Stabilization of Telomere Length and Karyotypic Stability Are Directly Correlated with the Level of hTERT Gene Expression in Primary Fibroblasts
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m205981200
ISSN1083-351X
AutoresWei Cui, Samena Aslam, Judy Fletcher, Diana Wylie, Michael Clinton, A. Clark,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoTelomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. However, telomere length is not maintained in all cell lines, even though in vitro telomerase activity is restored in all of them. Cell lines expressing higher levels of hTERT mRNA do not exhibit telomere erosion or genomic instability. By contrast, fibroblasts expressing lower levels of hTERT do exhibit telomere shortening, although the telomeres eventually stabilize at a shorter length. The shorter telomere lengths and the extent of karyotypic abnormalities are both functions of hTERT expression level. We conclude that telomerase activity is required to bypass senescence but is not sufficient to prevent telomere erosion and genomic instability at lower levels of expression. Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. However, telomere length is not maintained in all cell lines, even though in vitro telomerase activity is restored in all of them. Cell lines expressing higher levels of hTERT mRNA do not exhibit telomere erosion or genomic instability. By contrast, fibroblasts expressing lower levels of hTERT do exhibit telomere shortening, although the telomeres eventually stabilize at a shorter length. The shorter telomere lengths and the extent of karyotypic abnormalities are both functions of hTERT expression level. We conclude that telomerase activity is required to bypass senescence but is not sufficient to prevent telomere erosion and genomic instability at lower levels of expression. telomere repeat amplification protocol the catalytic component of telomerase human TERT nuclear transfer population doublings telomere restriction fragment reverse transcription-PCR quantitative RT-PCR fluorescencein situ hybridization glyceraldehyde-3-phosphate dehydrogenase horseradish peroxidase Telomeres are specialized structures found at the end of chromosomes, consisting of proteins and tandem G-rich repeats that are conserved in all vertebra as (TTAGGG)n (1Meyne J. Ratliff R.L. Moyzis R.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7049-7053Crossref PubMed Scopus (673) Google Scholar). They are essential elements that protect chromosomal ends from nuclease degradation, interchromosomal fusion, and improper recombination, thereby, contributing to genome stability. Telomerase is a specialized cellular ribonucleoprotein reverse transcriptase that synthesizes telomeric repeats onto chromosomal ends (2Greider C.W. Blackburn E.H. Nature. 1989; 337: 331-337Crossref PubMed Scopus (1303) Google Scholar, 3Lingner J. Hughes T.R. Shevchenko A. Mann M. Lundblad V. Cech T.R. Science. 1997; 276: 561-567Crossref PubMed Scopus (1041) Google Scholar). It consists of two main components that are required for core enzyme activity, which is measured by the telomeric repeat amplification protocol (TRAP)1 assay. These are a telomerase RNA, used as template for the addition of new telomeric repeats, and a catalytic protein subunit (TERT). In humans, telomerase is preferentially expressed in germ-line cells and in early embryonic tissues but is not detectable in somatic cells (4Wright W.E. Piatyszek M.A. Rainey W.E. Byrd W. Shay J.W. Dev. Genet. 1996; 18: 173-179Crossref PubMed Scopus (1146) Google Scholar). In the absence of telomerase activity, somatic cells undergo a progressive shortening of telomeres with each cell division, because DNA polymerase is unable to replicate the chromosomal ends completely. Eventually when the telomeres on one or more chromosomes become critically short, the cells stop dividing and enter replicative senescence (5Hemann M.T. Strong M.A. Hao L.Y. Greider C.W. Cell. 2001; 107: 67-77Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 6Lansdorp P.M. Mech. Ageing Dev. 2000; 118: 23-34Crossref PubMed Scopus (74) Google Scholar). Telomerase catalytic protein subunit (TERT) has been indicated as a key factor limiting telomerase activity in human somatic cells, because the RNA subunit is constitutively expressed at a low basal level in cells (7Feng 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. Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2079) Google Scholar). This was confirmed by experiments showing that forced expression of hTERT in human fibroblast, epithelial, endothelial, and osteoblast cells reconstitutes telomerase activity and extends their proliferative lifespan (8Bodnar 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 (4116) Google Scholar, 9Counter 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 (562) Google Scholar, 10Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (361) Google Scholar, 11Yang 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 (427) Google Scholar, 12Ouellette 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 (212) Google Scholar, 13Yudoh K. Matsuno H. Nakazawa F. Katayama R. Kimura T. J. Bone Miner. Res. 2001; 16: 1453-1464Crossref PubMed Scopus (93) Google Scholar). However, telomere lengths are not always maintained in these cells, despite the fact that telomerase activity is restored. Similarly, the telomeres of many tumor cell lines are shorter than those in normal cells despite the fact that they have high levels of telomerase activity. For example, telomeres in colorectal and ovarian carcinoma tissues are shorter than in the corresponding normal tissue from the same individual (14Hastie N.D. Dempster M. Dunlop M.G. Thompson A.M. Green D.K. Allshire R.C. Nature. 1990; 346: 866-868Crossref PubMed Scopus (1476) Google Scholar, 15Counter C.M. Hirte H.W. Bacchetti S. Harley C.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2900-2904Crossref PubMed Scopus (725) Google Scholar). It has been accepted generally that telomere length in human cells is controlled by a homeostatic mechanism that involves telomerase and other factors, such as the TTAGGG-repeat binding proteins, TRF1 and TRF2 (16Smogorzewska A. van Steensel B. Bianchi A. Oelmann S. Schaefer M.R. Schnapp G. de Lange T. Mol. Cell. Biol. 2000; 20: 1659-1668Crossref PubMed Scopus (627) Google Scholar), but it is not fully understood how these factors cooperate with each other or whether other factors are involved. We have shown recently that sheep fibroblasts are similar to human fibroblasts in several aspects of telomere biology and replicative senescence. 2W. Cui, D. Wylie, S. Aslam, A. Dinnyes, T. King, I. Wilmut, and A. J. Clark, manuscript in preparation. Thus, they have no detectable telomerase activity and undergo only a limited number of cell divisions before entering replicative senescence. Telomere lengths in these cells shorten with cell proliferation at a rate of 50–200 bp per cell division. Finally, forcing hTERT expression in transfected cells restores telomerase activity and extends their proliferative lifespan. In this report, we have followed the continuous growth of these cells for more than 15 months and investigated the telomere maintenance and karyotypic stability as a function of hTERT mRNA and protein levels, as well as telomerase catalytic activity in cell lysates. Our results show that the expression of hTERT reconstitutes telomerase catalytic activity, resulting in cell immortalization. At higher levels of hTERT expression full-length telomeres are maintained and karyotypic stability is preserved. At lower levels, telomere erosion and genomic stability occur to an extent that is directly determined by the level of hTERT expression. Sheep fibroblasts, BW6F2, were isolated from a day 35 Black Welsh eviscerated fetus as described previously (17Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H. Nature. 1997; 385: 810-813Crossref PubMed Scopus (4054) Google Scholar). Cells were cultured in Glasgow minimal essential medium (Sigma), supplemented with 2 mml-glutamine, 1 mm sodium pyruvate, 1× non-essential amino acids, and 10% fetal calf serum (Globe Farm, Surrey, UK) in a humidified incubator at 37 °C and 5% CO2. Plasmid GRN145 was kindly provided by the Geron Corp., which contains hTERT cDNA driven by myeloproliferative sarcoma virus promoter and puromycin-resistant gene driven by the SV40 promoter (8Bodnar 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 (4116) Google Scholar). An empty vector control was constructed by digesting GRN145 plasmid with EcoRI followed by re-ligation to remove the hTERT cDNA fragment. Linearized plasmids were electroporated at 250 microfarads/400 V to BW6F2 cells at passage 6. Cells were then plated into 96-well plates at 5000 cells per well. Puromycin (750 ng to 1 μg/ml) was added into medium 48 h after transfection to select puromycin-resistant colonies. Each colony was expanded to confluence in a T75 flask, from which 3 × 105 cells were plated into a T25 flask, and the growth curve was started at this point. The growth curve of the parental mass culture, BW6F2, was started at passage 6, which is the stage the transfection was carried out. Cells were sub-cultured twice weekly and maintained at log phase growth. Telomerase activity of cell extracts was analyzed by telomeric repeat amplification