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Brief Communication
Reconstitution of telomerase activity in normal human cells leads
to elongation of telomeres and extended replicative life span
Homayoun Vaziri and Samuel Benchimol
Normal somatic cells have a finite life span [1] and lose
and pBabest2-AS retroviruses were transfected into the telomeric DNA, present at the ends of chromosomes,
packaging cell line Phoenix-E and viral supernatants were each time they divide as a function of age in vivo or in
harvested. The normal human diploid fibroblast strain BJ, culture [2–4]. In contrast, many cancer cells and cell
previously transfected with the ecotropic virus receptor lines established from tumours maintain their telomere
gene [18], was infected with viral supernatants (multiplic- length by activation of an RNA–protein complex called
ity of infection = 4) at approximately 75–79 population telomerase, an enzyme originally discovered in
doublings (PDs). These cells have approximately 10–15 Tetrahymena [5], that synthesizes telomeric repeats
PDs remaining before reaching senescence. Colonies [6–8]. These findings have led to the formation of the
resistant to both G418 and hygromycin were selected, ‘telomere hypothesis’, which proposes that critical
isolated with cloning cylinders and expanded. BJ cells, shortening of telomeric DNA due to the end-replication
like other fibroblasts, do not normally have telomerase problem [9] is the signal for the initiation of cellular
activity [7], but they do express the RNA subunit (hTR) of senescence [10,11]. In yeast, the EST2 gene product, the
the telomerase complex [15]. We reasoned, therefore, that catalytic subunit of telomerase, is essential for telomere
BJ cells, which normally reach senescence after 87–90 maintenance in vivo [12–14]. The recent cloning of the
PDs, represented a suitable recipient cell strain in which cDNA encoding the catalytic subunit of human
to express hTERT, reconstitute telomerase activity, and telomerase (hTERT) [15,16] makes it possible to test the
test for elongated telomeres and extended life span.
telomere hypothesis. In this study, we expressed hTERT
in normal human diploid fibroblasts, which lack
The pBabe-infected and pBabest2-AS-infected BJ cells telomerase activity, to determine whether telomerase
formed sparse and small colonies (Figure 1a,b). In contrast, activity could be reconstituted leading to extension of
pBabest2-infected cells gave rise to many larger colonies replicative life span. Our results show that retroviral-
(Figure 1c). Ten drug-resistant colonies, each consisting of mediated expression of hTERT resulted in functional
approximately 250–350 cells, were isolated from each of telomerase activity in normal aging human cells.
the infected cultures and expanded. In this secondary Moreover, reconstitution of telomerase activity in vivo
plating assay, none of the 20 colonies derived from the led to an increase in the length of telomeric DNA and to
pBabe-infected or pBabest2-AS-infected cultures was extension of cellular life span. These findings provide
capable of dividing sufficiently to reach confluence even direct evidence in support of the telomere hypothesis,
after 14–20 days. These cells appeared to have reached indicating that telomere length is one factor that can
senescence after an estimated 87–90 PDs on the basis of determine the replicative life span of human cells.
increased cell size, failure to divide and increased endoge-nous β-galactosidase activity (Figure 2a,b) [19]. In contrast, Address: Ontario Cancer Institute and Department of Medical 9 of 10 colonies obtained from the pBabest2-infected Biophysics, University of Toronto, 610 University Avenue, Toronto,Ontario, M5G 2M9, Canada.
Correspondence: Homayoun VaziriE-mail: [email protected] Received: 9 January 1998
Revised: 26 January 1998
Accepted: 26 January 1998
Published: 16 February 1998
Current Biology 1998, 8:279–282
http://biomednet.com/elecref/0960982200800279
Colony-forming ability of BJ cells infected with retroviruses encoding the Results and discussion
human telomerase catalytic subunit, hTERT, and controls. BJ cells The cDNA encoding hTERT was subcloned in the retro- infected with (a) pBabest2-AS, (b) pBabe or (c) pBabest2 were
viral vector pBabe under the control of the promoter replated and selected in G418 and hygromycin. Cells were fixed and present in the Moloney murine leukemia virus long stained with methylene blue to detect colonies. The total number ofcolonies (containing ≥ 400 cells) in duplicate dishes was: pBabest2-AS, terminal repeat [17] in both sense (pBabest2) and anti- sense (pBabest2-AS) orientation. The pBabe, pBabest2 Current Biology, Vol 8 No 5
Endogenous β-galactosidase activity in BJ cells infected with retroviruses encoding the human telomerase catalytic subunit, hTERT, and controls.
