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W2053556250 abstract "Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250–400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres. Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250–400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres. mortality stage 1 mortality stage 2 the catalytic component of human telomerase telomere restriction fragment kilobase pair(s) polymerase chain reaction telomere repeat amplification protocol Normal human fibroblasts have a limited ability to proliferate in culture (1.Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4229) Google Scholar, 2.Hayflick L. Moorhead P.S. Exp. Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5381) Google Scholar). The use of conditionally expressed viral oncogenes led to the definition of two separate mechanisms regulating this phenomenon (3.Wright W.E. Pereira-Smith O.M. Shay J.W. Mol. Cell. Biol. 1989; 9: 3088-3092Crossref PubMed Scopus (422) Google Scholar). Mortality stage 1 (M1)1occurs when the functional activation of pathways requiring both p53 and pRB causes the growth arrest associated with cellular senescence (4.Shay J.W. Pereira-Smith O.M. Wright W.E. Exp. Cell Res. 1991; 196: 33-39Crossref PubMed Scopus (612) Google Scholar, 5.Hara E. Tsurui H. Shinozaki A. Nakada S. Oda K. Biochem. Biophys. Res. Commun. 1991; 179: 528-534Crossref PubMed Scopus (238) Google Scholar). Viral oncogenes that bind and inactivate p53 and pRB block M1 and permit continued cell division for an additional 20–40 doublings until an independent blockade to cell proliferation, the M2 mechanism, occurs. The balance of cell division and cell death at M2 (crisis) eventually tips in favor of cell death, so that the culture deteriorates and is generally lost. In human fibroblast cultures, some clones can spontaneously escape M2 and become immortal at a frequency of approximately 10−7 (6.Shay J.W. Wright W.E. Exp. Cell Res. 1989; 184: 109-118Crossref PubMed Scopus (237) Google Scholar).DNA polymerase α cannot replicate the very end of a linear chromosome (7.Olovnikov A.M. J. Theor. Biol. 1973; 41: 181-190Crossref PubMed Scopus (1402) Google Scholar, 8.Watson J.D. Nature. 1972; 239: 197-201Crossref PubMed Scopus (51) Google Scholar), and consequently the compensatory action of telomerase is required to maintain telomere length. Because telomerase is turned off in most human tissues during development (9.Wright W.E. Piatyszek M.A. Rainey W.E. Byrd W. Shay J.W. Dev. Genet. 1996; 18: 173-179Crossref PubMed Scopus (1130) Google Scholar) and cultured human fibroblasts lack telomerase activity (10.Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1921) Google Scholar, 11.Kim 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. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6496) Google Scholar), telomeres shorten progressively with ongoing cell divisions. A causal relationship between telomere shortening and proliferative limits was firmly established by the demonstration that telomere shortening controlled M2 (12.Wright W.E. Brasiskyte D. Piatyszek M.A. Shay J.W. EMBO J. 1996; 15: 1734-1741Crossref PubMed Scopus (142) Google Scholar). Telomerase was repressed in hybrids between normal young fibroblasts with long telomeres and SV40 T-antigen immortalized fibroblasts whose telomeres had been experimentally manipulated to an average size of either 2.5 or 5 kb. The 20 extra population doublings obtained in the hybrids with the 5-kb starting telomere length established that telomere length was the limiting factor (12.Wright W.E. Brasiskyte D. Piatyszek M.A. Shay J.W. EMBO J. 1996; 15: 1734-1741Crossref PubMed Scopus (142) Google Scholar). Since T-antigen would have blocked the M1 mechanism in these hybrids, these results showed that telomere shortening controlled the onset of the M2 mechanism. The demonstration that inhibiting telomerase activity by antisense inhibition of the integral RNA component of telomerase caused proliferative failure in HeLa cells also suggested a causal relationship between telomere shortening and M2 (13.Feng J. Funk W.D. Wang S.S. Weinrich S.L. Avilion A.A. Chiu C.P. Adams R.R. Chang E. Allsopp R.C., Yu, J. Le S. West M.D. Harley C.B. Andrews W.H. Greider C.W. Villeponteau B. Science. 1995; 269: 1236-1239Crossref PubMed Scopus (2060) Google Scholar). These conclusions were recently further extended by the observation that expressing an exogenous telomerase in cells infected with the viral oncoproteins that inactivate p53 and pRB prevented the occurrence of the M2 mechanism (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar, 15.Counter C.M. Hahn W.C. Wei W. 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 (558) Google Scholar, 16.Halvorsen T.L. Leibowitz G. Levine F. Mol. Cell. Biol. 1999; 19: 1864-1870Crossref PubMed Scopus (146) Google Scholar).The ability of an exogenous telomerase to extend the lifespan of normal human diploid cells (17.Vaziri H. Benchimol S. Curr. Biol. 1998; 8: 279-282Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar, 18.Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chui C.-P. Morin G.B. Harley C.B. Shay J.W. Lichsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar) established that telomere shortening also controlled the onset of the M1 mechanism of cellular senescence. The current synthesis of the relationship of telomere shortening to M1 and M2 is that the repression of telomerase results in telomere shortening until the M1 mechanism occurs. There are two current hypotheses for the induction of M1. 