行政院國家科學委員會補助專題研究計畫成果報告
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膀胱癌癌化及抗藥機轉研究：p53 及 neu
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計畫類別：●個別型計畫
□整合型計畫
計畫編號：NSC － 2320－B－ 006－ 148 －
執行期間： 89 年 8 月 1 日至 90 年 7 月 30 日
計畫主持人：賴明德
共同主持人：
本成果報告包括以下應繳交之附件：
執行單位：成功大學醫學院生化研究所
行政院國家科學委員會專題研究計畫成果報告
計畫編號：NSC 89-2320-B-006-148
執行期限：89 年 8 月 1 日至 90 年 7 月 31 日
主持人：賴明德 執行機構成功大學醫學院生化研究所
一、中文摘要
利用突變 p53 (V143A, N247I and R273L) 送 入 TCC-SUP, 我 們 發 現 可 增 加 對 cisplatin 之敏感度。本計劃研究其作用 機轉。(1) 活化 caspase 9, 使用 general caspase 抑制劑或 caspase 9 抑制劑可防 止細胞凋亡。(2) Bcl-2 下降(3) Noxa, p53R2, PIDD 無改變(4) actinomycin D 及 cycloheximide 無法抑制 cisplatin 造成 之 細 胞 凋 亡 ， (5) 突 變 p53 之 phosphorylation 微 量 上 揚 ， 但 acetylatoion 下降。此研究顯示突變 p53 不經由轉錄或轉譯促進 cisplatin 造成之 細胞凋亡。 關鍵詞：突變 p53，細胞凋亡，caspase 9 Abstract
It has been controversial whether the presence of mutant p53 increases or decreases the response to chemotherapy. In this report, we investigate the mechanism of cisplatin-induced apoptosis in two sensitive mutant p53 transfectants (V143A and N247I) and two resistant cell lines (parental cell and R273L mutant p53 transfectant). Five aspects of mechanisms were investigated. (1) Caspase activation, activation of caspase 9 was demonstrated by western blotting, and specific inhibitor for caspase 9 could inhibit apoptosis. Inhibitor for caspase 3 could partially inhibit the cisplatin-induced
and surface trafficking of Fas or Fas-L were not observed during cisplatin treatment. (4) Post-translational modification of p53, Ser15 of wild-type p53 was phosphorylated in response to cisplatin. On the other hand, phosphorylation of mutant p53 was weakly enhanced. Acetylation of wild-type p53 increased, while acetylation mutant p53 decreased during cisplatin treatment. (5) Transcriptional and translational independent, transcriptional inhibitor actinomycin D and translational inhibitor cycloheximide did not inhibit the apoptosis. These results indicated that phosphorylated and hypoacetylated mutant p53 could enhance cisplatin-induced apoptosis through activation of caspase 9, which was independent of transcriptional activation and translation.
Keywords: TCC-SUP, mutant p53, caspase 9,
Bcl-2
二、緣由與目的
Loss of p53 function, including point mutations and allelic loss of p53 gene, plays a major role in the development of many types of cancer (1). P53 is a short-lived protein and activated in response to a variety of stress, such as damaged DNA, hypoxia, and nucleotide depletion (2, 3). Active p53 can act as a transcriptional factor to turn on many apoptosis and cell-cycle-related genes. These genes include bax (4), IGF-BP3 (5), p53R2 (6), Noxa (7), PIDD (8), p53-AIP1(9), PUMA (10-11), PIG (12), PERP (13), and
apoptosis (20-21).
Activation of p53 involves both increasing protein expression level and post-translational modifications that include phosphorylation, acetylation, and
sumoylation. (22-24). Several serine and threonine residues, including Ser6, 9, 15, 20, 33, 37, 46, 315, 392 and Thr18, 81, are phosphorylated during activation. Acetylation of Lys320, 373, and 382 are observed in response to DNA damage. Post-translational modification of p53 occurs at most sites in response to stress; however, differences in responses to various agents were observed. For example, increase phosphorylation of Ser15 was seen in response to cisplatin, arsenite, and genistein, but not to
actinomycin D (25-27). Cadmium increases phosphorylation of p53 only at Ser15, but not at Ser6, 9, 20, 37, or 392 (28).
