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• W2080961798 abstract "Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53−/−HCT116 and p21−/− HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53−/− HCT116 and p21−/− HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21−/− HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT. Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53−/−HCT116 and p21−/− HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53−/− HCT116 and p21−/− HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21−/− HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT. The “p53 → p21 pathway” is activated in cells after DNA damage. Activation of this pathway temporarily arrests cells at the G1 and G2 checkpoints of the cell cycle, and terminates DNA replication and cell division (1.Bartek J. Lukas J. FEBS Lett. 2001; 490: 117-122Crossref PubMed Scopus (385) Google Scholar, 2.Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1294) Google Scholar, 3.Colman M.S. Afshari C.A. Barrett J.C. Mutat. Res. 2000; 462: 179-188Crossref PubMed Scopus (151) Google Scholar). These events provide the cells with enough time to repair damaged DNA and prevent accumulation of deleterious mutations in the genome that would otherwise be subsequently transferred to daughter cells (4.Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4503) Google Scholar, 5.Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6740) Google Scholar). DNA damage is sensed by the ataxia-telangiectasia mutated protein, which is a member of the phosphoinositol-3 lipid kinase family (6.Jeggo P.A. Carr A.M. Lehmann A.R. Trends Genet. 1998; 14: 312-316Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 7.Lavin M.F. Khanna K.K. Int. J. Radiat. Biol. 1999; 75: 1201-1214Crossref PubMed Scopus (123) Google Scholar). p53 is one of the key targets that are subjected to activation by ataxia-telangiectasia mutated catalyzed phosphorylation (7.Lavin M.F. Khanna K.K. Int. J. Radiat. Biol. 1999; 75: 1201-1214Crossref PubMed Scopus (123) Google Scholar). Activated p53, in turn, induces the expression of many proteins including p21, which is a universal inhibitor of the cyclin-dependent kinases (Cdks) 1The abbreviations used are: Cdkcyclin-dependent kinaseToptopoisomeraseMEFmouse enbryonic fibroblastsBrdUrd5-bromodeoxyuridineCPTcamptothecinPBSphosphate-buffered salineX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideβ-galβ-galactosidaseDDSDNA damage signal (8.Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3177) Google Scholar), and is required to arrest cells at the G1 and G2checkpoints of the cell cycle after DNA damage (9.Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1948) Google Scholar, 10.Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2538) Google Scholar, 11.Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar). cyclin-dependent kinase topoisomerase mouse enbryonic fibroblasts 5-bromodeoxyuridine camptothecin phosphate-buffered saline 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside β-galactosidase DNA damage signal DNA damaging agents including γ-irradiation and inhibitors of the nuclear topoisomerases I and II (Top1 and Top2) are widely included in therapies for cancer patients. The development of many types of human cancers (>50%) is associated with the loss of p53 or mutations in p53 (5.Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6740) Google Scholar). Therefore, the relationship between p53 status and the sensitivity of cancer cells to a variety of drugs, especially DNA damaging agents, has been extensively investigated (12.Lowe S.W. Curr. Opin. Oncol. 1995; 7: 547-553Crossref PubMed Scopus (200) Google Scholar, 13.Morgan S.E. Kastan M.B. Adv. Cancer Res. 1997; 71: 1-25Crossref PubMed Google Scholar, 14.Brown J.M. Wouters B.G. Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar). Initially, a relationship was established between p53 and drug sensitivity in mouse embryonic fibroblasts (MEFs) transformed by ras and E1Aoncogenes, showing that p53+/+ MEFs were more sensitive than p53−/− MEFs to the apoptotic effect of γ-irradiation or DNA damaging drugs (15.Lowe S.W. Ruley H.E. Jacks T. Housman D.E. Cell. 1993; 74: 957-967Abstract Full Text PDF PubMed Scopus (2962) Google Scholar). In contrast, p21 was not required for apoptosis of the MEFs after DNA damage (9.Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1948) Google Scholar). This led to the hypothesis that apoptosis of cancer cells after DNA damage was p53-dependent, but p21-independent, and that cancer cells containing a mutated p53 gene should be resistant to chemotherapies that utilize DNA damaging agents (12.Lowe S.W. Curr. Opin. Oncol. 1995; 7: 547-553Crossref PubMed Scopus (200) Google Scholar). However, this view was re-investigated when, by utilizing clonogenicity assays rather than short-term apoptosis assays, researchers were led to the conclusion that the p53 status was unrelated to the long-term survival of transformed MEFs after DNA damage (14.Brown J.M. Wouters B.G. Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar). Also, studies of the RKO human colon cancer cell line and its derivative cell lines with altered p53 status led to the conclusion that the long-term survival of the cells after DNA damage was independent of p53 (16.Slichenmyer W.J. Nelson W.G. Slebos R.J. Kastan M.B. Cancer Res. 1993; 53: 4164-4168PubMed Google Scholar). In contrast, the apoptotic effect of DNA damaging agents on the HCT116 human colon cancer cells was shown to be blocked by p21, which was expressed by a p53-dependent mechanism (17.Waldman T. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1996; 381: 713-716Crossref PubMed Scopus (719) Google Scholar, 18.Bunz F. Hwang P.M. Torrance C. Waldman T. Zhang Y. Dillehay L. Williams J. Lengauer C. Kinzler K.W. Vogelstein B. J. Clin. Invest. 1999; 104: 263-269Crossref PubMed Scopus (928) Google Scholar). Once again, subsequent results obtained from a clonogenicity assay led to the conclusion that the long-term survival of HCT116 cells after DNA damage was independent of either p53 or p21 (14.Brown J.M. Wouters B.G. Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar). Therefore, these studies collectively indicate that the requirement for p53 in the process of apoptosis depends on whether the cells are of rodent or human origin. In addition, contradictory conclusions can be drawn about the relationship between p53 and drug sensitivity because of variability in the experimental conditions applied to investigate the effect of DNA damaging agents (14.Brown J.M. Wouters B.G. Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar). Nevertheless, it remains to be elucidated why short-term survival, unlike long-term survival, of some cells after DNA damage is affected by p53 and/or p21. Furthermore, it should be noted that the loss of clonogenicity of cells does not necessarily result from loss of cell viability. In this context, it was demonstrated that normal human fibroblasts entered a state of senescence after DNA damage (19.Robles S.J. Adami G.R. Oncogene. 1998; 16: 1113-1123Crossref PubMed Scopus (381) Google Scholar). Therefore, it is possible that loss of clonogenicity of cancer cells after DNA damage can result from an irreversible arrest of the cell cycle rather than loss of viability. Pertinent to this is the demonstration that p53 and p21 play important roles in senescence development of normal human cells (20.Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1315) Google Scholar, 21.Wynford-Thomas D. J. Pathol. 1996; 180: 118-121Crossref PubMed Scopus (50) Google Scholar, 22.Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar, 23.Chen Q.M. Bartholomew J.C. Campisi J. Acosta M. Reagan J.D. Ames B.N. Biochem. J. 1998; 332: 43-50Crossref PubMed Scopus (358) Google Scholar, 24.Itahana K. Dimri G. Campisi J. Eur. J. Biochem. 