde as described previously (2). The antibodies used were as follows: anti-c-Jun (Becton Dickinson 610327), anti-H2AX pSer 139 (Upstate #07-164), and anti-ATM (Santa Cruz H248). All primary and secondary antibodies were used at a 1/100 dilution.
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RESULTS |
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Although low-oxygen culture overcame the acute growth arrest normally observed with hyperoxia, c-Jun–/– MEF still displayed a mild proliferation defect. After 90 days in culture, WT MEF had undergone approximately 15 additional cumulative population doublings compared to c-Jun–/– MEF (Fig. 1a and b). Despite this, these results demonstrate that c-Jun–/– MEF are not inherently incapable of proliferation and that the acute growth arrest which normally occurs under conventional culture conditions (19, 31, 37) is a specific consequence of hyperoxic stress.
Premature senescence of c-Jun–/– MEF is not associated with increased p53 expression or function. Growth arrest under hyperoxic conditions might be explained by a defect in the expression of one or more rate-limiting cell cycle components required for G1/S progression; however, by Western blotting we observed that the levels of cyclins D1, D2, D3, E, A, and Cdk2 were similar in early-passage WT and Jun–/– MEF cultured at 21% O2 (Fig. 1c). Previous studies, however, have established that premature senescence of c-Jun–/– MEF can be overcome when p53 is deleted, implying a functional link between Jun and p53 (31). To determine whether the growth arrest observed at 21% O2 was associated with increased p53 expression or function, WT and c-Jun–/– MEF were initially isolated in 3% O2, cultured at either 3 or 21% O2 for up to 3 weeks, and analyzed for p53 expression. This revealed that culture at 21% O2 did not result in a significant increase in p53 expression or in the product of a p53 target gene, p21/CIP1, in c-Jun–/– MEF compared to WT MEF under conditions where growth arrest was evident (Fig. 2a). To address the possibility that p53 might nevertheless be activated via posttranslational modification, we examined the levels of serine 15 phosphorylation of p53 (S15p), a modification induced in response to certain forms of DNA damage (34). We did not observe any large or consistent increase in the level of p53 S15p in either c-Jun–/– or WT MEF cultured at 21% O2, indicating that changes in p53 expression or phosphorylation are unlikely to be solely responsible for the premature growth arrest observed in primary c-Jun–/– MEF.
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To investigate whether p53 protein could be induced in response to damage and signal to its downstream targets, we treated early-passage WT and c-Jun–/– primary (1°) MEF with UV irradiation. This revealed that p53 levels were induced approximately twofold in both cell types following UV irradiation while p21 expression, although only very weakly induced, was again similar in both cell backgrounds (Fig. 2c). Taken together, these results demonstrate that neither p53 expression nor function is significantly elevated in primary c-Jun–/– MEF, implying that if c-Jun loss affects p53 function then it does so in a manner which cannot be detected as fluctuations in expression, phosphorylation, or transcriptional activity.
The results described here are at variance with previous reports which concluded that either p53 expression (31) or function (32) was elevated in the absence of c-Jun. The major difference between these studies and those reported here is that the previous studies predominantly examined established cell lines. To determine whether this could account for the differences, we established 3T3-like cell lines from our primary cultures and analyzed p53 and p21 levels. As shown in Fig. 2d, c-Jun-deficient 3T3-like cell lines display modestly elevated basal levels of p53 and its downstream target p21/CIP1 compared to their WT counterparts. Similar results were obtained with three independently derived cell lines. These results imply that modulation of p53 expression as a result of c-Jun loss is observed only after cells have acquired indefinite proliferative capacity.