protocol (TRAP) assay as described previously (18Kim 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 (6550) Google Scholar, 19Kim N.W. Wu F. Nucleic Acids Res. 1997; 25: 2595-2597Crossref PubMed Scopus (628) Google Scholar) with a TRAPeze Telomerase Detection kit (Intergen) using either radioactive or non-radioactive labeling of TS primer (19Kim N.W. Wu F. Nucleic Acids Res. 1997; 25: 2595-2597Crossref PubMed Scopus (628) Google Scholar). Each reaction product was resolved in 10% polyacrylamide gel and exposed to a PhosphorImager screen (AmershamBiosciences) and visualized by scanning. To quantify the activities, cell extracts from 0.01 μg to 1 μg were used for TRAP assay, and telomerase activity was calculated as the ratio of the intensity of telomere ladder over the intensity of 36 bp of internal control band. The TRAP assay was calibrated by quantifying activity in serial dilutions of cell extracts. Although the activities decreased with dilution in each cell line within a narrow range, the differences in activity between the various cell lines were consistent at each dilution. Genomic DNA from cultured cells was isolated by phenol/chloroform extraction using tubes containing phase lock gel (Eppendorf, Hamburg, Germany). Telomere length was determined by telomere restriction fragment (TRF) Southern blot analysis with either [γ-32P]ATP or digoxigenin labeled (TTAGGG)3 probe as described previously (20Harley C.B. Futcher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4632) Google Scholar). Mean TRF was calculated as described previously (12Ouellette 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 (212) Google Scholar). Sheep fibroblasts were harvested after sub-culturing in growth medium for 26 h and incubated in 0.075 m KCl at room temperature for about 10 min. Subsequently, cells were fixed in methanol-acetic acid (3:1, v/v) and were dropped onto clean microscopic slides. FISH was carried out as described before (21Lansdorp P.M. Verwoerd N.P. van de Rijke F.M. Dragowska V. Little M.T. Dirks R.W. Raap A.K. Tanke H.J. Hum. Mol. Genet. 1996; 5: 685-691Crossref PubMed Scopus (718) Google Scholar) with a fluorescein isothiocyanate-conjugated telomere peptide nucleic acid probe (Dako, Denmark). The slides were mounted in Vectashield containing 0.1 μg/ml 4′,6′-diamidino-2-phenylindole. For karyotype analysis, slides with metaphase spreads were stained in 5% Gurrs R66 Giemsa at room temperature for 8 min and mounted with DPX mountant. Thirty cells were analyzed from each culture. Cells were trypsinized and counted then washed with phosphate-buffered saline. Total RNA was extracted with RNAzol B and digested with DNase I. First-strand cDNA was synthesized from 5 μg of total RNA by reverse transcriptase in a 20-μl volume with oligo(Pd(T)15). Each PCR was carried out with 5 μl of the reverse transcription product and amplified for 26 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s. The primer sequences used are as follows: hTERT-W1, 5′-AGCGACTACTCCAGCTATG-3′; hTERT-W2, 5′-GTTCTTGGCTTTCAGGATGG-3′; sheep GAPDH-F, 5′-TGTCCGTTGTGGATCTGACC-3′; sheep GAPDH-R, 5′-CGTACCAGGAAATGAGCTTGAC-3′. Real-time qRT-PCR primers and TaqMan probes were selected for hTERT and sheep GAPDH using Primer Express software (Applied Biosystems). Forward primer for hTERT, 5′-TCAACCGCGGCTTCAAG-3′; the reverse primer, 5′-TCCAGAAACAGGCTGTGACACT-3′. The hTERT TaqMan probe was 5′-Fam-TTGCGACGCATGTTCCTCCCAG-Tamara-3′. Forward primer for sheep GAPDH, 5′-TTGTCTCCTGCGACTTC-AACA-3′; reverse primer, 5′-ACCAGGAAATGAGCTTGACAAAG-3′. The TaqMan probe for sheep GAPDH was 5′-Vic-CGACACTCACTCTTCTACCTTCGATGCTGG-Tamara-3′. Each reaction contained 1× TaqMan Universal PCR Master Mix, 300 nm forward and reverse primers, 200 nm TaqMan probe(s), and 1 μl of reverse transcription product diluted 1:30–60. Assays were performed in quadruplicate using an Applied Biosystems Model 7700 sequence detection system. All data were normalized to sheep GAPDH internal mRNA control (ΔΔCT analysis). The same real-time PCR was also performed with hTERT cDNA plasmid at concentration equivalent to 0.01, 0.1, 1, 10, 100, and 1000 copies per cell, and a standard curve was generated. The hTERT mRNA copy number was calculated with the standard curve. Protein extracts were prepared from logarithmically growing cells by lysis in buffer (25 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 1% Nonidet P-40, 1 mm EDTA, 2 mm EGTA). Protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce) according to the manufacturer's instruction. Protein (40 μg) was separated on a SDS 7.5% polyacrylamide gel and transferred onto nitrocellulose membrane. After blocking for 