Expression of Human Telomerase (hTERT) Does Not Prevent Stress-induced Senescence in Normal Human Fibroblasts but Protects the Cells from Stress-induced Apoptosis and Necrosis
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m202671200
ISSN1083-351X
AutoresVera Gorbunova, Andrei Seluanov, Olivia M. Pereira‐Smith,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoCells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, γ-irradiation, and H2O2. SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS. Cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, γ-irradiation, and H2O2. SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS. retinoblastoma stress-induced premature senescence human telomerase reverse transcriptase bromodeoxyuridine phosphate-buffered saline fluorescence-activated cell sorter senescence-associated β-galactosidase Normal human cells in culture do not divide indefinitely and after ∼60 population doublings enter irreversible growth arrest termed replicative senescence. Replicatively senescent cells are characterized by increased cell volume and a distinct flat morphology, the presence of SA-β-gal activity (1Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley 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 (5707) Google Scholar), elevated expression of p16Ink4a (p16) and p21Cip1/Waf1 (p21), and hypophosphorylated Rb.1 Replicative senescence is induced by progressive telomere shortening, which occurs at every cell division. When telomeres reach the critical length of less than 5 kb, the Rb and p53 pathways become activated and trigger the irreversible growth arrest (2Garkavtsev I. Hull C. Riabowol K. Exp. Gerontol. 1998; 33: 81-94Crossref PubMed Scopus (28) Google Scholar, 3Itahana K. Dimri G. Campisi J. Eur. J. Biochem. 2001; 268: 2784-2791Crossref PubMed Scopus (285) Google Scholar, 4Shay J.W. Wright W.E. Radiat. Res. 2001; 155: 188-193Crossref PubMed Scopus (106) Google Scholar, 5von Zglinicki T. Burkle A. Kirkwood T.B. Exp. Gerontol. 2001; 36: 1049-1062Crossref PubMed Scopus (161) Google Scholar). How the signal from short telomeres leads to activation of p53 and Rb pathways is not well understood. Replicative senescence in human fibroblasts can be overcome by overexpression of the catalytic subunit of telomerase (6Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Linchtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4105) Google Scholar). Telomerase elongates short telomeres, and the cells become immortalized. Cells subjected to sub-lethal stress may enter, within a short time, a state that closely resembles replicative senescence (reviewed in Ref.7Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (523) Google Scholar). Cells enter stress-induced senescence (SIPS) following DNA damage (UV and γ-irradiation) (7Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (523) Google Scholar, 8Di Leonardo A. Linke S.P. Clarkin K. Wahl G.M. 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Cells undergoing SIPS display all the major characteristics of replicatively senescent cells; they are large and flat, stain positive for SA-β-gal, accumulate p21, and contain hypophosphorylated Rb. Furthermore, it has been shown that the expression pattern in cells undergoing SIPS is similar to that of replicatively senescent cells (15Saretzki G. Feng J. von Zglinicki T. Villeponteau B. J. Gerontol. A Biol. Sci. Med. Sci. 1998; 53: B438-B442Crossref PubMed Scopus (59) Google Scholar). The molecular events that trigger SIPS are far from clear. It has been observed that telomeres shorten 5–10 times faster in cells grown under chronic hyperoxia (13von Zglinicki T.S.G. Docke W. Lotze C. Exp. Cell Res. 1995; 220: 186-193Crossref PubMed Scopus (713) Google Scholar, 16Vaziri H. West M.D. Allsopp R.C. Davison T.S., Wu, Y. Arrowsmith C.H. Poirier G.G. Benchimol S. EMBO J. 1997; 16: 6018-6033Crossref PubMed Scopus (332) Google Scholar). Accelerated telomere shortening was also observed after stress with SIPS-inducing concentrations of H2O2(17von Zglinicki T. Pilger R. Sitte N. Free Radic. Biol. Med. 2000; 28: 64-74Crossref PubMed Scopus (434) Google Scholar). It has been suggested that induction of the p53-dependent cell cycle arrest via generation