Mutation on p53 can exert a dominant negative effect on wild-type p53, thereby, mutant p53 may inhibit wild-type p53 mediated cell cycle arrest and apoptosis (29-30). The expression of mutant p53 is associated with later stages of tumor progression, poor prognosis, and drug resistance (1, 31-33). However, the effects of mutant p53 on drug resistance is more complex and unsettled (34, 35). Several studies indicated that the status of p53 has either no predictive value or better response to chemotherapy (36-40). For example, adjuvant chemotherapy benefited patients with altered p53, but did not benefit patients with wild-type p53 in their tumors in bladder cancer (41).
Since the nature of p53 mutations affect the cellular responses to stress (37, 38, 42), we have delivered several mutant p53 expression plasmids into TCCSUP bladder carcinoma cell line, and studied the response to outside stress in these transfectants (43). Our previous results indicated that p53
mutants (V143A, V173L, H179Q, and N247I) enhanced the sensitivity to cisplatin and doxorubicin, but not methotrexate. However, the DNA contact mutant p53R273L had no
effect on the sensitivity to cisplatin and doxorubicin. Cisplatin-induced cell death underwent apoptosis, while
doxorubicin-induced cell death probably occurred through a non-apoptotic pathway (43). Mutant p53 may enhance apoptosis by delaying a G2/M arrest through
transcriptional regulation (34); however TCCSUP and all mutant transfectants displayed similar changes of cell cycle distribution in response to various doses of cisplatin and doxorubicin (unpublished results). Therefore, the sensitivity to cisplatin and doxorubicin was not due to change of cell cycle distribution during drug treatment. The present study aimed to study the
mechanism of the enhancement of cisplatin-induced apoptosis by exogenous mutant p53. Our results indicated that mutant p53 was mainly phosphorylated at Ser15, but was deacetylated at lysine residues in
response to cisplatin. Moreover, mutant p53 enhanced cisplatin-induced apoptosis through activation of caspase 9, which was
independent of transcriptional activation and translation.
三、研究結果
Mutant p53 amplify cisplatin-induced apoptosis thr ough activation of caspase 9.
Cisplatin-induced cell death in TCCSUP and mutant p53 transfectants were mediated through apoptotic pathway as demonstrated by chromatin condensation, Annexin V assay, and appearance of DNA nick (43). Since apoptosis is usually initiated either by the activation of caspase 8 or 9, we first studied which initiator caspase is activated during cisplatin-induced apoptosis. TCCSUP parental cells and mutant p53 transfectants were treated with cisplatin for the indicated time, the cell death was measured at 12 hrs by Annexin V assay, and the cell lysates were harvested and analyzed with western blotting with antibody specific for active form of caspase 9 or caspase 8. Apoptosis was observed in TCCSUP-143-4 and TCCSUP-247-5 transfectants, but not in TCCSUP and TCCSUP-273-6 transfectant as described before (43). Activation of caspase 9 was observed 9 hrs after the treatment of cisplatin in TCCSUP-143-4 and
TCCSUP-247-5 mutant p53 transfectants, but not in TCCSUP parental cells and TCCSUP-273-6 transfectants (Fig. 1A). The control lane is the active form of caspase 9 supplied by manufacturer. On the other hand, no activation of caspase 8 was observed in all cell lines up to 24 hours of cisplatin treatment (Figure 1B). Activation of caspase 8 occurred in cell death (anoikis) by cell detachment (44). To serve as a positive control for caspase 8 activation, TCCSUP cells were grown over-confluent to induce anoikis and activation of caspase 8 was observed at 24 hours (Fig. 1B).
