telomeres and cancer

ANRV288-CB22-21 ARI 28 September 2006 22:6

Telomeres: Cancer toHuman AgingSheila A. Stewart1 and Robert A. Weinberg2,1Departments of Cell Biology and Physiology and of Medicine, WashingtonUniversity School of Medicine, St. Louis, Missouri 63110;email: [email protected] Institute for Biomedical Research and Department of Biology,Massachusetts Institute of Technology, Cambridge, Massachusetts 02142;email: [email protected]

Annu. Rev. Cell Dev. Biol. 2006. 22:53157

First published online as a Review inAdvance on July 7, 2006

The Annual Review of Cell and DevelopmentalBiology is online athttp://cellbio.annualreviews.org

This articles doi:10.1146/annurev.cellbio.22.010305.104518

Copyright c 2006 by Annual Reviews.All rights reserved

1081-0706/06/1110-0531$20.00

Corresponding author.

Key Words

senescence, telomerase, crisis, tumorigenesis

AbstractThe cell phenotypes of senescence and crisis operate to circumscribethe proliferative potential of mammalian cells, suggesting that bothare capable of operating in vivo to suppress the formation of tumors.The key regulators of these phenotypes are the telomeres, which arelocated at the ends of chromosomes and operate to protect the chro-mosomes from end-to-end fusions. Telomere erosion below a cer-tain length can trigger crisis. The relationship between senescenceand telomere function is more complex, however: Cell-physiologicalstresses as well as dysfunction of the complex molecular structuresat the ends of telomeric DNA can trigger senescence. Cells can es-cape senescence by inactivating the Rb and p53 tumor suppressorproteins and can surmount crisis by activating a telomere mainte-nance mechanism. The resulting cell immortalization is an essentialcomponent of the tumorigenic phenotype of human cancer cells.Here we discuss how telomeres are monitored and maintained andhow loss of a functional telomere inuences biological functions asdiverse as aging and carcinogenesis.

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Telomere:noncoding DNA andassociated proteinslocated at thetermini of linearchromosomes

Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 532TELOMERES AND CELLULAR

LIFESPAN . . . . . . . . . . . . . . . . . . . . . . 532Replicative Senescence . . . . . . . . . . . 532Cell Senescence: Man Versus

Mouse . . . . . . . . . . . . . . . . . . . . . . . . 534Telomere Dysfunction and

Senescence . . . . . . . . . . . . . . . . . . . . 535Stress-Induced Senescence . . . . . . . . 536Senescence: A Tumor Suppressor

Mechanism? . . . . . . . . . . . . . . . . . . 537Crisis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538

CELL SENESCENCE ANDHUMAN AGING . . . . . . . . . . . . . . . 540

TELOMERASE AND CANCER. . . . 540Telomeres and Cancer: Lessons

from the Mouse . . . . . . . . . . . . . . . 542EXTRATELOMERIC

FUNCTIONS OFTELOMERASE . . . . . . . . . . . . . . . . . 542

TELOMERE MAINTENANCE INTHE ABSENCE OFTELOMERASE . . . . . . . . . . . . . . . . . 544

TELOMERE-BINDINGPROTEINS . . . . . . . . . . . . . . . . . . . . . 545Telomeric Core Proteins . . . . . . . . . . 545Telomeres and DNA

Repair/Replication Proteins:Antagonistic or SynergisticRelationships? . . . . . . . . . . . . . . . . . 546

EPIGENETIC MODULATION OFTELOMERE LENGTH. . . . . . . . . 548

TELOMERE-BINDINGPROTEINS: BEYOND THETELOMERE . . . . . . . . . . . . . . . . . . . . 548

CONCLUDING REMARKS ANDFUTURE DIRECTIONS. . . . . . . . 549

INTRODUCTION

Since Barbara McClintocks original descrip-tion in the 1940s, telomeres have been rec-ognized as important capping structures thatplay a central role in distinguishing the true

ends of a linear chromosome from a bonade double-stranded DNA (dsDNA) break(McClintock 1941). In recent years, we havelearned a great deal about the complex nu-cleoprotein structures located at the ends ofchromosomes. Emerging from these studies isan understanding that telomere function is in-extricably linked to cell cycle control, cellularimmortalization, and tumorigenesis. We areonly now beginning to understand the molec-ular details of how a telomere is monitored,regulated, and modied and how these func-tions permit continued cell cycle progression.In this review, we attempt to cover a broadrange of topics concerning telomere biology,including information on the structure of atelomere, its relation to cellular lifespan, andits role in tumorigenesis. Each of these sub-jects warrants its own separate review, and sowe would like to apologize from the outset toour many colleagues whose work could not beincluded in the present one.

TELOMERES AND CELLULARLIFESPAN

Replicative Senescence

Early researchers in the area of replicativesenescence assumed that individual cells frommulticellular organisms possess an immor-talized growth phenotype, i.e., that they areable to proliferate indenitely. In the 1960s,however, Leonard Hayicks pioneering workchanged this paradigm and thereby initiateda new eld of study. Hayick found that -broblasts isolated from an individual possessonly a limited proliferative potential in vitro:A lineage of these cells could pass throughonly a predetermined number of growth-and-division cycles (Hayick 1965). Cells thathad reached the allowed limit of replicationand thus halted further proliferation weretermed senescent; such cells were character-ized by a at, extended shape and remainedactive metabolically but no longer divided.Indeed, we now know that such senescentcells, if properly treated, can remain viable for

532 Stewart Weinberg

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years (S. Stewart & R. Weinberg, unpublisheddata). Importantly, Hayicks work demon-strated that cells isolated from the same in-dividual on multiple occasions recapitulatedthis nite growth phenotype.

These seminal observations led to the hy-pothesis that cells possess an internal clockingmechanism that is capable of (a) tracking thenumber of cellular divisions through whichtheir lineage has passed and (b) halting anyfurther division after a predetermined numberof cell divisions (also known as the Hayicklimit). This work led to the further hypoth-esis that a limited proliferative capacity playsan important role in both aging and tumorsuppression (Hayick 1976). Since its originaldescription, the role of cell senescence in tu-mor suppression has received much support,whereas the role of cell senescence in organ-ismic aging remains unclear.

A link between cell senescence and thetelomere was suggested in the 1990s, whentwo researchers described what would even-tually become known as the telomere hypoth-esis. In the 1970s both Watson and Olovnikovhad described the end replication problem,in which they suggested that linear chro-mosomes would be unable to replicate theextreme 3 ends faithfully (Olovnikov 1973,Watson 1972). Thus, even if the initial RNAprimer were synthesized at the extreme 3 endof the template strand, a small portion of thechromosomal DNA would be lost followingcompletion of replication. Such underrepli-cation would create a problem if importantgenetic information were located at the endof the chromosome. This potentially catas-trophic problem was solved by the evolution-ary development of the telomere, which waseventually shown to be a repetitive sequenceof noncoding DNA (reviewed in Blackburn1991). In mammals, the telomere consists ofthousands of tandem repeats of the hexanu-cleotide sequence TTAGGG. Fluorescent insitu hybridization showing human telomeresis shown in Figure 1.