of nonspecific as well as telomere-specific DNA damage is the trigger of SIPS (7Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (523) Google Scholar). Chen et al. (18Chen Q.M. Prowse K.R., Tu, V.C. Purdom S. Linskens M.H. Exp. Cell Res. 2001; 265: 294-303Crossref PubMed Scopus (142) Google Scholar), however, did not observe telomere shortening in the cells undergoing SIPS after H2O2 treatment. It has also been shown that Ras-induced senescence, which represents another type of premature senescence, is telomere-independent and cannot be rescued by overexpression of hTERT (19Morales C.P. Holt I. Ouellette M. Kaur K.J. Yan Y. Wilson K.S. White M.A. Wright W.E. Shay J.W. Nat. Genet. 1999; 21: 115-118Crossref PubMed Scopus (681) Google Scholar, 20Wei S. Wei S. Sedivy J.M. Cancer Res. 1999; 59: 1539-1543PubMed Google Scholar). However, expression of oncogenic Ras triggers senescence via the mitogen-activated protein kinase pathway (21Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (765) Google Scholar), which is likely to be a pathway distinct from DNA damage-induced senescence. We therefore designed an experiment to test the hypothesis that accumulation of DNA breaks in telomeres and rapid telomere shortening was the primary cause of SIPS. We also aimed to test whether nonspecific DNA damage could cause senescence or whether the senescent phenotype was strictly telomere-dependent. We compared the induction of SIPS by UV and γ-irradiation and H2O2 in four different lines of normal human fibroblasts with and without hTERT. Our predictions were that if SIPS was triggered by telomere shortening, the presence of telomerase activity should render the cells more resistant to SIPS. On the other hand, if SIPS could be caused by nonspecific DNA damage, then overexpression of hTERT would have no effect. We observed no difference in the induction of SIPS between fibroblasts that did or did not express hTERT, as monitored by morphological changes, growth arrest, and SA-β-gal activity. This result suggests that SIPS is triggered by nonspecific DNA damage and most likely is telomere-independent. We also tested whether SIPS required p21 by inducing SIPS in p21−/− human fibroblasts. p21 accumulates in cells entering replicative senescence, and it has been demonstrated that p21−/− fibroblasts do not undergo replicative senescence (22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar). We observed that SIPS was attenuated in p21−/− cells, suggesting that SIPS and replicative senescence share some common pathways. However, the inhibition of SIPS in p21−/− cells was ∼50%, in contrast to replicative senescence, which is completely blocked in these cells. We have also found that hTERT-expressing cells were more resistant to apoptosis and necrosis induced by UV and γ-irradiation. A protective effect of telomerase on induction of apoptosis was recently reported (23Fu W. Begley J.G. Killen M.W. Mattson M.P. J. Biol. Chem. 1999; 274: 7264-7271Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 24Mattson M.P., Fu, W. Zhang P. Mech. Ageing Dev. 2001; 122: 659-671Crossref PubMed Scopus (55) Google Scholar, 25Ren J.G. Xia H.L. Tian Y.M. Just T. Cai G.P. Dai Y.R. FEBS Lett. 2001; 488: 133-138Crossref PubMed Scopus (70) Google Scholar, 26Xiang H. Wang J. Mao Y.W. Li D.W. Biochem. Biophys. Res. Commun. 2000; 278: 503-510Crossref PubMed Scopus (47) Google Scholar, 27Yang J. Chang E. Cherry A.M. Bangs C.D. Oei Y. Bodnar A. Bronstein A. Chiu C.P. Herron G.S. J. Biol. Chem. 1999; 274: 26141-26148Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). It has been proposed that telomerase may have additional functions (24Mattson M.P., Fu, W. Zhang P. Mech. Ageing Dev. 2001; 122: 659-671Crossref PubMed Scopus (55) Google Scholar) and may attenuate apoptosis by some interaction with the apoptotic machinery (26Xiang H. Wang J. Mao Y.W. Li D.W. Biochem. Biophys. Res. Commun. 