Stress-induced Premature Senescence in hTERT-expressing Ataxia Telangiectasia Fibroblasts
2004; Elsevier BV; Volume: 279; Issue: 3 Linguagem: Inglês
10.1074/jbc.m309457200
ISSN1083-351X
AutoresKazuhito Naka, Akira Tachibana, Kyoji Ikeda, Noboru Motoyama,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoIn addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G1 checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H2O2 in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated β-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21WAF1, and p16INK4A. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H2O2-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress. In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G1 checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H2O2 in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated β-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21WAF1, and p16INK4A. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H2O2-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress. Culture of primary cells for many generations eventually results in a loss of proliferative potential, a phenomenon referred to as replicative senescence or, more generally, as cellular senescence. Cellular senescence can also be induced by stressful conditions (1.Serrano M. Blasco M.A. Curr. Opin. Cell Biol. 2001; 13: 748-753Crossref PubMed Scopus (352) Google Scholar). Replicative senescence likely results from the shortening of telomeres to such an extent that the chromosome ends are not fully masked from recognition by the proteins responsible for double strand break repair. Whereas primary human fibroblasts, which lack telomerase activity, normally exhibit a finite life span in culture (2.Hayflick L. Moorhead P.S. Exp. Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5828) Google Scholar), ectopic expression of the catalytic subunit of human telomerase (hTERT) 1The abbreviations used are: hTERT, catalytic subunit of human telomerase; SIPS, stress-induced premature senescence; SA, senescence-associated; MAPK, mitogen-activated protein kinase; AT, ataxia telangiectasia; IR, ionizing radiation; PD, population doubling; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment; BrdUrd, bromodeoxyuridine; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RB, retinoblastoma. in these cells restores telomerase activity, stabilizes telomere length, and prevents replicative senescence (3.Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4200) Google Scholar). Although rodent cells in culture also undergo senescence, this phenotype is attributable to unsuitable conditions (culture shock) or to growth arrest mediated by p53 and p19Arf, not to telomere shortening per se. In contrast, the major signaling pathway responsible for senescence in human cells, including that due to telomere shortening, is mediated by RB and p16INK4A (4.Shay J.W. Wright W.E. Science. 2001; 291: 839-840Crossref PubMed Scopus (68) Google Scholar, 5.Kiyono T. Foster S.A. Koop J.I. McDougall J.K. Galloway D.A. Klingelhutz A.J. Nature. 1998; 396: 84-88Crossref PubMed Scopus (1094) Google Scholar). The introduction of activated oncogenes into primary cells triggers defense responses that prevent cell proliferation. Some oncogenes, such as those for c-Myc and E2F1, trigger apoptosis (6.Hermeking H. Eick D. Science. 1994; 265: 2091-2093Crossref PubMed Scopus (717) Google Scholar, 7.Kowalik T.F. DeGregori J. Leone G. Jakoi L. Nevins J.R. 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Cells subjected to other types of sublethal stress also enter a state that closely resembles replicative senescence and is referred to as stress-induced premature senescence (SIPS) (10.Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (555) Google Scholar). Cells subjected to DNA damage induced by ultraviolet or x-radiation (10.Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (555) Google Scholar, 11.Di Leonardo A. Linke S.P. Clarkin K. Wahl G.M. Genes Dev. 1994; 8: 2540-2551Crossref PubMed Scopus (1056) Google Scholar, 12.Medrano E.E. Im S. Yang F. Abdel-Malek Z.A. Cancer Res. 1995; 55: 4047-4052PubMed Google Scholar, 13.Oh C.W. Bump E.A. Kim J.S. Janigro D. Mayberg M.R. Radiat. Res. 2001; 156: 232-240Crossref PubMed Scopus (69) Google Scholar), to oxidative stress (induced by H2O2 or hyperoxia) (14.Chen Q.M. Bartholomew J.C. Campisi J. Acosta M. Reagan J.D. Ames B.N. Biochem. 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Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (555) Google Scholar). Overexpression of antioxidant proteins in human fibroblasts slows the rate of telomere shortening and extends their life span (18.Serra V. von Zglinicki T. Lorenz M. Saretzki G. J. Biol. Chem. 2003; 278: 6824-6830Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Accelerated telomere shortening caused by oxidative stress has thus been suggested as a cause of SIPS (10.Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (555) Google Scholar, 19.von Zglinicki T. Trends Biochem. Sci. 2002; 27: 339-344Abstract Full Text Full Text PDF PubMed Scopus (1925) Google Scholar, 20.von Zglinicki T. Petrie J. Kirkwood T.B. Nat. Biotechnol. 2003; 21: 229-230Crossref PubMed Scopus (69) Google Scholar). However, no difference in SIPS induction was detected between parental and hTERT-expressing human fibroblasts after exposure to H2O2 or to ultraviolet or x-radiation, and telomere shortening was not apparent in the hTERT-expressing cells during SIPS induction (21.Gorbunova V. Seluanov A. Pereira-Smith O.M. J. Biol. Chem. 2002; 277: 38540-38549Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). These observations thus indicate that, in contrast to replicative senescence, SIPS cannot be prevented by hTERT-mediated telomere elongation (21.Gorbunova V. Seluanov A. Pereira-Smith O.M. J. Biol. Chem. 2002; 277: 38540-38549Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). They further suggest that SIPS is triggered not only by telomere shortening but also by nonspecific genome-wide DNA damage. Senescence induced by oncogenic Ras is prevented by inhibition of the activity of the p38 (stress-activated) isoform of mitogen-activated protein kinase (p38 MAPK), suggesting that activation of p38 MAPK is essential for oncogenic stress-induced senescence (22.Wang W. Chen J.X. Liao R. Deng Q. Zhou J.J. Huang S. Sun P. Mol. Cell. Biol. 2002; 22: 3389-3403Crossref PubMed Scopus (328) Google Scholar). The activation of p38 MAPK also contributes to the onset of senescence induced by telomere shortening, oxidative stress, culture shock, or activation of Ras-Raf signaling (23.Iwasa H. Han J. Ishikawa F. Genes Cells. 2003; 8: 131-144Crossref PubMed Scopus (316) Google Scholar). However, the signaling pathway responsible for p38 MAPK activation during SIPS induction is not well characterized. Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive neurological degeneration, telangiectasia, growth retardation, specific immunodeficiency, high sensitivity to ionizing radiation (IR), an increased incidence of malignancy, and premature aging of the skin and hair (24.Sedgwick R.P. Boder E. de Jong J.M.B.V. Handbook of Clinical Neurology: Hereditary Neuropathies and Spinocerebellar Atrophies. 60. Elsevier Science, Amsterdam1991: 347-423Google Scholar, 25.Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (545) Google Scholar). Mitotic cells, such as fibroblasts and lymphoblasts, from individuals with AT exhibit a variety of anomalies in culture include an increased sensitivity to IR and radiomimetic agents, radioresistant DNA synthesis, chromosomal instability, a reduced life span, and an increased rate of telomere loss (26.Taylor A.M. Harnden D.G. Arlett C.F. Harcourt S.A. Lehmann A.R. Stevens S. 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Biol. 2001; 11: 962-966Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). ATM has recently been shown to contribute to telomere maintenance and to senescence signaling originating from telomeres (45.Wong K.K. Maser R.S. Bachoo R.M. Menon J. Carrasco D.R. Gu Y. Alt F.W. DePinho R.A. Nature. 2003; 421: 643-648Crossref PubMed Scopus (339) Google Scholar, 46.Chan S.W.