2001; 268: 2784-2791Crossref PubMed Scopus (286) Google Scholar). Accordingly, it is plausible that p53 and p21 are required for senescence development of human cancer cells after DNA damage. To address this issue, we investigated both long- and short-term effects of CPT-induced and Top1-mediated DNA damage on wild-type HCT116 (wt HCT116) and isogenic p53−/− p21+/+ (p53−/−) and p53+/+ p21−/− (p21−/−) HCT116 cells (17.Waldman T. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1996; 381: 713-716Crossref PubMed Scopus (719) Google Scholar, 18.Bunz F. Hwang P.M. Torrance C. Waldman T. Zhang Y. Dillehay L. Williams J. Lengauer C. Kinzler K.W. Vogelstein B. J. Clin. Invest. 1999; 104: 263-269Crossref PubMed Scopus (928) Google Scholar). Our results demonstrated that under treatment with a high concentration (250 nm) of CPT, wt HCT116, p53−/− HCT116, and p21−/− HCT116 cells underwent apoptosis indicating that apoptosis was independent of the cellular status of p53 and p21 status under this treatment. In contrast, treatment of p53−/− HCT116 and p21−/− HCT116 cells with 20 nm CPT resulted in them becoming apoptotic whereas wt HCT116 cells did not lose their viability but lost their clonogenicity. These results indicated that p53 and p21 were required to block apoptosis of HCT116 cells treated with 20 nm CPT. The loss of clonogenicity of wt HCT116 cells treated with 20 nm CPT was not due to the loss of long-term viability, but was rather the result of senescence development. Our results imply that the expression of p21 by a p53-dependent mechanism is required to fully develop senescent properties after DNA damage. Fetal bovine serum, X-gal, antibodies to BrdUrd (clone BU-33), Cy3- or fluorescein isothiocyanate-conjugated antibodies, and camptothecin (CPT) were obtained from Sigma. The pan-caspase inhibitor, Z-VAD-FMK, was obtained from BIOMOL Inc. (Plymouth Meeting, PA). Antibodies to p53 (FL-393; sc-6243), p21 (C19, sc-397), cyclin A (C19, sc-596), cyclin B1 (GNS1, sc-245), and Cdk2 (M2, sc-163) were obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Antibodies to Cdk1 (68516E), Rb (G3, 245), Top1 (556597), and Top2 (T90920) were obtained from BD Pharmingen (San Diego, CA); and the antibody (9284) to phosphoserine 15 of p53 was obtained from New England Biolabs, Inc. (Beverly, MA). The human autoimmune antibody to Top1 (614–451-5810) used for immunocytochemistry studies was obtained from TopoGEN, Inc. (Columbus, OH). The HCT116 human colon cancer cell lines (wt, p53−/−, and p21−/−) were generously provided by Dr. Bert Vogelstein (Johns Hopkins University). The cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum and antibiotics. All cell cultures were incubated at 37 °C in a humidified incubator containing 5% CO2. Apoptotic fractions in cultures of control and drug-treated cells were identified by flow cytometry analysis of cells stained with propidium iodide (25.Pantazis P. Early J.A. Mendoza J.T. DeJesus A.R. Giovanella B.C. Cancer Res. 1994; 54: 771-776PubMed Google Scholar). Aliquots of whole cell extracts containing 50 μg of protein were used for Western blot analysis as described (26.Han Z. Chatterjee D. Early J. Pantazis P. Hendrickson E.A. Wyche J.H. Cancer Res. 1996; 56: 1621-1628PubMed Google Scholar, 27.Han Z. Pantazis P. Wyche J.H. Kouttab N. Kidd V.J. Hendrickson E.A. J. Biol. Chem. 2001; 276: 38748-38754Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To detect senescence associated β-galactosidase (SA-β-galactosidase) activity, cells were washed in PBS, fixed in 0.5% glutaraldehyde in PBS at room temperature for 15 min, washed three times in PBS (pH 6.0), and then incubated for 20 h in X-gal/PBS solution (pH 6.0) as described (28.Wei W. Sedivy J.M. Exp. Cell Res. 1999; 253: 519-522Crossref PubMed Scopus (93) Google Scholar). DNA synthesis was determined by measuring BrdUrd incorporation into DNA as described (29.Wei W. Hemmer R.M. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 6748-6757Crossref PubMed Scopus (190) Google Scholar). Briefly, cells were incubated with BrdUrd (24 h for normal cells, and 72 h for CPT-treated cells), fixed for 20 min in 2% glutaraldehyde in PBS at room temperature, and the cell membranes were permeabilized with acetone and methanol (1:1). The cells were then incubated for 30 min in 2 m HCl followed by a 30-min incubation in 0.1 m sodium borate (pH 8.5), washed three times in PBS, incubated for 60 min in 1% bovine serum albumin/PBS, and followed by a 45-min incubation in PBS containing 2 μg/ml mouse monoclonal antibody to BrdUrd. The cells were then washed three times in PBS (10 min per wash), incubated with a Cy3-conjugated secondary antibody (1:200) against mouse IgG for 30 min, and washed three times in PBS (20 min per wash). BrdUrd-labeled nuclei were observed and photographed under a fluorescent microscope. The protocols for immunofluorescent detection of various cellular proteins have been described (30.Mohamood A.S. Gyles P. Balan K.V. Hollis V.W. Eckberg W.R. Asseffa A. Han Z. Wyche J.H. Anderson W.A. J. Submicrosc. Cytol. Pathol. 1997; 29: 1-17PubMed Google Scholar). Approximately 300 cells were seeded into each well of a 6-well cell culture plate, and then incubated in 5 ml of medium for 2 weeks or longer. Subsequently, the medium was removed, and the cells were fixed for 5 min in 5 ml of methanol. The methanol was removed, the wells were rinsed with water, the cell colonies stained for 10 min in 2 ml of 4% (w/v) methylene blue solution in PBS, washed once again with water, and then counted. To induce Top1-mediated DNA damage, wt HCT116, p21−/− HCT116 and p53−/− HCT116 cell lines were treated with CPT (31.Liu L.F. Desai S.D. Li T.K. Mao Y. Sun M. Sim S.P. Ann. N. Y. Acad. Sci. 2000; 922: 1-10Crossref PubMed Scopus (423) Google Scholar). Cells were exposed either to low (i.e. 20 nm) or high (i.e. 250 nm) concentrations of CPT, and the effects on cell proliferation and survival were monitored. Treatment of all three HCT116 cell lines with either concentration of CPT for 24 h was sufficient to induce cell cycle arrest (data not shown). Treatment with 250 nm CPT resulted in apoptosis of all three cell lines (Fig. 1 A), whereas treatment with 20 nm CPT resulted in significant apoptosis only of p53−/− HCT116 and p21−/− HCT116 cell lines (Fig. 1 A). Thus, the response of HCT116 cells to the apoptotic effect of low and high concentrations of CPT was differentially affected by the p53 and p21 status. Western blot analysis of proteins from the wt HCT116 cells, treated with 20 nm CPT, demonstrated accumulation and phosphorylation of Ser15 in p53, i.e. activation of p53, and expression of p21 (Fig. 1 B). In contrast, a very delayed expression of a small amount of p21 was detected in the CPT-treated p53−/− HCT116 cells (Fig. 1 B), indicating that most p21 expression in HCT116 cells after CPT treatment required a p53-dependent mechanism. Also, an accumulation and phosphorylation of Ser15 in p53, i.e. activation of p53, was observed in p21−/− HCT116 cells treated with 20 nm CPT (Fig. 1 B). Taken together, these results suggested that p53-dependent expression of p21 was required to block the apoptotic effect of 20 nm CPT on HCT116 cells. Since the effect of long-term treatment of cancer cells with low doses of CPT appears to be beneficial clinically (32.Gerrits C.J. de Jonge M.J. Schellens J.H. Stoter G. Verweij J. Br. J. Cancer. 1997; 76: 952-962Crossref PubMed Scopus (136) Google Scholar, 33.O'Leary J. Muggia F.M. Eur. J. Cancer. 