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Telomeres and telomerase in aging and cancer
Telomeres and telomerase in aging and cancer Calvin B Harley and Bryant Villeponteau G e r o n Corporation, M e n l o Park, USA Telomeres are maintained by the novel ribonucleoprotein enzyme telomerase. Telomerase activity is repressed in most somatic human cells, leading to telomere loss during replicative aging in vivo and in vitro. However, telomerase appears to be reactivated in essentially all human cancers. With the recent cloning of the RNA component of telomerase from several species, the stage is now set for critical tests of the role of telomeres and telomerase in aging and cancer. Current Opinion in Genetics and Development 1995, 5:249-255
Introduction Telomeres, the genetic elements at the ends o f linear chromosomes, are essential for proper chromosome structure and function. Because D N A synthesis occurs unidirectionally (5'--+3') and requires an ILNA primer for initiation, telomeres are not fully replicated by the conventional D N A polymerase complex. To overcome this 'end-replication' problem, most eukaryotic cells utilize a novel D N A polymerase, telomerase [1-3]. Telomerase is a ribonucleoprotein enzyme capable of extending chromosome ends with a specific telomeric D N A sequence by using a portion o f its integral tLNA component as the template. Telomere maintenance by telomerase was first characterized in the ciliate Tetrahymena, which has a large number oftelomeres and a correspondingly high level oftelomerase activity compared to vertebrate cells. Although telomerase has not been identified biochemically in yeast, telomere structure and function is perhaps best understood in Saccharomyces cerevisiae owing to the power of molecular genetics in this organism. As the work in ciliates and yeast has been covered extensively in previous reviews [4,5,6",7"], in this review, we will emphasize mammalian and especially human telomere biology.
Telomere structure and function All eukaryotic telomeres studied to date have simple arrays of tandem G-rich repeats that run 5'---)3' at chromosome ends and sometimes protrude as 3' overhangs. Single-stranded G-rich telomeric sequences can form intra- and inter-strand structures based on the G-quartet in vitro [8], but the extent to which these structures exist in vivo and their biological significance remains uncertain [7"]. Although telomere sequences can vary from species to species, a given organism has a characteristic repeat at all telomeres [4,9]. In Tetrahymena, for example, only the unique repeat T T G G G G was found at
chromosome ends, and slightly altered sequences were poorly tolerated when added by a mutated telomerase [101. Whereas ciliates vary in their choice o f telomere repeats, all vertebrates appear to have a conserved single telomere sequence (TTAGGG) at chromosome ends, although the subtelomeric sequences may vary [11,12]. In transfection experinaents with telomere-containing vectors that truncate human chromosomes, at least 500bp o f T T A G G G were needed to form new chromosomal ends, and closely related telomeres from the ciliates were very poor at seeding new ends [13"]. These data suggest that T T A G G G is the only allowable telomere repeat in vertebrates, consistent with the finding o f unique telomeric repeats in other species [101. In contrast, some species, such as S. cerevisiae, permit some degeneracy in their telomere sequence [7]. Chromatin structure is altered in mammalian telomeres. The first kb of T T A G G G sequence close to the end of the chromosome appears to have a diffuse and open chromatin structure that is not in a canonical nucleosomal array [14"]. This could promote binding by telomere-binding proteins and/or elongation by telomerase. The more internal part of the tdomere has an unusual shortened nucleosome repeat [14"',15], and it is not clear how far into the non-telomere sequence this shortened nucleosome repeat might spread. A similar shortened repeat has been observed in the chicken [~-globin genes [16], which reside close to the telomere in humans. If the altered chromatin spreads into a subtelomere region with the potential to be genetically active, then gene expression in the subtelomere region might be affected by changes in telomere length, as clearly happens in yeast [6",17,18]. T h i s telomere position effect could provide a mechanism to signal cell senescence. Other mechanisms to signal critical changes in telomere length are possible, however; for example, loss o f the protective function of the telomeric region could mimic a double-strand break in the chromosome,
Abbreviations TRBF--TTAGGG repeat binding factor; TRF--terminal restriction fragment. © Current Biology I.td ISSN 0959-437X
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Chromosomes and expression mechanisms initiating the cascade of events leading to the prolonged G 1 arrest characteristic of senescent cells [19]. Besides the histones that generate the basic nucleosome array, it is not clear what other proteins bind to mammalian telomeres. Telomeres co-fractionate with the nuclear matrix [20,21"] and some mammalian telomeres have been observed at the nuclear periphery, so that lamins may be one binding factor [22]. A second candidate is the T T A G G G repeat binding factor (TRBF) purified from human cells [23]. Recent results from telomere transfection studies indicate that T R B F binds tightly to T T A G G G in vitro, but poorly to closely related telomere sequences which do not seed telomere ends in vivo [13"]. These correlations suggest that T R B F may function in a manner similar to RAP1, the major telomere-binding protein in yeast, which is essential for telomere function and position effects [24-26].
Telomerase Telomerase activity was first characterized biochemically for Tetrahymena by Greider and Blackburn [1]. Four years later, a very similar biochemical activity was identified in the immortal human HeLa cell line by Morin [27]. The cloning of the 159 nucleotide IKNA component of Tetrahymena telomerase [3] clarified several aspects of the mechanism of action of this novel DNA polymerase (illustrated schematically for the human enzyme in Fig. 1). The predicted template region of the Tetrahymena 1KNA component is complementary to 1.5
repeats of the telomere sequence and was confirmed by the demonstration that mutations in this region led to the predicted alterations in telomere sequence when the mutated 1KNA was overexpressed in vivo [10]. Recent mutational analyses using in vitro reconstituted enzyme suggest that the templating 1KNA bases lie at the 5' end of the 9 nucleotide stretch, whereas the nucleotides at the 3' end of this region serve for 'alignment', allowing correct positioning of the 3' end of the telomeric DNA at the beginning of each elongation cycle [28"]. Thus, telomerase is capable of processive cycling between rounds of telomere polymerization (elongation) and translocation in which the enzyme complex remains associated with the DNA. Comparison of the R N A components from seven species of Tetrahymena led to a model for a common secondary structure, but only limited primary sequence conservation [29]. In a recent comparison of a larger group of ciliates, only a short 6bp sequence near the template was reported to be conserved [30"], but again a similar common secondary structure based on nucleotide covariation was reported. It will be of great interest to discover the relationships between the 1KNA structure and overall enzyme structure and function. Although yeast telomerase has still not been characterized biochemically, the 1KNA component of S. rerevisiae telomerase has been identified recently [31"]. Surprisingly, the yeast telomerase 1KNA component is much larger than that of the ciliates (1.3 kb versus - 150-200 bases). Given the relative lack of conservation in pri-
(a) Telomere binding Chromosomal DNA ~
5'
G G G T T A G
AUCCC
A A U Cm~e~ Telome RNA
Alignment
Fig. 1. Schematic representation of the 5'
(b) Polymerization
5' G G G T T A G G G T AU
TAG
C C C AAU
Ch
Elongation 3'
~
5'
(c) Translocation
- - G G G T
TAG
G G T TAG
Alignment 3' @ 19q5 Current Opiniorl it1 Geneti(s atKI Development
5'
predicted mechanism of action of human telomerase. This model is constructed in part on the basis of biochemical and molecular studies on Tetrahymena [ 1 - 3 , 2 8 " ] , biochemical studies on human telomerase [27], and data from the recent identification of a candidate for the human telomerase RNA component (I Feng, B Villeponteau et al., unpublished data). Associated protein components of the enzyme are not shown. (a) The 3' end of the chromosome aligns near the 3' end of the template region of the RNA. The entire template region is complementary to - 1.5 repeats of the human telomere sequence. (b) Telomere extension (polymerization) proceeds to the 5' end of the template region (i.e. to the C corresponding to the first G in the telomere repeat). (c) Telomerase can translocate to begin another round of telomere synthesis.