Growth arrest in c-Jun–/– MEF is associated with accumulation of cells in G2/M. To determine whether changes in cell cycle control accompany the decline in growth of c-Jun–/– MEF, we compared the cell cycle profiles of early-passage MEF cultured for up to 3 weeks at either 21 or 3% O2. The cell cycle distributions of WT MEF cultured at 21 and 3% O2 were extremely similar and did not change significantly over time (Fig. 3, lower panels). In contrast, c-Jun–/– MEF cultured at 21% O2 displayed a gradual increase in the proportion of G2/M cells with time compared to cells maintained at 3% O2 (Fig. 3). Cells with defects in mitogenic signaling processes are generally considered to accumulate in the G1 phase of the cell cycle, whereas arrest in the G2/M phase of the cell cycle is frequently indicative of a DNA damage response (1). These results demonstrate that premature senescence induced by hyperoxic stress in c-Jun–/– MEF was associated with accumulation in the G2/M phase of the cell cycle, suggesting that arrest might be associated with higher levels of endogenous DNA damage.
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WT MEF show very little endogenous DNA damage regardless of oxygen exposure during culture (Fig. 4a). This was also true for c-Jun–/– MEF cultured at 3% O2; however, c-Jun-deficient cells cultured at 21% O2 for 4 days contained a high proportion of cells with damaged DNA (Fig. 4a). This was specifically attributable to the absence of c-Jun, since ectopic expression of c-Jun at close to physiological levels in deficient cells by retroviral infection (Fig. 4b) restored the low basal level of DNA damage seen in WT MEF during hyperoxic culture (Fig. 4a, far right columns). These results demonstrate that under culture conditions where cell growth was blocked, c-Jun deficiency is associated with the accumulation of DNA damage.
To determine what type of damage we were detecting, a comet assay was performed under neutral conditions to detect double-strand breaks. We observed no similar excess of spontaneous endogenous double-strand breaks in c-Jun–/– MEF at 21% O2 under these conditions (data not shown), suggesting that the damage observed under denaturing conditions was most likely due to single-strand breaks generated by alkali-labile abasic sites or incomplete excision repair.
Prolonged persistence of induced DNA damage in the absence of c-Jun. To investigate whether the increased levels of spontaneous DNA damage in c-Jun–/– MEF was a consequence of more rapid DNA damage accumulation or a delay or defect in the rate of DNA repair, we used a neutral comet assay to detect the generation and repair of induced DNA damage in cells cultured at 3% O2 following exposure to IR. Shortly after exposure to 4 Gy of IR, essentially all cells contained high levels of damage, signified by large comet tails, regardless of genotype (Fig. 5a). Six hours after irradiation, however, DNA damage had been substantially repaired in both WT and c-Jun-reconstituted cultures as judged by the reappearance of a large proportion of cells lacking tails (Fig. 5a). Cultures of c-Jun–/– MEF, by contrast, did not contain any cells without tails at 6 h, although some repair had evidently occurred, as judged by a reduction in the proportion of large tails and an increase in the proportion of small tails (Fig. 5a). The persistence of irradiation-induced damage in c-Jun–/– MEF was also associated with a prolonged G2/M arrest, as documented by flow cytometry and more robust activation of the DNA damage-activated Chk1 protein kinase (data not shown). Oxidative damage induced by H2O2 treatment was also found to persist for longer in c-Jun–/– MEF compared to WT and reconstituted MEF (Fig. 5b).
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c-Jun co-localizes with H2AX and ATM at sites of DNA damage.
The Brca1 (8) and c-Myc (5) transcription factors have been shown to interact directly with components of the DNA repair machinery. To determine if this might also be true for c-Jun, we irradiated cells and examined whether c-Jun localized to sites of induced DNA damage. Irradiation rapidly induces phosphorylation of H2AX on serine 139 to generate H2AX, which subsequently accumulates in discrete foci at sites of DNA damage (3). We therefore examined the formation of H2AX foci in WT and c-Jun–/– MEF following IR treatment and compared the distribution of these structures with the distribution of c-Jun by immunocytochemistry.