2000; 278: 503-510Crossref PubMed Scopus (47) Google Scholar). We observed the strongest protective effect of hTERT in cells treated with UV and γ-irradiation, known inducers of DNA double-strand breaks, and almost no protection against H2O2 damage, which affects many other cell compartments in addition to DNA. This suggests that the protective mechanism works at the DNA level. We hypothesize that telomerase protects cells from apoptosis and necrosis by a healing process, such as adding telomeric repeats to broken DNA ends. WI-38, IMR-90, and LF1 are normal human lung fibroblasts. WI-38 fibroblasts were provided by J. Smith; IMR-90 cells were from the Coriell Institute for Medical Research, and LF1 fibroblasts were a kind gift from J. Sedivy (see Ref. 22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar). HCA2 human foreskin fibroblasts were isolated in our laboratory. The normal cells were used at population doubling of 34–37, 35–38, 20–23, and 26–30 for WI-38, IMR-90, LF1, and HCA2, respectively. WI-38-hTERT, IMR-90-hTERT, and HCA2-hTERT were a kind gift from J. Campisi. LF1-hTERT, LF1p21−/−, and LFp21−/− hTERT were kindly provided by J. Sedivy (20Wei S. Wei S. Sedivy J.M. Cancer Res. 1999; 59: 1539-1543PubMed Google Scholar, 28Wei W. Hemmer R.M. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 6748-6757Crossref PubMed Scopus (190) Google Scholar). Cells were grown in minimum essential media with Hanks' salts or minimum essential media with Earle's salts supplemented with 10% fetal calf serum, nonessential amino acids, and sodium pyruvate. Cells were seeded at 5 × 105–106cells per 100-mm tissue culture dish, grown for 2–3 days (until the first contacts between the cells became visible, but before confluence), and subjected to the following treatments. Cells were irradiated using Gammacell 1000 (Atomic Energy of Canada, Ltd.) at the dose of 5.5 kilorads (for most treatments), at 1 kilorad per 1.08 min. The culture medium was changed immediately after irradiation. Cells were washed once with phosphate-buffered saline (PBS) and irradiated in PBS using a UVB source with a light meter IL 700 (international light). Immediately after irradiation, PBS was replaced with fresh culture medium. Culture medium containing H2O2 was added to the cells and incubated for 2 h at 37 oC. Then cells were washed once with PBS and a fresh culture medium was added. Ten days after treatment cells were fixed and stained for SA-β-gal as described (1Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley 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 (5707) Google Scholar). Cells were counted under the microscope, and a minimum of 500 cells was counted for each coverslip. The percent β-galactosidase-positive cells from the total number of cells was calculated. Thymidine incorporation assay was performed as described (29Stein G.H. Yanishevsky R. Methods Enzymol. 1979; 58: 279-292Crossref PubMed Scopus (86) Google Scholar). Briefly, cells were grown on coverslips, 3–4 days after treatment, and [3H]thymidine was added to the cells. Cells were then incubated for an additional 36 h and fixed and subjected to autoradiography, and the percentage of cells incorporating tritiated thymidine was determined. Ten days after treatment genomic DNA was extracted from 2 × 106 cells, digested with the mixture of restriction enzymes (AluI, HaeIII, RsaI, andHinfI), and fractionated on 0.8% agarose gel. DNA fragments were transferred to nylon membrane (Hybond-N+) under alkali conditions, according to the manufacturer's instructions. Membranes were hybridized for 12 h at 55 °C with a radiolabeled (TTAGGG)4 probe, washed, and subjected to autoradiography. Cells were collected 48 and 72 h after post-treatment, stained with acridine orange, and analyzed by FACS (Beckman-Coulter, EPICS XL-MCL, using System II version 3.0) as described (30Darzynkiewicz Z. Methods Cell Biol. 1994; 41: 427-541Google Scholar). Apoptotic and necrotic fractions were identified as described (31Seluanov A. Gorbunova V. Falcovitz A. Sigal A. Milyavsky M. Zurer I. Shohat G. Goldfinger N. Rotter V. Mol. Cell. Biol. 2001; 21: 1552-1564Crossref PubMed Scopus (127) Google Scholar), using etoposide and actinomycin-treated young and old cells as standards. The cellular DNA fragmentation enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) was used for the detection of bromodeoxyuridine (BrdUrd)-labeled DNA released from necrotic cells into the cell medium. Briefly, 24 h before stress treatment cells were incubated with BrdUrd. At various time points after stress