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Telomere maintenance mechanisms and cellular immortalization
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Telomere maintenance mechanisms and cellular immortalization Lorel M Colgin and Roger R Reddel* Immortal cell populations are able to proliferate indefinitely. Immortalization is associated with activation of processes that compensate for the telomeric shortening that accompanies cell division in normal somatic cells. In many immortal cell lines, telomere maintenance is provided by the action of the ribonucleoprotein enzyme complex, telomerase. Some immortal cell lines have undetectable or very low levels of telomerase activity and there is evidence that these cells maintain their telomeres by an alternative mechanism. Addresses Children’s Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW 2145, Australia *e-mail: [email protected] Current Opinion in Genetics & Development 1999, 9:97–103 http://biomednet.com/elecref/0959437X00900097 © Elsevier Science Ltd ISSN 0959-437X Abbreviations ALT alternative lengthening of telomeres DSB double strand break Rb retinoblastoma RT reverse transcriptase TER telomerase RNA TERT telomerase reverse transcriptase TMM telomere maintenance mechanism
Introduction Telomeres, the ends of linear chromosomes, contain repetitive DNA sequences [1]. Human telomeres possess more than a thousand copies of the hexanucleotide T2AG3 at the end of each chromosome. At least half (perhaps most) human telomeres terminate in a single-stranded 3′ GT-rich overhang [2,3] that presumably has an important role in telomere structure and function. The overhang is thought to be produced by the inability of the conventional DNA replication machinery to copy the final few bases of the lagging strand (the ‘end-replication problem’) [4,5] and also by the action of a putative 5′–3′ exonuclease that recesses the CA-rich strand [2,6]. The recession of this strand results in telomere shortening in the next round of DNA synthesis because of the reduced terminal template for leading-strand synthesis. In unicellular eukaryotes and in the germline of multicellular organisms, replication-associated loss of telomeric DNA is counteracted in a variety of ways. The best studied of these is the ribonucleoprotein enzyme complex, telomerase, that uses an RNA template to add repeats onto the G-rich strand, thus extending the single-stranded 3′ overhang. The complementary C-rich strand is thought to be partly synthesized in by other replication machinery such as DNA polymerase-α [7,8]. In Drosophila melanogaster and other Dipterans, telomere shortening is counteracted by retroelements that transpose to the telomeres [9,10]. In the mosquito Anopheles gambiae, telomeres are maintained
primarily by a recombination mechanism [11] and this may also occur as a backup mechanism in yeast [12,13]. In normal human somatic cells — in which telomerase activity is present at either low or undetectable levels — telomere shortening is not counteracted, such that progressive telomere loss occurs with each cell division. According to the telomere hypothesis of senescence, telomere erosion eventually acts as the trigger for cells to senesce [4,14]. A corollary of this is that cell immortalization requires a mechanism for prevention of telomere attrition. This prediction appears to be confirmed by the observation (described in more detail below) that all immortalized human cell lines have a telomere-maintenance mechanism (TMM), either telomerase or an alternative mechanism — alternative lengthening of telomeres (ALT). Here we summarize recent progress in understanding the genes and mechanisms involved in telomere maintenance and their role in immortalization.
Telomerase genes The components of the multisubunit ribonucleoprotein enzyme, telomerase, are encoded by distinct genes. The catalytic subunit (TERT [telomerase reverse transcriptase]) of human telomerase was identified in 1997 [15–18], 12 years after the biochemical activity was first described in Tetrahymena thermophila [19]. As with the catalytic subunits of unicellular eukaryotes Euplotes aediculatus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Oxytricha trifallax and Tetrahymena [15,20,21,22•,23•], and of the mouse [24•], the human protein is a reverse transcriptase (RT) and has seven protein domains in common with retroviral and retrotransposon RTs (Figure 1). These conserved motifs are part of a protein fold that forms the active site of the enzyme and include three aspartates critical for catalysis (reviewed in [25]). There is an additional motif, T, that is telomerase specific, and another motif recently identified in ciliate TERTs [23•]. Telomerase differs from other RTs in that it carries its own template RNA. Genes encoding mammalian telomerase RNA (TER) have been cloned previously [26], as have genes for another protein, TEP1, that is part of the telomerase holoenzyme complex [27,28]. By analogy with Tetrahymena, where three protein subunits have now been identified [22•,23•,29], there may be one or more additional mammalian telomerase proteins. More needs to be learned about the size and composition of the telomerase holoenzyme. In Euplotes crassus, the size of the holoenzyme ranges from 280 kDa to 5 MDa, and its enzymatic properties vary, depending on the life-cycle stage [30•]. Adding to the potential complexity, several variant hTERT mRNAs have been identified, presumably arising
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Figure 1
T
1
2
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B'
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* * 1
1, 2, 3: insertion variants
α
β
* 2
α, β: deletion variants
non-coding
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Domains and variants of the human telomerase catalytic subunit, hTERT. Domains 1 and 2, and A through E are similar to those found in other RT molecules, and domain T is conserved among TERT proteins [15]. Variant cDNAs have been identified with deletions (α and β) from
or additions to the cDNA sequence [17]. Insertions 1 and 2 result in truncation of the protein, and insertion 3 encodes an alternative carboxyl terminus.
from alternative splicing [17] (Figure 1). Of the variants identified to date, one (α) has an in-frame deletion of 12 amino acids from RT domain A, another (β) has a 182nucleotide deletion that results in a premature stop codon, and another encodes an alternative carboxyl terminus [17]. It has been shown recently that telomerase activity in developing human heart, liver and kidney coincided with the expression of full-length hTERT mRNA, but some splice variants continued to be expressed in the absence of enzymatic activity [31•]. These data suggest that alternative splicing may play a role in regulating telomerase activity during development and differentiation.
TRF1 was the first mammalian telomere binding protein identified [46]. Homodimers of TRF1 bind double-stranded telomeric repeats, but not single-stranded DNA, through a Myb-type DNA-binding domain (reviewed in [47,48•]). TRF1 mediates parallel pairing of telomeric tracts [48•], is likely to have a role in telomere protection, and may be important for regulating telomere length through cis-mediated inhibition of telomerase [49].