Telomeres and telomerase in aging and cancer Harley mary sequence between the ciliate R N A components, it is not surprising that there is also no sequence homology to the yeast R N A . At present, it is not possible to determine whether similarities in secondary or tertiary structure exist between the telomerase RNAs of yeast and ciliates. The R N A component of human telomerase has been reported recently (J Feng, B Villeponteau et al., unpublished data). The candidate R N A is 560 nucleotides long and, as predicted, contains a template region (identified by mutational analysis) which is complementary to - 1.5 repeats o f the human telomere sequence. The candidate human telomerase R N A component has no homology to any o f the lower eukaryotic telomerase RNAs. The identification o f this clone means that the function of telomerase in human cancer cells may now be rigorously tested using antisense or ribozyme technology. Moreover, telomerase knockout experiments may be planned to help elucidate the role of telomerase in germline and somatic tissue development, stem cell renewal, and hyperproliferative disorders, including cancer. Although there has been significant success with the R N A component of telomerase, the protein components have been much more difficult to identify. The yeast protein EST1 is the only published candidate for a
protein component oftelomerase in any species [32,33]. As ciliates have -100-fold more telomerase than mammals, protein candidates from Tetrahymena are the closest to being characterized (K Collins, C Greider, personal communication). As mammalian telomerases may be present at only 100-200 copies per cell, cloning telomerase proteins in mammals will probably present a daunting task.
Telomere length and telomerase in human cells and tissues Because it is not currently possible to measure accurately the length of the true terminal telomeric DNA in vertebrate cells, estimates o f telomere length and changes in relative size on the basis o f terminal restriction fragment (TRF) measurements (Fig. 2) are relatively crude. At birth, the TRFs of normal human somatic cell chromosomes are - 1 0 - 1 2 k b [12,34-36], but the terminal T T A G G G repeats may comprise only 4-6 kb o f these restriction fragments. Although studies of identical and non-identical twins demonstrate that T R F length in humans is heritable [37], the extent of variation in telom-
(a) Young cells Rsa I
t
,
(b) Senescent cells Rsa l
t
i!!i
(c) Crisis
~a I
(d) Cancer: short TRF Rsa I
I| (e) Cancer: long TRF Rsa I
[]
Non-TTAGGG simple sequence
[]
Degenerate TTAGGG
[]
and Villeponteau
TIAGGG repeats
© ] ()or~ (]tlrrenl ()F)inior/ ill (]elltqJ(s and [)twglOl)rl/enl
Fig. 2. Simplified model of human terminal restriction fragment (TRF) structure. (a) An average TRF in cells from a newborn may have - 4 - 6 k b of pure ]q-AGGG repeats at the terminus and 3-5 kb of degenerate TTAGGG repeats and other simple sequence DNA distal to the first restriction enzyme site (e.g. Rsal). (b) In senescent cells, most of the pure TTAGGG repeats have been lost, leaving the degenerate ]q-AGGG repeats and non-telomeric sequence. As non-telomeric repeats do not seed new chromosome ends [13"*], it is unlikely that the degenerate TTAGGG repeats will fully function as telomeres. Assuming some heterogeneity in telomere length between chromosomes, the TTAGGG repeats on at least one chromosome end may be of insufficient length for telomere function. Thus, the cell could halt proliferation at senescence because of a critically short telomere. (c) If the cell has mutations or alterations in the cell cycle checkpoint pathway allowing senescence to be bypassed, then telomeres would continue to shorten until most of the pure and degenerate TTAGGG sequence was lost on essentially all chromosomes. At this point, 'crisis' occurs, resulting in massive genomic instability and cell death. (d,e) Rare cells which survive crisis reactivate telomerase and restore telomere function by adding pure TI-AGGG repeats to chromosome ends. Depending on the balance of telomere loss and telomerase activity, telomeres could be short or long.
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Chromosomes and expression mechanisms Table 1. Cell immortality and telomerase activity in human cells and tissues. Source
Phenotype
Tissue origin
Telomere dynamics
Cultured cells
Mortal (dividing) Mortal (non-dividing) Mortal (extended life-span) Immortal (transformed lines)* Immortal (tumor lines)
Various (skin, lung, vascular, hemopoietic, and ovary) Connective (skin and lung)
Shorten or nt tt
0 of 25+$
Stable
0 of 3+
(a)
Embryonic kidney Connective Blood Various (lung, kidney, prostate, and retina)
Shorten Shorten Shorten Stable or nt
0 of 5+
[41,46J
8 of 10+
[41,43"*,45,46]
Various (I 4 different tissue origins)
Stable or nt
0 of 70+
[41,43"',47"]
Immortal**
Testes Connective Epidermis Blood Vascular intima Brain Others (breast, prostate, uterus, intestine, kidney, liver, lung, muscle, and spleen)
Stable Shorten Shorten Shorten Shorten Stable nt
2 of 2+
[35,43"] [35,38,43"] [56] (b) [36,39,46,47"] [43"*] (a) [43"'] [43"]
Breast Primary node negative Ductal node positive Ovarian carcinoma Prostate BPH PIN3 Adenocarcinoma Neuroblastoma Head and neck Colon Polyp Tubular adenoma Carcinoma Uterine Fibroids Sarcoma
nt
Normal tissues
Mortal ~
Tumor tissues
?
Stable nt
Shorten nt nt Shorten or nt Shorten or nt nt
Telomerase activity
0 of >60+
References [35,36,38,41 ]
[43"] 1 of 4+ 14 of 15+ 7 of 7+ 1 3 2 5 14
of of of of of
10+ 5+ 2+ 5+ 16+
[47 "°] [43"]
[43"] [43""] [39,43"]
0 of 1+ 0 of 1+ 8 of 8+ [43"] 0 of 11 + 3 of 3+
*Sublines of T-antigen transformed and immortalized cells may become telomerase negative, perhaps through genetic instability [ 4 3 " ] ; it is not known whether these subclones have an indefinite replicative capacity. **The germline lineage is immortal, even if specific cell types may not be. tWhether normal tissues contain rare immortal stem cells that are not detected by current telomerase assays has not yet been determined. ,~tnt, not tested. $Some cultured hemopoetic cells may display weak telomerase activity detected with the PCR-based assay ([43"]; C-P Chiu, NW Kim, PM Lansdorp, CB Harley, unpublished data). (a) RC Allsopp, CB Harley, unpublished data. (b) KR Prowse, CB Harley, unpublished data. This table is adapted from Harley et al. [42"].
eric versus subtelomeric D N A lengths on T R F s on a single chromosome, between different chromosomes, and between different individuals is not well characterized. Gradual attrition of telomeric D N A with replicative age in vitro and as a function of donor age in vivo in human somatic, but not germline or cancer, cells [35-41] led to the hypothesis that telomerase was repressed in somatic tissues to reduce the probability of cancer in long-lived organisms, such as man (reviewed in [42"]). The recent development of a highly sensitive and re-
producible PCR-based assay for telomerase activity has made it possible to further examine this hypothesis by measuring telomerase activity in a variety of cells and tissues in vitro and in vivo [43"]. The data of Kim et al. [43"] confirm and extend earlier reports that telolnerase is repressed substantially in human fibrohlasts, embryonic kidney cells (in culture), lymphocytes, and epithelial cells [36,41,44-46,47"] (Table 1). As expected, telomerase was also found in both male and female reproductive tissues and a broad range of tumor cell lines and tissues,
Telomeres and telomerase in aging and cancer Harley and Villeponteau consistent with the apparently immortal characteristic of at least a subset of cells from these populations. The telomere hypothesis of cell aging and immortalization [38,40,48] (Fig. 3) is now supported by a large body o f circumstantial evidence. This model goes beyond associations, however, in proposing a causal link between telomere loss and aging, on the one hand, and telomerase reactivation, cell immortality, and cancer, on the other. To determine whether telomere loss and telomerase activity are simply biomarkers of these complex processes, or one of their fundamental causes, we await results from experiments designed to establish whether manipulation of telomere length or telomerase activity in human cells affects cell life-span, tissue aging, and cancer. A precedent for such a causal relationship exists in ciliates and yeast, as critical telomere loss and a 'senescent' phenotype can apparently be induced by mutations in the telomerase IkNA component or other genes involved in telomere maintenance [10,31"°,32]. TRF (kb) ~15 Fetal
I~ Germline cells: telomerase positive
.