treatments the supernatants of the cell cultures were collected, and DNA fragments were captured with an anti-DNA antibody and detected by an anti-BrdUrd antibody-peroxidase conjugate according to the manufacturer's instructions. Adherent fibroblasts were harvested and lysed in protein sample buffer, boiled for 10 min, and equal numbers of cells loaded on SDS-PAGE. The proteins were transferred to nitrocellulose membrane using a semidry transfer cell (Bio-Rad). Membranes were hybridized with the following antibodies: anti-p21 (WAF1(Ab1), Oncogene), anti-Rb (Rb Ab1(1F8), LabVision), anti-p53 (pAb1801 Calbiochem), and anti-p16 (p16 Ab1(DCS-50.1/A7) LabVision). Equivalent loading of lanes was verified by hybridization with anti-actin antibodies (Calbiochem). Introduction of telomerase into human fibroblasts allows them to escape replicative senescence (22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar). To test whether telomerase could protect the cells from SIPS, we compared the induction of SIPS in normal human fibroblasts cell lines with and without telomerase. We have used four lines of fibroblasts that have been frequently used to study senescence: fetal lung fibroblasts IMR-90, WI-38, and LF1 (22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar) and foreskin fibroblasts HCA2. The corresponding lines (IMR90-TERT, WI-38-TERT, LF1-TERT, and HCA2-TERT) with telomerase activity were obtained from the parental lines by infection with a retroviral vector containing an hTERT expression cassette. Cells were grown for at least 70 population doublings after infection to confirm the immortalized phenotype. Telomerase activity in the immortalized lines was tested by the telomeric repeat amplification protocol assay (32Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. W. S.J. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6539) Google Scholar). All the four immortalized lines had high telomerase activity (Fig.1). In order to induce premature senescence cells were treated with UV and γ-irradiation or H2O2. All these treatments have been reported to induce SIPS in human fibroblasts (7Toussaint O. Medrano E.E. von Zglinicki T. Exp. Gerontol. 2000; 35: 927-945Crossref PubMed Scopus (523) Google Scholar). We have found that doses of 5.5 kilorads for γ-irradiation, 2 J/m2 for UV irradiation, and 500 μmH2O2 are optimal for induction of SIPS by testing a wide range of doses (data not shown). The above doses irreversibly arrest cell division in more than 90% of the surviving cells. Three days following the treatment, all cells acquired an enlarged flat morphology typical of senescent cells. No difference was observed between the corresponding cell lines with and without hTERT. To obtain a quantitative comparison of induction of SIPS, we used SA-β-gal staining and measured the number of cells that synthesized DNA by tritiated thymidine incorporation. All the treatments induced SA-β-gal activity in more than 70% of the cells (Fig.2 A). There was some variability between the individual cell lines but no difference between the normal and hTERT expressing cells of the same line. Thymidine incorporation was strongly inhibited in all the cell lines, independent of telomerase activity (Fig. 2 B). We then tested whether telomerase activity was still present after the stress treatments. To this end cells from two telomerase positive cell lines IMR-90+hTERT and LF1+hTERT were analyzed by TRAP assay (32Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. W. S.J. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6539) Google Scholar) 24 h after stress. The results (Fig.3) indicate that telomerase activity is slightly reduced following stress. This small change in telomerase activity cannot account for induction of senescence, because a high level of telomerase activity was still present in these cells. For example, the telomerase activity in γ-irradiated and H2O2-treated LF1+hTERT cells was as high as that in untreated IMR-90+hTERT cells. We also determined telomere length in IMR-90, IMR-90+hTERT, LF1, and LF1+hTERT cell lines by Southern blot analysis 10 days after stress treatments (Fig.4). All the cell lines had long telomeres characteristic of young cells, and importantly, no