1 h in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 1% bovine serum albumin, blots were incubated with mouse monoclonal anti-hTERT antibody 1A4 (from Geron Corp., Menlo Park, CA) at 1:10,000 dilution in the blocking solution for 1 h at room temperature. The blots were washed and incubated with secondary antibody, horseradish peroxidase-conjugated sheep anti-mouse IgG, at 1:5000 dilution for 1 h. The signals were visualized with an ECL detection kit (Amersham Biosciences). Cells were cultured in glass chamber slides and fixed in 4% paraformaldehyde at room temperature for 9 min after washing with phosphate-buffered saline. The cells were incubated with blocking solution, TBS containing 0.1% bovine serum albumin for 10 min and then with mouse monoclonal anti-hTERT antibody 1A4 at 1:1000 dilution for 1 h at room temperature. After washing, the cells were incubated with secondary antibody, biotinylated goat anti-mouse IgG (1:500 dilution) for 1 h. The signals were detected by incubation with Streptavidin Texas red (Vector laboratories) at 1:200 dilutions for 20 min, and the slides were mounted with Vectashield containing 4′,6′-diamidino-2-phenylindole. Primary cultures of sheep fetal fibroblasts show similar characteristics to human fibroblasts in that there is no detectable telomerase activity by TRAP assay and the cells undergo only a limited number of cell divisions.2 Stable transfection and expression of human telomerase catalytic subunit hTERT in these cells reconstituted telomerase activity (Fig. 1 A) and extended their proliferative lifespan. The proliferative capacities of these colonies have been followed for more than 400 days of continuous culture. They maintained a steady growth rate, similar to young fibroblasts and have accumulated 400–500 population doublings (PDs) to date. This is between 8- and 25-fold more than the population doublings achieved by empty vector-transfected clones or clones without hTERT expression, which only grew for a maximum of 61 PDs and then senesced (Fig. 1 B). This is also more than 3-fold that of the parental mass culture, BW6F2, which senesced after 110 PDs (Fig.1 B). hTERT-expressing clones exhibited a similar morphology to young fibroblasts even at high PDs and were negative with senescence associated β-galactosidase staining.2 Therefore, we consider these cells to be functionally immortal. The most obvious function of active telomerase is to add telomeric repeats to telomere ends to prevent telomeres from shortening. From a number of recent reports, however, it is unclear whether reconstituted telomerase catalytic activity as measured by TRAP assay is always sufficient to maintain telomere lengths in somatic cells (8Bodnar 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 (4116) Google Scholar, 9Counter 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 (562) Google Scholar, 10Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (361) Google Scholar, 12Ouellette 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 (212) Google Scholar). In some experiments, overexpression of hTERT reconstituted telomerase catalytic activity, indefinitely extended proliferative lifespan, and maintained the telomere length (8Bodnar 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 (4116) Google Scholar, 9Counter 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 (562) Google Scholar, 22Vaziri H. Benchimol S. Curr. Biol. 1998; 8: 279-282Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar). By contrast, in other experiments, forced expression of hTERT, although extending proliferative lifespan, did not maintain telomere length (10Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (361) Google Scholar,12Ouellette 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 (212) Google Scholar). To assess the effects of reconstituted telomerase catalytic activity on telomere length in the hTERT-expressing sheep fibroblasts, the telomere lengths were measured by Southern blot with a (TTAGGG)3 probe, and the mean TRF was calculated. We measured telomere lengths at the beginning of the growth curve and after about 260–280 days in culture (Fig.2 A). The results showed that all clones have similar telomere length at early passage (PDs = 2). However, after more than 200 cell divisions, some clones (2-1, 2-5, and 2-7) maintained the telomere length (Fig. 2 A, lane 3–8), whereas others (1-1, 2-8, 2-12, and 2-13) exhibited telomere shortening (Fig. 2 A, lanes 1–2 and9–14). These results were confirmed by telomere FISH analysis (Fig. 2 B). Clones 1-1, 2-8, 2-12, and 2-13 had much lower telomere signals in compare with clones 2-1, 2-5, and 2-7. Telomere lengths in these