As shown in Fig. 7a, irradiation induced a marked increase in H2AX staining in WT MEF cultured in 21% O2, and strikingly, c-Jun was found to colocalize with a significant proportion of the H2AX foci shortly after DNA damage (Fig. 7a). Limited colocalization was also observed between c-Jun and the smaller number of H2AX foci present in unirradiated cells (Fig. 7a). H2AX staining also increased in c-Jun–/– MEF cultured in 21% O2 after irradiation, although the basal level appeared to be somewhat higher, as would be expected in cells bearing higher levels of spontaneous DNA damage (Fig. 7b). c-Jun–/– MEF, however, were completely devoid of c-Jun staining, thereby validating the specificity of the anti-c-Jun antibody (Fig. 7b).
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DISCUSSION |
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Our results are consistent with a recent report which demonstrates that normal mouse cell senescence occurs as a consequence of the accumulation of DNA damage resulting from hyperoxic culture conditions (26). This study also established that premature senescence resulting from deletion of the DNA repair protein DNA-PKcs can be overcome by culture in 3% O2, indicating that diminished repair can exacerbate oxygen-dependent premature senescence, presumably by accelerating the rate at which oxidative damage accumulates during hyperoxic culture. Since DNA damage leads to multiple checkpoint-mediated cell cycle arrests (25), one might expect senescent MEF to arrest at both the G1/S and G2/M transitions of the cell cycle. Consistent with this prediction, we observed accumulation of G2/M-arrested cells in our senescent cultures of c-Jun–/– MEF, and very recently Wada et al. have also reported G2/M arrest in senescent cultures of MEF deficient for either c-Jun or the upstream JNK pathway regulator MKK7 (35). These findings are unexpected, since G2/M arrest was not detected in earlier studies of c-Jun-deficient cells, which instead documented arrest primarily in the G1/S phase of the cell cycle (31, 37). Although the reasons for this apparent discrepancy are not completely clear, G2/M-arrested cells constitute only a subpopulation of the senescent cultures (Fig. 3) (35), which may have been obscured as a result of variations in cell culture conditions and/or other experimental methodologies in these earlier studies.
The absence of any obvious relationship between DNA damage accumulation and activation of p53 was also unexpected. Previous reports have demonstrated that growth arrest of c-Jun–/– MEF was dependent on p53, and since c-Jun–/– MEF accumulate DNA damage it might be anticipated that this would activate p53. However, we analyzed p53 expression, phosphorylation, and transcriptional activity and observed no evidence of activation in early-passage 1° c-Jun–/– MEF, suggesting that if c-Jun deficiency modifies p53 function it does so in a manner that cannot be detected by conventional criteria. Historically, p53 function has generally been studied following acute insults which result in high levels of DNA damage. We, in contrast, have examined spontaneously occurring DNA damage resulting from chronic exposure to hyperoxia. c-Jun–/– MEF presumably undergo senescence after a certain threshold of spontaneous oxidative damage has accumulated; however, this is evidently not sufficient to trigger a conventional p53 response. Our observations, however, do not exclude the possibility that low-level chronic oxidative damage modulates p53 activity in a manner distinct from acute DNA damage. In fact, this seems likely and furthermore can provide an alternative explanation for the previously documented genetic interaction between c-Jun and p53 in senescence (31).
One potential mechanism that could account for the accumulation of DNA damage in c-Jun–/– MEF would be if c-Jun was required for the expression of genes that protect cells from DNA damage or are required for efficient DNA repair. We considered the possibility that c-Jun might regulate genes involved in redox homeostasis, since imbalances in reactive oxygen species production and removal could lead to elevated levels of DNA damage. However, using a redox-sensitive indicator dye (CM-H2DCF-DA), we found no significant difference in intracellular redox potential between WT and c-Jun–/– MEF, suggesting that excess reactive oxygen species production is unlikely to be a major cause of DNA damage accumulation (data not shown).