Expression of hTER and hTERT in rabbit reticulocyte lysates [32•] and transfection of hTERT into hTER-positive telomerase-negative cells [32•–36•] results in telomerase activity. These hTERT transfection data suggest that the availability of hTERT may be rate-limiting for the assembly of catalytically active telomerase. Control of hTERT expression appears to be primarily transcriptional [16,17], although phosphorylation may affect telomerase activity at least indirectly [37–39] and perhaps directly [40•]. Myc induces telomerase activity by increasing hTERT transcription [41•] and the cloning and analysis of hTERT gene upstream sequences has identified myc binding sites, as well as hormone-response elements and potential binding sites for other transcription factors that may be involved in controlling hTERT expression [42•].
Telomere-binding proteins Studies of telomerase-positive immortalized cells [43,44] and of telomerase-negative normal cells [45] reveal evidence for the existence of factors other than telomerase that regulate telomere length. There is a rapidly increasing list of proteins reported to bind to the telomere and/or affect telomere length (only a few of which are highlighted here). Telomerebinding proteins may affect telomere maintenance by modulating the activity of telomerase, or by protecting chromosome ends against loss of telomere repeats.
TRF2 is a distantly related homolog of TRF1 that also binds double-stranded telomere repeats and is ubiquitously expressed [50]. TRF2 has the same general protein architecture as TRF1 except that its amino terminus carries mostly basic residues instead of the acidic residues seen in TRF1. Unexpectedly, overexpression of dominant negative alleles of TRF2 in human cells caused loss of the terminal single-stranded 3′ overhang, an increase in chromosome end-to-end fusions, and irreversible growth arrest in a senescence-like state [51••]. It is not known whether TRF2 serves to protect the G-rich overhang directly — which is unlikely, as TRF2 cannot bind single-stranded DNA in vitro — whether it stimulates the formation of G tails by recruiting an exonuclease or other factor, or whether TRF2 recruits other telomere binding proteins that protect G tails. A recently identified putative telomere-binding protein is UP1, the amino-terminal fragment of heterogeneous nuclear ribonucleoprotein A1 [52]. UP1 appears to bind both telomeric repeats and telomerase and it has therefore been proposed that proteolytic processing of A1 to UP1 may recruit telomerase to the ends of telomeres.
Double strand break repair proteins The Ku heterodimer is critically important for end-joining of DNA double-stranded breaks (DSBs). In addition, in the yeast S. cerevisiae, there is evidence that it has an important role in telomere maintenance where end-to-end fusion is not desirable, thus indicating that its role is context-dependent
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The Rad50/Mre11/Xrs2 protein complex is also involved in both DSB-end-joining repair and telomere maintenance in yeast [60••,61•]. The complex may be an exonuclease or have a structural function, and it has been proposed that it may be required for preparation of telomeric ends or their presentation to telomerase to facilitate its enzymatic activity [62]. An additional yeast telomere-binding protein, Cdc13p, binds single-stranded telomeric DNA and may have a dual role in protecting the end and facilitating access of telomerase [63,64]. Interestingly, epistasis analysis showed that Rad50/Mre11/Xrs2 functions in the same telomere-maintenance pathway as telomerase, but Ku and Cdc13 function in separate pathways [60••].
Telomere maintenance and immortalization Many normal human cells and tissues do not have detectable telomerase activity but the list of known exceptions is growing [65–70]. Where telomerase is present in normal cells, it is generally thought to be insufficient for prevention of telomere erosion [65]. Recent evidence suggests, however, that activation of telomerase may cause telomere lengthening in normal B lymphocytes in germinal centers [71•], possibly providing a temporary extension of proliferative capacity during clonal expansion. Mammalian telomerase activity was first discovered in the archetypal immortalized human cell line, HeLa [72]. Immortalization of human cells by viral genes usually proceeds via two stages (Figure 2). In the first stage, there is a finite extension of proliferative potential accompanied by telomere shortening, ending in a period of culture crisis where the culture in on longer expanding due to increased cell death of senescence. In the second stage, which does not always occur, a small number of cells escape from crisis and acquire an apparently unlimited ability to proliferate and telomere length is maintained [73]. Escape from crisis may be associated with acquisition of telomerase activity [74–77] and crisis may be prevented by transduction of pre-crisis cells with hTERT cDNA [78•]. In some cases, cells that escape from crisis have no detectable telomerase activity [77]. Southern analysis with
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Figure 2
ALT Germline length hTERT transduction
Length
Telomerase Crisis
Senescence
(reviewed in [53,54]). Crosslinking experiments showed that Ku binds telomeric DNA [55•], and yeast cells defective in either of the Ku genes (YKU70 or YKU80) lose most but not all of their telomeric repeats [56,57]. In addition to preventing telomere repeat attrition, Ku plays a role in telomere end structure. Telomeric G tails are only detected in yeast during S-phase but in strains defective in YKU80, these ends are present constitutively [55•,58•]. The Ku heterodimer may regulate an exonuclease that generates these G tails, normally keeping their production confined to S-phase, or participate in protecting G tails from degradation [54]. Ku may also be involved in clustering of telomeres and possibly interaction with the nuclear envelope, as deletion of YKU70 or YKU80 disrupts the normal clustering and localization of yeast telomeres [59•].