Various inhibitors for different caspases were assayed for their abilities to inhibit cisplatin-induced apoptosis in mutant p53 transfectant TCCSUP-143-4 cell line. Figure 2 showed that the general caspase inhibitor (VAD-fmk) could completely inhibit the apoptosis. The caspase 9 inhibitor
(LEHD-CHO) could significantly block the apoptosis as measured by Annexin-V assay. In contrast, inhibitor for caspase 8
(IETD-CHO) had no effect on
cisplatin-induced apoptosis. The inhibitor for caspase 3 (DEVD-fmk) had only partial protecting effect, suggesting that caspase 9 may activate additional execution caspases besides caspase 3 to carry out apoptosis. Inhibitors for other caspases, including caspase 1 (YVAD-CHO), 2
(Z-VDVAD-FMK), and 6 (VEID-CHO), did not interfere with cisplatin-induced cell death in mutant p53 transfectants at all.
Mutant p53 did not alter sur face Fas and Fas-L expr ession dur ing
cisplatin-tr eatment. Expression of p53
could alter surface Fas expression in response to chemotherapy or actinomycin D in certain cell types (45). The expression of surface Fas
obtained at 10 hrs after cisplatin treatment (data not shown). In addition, anti-Fas antibody can not induce cell death in
TCCSUP and all transfectants (Fig. 3C). As a positive control, anti-Fas antibody induced cell death in Jurkat cells.
Down-r egulation of Bcl-2 in
cisplatin-induced apoptosis. Caspase 9 is
usually activated by the change of the expression of mitochondrial Bcl-2 family member, and wild-type p53 can transactivate the expression of bax and repress the
expression of Bcl-2. Therefore, we measured the expression of three mitochondrial Bcl-2 family members during treatment of cisplatin. Western blotting revealed that the expression of Bax and Bcl-XL was not significantly
altered in all cell lines under treatment. On the other hand, the expression of Bcl-2 reduced significantly in two
cisplatin-sensitive mutant p53 transfectants (TCCSUP-143-4 and TCCSUP-247-5), but not in the cisplatin-resistant cell lines (TCCSUP and TCCSUP-273-6). The down regulation of Bcl-2 was observed 18 hrs after the treatment of cisplatin in TCCSUP-247-5 cells. The disappearance of Bcl-2 was later than the apoptosis detected by Annexin-V assay, suggesting that repression of Bcl-2 may be the consequence of apoptosis.
P53-inducible genes (Noxa, p53R2, and PIDD) wer e not activated dur ing cisplatin tr eatment. Recently, several novel
p53-inducible genes have been identified, the expression of three novel p53-inducible genes, Noxa, p53R2, PIDD, was determined by RT-PCR in these transfectants and parental cells. The expression of these genes were not altered in two cisplatin-sensitive transfectants, TCCSUP-143-4 and
indicated that the mutant p53 did not enhance cisplatin-induced apoptosis through
influencing the expression of p53-targeted genes examined here.
Both wild-type and mutant p53 was phosphor ylated at ser ine15 in r esponse to cisplatin. Post-translational modification of
p53 plays an important role in the regulation of p53 function. Firstly, we studied the change of phosphorylation of p53 in response to cisplatin. TCCSUP and all transfectants were treated with cisplatin, and cell lysates were harvested at early time points and analyzed by western blotting with antibodies specific for several phosphorylated serine residues in p53. Strong constitutive
phosphorylation at serine 6 was observed in all cell lines, while weak constitutive phosphorylation on serine 392 was observed (Figure 6A). Phosphorylation on serine 20 residue was very weak and only appeared 6 hours after cisplatin treatment.
Phosphorylation on serine 15 of p53 increased significantly 3 hr after cisplatin treatment in all cell lines. Since the TCCSUP mutant p53 transfectants contain endogenous p53 and exogenous full-length mutant p53, it is necessary to distinguish which form is phosphorylated. Cell lysates were first immunoprecipitated with antibodies specific for wild type p53 and mutant p53
respectively, and the immunoprecipitation products was analyzed by western blotting with antibody specific for Ser15 of p53. The expression of wild-type p53 increased during cisplatin-treatment while the expression of mutant p53 is unaltered (Fig. 6B). Both wild-type and mutant p53 were
phosphorylated in response to cisplatin; however, the mutant p53 are less
phosphorylated/ per molecule comparing with wild-type p53.