-L. Blackburn E.H. Mol. Cell. 2003; 11: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In addition to its role as a damage sensor for cell cycle checkpoints and DNA repair, ATM mediates up-regulation of ATF3, a target of p38 MAPK, in cells exposed to IR (47.Kool J. Hamdi M. Cornelissen-Steijger P. van der Eb A.J. Terleth C. van Dam H. Oncogene. 2003; 22: 4235-4242Crossref PubMed Scopus (69) Google Scholar), suggesting that it acts as a trigger for stress-related MAPK signaling by activating c-Abl (32.Baskaran R. Wood L.D. Whitaker L.L. Canman C.E. Morgan S.E. Xu Y. Barlow C. Baltimore D. Wynshaw-Boris A. Kastan M.B. Wang J.Y.J. Nature. 1997; 387: 516-519Crossref PubMed Scopus (487) Google Scholar). ATM has thus been proposed to function as a stress sensor in SIPS (48.Barzilai A. Rotman G. Shiloh Y. DNA Repair (Amst.). 2002; 1: 3-25Crossref PubMed Scopus (318) Google Scholar). It has not been possible to study SIPS in AT cells, however, because of their premature replicative senescence. To evaluate the possible role of ATM in SIPS, we therefore established hTERT-expressing AT fibroblasts and examined whether SIPS is induced in these cells. Cell Culture and Infection—Primary human dermal fibroblasts established from a normal individual (YMM) or from individuals with AT (AT1KY, AT2KY, AT4KY, AT5KY, AT6KY, and AT1OS) were cultured under a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen, Rockville, MD) supplemented with 15% heat-inactivated fetal bovine serum (Invitrogen) (49.Ejima Y. Sasaki M.S. Hum. Genet. 1998; 102: 403-408Crossref PubMed Scopus (26) Google Scholar). The retroviral vector pMX-puro-hTERT (23.Iwasa H. Han J. Ishikawa F. Genes Cells. 2003; 8: 131-144Crossref PubMed Scopus (316) Google Scholar) and Phenix-A retroviral packaging cells were kindly provided by F. Ishikawa (Kyoto University), T. Kitamura (University of Tokyo), and G. P. Nolan (Stanford University). For retrovirus production, we transfected Phenix-A cells with pMX-puro-hTERT with the use of FuGENE 6 (Roche Applied Science, Mannheim, Germany). The resulting retroviruses were used to infect fibroblasts, which were then selected in medium containing puromycin (0.5 μg/ml) (Sigma, St. Louis, MO). To examine the kinetics of cell proliferation, we plated hTERT-expressing and parental primary fibroblasts (2 × 105 cells) in 95-mm-diameter culture dishes and determined the cell number with a Coulter Counter (Beckman Coulter, Fullerton, CA) at each passage. The number of population doublings (PD value) was calculated from PD = log(Nf/N0)/log 2, where Nf is the final cell number and N0 is the initial number of seeded cells. Determination of Telomerase Activity and Telomere Length—Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) with the use of a TPAPeze kit (Intergen, Purchase, NY). Telomere length was measured with a terminal restriction fragment (TRF) assay. Genomic DNA (5 μg) was thus digested with HinfI and RsaI, and the resulting fragments were subjected to Southern blot analysis with a 32P-labeled telomeric oligonucleotide probe, (TTAGGG)4; hybridization was performed for 12–15 h at 37 °C in a solution containing 0.75 m NaCl, 30 mm sodium citrate, and 1% SDS. Signals were visualized by autoradiography. Cell Cycle Analysis—Cells were labeled for the indicated times with 10 μm bromodeoxyuridine (BrdUrd), fixed with 70% ethanol, and stained with a fluorescein isothiocyanate-conjugated mouse monoclonal antibody to BrdUrd (BD Pharmingen, San Diego, CA) and with propidium iodide (Sigma). The cellular content of DNA was determined by flow cytometry with a FACSCalibur instrument, and analysis of the resulting data was carried out with CELL Quest software (BD Biosciences, San Jose, CA). Clonogenic Assay—Clonogenic assays were performed as described previously (26.Taylor A.M. Harnden D.G. Arlett C.F. Harcourt S.A. Lehmann A.R. Stevens S. Bridges B.A. Nature. 