develop~ ment? ~ ~ .
cells Normalsomatic cells:
N
lomerase negative
" ~ ' ~ % . Transformi ~" ng event
Precrisis cells: ~ . ~ Jtelomer~. ~se neg,~tlve "ve ~ In]mortal tumor cells: S" telomerase positive
1 Birth
[ M1
-T M2
Replicative age
Hayflick limit .....
© 19~5 Current Opinion i~ Genetics and Develop~nt
Fig. 3. The telomere hypothesis of cell aging and immortalization. The terminal restriction fragment (TRF) length is plotted schematically against replicative age. Telomerase is active in germline cells, maintaining long stable telomeres, but is repressed in most normal somatic cells, resulting in telomere loss in dividing cells. Telomerase activity and telomere length have not been well characterized during embryonic and fetal development, or for true somatic stem cells. At mortality phase 1 (M1, or the Hayflick Limit), presumed critical telomere loss occurs on one or perhaps a few chromosomes, signaling irreversible cell cycle arrest. Transformation events may allow somatic cells to bypass M1 [55] without activating telomerase [41 ]. When telomeres become critically short on a large number of chromosomes, cells enter crisis (M2). Rare clones which activate telomerase escape M2, stabilize chromosomes and acquire an indefinite growth capacity. (Adapted from [42"].)
Do stem cells lose telomeric D N A with age? Telomere loss as a function o f replicative aging in culture or as a function of donor age in vivo has been documented in a variety of human cells and tissues which undergo renewal, including candidate hematopoietic stem cells [49"[. However, weak telomerase activity has been detected with the PCR.-based assay in some preparations
of candidate stem cells stimulated for growth and differentiation in vitro, and in peripheral blood lymphocytes (C-P Chiu, N W Kim, CB Harley, PM Lansdorp, unpublished data). Given that telomeric DNA is lost with age in vitro and in vivo in these cells [36,49"], the biological significance of this activity is uncertain.
Telomeres and telomerase in mice Compared to humans, many laboratory strains o f mice and other rodents have relatively large arrays o f T T A G G G repeats near chromosomal ends, as well as at internal sites. Thus, it has been difficult to assess the significance of reports that telomere loss does not occur as a function of age in mice [50,51], as loss of true telomeric DNA may be obscured by large non-telomeric arrays o f TTAGGG. A predominantly non-processive telomerase activity was originally reported for immortal murine cells in culture [52]; however, the apparent difference in processivity between murine and human telomerase may be subject to assay conditions and, therefore, less distinct than thought originally [53]. Unlike the situation in humans, telomerase activity can be readily detected in a variety of normal rodent tissues in viw~ [53]. This observation may account for the high frequency of spontaneous imnmrtalization of rodent cells in vitro and the extremely high frequency o f cancer in mice in vivo on a per cell per year basis (reviewed in [42°]).
Implications for human health Iftelomere loss and telomerase activation are causally involved in cellular aging and immortalization, and these events in turn contribute to pathologies of aging, including cancer, then telomeres and telomerase present exciting new targets for drug discovery and diagnostics. Delaying critical telomere loss therapeutically through transient reactivation of telomerase or otherwise slowing the rate of telomere loss, for example, could extend cell life-span and reduce the impact o f replicative senescence in ex vivo applications as well as diseases in vivo. Inhibition ' o f telomerase, on the other hand, could provide a safe and effective therapy for nearly all cancers. The high degree o f specificity in telomerase expression in tumor and germline cells versus normal somatic cells suggests that the therapeutic index, or window for safe drug application, could be large. Nmnerous hurdles remain before these ideas can be tested in human clinical trials, however, including the discovery and development of specific modulators of telomere length or telomerase, and elucidation of their effects on normal and abnormal growth and development in animals.
Conclusions As telomeres perform an essential genetic function for chronmsome structure and stability, it is clear that elu-
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Chromosomesand expressionmechanisms cidation of the dynamics of telomere maintenance and factors which modulate telomere loss or re-acquisition will have profound implications for our understanding of the fate of a cell. A recent research news article in Science [54] highlights the current excitement in telomere biology arising from discoveries in simple eukaryotes and basic and applied research in humans. The cloning of the R N A component of telomerase from yeast and humans, and the promise of identifying telomerase protein components and the regulatory pathways for telomere maintenance, will contribute not only to increased research and development activity in this area, but also to the critical testing of hypotheses linking telomere loss to age-related diseases and telomerase reactivation to cancer. The implications for novel drug discovery and the benefits to human health are potentially dramatic.
11.
Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne l, Ratliff RL, Wu JR: A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Nat/ Acad Sci USA 1988, 85:6622-6626.
12.
AIIshire RC, Dempster M, Hastie ND: Human telomeres contain at least three types of G-rlch repeat distributed non-randomly. Nucleic Acids Res 1989, 17:4611-4627.
Hanish JP, Yanowitz IL, De Lange T: Stringent sequence requirements for the formation of human lelomeres. Proc Nat/ Acad 5ci USA 1994, 91:8861-8865. This paper shows that telomere formation in human cells has stringent requirements for the TTAGGG repeat. 13. *,
14. Tommerup H, Dousmanis A, De Lange T: Unusual chromalin •. in human lelomeres. Mol Cell Biol 1994, 14:5777-5785. The authors report that human teiomeres have an unusual chromatin structure characterized by diffuse micrococcal nuciease patterns near the end of the telomere and a shortened nucleosome repeat near the subtelomere region. 15.
Makarov VL, Lejnine S, Bedoyan J, Langmore JP: Nucleosomal organization of telomere-specific chromatin in rat. Cell 1993, 73:775-787.
We would like thank our many colleagues at Geron and elsewhere for critical contributions to the literature and concepts described in this review. It is impractical to acknowledge them all here, but many of their names are listed in the publications below. Finally, we apologize for being unable to do justice to the elegant work on simpler eukaryotes because of the need to focus on human telomere biology.
16.
Villeponteau B, Brawley ), Martinson HG: Nucleosome spacing is compressed in active chromatin domains of chick erythroid cells. Biochemistry 1992, 31:1554-1 563.
17.
Gottschling DE, Aparicio OM, Billington BL, Zakian VA: Posilion effect at S. cerevlsiae telomeres: reversible repression of Pol II transcription. Cell 1990, 63:751-762.
18.
Gottschling DE: Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc Nat/ Acad 5ci USA 1992, 89:4062-4065.
References and recommended reading
19.
Di Leonardo A, Linke SP, Clarkin K, Wahl GM: DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cipl in normal human fibroblasts. Genes Dev 1994, 8:2540-2551.
20.
De Lange T: Human telomeres are attached to the nuclear matrix. EMBO J 1992, 11:717-724.
Acknowledgements
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest I.
Greider CW, Blackburn EH: Identification of a specific lelomere terminal transferase adivity in Telrahymena extracts. Cell 1985, 43:405-413.
2.
Greider CW, Blackburn EH: The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 1987, 51:887-898.