Incr ease acetylation of wild-type p53 but decr ease acetylation of mutant p53 in r esponse to cisplatin tr eatment. We next
investigate the acetylation of C-terminal residues of p53, which may be crucial for the full p53-mediated response to genotoxic stress. The cell lysates harvested 3 and 6
hours after cisplatin treatment were immunoprecipitated with antibody specific for p53, and analyzed with antibody specific for acetyl-lysine (Figure 6C). The acetylation of p53 increase significantly only in the TCCSUP parental cells, but not in the other three mutant transfectants. To further
differentiate the acetylation on wild-type p53 from mutant p53, the cell lysates were immunoprecipitated with
conformation-specific antibodies for wild-type p53 and mutant p53 respectively. The immunoprecipitation products were subject to similar analysis on acetyl-lysine. The acetylation of wild-type p53 increased 3-6 hrs after cisplatin treatment; however, the acetylation of mutant p53 decreased during the treatment of cisplatin (Fig. 6D). No change of total acetylation of p53 in mutant p53 transfectants (Fig. 6C) can be explained by the sum of increase acetylation of
endogenous wild-type p53 and decrease acetylation of exogenous mutant p53. Selective deacetylation of mutant p53 in response to cisplatin was observed in all mutant transfectants including V143A, N247I, and R273L. The state of acetylation might not play an essential role in enhancing cisplatin-induced apoptosis by V143A and N247I mutant p53.
Tr anscr iptional activation and tr anslation ar e not r equir ed for mutant p53-enhanced apoptosis. Acetylation of p53 is important
for transcriptional activation ability of p53; however, the mutant p53 in our transfectants was deacetylated in response to cisplatin. It prompted us to study whether transcription or translation is required for the
cisplatin-induced apoptosis in our transfectants with the use of translational inhibitor cycloheximide and transcriptional inhibitor actinomycin D. Cycloheximide alone induced little apoptosis as measured by Annexin-V assay in all cell lines (Figure 7). Addition of cycloheximide did not inhibit the cisplatin-induced apoptosis. In addition, cycloheximide enhanced the
cisplatin-induced apoptosis in all cell lines. The effect was more prominent in the TCCSUP-143-4 and TCCSUP-247-5 mutant
transfectants (Figure 7). Addition of increasing concentrations of transcriptional inhibitor actinomycin D to all cell lines did not prohibit the cell from cisplatin-induced apoptosis, but on the contrary enhance apoptosis (Figure 8). Treatment for
actinomycin D alone did not induce apoptosis in all cell lines in the 12-hour period (data not shown). These results indicated that enhanced apoptosis by mutant p53 was unlikely to mediate through transcriptional activation and translation of p53-related target genes.
The effects of histone deacetylase inhibitor on the cisplatin-induced apoptosis.
Cisplatin-induced apoptosis was
accompanied with Bcl-2 downregulation in the TCCSUP-143-4 and TCCSUP-247-5 mutant p53 transfectants (Figure 4). The down regulation of Bcl-2 may be due to protein turnover or transcriptional repression. Since transcriptional repression is frequently mediated by histone deacetylase, we tested the effect of histone deacetylase inhibitor trichostatin A (TSA) on the expression of Bcl-2. Figure 9A showed that pre-treated the TCCSUP-143-4 cells with TSA for 2 hr could reverse the down-regulation of Bcl-2 by cisplatin. However, the presence of TSA did not block the cisplatin-induced apoptosis (Figure 9B), it even enhanced
cisplatin-induced apoptosis at the concentration of 200 nM. TSA lost its enhancing effect on cisplatin-induced apoptosis when added 4 hours after the cisplatin treatment (data not shown). TSA by itself had no effect on the TCCSUP and all mutant p53 transfectants at the concentration of 50-200 nM (data not shown). Another histone deacetylase inhibitor sodium butyrate, similar to TSA, did not inhibit
cisplatin-induced apoptosis in all
in human cancer. Science 253, 49-53.
2. Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell
88, 323-331.
3. Ryan, K. M., Phillips, A. C., and Vousden, K. H. (2001) Regulation and function of the p53 tumor suppressor protein. Curr. Opin. Cell Biol. 13, 332-337.
4. Miyashita, T., and Reed, J. C. (1995) Tumor suppressor p53 is a direct
transcriptional activator of the human bax gene. Cell 80, 293-299.
5. Buckbinder, L., Talbott, R.,
Velasco-Miguel, S., Takenaka, I., Faha, B., Seizinger, B. R., and Kley, N. (1995) Induction of the growth inhibitor
IGF-binding protein 3 by p53. Nature. 377,
646-649.
6. Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y., and Nakamura, Y. (2000) A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42-49.
7. Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T., Taniguchi, T., and Tanaka, N. (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288,
1053-1058.
8. Lin, Y., Ma, W., and Benchimol, S. (2000) Pidd, a new death-domain-containing protein, is induced by p53 and promotes
p53-dependent apoptosis, and its
regulation by Ser-46-phosphorylated p53.
Cell 102, 849-862.
10. Nakano, K., and Vousden K. H. (2001) PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell. 7, 683-694
11. Yu, J., Zhang, L., Hwang, P. M., Kinzler, K. W., and Vogelstein, B. (2001) PUMA induces the rapid apoptosis of colorectal cancer cells. Mol. Cell. 7,
673-82.
12. Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W., and Vogelstein, B. (1997) A model for p53-induced apoptosis.
Nature 389, 300-305.
13. Attardi, L. D., Reczek, E. E., Cosmas, C., Demicco, E. G., McCurrach, M. E., Lowe, S. W,. Jacks, T. (2000) PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family.
Genes Dev. 14, 704-718.
14. Moll, U. M. and Zaika, A. (2001) Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett. 493, 65-69.
15. Zhao, R., Gish, K., Murphy, M., Yin, Y., Notterman, D., Hoffman, W. H., Tom, E., Mack, D. H., and Levine, A. J. (2000) Analysis of p53-regulated gene expression patterns using oligonucleotide arrays.
Genes Dev. 14, 981-993.
16. Miyashita, T., Harigai, M., Hanada, M., and Reed, J. C. (1994) Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res. 54,
3131-3135.
17. Murphy, M., Ahn, J., Walker, K. K., Hoffman, W. H., Evans, R. M., Levine, A. J., and George, D. L. (1999)
Transcriptional repression by wild-type p53 utilizes histone deacetylases, mediated
by interaction with mSin3a. Genes Dev. 13,
2490-2501.
18. Caelles, C., Helmberg, A., and Karin, M. (1994) p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 370, 220-223.
19. Wagner, A. J., Kokontis, J. M., and Hay, N. (1994) Myc-mediated apoptosis
requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1.
Genes Dev. 8, 2817-2830.
20. Haupt, Y., Rowan, S., Shaulian, E., Vousden, K. H., and Oren, M. (1995) Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev.
9, 2170-2183.
21. Chen, X., Ko, L. J., Jayaraman, L., and Prives, C. (1996) p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev. 10, 2438-2451.
22. Ryan, K. M., Phillips, A. C., and Vousden, K. H. (2001) Regulation and function of the p53 tumor suppressor protein. Curr. Opin. Cell Biol. 13,
332-337.
23. Woods, D. B., Vousden, K. H. (2001) Regulation of p53 function. Exp. Cell Res.
264, 56-66.
24. Appella, E., and Anderson CW. (2001) Post-translational modifications and activation of p53 by genotoxic stresses.
Eur. J. Biochem. 268, 2764-2772.
25. Persons, D. L., Yazlovitskaya, E. M., and Pelling, J. C. (2000) Effect of extracellular signal-regulated kinase on
p53 accumulation in response to cisplatin. J. Biol. Chem. 275, 35778-3585.
Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts. Cancer Res. 60, 6346-6352.