To determine the fate of the chromosomeends, Harley, Greider, and colleagues began to

Figure 1Telomere and centromere localization of metaphase chromosomes byuorescence in situ hybridization (FISH). Use of FISH, together withtelomere- and centromere-specic probes, reveals the telomeres at the endsof each HeLa cell metaphase chromosome (red ) and a centromere locatedin its middle (green).

Tumorigenesis:process by whichnormal human cellsare transformed intotumor cells

Senescence: p53-and Rb-dependentpermanent growtharrest

TRF: telomererestriction fragment

examine the telomeric DNA of human chro-mosomes during successive rounds of DNAreplication and cellular division (Allsopp et al.1992). To do so, they utilized four-base-pairrestriction enzymes to digest nontelomericchromosomal DNA into small pieces (withan average DNA size of 126 base pairs); thetelomeric DNA remained intact because itsrepeating hexanucleotide sequences were notrecognized by these enzymes. The researchersthen analyzed the size of this surviving telom-eric DNA by Southern blot analysis, a proce-dure now termed the TRF (telomere restric-tion fragment) Southern blot.

Harley, Greider, and colleagues (Allsoppet al. 1992) initially observed a smear rep-resenting the heterogeneous lengths of thetelomeres within their cell populations. Ex-tending these analyses, they then examined apopulation of cells throughout its replicativelifespan and demonstrated that mean telom-ere lengths were reduced progressively with

www.annualreviews.org Telomeres: Cancer to Human Aging 533

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LT: SV40 large Tantigen

Figure 2The telomere hypothesis. Telomere length (ordinate) is progressively lostduring successive rounds of cellular division (abscissa), eventually leadingto p53- and Rb-dependent permanent growth arrest, referred to assenescence. Inactivation of p53 and Rb function allows continued cellulardivision and further telomere shortening. Telomeres eventually erode to alength at which they are unable to protect chromosome ends, resulting incrisis, i.e., end-to-end chromosome fusions and apoptotic cell death. Rareclones (1 in 107) may emerge from a population of cells in crisis. Theseclones maintain stable telomere lengths through the activation of atelomere maintenance mechanism, i.e., human telomerase catalyticsubunit (hTERT) expression or the alternative lengthening of telomeres(ALT) mechanism.

each subsequent division (Figure 2), exactly asWatsons (1972) and Olovnikovs (1973) mod-els had predicted. Importantly, when cells iso-lated from the same individual were followedin several independent cultures, these cells en-tered into senescence with roughly the sameaverage telomere lengths. This observationsuggested that telomere shortening serves asthe genetic clocking mechanism originally de-scribed by Hayick (1965) and that the telo-meres tracked the number of cellular divi-sions through which an individual cell lineagehad passed. In addition, it was hypothesizedthat once the telomeres shortened to a certainpredetermined length, these DNA sequenceswere responsible for triggering entrance intosenescence (Greider & Blackburn 1996 andreferences therein).

Since these path-nding discoveries, wehave learned that telomeres can indeed con-trol replicative lifespan. However, they do so

through mechanisms that are more complexthan simple maintenance of telomere lengthabove a critical threshold level. Instead, itis the maintenance of a properly functional,or capped, telomere structure (which can beinuenced by telomere length) that is criti-cal to continued cellular division (reviewed inBlackburn 2000). Indeed, as described below,the induction of replicative senescence can of-ten be separated from the erosion of telomerelength below a certain threshold level.

Cell Senescence: Man Versus Mouse

The permanent growth arrest that character-izes replicative senescence is dependent on thedownstream effectors p53 and Rb, which im-pose this state once telomeric DNA has un-dergone certain changes (Shay et al. 1991).The key role of these two tumor suppressorpathways in senescence was demonstrated byexpressing in presenescent cells the SV40 viralLarge T antigen (LT) or the human papillo-mavirus (HPV) proteins E6 plus E7. LT func-tionally eliminates p53 and Rb, as do HPVE6 plus E7. Analyses of human cells that weredestined to undergo senescence demonstratedthat abrogation of both the p53 and Rb path-ways was necessary for cells to bypass senes-cence (Shay et al. 1991), whereas inactivationof either p53 or Rb did not sufce to by-pass senescence. This was not found, how-ever, to be the case for murine cells, whichneed lose only the function of either the p53or Rb pathways to bypass senescence (Sherr& DePinho 2000 and references therein). It isstill unclear why, to escape senescence, bothpathways must be inactivated in human cells,whereas murine cells need lose function ofonly one pathway. Nonetheless, these obser-vations indicate that these regulatory path-ways are organized differently in cells of thetwo species.

The importance of the p53 and Rb tumorsuppressor pathways is not the only charac-teristic that distinguishes human and murinesenescence. Unlike in human cells, progres-sive telomere shortening is not observed in


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cultured mouse embryonic broblasts (MEFs)(Blasco et al. 1997, Zijlmans et al. 1997). Inaddition, human embryonic broblasts typ-ically divide 50100 times before enteringsenescence. In contrast, MEFs divide only ap-proximately ten times before entering senes-cence. Because murine telomeres are onaverage 50 kb, whereas their human counter-parts are approximately 1520 kb (Blasco et al.1997, Zijlmans et al. 1997), the above obser-vations raised questions about the importanceof telomere erosion to replicative senescencein the mouse.

To explain these apparent discrepancies,some suggested that murine cells were moresensitive to in vitro growth conditions thatresulted in nontelomeric damage and a dis-tinct type of senescence often termed stress-induced senescence (Itahana et al. 2004). In-deed, this was found to be the case: Murinecells are exquisitely sensitive to high oxy-gen tensions, notably those experienced understandard conditions of cell culture (20%).Accordingly, when MEFs are propagatedat physiological (rather than ambient) oxy-gen tensions (3%), they do not undergoearly senescence in vitro (Parrinello et al.2003). These observations indicated that cell-physiological mechanisms unrelated to telo-

mere length are able to induce senescence inculture.

Telomere Dysfunction andSenescence

Telomere shortening is thought to lead to lossof structural integrity of the telomere nucleo-protein, resulting in activation of the p53and Rb tumor suppressor pathways and cel-lular senescence (Shay & Wright 2005). Thetelomere is a complex nucleoprotein that con-tains both single- and double-stranded DNAand associated proteins that interact to main-tain a stable structure. Electron microscopyhas revealed that the telomeric DNA formsa higher-order structure (Grifth et al. 1999),in which the overhanging, single-stranded tailof the G-rich strand inserts itself back into thedouble-stranded telomeric repeats, therebyyielding a displacement loop that is known asa T-loop (owing to the presence of the telom-eric DNA repeat sequence) (Figure 3).

TRF2 is a telomere-associated protein thatfacilitates T-loop formation (Stansel et al.2001), and overexpression of a dominant-negative form of TRF2 (TRF2BM) isthought to result in T-loop loss in vivo. More-over, introduction of TRF2BM results in

Figure 3The T-loopschematic.Representation ofthe T-loop, in whichthe single-stranded,G-rich overhanginserts into thedouble-strandedtelomeric DNA,creating adisplacement loop.The DNA andassociated telomericproteins create acapped, orfunctional, telomere.