Mean telomere length
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Normal human somatic cells are usually telomerase-negative and their proliferation is accompanied by a progressive decrease in telomere length that is thought to result eventually in senescence (i.e. permanent proliferative arrest). Cells transduced with hTERT express telomerase, elongate their telomeres to germline length, and escape from senescence. Cells transduced with viral oncogenes continue to proliferate beyond the senescence barrier and their telomeres continue to shorten until culture crisis ensues. Most cells in crisis either senesce or die but rare immortalized cells arise that have activated a telomeremaintenance mechanism — either by employing telomerase or ALT. ALT cells have telomeres of heterogeneous length, ranging from short to very long, with the mean length often being greater than that of germline cells. The telomeres of telomerase-positive immortal cells tend to be short or in the normal range.
a telomere-specific probe showed that telomerasenegative immortalized cells have a characteristic pattern of terminal restriction fragment length that ranges from very short to abnormally long [77,79,80•]. In situ hybridization analyses showed that this heterogeneity of telomere length occurs within individual cells [81,82]. Recent evidence indicates that telomeric DNA fragments may be released from the chromosomes in such cells [83,84]. In clonally derived cell lines immortalized in vitro it was shown that the acquisition of abnormally long telomeres coincided with escape from crisis [77] (Figure 2), demonstrating the existence of one or more ALT mechanisms. It is of interest that mouse cells rendered telomerase-negative by targeted disruption of the TER gene undergo immortalization readily [85], possibly through the efficient utilization of an ALT mechanism. Human fibroblasts and other cells of mesenchymal origin seem more likely to utilize the ALT pathway for immortalization than epithelial cells, although a minority of in vitro immortalized epithelial cell lines, carcinoma-derived cell lines, and carcinomas do exhibit ALT [77,80•]. Low levels of telomerase may be detected in some normal human epithelial cells [86,87] but activity is usually undetectable
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in normal fibroblasts. It is therefore possible that upregulation of telomerase at immortalization occurs more readily in epithelial cells than in fibroblasts. All immortalized human cell lines examined to date have either telomerase or an ALT TMM [72,74,76,77,80•,88]. Inhibition of telomerase with antisense hTER or repression of either telomerase or ALT activity by chromosome transfer resulted in telomere shortening and re-imposition of a finite proliferative potential [26,89,90]. These observations, together with the close temporal correlation between the activation of these mechanisms and escape from crisis, are strong circumstantial evidence for a role of TMMs in immortalization. It is not clear, however, whether expression of a TMM alone is sufficient for immortalization. Transduction of telomerase-negative normal human retinal pigment epithelial cells and fibroblasts with hTERT cDNA under the control of a heterologous promoter resulted in expression of telomerase enzyme activity, in prevention or reversal of the telomere shortening that normally accompanies cell division, and in continued cell division beyond the population doubling levels at which the control cultures became senescent [91••,92••]. Follow-up studies have shown that the hTERT-transfected fibroblasts have continued dividing for 200 population doublings more than the control cultures [93•] and have normal checkpoint controls [93•,94•]. It will be important to determine whether these cells eventually become senescent despite well-maintained telomeres and a remarkable extension of lifespan, or whether genetic changes equivalent to the effects of viral oncoproteins are also required for indefinite proliferation. It has been proposed that there may be mitotic clocks other than or in addition to telomere shortening and that each of these must be inactivated in order for immortalization to occur [95]. Consistent with this hypothesis, it has been observed that some types of hamster cells express telomerase and maintain their telomeres but still undergo senescence [96,97•]. For human keratinocytes and breast epithelial cells grown in defined media, it has been found that inactivation of the retinoblastoma (Rb) gene ‘pathway’ is required for telomerase-induced lifespan extension [98•]; it is not clear to what extent this requirement is cell-type specific or determined by the in vitro growth conditions. In contrast, hTERT-transfected retinal pigment epithelial cells have a substantial extension of their proliferative capacity but appear to have an intact Rb pathway in that they express p16INK4, and have hypophosphorylated pRb when confluent [94•]. It is not yet possible to induce ALT activity experimentally because the genes involved in this/these mechanism(s) are unknown. It has been suggested that ALT cells may use a variant or mutant telomerase that is not detected in existing biochemical assays and not subject to the usual feedback controls so that it produces telomeres with abnormal lengths [77]. On the basis of the non-telomerase TMMs found in
other species, ALT could also be caused by retrotransposition or recombination. Retrotransposition of a sequence unrelated to the telomere repeat seems unlikely in ALT cells where the total amount of telomeric sequence hybridizing to a (TTAGGG)3 probe increases [99]. The rapid lengthening of telomeres seen in a telomerase-negative cell line [79] is consistent with recombination. If ALT is recombinational, the Ku and related genes will be interesting candidates in view of their role in mammalian recombination, including V(D)J joining (reviewed in [100]) and their presence and role at the yeast telomere. The mammalian homologues of yeast genes in other epistasis groups for telomere maintenance [60] are also of great interest.
Conclusions In the past year, it has become possible for the first time to induce telomerase activity in mammalian cells by the transfer of TERT cDNA and thus to begin experiments that directly address the role of telomere maintenance in control of proliferative potential. These experiments may have major implications for our understanding of both ageing and cancer. Continuing challenges for telomerase research include elucidating the composition of telomerase complexes, the role of TERT variants, the interactions between telomerase and other components of the telomere, control of TERT transcription, and how all of these processes are regulated in development. Additional challenges for understanding telomere biology include understanding maintenance of telomere integrity in normal cells and mechanisms of telomere length maintenance in telomerase-negative immortalized cells.
Acknowledgements The authors are supported by project grants from the National Health and Medical Research Council of Australia and the Carcinogenesis Fellowship of the New South Wales Cancer Council, and thank Vicki Lundblad and Silvia Bacchetti for their comments on the manuscript.
