 1997 Oxford University Press

Human Molecular Genetics, 1997, Vol. 6, No. 6

921–926

The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit Tracy M. Bryan1, Lidija Marusic2, Silvia Bacchetti2, Masayoshi Namba3 and Roger R. Reddel1,* 1Children’s

Medical Research Institute, 214 Hawkesbury Rd, Westmead, Sydney, NSW 2145, Australia, 2Cancer Research Group, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada and 3Okayama University Medical School, Department of Cell Biology, Institute of Cellular and Molecular Biology, 2-5-1 Shikata, Okayama 700, Japan Received January 31, 1997; Revised and Accepted March 25, 1997

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR– adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the

expression of hTR, and does not involve mutations in the hTR sequence. INTRODUCTION Normal human cells have a finite lifespan in vitro, after which they cease dividing and undergo senescence (1). Tumour cells, however, will often divide indefinitely in vitro, and hence have overcome the normal limits to proliferation and are immortal. The molecular basis of senescence and immortalisation is not well understood, but one hypothesis for which there has recently been a large amount of evidence involves the shortening of telomeres. Human telomeres consist of large tracts of the 6 bp repeat TTAGGG (reviewed in ref. 2), which diminish in size as somatic human cells age, both in vitro and in vivo (3–8). Germline cells, however, do not exhibit telomere shortening (9–11), and have been shown to have active telomerase (13), an enzyme which adds telomeric repeats to telomeres (12). It has been proposed that the expression of telomerase leads to stabilisation of telomere length and is a requirement for the immortalisation of human cells (14,15). Using a very sensitive PCR-based assay (13,16) telomerase activity has been detected in most tumour-derived cell lines and 70–90% of human tumours of different types (17). Many in vitro-immortalised human cell lines, however, lack telomerase activity and all have very long and heterogeneous telomeres, up to about 20–50 kb (13,18–20), distinct from those of telomerase-positive lines. This suggests that their telomeres have been lengthened by a novel mechanism, which we have called ALT (Alternative Lengthening of Telomeres) (21). ALT has been identified in about one third of cell lines immortalised in vitro using simian virus 40 (SV40), human papillomavirus (HPV) and chemical carcinogens or spontaneously immortalised cells (21). ALT has also recently been detected in four tumour-derived cell lines and a small number of tumour samples,

*To whom correspondence should be addressed at: Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia. Tel: +61 2 9687 2800; Fax: +61 2 9687 2120; Email: [email protected]

922

Human Molecular Genetics, 1997, Vol. 6, No. 6

indicating that ALT is a phenomenon involved in in vivo tumour development, albeit at a much lower frequency than in in vitro-derived cell lines (T.M.Bryan, A.Englezou, L.Dalla-Pozza, M.Dunham and R.R.Reddel, manuscript in preparation). The mechanism of telomere lengthening in ALT is currently unknown. It does not, however, appear to be due to a burst of telomerase activity that has subsequently been switched off since a gradual increase in telomere length in two ALT cell lines occurred in the absence of telomerase activity (19,20). It also does not appear to be a result of movement of transposons to telomeres, the telomere-maintenance mechanism utilised by Drosophila, since the telomeric sequences hybridise strongly to TTAGGG repeat probes (18,20). Another possible explanation for ALT involves the RNA subunit of telomerase. Telomerase is a ribonucleoprotein, consisting of one RNA and at least two protein subunits, all of which are encoded by separate genes. The gene for the RNA subunit has been cloned from more than 20 ciliate species (22–27), the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis (28,29), mice (30) and humans (31). These RNAs all contain a sequence that is complementary to about 1.5 telomere repeats and has been shown to act as the template for the addition of nucleotides to the G-rich strand of the telomere. Two different mutations within this template region in the telomerase RNA of Tetrahymena thermophila resulted in an altered repeat being added to telomeres, which became longer and had a broader size distribution (32). Similarly, telomerase RNA mutations in K.lactis caused ‘runaway telomere elongation’ of up to 100 times the normal telomere length (29). It is proposed that this elongation is caused by the inability of a negative regulator of telomerase to bind to altered telomere repeats, leading to unregulated telomerase action. The similarity between these long and heterogeneous telomeres in ciliates and yeast and those in human ALT cell lines suggested that ALT may be due to a mutation in the gene for the human telomerase RNA, hTR. hTR was recently cloned and sequenced; it encodes an RNA of 445 nucleotides (nt) and includes 11 nt complementary to TTAGGG (31,33). We hypothesised that the putative mutation, as well as leading to dysregulated telomere lengthening, makes telomerase undetectable in the in vitro assay for telomerase activity. To test this, we isolated and sequenced hTR from a set of human cell lines with ALT activity.