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RNAi: RNAinterference

siRNA: smallinterfering RNA

the rapid induction of senescence in wild-type human broblasts (Karlseder et al. 2002).As in replicative senescence, TRF2BM-induced senescence activates both the p53and Rb pathways. Importantly, cells under-going TRF2BM-induced senescence dis-play reduction in the G-rich single-strandedoverhang without an associated loss of over-all double-stranded telomeric DNA repeats.Because TRF2 is critical to T-loop forma-tion, maintenance of a specic telomere-associated molecular structurethe T-loopmay be critical for continued cellularproliferation and the avoidance of senescence.Moreover, such observations dissociate thesignal that triggers entrance into senescencefrom the overall length of telomeric DNA.

The unique structure of the telomere, de-scribed in part above, is thought to distinguishit from the bona de dsDNA breaks that occa-sionally affect chromosomal DNA. If so, lossof telomere integrity should erase these differ-ences, causing the degraded telomere to ap-pear much like a dsDNA break and leading tothe same DNA damage response that usuallyfollows the formation of dsDNA breaks, in-cluding activation of the p53 damage responsepathway. Indeed, researchers have reportedsuch a nding, supporting the importance oftelomere integrity in the suppression of theDNA damage response.

Overexpression of TRF2BM in humancells results in activation of the ATM and ATRkinases (which signals the presence of dsDNAbreaks in a cell) as well as the downstream ef-fectors, p53 and p16 (Karlseder et al. 1999,2002). These observations suggest that un-capped or dysfunctional telomeres can elicita classic DNA damage response, raising thepossibility that a similar mechanism operatesduring replicative senescence, when loss oftelomere integrity results in an inability toprotect the chromosomal ends. In agreementwith this notion, some have demonstratedthat human broblasts undergoing replicativesenescence exhibit DNA damage foci at theirtelomeres (dAdda di Fagagna et al. 2003).These foci, referred to as TIFs (telomere

dysfunctioninduced foci), contain many ofthe classic DNA damage response proteins,including H2A.X, 53BP1, MDC1, NBS1,and phosphorylated SMC1.

TIFs have been found in the majority ofcells undergoing replicative or TRF2BM-induced senescence (dAdda di Fagagna et al.2003, Takai et al. 2003), suggesting that a con-tinuing DNA damage response is responsi-ble for maintaining the senescent phenotype.Indeed, RNA interference (RNAi) and mi-croinjection studies support this contention.For example, delivery of small interferingRNA (siRNA) constructs targeting Chk1/2 ormicroinjection of nonfunctional, dominant-interfering ATM or ATR kinase proteins re-sulted in S phase entry (indicating emergencefrom senescence), in spite of the presenceof defective telomeres in a cell (dAdda diFagagna et al. 2003, Takai et al. 2003). Thisdemonstrates that the classic DNA damageresponse mechanism mediates the G1 arrestobserved in senescent cells.

Although the data above clearly supporta role for the DNA damage response intelomere dysfunction and replicative senes-cence, many senescing cells within a popu-lation do not display TIFs. In these popula-tions, those cells that did not display TIFsinstead expressed high levels of the cell cy-cle inhibitor p16INK4A (Herbig et al. 2004).This observation suggested that senescingpopulations of cells are biologically heteroge-neous: Some cells experience telomere dys-function, whereas others are affected by atelomere-independent mechanism that in-duces an arrest phenotype indistinguishablefrom senescencethe stress-induced senes-cent state, which is discussed in more detailbelow.

Stress-Induced Senescence

Recently reported data have highlighted anumber of conditions that can lead to stress-induced senescence. These include low serumor growth factor concentrations, exposure tohigh levels of DNA damage, inappropriate


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conditions of growth (including those thatinduce the expression of the p16INK4A andp21Waf1 cell cycle inhibitors), and high oxida-tive stress levels (Ramirez et al. 2001, Sherr& DePinho 2000, Wei et al. 2001, Wright &Shay 2002, and references in all). Exposureto these various agents or signals results in aphenotype that is indistinguishable from thatshown by cells that have reached the Hayicklimit and enter replicative senescence.

Ectopic expression of the human telom-erase catalytic subunit (hTERT)the cellularenzyme that functions to restore and extendtelomeric DNAbypasses replicative senes-cence (Bodnar et al. 1998, Vaziri & Benchimol1998). In contrast, overexpression of hTERThas no detectable effect on stress-inducedsenescence, causing some to argue thatreplicative senescence and stress-inducedsenescence are distinct biological processes.This would imply that stress-induced senes-cence represents a response to stimuli orconditions that originate outside of the cell(i.e., extrinsic), whereas replicative senescenceis exclusively a cell-autonomous mechanism(i.e., intrinsic). More work in this area will beneeded to answer this possibility denitively.

Senescence: A Tumor SuppressorMechanism?

Senescence limits the proliferative potentialof a cell and consequently has been argued tofunction as a potent tumor-suppressing mech-anism (reviewed in Campisi et al. 2001). Morespecically, senescence may prevent the out-growth of cell populations at risk of evolv-ing into neoplasias. Several lines of evidencesupport this notion. For example, as detailedabove, damaged telomeres are a potent in-ducer of senescence; these may arise in celllineages that have passed through an exces-sive number of growth-and-division cycles,which may occur during the long, multistepformation of neoplastic cell populations. Inaddition, as already mentioned above, certaintypes of DNA damage; inappropriate growthstimuli, including oncogene activation; and

hTERT: humantelomerase catalyticsubunit

perturbations in chromatin structure can alllead to stress-induced senescence (Sherr &DePinho 2000, Wei et al. 2001, Wright &Shay 2002, and references in all).

Molecular proof demonstrating that senes-cence blocks the growth of neoplastic cellsin vivo recently was supplied through studyof both murine cancer models and humancancer. Thus, some researchers have utilizedthe EN-Ras mouse model to demonstratethat N-Ras overexpression leads to a senes-cent growth arrest that delays the appearanceof tumors (Braig et al. 2005). Loss of wild-type p53 in this model results in the bypassof senescence and rapidly developed invasiveT cell lymphomas. This study demonstratedthat senescence delayed the appearance of ma-lignant cells (Braig et al. 2005). In anotherstudy, loss of the tumor suppressor PTEN inthe prostate resulted in a p53-mediated senes-cent growth arrest that, once again, blockedfurther tumor progression (Chen et al.2005).

Further support of the role of senescencein tumor prevention comes from the nd-ings of other investigators who have demon-strated that human nevi display a senescentphenotype (Michaloglou et al. 2005). Neviare benign growths of melanocyte origin andoften contain mutations in the BRAF onco-gene, which is a protein kinase that func-tions downstream of Ras. Interestingly, over-expression of oncogenic BRAF V600E inmelanocytes leads to senescence, suggestingthat the expression of this oncogene resultsin senescence in vivo and inhibits transforma-tion. Yet other researchers have utilized theconditional K-Ras V12 transgenic mouse todemonstrate that nontumorigenic lung ade-nomas are senescent, displaying the classicmarkers of this state, including senescence-associated -galactosidase (SA-gal) and highlevels of p16INK4A expression (Collado et al.2005). In this model, progression to adeno-carcinoma resulted in loss of all the senes-cence markers, suggesting that progressionrequired premalignant cells to bypass senes-cence. Taken together, these studies strongly


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Crisis: cellulardeath characterizedby end-to-endchromosomal fusions

ALT: alternativelengthening oftelomeres

Telomerase:cellular,RNA-dependentDNA polymerasethat adds telomericrepeats to the ends ofchromosomes

support a role for senescence in blocking theformation of tumors.