3.
Greider C, Blackburn E: A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 1989, 337:331-337.
4.
Blackburn EH: Telomerases. 61:113-129.
S.
Shippen DE: Telomeres and telomerases. Curr Opin Genet Dev 1993, 3:759-763.
Markova D, Donev R, Patriotis C, Djondjurov L: Interphase chromosomes of Friend-S cells are attached to the matrix structures through the centromeric/telomeric regions. DNA Cell Biol 1994, 13:941-951. Provides further support for the hypothesis that telomeres bind nuclear matrix. 21. •
22.
Shoeman RL, Traub P: The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentln. J Biol Chem 1990, 265:9055-9061.
23.
Zhong Z, Shiue L, Kaplan S, De Lange T: A mammalian factor that binds telomeric ]qAGGG repeats in vitro. Mol Cell Biol 1992, 12:4834~1843.
24.
Conrad MN, Wright JH, Wolf AJ, Zakian VA: RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell 1990, 63:739-750.
25.
7. Blackburn EH: Telomeres: no end in sight. Cell 1994, • 77:621~23. An excellent mini-review describing the recent data on yeast and other lower eukaryotes.
Kyrion G, Boakye KA, Lustig AI: C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. Mol Cell Biol 1992, 12:5159-5173.
26.
Kyrion G, Liu K, Liu C, Lustig AJ: RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevlsiae. Genes Dev 1993, 7:1146-1159.
8.
Fang G, Cech TR: Characterization of a G-quartet formation reaction promoted by the beta-subunit of the Oxytricha telomere-binding protein. Biochemistry 1993, 32:11646-11657.
27.
Morin GB: The human telomere terminal transferase enzyme is a ribonucleoprolein that synthesizes TTAGGG repeats. Cell 1989, 59:521-529.
9.
Zakian VA: Structure and function of telomeres. Annu Rev Genet 1989, 23:579-604.
10.
Yu GL, Bradley JD, Attardi LD, Blackburn EH: In vivo alteration of telomere sequences and senescence caused by mutated Tetrahvmena telomerase RNAs. Nature 1990. 344:126-132.
Annu
Rev
Biochem
1992,
Palladino F, Gasser SM: Telomere maintenance and gene repression: a common end? Curr Opin Cell Biol 1994, 6:373-379. This review describes the factors influencing nuclear architecture and position-effect variegation. 6. •
Autexier C, Greider CW: Functional reconstitutlon of wildtype and mutant Tetrahymena telomerase. Genes Dev 1994, 8:563-575. Presents the first in vitro reconstitution of telomerase activity using wildtype and mutant telomerase RNA added to nuclease-treated extracts and 28. ,,
Telomeres and t e l o m e r a s e in aging and cancer H a r l e y and Villeponteau shows that the template region of the RNA can be dissected into an 'alignment' region and a true 'template' region. 29.
Romero DP, Blackburn EH: A conserved secondary structure for telomerase RNA. Cell ]991, 67:343-353.
30. •
Lingner 1, Hendrick LL, Cech TR: Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev 1994, 8:1984-1988. Presents a careful secondary structure analysis of ciliate telomerase RNA sequences and proposes a common structure on the basis of nucleotide covariations. 31. ••
Singer MS, Gottschling DE: TLCl: template RNA component of Saccharomyces cerevislae telomerase. Science 1994, 266:404-409. Identification of the S. cerevisiae telomerase RNA component and demonstration that disrupting the corresponding gene leads to telomere shortening, decreased growth rate, and loss of cell viability. 32.
Lundblad V, Szostak JW: A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 1989, 57:633-643.
33.
Lundblad V, Blackburn EH: RNA-dependent polymerase motifs in ESTI: tentative identification of a protein component of an essential yeast telomerase. Cell ]990, 60:529-530.
34.
De Lange T, Shiue L, Myers RM, Cox DR, Naylor SL, Killery AM, Varmus HE: Structure and variability of human chromosome ends. Mol Cell Biol 1990, 10:518-527.
35.
AIIsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB: Telomere length predicts replicative capacity of human fibroblasts. Proc Nat/Acad Sci USA 1992, 89:10114-10118.
36.
Vaziri H, Schachter F, Uchida I, We] L, Zhu X, Effros R, Cohen D, Harley CB: Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet ]993, 52:661-667.
37.
Slagboom PE, Droog S, Boomsma DI: Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet 1994, 55:876-882.
38.
Harley CB, Futcher AB, Greider CW: Telomeres shorten during ageing of human fibroblasts. Nature 1990, 345:458-460.
39.
Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, AIIshire RC: Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990, 346:866-868.
40.
Harley CB: Telomere loss: mitotic clock or genetic time bomb? Murat Res 1991, 256:271-282.
41.
Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S: Telomere shortening associated with chromosome inslability is arrested in immortal cells which express telomerase activity. EMBO J 1992, 11:1921-1929.
Harley CB, Kim NW, Prowse KR, Weinrich SL, Hirsch KS, West MD, Bacchetti S, Hirte HW, Counter CM, Greider CW et al.: Telomerase, cell immortality, and cancer. Cold Spring Harb Syrup Quant Biol 1994, 59:307-315. A statement of the apparent relationship between telomerase activation, cell immortality, and cancer, and a review of the relevant data, together with a theoretical model demonstrating the effect of telomerase inhibition on tumor growth.
association of human telomerase activity with immortal cells and cancer. Science 1994, 266:2011-2014. Description of 'TRAP' (telomere repeat amplification protocol), a highly sensitive and reproducible telomerase assay, and a broad survey of normal and tumor cells and tissues. This paper demonstrates that telomerase activity correlates highly with malignant, but not benign, cancers and is present in germline tissues, but not normal somatic tissues tested. 44.
Shay IW, Wright WE, Werbin H: Toward a molecular understanding of human breast cancer: a hypothesis. Breast Cancer Res Treat 1993, 25:83-94.
45.
Shay JW, Wright WE, Werbin H: Loss of telomeric DNA during aging may predispose cells to cancer. Int J Oncol 1993, 3:559-563.
46.
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.
47. •.
Counter CM, Hirte HW, Bacchetti S, Harley CB: Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci USA 1994, 91:2900-2904. The first demonstration that telomerase can be detected in a human cancer, but not the surrounding normal cells, nor the cell type (ovarian epithelial cells) from which the cancer arose. 48. 49. •
Vaziri H, Dragowska W, AIIsopp RC, Thomas TE, Harley CB, Lansdorp PM: Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acacl Sci USA 1994, 91:9857-9860. This paper presents evidence that telomeres decrease with age in vitro and as a function of donor age in vivo in candidate hematopoietic stem cells from humans (CD34 + and CD381o). These data suggest that if telomerase is present in stem cells, it does not fully renew telomeres with replicative aging, or the stem cell population otherwise decreases with age. 50.
Kipling D, Cooke HJ: Hypervariable ultra-long telomeres in mice. Nature 1990, 347:400-402.
51.
Starling JA, Maule J, Hastie ND, AIIshire RC: Extensive telomere repeat arrays in mouse are hypervariable. Nucleic Acids Res 1990, 18:6881~888.
52.
Prows• KR, Avilion An,, Greider CW: Identification of a nonprocesslve telomerase activity from mouse cells. Proc Natl Acad Sci USA ]993, 90:]493-1497.
53.
Prows• KR, Greider CW: Developmenlal and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci USA 1995, 92:in press.
54.
Marx J: Chromosome 265:1656-1658.
55.
Shay JW, Per•ira-Smith OM, Wright WE: A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res 1991, 196:33~.1.
56.
Lindsey J, McGill N, Lindsey L, Green D, Cooke H: In vivo loss of lelomeric repreats with age in humans. Murat Res 1991, 225:45-48.