The prolonged persistence of DNA damage induced by IR or H2O2 in c-Jun–/– MEF is however strongly suggestive of a defect in DNA repair. Recently, the c-Myc proto-oncogene has been shown to play a role in regulating the expression of two genes involved in DNA repair: the human Werner syndrome protein gene (WRN gene) (13) and, more recently, the Nbs1 gene (5). Furthermore, a number of studies have implicated the JNK signaling pathway acting via c-Jun or the related transcription factor ATF2 in modulation of DNA repair and/or cell survival in response to various forms of genotoxic damage (17, 27, 28). Consistent with these observations, we found that mutant forms of c-Jun which are deficient for DNA binding or transactivation or which were nonresponsive to JNK regulation were unable to restore normal regulation of basal or induced DNA damage in c-Jun–/– MEF. c-Jun is involved in regulating the activity of many individual gene promoters through AP-1 response elements, and although there is limited evidence that some of these might influence DNA repair (12, 28, 30), a direct link between specific c-Jun target genes and repair processes remains to be established.
Alternatively, it is also conceivable that c-Jun might have a direct role in DNA repair processes per se. The finding that c-Jun, like ATM and H2AX, localizes to sites of DNA damage is broadly consistent with this idea. Furthermore c-Jun is known to interact physically or functionally with two proteins involved in DNA repair: redox factor 1 (Ref-1; also known as APEX), an endonuclease specific for abasic sites (38), and thymine DNA glycosylase (4), both of which are involved in base excision repair (BER). BER is responsible for the repair of modified lesions on DNA, including those produced by oxidative damage (14). We investigated whether c-Jun–/– MEF displayed deficiencies in BER by using in vitro assays; however, extracts from c-Jun–/– MEF were as active as those from WT MEF in the recognition and excision of oxidatively modified bases from DNA (data not shown).
Precedents exist for interactions between other transcription factors and components of the repair machinery, particularly in the context of transcription-coupled repair (20). Transcriptional activators may stimulate DNA repair at sites of active transcription either by facilitating access through chromatin remodeling or by directly recruiting repair proteins to actively transcribed genes (11). c-Jun binds to a DNA recognition sequence common to many gene promoters and theoretically could promote transcription-coupled repair by either of these mechanisms. Such a relatively subtle role could explain why c-Jun–/– MEF are capable of a low level of repair and why the repair defect can be tolerated under permissive conditions. In low oxygen, where damage is presumably minimal, c-Jun–/– MEF may be able to repair at a rate which is compatible with proliferation, but when switched to a more damaging hyperoxic environment, the cells may be unable to prevent the accumulation of unrepaired lesions in essential genes.
Each of these scenarios could be reconciled with the current data; however, further studies including direct measurement of repair capacity per se will be required to determine conclusively whether c-Jun regulates DNA repair, and if so, to understand the molecular mechanism. The present study nevertheless demonstrates a novel and previously unrecognized role for c-Jun in sustaining cell proliferation by limiting the genotoxic consequences of hyperoxic culture.
Mutations which impair the recognition or repair of DNA damage frequently predispose to cancer, and therefore loss, as opposed to gain, of c-Jun function might conceivably promote neoplasia through accumulation of genetic damage. Although we are not aware of any specific evidence to support this scenario, it is intriguing that certain other AP-1 family proteins, such as JunB and c-Fos, have been implicated in tumor suppression (9). Further work will be required to establish if these other AP-1 proteins share c-Jun's capacity to protect cells from genotoxic stress, and if so, to determine whether this function can influence carcinogenesis.
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ACKNOWLEDGMENTS |
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This work was supported by Cancer Research UK.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Black, E. J., W. Clark, and D. A. Gillespie. 2000. Transient deactivation of ERK signalling is sufficient for stable entry into G0 in primary avian fibroblasts. Curr. Biol. 10:1119-1122.[CrossRef][Medline]
3. Burma, S., B. P. Chen, M. Murphy, A. Kurimasa, and D. J. Chen. 2001. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276:42462-42467.
4. Chevray, P. M., and D. Nathans. 1992. Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc. Natl. Acad. Sci. USA 89:5789-5793.