1975; 258: 427-429Crossref PubMed Scopus (816) Google Scholar). Cells in the exponential phase of growth were plated on 95-mm culture dishes, incubated for 24 h, and then irradiated at room temperature, at the indicated doses, with a 137Cs γ-ray source at a rate of 1.143 Gy/min. The number of cells per dish was chosen to ensure that ∼100 colonies would survive the particular treatment. After 2 weeks, cells were fixed with methanol:acetic acid (3:1, v/v) and stained with crystal violet. Only colonies containing >50 cells were judged to be derived from viable clonogenic cells. Induction and Analysis of SIPS—For x-irradiation, cells (5 × 105) were seeded in 95-mm dishes, incubated for 3 days, and exposed to 55 Gy of x-radiation at a rate of 7.32 Gy/min (21.Gorbunova V. Seluanov A. Pereira-Smith O.M. J. Biol. Chem. 2002; 277: 38540-38549Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). For H2O2 treatment, cells were exposed to 500 μm H2O2 for 2 h at 37 °C. All cells were then washed twice with phosphate-buffered saline, cultured for 10 days in culture medium, fixed, stained for SA β-galactosidase activity as described (50.Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelly C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5936) Google Scholar), and counterstained with propidium iodide. Immunoblot Analysis—Cells were lysed in a solution containing 50 mm Tris-HCl (pH 7.4), 125 mm NaCl, 0.1% Nonidet P-40 (Sigma), 5 mm EDTA, 0.1 m NaF, and a mixture of protease inhibitors (Complete, Roche Applied Science). The protein concentration of the lysate was determined with the BCA protein assay reagent (Pierce, Rockford, IL), after which samples (50 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis with mouse monoclonal antibodies to ATM (2C-1; GeneTex, San Antonio, TX), sheep polyclonal antibodies to Chk2 (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal antibodies to p53 (Ab-6; Oncogene Research, Cambridge, MA), mouse monoclonal antibodies to p21WAF1 (F-5; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antibodies to α -tubulin (B-5-1-2, Sigma), rabbit polyclonal antibodies to p16INK4A (C-20, Santa Cruz Biotechnology), rabbit polyclonal antibodies to p38 MAPK (Santa Cruz Biotechnology), or mouse monoclonal antibodies to phosphorylated (Thr180 and Tyr182) p38 MAPK (28B10, Santa Cruz Biotechnology). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and the ECL Plus system (Amersham Bioscience, Piscataway, NJ). Establishment of hTERT-expressing AT Fibroblasts—Primary fibroblasts derived from individuals with AT exhibit a variety of abnormalities, including defective cell cycle checkpoint function in response to DNA damage as well as rapid shortening of telomere length and consequent premature replicative senescence. To examine the role of ATM in SIPS, we established primary fibroblasts from a normal individual (normal/TERT) and from AT patients (AT/TERT) that stably express hTERT as a result of infection with a recombinant retrovirus. The cells were selected and expanded as polyclonal populations. Telomerase activity was detected by the TRAP assay in AT1KY/TERT, AT2KY/TERT, AT4KY/TERT, AT5KY/TERT, AT6KY/TERT, and AT1OS/TERT cells as well as in normal/TERT cells but not in the corresponding parental cells (Fig. 1A; data not shown). Reverse transcription and polymerase chain reaction analysis also revealed the presence of hTERT mRNA in AT/TERT and normal/TERT cells but not in the corresponding parental cells (data not shown). The TRF assay showed that the telomere length of AT/TERT cells was extended to >12 kb, compared with ∼4–11 kb for parental AT cells (Fig. 1B). Whereas parental AT cells underwent replicative senescence after ∼24 PDs (Fig. 2A), AT/TERT cells survived for >70 to 100 PDs (Fig. 2B, data not shown). These results indicated that the telomere elongation induced by expression of hTERT allowed AT cells to overcome premature replicative senescence.Fig. 2Proliferation of parental (A) and hTERT-expressing (B) AT fibroblasts. Cells were plated at a density of 2 × 105 per 95-mm dish and passaged (after 7–9 days) before they achieved confluence. The number of cells was determined at each passage, and the PD value was calculated. For parental cells, time zero was defined as the time of initial plating; for hTERT-expressing cells, it was defined as the beginning of retroviral infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Defective Checkpoint Function in AT/TERT Cells Exposed to IR—AT fibroblasts show defective cell cycle checkpoint responses to DNA damage induced by IR (51.Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr, A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2994) Google Scholar) or to oxidative stress (52.Shackelford R.E. Innes C.L. Sieber S.O. Heinloth A.N. Leadon S.A. Paules R.S. J. Biol. Chem. 2001; 276: 21951-21959Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To examine whether the abnormality of the G1 cell cycle checkpoint in AT fibroblasts was rescued by hTERT expression, we exposed asynchronous normal/TERT or AT/TERT cells to IR and determined the numbers of cells in G1, S, and G2-M phases of the cell cycle 8 h thereafter by flow cytometry. The proportion of normal/TERT cells in S phase (BrdUrd-positive) was greatly reduced 8 h after irradiation, whereas IR had no effect on the proportion of AT/TERT cells in S phase (Fig. 3A). The IR-induced G1 checkpoint is triggered by sequential signaling by ATM, Chk2, p53, and p21WAF1 (40.Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (357) Google Scholar). Although ATM was detected in normal/TERT cells, this protein was not apparent in AT/TERT cells by immunoblot analysis (Fig. 3B). The IR-induced phosphorylation and activation of Chk2, as revealed by a decrease in the electrophoretic mobility of the protein, were also evident in normal/TERT cells but not in AT/TERT cells (Fig. 3B). Consistent with these observations, up-regulation of p21WAF1 in response to IR was apparent in normal/TERT cells but not in AT/TERT cells (Fig. 3B). Activation of the ATM-Chk2-p53-p21WAF1 signaling pathway by IR was thus impaired in the AT/TERT cells. Like primary AT fibroblasts, AT/TERT cells also exhibit a defective G1 cell cycle checkpoint as a result of their ATM deficiency. Radiosensitivity of AT/TERT Cells—AT fibroblasts are markedly more sensitive to IR than are normal cells. To examine whether hTERT expression affected the radiosensitivity of AT cells, we performed a clonogenic survival assay after exposure of AT/TERT, normal/TERT, and the corresponding parental cells to various doses of IR. Consistent with previous report, both AT/TERT cells and primary AT fibroblasts were hypersensitive to IR in comparison with normal fibroblast and normal/TERT cells (Fig. 4) (41.Wood L.D. Halvorsen T.L. Dhar S. Baur J.A. Pandita R.K. Wright W.E. Hande M.P. Calaf G. Hei T.K. Levine F. Shay J.W. Wang J.J. Pandita T.K. Oncogene. 2001; 20: 278-288Crossref PubMed Scopus (88) Google Scholar). ATM is thus essential for signaling that results in DNA repair after exposure of cells to IR. SIPS in AT/TERT Fibroblasts—We have shown that, like that of the parental cells, the response of AT/TERT cells to DNA damage is impaired. If the DNA damage signaling pathway also contributes to SIPS, then AT/TERT cells would also be expected to be resistant to SIPS. To examine this possibility, we compared the responses of normal/TERT and AT/TERT fibroblasts to SIPS inducers (21.Gorbunova V. Seluanov A. Pereira-Smith O.M. J. Biol. Chem. 2002; 277: 38540-38549Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). 5 or 10 days after exposure to IR or H2O2, normal/TERT cells exhibited a senescence phenotype, including a flattened and enlarged morphology (Fig. 5, A–F, data not shown). Similar morphological changes were also evident in AT4KY/TERT and AT6KY/TERT cells (Fig. 5, G–L, data not shown). Furthermore, normal/TERT cells stained intensely for SA β-galactosidase activity10 days after exposure to IR or H2O2 (Fig. 5, A–C), as did AT4KY/TERT and AT6KY/TERT cells (Fig. 5, G–I, data not shown). We also examined DNA synthesis in the treated cells by measuring BrdUrd incorporation. The proportion of BrdUrd-positive cells was reduced by >95% 3 days after exposure of normal/TERT, AT4KY/TERT, or AT6KY/TERT cells to IR or to H2O2 (Fig. 6). These results thus indicate that, despite the defects in cell cycle checkpoint and other DNA damage responses in AT/TERT cells, SIPS was induced similarly in AT/TERT cells and normal/TERT cells. Induction or Phosphoryl
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