42. •
43. ••
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific
Olovnikov AM: A theory of marginotomy. J Theoret Biol 1973, 41:181-190.
ends
catch
fire.
Science
1994,
CB Harley and B ViUeponteau, Geron Corporation, 200 Constitution Drive, Menlo Park, California 94025, USA.
255
Introduction Telomeres, the genetic elements at the ends o f linear chromosomes, are essential for proper chromosome structure and function. Because D N A synthesis occurs unidirectionally (5'--+3') and requires an ILNA primer for initiation, telomeres are not fully replicated by the conventional D N A polymerase complex. To overcome this 'end-replication' problem, most eukaryotic cells utilize a novel D N A polymerase, telomerase [1-3]. Telomerase is a ribonucleoprotein enzyme capable of extending chromosome ends with a specific telomeric D N A sequence by using a portion o f its integral tLNA component as the template. Telomere maintenance by telomerase was first characterized in the ciliate Tetrahymena, which has a large number oftelomeres and a correspondingly high level oftelomerase activity compared to vertebrate cells. Although telomerase has not been identified biochemically in yeast, telomere structure and function is perhaps best understood in Saccharomyces cerevisiae owing to the power of molecular genetics in this organism. As the work in ciliates and yeast has been covered extensively in previous reviews [4,5,6",7"], in this review, we will emphasize mammalian and especially human telomere biology.
Telomere structure and function All eukaryotic telomeres studied to date have simple arrays of tandem G-rich repeats that run 5'---)3' at chromosome ends and sometimes protrude as 3' overhangs. Single-stranded G-rich telomeric sequences can form intra- and inter-strand structures based on the G-quartet in vitro [8], but the extent to which these structures exist in vivo and their biological significance remains uncertain [7"]. Although telomere sequences can vary from species to species, a given organism has a characteristic repeat at all telomeres [4,9]. In Tetrahymena, for example, only the unique repeat T T G G G G was found at
chromosome ends, and slightly altered sequences were poorly tolerated when added by a mutated telomerase [101. Whereas ciliates vary in their choice o f telomere repeats, all vertebrates appear to have a conserved single telomere sequence (TTAGGG) at chromosome ends, although the subtelomeric sequences may vary [11,12]. In transfection experinaents with telomere-containing vectors that truncate human chromosomes, at least 500bp o f T T A G G G were needed to form new chromosomal ends, and closely related telomeres from the ciliates were very poor at seeding new ends [13"]. These data suggest that T T A G G G is the only allowable telomere repeat in vertebrates, consistent with the finding o f unique telomeric repeats in other species [101. In contrast, some species, such as S. cerevisiae, permit some degeneracy in their telomere sequence [7]. Chromatin structure is altered in mammalian telomeres. The first kb of T T A G G G sequence close to the end of the chromosome appears to have a diffuse and open chromatin structure that is not in a canonical nucleosomal array [14"]. This could promote binding by telomere-binding proteins and/or elongation by telomerase. The more internal part of the tdomere has an unusual shortened nucleosome repeat [14"',15], and it is not clear how far into the non-telomere sequence this shortened nucleosome repeat might spread. A similar shortened repeat has been observed in the chicken [~-globin genes [16], which reside close to the telomere in humans. If the altered chromatin spreads into a subtelomere region with the potential to be genetically active, then gene expression in the subtelomere region might be affected by changes in telomere length, as clearly happens in yeast [6",17,18]. T h i s telomere position effect could provide a mechanism to signal cell senescence. Other mechanisms to signal critical changes in telomere length are possible, however; for example, loss o f the protective function of the telomeric region could mimic a double-strand break in the chromosome,
Abbreviations TRBF--TTAGGG repeat binding factor; TRF--terminal restriction fragment. © Current Biology I.td ISSN 0959-437X
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Chromosomes and expression mechanisms initiating the cascade of events leading to the prolonged G 1 arrest characteristic of senescent cells [19]. Besides the histones that generate the basic nucleosome array, it is not clear what other proteins bind to mammalian telomeres. Telomeres co-fractionate with the nuclear matrix [20,21"] and some mammalian telomeres have been observed at the nuclear periphery, so that lamins may be one binding factor [22]. A second candidate is the T T A G G G repeat binding factor (TRBF) purified from human cells [23]. Recent results from telomere transfection studies indicate that T R B F binds tightly to T T A G G G in vitro, but poorly to closely related telomere sequences which do not seed telomere ends in vivo [13"]. These correlations suggest that T R B F may function in a manner similar to RAP1, the major telomere-binding protein in yeast, which is essential for telomere function and position effects [24-26].
Telomerase Telomerase activity was first characterized biochemically for Tetrahymena by Greider and Blackburn [1]. Four years later, a very similar biochemical activity was identified in the immortal human HeLa cell line by Morin [27]. The cloning of the 159 nucleotide IKNA component of Tetrahymena telomerase [3] clarified several aspects of the mechanism of action of this novel DNA polymerase (illustrated schematically for the human enzyme in Fig. 1). The predicted template region of the Tetrahymena 1KNA component is complementary to 1.5
repeats of the telomere sequence and was confirmed by the demonstration that mutations in this region led to the predicted alterations in telomere sequence when the mutated 1KNA was overexpressed in vivo [10]. Recent mutational analyses using in vitro reconstituted enzyme suggest that the templating 1KNA bases lie at the 5' end of the 9 nucleotide stretch, whereas the nucleotides at the 3' end of this region serve for 'alignment', allowing correct positioning of the 3' end of the telomeric DNA at the beginning of each elongation cycle [28"]. Thus, telomerase is capable of processive cycling between rounds of telomere polymerization (elongation) and translocation in which the enzyme complex remains associated with the DNA. Comparison of the R N A components from seven species of Tetrahymena led to a model for a common secondary structure, but only limited primary sequence conservation [29]. In a recent comparison of a larger group of ciliates, only a short 6bp sequence near the template was reported to be conserved [30"], but again a similar common secondary structure based on nucleotide covariation was reported. It will be of great interest to discover the relationships between the 1KNA structure and overall enzyme structure and function. Although yeast telomerase has still not been characterized biochemically, the 1KNA component of S. rerevisiae telomerase has been identified recently [31"]. Surprisingly, the yeast telomerase 1KNA component is much larger than that of the ciliates (1.3 kb versus - 150-200 bases). Given the relative lack of conservation in pri-
(a) Telomere binding Chromosomal DNA ~
5'
G G G T T A G
AUCCC
A A U Cm~e~ Telome RNA
Alignment
Fig. 1. Schematic representation of the 5'
(b) Polymerization
5' G G G T T A G G G T AU
TAG
C C C AAU
Ch
Elongation 3'
~
5'
(c) Translocation
- - G G G T
TAG
G G T TAG
Alignment 3' @ 19q5 Current Opiniorl it1 Geneti(s atKI Development
5'
predicted mechanism of action of human telomerase. This model is constructed in part on the basis of biochemical and molecular studies on Tetrahymena [ 1 - 3 , 2 8 " ] , biochemical studies on human telomerase [27], and data from the recent identification of a candidate for the human telomerase RNA component (I Feng, B Villeponteau et al., unpublished data). Associated protein components of the enzyme are not shown. (a) The 3' end of the chromosome aligns near the 3' end of the template region of the RNA. The entire template region is complementary to - 1.5 repeats of the human telomere sequence. (b) Telomere extension (polymerization) proceeds to the 5' end of the template region (i.e. to the C corresponding to the first G in the telomere repeat). (c) Telomerase can translocate to begin another round of telomere synthesis.