27. Ye, R., Bodero, A., Zhou, B. B., Khanna, K. K., Lavin, M. F., and Lees-Miller, S. P. (2001) The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner. J. Biol. Chem. 276, 4828-4833.
28. Matsuoka, M., and Igisu, H. (2001) Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells. Biochem. Biophys. Res. Commun. 282, 1120-1125.
29. Vogelstein, B., and Kinzler, K. W. (1992) p53 function and dysfunction. Cell.
70, 523-526.
30. Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell
88, 323-331.
31. Harris, C. C., and Hollstein, M. N. (1993) Clinical implications of the p53 tumor suppressor gene. N. Engl. J. Med.
329, 1318-1327.
32. Fan. S., El-Deiry, W. S., Bae, I., Freeman, J., Jondle, D., Bhatia, K.,
Fornace, Jr A. J., Magrath, I., Kohn, K. W., and O’Connor P. M. (1994) P53 mutations are associated with decreased sensitivity to DNA damaging agents. Cancer Res. 54,
5824-5830.
33. Righetti, S. C., Perego, P., Corna, E., Pierotti, M. A., and Zunino, F. (1999) Emergence of p53 mutant cisplatinresistant
Res. 59, 1391-1399.
35. Slaton, J. W., Benedict, W. F., and Dinney, C. P. (2001) P53 in bladder cancer: mechanism of action, prognostic value, and target for therapy. Urology 57,
852-859.
36. Zhang, X., Jin, L., and Takenaka, I. (1998) MVAC chemotherapy-induced apoptosis and p53 alterations in the rat model of bladder cancer. Urology 52,
925-931.
37. Kaneuchi, M., Yamashita, T., Shindoh, M., Segawa, K., Takahashi, S., Furuta, I., Fujimoto, S., and Fujinaga, K. (1999) induction of apoptosis by the p53-273L (arg-> leu) mutant in HSC3 cells without transactivation of p21Waf1/Cip1/Sdi1 and bax. Mol. Carcinog. 26, 44-52.
38. Saller, E., Tom, E., Brunori, M., Otter, M., Estreicher, A., Mack, D. H., and Iggo, R. (1999) Increased apoptosis induction by 121F mutant p53. EMBO J. 18,
4424-4437.
39. Ceraline, J., Deplanque, G., Duclos, B., Limacher, J. M., Hajri, A., Noel, F., Orvain, C., Frebourg, T., Klein-Sover, C., and Bergerat, J. P. (1998) Inactivation of p53 in normal human cells increases G2/M arrest and sensitivity to DNA-damaging agents. Int. J. Cancer 75, 432-438.
40. Bunz, F., Hwang, P. M., Torrance, C., Waldman, T., Zhang, Y., Dillehay, L., Williams, J., Lengauer, C., Kinzler, K., and Vogelstein, B. (1999) Disruption of
123-124.
42. Smith, P. D., Crossland, S., Parker, G., Osin, P., Brooks, L., Waller, J., Philp, E., Crompton, M. R., Gusterson, B. A., Allday, M. J., and Crook, T. (1999) Novel p53 mutatants selected in BRCA-associated tumours which dissociate transformation suppression from other wild-type p53 functions. Oncogene 18, 2451-2459.
43. Chang, F. L., and Lai, M. D. (2001) Various forms of mutant p53 confer sensitivity to cisplatin and doxorubicin in bladder cancer cells. J. Urol. 166,
304-310.
44. Rytomaa, M., Martins, L. M., and Downward, J. (1999) Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis. Curr. Biol.
9, 1043-1046.
45. Bennett, M., Macdonald, K., Chan, S. W., Luzio, J. P., Simari, R., and Weissberg, P. (1998) Cell surface trafficking of Fas: a rapid mechanism of p53-mediated
apoptosis. Science 282, 290-293.
46. Khan, Q. A., Vousden, K. H., and Dipple, A. (1997) Cellular response to DNA damage from a potent carcinogen involves stabilization of p53 without induction of p21(waf1/cip1).