5. Chiang, Y. C., S. C. Teng, Y. N. Su, F. J. Hsieh, and K. J. Wu. 2003. c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair. J. Biol. Chem. 278:19286-19291.
6. Clark, W., and D. A. Gillespie. 1997. Transformation by v-Jun prevents cell cycle exit and promotes apoptosis in the absence of serum growth factors. Cell Growth Differ. 8:371-380.[Abstract]
7. Cressman, V. L., D. C. Backlund, A. V. Avrutskaya, S. A. Leadon, V. Godfrey, and B. H. Koller. 1999. Growth retardation, DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice. Mol. Cell. Biol. 19:7061-7075.
8. Deng, C. X., and R. H. Wang. 2003. Roles of BRCA1 in DNA damage repair: a link between development and cancer. Hum. Mol. Genet. 12:R113-R123.
9. Eferl, R., and E. F. Wagner. 2003. AP-1: a double-edged sword in tumorigenesis. Nat. Rev. Cancer 3:859-868.[CrossRef][Medline]
10. Frank, K. M., N. E. Sharpless, Y. Gao, J. M. Sekiguchi, D. O. Ferguson, C. Zhu, J. P. Manis, J. Horner, R. A. DePinho, and F. W. Alt. 2000. DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Mol. Cell 5:993-1002.[CrossRef][Medline]
11. Frit, P., K. Kwon, F. Coin, J. Auriol, S. Dubaele, B. Salles, and J. M. Egly. 2002. Transcriptional activators stimulate DNA repair. Mol. Cell 10:1391-1401.[CrossRef][Medline]
12. Gjerset, R. A., S. Lebedeva, A. Haghighi, S. T. Turla, and D. Mercola. 1999. Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification. Cell Growth Differ. 10:545-554.
13. Grandori, C., K. J. Wu, P. Fernandez, C. Ngouenet, J. Grim, B. E. Clurman, M. J. Moser, J. Oshima, D. W. Russell, K. Swisshelm, S. Frank, B. Amati, R. Dalla-Favera, and R. J. Monnat, Jr. 2003. Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev. 17:1569-1574.
14. Grollman, A. P., and M. Moriya. 1993. Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet. 9:246-249.[CrossRef][Medline]
15. Halliwell, B. 2003. Oxidative stress in cell culture: an under-appreciated problem? FEBS Lett. 540:3-6.[CrossRef][Medline]
16. Hartmann, A., E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A. Collins, A. Smith, G. Speit, V. Thybaud, and R. R. Tice. 2003. Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop. Mutagenesis 18:45-51.
17. Hayakawa, J., C. Depatie, M. Ohmichi, and D. Mercola. 2003. The activation of c-Jun NH2-terminal kinase (JNK) by DNA-damaging agents serves to promote drug resistance via activating transcription factor 2 (ATF2)-dependent enhanced DNA repair. J. Biol. Chem. 278:20582-20592.
18. Hennigan, R. F., and P. J. Stambrook. 2001. Dominant negative c-jun inhibits activation of the cyclin D1 and cyclin E kinase complexes. Mol. Biol. Cell 12:2352-2363.
19. Johnson, R. S., B. van Lingen, V. E. Papaioannou, and B. M. Spiegelman. 1993. A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture. Genes Dev. 7:1309-1317.
20. Leadon, S. A. 1999. Transcription-coupled repair of DNA damage: unanticipated players, unexpected complexities. Am. J. Hum. Genet. 64:1259-1263.[CrossRef][Medline]
21. Maclaren, A., W. Clark, E. J. Black, D. Gregory, H. Fujii, and D. A. Gillespie. 2003. v-Jun stimulates both cdk2 kinase activity and G1/S progression via transcriptional repression of p21 CIP1. Oncogene 22:2383-2395.[CrossRef][Medline]
22. MacLaren, A., W. Clark, and D. A. Gillespie. 2000. v-Jun sensitizes cells to apoptosis by a mechanism involving mitochondrial cytochrome C release. Oncogene 19:5906-5918.[CrossRef][Medline]
23. Maki, Y., T. J. Bos, C. Davis, M. Starbuck, and P. K. Vogt. 1987. Avian sarcoma virus 17 carries the jun oncogene. Proc. Natl. Acad. Sci. USA 84:2848-2852.