RESULTS AND DISCUSSION Sequence analysis of hTR We used PCR to amplify 598 base pairs (bp) of the hTR gene, using the primers indicated in Figure 1. This segment of the gene contains the whole 445 nt transcribed region (33). The location of the 11 nt template region is also indicated in Figure 1. The 598 bp segment was amplified from genomic DNA of six cell lines with telomerase activity and seven cell lines with no telomerase and the long telomeres characteristic of ALT (Table 1). Both strands of the PCR product were sequenced, using a total of seven internal hTR primers (see Materials and Methods).

Figure 1. Sequence of the first 598 bp of the hTR gene, showing changes from published sequence (31) in lower case bold. The position of a polymorphism is indicated by an asterisk (*) and the telomerase template region is enclosed within a box. Primers used to amplify this fragment of hTR are underlined.

Table 1. Sequencing of hTR in immortal human cell lines with either telomerase activity or ALT Cell line

Telomerase statusa

Sequenceb

Alleles at nt 514

SUSM-1

ALT

wt

A/A

WI38 VA13/2RA

ALT

wt

G/G

BET-3M

ALT

wt

A/G

GM847

ALT

G to G/A @ 228

A/A

MeT-4A

ALT

wt

A/G

IIICF/c

ALT

wt

A/G

IIICF-T/A6

ALT

wt

A/G

HeLa

telomerase

wt

G/G

293

telomerase

wt

A/A

BET-3a

telomerase

wt

A/G

BET-3b

telomerase

wt

A/G

GM639

telomerase

wt

A/A

T24

telomerase

wt

A/G

aTelomerase

status was reported in (20) and (45). Cell lines with ALT are defined as those lacking telomerase activity and having long (>20 kb) telomeres. bwt indicates sequence that is identical to that in Figure 1, except for the polymorphism at nt 514.

All cell lines had identical sequence, including a wild-type (wt) template region, with two exceptions. One was a G/A heterozygosity at nt 228 in the cell line GM847. We consider it unlikely that this change has functional significance for ALT since it occurs in only one of seven ALT cell lines. This change is not represented on Figure 1 since it is not known whether this is a mutation or an uncommon polymorphism in the population. The other sequence difference was a G/A polymorphism at nt 514. The allele present in each of the cell lines is shown in Table 1, and does not correlate with telomerase status and hence is unlikely to

923 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 6

Figure 2. Polymorphism in hTR gene at nt 514 (position numbers refer to the sequence in Figure 1 rather than the published sequence). The 598 bp PCR fragment shown in Figure 1 was digested with restriction enzyme BsrDI, which cuts at nt 514 only if this nucleotide is an A. Thus, an A allele results in a band of size 518 bp whereas a G allele results in an uncut band of 598 bp, and heterozygotes (A/G) have both bands. The results of 11 normal individuals are shown, of 47 tested. Size markers are in the lane marked M.

be involved in any changes in telomere structure. We amplified hTR from genomic DNA of 47 normal individuals to determine the frequency of this polymorphism. The recognition site for the restriction enzyme BsrDI is created if the G allele is replaced with an A allele. Therefore when the PCR product is digested with BsrDI, a G allele results in an undigested fragment of 598 bp whereas an A allele produces fragments of 518 and 80 bp, which are distinguishable on an agarose gel (Fig. 2). The frequency of the G and A alleles in the population examined was determined to be 0.3 and 0.7, respectively. In addition to this polymorphism, there were five differences in the sequence that we obtained from that originally published (31). These are indicated in Figure 1. After this study was completed we learned that a corrected sequence has been deposited in GenBank, accession number U86046.

923

Figure 3. Northern blot analysis of transcription from the hTR gene. RNA (5 µg) from the cell lines indicated was electrophoresed on a 1% agarose formaldehyde gel and probed either with a 32P-labelled cloned hTR fragment probe (top panel) or an oligonucleotide probe to 18S rRNA as a loading control (bottom panel). The telomerase status of the cell lines is given in Table 1, except for: HFF5 and WI38 are normal fibroblast strains and are telomerase-negative; IIICF-T/B1 and IIICF-T/C3 are immortalised fibroblast cell lines and are telomerase-negative, ALT-positive. VA13/2RA is cell line WI38 VA13/2RA.