Crisis

Cell populations that succeed in bypassingsenescence through the inactivation of the Rband p53 signaling pathways continue to di-vide until their telomeres become criticallyshort (Figure 2) and no longer protect thechromosome ends from the cell machinerycharged with the detection and repair of ds-DNA breaks. When this occurs, the popula-tion of cells enters a second proliferative blockreferred to as crisis, which is characterizedby short telomeres, end-to-end chromosomalfusions, anaphase bridges, and cell death byapoptosis (Shay & Wright 2005, Wright &Shay 1992). This second arrest state is distinctfrom senescence in two fundamental ways:(a) Rampant genomic instability is observed,which is associated with (b) widespread celldeath.

On occasion, however, a rare clone ofcells (1 in 107 human cells) can emergefrom a population of cells in crisis; such cellclones are invariably immortal. Analyses oftelomeric DNA in such variant clones in-dicate that telomere lengths are maintainedin the face of ongoing rounds of DNAreplication and cellular division (Wright &Shay 1992). Moreover, unlike populations ofprecrisis cells, the great majority of these im-mortalized cell populations demonstrate ex-pression of the telomerase reverse transcrip-tase (hTERT) enzyme, which was mentionedin passing earlier. Those cell clones that donot activate hTERT expression resort insteadto activating a poorly understood telomeremaintenance mechanism termed alternativelengthening of telomeres (ALT), which, at themolecular level, may in fact represent sev-eral distinct mechanisms (discussed below)(Figure 2). Taken together, these observa-tions provide strong support for the hypothe-ses that escape from crisis and entrance intoan immortal growth state depends on acqui-

sition of telomere maintenance functions andthat this can be achieved by activation of eithertelomerase function or an ALT mechanism.

The causal role of telomerase in cellularimmortalization was demonstrated formallyfollowing the cloning of the gene encodinghTERT (Meyerson et al. 1997, Nakamuraet al. 1997). Telomerase is a cellular reversetranscriptase that utilizes an associated RNAmolecule as a template to add telomeric re-peats to the ends of telomeres, thereby ex-tending them. By studying structure-functionrelationships in the distantly related HIV-1 reverse transcriptase, two research groupswere able to create a dominant-negative al-lele of hTERT (DN-hTERT) and demon-strate its essential role in both telomere main-tenance and cellular immortality (Hahn et al.1999b, Zhang et al. 1999). Thus, inhibitionof hTERT activity following DN-hTERTexpression in already immortalized cells re-sulted in the progressive loss of telomericrepeats during successive rounds of DNAreplication and cellular division. Eventuallytelomeres became critically shortened, and asobserved in crisis, end-to-end chromosomalfusions became evident and were followed bywidespread cell death. Strikingly, the preex-isting length of telomeres at the beginning ofeach of these experiments primarily dictatedthe time required for cell populations to entercrisis. For example, when the DN-hTERT al-lele was introduced into cells that initially pos-sessed telomeres in the range of 23 kb, thesecells entered crisis following fewer cell divi-sions than did cells possessing initial telomerelengths of 68 kb (Figure 4).

The critical role of telomere maintenancein cellular immortality was further supportedby the introduction of wild-type hTERTinto precrisis broblasts, endothelial cells, andretinal pigment epithelial cells (Bodnar et al.1998, Rufer et al. 1998, Vaziri & Benchimol1998, Yang et al. 1999). Introduction ofhTERT resulted in telomere stabilizationand cellular immortality. Indeed, resultingtelomerase-expressing broblasts have been


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Tel

om

ere

len

gth

Time

STOP

ALT-dependent telomere maintenance

Telomerase-dependent telomere maintenance

Inhibit TERT activity(introduction of DN-hTERT)

Senescence Crisis

Figure 4Telomere length dictates entrance into crisis. Immortal cells that have bypassed senescence and crisismaintain stable telomeres. Cell clones can possess different telomere lengths, as the graph shows.Introduction of a dominant-negative hTERT allele (DN-hTERT) into immortal telomerase-positivecells (see text for details) inhibits telomerase activity, resulting in a growth arrest reminiscent of crisis,which is characterized by telomere loss, end-to-end chromosomal fusions, and apoptotic cell death.Initial telomere lengths in an individual cell clone dictate the timing of the entrance into crisis, followinginactivation of telomerase activity. In other words, cells that maintain longer telomeres require morerounds of DNA replication and cellular division before telomeres reach a critically short length thatresults in crisis. Cells that maintain their telomeres through the ALT mechanism are unaffected byintroduction of the DN-hTERT allele.

maintained in culture for years, further sup-porting the idea of telomere maintenance asa prerequisite for cellular immortality. Impor-tantly, cells immortalized through hTERT ex-pression appear normal and are responsive togrowth stimuli and growth inhibitors, indi-cating that whereas hTERT can immortalizecells, it does not, on its own, cause cell trans-formation (Hahn et al. 1999a, Jiang et al. 1999,Morales et al. 1999).

Fibroblasts immortalized by ectopic ex-pression of hTERT display gene expressionproles that are very similar to that of theirnormal early-passage counterparts. This ob-servation soon led to the notion that hTERTimmortalization may allow experimenters toimmortalize human cells while maintainingtheir normal karyotype and gene expressionproles. Such cell lines should, in principle,allow one to study normal cellular processesin the absence of the genomic instability in-

herent in the previous models of cell immor-talization that utilized viral oncogenes such asSV40 LT to effect immortalization. However,this initial excitement regarding the possibili-ties of hTERT-mediated cell immortalizationfaded after it was shown that many epithelialcell types did not become immortalized in re-sponse to hTERT (Rheinwald et al. 2002) andthat, after extended in vitro culture, hTERT-immortalized cells do indeed display changesin their gene expression proles (Choi et al.2001; S. Stewart, unpublished data). In fact, itis difcult to know whether these changes aredue to telomerase expression or to the long-term growth of cells in tissue culture dishesin the presence of bovine serum. Despitethese potential pitfalls, many groups haveutilized hTERT-mediated immortalization tostudy the normal biological functions of cellsshowing only minimal genomic instabilityin vitro.


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CELL SENESCENCE ANDHUMAN AGING

Two major hypotheses have attempted to ex-plain the molecular basis of cellular and thusorganismic aging (reviewed in Aviv 2004,Ben-Porath & Weinberg 2004, and Vijg &Suh 2005). The rst suggests that aging isthe result of the slow accumulation of dam-age that leads to cellular and eventually tissuedeterioration. The second suggests that ag-ing is the result of a cellular program that isgoverned by a biological clock, such as telom-ere length. Examination of human cells prop-agated in culture has indeed shed light on thecellular bases of aging.

