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Reversible Manipulation of Telomerase Expression and Telomere Length

2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês

10.1074/jbc.m203747200

1083-351X

Miguel A. Rubio, Sahn-Ho Kim, Judith Campisi,

Genetics, Aging, and Longevity in Model Organisms

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERTflanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, afterhTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence. Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERTflanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, afterhTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence. population doublings ionizing radiation telomerase repeat amplification protocol terminal restriction fragment reverse transcription cytomegalovirus labeling index hygromycin l-histidol puromycin Most eukaryotic cells do not divide indefinitely owing to a process termed replicative senescence. Replicative senescence was formally described more than four decades ago for cultures of normal human fibroblasts (1Hayflick L. Moorhead P.S. Exp. Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5589) Google Scholar). Since that time, many cell types from many animal species have been shown to undergo replicative senescence, both in culture and in vivo (reviewed in Refs. 2Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (158) Google Scholar and 3Campisi J. Dimri G.P. Hara E. Schneider E. Rowe J. Handbook of the Biology of Aging. Fourth Ed. Academic Press, New York1996: 121-149Google Scholar). Recent data suggest that, at least in mammalian cells, replicative senescence is an example of a more general process, termed cellular senescence. Cellular senescence arrests cell proliferation (used here interchangeably with growth) in response to insults that have the potential to cause neoplastic transformation. These and other findings suggest that cellular senescence is important for suppressing tumorigenesis in organisms with renewable tissues (4Smith J.R. Pereira-Smith O.M. Science. 1996; 273: 63-67Crossref PubMed Scopus (471) Google Scholar, 5Campisi J. Trends Cell Biol. 2001; 11: 27-31Abstract Full Text PDF PubMed Scopus (424) Google Scholar). Cellular senescence has also been proposed to cause or contribute to aging in selected mammalian tissues (4Smith J.R. Pereira-Smith O.M. Science. 1996; 273: 63-67Crossref PubMed Scopus (471) Google Scholar, 6Campisi J. Cell. 1996; 84: 497-500Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 7Campisi J. In Vivo. 2000; 14: 183-188PubMed Google Scholar). Cellular senescence entails an essentially irreversible arrest of cell growth. In replicative senescence, this growth arrest occurs as a consequence of cell division. The number of divisions that normal cells complete before they senesce depends on the cell type and the species, age, and genotype of the donor (2Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (158) Google Scholar, 3Campisi J. Dimri G.P. Hara E. Schneider E. Rowe J. Handbook of the Biology of Aging. Fourth Ed. Academic Press, New York1996: 121-149Google Scholar). This number can be large; for example, >80 population doublings (PD)1 for fetal or neonatal human fibroblast cultures. In the last decade, it has become clear that human cells sense the number of divisions they have completed through the telomeres, which shorten progressively with each cell cycle owing to the inability of DNA polymerases to replicate the ends of linear chromosomes. Telomeres are the repetitive DNA sequence and associated proteins that cap the ends of linear chromosomes. Telomeres allow cells to distinguish chromosome ends from double-strand DNA breaks and are essential for chromosome stability (8Blackburn E.H. Nat. Struct. Biol. 2000; 7: 847-850Crossref PubMed Scopus (135) Google Scholar). Because the DNA replication machinery cannot completely replicate termini, 50–200 bp of telomeric DNA are lost during each S phase. This loss can be prevented by telomerase, the enzyme that adds telomeric DNA to termini de novo. Most human cells do not express telomerase, and