Northern blot analysis of hTR expression The fact that we were able to amplify hTR from genomic DNA of all seven ALT cell lines indicates that this gene has not suffered a homozygous deletion in these cell lines. Furthermore, the gene has wt sequence in all seven cell lines, ruling out the possibility that a mutant hTR is responsible for ALT. We were therefore interested to determine whether this wt hTR is being expressed in these cell lines. hTR was amplified by PCR from the cell line HeLa, purified and cloned into plasmid pGEM-T. This cloned hTR fragment was labelled with 32P and used as a probe on a Northern blot of RNA from two telomerase-positive cell lines (293 and HeLa), two normal fibroblast cell strains (HFF5 and WI38) and nine ALT cell lines (Fig. 3 and data not shown). The normal cell strains expressed a low level of hTR. Most of the immortal cell lines produced higher but varying amounts of hTR, regardless of telomerase status. However, two ALT cell lines (SUSM-1 and WI38 VA13/2RA) produced no hTR RNA that was detectable on a Northern blot (Fig. 3). WI38 is the cell strain from which WI38 VA13/2RA was derived; there has therefore been a loss of hTR expression during immortalisation of this cell line. These two cell lines lacked detectable hTR expression even when the amount of RNA on the blot was increased from 10 to 40 µg (Fig. 4, first four lanes), whereas 1 µg of 293 RNA was sufficient to detect

Figure 4. Northern blot analysis of transcription of hTR from a recombinant adenovirus vector. RNA from the cell lines indicated was electrophoresed on a 1.2% agarose formaldehyde gel and probed with a digoxigenin-labelled hTR RNA probe. Saos-2 and 293 are uninfected positive controls for hTR expression. ‘+hTR’ indicates infection with the recombinant hTR adenovirus. VA13/2RA is cell line WI38 VA13/2RA.

expressed hTR (Fig. 4, last two lanes). A band smaller than hTR is visible on this Northern blot for all cell lines except WI38 VA13/2RA (the band in SUSM-1 is the same size as the others, although it appears larger due to a ‘smile’ in the gel). The identity of this band is unknown, but may represent an artefact of the in vitro-transcribed RNA probe (the probe used in Figure 3, in contrast, was a cloned DNA fragment). The lack of hTR expression in two ALT cell lines rules out its involvement in ALT telomere lengthening, at least in these cases. To our knowledge, all other cell lines and strains examined have been found to express at least a small amount of hTR (34). This unusual repression of hTR raised the question of whether these cell lines may be expressing a totally different RNA as a telomerase template. If this is the case, one might expect that these cell lines retain active protein subunits of telomerase, assuming that such proteins could interact with a different RNA molecule.

924

Human Molecular Genetics, 1997, Vol. 6, No. 6 subunits, although testing the expression of these protein subunits awaits the cloning of their genes. It remains a possibility, however, that ALT-positive cell lines express a different form of telomerase protein(s) that interact with a different RNA template molecule and not with hTR. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence. A likely mechanism for ALT is non-reciprocal recombination between telomeres, as has been observed in several species of yeast (35–38). This possibility is under investigation. MATERIALS AND METHODS Cell lines and culture conditions

Figure 5. Telomerase assay of ALT cell lines expressing exogenous hTR. The PCR-based TRAP assay (13) was used to analyse mock-infected SUSM-1 (lane 1), hTR-infected SUSM-1 (lane 2), mock-infected WI38 VA13/2RA (lane 3), hTR-infected WI38 VA13/2RA (lane 4) and a 293 positive control (lane 5).