cell lines were re-analyzed after further population doublings. In each line, the telomeres became stabilized at a characteristic length, although there was considerable variation between individual cell lines (Fig. 2 C and TableI). Clones 2-1, 2-5, and 2-7 maintained their telomere lengths at ∼21.0 kb throughout culture. Telomere lengths were the same as they were at the earlier passages (Fig.2 C, lanes 1–6) and, moreover, were the same as those in early passage primary parental cells (Fig. 2 C,lane 15). Clones 1-1, 2-8, and 2-12, in which the telomeres had already shortened, did not exhibit further shortening of telomeres and showed no significant difference in telomere length at the two later passages (Fig. 2 C, lanes 7–12). By contrast, clone 2-13 continued to exhibit further shortening (Fig.2 C, lane 13 versus 14), but this subsequently stabilized after approximately four hundred population doublings (data not shown). In the cell lines that exhibited telomere shortening, the new, stabilized telomere lengths varied considerably, with mean TRF at about 8.4, 10.8, 14.6, and 6.0 kb in clones 1-1, 2-8, 2-12, and 2-13, respectively (Table I). The stabilized mean TRF in clones 1-1 and 2-13 were substantially shorter than that in parental mass culture, BW6F2, at the time of senescence (mean TRF = 11.3 kb) (Fig. 2 C, lane 16) even though these cell lines still grew vigorously.Table ISummary of hTERT-expressing sheep fibroblast clonesCell cloneMean TRF1-aRepresents the mean TRF at which telomeres are stabilized in each cell line.TRAP assay1-bDefines TRAP activity in human tumor cell line 293 as 100% and PDs as defined in the growth curves.Relative hTERT mRNA expression1-cThe expression levels were measured by qRT-PCR, normalized to the internal GAPDH mRNA level and calculated relative to clone 2–13.hTERT mRNA Per cell1-dhTERT mRNA per cell was measured by qRT-PCR and calculated by reference to a standard curve for hTERT plasmid (see "Experimental Procedures" for details).PDs = 0PDs > 200kb%1–18.430482.60.342–120.751465873.5375.082–521.039521278.387.242–721.230525752.6345.142–810.852398.1%0.582–1214.660559.30.652–136.038301.00.08BW6F2 (young)20.9000.02BW6F2 (senescent)11.30ND1-eND, not done.ND1-a Represents the mean TRF at which telomeres are stabilized in each cell line.1-b Defines TRAP activity in human tumor cell line 293 as 100% and PDs as defined in the growth curves.1-c The expression levels were measured by qRT-PCR, normalized to the internal GAPDH mRNA level and calculated relative to clone 2–13.1-d hTERT mRNA per cell was measured by qRT-PCR and calculated by reference to a standard curve for hTERT plasmid (see "Experimental Procedures" for details).1-e ND, not done. Open table in a new tab We next addressed whether the telomere length maintenance was correlated with the levels of telomerase catalytic activity in extracts from these various cell lines. Telomerase catalytic activity was measured by TRAP assay in these clones at early and later passages, and their relative levels were calculated to that in human tumor cell line 293 as described under "Experimental Procedures." All the clones showed relatively high levels of telomerase catalytic activity, between 30 and 60% of that measured in 293 cells (Table I). The range was narrow, and there was no more than a 2-fold difference in telomerase activity between the highest and lowest expressing cell lines. No consistent increase or decrease of telomerase activity was observed after cell passages. The TRAP activity measured in later passage cells, when telomere length had been maintained or was stabilized at a shorter length, showed a moderately strong correlation with the mean TRFs at this stage (r = 0.696). Expression of hTERT mRNA in the later passages (PDs > 250) was first analyzed by semi-quantitative reverse transcription-PCR (RT-PCR) in each of the cell lines. In contrast to the TRAP assays the RT-PCR showed there was a broad range of steady-state hTERT mRNA levels (Fig.3 A). Thus in lines 2-1, 2-5, and 2-7 hTERT transcripts were readily detected after 26 PCR cycles, whereas they were only just detectable in lines 2-8 and 2-12. In lines 1-1 and 2-13 as well as in human 293 cells (not shown) the steady-state mRNA level of hTERT were so low that the transcript could not be detected under these conditions. However, when the RT-PCR was repeated using 35 cycles of amplification all samples, including 1-1 and 2-13 and human 293 cells showed detectable hTERT transcripts. To confirm these results and to quantitate the steady-state mRNA levels accurately, we carried out real-time quantitative RT-PCR (qRT-PCR) on these samples. The results are shown in Fig. 