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48. Griffith J, Bianchi A, de Lange T: TRF1 promotes parallel pairing of • telomeric tracts in vitro. J Mol Biol 1998, 278:79-88. Electron microscopy and mass analysis suggests that dimers and tetramers of the telomere-specific TRF1 protein bind to hexanucleotide tandem repeats. The authors also show that two telomeric tracts tend to exhibit parallel pairing along a TRF1-covered segment. 49. van Steensel B, de Lange T: Control of telomere length by the human telomeric protein TRF1. Nature 1997, 385:740-743. 50. Broccoli D, Smogorzewska A, Chong L, de Lange T: Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat Genet 1997, 17:231-235. 51. van Steensel B, Smogorzewska A, de Lange T: TRF2 protects •• human telomeres from end-to-end fusions. Cell 1998, 92:401-413. Expression of a dominant negative mutant of the telomere-binding protein TRF2 in a human telomerase-positive fibrosarcoma cell line induced loss of telomeric G-rich strand overhangs, end-to-end fusion of telomeres, and a rapid senescence-like growth arrest. Suggests that TRF2 may protect chromosome ends by maintaining the correct structure at telomeric termini, and raises the possibility that senescence of normal cells may be associated with loss of TRF2 from shortened telomeres. 52. LaBranche H, Dupuis S, Ben-David Y, Bani M-R, Wellinger RJ, Chabot B: Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase. Nat Genet 1998, 19:199-202. 53. Weaver DT: Telomeres: moonlighting by DNA repair proteins. Curr Biol 1998, 8:R492-R494. 54. Bertuch A, Lundblad V: Telomeres and double-strand breaks: trying to make ends meet. Trends Cell Biol 1998, 8:339-342. 55. Gravel S, Larrivée M, Labrecque P, Wellinger RJ: Yeast Ku as a • regulator of chromosomal DNA end structure. Science 1998, 280:741-744. In yeast, 3′ overhangs at the ends of telomeres are normally present only during S phase but in strains lacking Ku these overhangs are longer and pre-
57.
Boulton SJ, Jackson SP: Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res 1996, 24:4639-4648.
58. Polotnianka RM, Li J, Lustig AJ: The yeast Ku heterodimer is • essential for protection of the telomere against nucleolytic and recombinational activities. Curr Biol 1998, 8:831-834. Ku genes are involved in double strand break repair and, in mammals, V(D)J recombination. This paper reports that mutations in yeast Ku increase the length of 3′ overhangs on telomeres and cause loss of the cell-cycle dependence of overhangs. Ku mutants also show chromosomal instability. Genetic analyses indicate that Ku interacts with the single-stranded telomeric DNA binding protein Cdc13p. 59. Laroche T, Martin SG, Gotta M, Gorham HC, Pryde FE, Louis EJ, • Gasser SM: Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres. Curr Biol 1998, 8:653-656. Deletion of either the yeast Ku70 or Ku80 genes disrupts the normal clustering of telomeres and causes dispersion of the telomere-specific binding protein, Rap1p. The telomere-associated silencing factors, Sir3p and Sir4p, are also dispersed and telomeric silencing is lost. The Ku proteins are implicated in the spatial organization of telomeres within the nucleus. 60. Nugent CI, Bosco G, Ross LO, Evans SK, Salinger AP, Moore JK, •• Haber JE, Lundblad V: Telomere maintenance is dependent on activities required for end repair of double-strand breaks. Curr Biol 1998, 8:657-660. Yeast Ku is identified as a third epistasis group required for telomere maintenance. The other two groups contain telomerase and the single-stranded DNA-binding protein Cdc13p. Although the Rad50/Mre11/Xrs2 complex is required along with Ku for double strand break repair, it is in the telomerase epistasis group for telomere maintenance. The authors suggest that telomere termini are identified by the cell as double-strand breaks but that there are mechanisms for preventing their ‘repair’. 61. Boulton SJ, Jackson SP: Components of the Ku-dependent non • homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J 1998, 17:1819-1828. Yeast cells deficient in Ku are unable to silence a URA3 gene placed at the telomere. TEL1 — a homolog of DNA-PK, which complexes with Ku to function in mammalian DSB repair — is not involved in telomeric silencing or yeast Ku-dependent DSB repair. The authors also show that the yeast RAD50/MRE11/XRS2 complex and Ku have different roles in DSB repair, and although the Rad50 complex is needed for telomere maintenance, it has little effect on silencing. 62. Tsubouchi H, Ogawa H: A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. Mol Cell Biol 1998, 18:260-268. 63. Nugent CI, Hughes TR, Lue NF, Lundblad V: Cdc13p: a singlestrand telomeric DNA binding protein with a dual role in yeast telomere maintenance. Science 1996, 274:249-252. 64. Lin J-J, Zakian VA: The Saccharomyces CDC13 protein is a singlestrand TG1-3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo. Proc Natl Acad Sci USA 1996, 93:13760-13765. 65. Broccoli D, Young JW, de Lange T: Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci USA 1995, 92:9082-9086. 66. Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S: Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995, 85:2315-2320. 67.
Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, Shay JW, Ishioka S, Yamakido M: Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995, 155:3711-3715.
68. Härle-Bachor C, Boukamp P: Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA 1996, 93:6476-6481. 69. Yasumoto S, Kunimura C, Kikuchi K, Tahara H, Ohji H, Yamamoto H, Ide T, Utakoji T: Telomerase activity in normal human epithelial cells. Oncogene 1996, 13:433-439.
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70. Hsiao R, Sharma HW, Ramakrishnan S, Keith E, Narayanan R: Telomerase activity in normal human endothelial cells. Anticancer Res 1997, 17:827-832. 71. Weng N-P, Hathcock KS, Hodes RJ: Regulation of telomere length • and telomerase in T and B cells: a mechanism for maintaining replicative potential. Immunity 1998, 9:151-157. A review of the current understanding of telomere-length regulation in T and B cells. Of special interest is the finding of telomerase activity capable of maintaining telomere lengths in germinal center B cells. 72. Morin GB: The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 1989, 59:521-529. 73. Girardi AJ, Jensen FC, Koprowski H: SV40-induced transformation of human diploid cells: crisis and recovery. J Cell Comp Physiol 1965, 65:69-84. 74. Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S: Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1992, 11:1921-1929. 75. Klingelhutz AJ, Barber SA, Smith PP, Dyer K, McDougall JK: Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells. Mol Cell Biol 1994, 14:961-969. 76. Counter CM, Botelho FM, Wang P, Harley CB, Bacchetti S: Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes. J Virol 1994, 68:3410-3414. 77.
Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR: Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995, 14:4240-4248.