1998; 34: 1500-1508Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), the HCT116 cell lines were treated with 20 nm CPT in subsequent experiments. Treatment of wt HCT116 cells with CPT for 24, 48, 72, and 96 h inhibited, in a time-dependent fashion, expression of cyclin A, cyclin B1, Cdk1, E2F1, and activated, as shown by dephosphorylation, by the Rb protein (Fig. 2 A). Of interest, an initial decrease in Top1 expression was observed at 24 h of CPT treatment, but no further decrease was detected thereafter (Fig. 2 A). In contrast, Top2 expression was dramatically down-regulated after 48 h of CPT treatment, and virtually no expression was detected at 72 and 96 h of treatment (Fig. 2 A). The CPT-induced suppression of the expression of cyclin A, cyclin B1, Cdk1, E2F1, and Top2 was irreversible, because it remained unaltered for a prolonged period of time after CPT was removed from the culture medium as assessed by immunofluorescence studies of the cells (Fig. 2 B). Although the expression level of Cdk2 was not significantly altered during CPT treatment (Fig. 2 A), it was reduced to an undetectable level in the senescent cells after the treatment was terminated (Fig. 2 B). Thus, it appeared that down-regulation of Cdk2 was a very late event. After a 96-h treatment of wt HCT116 cells with CPT, the size of the cells had dramatically increased, the nuclei were enlarged and prominent, and the nucleolar content was highly heterochromatic (Fig. 3 A). Also, the CPT-treated cells stained positive, at pH 6.0 for SA-β-gal activity in the cytoplasm (Fig. 3 A). SA-β-gal has been identified as a specific marker for senescent cells (29.Wei W. Hemmer R.M. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 6748-6757Crossref PubMed Scopus (190) Google Scholar, 34.Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5738) Google Scholar, 35.Dimri G.P. Itahana K. Acosta M. Campisi J. Mol. Cell. Biol. 2000; 20: 273-285Crossref PubMed Scopus (339) Google Scholar, 36.Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar). This suggested that the CPT-treated wt HCT116 cells had acquired properties of senescence. To confirm this, wt HCT116 cells were treated with CPT for 72 h, and then cultured in CPT-free medium for 6 days. Subsequently, control and CPT-treated cells were assayed for their ability to synthesize DNA (i.e. to incorporate BrdUrd into DNA), proliferate, and form colonies. The results showed that there was practically no BrdUrd incorporation into the DNA of the CPT-treated cells (Fig. 3 B), and concurrently these cells had completely lost their clonogenicity (Fig. 3 C). The changes described above (Figs.2 and 3) are characteristic of senescent cells (20.Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1315) Google Scholar, 23.Chen Q.M. Bartholomew J.C. Campisi J. Acosta M. Reagan J.D. Ames B.N. Biochem. J. 1998; 332: 43-50Crossref PubMed Scopus (358) Google Scholar, 29.Wei W. Hemmer R.M. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 6748-6757Crossref PubMed Scopus (190) Google Scholar, 35.Dimri G.P. Itahana K. Acosta M. Campisi J. Mol. Cell. Biol. 2000; 20: 273-285Crossref PubMed Scopus (339) Google Scholar, 36.Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar, 37.Stein G.H. Beeson M. Gordon L. Science. 1990; 249: 666-669Crossref PubMed Scopus (334) Google Scholar, 38.Richter K.H. Afshari C.A. Annab L.A. Burkhart B.A. Owen R.D. Boyd J. Barrett J.C. Cancer Res. 1991; 51: 6010-6013PubMed Google Scholar, 39.Dimri G.P. Hara E. Campisi J. J. Biol. Chem. 1994; 269: 16180-16186Abstract Full Text PDF PubMed Google Scholar, 40.Wong H. Riabowol K. Exp. Gerontol. 1996; 31: 311-325Crossref PubMed Scopus (86) Google Scholar, 41.Vaziri H. Benchimol S. Oncogene. 