telomere shortening was observed in the cells undergoing SIPS compared with control cells, suggesting that SIPS was not caused by rapid telomere shortening.Figure 4Telomere length in normal and hTERT-immortalized fibroblast cell lines after stress treatments.Genomic DNA was extracted from cells 10 days after stress treatments and analyzed by Southern blot as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also compared the induction of p21 and the p53 and Rb status in the wild type and hTERT-expressing cells undergoing SIPS. Adherent cells were collected 48 h after treatment and analyzed by Western blot. p21 levels were elevated in all the cells undergoing SIPS (Fig.5 A), and there was no difference in the level of p21 induction between the parental and hTERT-expressing cell lines. Rb protein levels were decreased, and Rb was mainly in the unphosphorylated state in the cells treated with γ-irradiation and H2O2 (Fig. 5 B). Similar to results with p21 induction, there was no difference in the Rb status between the cells with or without telomerase. The level of p53 was strongly increased after UV treatment (Fig. 5 C), and a similar level of p53 induction was observed between the cells with or without telomerase. The level of p16 (Fig. 5 D) was not up-regulated in the cells undergoing SIPS. Our results demonstrate that there is no difference in the induction of SIPS between the cells with or without telomerase activity, as assayed by SA-β-gal staining, thymidine incorporation, and the expression pattern of the major proteins regulating cell cycle arrest. p21 plays a key role in the regulation of replicative senescence, and human fibroblasts with the knock out of p21 escape replicative senescence (22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar). In order to investigate the role of p21 in SIPS, we examined the induction of SIPS in p21−/− human fibroblasts. We have used p21−/− cells that were derived from LF1 normal human fibroblasts by double knock out (22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar). LF1, LFp21−/−, and LF1p21−/−hTERT cells were subjected to UV, γ-irradiation, and H2O2 as described above. Following the various treatments, some of the p21−/− cells became enlarged and elongated but not as flattened as senescent wild type LF1 cells. Induction of SA-β-gal activity (Fig.6 A) and inhibition of DNA synthesis (Fig. 6 B) in p21−/− cells were also reduced ∼2-fold compared with the parental LF1 strain. H2O2 and γ-irradiation stress caused a decrease in levels of Rb in p21−/− cells, similar to that seen in wild type cells (Fig. 7 A). Therefore, p21−/− cells under stress displayed an intermediate phenotype with some but not all the features of senescent cells. The level of p16 protein was elevated in both treated and untreated p21−/− fibroblasts (Fig. 7 B), which has been observed previously (22Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar, 28Wei W. Hemmer R.M. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 6748-6757Crossref PubMed Scopus (190) Google Scholar). Our results indicate that p21 is involved in SIPS in human fibroblasts; however, it is not absolutely required, and there are alternative pathways for induction of SIPS.Figure 7Status of Rb and p16 in p21−/− human fibroblasts during stress-induced senescence. Cells were subjected to stress with 2 J/m2 UVB and 5.5 kilorads of γ-irradiation or treated with 500 mmH2O2 for 2 h. Adherent cells were collected 48 h after stress, and equal numbers of cells (105 for Rb and 5 × 105 for p16) were used for Western blot analysis with the antibodies against Rb (A) and p16 (B). The membranes were then hybridized with anti-actin antibodies (C) to demonstrate equal loading of samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although we did not detect any difference in the stress-induced senescence between wild type normal cells and cells with introduced telomerase, microscopic examination revealed some difference in survival following stress. Cells expressing hTERT showed better survival after treatment with UV and γ-irradiation. In order to determine what type of cell death is rescued by telomerase, we analyzed the induction of apoptosis and necrosis. Apoptosis and necrosis were assayed