78. Counter CM, Hahn WC, Wei W, Dickinson Caddle S, • Beijersbergen RL, Lansdorp PM, Sedivy JM, Weinberg RA: Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc Natl Acad Sci USA 1998, 95:14723-14728. Expression of hTERT cDNA in SV40-transformed, pre-crisis (non-immortalized) human embryonic kidney cells results in telomere maintenance, and allows cells to continue growing without entering crisis. hTERT that is modified by the addition of a hemagglutinin epitope tag at its carboxyl terminus does not prevent telomere shortening, even though it is active in the in vitro TRAP assay and cells expressing this form of hTERT do enter crisis. 79. Murnane JP, Sabatier L, Marder BA, Morgan WF: Telomere dynamics in an immortal human cell line. EMBO J 1994, 13:4953-4962. 80. Bryan TM, Englezou A, Dalla-Pozza L, Dunham MA, Reddel RR: • Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat Med 1997, 3:1271-1274. A demonstration that a mechanism for alternative lengthening of telomeres (ALT), characterized by telomeres of heterogeneous length (ranging from short to very long) and absence of detectable telomerase activity, is present in some tumor-derived cell lines and tumors. ALT is therefore not confined to cell lines immortalized in vitro. 81. Henderson S, Allsopp R, Spector D, Wang S-S, Harley C: In situ analysis of changes in telomere size during replicative aging and cell transformation. J Cell Biol 1996, 134:1-12. 82. Lansdorp PM, Poon S, Chavez E, Dragowska V, Zijlmans M, Bryan T, Reddel R, Egholm M, Bacchetti S, Martens U: Telomeres in the hematopoietic system. Ciba Found Symp 1997, 211:209-218. 83. Tokutake Y, Matsumoto T, Watanabe T, Maeda S, Tahara H, Sakamoto S, Niida H, Sugimoto M, Ide T, Furuichi Y: Extra-chromosome telomere repeat DNA in telomerase-negative immortalized cell lines. Biochem Biophys Res Commun 1998, 247:765-772. 84. Ogino H, Nakabayashi K, Suzuki M, Takahashi E, Fujii M, Suzuki T, Ayusawa D: Release of telomeric DNA from chromosomes in immortal human cells lacking telomerase activity. Biochem Biophys Res Commun 1998, 248:223-227. 85. Blasco MA, Lee H-W, Hande MP, Samper E, Lansdorp PM, DePinho RA, Greider CW: Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 1997, 91:25-34. 86. Ramakrishnan S, Eppenberger U, Mueller H, Shinkai Y, Narayanan R: Expression profile of the putative catalytic subunit of the telomerase gene. Cancer Res 1998, 58:622-625. 87.
Kolquist KA, Ellisen LW, Counter CM, Meyerson M, Tan LK, Weinberg RA, Haber DA, Gerald WL: Expression of TERT in early
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premalignant lesions and a subset of cells in normal tissues. Nat Genet 1998, 19:182-186. 88. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266:2011-2015. 89. Nakabayashi K, Ogata T, Fujii M, Tahara H, Ide T, Wadhwa R, Kaul SC, Mitsui Y, Ayusawa D: Decrease in amplified telomeric sequences and induction of senescence markers by introduction of human chromosome 7 or its segments in SUSM-1. Exp Cell Res 1997, 235:345-353. 90. Horikawa I, Oshimura M, Barrett JC: Repression of the telomerase catalytic subunit by a gene on human chromosome 3 that induces cellular senescence. Mol Carcinog 1998, 22:65-72. 91. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu C-P, Morin GB, •• Harley CB, Shay JW, Lichtsteiner S, Wright WE: Extension of lifespan by introduction of telomerase into normal human cells. Science 1998, 279:349-352. Stable expression of exogenous hTERT cDNA in telomerase-negative normal retinal pigment epithelial cells and foreskin fibroblasts induces telomerase activity. A normal karyotype is maintained and cells exceed their normal replicative lifespan by at least 20 population doublings. These findings demonstrate that telomerase activity permits escape from senescence. 92. Vaziri H, Benchimol S: Reconstitution of telomerase activity in •• normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 1998, 8:279-282. Retrovirus-mediated expression of hTERT in normal human diploid fibroblasts results in telomerase activity and telomere maintenance in these cells and extends replicative lifespan for at least 26 population doublings, demonstrating that telomerase activity permits escape from senescence. 93. Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, • White MA, Wright WE, Shay JW: Lack of cancer-associated changes in human fibroblasts after immortalization with telomerase. Nat Genet 1999, in press. hTERT-transfected fibroblasts proliferate for at least 200 population doublings more than the controls. Cell-cycle checkpoints activated by low serum concentration, confluence, and UV-B irradiation appear to be intact. As shown previously by others in normal cells, oncogenic ras is able to induce a senescence-like state. The frequency of karyotypic abnormalities was not greater than that seen in late-passage normal cells. 94. Jiang X-R, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, • Beeche M, Bodnar AG, Wahl GM, Tlsty TD, Chiu C-P: Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 1999, in press. At twice the normal number of population doublings, human fibroblasts and retinal pigment epithelial cells expressing hTERT cDNA are able to undergo growth arrest in response to low-serum concentration, confluence, and cellcycle blocking chemicals. The cells retain anchorage dependence. 95. Reddel R: A reassessment of the telomere hypothesis of senescence. Bioessays 1999, in press. 96. Newbold RF: Genetic control of telomerase and replicative senescence in human and rodent cells. Ciba Found Symp 1997, 211:177-189. 97. •
Carman TA, Afshari CA, Barrett JC: Cellular senescence in telomerase-expressing syrian hamster embryo cells. Exp Cell Res 1998, 244:33-42. Syrian hamster embryo cells were found to express telomerase and to undergo senescence even though their telomeres had not shortened. The data suggest that the mitotic clock in these cells does not involve telomere shortening. 98. Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, • Klingelhutz AJ: Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 1998, 396:84-88. The authors show that induction of telomerase activity is not sufficient to immortalize human keratinocyte or mammary epithelial cells. Inactivation of the pRb/p16 pathway — but not p53 or p19ARF — is an additional equirement for hTERT-induced lifespan extension in these cells. 99. Rogan EM, Bryan TM, Hukku B, Maclean K, Chang AC-M, Moy EL, Englezou A, Warneford SG, Dalla-Pozza L, Reddel RR: Alterations in p53 and p16INK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts. Mol Cell Biol 1995, 15:4745-4753. 100. Lieber MR, Grawunder U, Wu X, Yaneva M: Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of doublestrand breaks. Curr Opin Genet Dev 1997, 7:99-104.