1999; 18: 7676-7680Crossref PubMed Scopus (44) Google Scholar), and thus indicated that the CPT-treated wt HCT116 cells became senescent and concurrently lost their clonogenicity. The results described above demonstrated that the presence of p21 in CPT-treated wt HCT116 cells enabled them to escape apoptosis and enter senescence, thus maintaining their viability. Therefore, the question raised by this observation was whether p21 selectively blocked the apoptotic pathway activated by DNA damage or whether it impaired the process of apoptosis in general. To investigate this, the CPT-treated wt HCT116 cells were also treated with 1 μm staurosporine to induce apoptosis by a pathway that is not associated with DNA damage (26.Han Z. Chatterjee D. Early J. Pantazis P. Hendrickson E.A. Wyche J.H. Cancer Res. 1996; 56: 1621-1628PubMed Google Scholar, 42.Ishizaki Y. Voyvodic J.T. Burne J.F. Raff M.C. J. Cell Biol. 1993; 121: 899-908Crossref PubMed Scopus (146) Google Scholar, 43.Jacobson M.D. Burne J.F. Raff M.C. Biochem. Soc. Trans. 1994; 22: 600-602Crossref PubMed Scopus (37) Google Scholar, 44.Weil M. Jacobson M.D. Coles H.S. Davies T.J. Gardner R.L. Raff K.D. Raff M.C. J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (356) Google Scholar). The cells were sensitive to the apoptotic effect of staurosporine at all time points tested (Fig. 4). These results indicated that the CPT-treated wt HCT116 cells were selectively defective, in the presence of p21, in the apoptotic pathway activated by DNA damage signals. The findings described above raised the issue of how p21 affects senescence of CPT-treated wt HCT116 cells. Is p21 required to simply block apoptosis and thus provide the cells time to develop into senescence? Or, is p21 required for blocking both apoptosis and inducing senescence? To investigate whether the CPT-treated p21−/− HCT116 cells were able to enter senescence while the apoptotic pathway was blocked, the cells were treated for 48 h, and 96 h with CPT in presence of 100 μm Z-VAD-FMK, a pan-caspase inhibitor that blocks apoptosis of cells subjected to various apoptotic conditions. The presence of Z-VAD-FMK blocked apoptosis (Fig. 5 A). At the end of the treatment, the cells stained positive for the presence of SA-β-gal activity (Fig. 5 B), suggesting that they had become senescent. However, the expression and nuclear localization of cyclin A, cyclin B1, Cdk1, Cdk2, E2F1, and Top2 remained essentially unaltered (Figs. 5 C and 6). Furthermore, these cells rapidly underwent apoptosis upon withdrawal of CPT and Z-VAD-FMK from the medium (Fig. 5 D). Collectively, these results indicated that blocking apoptosis alone in the CPT-treated p21−/− HCT116 cells was insufficient to allow them to develop full senescent properties. Therefore, p21 appeared to be required not only to block apoptosis but also induce senescence of HCT116 cells after CPT treatment.Figure 6Nuclear localization of proteins in CPT-treated p21−/− HCT116 cells.p21−/− HCT116 cells were treated with 20 nmCPT in the presence of 100 μm Z-VAD-FMK for 96 h, and then fixed, and subjected to immunofluorescence detection of the proteins indicated. Nuclear DNA was stained with 4,6-diamidino-2-phenylindole (DAPI).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Treatment of wt HCT116 cells with 250 nm CPT induced the stable accumulation of p53, transient phosphorylation of Ser15 on p53, and the up-regulation of p21 expression (Fig. 7). This treatment did not significantly alter the expression level of Cdk1 and Cdk2, but induced down-regulation of the expression of cyclin A, cyclin B1, Rb, and Top2, and degradation of Top1 (Fig. 7). However, the E2F1 expression level was increased in the cells following drug treatment (Fig. 7). Nevertheless, the elevated presence of p21 suggested that it might be possible for wt HCT116 cells to develop along the senescent pathway following treatment with 250 nm CPT if the apoptotic pathway could be blocked. However, although the apoptotic effect of 250 nm CPT on the wt HCT116 cells was blocked by the presence of 100 μm Z-VAD-FMK (Fig. 8 A), the cells did not develop any senescent properties, including the expression of SA-β-gal activity (Fig. 8 A). Furthermore, the cells rapidly underwent apoptosis after withdrawal of both Z-VAD-FMK and CPT from the medium (Fig. 8 B). Thus, even in the presence of elevated p21 levels, wt HCT116 cells were unable to escape apoptosis induced by a high concentration of CPT and enter senescence.Figure 8The apoptotic fate of wt HCT116 cells treated with 250 nm CPT. A, wt HCT116 cells were left untreated or treated for 72 h with 250 nm CPT in the absence or presence of 100 μm Z-VAD-FMK and then examined for induction of apoptosis and expression of SA-β-gal. B, alternatively, the cells were incubated in Z-VAD-FMK/CPT-free medium and subsequently apoptosis was determined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we demonstrated that Top1-mediated DNA damage induced by 20 nm CPT resulted in the apoptotic death of p53−/− and the p21−/− HCT116 cells, both of which were deficient in p21 expression (Fig. 1 B). In contrast, wt HCT116 cells that expressed robust levels of p21 became senescent, and remained viable for a prolonged period of time (Figs. 1, 3, and 4). Furthermore, we demonstrated that the CPT-treated wt HCT116 cells that remained viable were still sensitive to the apoptotic effect of staurosporine (Fig. 4). Staurosporine is an inhibitor of many protein kinases and triggers apoptotic signals in the absence of DNA damage (26.Han Z. Chatterjee D. Early J. Pantazis P. Hendrickson E.A. Wyche J.H. Cancer Res. 1996; 56: 1621-1628PubMed Google Scholar, 42.Ishizaki Y. Voyvodic J.T. Burne J.F. Raff M.C. J. Cell Biol. 1993; 121: 899-908Crossref PubMed Scopus (146) Google Scholar, 44.Weil M. Jacobson M.D. Coles H.S. Davies T.J. Gardner R.L. Raff K.D. Raff M.C. J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (356) Google Scholar). Therefore, our results indicated that the ability of p21 to block apoptosis was selectively associated with the apoptotic pathway activated by a nuclear DNA damage signal (DDS) in CPT-treated cells (Fig. 9). Although p21 expression was also induced in wt HCT116 cells treated with 250 nm CPT (Fig. 7), it failed to prevent the cells from undergoing apoptosis (Figs. 1 A, 3, and 8). Since it has been shown that the extent of CPT-induced DNA damage in cells is proportional to the concentration of CPT used (45.Kaufmann W.K. Boyer J.C. Estabrooks L.L. Wilson S.J. Mol. Cell. Biol. 1991; 11: 3711-3718Crossref PubMed Scopus (61) Google Scholar), it is very likely that the amount of DNA damage in HCT116 cells caused by 250 nm CPT was more extensive than that caused by 20 nm CPT. Thus, when HCT116 cells (wt, p53−/−, and p21−/−) were exposed to only 20 nm CPT a limited amount of DNA damage should have been produced, resulting in the production of a low strength DDS which was nonetheless sufficient to induce apoptosis. In wt HCT116 cells, however, this low dose of CPT resulted in the p53-dependent induction of p21 (Fig. 1). As a consequence of p21 induction, the commitment to apoptosis was blocked and senescence was instead induced (Fig. 9). When wt HCT116 cells were treated with 250 nm CPT, extensive DNA damage presumably occurred in the cells, which, in turn, resulted in the production of a high strength DDS. Although the high strength DDS induced expression of p21 in wt HCT116 cells by a p53-dependent mechanism, the presence of elevated p21 failed to override the apoptotic effect of the signal (Fig. 9). This observation also suggests that the mechanism mediating the apoptotic effect of 250 nm CPT (or high strength DDS) on wt HCT116 cells was in this instance not regulated by p21 but by another, unidentified, factor(s) that was defective in the cells. The fact that blocking" @default.
• W2080961798 created "2016-06-24" @default.
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• W2080961798 date "2002-05-01" @default.
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• W2080961798 title "Role of p21 in Apoptosis and Senescence of Human Colon Cancer Cells Treated with Camptothecin" @default.
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