In that case, providing them with a wt hTR transcript may restore activity in the telomerase assay. Introduction of hTR into ALT cell lines with no detectable hTR expression To test whether it is possible to regain telomerase activity in SUSM-1 and WI38 VA13/2RA by restoring hTR expression, hTR was introduced into these cell lines via an adenovirus vector. The cells were infected at high multiplicity with the recombinant virus and tested for expression of introduced hTR by Northern blot (Fig. 4). Both cell lines expressed high amounts of hTR after infection. The cells were then lysed and the extract used in the PCR-based assay for telomerase activity (TRAP). No activity was detected in either cell line (Fig. 5). TRAP activity has been detected in telomerase-positive cell lines infected with adenoviruses containing mutant hTRs, using substrate primers appropriate for the mutations (L.Maru
ic et al., manuscript in preparation). This indicates that it is possible to reconstitute in vitro telomerase activity by transient expression of exogenous copies of hTR if active telomerase protein subunits are present. It remains a possibility, however, that SUSM-1 and WI38 VA13/2RA do have wt telomerase protein subunits that are present in an inactive form. Our results demonstrate that ALT is independent of the RNA subunit of human telomerase. The extreme telomere lengthening in cell lines with ALT is not due to a mutated telomerase RNA, as has been observed in ciliates and yeast. In fact, at least two ALT cell lines completely lack hTR expression. This lack of hTR is not, however, the sole reason for their lack of telomerase activity. Introduction of hTR did not restore telomerase activity and it is therefore unlikely that they contain active telomerase protein

GM847 and GM639 (SV40-immortalised skin fibroblasts) were a gift from Dr O. Pereira-Smith. WI38 (normal lung fibroblasts), WI-38 VA13/2RA (SV40-immortalised WI38), HeLa (cervical carcinoma cell line), T24 (bladder carcinoma cell line) and 293 (adenovirus 5-transformed human embryonic kidney) were obtained from the American Type Culture Collection. HFF5 is a normal fibroblast cell strain established from foreskin fibroblasts obtained from Ralph Böhmer, Ludwig Institute of Cancer Research, Melbourne. SUSM-1 are 4-nitroquinoline (4NQO)immortalised liver fibroblasts (39). BET-3M, BET-3a and BET-3b were established by transfection of human bronchial epithelial cells with the SV40 early region plasmid, pRSV-T (40,41). MeT-4A is a human mesothelial cell line immortalised with pRSV-T (E.Duncan et al., unpublished data). IIICF cells are fibroblasts from an individual with Li–Fraumeni Syndrome (42). IIICF-T/A6, IIICF-T/B1 and IIICF-T/C3 were immortalised with pRSV-T (47). IIICF/c cells spontaneously immortalised in vitro (19). IIICF cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% foetal bovine serum (FBS) and gentamicin. All other fibroblasts and 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and gentamicin. Epithelial cells were maintained in Laboratory of Human Carcinogenesis-9 (LHC-9) medium in flasks coated with a matrix of collagen and fibronectin (43). Mesothelial cells were maintained in Laboratory of Human Carcinogenesis-Mesothelial medium (LHC-MM) (44), also in coated flasks. Cultures were maintained in a humidified 37
C incubator with 5% (fibroblasts) or 3.5% (epithelial and mesothelial cells) CO2 in air. PCR and sequencing of hTR Primers used for amplification of hTR were A307 (5′-GGGTTGCGGAGGGTGGGC) and A308 (5′-CGACTTTGGAGGTGCCTTCA). Genomic DNA (500 ng) from each cell line or individual was added to 250 ng of each primer in a buffer containing final concentrations of 10 mM Tris-Cl pH 9.2, 1.5 mM MgCl2, 75 mM KCl, 0.2 mM dNTPs (Boehringer Mannheim), 15% glycerol and 2.5 U Taq polymerase (Boehringer Mannheim) in a final volume of 50 µl. After placement in a hot (94
C) PCR machine, the reactions were cycled 35 times at 94
C for 30 s, 55
C for 30 s and 72
C for 1 min, with a final elongation step of 72
C for 2 min. PCR products were purified with PCR Spinclean DNA columns (Progen). They were sequenced with the fmol DNA Cycle Sequencing kit (Promega), using 6 ng template and 9 ng