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Telomere maintenance mechanisms and cellular immortalization Lorel M Colgin and Roger R Reddel* Immortal cell populations are able to proliferate indefinitely. Immortalization is associated with activation of processes that compensate for the telomeric shortening that accompanies cell division in normal somatic cells. In many immortal cell lines, telomere maintenance is provided by the action of the ribonucleoprotein enzyme complex, telomerase. Some immortal cell lines have undetectable or very low levels of telomerase activity and there is evidence that these cells maintain their telomeres by an alternative mechanism. Addresses Children’s Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW 2145, Australia *e-mail: [email protected] Current Opinion in Genetics & Development 1999, 9:97–103 http://biomednet.com/elecref/0959437X00900097 © Elsevier Science Ltd ISSN 0959-437X Abbreviations ALT alternative lengthening of telomeres DSB double strand break Rb retinoblastoma RT reverse transcriptase TER telomerase RNA TERT telomerase reverse transcriptase TMM telomere maintenance mechanism
Introduction Telomeres, the ends of linear chromosomes, contain repetitive DNA sequences [1]. Human telomeres possess more than a thousand copies of the hexanucleotide T2AG3 at the end of each chromosome. At least half (perhaps most) human telomeres terminate in a single-stranded 3′ GT-rich overhang [2,3] that presumably has an important role in telomere structure and function. The overhang is thought to be produced by the inability of the conventional DNA replication machinery to copy the final few bases of the lagging strand (the ‘end-replication problem’) [4,5] and also by the action of a putative 5′–3′ exonuclease that recesses the CA-rich strand [2,6]. The recession of this strand results in telomere shortening in the next round of DNA synthesis because of the reduced terminal template for leading-strand synthesis. In unicellular eukaryotes and in the germline of multicellular organisms, replication-associated loss of telomeric DNA is counteracted in a variety of ways. The best studied of these is the ribonucleoprotein enzyme complex, telomerase, that uses an RNA template to add repeats onto the G-rich strand, thus extending the single-stranded 3′ overhang. The complementary C-rich strand is thought to be partly synthesized in by other replication machinery such as DNA polymerase-α [7,8]. In Drosophila melanogaster and other Dipterans, telomere shortening is counteracted by retroelements that transpose to the telomeres [9,10]. In the mosquito Anopheles gambiae, telomeres are maintained
primarily by a recombination mechanism [11] and this may also occur as a backup mechanism in yeast [12,13]. In normal human somatic cells — in which telomerase activity is present at either low or undetectable levels — telomere shortening is not counteracted, such that progressive telomere loss occurs with each cell division. According to the telomere hypothesis of senescence, telomere erosion eventually acts as the trigger for cells to senesce [4,14]. A corollary of this is that cell immortalization requires a mechanism for prevention of telomere attrition. This prediction appears to be confirmed by the observation (described in more detail below) that all immortalized human cell lines have a telomere-maintenance mechanism (TMM), either telomerase or an alternative mechanism — alternative lengthening of telomeres (ALT). Here we summarize recent progress in understanding the genes and mechanisms involved in telomere maintenance and their role in immortalization.
Telomerase genes The components of the multisubunit ribonucleoprotein enzyme, telomerase, are encoded by distinct genes. The catalytic subunit (TERT [telomerase reverse transcriptase]) of human telomerase was identified in 1997 [15–18], 12 years after the biochemical activity was first described in Tetrahymena thermophila [19]. As with the catalytic subunits of unicellular eukaryotes Euplotes aediculatus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Oxytricha trifallax and Tetrahymena [15,20,21,22•,23•], and of the mouse [24•], the human protein is a reverse transcriptase (RT) and has seven protein domains in common with retroviral and retrotransposon RTs (Figure 1). These conserved motifs are part of a protein fold that forms the active site of the enzyme and include three aspartates critical for catalysis (reviewed in [25]). There is an additional motif, T, that is telomerase specific, and another motif recently identified in ciliate TERTs [23•]. Telomerase differs from other RTs in that it carries its own template RNA. Genes encoding mammalian telomerase RNA (TER) have been cloned previously [26], as have genes for another protein, TEP1, that is part of the telomerase holoenzyme complex [27,28]. By analogy with Tetrahymena, where three protein subunits have now been identified [22•,23•,29], there may be one or more additional mammalian telomerase proteins. More needs to be learned about the size and composition of the telomerase holoenzyme. In Euplotes crassus, the size of the holoenzyme ranges from 280 kDa to 5 MDa, and its enzymatic properties vary, depending on the life-cycle stage [30•]. Adding to the potential complexity, several variant hTERT mRNAs have been identified, presumably arising
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Figure 1
T
1
2
A
B'
C
D
E
* * 1
1, 2, 3: insertion variants
α
β
* 2
α, β: deletion variants
non-coding
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Current Opinion in Genetics & Development
Domains and variants of the human telomerase catalytic subunit, hTERT. Domains 1 and 2, and A through E are similar to those found in other RT molecules, and domain T is conserved among TERT proteins [15]. Variant cDNAs have been identified with deletions (α and β) from
or additions to the cDNA sequence [17]. Insertions 1 and 2 result in truncation of the protein, and insertion 3 encodes an alternative carboxyl terminus.
from alternative splicing [17] (Figure 1). Of the variants identified to date, one (α) has an in-frame deletion of 12 amino acids from RT domain A, another (β) has a 182nucleotide deletion that results in a premature stop codon, and another encodes an alternative carboxyl terminus [17]. It has been shown recently that telomerase activity in developing human heart, liver and kidney coincided with the expression of full-length hTERT mRNA, but some splice variants continued to be expressed in the absence of enzymatic activity [31•]. These data suggest that alternative splicing may play a role in regulating telomerase activity during development and differentiation.