Cells were infected with (a) pBabest2-AS (assessed at PD ~87), (b) pBabe (assessed at PD ~87) and (c) pBabest2 (assessed at PD 117).
cultures reached confluence in the secondary plating assay.
and PD 123 (Figure 3). Extracts prepared from varying To test for extended proliferative potential, single cells numbers of cells indicated that the level of telomerase were isolated by limiting dilution from the nine surviving activity in the TIELF cells was comparable to that of the clones and grown in a tertiary plating assay. These cells established adenovirus5-transformed human cell line 293 have now been in continuous culture and have reached a (Figure 3). Telomerase activity was sensitive to RNase and minimum of 116 PDs (Table 1). The mean PD number for the 10 pBabest2-infected clones was 28 PDs higher thanthe 20 control clones (p < 10–5 by student’s t-test). No evi- Telomere length was measured using the terminal restric- dence of β-galactosidase activity has been detected in tion fragment (TRF) length assay as described previously these dividing cultures (Figure 2c). To date, these cultures [4]. Genomic DNA was extracted from BJ cells and from show no sign of senescence and continue to divide. The two independent TIELF clones at several PDs and the cells show no sign, so far, of aneuploidy and, like normal mean TRF length was determined. As expected, control fibroblasts, they arrest their growth in response to low BJ cells lose telomeric DNA with each PD at a rate of serum and undergo contact inhibition at high density –76 bp/PD (Figure 4a,b), similar to rates that we reported BJ cells infected with pBabest2 had reconstitutedtelomerase activity as detected by the telomeric repeatamplification protocol (TRAP) assay (Figure 3), or by theconventional primer extension assay (data not shown),whereas BJ cells infected with pBabe or pBabest2-AS hadno detectable activity (Figure 3). Reconstitution of telom-erase activity has also been observed in other telomerase-negative cells [20]. We refer to the pBabest2-infectedclones as TIELF cells (for telomerase-induced extendedlife span human fibroblasts). TIELF cells continuouslyexpressed telomerase activity over time at PD 111, PD 117 Reconstitution of telomerase activity in BJ human diploid fibroblasts.
Extended life span of telomerase-positive clones.
TRAP assays were performed as described previously [24]. Lane 1,RNase-treated 293 cell extract serving as a negative control; lanes 2–5, 293 cell extracts corresponding to 105, 104, 103 and 102 cells,respectively; lane 6, RNase-treated TIELF cell extract; lanes 7–9, cell extracts derived from 103 TIELF cells at PD 111, PD 117 and PD 123,respectively; lane 10, TIELF cell extract heated at 80°C for 3 min; lanes 11,12, cell extract corresponding to 100 and 50 TIELF cells, respectively, at PD 123; lanes 13,14, cell extracts corresponding to105 BJ cells infected with pBabest2-AS and pBabe, respectively.
*PDs reached by pBabe and pBabest2-AS represent the maximum PD.
Brief Communication
Analysis of TRFs in BJ and BJ-derived TIELFcells. Genomic DNA, purified by DNAzol (BRL), was digested with HinfI and RsaI, quantitated in triplicate by fluorometry and 1 µg DNA per lane was resolved on a 0.5% described [4]. (a) Lanes 1–3, DNA size
markers; lanes 4,5, blank; lane 6, young BJ cells at PD 29; lane 7, BJ cells at PD 51; lane 8, old BJ cells at PD 87; lanes 9–12, TIELF1 cells with increasing PDs as indicated; lanes 13,14, BJ cells infected with control (C)pBabest2-AS and pBabe, respectively, at PD ~50–55. (b) Quantitative analysis of telomeric
DNA in normal BJ cells and the TIELF cells.