thus their telomeres shorten with each division (9Levy M.Z. Allsopp R.C. Futcher A.B. Greider C.W. Harley C.B. J. Mol. Biol. 1992; 225: 951-960Crossref PubMed Scopus (897) Google Scholar). Telomere shortening was first demonstrated in cultured human fibroblasts, which completely senesced with an average telomere length of 4–7 kb (reduced from 10–15 kb in the germ line) (10Harley C.B. Futcher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4665) Google Scholar). These findings led to the hypothesis that human cells undergo a senescence growth arrest when their telomeres reach a critically short length (11Chiu C.P. Harley C.B. Proc. Soc. Exp. Biol. Med. 1997; 214: 99-106Crossref PubMed Scopus (175) Google Scholar). This hypothesis was strengthened by subsequent studies showing that ectopic expression of hTERT, the rate-limiting and catalytic subunit of telomerase, can prevent telomere erosion and replicative senescence (12Bodnar 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 (4147) Google Scholar, 13Vaziri H. Benchimol S. Curr. Biol. 1998; 8: 279-282Abstract Full Text Full Text PDF PubMed Scopus (867) Google Scholar). Recent findings have refined, and added complexity to, this hypothesis. First, telomere-associated proteins have been identified that regulate telomere length indirectly. Some of these proteins appear to alter the telomeric structure and hence the ability of telomerase to access the telomere (14Evans S.K. Lundblad V. J. Cell Sci. 2000; 113: 3357-3364Crossref PubMed Google Scholar, 15McEachern M.J. Krauskopf A. Blackburn E.H. Annu. Rev. Genet. 2000; 34: 331-358Crossref PubMed Scopus (612) Google Scholar, 16Gasser S. Science. 2000; 288: 1377-1379Crossref PubMed Scopus (61) Google Scholar, 17Campisi J. Kim S. Lim C. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Crossref PubMed Scopus (311) Google Scholar). One such protein is TIN2. TIN2 negatively regulates telomere length in a telomerase-dependent fashion but does not act directly on the enzyme (18Kim S.H. Kaminker P. Campisi J. Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (429) Google Scholar). Thus, telomerase expression alone may not be sufficient to prevent replicative senescence. Second, short telomeres may be more prone than long telomeres to structural dysfunction, and telomere function, rather than length, may control cellular senescence. Indeed, human fibroblasts that express ectopic telomerase can proliferate indefinitely with decidedly subsenescent telomere lengths (19Ouellette M.M. Liao M. Herbert B.S. Johnson M. Holt S.E. Liss H.S. Shay J.W. Wright W.E. J. Biol. Chem. 2000; 275: 10072-10076Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Likewise, in telomerase-deficient mice, the shortest telomeres appear to be responsible for the telomere dysfunction that compromises cellular and organismal survival (20Hemann M.T. Strong M.A. Hao L.Y. Greider C.W. Cell. 2001; 107: 67-77Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). These findings suggest that telomerase can prevent cellular senescence by preferentially capping and acting on the shortest telomeres and that the senescence response is not induced by telomere length but, rather, by a dysfunctional telomere structure (21Blackburn E.H. Nature. 2000; 408: 53-56Crossref PubMed Scopus (1104) Google Scholar, 22Blackburn E.H. Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1772) Google Scholar). Third, both replicative and cellular senescence induce similar phenotypes, but cellular senescence can occur after very few doublings and in response to stimuli that likely act independently of telomeres (17Campisi J. Kim S. Lim C. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Crossref PubMed Scopus (311) Google Scholar, 23Morales C.P. Holt S.E. 