Telomeres and telomerase in aging and cancer Harley mary sequence between the ciliate R N A components, it is not surprising that there is also no sequence homology to the yeast R N A . At present, it is not possible to determine whether similarities in secondary or tertiary structure exist between the telomerase RNAs of yeast and ciliates. The R N A component of human telomerase has been reported recently (J Feng, B Villeponteau et al., unpublished data). The candidate R N A is 560 nucleotides long and, as predicted, contains a template region (identified by mutational analysis) which is complementary to - 1.5 repeats o f the human telomere sequence. The candidate human telomerase R N A component has no homology to any o f the lower eukaryotic telomerase RNAs. The identification o f this clone means that the function of telomerase in human cancer cells may now be rigorously tested using antisense or ribozyme technology. Moreover, telomerase knockout experiments may be planned to help elucidate the role of telomerase in germline and somatic tissue development, stem cell renewal, and hyperproliferative disorders, including cancer. Although there has been significant success with the R N A component of telomerase, the protein components have been much more difficult to identify. The yeast protein EST1 is the only published candidate for a
protein component oftelomerase in any species [32,33]. As ciliates have -100-fold more telomerase than mammals, protein candidates from Tetrahymena are the closest to being characterized (K Collins, C Greider, personal communication). As mammalian telomerases may be present at only 100-200 copies per cell, cloning telomerase proteins in mammals will probably present a daunting task.
Telomere length and telomerase in human cells and tissues Because it is not currently possible to measure accurately the length of the true terminal telomeric DNA in vertebrate cells, estimates o f telomere length and changes in relative size on the basis o f terminal restriction fragment (TRF) measurements (Fig. 2) are relatively crude. At birth, the TRFs of normal human somatic cell chromosomes are - 1 0 - 1 2 k b [12,34-36], but the terminal T T A G G G repeats may comprise only 4-6 kb o f these restriction fragments. Although studies of identical and non-identical twins demonstrate that T R F length in humans is heritable [37], the extent of variation in telom-
(a) Young cells Rsa I
t
,
(b) Senescent cells Rsa l
t
i!!i
(c) Crisis
~a I
(d) Cancer: short TRF Rsa I
I| (e) Cancer: long TRF Rsa I
[]
Non-TTAGGG simple sequence
[]
Degenerate TTAGGG
[]
and Villeponteau
TIAGGG repeats
© ] ()or~ (]tlrrenl ()F)inior/ ill (]elltqJ(s and [)twglOl)rl/enl
Fig. 2. Simplified model of human terminal restriction fragment (TRF) structure. (a) An average TRF in cells from a newborn may have - 4 - 6 k b of pure ]q-AGGG repeats at the terminus and 3-5 kb of degenerate TTAGGG repeats and other simple sequence DNA distal to the first restriction enzyme site (e.g. Rsal). (b) In senescent cells, most of the pure TTAGGG repeats have been lost, leaving the degenerate ]q-AGGG repeats and non-telomeric sequence. As non-telomeric repeats do not seed new chromosome ends [13"*], it is unlikely that the degenerate TTAGGG repeats will fully function as telomeres. Assuming some heterogeneity in telomere length between chromosomes, the TTAGGG repeats on at least one chromosome end may be of insufficient length for telomere function. Thus, the cell could halt proliferation at senescence because of a critically short telomere. (c) If the cell has mutations or alterations in the cell cycle checkpoint pathway allowing senescence to be bypassed, then telomeres would continue to shorten until most of the pure and degenerate TTAGGG sequence was lost on essentially all chromosomes. At this point, 'crisis' occurs, resulting in massive genomic instability and cell death. (d,e) Rare cells which survive crisis reactivate telomerase and restore telomere function by adding pure TI-AGGG repeats to chromosome ends. Depending on the balance of telomere loss and telomerase activity, telomeres could be short or long.
251
252
Chromosomes and expression mechanisms Table 1. Cell immortality and telomerase activity in human cells and tissues. Source
Phenotype
Tissue origin
Telomere dynamics
Cultured cells
Mortal (dividing) Mortal (non-dividing) Mortal (extended life-span) Immortal (transformed lines)* Immortal (tumor lines)
Various (skin, lung, vascular, hemopoietic, and ovary) Connective (skin and lung)
Shorten or nt tt
0 of 25+$
Stable
0 of 3+
(a)
Embryonic kidney Connective Blood Various (lung, kidney, prostate, and retina)
Shorten Shorten Shorten Stable or nt
0 of 5+
[41,46J
8 of 10+
[41,43"*,45,46]
Various (I 4 different tissue origins)
Stable or nt
0 of 70+
[41,43"',47"]
Immortal**
Testes Connective Epidermis Blood Vascular intima Brain Others (breast, prostate, uterus, intestine, kidney, liver, lung, muscle, and spleen)
Stable Shorten Shorten Shorten Shorten Stable nt
2 of 2+
[35,43"] [35,38,43"] [56] (b) [36,39,46,47"] [43"*] (a) [43"'] [43"]
Breast Primary node negative Ductal node positive Ovarian carcinoma Prostate BPH PIN3 Adenocarcinoma Neuroblastoma Head and neck Colon Polyp Tubular adenoma Carcinoma Uterine Fibroids Sarcoma
nt
Normal tissues
Mortal ~
Tumor tissues
?
Stable nt
Shorten nt nt Shorten or nt Shorten or nt nt
Telomerase activity
0 of >60+
References [35,36,38,41 ]
[43"] 1 of 4+ 14 of 15+ 7 of 7+ 1 3 2 5 14
of of of of of
10+ 5+ 2+ 5+ 16+
[47 "°] [43"]
[43"] [43""] [39,43"]
0 of 1+ 0 of 1+ 8 of 8+ [43"] 0 of 11 + 3 of 3+
*Sublines of T-antigen transformed and immortalized cells may become telomerase negative, perhaps through genetic instability [ 4 3 " ] ; it is not known whether these subclones have an indefinite replicative capacity. **The germline lineage is immortal, even if specific cell types may not be. tWhether normal tissues contain rare immortal stem cells that are not detected by current telomerase assays has not yet been determined. ,~tnt, not tested. $Some cultured hemopoetic cells may display weak telomerase activity detected with the PCR-based assay ([43"]; C-P Chiu, NW Kim, PM Lansdorp, CB Harley, unpublished data). (a) RC Allsopp, CB Harley, unpublished data. (b) KR Prowse, CB Harley, unpublished data. This table is adapted from Harley et al. [42"].
eric versus subtelomeric D N A lengths on T R F s on a single chromosome, between different chromosomes, and between different individuals is not well characterized. Gradual attrition of telomeric D N A with replicative age in vitro and as a function of donor age in vivo in human somatic, but not germline or cancer, cells [35-41] led to the hypothesis that telomerase was repressed in somatic tissues to reduce the probability of cancer in long-lived organisms, such as man (reviewed in [42"]). The recent development of a highly sensitive and re-
producible PCR-based assay for telomerase activity has made it possible to further examine this hypothesis by measuring telomerase activity in a variety of cells and tissues in vitro and in vivo [43"]. The data of Kim et al. [43"] confirm and extend earlier reports that telolnerase is repressed substantially in human fibrohlasts, embryonic kidney cells (in culture), lymphocytes, and epithelial cells [36,41,44-46,47"] (Table 1). As expected, telomerase was also found in both male and female reproductive tissues and a broad range of tumor cell lines and tissues,
Telomeres and telomerase in aging and cancer Harley and Villeponteau consistent with the apparently immortal characteristic of at least a subset of cells from these populations. The telomere hypothesis of cell aging and immortalization [38,40,48] (Fig. 3) is now supported by a large body o f circumstantial evidence. This model goes beyond associations, however, in proposing a causal link between telomere loss and aging, on the one hand, and telomerase reactivation, cell immortality, and cancer, on the other. To determine whether telomere loss and telomerase activity are simply biomarkers of these complex processes, or one of their fundamental causes, we await results from experiments designed to establish whether manipulation of telomere length or telomerase activity in human cells affects cell life-span, tissue aging, and cancer. A precedent for such a causal relationship exists in ciliates and yeast, as critical telomere loss and a 'senescent' phenotype can apparently be induced by mutations in the telomerase IkNA component or other genes involved in telomere maintenance [10,31"°,32]. TRF (kb) ~15 Fetal
I~ Germline cells: telomerase positive
.
develop~ ment? ~ ~ .
cells Normalsomatic cells:
N
lomerase negative
" ~ ' ~ % . Transformi ~" ng event
Precrisis cells: ~ . ~ Jtelomer~. ~se neg,~tlve "ve ~ In]mortal tumor cells: S" telomerase positive
1 Birth
[ M1
-T M2
Replicative age
Hayflick limit .....