3 B and tabulated in Table I. They show that the steady-state levels of hTERT mRNA vary over an enormous, 6000-fold range in these lines. Assigning a relative value of 1 to the lowest expressor (2-13) we grouped the lines into three classes according to the level of expression: very low (1–3, in lines 1-1 and 2-13); low (8–10, lines 2-8 and 2-12), and high (1000–6000, in lines 2-1, 2-5, and 2-7) and corresponded to the result obtained by semi-quantitative RT-PCR (Fig.3 A). We also independently measured the copy number of hTERT mRNA per cell in these clones using real-time quantitative PCR with diluted hTERT cDNA plasmid as standard curve. The results are shown in Table I. The low and very low hTERT expression clones had less than one copy of hTERT mRNA transcript per cell, whereas the highly expressing clones were estimated to contain 80–400 copies per cell. hTERT protein levels were measured in the cell extracts of each of the lines at both early (∼4 PDs), middle (105–165 PDs), and later passages (270–360 PDs) by Western blotting using an antibody against hTERT. In all Western blotting experiments clones 2-1, 2-5, and 2-7 showed detectable levels of the correctly sized 127-kDa hTERT protein, whereas, no detectable level of hTERT protein was found in the other clones (Fig. 4 A). Human tumor cell line 293, which has high telomerase catalytic activity, did not exhibit detectable levels of hTERT protein (Fig. 4 A,lane 9). Although initially surprising to us, this observation is consistent with recent reports using a different hTERT antibody in Western blotting experiments, which, again, failed to detect hTERT protein in 293 cells, despite high levels of telomerase catalytic activity (23Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (78) Google Scholar, 24Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (120) Google Scholar). Protein expression was also investigated by immunocytochemistry. Cell lines 2-1, 2-5, and 2-7, in which hTERT was detectable Western blotting showed strong nuclear staining, whereas cells from the other clones, including human tumor 293 were negative (Fig. 4 B). Our measurements on hTERT mRNA and protein levels are fully consistent, and the three cell lines expressing high steady-state levels of hTERT mRNA were the only ones with detectable level of hTERT protein. Indeed, the lowest expressor of these three lines (2-5, which had about 1/5 the steady-state hTERT mRNA level as compared with the other two, Table I) also had the lowest level of detectable hTERT protein (Fig. 4 A). We conclude that there is a large range of hTERT expression in these cell lines that is reflected in both the steady-state mRNA and protein levels but not in the telomerase catalytic activity. Shortening of telomeres during extended culture correlated with the hTERT mRNA levels. Thus lines 2-8 and 2-12, which had low levels of hTERT mRNA and undetectable levels of hTERT protein, exhibited telomere shortening, and the telomeres were stabilized with a mean TRF greater than 10 kb. Lines 1-1 and 2-13, having even lower levels of hTERT mRNA, also exhibited telomere shortening. Their telomeres shortened to an even greater extent than 2-8 or 2-12 and only stabilized when the mean TRF was below 10 kb. In these four lowly expressing lines there was a very strong correlation (r = 0.940) between the steady-state mRNA levels and the stabilized telomere length. By contrast, the cell lines exhibiting high steady-state hTERT mRNA levels and detectable hTERT protein (2-1, 2-5, and 2-7) did not undergo telomere shortening and maintained full-length telomere throughout the extended culture period. Recent reports in yeast and plants suggest that the telomeres play an important role in genome stability (25Hackett J.A. Feldser D.M. Greider C.W. Cell. 2001; 106: 275-286Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 26Riha K. McKnight T.D. Griffing L.R. Shippen D.E. Science. 2001; 291: 1797-1800Crossref PubMed Scopus (187) Google Scholar). To assess the effects of hTERT expression on the genome stability of the stably transfected sheep fibroblast, we examined the chromosomes in these cell lines by cytogenetic analysis (Table II). At beginning of their proliferative lifespan, all hTERT-transfected clones showed normal karyotype (Fig. 5 A). However, after about 220 population doublings, clones 1-1 and 2-13, started to exhibit a high frequency of abnormal karyotype (20% or more cells). These abnormalities included abnormal sub-metrocentric, dicentric, and ring chromosomes (Fig. 5, B–D), which likely resulted from chromosomal end-to-end fusions. The frequency of thes
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