1) One or a few of the 92 telomeres in a normal cell have shortened sufficiently so that their ends are no longer masked and they generate a DNA damage signal (19.Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1081) Google Scholar), and 2) there might be regulatory loci in subtelomeric regions that are silenced when telomeres are long but which are able to be expressed upon sufficient telomere shortening (20.Wright W.E. Shay J.W. Trends Genet. 1992; 8: 193-197Abstract Full Text PDF PubMed Scopus (237) Google Scholar). If the consequent growth arrest is circumvented by blocking the actions of p53 and pRB/p16, cells can continue to proliferate and telomeres continue to shorten until they become so short that they are no longer hidden from the DNA repair apparatus and end-to-end fusions result in the M2 mechanism in which apoptosis balances cell division. Cells then escape the M2 mechanism only if they develop a method for maintaining telomeres, either through the derepression of telomerase (10.Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1921) Google Scholar) or by the activation of an alternative pathway that probably involves recombination (21.Pluta A.F. Zakian V.A. Nature. 1989; 337: 429-433Crossref PubMed Scopus (139) Google Scholar, 22.Lundblad V. Blackburn E.H. Cell. 1993; 73: 347-360Abstract Full Text PDF PubMed Scopus (788) Google Scholar, 23.Bryan T.M. Englezou A. Gupta J. Bacchetti S. Reddel R.R. EMBO J. 1995; 14: 4240-4248Crossref PubMed Scopus (1094) Google Scholar).In this report, we describe the long term behavior of some of the human fibroblasts originally described as showing an extended lifespan following the introduction of an exogenous hTERT (18.Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chui C.-P. Morin G.B. Harley C.B. Shay J.W. Lichsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar). Our results suggest there may be cis-acting mechanisms to preferentially recruit telomerase to maintain the shortest telomeres under conditions of limiting telomerase activity. The consequent reduction of average telomere length to sizes less than that observed in senescent cells argues against a role for telomere positional effects controlling subtelomeric loci that cause the growth arrest observed at M1. Additional observations suggest that telomerase does not have a capping function on very short telomeres that is independent of its catalytic activity.DISCUSSIONThese results show that normal human fibroblasts expressing a transfected hTERT cDNA gradually showed reduced telomerase activity and decreasing telomere lengths. After 150–300 population doublings, the telomeres stabilized at subsenescent lengths and in some cases have remained at that size for over 150 additional doublings, and thus the cells are still functionally immortal. Analysis of these cells suggests several important interpretations. 1) The observed change in the distribution of telomere sizes implies the presence of cis-acting factors that preferentially recruit telomerase to act on the shortest telomeres; 2) the ability of cells with subsenescent telomere length to proliferate for 20 doublings following the abolition of telomerase activity argues against telomerase having a “capping” function independent of catalytic activity; and 3) the proliferation of normal cells with subsenescent telomere lengths provides evidence against the induction of growth arrest by subtelomeric regulatory loci silenced by long telomeres.The cells used in the present study had been transfected with a plasmid-based hTERT expression vector and showed a progressive decrease in telomerase activity over time. Although the resumption of telomere shortening was thus expected, the stabilization of telomeres at lengths approximately 2–4 kb shorter than that normally observed in senescent cells was surprising. The size of the shortest telomeres was maintained in multiple different clones over many months during which the longest telomeres continued to shorten. Despite the fact that all of the telomeres were sufficiently short to be expected to provide cis-acting signals, under conditions of limiting telomerase activity the shortest telomeres were preferentially maintained. Possible explanations include a more efficient recruitment of telomerase to the shortest telomeres, and loss of cells with the shortest telomeres and selection of the survivors.A very large number of proteins have been found to influence telomere length in yeast (reviewed in Ref. 36.Muniyappa K. Kironmai K.M. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 297-336Crossref PubMed Scopus (25) Google Scholar), and many of them are telomere-binding proteins. The most compelling evidence for cis-regulation of telomere length is for Rap1, where it has been shown that length is controlled by the number of Rap1 binding sites (37.Ray A. Runge K.W. Mol. Cell. Biol. 1999; 19: 31-45Crossref PubMed Scopus (66) Google Scholar, 38.Marcand S. Gilson E. Shore D. Science. 