As described above, when grown in cul-ture, normal human cells will undergo a lim-ited number of divisions before entering astate of replicative senescence in which theyremain viable but are unable to divide fur-ther (reviewed in Campisi 1996). Some haveproposed that this limited replicative capac-ity contributes to the phenotypes associatedwith aging, such as reduced wound healingand weakened immune systems (Effros 2000,Paradis et al. 2001, Rubin 2002). Simi-larly, diseases characterized by high cellularturnover, such as acquired immunodeciencydisease (AIDS) and cirrhosis, are thought to beat least partially the result of cellular mortality(Effros 2000, Paradis et al. 2001, Rubin 2002),i.e., the limited ability of tissues to regeneratethemselves through cell proliferation.

The role of telomere homeostasis and cellsenescence in human aging has been sup-ported by studies demonstrating a relation-ship between donor age and telomere lengths,correlations between in vitro growth capacityand donor age, and reduced in vitro growthcapacity of cells isolated from patients suf-fering from various types of progeria (condi-tions characterized by premature aging) whencompared with normal, age-matched controlcells (Dimri et al. 1995, Faragher et al. 1993,Martin et al. 1970). Although these observa-tions are intriguing, their correlative naturemakes it difcult to conclude that telomere

loss is the central driving force behind humancell and thus tissue aging.

Arguably, some of the most persuasivedata on this point come from patients suffer-ing from dyskeratosis congenita (Dokal 2001,Mason et al. 2005). There are two forms of thisdisease (Dokal 2001; Vulliamy et al. 2001a,b).The rst form is autosomal recessive and re-sults from mutations in the human TERCgene (which encodes the RNA subunit of thetelomerase holoenzyme). The second is an X-linked autosomal dominant form of the dis-ease and is the result of a mutation in thedyskerin gene, which compromises ribosomebiosynthesis and seems also to affect assem-bly of the telomerase holoenzyme, resultingin loss of enzyme function.

Analyses of cells from dyskeratosis con-genita patients reveal telomere shorteningand dysfunction when compared with telom-eres in the cells of age-matched controls.Patients suffering from this disease manifestseveral distinct abnormalities, including ab-normal skin pigmentation, nail dystrophy,mucosal leukoplakia, bone marrow failure,and cancer predisposition (Dokal 2001). In-terestingly, this disease also appears to demon-strate anticipation (symptoms increasing inseverity with each succeeding organismic gen-eration), although more patient families mustbe analyzed to conrm this (Vulliamy et al.2004). Many of these symptoms are reminis-cent of aged humans and, as discussed below,are also observed in mice lacking telomerasefunction. Nevertheless, the fact that aspectsof human aging can be phenocopied by spe-cic defects in the telomere-maintenance ma-chinery does not prove that telomere erosionis normally a primary causal force that driveshuman aging.

TELOMERASE AND CANCER

The development of neoplasia is a multi-step process that involves accumulation of anumber of genetic and epigenetic alterationsthat collaborate to produce transformed cells.


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In addition, studies of human tumor cellgenomes reveal an ever-increasing numberof genetic abnormalities that can contributeto tumorigenesis, underscoring the complex-ity of this process. Despite this complexity,early studies in murine and avian models sug-gested that cancer may be the result of onlya few mutations, raising the possibility thatmany of the observed mutations in humancancer cells were not responsible for the trans-formed phenotype (Hanahan & Weinberg2000, Land et al. 1983). For example, in the1980s, researchers demonstrated that the in-troduction of two cooperating oncogenes wassufcient to transform murine cells (Landet al. 1983, Ruley & Fried 1983). Subsequentstudies in transgenic mice corroborated theseoriginal observations. Despite such successesin transforming rodent cells, similar experi-ments consistently failed to transform humancells to a tumorigenic state, calling into ques-tion the relevance of animal models to humancarcinogenesis.

In fact, epidemiologic studies in humansystems have suggested that four to six rate-limiting events are required for cancer for-mation (Armitage & Doll 1954, 2004; Ruley& Fried 1983). These original estimates wereeventually echoed by experiments that fol-lowed the cloning of the hTERT gene, whichencodes the catalytic subunit of the telom-erase holoenzyme. Because hTERT is ex-pressed at signicant levels in 90% of humantumors and because normal cells express ex-tremely low levels of this enzyme, it seemedreasonable that telomerase activity would berequired to transform a human cell. Indeed,Hahn et al. (1999a, 2002) demonstrated that anormal human cell can be converted to a fullytumorigenic cell with a set of introduced, de-ned genetic elements, including a clone ofthe hTERT gene. In particular, inactivationof the p53 and Rb tumor suppressor path-ways through expression of the SV40 LT, ab-rogation of a PP2A (protein phosphatase 2A)function through expression of SV40 small tantigen (ST), mitogenic stimulation through

Figure 5In vitro transformation of human cells. Normal human cells can betransformed with a set of introduced, dened genetic elements. Specically,introduction of clones specifying the SV40 Large T and small t antigens(which functionally inactivate p53, Rb, and PP2A), oncogenic H-Ras, andthe hTERT catalytic component of the telomerase holoenzyme results inanchorage-independent growth and tumor formation inimmunocompromised mice.

oncogenic H-Ras expression, and cellular im-mortalization achieved through hTERT ex-pression together produced a transformed hu-man cell capable of colony formation in softagar and tumor formation in immunocom-promised host mice (Hahn et al. 1999a, 2002)(Figure 5).

The study by Hahn et al. (1999a) under-scored the requirement for cellular immor-talization in the transformation process. Inaddition, a subsequent study demonstratedthat aneuploidy was not a prerequisite for thetumorigenic cell phenotype (Zimonjic et al.2001). Since this original report by Hahn et al.(1999a), numerous groups have applied thissame transformation strategy and found that,indeed, these ve changes sufce to transforma variety of human cell types. Current effortsare now focused on humanizing the mu-tations, i.e., utilizing mutant alleles that arecommonly found in human tumors. For ex-ample, in humans, SV40 LT is not associatedwith tumorigenesis, whereas p53 mutationsand loss of p16INK4A are commonly observed.Moreover, by introducing genetic mutationsthat are documented to be present in humantumors, researchers are now building cellularmodels that may be useful in drug discoveryand development.


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Telomeres and Cancer: Lessons fromthe Mouse

As mentioned above, normal human somaticcells express low to undetectable levels oftelomerase activity that are insufcient tomaintain telomere lengths, whereas morethan 90% of human tumor cell populationsthat have been assayed express high levelsof telomerase activity and maintain telomerelengths (Hahn 2005, Shay & Bacchetti 1997).Because tumor cells typically display telom-ere lengths that are shorter than neighboring,normal cells, telomerase activation is thoughtto be a relatively late event in the transforma-tion process that, once achieved, may stabilizeexisting telomere lengths but fails to restoretelomeres to the lengths seen in the nearbynormal cells (reviewed in Maser & DePinho2002).

Telomere loss and the ensuing genomic in-stability are believed to be driving forces inthe transformation process. This notion hasreceived substantial support from the studies,noted above, that utilize telomerase knock-out mice, which display progressive telom-ere shortening, similar to the behavior oftelomeres in normal human cells (Blasco et al.1997). As mentioned above, these animals donot exhibit any apparent pathological phe-notypes in the early generations, presumablybecause their cells still possess large telom-ere reserves. However, late-generation micedo display pathologies associated with ag-ing and demonstrate an enhanced transforma-tion phenotype. Signicantly, when mated tocancer-prone mice, the telomerase-negativeanimals recapitulate some of the pheno-types observed in human neoplasias (Maser &DePinho 2002).