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 (683) Google Scholar, 24Serrano M. Blasco M.A. Curr. Opin. Cell Biol. 2001; 13: 748-753Crossref PubMed Scopus (350) Google Scholar, 25Wei S. Wei S. Sedivy J.M. Cancer Res. 1999; 59: 1539-1543PubMed Google Scholar, 26von Zglinicki T. Saretzki G. Döcke W. Lotze C. Exp. Cell Res. 1995; 220: 186-193Crossref PubMed Scopus (715) Google Scholar). Some of these stimuli, however, may damage telomeres. For example, human fibroblasts cultured under hyperoxia senesce very rapidly but accumulate single-strand breaks at the telomeres (26von Zglinicki T. Saretzki G. Döcke W. Lotze C. Exp. Cell Res. 1995; 220: 186-193Crossref PubMed Scopus (715) Google Scholar). Conversely, telomeres may influence the sensitivity of cells and organisms to DNA-damaging agents such as ionizing radiation (IR). Thus, there was an inverse correlation between telomere length and chromosomal radiosensitivity in the lymphocytes of some breast cancer patients (27McIlrath J. Bouffler S.D. Samper E. Cuthbert A. Wojcik A. Szumiel I. Bryant P.E. Riches A.C. Thompson A. Blasco M.A. Newbold R.F. Slijepcevic P. Cancer Res. 2001; 61: 912-915PubMed Google Scholar), and short telomeres enhanced IR-induced lethality in telomerase-deficient mice (28Goytisolo F.A. Samper E. Martin-Caballero J. Finnon P. Herrera E. Flores J.M. Bouffler S.D. Blasco M.A. J. Exp. Med. 2000; 192: 1625-1636Crossref PubMed Scopus (205) Google Scholar, 29Wong K.K. Chang S. Weller S.R. Ganesau S. Chaudhuri J. Zhu C. Artandi S.E. Rudolph K.L. Gottlieb G.J. Chin L. Alt F.W. DePinho R.A. Nat. Genet. 2000; 26: 85-88Crossref PubMed Scopus (286) Google Scholar). In addition, cells from organisms with defects in telomere maintenance are frequently radiosensitive (30Metcalfe J.A. Parkhill J. Campbell L. Stacey M. Biggs P. Byrd P.J. Taylor A.M. Nat. Genet. 1996; 13: 350-353Crossref PubMed Scopus (297) Google Scholar, 31Hande P. Slijepcevic P. Silver A. Bouffler S. van Buul P. Bryant P. Lansdorp P. Genomics. 1999; 56: 221-223Crossref PubMed Scopus (81) Google Scholar, 32Samper E. Goytisolo F.A. Slijepcevic P. van Buul P.P. Blasco M.A. EMBO Rep. 2000; 1: 244-252Crossref PubMed Scopus (285) Google Scholar). Moreover, telomerase has been reported to protect some cells from the lethality that results from severe damage to telomeres (33Lu C. Fu W. Mattson M.P. Brain Res. Dev. Brain Res. 2001; 131: 167-171Crossref PubMed Scopus (56) Google Scholar, 34Ludwig A. Saretzki G. Holm P.S. Tiemann F. Lorenz M. Emrich T. Harley C.B. von Zglinicki T. Cancer Res. 2001; 61: 3053-3061PubMed Google Scholar, 35Ren 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). The above findings cast doubt on the relevance of telomere lengthper se in signaling the replicative senescence of human cells. They also suggest that telomeres can act as sensors or transducers of DNA damage signals and that telomerase can repair or protect cells from telomeric damage. One caveat to these possibilities is that cell types may differ in their responses to telomere dysfunction and/or DNA damage. Moreover, given the significant differences in telomere biology between rodents and humans (36Weng N.P. Hodes R.J. J. Clin. Immunol. 2000; 20: 257-267Crossref PubMed Google Scholar, 37Wright W.E. Shay J.W. Nat. Med. 2000; 6: 849-851Crossref PubMed Scopus (334) Google Scholar), it may not be possible to extrapolate findings in rodent cells to human cells. To explore the relationships between telomere length, replicative senescence, and IR sensitivity, we created essentially isogenic human cell populations that have varying average telomere lengths. We started with normal human fibroblasts and used genetically defined manipulations. We show that telomere length does indeed influence replicative senescence but that human fibroblasts can senesce with telomeres that are significantly shorter or longer than expected. Our results support the idea that telomerase acts preferentially on short telomeres and that telomere structure, rather than length, causes replicative senescence. Finally, our results suggest that telomere length, but not telomerase, influences the response of human cells to IR. Normal human fibroblast strains 82-6 and BJ (HCA2) were obtained from J. Oshima (University of Washington, Seattle) and J. Smith (University of Texas, San Antonio), respectively. Cells were cultured in Dulbecco's modified Eagle's medium and 10% fetal calf serum, as described previously (38Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O.M. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5788) Google Scholar), until they reached 70–80% confluence, whereupon they were subcultured at 1–3 × 103/cm2. To determine the fraction of senescent cells, cells were plated at 103/cm2 and labeled with [3H]thymidine for 72 h; the percentage of labeled nuclei was determined by autoradiography and/or the cells were stained for the senescence-associated β-galactosidase, as described previously (38Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O.M. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5788) Google Scholar). The labeling index (LI) refers to the percentage of cells that incorporated [3H]thymidine during the 72-h labeling interval. Cultures were considered completely senescent when the LI reached 1 mm in diameter were counted. We designed a retroviral-based system to allow conditional expression of telomerase and selection for cells that received or lost telomerase (Fig. 1). The system is based on the well-characterized ability of Cre recombinase to site-specifically excise DNA sequences that are flanked byloxP sites (44Sternberg N. Hamilton D. J. Mol. Biol. 1981; 150: 467-486Crossref PubMed Scopus (511) Google Scholar). The starting vector was pBABE-PURO (40Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1903) Google Scholar), into which we introduced three modifications. First, we cloned into the multiple cloning site the cDNA encoding the catalytic subunit of human telomerase (hTERT) (41Counter C.M. Hahn W.C. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Crossref PubMed Scopus (564) Google Scholar). Second, we introduced a histidinol resistance gene (HISD) from pLXSHD (39Miller A.D. Rosman G.J. Biotechniques. 1989; 7: 980-988PubMed Google Scholar) between the SV40 early promoter (SV40p) and puromycin resistance gene (PURO). This vector was designated pBTSH. Third, we introduced loxP sites upstream of the hTERT cDNA and between the HISD and PURO genes. This vector was designated pBloxTSH. Thus, pBTSH and pBloxTSH were identical except for the presence of loxP sites. We also used pLXSH (39Miller A.D. Rosman G.J. Biotechniques. 1989; 7: 980-988PubMed Google Scholar) to create a retrovirus (pLCRESH) carrying Cre and the hygromycin resistance gene (HYGRO). We expected normal telomerase-negative cells infected with either pBTSH or pBloxTSH to acquire telomerase activity and histidinol resistance. After superinfection with pLCRESH, we expected pBloxTSH-infected cells to acquire hygromycin resistance but lose telomerase activity and histidinol sensitivity, owing to Cre-mediated excision of the hTERT and HISD genes. Simultaneously, these cells should acquire puromycin resistance because the excision brings the PURO gene under the control of the retroviral 5′ promoter (long terminal repeat). pBTSH-infected cells, by contrast, were predicted to acquire hygromycin resistance but no other changes after superinfection with pLCRESH (Fig. 1). We infected normal human fibroblasts with these retroviruses, using a protocol that produced 80–90% infected cells. Thus, the manipulations we describe were applied to mass cultures, obviating the need for single cell cloning. We infected early passage (PD 20, LI = 74%) normal human fibroblasts (strain 82-6) with pLXSHD, pBTSH, or pBloxTSH and assessed uninfected and infected populations for their ability to survive and proliferate in the presence of l-histidinol. As expected, uninfected cells were histidinol-sensitive, whereas the infected cells were histidinol-resistant (Table I). We also assessed these populations for telomerase activity, using a telomerase repeat amplification protocol (TRAP). After histidinol selection, cells infected with the hTERT-expressing viruses, either pBloxTSH or pBTSH, showed robust telomerase activity (Table I; see also Fig.3 A below). Uninfected cells, and cells infected with pLXSHD, remained telomerase-negative (Table I).Table IPhenotypes of infected human fibroblastsVirus 1Plasmid1-aCMV-based vectors. or virus 2HISD1-bS, sensitive; R, resistant.PUROHYGROTRAPSenescencePD1-cNumber of PD, after the last infection, at which senescence occurred.SSS−+20–25pLXSHDRSS−+20pBTSHRSS+−pBloxTSHRSS+−pBTSHpLXSHRSR+−pBTSHpLCRESHRSR+−pBloxTSHpLXSHRSR+−pBloxTSHpLCRESHSRR−+42pBloxTSHpCMV-1RSS+−pBloxTSHpCMV-CRESRS−+>301-dApproximation. PD can only be estimated because transfection efficiency is low (<10%).1-eTwo out of three transfection experiments.pBloxTSHpCMV-CRES/RRS−/+1-fOne out of three transfection experiments.