Normal BJ cells lost telomeric DNA at a rate of –76 bp/PD, r = –0.98. Two TIELF cell clones increased their telomere length at a rate of +40 bp/PD, r = –0.96 and +94 bp/PD, previously in these cells [21]. However, TIELF1 and applications. Ectopic expression of telomerase in normal TIELF2, two clones derived from pBabest2-infected BJ cells may be successfully used in gene therapy to increase cells, acquired very long telomeres rapidly in the initial the life span of cells carrying the desired transgene.
expansion phase that continued to elongate with increas- Similar approaches can also be used for treatment of ing PD number; the approximate rates were +40 bp/PD and +94 bp/PD in clones TIELF1 and TIELF2, respec-tively (Figure 4a,b). An increase in the size and intensityof the TRF signal from TIELF1 is evident from the Materials and methods
Cell cultureThe neonatal human fibroblast cell strain (BJ) attained a maximum lifespan of approximately 87–90 PDs under our conditions. Cells were Our results provide direct evidence for the telomere grown in α-minimal essential medium supplemented with 10% foetal hypothesis. They show that forced expression of hTERT bovine serum (FBS). Older cells were grown in medium supplemented cDNA in normal human cells results in telomerase with 15% FBS. Cells were split at a ratio of 1:4 or 1:8 at early passageor at a ratio of 1:2 in later passages. Phosphate-buffered saline con- activity, elongation of telomere length and an extended tained no calcium or magnesium. Plating efficiency for BJ cells was life span. Thus, normal human cells can bypass the > 90%. PD number was calculated by the count/split method or as Hayflick limit and increase their replicative life span upon PD = log(Nf/N0)/log2, where Nf is the final cell number and N0 is the expression of telomerase activity. It is notable that the length of telomeric DNA was not merely maintained but increased in TIELF cells. After submission of this manu- The retroviral constructs were packaged using the highly efficient and script, similar results were reported by Bodnar et al. [22].
helper-free cell lines Phoenix-A and Phoenix-E (ATCC). Packaging In contrast to their study, in which young/midlife cells cells were transfected when approximately 80% confluent. Phoenix were used to extend life span, our study used older cells cells were incubated in 25 µM chloroquine 5 min prior to transfection that had completed 80% of their life span and, hence, with 10 µg retroviral plasmid DNA by the calcium phosphate tech-nique. At 72 h post-transfection, the virus-containing medium was col- demonstrates that senescence can be prevented even in lected and the virus titre determined using NIH3T3 cells. Titres of old cells. Our results are reminiscent of studies with germ 2–4 × 106 transducing units per ml were obtained. BJ cells were cells, which, unlike somatic cells, express telomerase infected in the presence of polybrene (4 µg/ml) using viral super- activity, have long TRFs (~15 kb) and show a net increase natants at a multiplicity of infection = 4. Throughout this work, thetransfection efficiencies were monitored by a cytomegalovirus– in the length of the telomeric DNA with age [23]. These enhanced green fluorescent protein (CMV–EGFP) construct. BJ cells results suggest that expression of telomerase in normal were incubated for 20 h at 32°C in virus-containing medium. Fresh human cells might be associated with processes related to media was added and the cells were incubated for a further 24 h in de-differentiation and could lead to generation of cells virus-free media prior to trypsinization and plating in media supple-mented with G418 (400 µg/ml) and hygromycin (20–50 µg/ml). Drug- with the stem cell property of indefinite self renewal.
resistant colonies were isolated with cloning rings approximately 2–3 TIELF cells could replace genetically unstable, estab- weeks later. Throughout this work, BJ cells carrying pM5-Eco (the lished cell lines currently used in a wide variety of Current Biology, Vol 8 No 5
Acknowledgements
23. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher This work was supported by grants from the Medical Research Council of AB, et al.: Telomere length predicts replicative capacity of human
fibroblasts. Proc Natl Acad Sci USA 1992, 89:10114-10118.
Canada and the National Cancer Institute of Canada. We thank Greg Morin 24. Kim NW, Wu F: Advances in quantitation and characterization of
and Calvin Harley for the hTRT cDNA and Garry Nolan for providing the telomerase activity by telomeric repeat amplification protocol
retroviral protocols used in this study.
(TRAP). Nucleic Acids Res 1997, 25:2595-2597.
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