by acridine orange staining followed by FACS analysis (Figs.8 and 9). This method is very sensitive and allows one to differentiate between apoptosis and necrosis in the same sample (31Seluanov A. Gorbunova V. Falcovitz A. Sigal A. Milyavsky M. Zurer I. Shohat G. Goldfinger N. Rotter V. Mol. Cell. Biol. 2001; 21: 1552-1564Crossref PubMed Scopus (127) Google Scholar) (Fig. 8). In addition, induction of necrosis was confirmed by measuring the release of DNA into the media by necrotic cells (Fig.10).Figure 9Induction of apoptosis and necrosis in normal and hTERT-immortalized fibroblasts following stress. Cells were subjected to 2 J/m2 UVB (A) and 5.5 kilorads of γ-irradiation (B) or treated with 500 mmH2O2 for 2 h (C). 48 and 72 h after stress, the cells were stained with acridine orange and analyzed by FACS, which allowed the measurement of both apoptosis and necrosis. All the experiments were repeated at leas three times, and standard errors are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Induction of necrosis in normal and hTERT-immortalized fibroblast cell lines after stress treatments.Necrosis was analyzed by the release of DNA from necrotic cells into the medium, and DNA was quantified with a cellular DNA fragmentation enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) as described under "Experimental Procedures." The percentage of DNA released from the total DNA is shown. Experiments were repeated three times, and S.D. values are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) UV irradiation induced primarily an apoptotic response (Fig.9 A), and a concomitant accumulation of p53 following treatment (Fig. 5 C). UV is known to be a strong inducer of apoptosis (31Seluanov A. Gorbunova V. Falcovitz A. Sigal A. Milyavsky M. Zurer I. Shohat G. Goldfinger N. Rotter V. Mol. Cell. Biol. 2001; 21: 1552-1564Crossref PubMed Scopus (127) Google Scholar, 33Norbury C.J. Hickson I.D. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 367-401Crossref PubMed Scopus (415) Google Scholar), and we have here detected a low level of necrosis. Although there was a significant variation between the four cell types in their resistance to stress, and the ratio between apoptosis and necrosis, expression of hTERT reduced cell death, both apoptotic and necrotic, in all the cell lines. γ-Irradiation induced a very low level of apoptosis (Fig.9 B), consistent with a previous report (8Di Leonardo A. Linke S.P. Clarkin K. Wahl G.M. Genes Dev. 1994; 8: 2540-2551Crossref PubMed Scopus (1025) Google Scholar) on the lack of γ-irradiation-induced apoptosis in fibroblasts. The major type of cell death that we observed was necrosis (Figs. 9 C and 10). To our knowledge, this is the first demonstration of γ-irradiation-induced necrosis in fibroblasts. As was the case with UV treatment, cells containing telomerase activity were significantly more resistant to both types of cell death after γ-irradiation. Induction of necrosis was observed after H2O2treatment, with no difference between the cells with or without telomerase (Figs. 9 C and 10). The differences in resistance to H2O2 between the different cell lines had a pattern similar to UV resistance, with LF1 and HCA2 being more resistant to stress than WI-38 and IMR-90. Necrosis has been reported previously to occur at the higher doses of H2O2(more that 5 mm); however, our method of detection of necrosis is much more sensitive than the methods used in earlier studies (55Teramoto S.T.T. Matsui H. Ohga E. Matsuse T. Ouchi Y. Jpn. J. Pharmacol. 1999; 79: 33-40Crossref PubMed Scopus (116) Google Scholar). In summary, telomerase activity protected cells from apoptosis and necrosis induced by UV and γ-irradiation but not by H2O2. We have demonstrated that expression of catalytically active telomerase does not prevent stress-induced senescence. This was observed in different cell lines of normal human fibroblasts following UV and γ-irradiation and H2O2 treatments. This allows us to rule out cell line-specific variations, which frequently cause discrepancies between different studies. Cells undergoing SIPS displayed the characteristics of replicative senescence such as SA-β-gal activity, lack of
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