1997; 275: 986-990Crossref PubMed Scopus (424) Google Scholar). Preferential action of telomerase on the shortest telomeres has recently been demonstrated in yeast (39.Marcand S. Brevet V. Gilson E. EMBO J. 1999; 18: 3509-3519Crossref PubMed Scopus (167) Google Scholar). Results using hTRF1, the human orthologue of Rap1, have also implicated it as a cis-acting factor influencing human telomere length control (40.van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1050) Google Scholar). Our results suggest that the cis-acting telomere-binding proteins present in normal human cells are not only able to cause telomerase to act on the telomeres, but do so in a quantitative fashion that preferentially recruits it to the shortest telomeres despite the presumed presence of signals from other very short but nonetheless longer telomeres.An alternate interpretation is that telomerase is randomly acting on all telomeres, and that selection is producing the observed result. Cells in which telomerase acted on long but not short telomeres would become senescent and be lost from the population, while cells in which telomerase acted on short telomeres would continue to divide. When analyzing the entire population, the effect of this selection would be the apparent preservation of short telomere lengths while long telomeres shortened. Experiments in which chromosomes are broken by insertion of a plasmid with telomeric repeats on one end have shown that the telomere on the “healed chromosome” elongates while the length of the endogenous telomeres remain unaffected (41.Sprung C.N. Sabatier L. Murnane J.P. Exp. Cell Res. 1999; 247: 29-37Crossref PubMed Scopus (28) Google Scholar). Under conditions in which telomerase is not limiting, this shows that in human cells telomerase can preferentially be recruited to act on a telomere that is too short. We believe that it is likely that the same mechanisms that recruit telomerase to act on these “too short” healing chromosomes would act to preferentially recruit limiting amounts of telomerase to the shortest chromosomes, and thus prefer recruitment rather than selection as an explanation for these observations.Telomerase has been proposed to perform a capping function on short telomeres that requires catalytic activity (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar). Telomerase activity became undetectable in two clones following the introduction of the D869A hTERT mutant in B14 cells. These cells with very short telomeres divided for 20 additional doublings in the presence of the mutant hTERT before undergoing a growth arrest. The telomere shortening that occurred during these 20 doublings demonstrates that catalytically active telomerase was not present for a significant fraction of time on most of the telomeres. The replacement of wild-type telomerase with the dominant-negative mutant argues against a “capping” role for the telomerase protein on short telomeres that requires catalytic activity but is independent of the actual addition of TTAGGG repeats to the ends of the chromosomes. The ability of limiting amounts of catalytically active telomerase to preferentially maintain the shortest telomeres, so that average size decreases while minimum size does not, provides a sufficient explanation for the presence of subsenescent (this report) or subcrisis (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar) telomere lengths.We have previously proposed that genes regulating cellular senescence might be located in subtelomeric regions, and that their expression might be controlled by changes in telomere positional effects as telomeres shortened (20.Wright W.E. Shay J.W. Trends Genet. 1992; 8: 193-197Abstract Full Text PDF PubMed Scopus (237) Google Scholar). The present result demonstrates that these cultures continue to proliferate vigorously even though telomere sizes decreased to well below their normal lengths at senescence. This provides evidence against the re-expression of previously silenced genes that induce a growth arrest when telomeres become sufficiently short, and favors the hypothesis that it is the generation of a DNA damage signal from an insufficiently long telomere(s) that causes M1 (19.Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1081) Google Scholar). Several previous reports have failed to find evidence of telomere positional effects in vertebrate cells (42.Bayne R.A. Broccoli D. Taggart M.H. Thomson E.J. Farr C.J. Cooke H.J. Hum. Mol. Genet. 1994; 3: 539-546Crossref PubMed Scopus (49) Google Scholar, 43.Sprung C.N. Sabatier L. Murnane J.P. Nucleic Acids Res. 1996; 24: 4336-4340Crossref PubMed Scopus (27) Google Scholar). Although we now think it unlikely that telomere positional effects regulate the onset of M1, we continue to entertain the possibility that telomere shortening might regulate gene expression in ways that permit the counting of cell divisions to be used as a mechanism for timing decades-long processes during the human life span (44.Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5: 293-296Abstract Full Text PDF PubMed Scopus (127) Google Scholar).The concept that short telomeres increase the efficiency with which they recruit telomerase leads to the speculation that very efficient inhibition of telomerase might be required for anti-telomerase cancer therapy to be successful. It also raises the possibility that a combination of interventions inhibiting both the catalytic activity of telomerase as well as its ability to be recruited to telomeres might be much more successful than either alone. It is important to remember that (in contrast to germline cells) adult human somatic cells are not biologically programmed to maintain telomere length, and that the expression and function of an unknown number of accessory factors may have been altered in different somatic cells that have repressed telomerase. We anticipate that the factors that modify telomerase, recruit it to the telomeres, cause it to catalyze the addition of telomeric repeats, and regulate the number of repeats added at one time will show significant variability in levels and efficiencies between different normal cell types, and that this variability will be compounded in cancer cells. The consequences of expressing telomerase in an “inappropriate” biological context, either via an exogenous cDNA or through the mutational inactivation of repressive pathways, are thus likely to be diverse as well. Disentangling these multiple mechanisms should increase our ability to alter telomere length regulation for modifying the time course of replicative aging and in the treatment of cancer. Normal human fibroblasts have a limited ability to proliferate in culture (1.Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4229) Google Scholar, 2.Hayflick L. Moorhead P.S. Exp. Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5381) Google Scholar). The use of conditionally expressed viral oncogenes led to the definition of two separate mechanisms regulating this phenomenon (3.Wright W.E. Pereira-Smith O.M. Shay J.W. Mol. Cell. Biol. 1989; 9: 3088-3092Crossref PubMed Scopus (422) Google Scholar). Mortality stage 1 (M1)1occurs when the functional activation of pathways requiring both p53 and pRB causes the growth arrest associated with cellular senescence (4.Shay J.W. Pereira-Smith O.M. Wright W.E. Exp. Cell Res. 1991; 196: 33-39Crossref PubMed Scopus (612) Google Scholar, 5.Hara E. Tsurui H. Shinozaki A. Nakada S. Oda K. Biochem. Biophys. Res. Commun. 1991; 179: 528-534Crossref PubMed Scopus (238) Google Scholar). Viral oncogenes that bind and inactivate p53 and pRB block M1 and permit continued cell division for an additional 20–40 doublings until an independent blockade to cell proliferation, the M2 mechanism, occurs. The balance of cell division and cell death at M2 (crisis) eventually tips in favor of cell death, so that the culture deteriorates and is generally lost. In human fibroblast cultures, some clones can spontaneously escape M2 and become immortal at a frequency of approximately 10−7 (6.Shay J.W. Wright W.E. Exp. Cell Res. 1989; 184: 109-118Crossref PubMed Scopus (237) Google Scholar). DNA polymerase α cannot replicate the very end of a linear chromosome (7.Olovnikov A.M. J. Theor. Biol. 1973; 41: 181-190Crossref PubMed Scopus (1402) Google Scholar, 8.Watson J.D. Nature. 1972; 239: 197-201Crossref PubMed Scopus (51) Google Scholar), and consequently the compensatory action of telomerase is required to maintain telomere length. Because telomerase is turned off in most human tissues during development (9.Wright W.E. Piatyszek M.A. Rainey W.E. Byrd W. Shay J.W. Dev. Genet. 1996; 18: 173-179Crossref PubMed Scopus (1130) Google Scholar) and cultured human fibroblasts lack telomerase activity (10.Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1921) Google Scholar, 11.Kim 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. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6496) Google Scholar), telomeres shorten progressively with ongoing cell divisions. A causal relationship between telomere shortening and proliferative limits was firmly established by the demonstration that telomere shortening controlled M2 (12.Wright W.E. Brasiskyte D. Piatyszek M.A. Shay J.W. EMBO J. 1996; 15: 1734-1741Crossref PubMed Scopus (142) Google Scholar). Telomerase was repressed in hybrids between normal young fibroblasts with long telomeres and SV40 T-antigen immortalized fibroblasts whose telomeres had been experimentally manipulated to an average size of either 2.5 or 5 kb. The 20 extra population doublings obtained in the hybrids with the 5-kb starting telomere length established that telomere length was the limiting factor (12.Wright W.E. Brasiskyte D. Piatyszek M.A. Shay J.W. EMBO J. 1996; 15: 1734-1741Crossref PubMed Scopus (142) Google Scholar). Since T-antigen would have blocked the M1 mechanism in these hybrids, these results showed that telomere shortening controlled the onset of the M2 mechanism. The demonstration that inhibiting telomerase activity by antisense inhibition of the integral RNA component of telomerase caused proliferative failure in HeLa cells also suggested a causal relationship between telomere shortening and M2 (13.Feng J. Funk W.D. Wang S.S. Weinrich S.L. Avilion A.A. Chiu C.P. Adams R.R. Chang E. Allsopp R.C., Yu, J. Le S. West M.D. Harley C.B. Andrews W.H. Greider C.W. Villeponteau B. Science. 1995; 269: 1236-1239Crossref PubMed Scopus (2060) Google Scholar). These conclusions were recently further extended by the observation that expressing an exogenous telomerase in cells infected with the viral oncoproteins that inactivate p53 and pRB prevented the occurrence of the M2 mechanism (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar, 15.Counter C.M. Hahn W.C. Wei W. 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 (558) Google Scholar, 16.Halvorsen T.L. Leibowitz G. Levine F. Mol. Cell. Biol. 1999; 19: 1864-1870Crossref PubMed Scopus (146) Google Scholar). The ability of an exogenous telomerase to extend the lifespan of normal human diploid cells (17.Vaziri H. Benchimol S. Curr. Biol. 1998; 8: 279-282Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar, 18.Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chui C.-P. Morin G.B. Harley C.B. Shay J.W. Lichsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar) established that telomere shortening also controlled the onset of the M1 mechanism of cellular senescence. The current synthesis of the relationship of telomere shortening to M1 and M2 is that the repression of telomerase results in telomere shortening until the M1 mechanism occurs. There are two current hypotheses for the induction of M1. 1) One or a few of the 92 telomeres in a normal cell have shortened sufficiently so that their ends are no longer masked and they generate a DNA damage signal (19.Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1081) Google Scholar), and 2) there might be regulatory loci in subtelomeric regions that are silenced when telomeres are long but which are able to be expressed upon sufficient telomere shortening (20.Wright W.E. Shay J.W. Trends Genet. 1992; 8: 193-197Abstract Full Text PDF PubMed Scopus (237) Google Scholar). If the consequent growth arrest is circumvented by blocking the actions of p53 and pRB/p16, cells can continue to proliferate and telomeres continue to shorten until they become so short that they are no longer hidden from the DNA repair apparatus and end-to-end fusions result in the M2 mechanism in which apoptosis balances cell division. Cells then escape the M2 mechanism only if they develop a method for maintaining telomeres, either through the derepression of telomerase (10.Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1921) Google Scholar) or by the activation of an alternative pathway that probably involves recombination (21.Pluta A.F. Zakian V.A. Nature. 1989; 337: 429-433Crossref PubMed Scopus (139) Google Scholar, 22.Lundblad V. Blackburn E.H. Cell. 1993; 73: 347-360Abstract Full Text PDF PubMed Scopus (788) Google Scholar, 23.Bryan T.M. Englezou A. Gupta J. Bacchetti S. Reddel R.R. EMBO J. 1995; 14: 4240-4248Crossref PubMed Scopus (1094) Google Scholar). In this report, we describe the long term behavior of some of the human fibroblasts originally described as showing an extended lifespan following the introduction of an exogenous hTERT (18.Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chui C.-P. Morin G.B. Harley C.B. Shay J.W. Lichsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar). Our results suggest there may be cis-acting mechanisms to preferentially recruit telomerase to maintain the shortest telomeres under conditions of limiting telomerase activity. The consequent reduction of average telomere length to sizes less than that observed in senescent cells argues against a role for telomere positional effects controlling subtelomeric loci that cause the growth arrest observed at M1. Additional observations suggest that telomerase does not have a capping function on very short telomeres that is independent of its catalytic activity. DISCUSSIONThese results show that normal human fibroblasts expressing a transfected hTERT cDNA gradually showed reduced telomerase activity and decreasing telomere lengths. After 150–300 population doublings, the telomeres stabilized at subsenescent lengths and in some cases have remained at that size for over 150 additional doublings, and thus the cells are still functionally immortal. Analysis of these cells suggests several important interpretations. 1) The observed change in the distribution of telomere sizes implies the presence of cis-acting factors that preferentially recruit telomerase to act on the shortest telomeres; 2) the ability of cells with subsenescent telomere length to proliferate for 20 doublings following the abolition of telomerase activity argues against telomerase having a “capping” function independent of catalytic activity; and 3) the proliferation of normal cells with subsenescent telomere lengths provides evidence against the induction of growth arrest by subtelomeric regulatory loci silenced by long telomeres.The cells used in the present study had been transfected with a plasmid-based hTERT expression vector and showed a progressive decrease in telomerase activity over time. Although the resumption of telomere shortening was thus expected, the stabilization of telomeres at lengths approximately 2–4 kb shorter than that normally observed in senescent cells was surprising. The size of the shortest telomeres was maintained in multiple different clones over many months during which the longest telomeres continued to shorten. Despite the fact that all of the telomeres were sufficiently short to be expected to provide cis-acting signals, under conditions of limiting telomerase activity the shortest telomeres were preferentially maintained. Possible explanations include a more efficient recruitment of telomerase to the shortest telomeres, and loss of cells with the shortest telomeres and selection of the survivors.A very large number of proteins have been found to influence telomere length in yeast (reviewed in Ref. 36.Muniyappa K. Kironmai K.M. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 297-336Crossref PubMed Scopus (25) Google Scholar), and many of them are telomere-binding proteins. The most compelling evidence for cis-regulation of telomere length is for Rap1, where it has been shown that length is controlled by the number of Rap1 binding sites (37.Ray A. Runge K.W. Mol. Cell. Biol. 1999; 19: 31-45Crossref PubMed Scopus (66) Google Scholar, 38.Marcand S. Gilson E. Shore D. Science. 