−1-fOne out of three transfection experiments.82–6 human fibroblasts were either mock infected (−) or infected with the indicated retroviruses. Cells were assessed for sensitivity tol-histidinol (HISD), puromycin (PURO) and hygromycin (HYGRO), telomerase activity (TRAP), and population doubling (PD) level at replicative senescence, as described in Experimental Procedures.1-a CMV-based vectors.1-b S, sensitive; R, resistant.1-c Number of PD, after the last infection, at which senescence occurred.1-d Approximation. PD can only be estimated because transfection efficiency is low ( 100 PD and appear to be replicatively immortal. We superinfected pBloxTSH- and pBTSH-infected cells with pLCRESH or pLXSH (control), both of which conferred hygromycin resistance, as expected (Table I). After selection in hygromycin, the cells were retested for histidinol sensitivity and telomerase and tested for puromycin sensitivity. As expected, pLXSH did not alter the histidinol or puromycin sensitivity, that is, pBloxTSH- and pBTSH-infected cells remained histidinol-resistant and puromycin-sensitive. In addition, pLCRESH did not alter the histidinol and puromycin response of pBTSH-infected cells. However, pLCRESH completely reversed the antibiotic response of pBloxTSH-infected cells, abolishing resistance to histidinol and conferring puromycin resistance (Table I). Moreover, only pBloxTSH + pLCRESH doubly infected cells lost telomerase activity (Table I and Fig. 3 A). This loss appeared to be complete, because lysates from these cells had no detectable TRAP activity, even when a 10-fold greater number of cells was assayed (Fig.3 A). Moreover, loss of telomerase activity resulted in progressive telomere shortening (see Fig. 4). Most important, pBloxTSH + pLCRESH doubly infected cells, but none of the other populations, underwent replicative senescence (Fig. 3 B). The cells senesced 42 PD after hygromycin selection (Table I). Thus, after expression and excision of hTERT, cells underwent an additional 20 PD before senescing. Consistent with this additional proliferative potential, the cells senesced with shorter telomeres, compared with those of senescent cells that never expressed hTERT (Fig. 4). Twenty-one PD after hTERT was excised from pBloxTSH cells by pLCRESH, seven cell clones were isolated. All the clones underwent senescence after an additional 15–18 PD (data not shown), consistent with the results obtained with the mass-infected population. These results indicate that all the retroviruses conferred the predicted phenotypes on cells. Moreover, they demonstrate that sequential infection with pBloxTSH and pLCRESH can efficiently and reversibly immortalize uncloned populations of normal human fibroblasts. We observed no deleterious effects owing to stable expression of Cre. However, Cre was reported to be mildly to moderately toxic in some cells (45Loonstra A. Vooijs M. Beverloo H.B. Allak B.A. van Drunen E. Kanaar R. Berns A. Jonkers J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9209-9214Crossref PubMed Scopus (458) Google Scholar). We therefore constructed a CRE expression vector (pCMV-CRE) suitable for transient transfection and expression. We then transfected pBloxTSH-infected fibroblasts with control (pCMV-1) or expression (pCMV-CRE) vector DNA, using a protocol by which about 10% of the cells transiently take up and express the transfected DNA. Cultures that received the control vector did not survive puromycin selection, but numerous puromycin-resistant cells grew out of cultures that received pCMV-CRE (Table I). After transfection and puromycin selection, 103 cells were replated. In two experiments, these cultures of pooled puromycin-resista