925 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 6 [γ-32P]ATP end-labelled primer, cycling the reactions for 30 cycles at 95
C for 30 s, 55
C for 30 s and 70
C for 1 min. Sequencing primers used were: A309 (5′-ACGTCTCCTGCCAATTTG), A310 (5′-ATGAACGGTGGAAGGCGG), A311 (5′-GACTCGCTCCGTTCCTCT), A319 (5′-AGGGTGGGCCTGGGAGGG), A493 (5′-CCTTCCACCGTTCATTCTAG), A494 (5′-CGAAGAGTTGGGCTCTGTC) and A495 (5′-GGACTCGGCTCACACATG). To eliminate ‘stops’ (bands in all lanes), completed sequencing reactions were treated with 1 µl of a solution containing 5 U/µl terminal deoxytransferase (Boehringer Mannheim), 40 mM Tris-Cl pH 7.5, 20 mM NaCl, 20 mM MgCl2 and 1 mM dNTPs (Boehringer Mannheim) at 37
C for 60 min. Restriction enzyme analysis of sequence changes Spinclean-purified hTR PCR fragments (see above) were digested separately with the following restriction enzymes: SmaI, StuI (Boehringer Mannheim), BsrDI and EaeI (New England Biolabs). Digested DNA was electrophoresed on 3.5% or 2% MetaPhor agarose gels (FMC Bioproducts) at 4 V/cm for 16 h and stained with ethidium bromide. Northern blot analysis of hTR Figure 3. RNA was isolated from cells using a Total RNA Isolation Kit (Advanced Biotechnologies). RNA (5 µg) was subjected to electrophoresis on a 1% agarose formaldehyde gel at 6 V/cm for 4 h, transferred to Hybond-N membrane (Amersham) by capillary transfer and UV cross-linked. A 598 bp PCR fragment of HeLa hTR (see above) was ligated into plasmid pGEM-T (Promega) and propagated in the SURE strain of Escherichia coli (Stratagene). The insert was released by digestion with NcoI and NsiI (Boehringer Mannheim) and electrophoresed on a 3.5% NuSieve agarose gel (FMC Bioproducts). The hTR fragment was excised, purified with PCR Spinclean columns (Progen), labelled with [α-32P]dATP with the Gigaprime labelling kit (Bresatec) and used as a probe on Northern blots. Hybridisation and washing were carried out according to the membrane manufacturer’s instructions, at 65
C. RNA loading was confirmed by hybridisation at 55
C with a [γ-32P]ATP end-labelled 18S ribosomal RNA oligonucleotide probe (5′-GCATATGCTACTGGCAGGATCAACCAGGTA). Figure 4. Total RNA was isolated with TRIZOL (Gibco/BRL), electrophoresed on 1.2% agarose formaldehyde gels and transferred to nylon membranes (BMC). Hybridisation was with a digoxigenin-labelled RNA probe according to BMC protocols and detection was by chemiluminescence (BMC). Infection of cell lines with recombinant adenovirus Cells were infected with virus (100 p.f.u./cell) for 45 min at room temperature, refed with growth medium and harvested after 48 h. Permissive 293 cells were infected with 10 p.f.u./cell and harvested after 24 h. Telomerase assay Telomerase activity was detected by the PCR-based TRAP method (13) with the following modifications: cell extracts were prepared by three cycles of freezing and thawing in the absence of detergent (46); the TS oligonucleotide was end-labelled with