Late-generation mTERC/ mice developlymphomas and teratocarcinomas with highfrequency, and these are associated withextensive anaphase bridge formation andgenetic instability (reviewed in Maser &DePinho 2002). Such anaphase bridges areexpected to form following loss of telom-eres and resulting end-to-end fusions of chro-

matids. When the mTERC/ mice are matedto Ink4a/ mice, the spectrum of tumor typesis altered slightly, and mice develop lym-phomas and brosarcomas (Greenberg et al.1999). Mice decient in both mTERC andp53 develop an even more varied tumor spec-trum, displaying lymphomas, sarcomas, andadenocarcinomas (Artandi et al. 2000). Im-portantly, tumor formation in late-generationmTERC/Ink4a/ and mTERC/p53/

mice is suppressed, suggesting that telomerereserves decline to a level that is incompatiblewith further cellular proliferation.

Of greater interest is the tumor spectrumobserved in mTERC/p53+/ mice. Thesemice still develop lymphomas and sarcomas,but they also develop epithelial carcinomasthat are similar to the majority of human tu-mors (Artandi et al. 2000). Again, tumor de-velopment is reduced in late-generation mice,but the altered tumor spectrum suggests thattumors in these animals more closely resemblethose found in humans. The breakage-fusion-bridge cycles occurring in these mice follow-ing telomere collapse presumably contributeto generalized instability in their cells, whichin turn favors the inception of the observedtumors.

EXTRATELOMERICFUNCTIONS OF TELOMERASE

The ability of the telomerase enzyme to re-verse telomere attrition, impart cellular im-mortality, and contribute to tumorigenesis in-dicates that this enzymes effects derive fromits ability to add telomeric repeats to theends of the chromosomal DNA and therebystabilize telomere lengths and the structuralintegrity of entire chromosomes. Althoughthis model has been borne out by numer-ous studies, it has also become clear that therole of telomerase in cellular biology is morecomplex then simply one of telomere lengthmaintenance.

Accumulating evidence suggests thattelomerase also inuences normal cellular


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physiology, even in cells that have long telom-eres. For example, studies have demonstratedthat mice lacking functional telomerase areunable to maintain proper tissue homeostasis,particularly in tissues of high cell turnover,such as the bone marrow, skin, liver, andgastrointestinal tract (Herrera et al. 1999,Lee et al. 1998, Rudolph et al. 1999). Thesephenotypes may be exacerbated in aged miceor following an infectious challenge, even inanimals that do not demonstrate criticallyshort telomeres (Rudolph et al. 2000).

Other studies have demonstrated that ec-topic expression of telomerase in cells thatare already telomerase positive results in in-creased resistance to apoptosis (Holt et al.1999). Another study has demonstrated thatectopic expression of wild-type hTERT al-lows nontumorigenic ALT cells (cells thatpossess long telomeres but do not express en-dogenous hTERT) to form tumors (Stewartet al. 2002). Furthermore, use of an hTERTmutant that is unable to extend telomerelengths recapitulated this phenotype, leadingto the argument that the tumorigenic phe-notype in this model was not the result oftelomere elongation (Stewart et al. 2002). Inagreement with this study, mice with rela-tively long telomeres displayed an enhancedtumorigenesis rate once an mTERT trans-gene was expressed in certain tissues (Artandiet al. 2002). Once again, it is difcult to un-derstand how increased telomerase expressioncould enhance tumorigenesis simply by in-creasing the lengths of already long telomeres.

More recently, telomerase expression hasbeen found to inuence hair growth in a trans-genic mouse model (Sarin et al. 2005). Ectopicexpression of mTERT in the skin epitheliumcaused a rapid transition from the telogenphase (resting phase of the hair follicle cy-cle) to the anagen phase (active growth phase).This transition resulted in robust hair growth.Examination of the stem cells localized to thebulge region of the hair follicle revealed thatthis transition was due to increased prolifera-tion of the normally quiescent, multipotentstem cells. Importantly, stem cell prolifera-

tion did not depend on the concomitant pres-ence of mTERC (the RNA component of thetelomerase enzyme), indicating that this pro-liferation did not depend on telomere elonga-tion by the ectopically expressed telomeraseenzyme.

Recently reported data also suggest thattelomerase may play an important role inmaintaining chromosomal structure in do-mains other than telomeres (Masutomi et al.2003). Thus, normal human broblasts tran-siently express low but detectable levels oftelomerase during each S phase. In spite ofthis expression, telomeres shorten during eachround of DNA replication and cell division,indicating that this transient telomerase ex-pression does not sufce to prevent telomereerosion. This raised the question of the pre-cise function of telomerase expression in thesecells.

RNAi-directed knockdown of hTERT innormal, presenescent broblasts led to loss ofthe single-stranded overhang and to an ac-celerated entrance into senescence, suggest-ing that telomerase expression is required torebuild the overhang and allow proper telom-ere assembly and function, even when telom-eres are quite long. Further work is neededto demonstrate conclusively that this is in-deed the role of telomerase expression in thesecells, and it is not clear whether this pro-posed function would explain the apparenttelomere-independent roles of telomerase innormal cells. In any event, it is already appar-ent that telomerase plays a key role in nor-mal cell physiology that is independent of itseffects on overall telomere length.

A recent study has added further complex-ity by suggesting that hTERT plays a keyrole in DNA repair (Masutomi et al. 2005).RNAi-directed knockdown of hTERT in nor-mal human cells led to abrogation of theusual DNA damage response, following ra-diation, in the absence of apparent effects onthe telomeres. Analysis of cells following ra-diation treatment revealed that they did notform the DNA damage foci that are usu-ally seen following irradiation. Accordingly,


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APB:ALT-associatedPML body

PML:promyelocyticleukemia

normal cells devoid of hTERT were radiosen-sitive and demonstrated chromatin fragmen-tation in response to irradiation. The pre-cise role that hTERT plays in the repair ofradiation-induced dsDNA breaks remains tobe claried. Nonetheless, it is clear once againthat this enzyme inuences cellular processesthat are unrelated to maintenance of telomerelength.

TELOMERE MAINTENANCE INTHE ABSENCE OFTELOMERASE

As discussed above, more than 90% of allhuman tumors utilize telomerase for telom-ere maintenance, whereas the telomerase-independent telomere maintenance (ALT)mechanism mentioned above is observed inonly 710% of tumors (Bryan et al. 1997, Shay& Bacchetti 1997). When tumors are analyzedby tissue type, the ALT mechanism appearsto be more prevalent in tumors arising frommesenchymal tissues and the central nervoussystem, raising the possibility that there areintrinsic differences in cells isolated from vari-ous tissue lineages that dictate whether telom-erase or ALT is activated as a means of escap-ing crisis during multistep tumor progression.Uncovering these putative differences will bean important advance in our understanding ofthe cellular immortalization process.

Several markers have been associated withthe ALT phenotype. In particular, individ-ual telomeres in ALT cells are highly hetero-geneous in length and are subject to rapiddecreases and increases in this length. In-deed, some chromosomes that completelylack telomeric repeats can be detected in apopulation of ALT cells (Londono-Vallejoet al. 2004). Some have proposed that therapid changes in telomere lengths are due tothe ALT mechanism itself.