TRF1 was the first mammalian telomere binding protein identified [46]. Homodimers of TRF1 bind double-stranded telomeric repeats, but not single-stranded DNA, through a Myb-type DNA-binding domain (reviewed in [47,48•]). TRF1 mediates parallel pairing of telomeric tracts [48•], is likely to have a role in telomere protection, and may be important for regulating telomere length through cis-mediated inhibition of telomerase [49].
Expression of hTER and hTERT in rabbit reticulocyte lysates [32•] and transfection of hTERT into hTER-positive telomerase-negative cells [32•–36•] results in telomerase activity. These hTERT transfection data suggest that the availability of hTERT may be rate-limiting for the assembly of catalytically active telomerase. Control of hTERT expression appears to be primarily transcriptional [16,17], although phosphorylation may affect telomerase activity at least indirectly [37–39] and perhaps directly [40•]. Myc induces telomerase activity by increasing hTERT transcription [41•] and the cloning and analysis of hTERT gene upstream sequences has identified myc binding sites, as well as hormone-response elements and potential binding sites for other transcription factors that may be involved in controlling hTERT expression [42•].
Telomere-binding proteins Studies of telomerase-positive immortalized cells [43,44] and of telomerase-negative normal cells [45] reveal evidence for the existence of factors other than telomerase that regulate telomere length. There is a rapidly increasing list of proteins reported to bind to the telomere and/or affect telomere length (only a few of which are highlighted here). Telomerebinding proteins may affect telomere maintenance by modulating the activity of telomerase, or by protecting chromosome ends against loss of telomere repeats.
TRF2 is a distantly related homolog of TRF1 that also binds double-stranded telomere repeats and is ubiquitously expressed [50]. TRF2 has the same general protein architecture as TRF1 except that its amino terminus carries mostly basic residues instead of the acidic residues seen in TRF1. Unexpectedly, overexpression of dominant negative alleles of TRF2 in human cells caused loss of the terminal single-stranded 3′ overhang, an increase in chromosome end-to-end fusions, and irreversible growth arrest in a senescence-like state [51••]. It is not known whether TRF2 serves to protect the G-rich overhang directly — which is unlikely, as TRF2 cannot bind single-stranded DNA in vitro — whether it stimulates the formation of G tails by recruiting an exonuclease or other factor, or whether TRF2 recruits other telomere binding proteins that protect G tails. A recently identified putative telomere-binding protein is UP1, the amino-terminal fragment of heterogeneous nuclear ribonucleoprotein A1 [52]. UP1 appears to bind both telomeric repeats and telomerase and it has therefore been proposed that proteolytic processing of A1 to UP1 may recruit telomerase to the ends of telomeres.
Double strand break repair proteins The Ku heterodimer is critically important for end-joining of DNA double-stranded breaks (DSBs). In addition, in the yeast S. cerevisiae, there is evidence that it has an important role in telomere maintenance where end-to-end fusion is not desirable, thus indicating that its role is context-dependent
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The Rad50/Mre11/Xrs2 protein complex is also involved in both DSB-end-joining repair and telomere maintenance in yeast [60••,61•]. The complex may be an exonuclease or have a structural function, and it has been proposed that it may be required for preparation of telomeric ends or their presentation to telomerase to facilitate its enzymatic activity [62]. An additional yeast telomere-binding protein, Cdc13p, binds single-stranded telomeric DNA and may have a dual role in protecting the end and facilitating access of telomerase [63,64]. Interestingly, epistasis analysis showed that Rad50/Mre11/Xrs2 functions in the same telomere-maintenance pathway as telomerase, but Ku and Cdc13 function in separate pathways [60••].
Telomere maintenance and immortalization Many normal human cells and tissues do not have detectable telomerase activity but the list of known exceptions is growing [65–70]. Where telomerase is present in normal cells, it is generally thought to be insufficient for prevention of telomere erosion [65]. Recent evidence suggests, however, that activation of telomerase may cause telomere lengthening in normal B lymphocytes in germinal centers [71•], possibly providing a temporary extension of proliferative capacity during clonal expansion. Mammalian telomerase activity was first discovered in the archetypal immortalized human cell line, HeLa [72]. Immortalization of human cells by viral genes usually proceeds via two stages (Figure 2). In the first stage, there is a finite extension of proliferative potential accompanied by telomere shortening, ending in a period of culture crisis where the culture in on longer expanding due to increased cell death of senescence. In the second stage, which does not always occur, a small number of cells escape from crisis and acquire an apparently unlimited ability to proliferate and telomere length is maintained [73]. Escape from crisis may be associated with acquisition of telomerase activity [74–77] and crisis may be prevented by transduction of pre-crisis cells with hTERT cDNA [78•]. In some cases, cells that escape from crisis have no detectable telomerase activity [77]. Southern analysis with
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Figure 2
ALT Germline length hTERT transduction
Length
Telomerase Crisis
Senescence
(reviewed in [53,54]). Crosslinking experiments showed that Ku binds telomeric DNA [55•], and yeast cells defective in either of the Ku genes (YKU70 or YKU80) lose most but not all of their telomeric repeats [56,57]. In addition to preventing telomere repeat attrition, Ku plays a role in telomere end structure. Telomeric G tails are only detected in yeast during S-phase but in strains defective in YKU80, these ends are present constitutively [55•,58•]. The Ku heterodimer may regulate an exonuclease that generates these G tails, normally keeping their production confined to S-phase, or participate in protecting G tails from degradation [54]. Ku may also be involved in clustering of telomeres and possibly interaction with the nuclear envelope, as deletion of YKU70 or YKU80 disrupts the normal clustering and localization of yeast telomeres [59•].