1997; 275: 986-990Crossref PubMed Scopus (424) Google Scholar). Preferential action of telomerase on the shortest telomeres has recently been demonstrated in yeast (39.Marcand S. Brevet V. Gilson E. EMBO J. 1999; 18: 3509-3519Crossref PubMed Scopus (167) Google Scholar). Results using hTRF1, the human orthologue of Rap1, have also implicated it as a cis-acting factor influencing human telomere length control (40.van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1050) Google Scholar). Our results suggest that the cis-acting telomere-binding proteins present in normal human cells are not only able to cause telomerase to act on the telomeres, but do so in a quantitative fashion that preferentially recruits it to the shortest telomeres despite the presumed presence of signals from other very short but nonetheless longer telomeres.An alternate interpretation is that telomerase is randomly acting on all telomeres, and that selection is producing the observed result. Cells in which telomerase acted on long but not short telomeres would become senescent and be lost from the population, while cells in which telomerase acted on short telomeres would continue to divide. When analyzing the entire population, the effect of this selection would be the apparent preservation of short telomere lengths while long telomeres shortened. Experiments in which chromosomes are broken by insertion of a plasmid with telomeric repeats on one end have shown that the telomere on the “healed chromosome” elongates while the length of the endogenous telomeres remain unaffected (41.Sprung C.N. Sabatier L. Murnane J.P. Exp. Cell Res. 1999; 247: 29-37Crossref PubMed Scopus (28) Google Scholar). Under conditions in which telomerase is not limiting, this shows that in human cells telomerase can preferentially be recruited to act on a telomere that is too short. We believe that it is likely that the same mechanisms that recruit telomerase to act on these “too short” healing chromosomes would act to preferentially recruit limiting amounts of telomerase to the shortest chromosomes, and thus prefer recruitment rather than selection as an explanation for these observations.Telomerase has been proposed to perform a capping function on short telomeres that requires catalytic activity (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar). Telomerase activity became undetectable in two clones following the introduction of the D869A hTERT mutant in B14 cells. These cells with very short telomeres divided for 20 additional doublings in the presence of the mutant hTERT before undergoing a growth arrest. The telomere shortening that occurred during these 20 doublings demonstrates that catalytically active telomerase was not present for a significant fraction of time on most of the telomeres. The replacement of wild-type telomerase with the dominant-negative mutant argues against a “capping” role for the telomerase protein on short telomeres that requires catalytic activity but is independent of the actual addition of TTAGGG repeats to the ends of the chromosomes. The ability of limiting amounts of catalytically active telomerase to preferentially maintain the shortest telomeres, so that average size decreases while minimum size does not, provides a sufficient explanation for the presence of subsenescent (this report) or subcrisis (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar) telomere lengths.We have previously proposed that genes regulating cellular senescence might be located in subtelomeric regions, and that their expression might be controlled by changes in telomere positional effects as telomeres shortened (20.Wright W.E. Shay J.W. Trends Genet. 1992; 8: 193-197Abstract Full Text PDF PubMed Scopus (237) Google Scholar). The present result demonstrates that these cultures continue to proliferate vigorously even though telomere sizes decreased to well below their normal lengths at senescence. This provides evidence against the re-expression of previously silenced genes that induce a growth arrest when telomeres become sufficiently short, and favors the hypothesis that it is the generation of a DNA damage signal from an insufficiently long telomere(s) that causes M1 (19.Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1081) Google Scholar). Several previous reports have failed to find evidence of telomere positional effects in vertebrate cells (42.Bayne R.A. Broccoli D. Taggart M.H. Thomson E.J. Farr C.J. Cooke H.J. Hum. Mol. Genet. 1994; 3: 539-546Crossref PubMed Scopus (49) Google Scholar, 43.Sprung C.N. Sabatier L. Murnane J.P. Nucleic Acids Res. 1996; 24: 4336-4340Crossref PubMed Scopus (27) Google Scholar). Although we now think it unlikely that telomere positional effects regulate the onset of M1, we continue to entertain the possibility that telomere shortening might regulate gene expression in ways that permit the counting of cell divisions to be used as a mechanism for timing decades-long processes during the human life span (44.Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5: 293-296Abstract Full Text PDF PubMed Scopus (127) Google Scholar).The concept that short telomeres increase the efficiency with which they recruit telomerase leads to the speculation that very efficient inhibition of telomerase might be required for anti-telomerase cancer therapy to be successful. It also raises the possibility that a combination of interventions inhibiting both the catalytic activity of telomerase as well as its ability to be recruited to telomeres might be much more successful than either alone. It is important to remember that (in contrast to germline cells) adult human somatic cells are not biologically programmed to maintain telomere length, and that the expression and function of an unknown number of accessory factors may have been altered in different somatic cells that have repressed telomerase. We anticipate that the factors that modify telomerase, recruit it to the telomeres, cause it to catalyze the addition of telomeric repeats, and regulate the number of repeats added at one time will show significant variability in levels and efficiencies between different normal cell types, and that this variability will be compounded in cancer cells. The consequences of expressing telomerase in an “inappropriate” biological context, either via an exogenous cDNA or through the mutational inactivation of repressive pathways, are thus likely to be diverse as well. Disentangling these multiple mechanisms should increase our ability to alter telomere length regulation for modifying the time course of replicative aging and in the treatment of cancer. These results show that normal human fibroblasts expressing a transfected hTERT cDNA gradually showed reduced telomerase activity and decreasing telomere lengths. After 150–300 population doublings, the telomeres stabilized at subsenescent lengths and in some cases have remained at that size for over 150 additional doublings, and thus the cells are still functionally immortal. Analysis of these cells suggests several important interpretations. 1) The observed change in the distribution of telomere sizes implies the presence of cis-acting factors that preferentially recruit telomerase to act on the shortest telomeres; 2) the ability of cells with subsenescent telomere length to proliferate for 20 doublings following the abolition of telomerase activity argues against telomerase having a “capping” function independent of catalytic activity; and 3) the proliferation of normal cells with subsenescent telomere lengths provides evidence against the induction of growth arrest by subtelomeric regulatory loci silenced by long telomeres. The cells used in the present study had been transfected with a plasmid-based hTERT expression vector and showed a progressive decrease in telomerase activity over time. Although the resumption of telomere shortening was thus expected, the stabilization of telomeres at lengths approximately 2–4 kb shorter than that normally observed in senescent cells was surprising. The size of the shortest telomeres was maintained in multiple different clones over many months during which the longest telomeres continued to shorten. Despite the fact that all of the telomeres were sufficiently short to be expected to provide cis-acting signals, under conditions of limiting telomerase activity the shortest telomeres were preferentially maintained. Possible explanations include a more efficient recruitment of telomerase to the shortest telomeres, and loss of cells with the shortest telomeres and selection of the survivors. A very large number of proteins have been found to influence telomere length in yeast (reviewed in Ref. 36.Muniyappa K. Kironmai K.M. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 297-336Crossref PubMed Scopus (25) Google Scholar), and many of them are telomere-binding proteins. The most compelling evidence for cis-regulation of telomere length is for Rap1, where it has been shown that length is controlled by the number of Rap1 binding sites (37.Ray A. Runge K.W. Mol. Cell. Biol. 1999; 19: 31-45Crossref PubMed Scopus (66) Google Scholar, 38.Marcand S. Gilson E. Shore D. Science. 1997; 275: 986-990Crossref PubMed Scopus (424) Google Scholar). Preferential action of telomerase on the shortest telomeres has recently been demonstrated in yeast (39.Marcand S. Brevet V. Gilson E. EMBO J. 1999; 18: 3509-3519Crossref PubMed Scopus (167) Google Scholar). Results using hTRF1, the human orthologue of Rap1, have also implicated it as a cis-acting factor influencing human telomere length control (40.van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1050) Google Scholar). Our results suggest that the cis-acting telomere-binding proteins present in normal human cells are not only able to cause telomerase to act on the telomeres, but do so in a quantitative fashion that preferentially recruits it to the shortest telomeres despite the presumed presence of signals from other very short but nonetheless longer telomeres. An alternate interpretation is that telomerase is randomly acting on all telomeres, and that selection is producing the observed result. Cells in which telomerase acted on long but not short telomeres would become senescent and be lost from the population, while cells in which telomerase acted on short telomeres would continue to divide. When analyzing the entire population, the effect of this selection would be the apparent preservation of short telomere lengths while long telomeres shortened. Experiments in which chromosomes are broken by insertion of a plasmid with telomeric repeats on one end have shown that