925

[γ-32P]ATP by T4 polynucleotide kinase; and the reverse primer and Taq polymerase were added after telomerase elongation and warming of the reactions to 92
C (hot start). ACKNOWLEDGEMENTS We thank Melissa Dunham and Anna Englezou for excellent technical assistance. We also thank Dr Cal Harley for informative discussions. These studies were supported by a fellowship from the NSW State Cancer Council (to R.R.R.), a National Health and Medical Research Council of Australia Biomedical Research Scholarship (to T.M.B.), and grants from the Medical Research Council and the National Cancer Institute of Canada (NCIC) and Geron Corporation (USA) (to S.B.). S.B. is a Terry Fox Cancer Research Scientist of the NCIC. REFERENCES 1. Hayflick,L. and Moorhead,P.S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Res., 25, 585–621. 2. Blackburn,E.H. (1991) Structure and function of telomeres. Nature, 350, 569–573. 3. Harley,C.B., Futcher,A.B. and Greider,C.W. (1990) Telomeres shorten during ageing of human fibroblasts. Nature, 345, 458–460. 4. Hastie,N.D., Dempster,M., Dunlop,M.G., Thompson,A.M., Green,D.K. and Allshire,R.C. (1990) Telomere reduction in human colorectal carcinoma and with ageing. Nature, 346, 866–868. 5. Lindsey,J., McGill,N.I., Lindsey,L.A., Green,D.K. and Cooke,H.J. (1991) In vivo loss of telomeric repeats with age in humans. Mutat. Res., 256, 45–48. 6. Vaziri,H., Schächter,F., Uchida,I., Wei,L., Zhu,X., Effros,R., Cohen,D. and Harley,C.B. (1993) Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am. J. Hum. Genet., 52, 661–667. 7. Lansdorp,P.M., Verwoerd,N.P., Van de Rijke,F.M., Dragowska,V., Little,M.-T., Dirks,R.W., Raap,A.K. and Tanke,H.J. (1996) Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet., 5, 685–691. 8. Henderson,S., Allsopp,R., Spector,D., Wang,S.-S. and Harley,C. (1996) In situ analysis of changes in telomere size during replicative aging and cell transformation. J. Cell Biol., 134, 1–12. 9. Cooke,H.J. and Smith,B.A. (1986) Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harbor Symp. Quant. Biol., 51, 213–219. 10. Allshire,R.C., Gosden,J.R., Cross,S.H., Cranston,G., Rout,D., Sugawara,N., Szostak,J.W., Fantes,P.A. and Hastie,N.D. (1988) Telomeric repeat from T. thermophila cross hybridizes with human telomeres. Nature, 332, 656–659. 11. De Lange,T., Shiue,L., Myers,R.M., Cox,D.R., Naylor,S.L., Killery,A.M. and Varmus,H.E. (1990) Structure and variability of human chromosome ends. Mol. Cell. Biol., 10, 518–527. 12. Greider,C.W. and Blackburn,E.H. (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell, 43, 405–413. 13. Kim,N.W., Piatyszek,M.A., Prowse,K.R., Harley,C.B., West,M.D., Ho,P.L.C., Coviello,G.M., Wright,W.E., Weinrich,S.L. and Shay,J.W. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science, 266, 2011–2015. 14. Harley,C.B. (1991) Telomere loss: Mitotic clock or genetic time bomb? Mutat. Res., 256, 271–282. 15. Harley,C.B., Vaziri,H., Counter,C.M. and Allsopp,R.C. (1992) The telomere hypothesis of cellular aging. Exp. Gerontol., 27, 375–382. 16. Piatyszek,M.A., Kim,N.W., Weinrich,S.L., Hiyama,K., Hiyama,E., Wright,W.E. and Shay,J.W. (1995) Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods Cell Sci., 17, 1–15. 17. Bacchetti,S. and Counter,C.M. (1995) Telomeres and telomerase in human cancer (Review). Int. J. Oncology, 7, 423–432. 18. Murnane,J.P., Sabatier,L., Marder,B.A. and Morgan,W.F. (1994) Telomere dynamics in an immortal human cell line. EMBO J., 13, 4953–4962. 19. Rogan,E.M., Bryan,T.M., Hukku,B., Maclean,K., Chang,A.C.-M., Moy,E.L., Englezou,A., Warneford,S.G., Dalla-Pozza,L. and Reddel,R.R. (1995) Alterations in p53 and p16INK4 expression and telomere length during spontaneous immortalization of Li–Fraumeni syndrome fibroblasts. Mol. Cell. Biol., 15, 4745–4753.