In yeast, a mechanism similar to the ALTmechanism in mammalian cells becomes acti-vated in response to the absence of telomerase,and in these cells, telomerase-independenttelomere maintenance depends on homolo-

gous recombination (Lundblad & Blackburn1993). In fact, recently reported data dosupport homologous recombination as akey mechanism in mammalian ALT cells(see below) (Londono-Vallejo et al. 2004,Varley et al. 2002). In addition to highly vari-able telomere lengths, mammalian ALT cellsalso contain promyelocytic leukemia bodies(APBs), which are not observed in telomerase-positive cells (Yeager et al. 1999). APBscontain the promyelocytic leukemia (PML)protein; telomeric DNA; telomere-bindingproteins, including TRF1 and -2; and numer-ous proteins involved in genetic recombina-tion (Yeager et al. 1999).

One study that has suggested that ALTcells may utilize a recombination-based mech-anism demonstrated that a unique DNA se-quence inserted into the subtelomeric re-gion of one chromosome in an ALT cell linewas rapidly transmitted to other telomeres(Dunham et al. 2000). This suggested thatALT occurs either through interchromoso-mal homologous recombination or througha copy-choice mechanism of template switch-ing during DNA replication. From this ob-servation, one may speculate that ALT cellsexhibit a higher incidence of generalized ho-mologous recombination than telomerase-positive cells. One study addressed this pos-sibility directly and found no evidence thatsupported such a contention (Bechter et al.2003). This may indicate that the elevatedrecombination rates in ALT cells are lim-ited to the telomeres and do not representa global change in homologous recombina-tion. Although it is not clear how changes intelomere-specic recombination may occur,it is attractive to speculate that a telomere-binding protein, such as TRF2, may be alteredin ALT cells, resulting in telomere-specic re-combination events.

Three additional studies have supportedthe hypothesis that ALT is recombinationbased. The rst demonstrated that Sp100 caninuence the presence of APBs and telom-ere maintenance ( Jiang et al. 2005). Sp100is a component of PML nuclear bodies.


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Overexpression of this protein sequestersMRE11, Rad50, and NBS1 away from APBs,resulting in progressive telomere shortening.Importantly, ectopic expression of Sp100 alsosuppressed the rapid telomere-length uctua-tions that are characteristic of the ALT pheno-type, suggesting that the ALT mechanism wassuppressed in these cells. The second studydemonstrated that expression of a TRF2 mu-tant (TRF2B) protein resulted in massivetelomeric deletions that were dependent onthe activities of XRCC3, a protein that is in-volved in Holliday junction resolution (Y. Liuet al. 2004). Interestingly, the telomericdeletions corresponded to the appearanceof T-loop-sized telomeric circles, suggest-ing that homologous recombination con-tributed to the telomeric loss associated withTRF2B expression. Electron microscopicanalysis of ALT cells revealed the presenceof telomeric circles (Cesare & Grifth 2004,Tokutake et al. 1998) like those observed fol-lowing TRF2B expression, suggesting that asimilar mechanism was active and responsiblefor telomere maintenance.

The third and most recent study regard-ing the recombination-based mechanisms ofALT has suggested that chromatin modu-lation may be a key determinant of telom-erase activation or lack thereof. The chro-matin environments surrounding the hTERTand hTERC promoters were compared amongtelomerase-positive human tumor cell lines,normal human cells, and human cells uti-lizing ALT (Atkinson et al. 2005). In ALTcells, histone H3 and H4 were hypoacety-lated, and H3, lysine 9 and H4, lysine 20were methylated. In contrast, hTERT expres-sion was associated with hyperacetylation ofH3 and H4 and methylation of H3, lysine 4.Treatment of normal cells and ALT cellswith 5-azadeoxycytidine and trichostatin Aresulted in chromatin remodeling and acti-vation of hTERC and hTERT expression,supporting a role for the chromatin envi-ronment in governing telomerase expression.This study suggests that normal cells sup-press telomerase activity through chromatin

hTERC: humantelomerase RNAsubunit

modication, which needs to be reversed forcells to activate telomerase during tumorige-nesis. This raises the question of why ALTcells do not simply remodel the chromatinsurrounding these promoters and thereby ac-tivate telomerase function. Further studies re-garding control of chromatin modulation mayhelp explain whether certain cell types aremore adept at remodeling their chromatin andmay thereby reveal the controls governing ac-tivation of ALT versus telomerase.

TELOMERE-BINDINGPROTEINS

Over the past ten years, a substantial list oftelomere-specic proteins has been assem-bled, and it is now appreciated that theseproteins, together with the telomeric DNA,form a functional, or capped, telomere. In fact,as Blackburn (2000) points out, the telom-eric proteins and DNA operate in a mutu-ally reinforcing fashion to maintain a properlyfolded and functional structure. This sectiondetails the known functions of the core telom-eric proteins, collectively referred to as eitherthe Shelterin complex (de Lange 2005) or thetelosome (D. Liu et al. 2004a), as well as otherproteins that are important to telomere func-tion but are known to play other roles in DNAmaintenance distinct from repairing or main-taining telomeric DNA.

Telomeric Core Proteins

The rst mammalian telomere-binding pro-tein, TRF1 (telomere repeatbinding factor),was initially described in 1992 (Zhong et al.1992). This protein was identied on the ba-sis of its binding specicity for the vertebratetelomeric hexanucleotide repeats, TTAGGG.A second protein, TRF2, was later identi-ed as a TRF1 paralog (Bilaud et al. 1997,Broccoli et al. 1997). Like TRF1, TRF2 dis-plays high sequence specicity for telomericrepeats and contains a Myb DNA-bindingdomain. Later, yeast two-hybrid experi-ments demonstrated that the Tin2 and Rap1


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MRN: complexconsisting ofMRE11, Rad50, andNBS1

proteins also interact with TRF2 (Kim et al.1999, Li et al. 2000). More recently, sev-eral groups have used a variety of ap-proaches to identify TPP1 (also referred toas TINT1, PTOP, and PIP1) as a Tin2-or Pot1-interacting protein. (Houghtalinget al. 2004, D. Liu et al. 2004b, Ye et al.2004). Finally, Pot1, the telomeric single-strand-binding protein, was identied by se-quence homology to telomere-binding pro-teins in unicellular organisms (Baumann &Cech 2001) and appears to be evolutionarilythe most well-conserved protein of the group.Below, we highlight only some of the impor-tant functions of these proteins. The litera-ture on telomere-binding proteins has grownrapidly in recent years, and a single reviewcould easily be dedicated to them.

Telomeres and DNARepair/Replication Proteins:Antagonistic or SynergisticRelationships?

In addition to the known core telomere-binding proteins, DNA repair proteins arealso found at the telomeres. A simplistic viewmight suggest that the presence of DNA re-pair enzymes would compromise the telom-eres task of concealing the chromosome endsfrom the DNA repair machinery. The avail-able evidence, on the contrary, suggests thatthe presence of these repair proteins is vi-tal to telomere maintenance. These proteinsinclude the Ku complex, the MRN com-plex (including MRE11, Rad50 and NBS1),XPF/ERCC1, ATM, BLM/WRN, Rad51D,and Rad54 (reviewed in de Lange 2005). Theprecise function of each of these proteins atthe telomere is only now being uncovered, andfar more work is required. We briey discusssome of the known functions below.