the telomere on the “healed chromosome” elongates while the length of the endogenous telomeres remain unaffected (41.Sprung C.N. Sabatier L. Murnane J.P. Exp. Cell Res. 1999; 247: 29-37Crossref PubMed Scopus (28) Google Scholar). Under conditions in which telomerase is not limiting, this shows that in human cells telomerase can preferentially be recruited to act on a telomere that is too short. We believe that it is likely that the same mechanisms that recruit telomerase to act on these “too short” healing chromosomes would act to preferentially recruit limiting amounts of telomerase to the shortest chromosomes, and thus prefer recruitment rather than selection as an explanation for these observations. Telomerase has been proposed to perform a capping function on short telomeres that requires catalytic activity (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar). Telomerase activity became undetectable in two clones following the introduction of the D869A hTERT mutant in B14 cells. These cells with very short telomeres divided for 20 additional doublings in the presence of the mutant hTERT before undergoing a growth arrest. The telomere shortening that occurred during these 20 doublings demonstrates that catalytically active telomerase was not present for a significant fraction of time on most of the telomeres. The replacement of wild-type telomerase with the dominant-negative mutant argues against a “capping” role for the telomerase protein on short telomeres that requires catalytic activity but is independent of the actual addition of TTAGGG repeats to the ends of the chromosomes. The ability of limiting amounts of catalytically active telomerase to preferentially maintain the shortest telomeres, so that average size decreases while minimum size does not, provides a sufficient explanation for the presence of subsenescent (this report) or subcrisis (14.Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar) telomere lengths. We have previously proposed that genes regulating cellular senescence might be located in subtelomeric regions, and that their expression might be controlled by changes in telomere positional effects as telomeres shortened (20.Wright W.E. Shay J.W. Trends Genet. 1992; 8: 193-197Abstract Full Text PDF PubMed Scopus (237) Google Scholar). The present result demonstrates that these cultures continue to proliferate vigorously even though telomere sizes decreased to well below their normal lengths at senescence. This provides evidence against the re-expression of previously silenced genes that induce a growth arrest when telomeres become sufficiently short, and favors the hypothesis that it is the generation of a DNA damage signal from an insufficiently long telomere(s) that causes M1 (19.Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1081) Google Scholar). Several previous reports have failed to find evidence of telomere positional effects in vertebrate cells (42.Bayne R.A. Broccoli D. Taggart M.H. Thomson E.J. Farr C.J. Cooke H.J. Hum. Mol. Genet. 1994; 3: 539-546Crossref PubMed Scopus (49) Google Scholar, 43.Sprung C.N. Sabatier L. Murnane J.P. Nucleic Acids Res. 1996; 24: 4336-4340Crossref PubMed Scopus (27) Google Scholar). Although we now think it unlikely that telomere positional effects regulate the onset of M1, we continue to entertain the possibility that telomere shortening might regulate gene expression in ways that permit the counting of cell divisions to be used as a mechanism for timing decades-long processes during the human life span (44.Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5: 293-296Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The concept that short telomeres increase the efficiency with which they recruit telomerase leads to the speculation that very efficient inhibition of telomerase might be required for anti-telomerase cancer therapy to be successful. It also raises the possibility that a combination of interventions inhibiting both the catalytic activity of telomerase as well as its ability to be recruited to telomeres might be much more successful than either alone. It is important to remember that (in contrast to germline cells) adult human somatic cells are not biologically programmed to maintain telomere length, and that the expression and function of an unknown number of accessory factors may have been altered in different somatic cells that have repressed telomerase. We anticipate that the factors that modify telomerase, recruit it to the telomeres, cause it to catalyze the addition of telomeric repeats, and regulate the number of repeats added at one time will show significant variability in levels and efficiencies between different normal cell types, and that this variability will be compounded in cancer cells. The consequences of expressing telomerase in an “inappropriate” biological context, either via an exogenous cDNA or through the mutational inactivation of repressive pathways, are thus likely to be diverse as well. Disentangling these multiple mechanisms should increase our ability to alter telomere length regulation for modifying the time course of replicative aging and in the treatment of cancer." @default.
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W2053556250 title "Subsenescent Telomere Lengths in Fibroblasts Immortalized by Limiting Amounts of Telomerase" @default.
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