24. Nussenzweig, A., C. Chen, V. da Costa Soares, M. Sanchez, K. Sokol, M. C. Nussenzweig, and G. C. Li. 1996. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382:551-555.[CrossRef][Medline]
25. Nyberg, K. A., R. J. Michelson, C. W. Putnam, and T. A. Weinert. 2002. Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36:617-656.[CrossRef][Medline]
26. Parrinello, S., E. Samper, A. Krtolica, J. Goldstein, S. Melov, and J. Campisi. 2003. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat. Cell Biol. 5:741-747.[CrossRef][Medline]
27. Potapova, O., S. Basu, D. Mercola, and N. J. Holbrook. 2001. Protective role for c-Jun in the cellular response to DNA damage. J. Biol. Chem. 276:28546-28553.
28. Potapova, O., A. Haghighi, F. Bost, C. Liu, M. J. Birrer, R. Gjerset, and D. Mercola. 1997. The Jun kinase/stress-activated protein kinase pathway functions to regulate DNA repair and inhibition of the pathway sensitizes tumor cells to cisplatin. J. Biol. Chem. 272:14041-14044.
29. Ries, S., C. Biederer, D. Woods, O. Shifman, S. Shirasawa, T. Sasazuki, M. McMahon, M. Oren, and F. McCormick. 2000. Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF. Cell 103:321-330.[CrossRef][Medline]
30. Scherer, S. J., S. M. Maier, M. Seifert, R. G. Hanselmann, K. D. Zang, H. K. Muller-Hermelink, P. Angel, C. Welter, and M. Schartl. 2000. p53 and c-Jun functionally synergize in the regulation of the DNA repair gene hMSH2 in response to UV. J. Biol. Chem. 275:37469-37473.
31. Schreiber, M., A. Kolbus, F. Piu, A. Szabowski, U. Mohle-Steinlein, J. Tian, M. Karin, P. Angel, and E. F. Wagner. 1999. Control of cell cycle progression by c-Jun is p53 dependent. Genes Dev. 13:607-619.
32. Shaulian, E., M. Schreiber, F. Piu, M. Beeche, E. F. Wagner, and M. Karin. 2000. The mammalian UV response: c-Jun induction is required for exit from p53-imposed growth arrest. Cell 103:897-907.[CrossRef][Medline]
33. Sherr, C. J., and R. A. DePinho. 2000. Cellular senescence: mitotic clock or culture shock? Cell 102:407-410.[CrossRef][Medline]
34. Siliciano, J. D., C. E. Canman, Y. Taya, K. Sakaguchi, E. Appella, and M. B. Kastan. 1997. DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev. 11:3471-3481.
35. Wada, T., N. Joza, H. Y. Cheng, T. Sasaki, I. Kozieradzki, K. Bachmaier, T. Katada, M. Schreiber, E. F. Wagner, H. Nishina, and J. M. Penninger. 2004. MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence. Nat. Cell Biol. 6:215-226.[Medline]
36. Weiss, R. S., T. Enoch, and P. Leder. 2000. Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress. Genes Dev. 14:1886-1898.
37. Wisdom, R., R. S. Johnson, and C. Moore. 1999. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 18:188-197.[CrossRef][Medline]
38. Xanthoudakis, S., and T. Curran. 1992. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J. 11:653-665.[Medline]
39. Xu, Y., E. M. Yang, J. Brugarolas, T. Jacks, and D. Baltimore. 1998. Involvement of p53 and p21 in cellular defects and tumorigenesis in Atm–/– mice. Mol. Cell. Biol. 18:4385-4390.
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