Mean telomere length
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Normal human somatic cells are usually telomerase-negative and their proliferation is accompanied by a progressive decrease in telomere length that is thought to result eventually in senescence (i.e. permanent proliferative arrest). Cells transduced with hTERT express telomerase, elongate their telomeres to germline length, and escape from senescence. Cells transduced with viral oncogenes continue to proliferate beyond the senescence barrier and their telomeres continue to shorten until culture crisis ensues. Most cells in crisis either senesce or die but rare immortalized cells arise that have activated a telomeremaintenance mechanism — either by employing telomerase or ALT. ALT cells have telomeres of heterogeneous length, ranging from short to very long, with the mean length often being greater than that of germline cells. The telomeres of telomerase-positive immortal cells tend to be short or in the normal range.
a telomere-specific probe showed that telomerasenegative immortalized cells have a characteristic pattern of terminal restriction fragment length that ranges from very short to abnormally long [77,79,80•]. In situ hybridization analyses showed that this heterogeneity of telomere length occurs within individual cells [81,82]. Recent evidence indicates that telomeric DNA fragments may be released from the chromosomes in such cells [83,84]. In clonally derived cell lines immortalized in vitro it was shown that the acquisition of abnormally long telomeres coincided with escape from crisis [77] (Figure 2), demonstrating the existence of one or more ALT mechanisms. It is of interest that mouse cells rendered telomerase-negative by targeted disruption of the TER gene undergo immortalization readily [85], possibly through the efficient utilization of an ALT mechanism. Human fibroblasts and other cells of mesenchymal origin seem more likely to utilize the ALT pathway for immortalization than epithelial cells, although a minority of in vitro immortalized epithelial cell lines, carcinoma-derived cell lines, and carcinomas do exhibit ALT [77,80•]. Low levels of telomerase may be detected in some normal human epithelial cells [86,87] but activity is usually undetectable
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in normal fibroblasts. It is therefore possible that upregulation of telomerase at immortalization occurs more readily in epithelial cells than in fibroblasts. All immortalized human cell lines examined to date have either telomerase or an ALT TMM [72,74,76,77,80•,88]. Inhibition of telomerase with antisense hTER or repression of either telomerase or ALT activity by chromosome transfer resulted in telomere shortening and re-imposition of a finite proliferative potential [26,89,90]. These observations, together with the close temporal correlation between the activation of these mechanisms and escape from crisis, are strong circumstantial evidence for a role of TMMs in immortalization. It is not clear, however, whether expression of a TMM alone is sufficient for immortalization. Transduction of telomerase-negative normal human retinal pigment epithelial cells and fibroblasts with hTERT cDNA under the control of a heterologous promoter resulted in expression of telomerase enzyme activity, in prevention or reversal of the telomere shortening that normally accompanies cell division, and in continued cell division beyond the population doubling levels at which the control cultures became senescent [91••,92••]. Follow-up studies have shown that the hTERT-transfected fibroblasts have continued dividing for 200 population doublings more than the control cultures [93•] and have normal checkpoint controls [93•,94•]. It will be important to determine whether these cells eventually become senescent despite well-maintained telomeres and a remarkable extension of lifespan, or whether genetic changes equivalent to the effects of viral oncoproteins are also required for indefinite proliferation. It has been proposed that there may be mitotic clocks other than or in addition to telomere shortening and that each of these must be inactivated in order for immortalization to occur [95]. Consistent with this hypothesis, it has been observed that some types of hamster cells express telomerase and maintain their telomeres but still undergo senescence [96,97•]. For human keratinocytes and breast epithelial cells grown in defined media, it has been found that inactivation of the retinoblastoma (Rb) gene ‘pathway’ is required for telomerase-induced lifespan extension [98•]; it is not clear to what extent this requirement is cell-type specific or determined by the in vitro growth conditions. In contrast, hTERT-transfected retinal pigment epithelial cells have a substantial extension of their proliferative capacity but appear to have an intact Rb pathway in that they express p16INK4, and have hypophosphorylated pRb when confluent [94•]. It is not yet possible to induce ALT activity experimentally because the genes involved in this/these mechanism(s) are unknown. It has been suggested that ALT cells may use a variant or mutant telomerase that is not detected in existing biochemical assays and not subject to the usual feedback controls so that it produces telomeres with abnormal lengths [77]. On the basis of the non-telomerase TMMs found in
other species, ALT could also be caused by retrotransposition or recombination. Retrotransposition of a sequence unrelated to the telomere repeat seems unlikely in ALT cells where the total amount of telomeric sequence hybridizing to a (TTAGGG)3 probe increases [99]. The rapid lengthening of telomeres seen in a telomerase-negative cell line [79] is consistent with recombination. If ALT is recombinational, the Ku and related genes will be interesting candidates in view of their role in mammalian recombination, including V(D)J joining (reviewed in [100]) and their presence and role at the yeast telomere. The mammalian homologues of yeast genes in other epistasis groups for telomere maintenance [60] are also of great interest.
Conclusions In the past year, it has become possible for the first time to induce telomerase activity in mammalian cells by the transfer of TERT cDNA and thus to begin experiments that directly address the role of telomere maintenance in control of proliferative potential. These experiments may have major implications for our understanding of both ageing and cancer. Continuing challenges for telomerase research include elucidating the composition of telomerase complexes, the role of TERT variants, the interactions between telomerase and other components of the telomere, control of TERT transcription, and how all of these processes are regulated in development. Additional challenges for understanding telomere biology include understanding maintenance of telomere integrity in normal cells and mechanisms of telomere length maintenance in telomerase-negative immortalized cells.
Acknowledgements The authors are supported by project grants from the National Health and Medical Research Council of Australia and the Carcinogenesis Fellowship of the New South Wales Cancer Council, and thank Vicki Lundblad and Silvia Bacchetti for their comments on the manuscript.
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