© 19~5 Current Opinion i~ Genetics and Develop~nt
Fig. 3. The telomere hypothesis of cell aging and immortalization. The terminal restriction fragment (TRF) length is plotted schematically against replicative age. Telomerase is active in germline cells, maintaining long stable telomeres, but is repressed in most normal somatic cells, resulting in telomere loss in dividing cells. Telomerase activity and telomere length have not been well characterized during embryonic and fetal development, or for true somatic stem cells. At mortality phase 1 (M1, or the Hayflick Limit), presumed critical telomere loss occurs on one or perhaps a few chromosomes, signaling irreversible cell cycle arrest. Transformation events may allow somatic cells to bypass M1 [55] without activating telomerase [41 ]. When telomeres become critically short on a large number of chromosomes, cells enter crisis (M2). Rare clones which activate telomerase escape M2, stabilize chromosomes and acquire an indefinite growth capacity. (Adapted from [42"].)
Do stem cells lose telomeric D N A with age? Telomere loss as a function o f replicative aging in culture or as a function of donor age in vivo has been documented in a variety of human cells and tissues which undergo renewal, including candidate hematopoietic stem cells [49"[. However, weak telomerase activity has been detected with the PCR.-based assay in some preparations
of candidate stem cells stimulated for growth and differentiation in vitro, and in peripheral blood lymphocytes (C-P Chiu, N W Kim, CB Harley, PM Lansdorp, unpublished data). Given that telomeric DNA is lost with age in vitro and in vivo in these cells [36,49"], the biological significance of this activity is uncertain.
Telomeres and telomerase in mice Compared to humans, many laboratory strains o f mice and other rodents have relatively large arrays o f T T A G G G repeats near chromosomal ends, as well as at internal sites. Thus, it has been difficult to assess the significance of reports that telomere loss does not occur as a function of age in mice [50,51], as loss of true telomeric DNA may be obscured by large non-telomeric arrays o f TTAGGG. A predominantly non-processive telomerase activity was originally reported for immortal murine cells in culture [52]; however, the apparent difference in processivity between murine and human telomerase may be subject to assay conditions and, therefore, less distinct than thought originally [53]. Unlike the situation in humans, telomerase activity can be readily detected in a variety of normal rodent tissues in viw~ [53]. This observation may account for the high frequency of spontaneous imnmrtalization of rodent cells in vitro and the extremely high frequency o f cancer in mice in vivo on a per cell per year basis (reviewed in [42°]).
Implications for human health Iftelomere loss and telomerase activation are causally involved in cellular aging and immortalization, and these events in turn contribute to pathologies of aging, including cancer, then telomeres and telomerase present exciting new targets for drug discovery and diagnostics. Delaying critical telomere loss therapeutically through transient reactivation of telomerase or otherwise slowing the rate of telomere loss, for example, could extend cell life-span and reduce the impact o f replicative senescence in ex vivo applications as well as diseases in vivo. Inhibition ' o f telomerase, on the other hand, could provide a safe and effective therapy for nearly all cancers. The high degree o f specificity in telomerase expression in tumor and germline cells versus normal somatic cells suggests that the therapeutic index, or window for safe drug application, could be large. Nmnerous hurdles remain before these ideas can be tested in human clinical trials, however, including the discovery and development of specific modulators of telomere length or telomerase, and elucidation of their effects on normal and abnormal growth and development in animals.
Conclusions As telomeres perform an essential genetic function for chronmsome structure and stability, it is clear that elu-
253
254
Chromosomesand expressionmechanisms cidation of the dynamics of telomere maintenance and factors which modulate telomere loss or re-acquisition will have profound implications for our understanding of the fate of a cell. A recent research news article in Science [54] highlights the current excitement in telomere biology arising from discoveries in simple eukaryotes and basic and applied research in humans. The cloning of the R N A component of telomerase from yeast and humans, and the promise of identifying telomerase protein components and the regulatory pathways for telomere maintenance, will contribute not only to increased research and development activity in this area, but also to the critical testing of hypotheses linking telomere loss to age-related diseases and telomerase reactivation to cancer. The implications for novel drug discovery and the benefits to human health are potentially dramatic.
11.
Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne l, Ratliff RL, Wu JR: A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Nat/ Acad Sci USA 1988, 85:6622-6626.
12.
AIIshire RC, Dempster M, Hastie ND: Human telomeres contain at least three types of G-rlch repeat distributed non-randomly. Nucleic Acids Res 1989, 17:4611-4627.
Hanish JP, Yanowitz IL, De Lange T: Stringent sequence requirements for the formation of human lelomeres. Proc Nat/ Acad 5ci USA 1994, 91:8861-8865. This paper shows that telomere formation in human cells has stringent requirements for the TTAGGG repeat. 13. *,
14. Tommerup H, Dousmanis A, De Lange T: Unusual chromalin •. in human lelomeres. Mol Cell Biol 1994, 14:5777-5785. The authors report that human teiomeres have an unusual chromatin structure characterized by diffuse micrococcal nuciease patterns near the end of the telomere and a shortened nucleosome repeat near the subtelomere region. 15.
Makarov VL, Lejnine S, Bedoyan J, Langmore JP: Nucleosomal organization of telomere-specific chromatin in rat. Cell 1993, 73:775-787.
We would like thank our many colleagues at Geron and elsewhere for critical contributions to the literature and concepts described in this review. It is impractical to acknowledge them all here, but many of their names are listed in the publications below. Finally, we apologize for being unable to do justice to the elegant work on simpler eukaryotes because of the need to focus on human telomere biology.
16.
Villeponteau B, Brawley ), Martinson HG: Nucleosome spacing is compressed in active chromatin domains of chick erythroid cells. Biochemistry 1992, 31:1554-1 563.
17.
Gottschling DE, Aparicio OM, Billington BL, Zakian VA: Posilion effect at S. cerevlsiae telomeres: reversible repression of Pol II transcription. Cell 1990, 63:751-762.
18.
Gottschling DE: Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc Nat/ Acad 5ci USA 1992, 89:4062-4065.
References and recommended reading
19.
Di Leonardo A, Linke SP, Clarkin K, Wahl GM: DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cipl in normal human fibroblasts. Genes Dev 1994, 8:2540-2551.
20.
De Lange T: Human telomeres are attached to the nuclear matrix. EMBO J 1992, 11:717-724.
Acknowledgements
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest I.
Greider CW, Blackburn EH: Identification of a specific lelomere terminal transferase adivity in Telrahymena extracts. Cell 1985, 43:405-413.
2.
Greider CW, Blackburn EH: The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 1987, 51:887-898.
3.
Greider C, Blackburn E: A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 1989, 337:331-337.
4.
Blackburn EH: Telomerases. 61:113-129.
S.
Shippen DE: Telomeres and telomerases. Curr Opin Genet Dev 1993, 3:759-763.