926

Human Molecular Genetics, 1997, Vol. 6, No. 6

20. Bryan,T.M., Englezou,A., Gupta,J., Bacchetti,S. and Reddel,R.R. (1995) Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J., 14, 4240–4248. 21. Bryan,T.M. and Reddel,R.R. (1997) Telomere dynamics and telomerase activity in in vitro-immortalised human cells. Eur. J. Cancer, in press. 22. Greider,C.W. and Blackburn,E.H. (1989) A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature, 337, 331–337. 23. Romero,D.P. and Blackburn,E.H. (1991) A conserved secondary structure for telomerase RNA. Cell, 67, 343–353. 24. Lingner,J., Hendrick,L.L. and Cech,T.R. (1994) Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev., 8, 1984–1998. 25. Melek,M., Davis,B.T. and Shippen,D.E. (1994) Oligonucleotides complementary to the Oxytricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization. Mol. Cell. Biol., 14, 7827–7838. 26. McCormick-Graham,M. and Romero,D.P. (1995) Ciliate telomerase RNA structural features. Nucleic Acids Res., 23, 1091–1097. 27. McCormick-Graham,M. and Romero,D.P. (1996) A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium. Mol. Cell. Biol., 16, 1871–1879. 28. Singer,M.S. and Gottschling,D.E. (1994) TLC1: Template RNA component of Saccharomyces cerevisiae telomerase. Science, 266, 404–409. 29. McEachern,M.J. and Blackburn,E.H. (1995) Runaway telomere elongation caused by telomerase RNA gene mutations. Nature, 376, 403–409. 30. Blasco,M.A., Funk,W., Villeponteau,B. and Greider,C.W. (1995) Functional characterization and developmental regulation of mouse telomerase RNA. Science, 269, 1267–1270. 31. 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.H., Le,S.Y., West,M.D., Harley,C.B., Andrews,W.H., Greider,C.W. and Villeponteau,B. (1995) The RNA component of human telomerase. Science, 269, 1236–1241. 32. Yu,G.-L., Bradley,J.D., Attardi,L.D. and Blackburn,E.H. (1990) In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature, 344, 126–132. 33. Zaug,A.J., Lingner,J. and Cech,T.R. (1996) Method for determining RNA 3′ ends and application to human telomerase RNA. Nucleic Acids Res., 24, 532–533. 34. Avilion,A.A., Piatyszek,M.A., Gupta,J., Shay,J.W., Bacchetti,S. and Greider,C.W. (1996) Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res., 56, 645–650.

35. Pluta,A.F. and Zakian,V.A. (1989) Recombination occurs during telomere formation in yeast. Nature, 337, 429–433. 36. Wang,S.-S. and Zakian,V.A. (1990) Telomere-telomere recombination provides an express pathway for telomere acquisition. Nature, 345, 456–458. 37. Lundblad,V. and Blackburn,E.H. (1993) An alternative pathway for yeast telomere maintenance rescues est1– senescence. Cell, 73, 347–360. 38. McEachern,M.J. and Blackburn,E.H. (1996) Cap-prevented recombination between terminal telomeric repeat arrays (telomere CPR) maintains telomeres in Kluyveromyces lactis lacking telomerase. Genes Dev., 10, 1822–1834. 39. Namba,M., Nishitani,K., Fukushima,F., Kimoto,T., Utsunomiya,J. and Hayflick,L. (1981) Neoplastic transformation of human diploid fibroblasts treated with chemical carcinogens and Co-60 gamma rays. GANN Monogr. Cancer Res., 27, 221–230. 40. De Silva,R. and Reddel,R.R. (1993) Similar simian virus 40-induced immortalization frequency of fibroblasts and epithelial cells from human large airways. Cell. Mol. Biol. Res., 39, 101–110. 41. De Silva,R., Whitaker,N.J., Rogan,E.M. and Reddel,R.R. (1994) HPV-16 E6 and E7 genes, like SV40 early region genes, are insufficient for immortalization of human mesothelial and bronchial epithelial cells. Exp. Cell Res., 213, 418–427. 42. Warneford,S.G., Witton,L.J., Townsend,M.L., Rowe,P.B., Reddel,R.R., Dalla-Pozza,L. and Symonds,G. (1992) Germ-line splicing mutation of the p53 gene in a cancer-prone family. Cell Growth Differ., 3, 839–846. 43. Lechner,J.F. and LaVeck,M.A. (1985) A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Culture Methods, 9, 43–48. 44. LaVeck,M.A., Somers,A.N.A., Moore,L.L., Gerwin,B.I. and Lechner,J.F. (1988) Dissimilar peptide growth factors can induce normal human mesothelial cell multiplication. In Vitro Cell. Dev. Biol., 24, 1077–1084. 45. Whitaker,N.J., Bryan,T.M., Bonnefin,P., Chang,A.C.-M., Musgrove,E.A., Braithwaite,A.W. and Reddel,R.R. (1995) Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells. Oncogene, 11, 971–976. 46. Zhu,X., Kumar,R., Mandal,M., Sharma,N., Sharma,H.W., Dhingra,U., Sokoloski,J.A., Hsiao,R.S. and Narayanan,R. (1996) Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc. Natl. Acad. Sci. USA, 93, 6091–6095. 47. Maclean,K., Rogan,E.M., Whitaker,N.J., Chang,A.C.-M., Rowe,P.B., Dalla-Pozza,L., Symonds,G. and Reddel,R.R. (1994) In vitro transformation of Li–Fraumeni syndrome fibroblasts by SV40 large T antigen mutants. Oncogene, 9, 719–725.