Markova D, Donev R, Patriotis C, Djondjurov L: Interphase chromosomes of Friend-S cells are attached to the matrix structures through the centromeric/telomeric regions. DNA Cell Biol 1994, 13:941-951. Provides further support for the hypothesis that telomeres bind nuclear matrix. 21. •
22.
Shoeman RL, Traub P: The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentln. J Biol Chem 1990, 265:9055-9061.
23.
Zhong Z, Shiue L, Kaplan S, De Lange T: A mammalian factor that binds telomeric ]qAGGG repeats in vitro. Mol Cell Biol 1992, 12:4834~1843.
24.
Conrad MN, Wright JH, Wolf AJ, Zakian VA: RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell 1990, 63:739-750.
25.
7. Blackburn EH: Telomeres: no end in sight. Cell 1994, • 77:621~23. An excellent mini-review describing the recent data on yeast and other lower eukaryotes.
Kyrion G, Boakye KA, Lustig AI: C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. Mol Cell Biol 1992, 12:5159-5173.
26.
Kyrion G, Liu K, Liu C, Lustig AJ: RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevlsiae. Genes Dev 1993, 7:1146-1159.
8.
Fang G, Cech TR: Characterization of a G-quartet formation reaction promoted by the beta-subunit of the Oxytricha telomere-binding protein. Biochemistry 1993, 32:11646-11657.
27.
Morin GB: The human telomere terminal transferase enzyme is a ribonucleoprolein that synthesizes TTAGGG repeats. Cell 1989, 59:521-529.
9.
Zakian VA: Structure and function of telomeres. Annu Rev Genet 1989, 23:579-604.
10.
Yu GL, Bradley JD, Attardi LD, Blackburn EH: In vivo alteration of telomere sequences and senescence caused by mutated Tetrahvmena telomerase RNAs. Nature 1990. 344:126-132.
Annu
Rev
Biochem
1992,
Palladino F, Gasser SM: Telomere maintenance and gene repression: a common end? Curr Opin Cell Biol 1994, 6:373-379. This review describes the factors influencing nuclear architecture and position-effect variegation. 6. •
Autexier C, Greider CW: Functional reconstitutlon of wildtype and mutant Tetrahymena telomerase. Genes Dev 1994, 8:563-575. Presents the first in vitro reconstitution of telomerase activity using wildtype and mutant telomerase RNA added to nuclease-treated extracts and 28. ,,
Telomeres and t e l o m e r a s e in aging and cancer H a r l e y and Villeponteau shows that the template region of the RNA can be dissected into an 'alignment' region and a true 'template' region. 29.
Romero DP, Blackburn EH: A conserved secondary structure for telomerase RNA. Cell ]991, 67:343-353.
30. •
Lingner 1, Hendrick LL, Cech TR: Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev 1994, 8:1984-1988. Presents a careful secondary structure analysis of ciliate telomerase RNA sequences and proposes a common structure on the basis of nucleotide covariations. 31. ••
Singer MS, Gottschling DE: TLCl: template RNA component of Saccharomyces cerevislae telomerase. Science 1994, 266:404-409. Identification of the S. cerevisiae telomerase RNA component and demonstration that disrupting the corresponding gene leads to telomere shortening, decreased growth rate, and loss of cell viability. 32.
Lundblad V, Szostak JW: A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 1989, 57:633-643.
33.
Lundblad V, Blackburn EH: RNA-dependent polymerase motifs in ESTI: tentative identification of a protein component of an essential yeast telomerase. Cell ]990, 60:529-530.
34.
De Lange T, Shiue L, Myers RM, Cox DR, Naylor SL, Killery AM, Varmus HE: Structure and variability of human chromosome ends. Mol Cell Biol 1990, 10:518-527.
35.
AIIsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB: Telomere length predicts replicative capacity of human fibroblasts. Proc Nat/Acad Sci USA 1992, 89:10114-10118.
36.
Vaziri H, Schachter F, Uchida I, We] L, Zhu X, Effros R, Cohen D, Harley CB: Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet ]993, 52:661-667.
37.
Slagboom PE, Droog S, Boomsma DI: Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet 1994, 55:876-882.
38.
Harley CB, Futcher AB, Greider CW: Telomeres shorten during ageing of human fibroblasts. Nature 1990, 345:458-460.
39.
Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, AIIshire RC: Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990, 346:866-868.
40.
Harley CB: Telomere loss: mitotic clock or genetic time bomb? Murat Res 1991, 256:271-282.
41.
Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S: Telomere shortening associated with chromosome inslability is arrested in immortal cells which express telomerase activity. EMBO J 1992, 11:1921-1929.
Harley CB, Kim NW, Prowse KR, Weinrich SL, Hirsch KS, West MD, Bacchetti S, Hirte HW, Counter CM, Greider CW et al.: Telomerase, cell immortality, and cancer. Cold Spring Harb Syrup Quant Biol 1994, 59:307-315. A statement of the apparent relationship between telomerase activation, cell immortality, and cancer, and a review of the relevant data, together with a theoretical model demonstrating the effect of telomerase inhibition on tumor growth.
association of human telomerase activity with immortal cells and cancer. Science 1994, 266:2011-2014. Description of 'TRAP' (telomere repeat amplification protocol), a highly sensitive and reproducible telomerase assay, and a broad survey of normal and tumor cells and tissues. This paper demonstrates that telomerase activity correlates highly with malignant, but not benign, cancers and is present in germline tissues, but not normal somatic tissues tested. 44.
Shay IW, Wright WE, Werbin H: Toward a molecular understanding of human breast cancer: a hypothesis. Breast Cancer Res Treat 1993, 25:83-94.
45.
Shay JW, Wright WE, Werbin H: Loss of telomeric DNA during aging may predispose cells to cancer. Int J Oncol 1993, 3:559-563.
46.
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.
47. •.
Counter CM, Hirte HW, Bacchetti S, Harley CB: Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci USA 1994, 91:2900-2904. The first demonstration that telomerase can be detected in a human cancer, but not the surrounding normal cells, nor the cell type (ovarian epithelial cells) from which the cancer arose. 48. 49. •
Vaziri H, Dragowska W, AIIsopp RC, Thomas TE, Harley CB, Lansdorp PM: Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acacl Sci USA 1994, 91:9857-9860. This paper presents evidence that telomeres decrease with age in vitro and as a function of donor age in vivo in candidate hematopoietic stem cells from humans (CD34 + and CD381o). These data suggest that if telomerase is present in stem cells, it does not fully renew telomeres with replicative aging, or the stem cell population otherwise decreases with age. 50.
Kipling D, Cooke HJ: Hypervariable ultra-long telomeres in mice. Nature 1990, 347:400-402.
51.
Starling JA, Maule J, Hastie ND, AIIshire RC: Extensive telomere repeat arrays in mouse are hypervariable. Nucleic Acids Res 1990, 18:6881~888.
52.
Prows• KR, Avilion An,, Greider CW: Identification of a nonprocesslve telomerase activity from mouse cells. Proc Natl Acad Sci USA ]993, 90:]493-1497.
53.
Prows• KR, Greider CW: Developmenlal and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci USA 1995, 92:in press.
54.
Marx J: Chromosome 265:1656-1658.
55.
Shay JW, Per•ira-Smith OM, Wright WE: A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res 1991, 196:33~.1.
56.
Lindsey J, McGill N, Lindsey L, Green D, Cooke H: In vivo loss of lelomeric repreats with age in humans. Murat Res 1991, 225:45-48.
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43. ••
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific
Olovnikov AM: A theory of marginotomy. J Theoret Biol 1973, 41:181-190.
ends
catch
fire.
Science
1994,
CB Harley and B ViUeponteau, Geron Corporation, 200 Constitution Drive, Menlo Park, California 94025, USA.
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