The Ku complex is central to the nonho-mologous end-joining machinery and bindsto the telomere through an interaction withTRF2 (Song et al. 2000). Loss of Ku ac-tivity in humans leads to immunodeciencyand cancer predisposition (Doherty & Jackson

2001). Deletion of the Ku homologs, Ku70or Ku86, in the mouse results in prematureaging, aberrant telomere lengths, and loss oftelomere capping (Espejel & Blasco 2002,Espejel et al. 2002, Goytisolo et al. 2001, Jacoet al. 2004, Samper et al. 2000). Rad54 is in-volved in homologous recombination, and itsabsence in murine cells results in short telom-eres and loss of capping function (Jaco et al.2003). Rad51D loss yields a similar outcome(Tarsounas et al. 2004).

Loss of XPF/ERCC1 results in the cancerpredisposition syndrome termed xerodermapigmentosum (Boulikas 1996, Lehmann2003, Wood 1999). A biochemical screenhas demonstrated that this nucleotide ex-cision repair complex associates with thetelomere through an interaction with thetelomere-binding protein TRF2 (Zhu et al.2003). Importantly, it was suggested thatXPF/ERCC1 is able to modulate the 3 single-stranded telomeric overhang during telom-ere uncapping. As described above, introduc-tion of the dominant-negative allele of TRF2(TRF2DN) results in rapid telomere uncap-ping and, in the absence of functional p53and Rb, leads to end-to-end chromosomal fu-sions (Karlseder et al. 1999). These fusions,as mentioned above, are the result of non-homologous end joining; however, prior toend-to-end chromosomal fusions, the telom-ere single-stranded overhang must be pro-cessed, a task carried out by XPF/ERCC1.When the TRF2DN allele was introducedinto cells from mice decient in ERCC1, thecells displayed fewer chromosomal fusions,supporting XPF/ERRC1s role in telomereprocessing (Zhu et al. 2003). Still, this leavesunanswered the question of XPF/ERCC1snormal function at the telomere. One possi-bility is that XPF/ERCC1 is required to pro-cess the telomere overhang to aid in disassem-bling the T-loop, thereby providing access tothe replication machinery during the S phaseof the cell cycle.

WRN and BLM are ReqQ helicases(Nakura et al. 2000). As noted in the sup-plemental material (Supplemental Text and


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Supplemental Figure 1; follow the Sup-plemental Material link from the An-nual Reviews home page at http://www.annualreviews.org), mutations of their en-coding genes result in progeria and cancerpredisposition (Dyer & Sinclair 1998). In-terestingly, loss of these genes in the mouseresults in premature aging phenotypes onlywhen mice lacking these genes are bred withTERC knockout animals (Chang et al. 2004,Lombard et al. 2000). In other words, the phe-notype is only seen when telomere lengths arelimiting, a situation that is thought to recapit-ulate the human condition of progeria. BLMand WRN are structure-specic helicases thatresolve complex structures, such as Hollidayjunctions, which are reminiscent of theT-loops located at the ends of the telomere.Both proteins are recruited to the telomerethrough an interaction with TRF2 (Opreskoet al. 2002, Stavropoulos et al. 2002).

BLMs role at the telomere remains ob-scure. More is known about WRN: A re-cent report indicates that it is indispensablefor telomere replication, playing a centralrole in lagging-strand synthesis at the telom-ere (Crabbe et al. 2004). When WRN ac-tivity was compromised, cells displayed largesister telomere losses and subsequent chro-mosomal fusions. Furthermore, cell cycleanalysis demonstrated that WRN specicallylocalized to the telomere only during S phaseof the cell cycle, supporting its role in telom-ere replication. This observation raises thepossibility that WRN activity is requiredto resolve complex structures, such as theG-quadruplexes hypothesized by some inves-tigators to form at telomeric sequences. If thiswere indeed WRNs role, then loss of WRNactivity, if not properly resolved, might resultin stalled replication forks that would lead totelomere loss and fusions.

Cells from WRN patients do display sis-ter chromatid telomere losses that are rescuedby telomerase expression (Crabbe et al. 2004).How might telomerase rescue such a defect?Teixeira et al. (2004) reported that telom-erase shows a preference for shorter telom-

eres; these authors suggest that in WRN cellstelomerase may protect chromosomes under-going sister telomere loss by adding telomericrepeats back to them before they can be pro-cessed by the DNA repair machinery. Conr-mation of this model requires additional work.

ATM is a PI3 kinase homolog that plays acentral role in DNA damage sensing and re-pair (Khanna & Jackson 2001). Indeed, ATMactivity is required to initiate many DNA re-pair processes and to activate p53 functionin response to DNA damagea role men-tioned earlier in this review. Loss of ATMin the human results in ataxia telangiecta-sia, whereas loss in the mouse results in ra-diosensitivity (Shiloh & Kastan 2001). Whenbred to mTERC knockout mice, mice showingATM loss have premature aging phenotypes,again highlighting a requirement for ade-quate telomere reserves to forestall these pro-cesses (Wong et al. 2003). ATM interacts withthe telomere through the TRF2 protein, andearly work suggested that TRF2 suppressedATM function at the telomere (Karlseder et al.2004). However, ATM may have an alterna-tive role at the telomere: In one study, ATMactivity was required during the G2 phase ofthe cell cycle (Verdun et al. 2005). During thistime, ATM activated a DNA damage responsemechanism at the telomere, leading to recruit-ment of MRE11 and NBS1. Degradation ofMRE11 or NBS1 or loss of ATM functionresulted in telomere dysfunction, indicatingthat this recruitment was critical to normaltelomere maintenance. This led, furthermore,to the proposal that a localized DNA damageresponse is required to process the telomereend and allow it to fold properly, resulting in acapped, functional conguration. This obser-vation appears to contradict the widely heldnotion that the telomere operates to disguiseor protect the telomere from the DNA dam-age response and thereby highlights the com-plex nature of telomere maintenance that weare only now beginning to understand. Thecoming years are likely to supply us with amore detailed understanding of how the DNArepair enzymes work to maintain telomere


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homeostasis and how these functions becomecompromised in tumorigenesis and aging.

EPIGENETIC MODULATION OFTELOMERE LENGTH

Telomere homeostasis is the result of a deli-cate balance of sufcient double-stranded andsingle-stranded telomeric DNA and the cor-rect complement of telomere-binding pro-teins. More recent work has also suggestedthat modulation of the histones associatedwith telomeric DNA can govern telomeremaintenance as well. Like many regions of thebulk genomic DNA, telomeric DNA is associ-ated with arrays of nucleosomes (Tommerupet al. 1994) that are characteristic of hete-rochromatin. In many respects, the telom-eric heterochromatin is similar to heterochro-matin found around pericentric regions and,as such, demonstrates an enriched bindingof the heterochromatin protein 1 (HP1) iso-forms HP1, HP1, and HP1 (Garcia-Caoet al. 2004, Peters et al. 2001, Schotta et al.2004). In addition, telomeric DNA possesseshigh levels of histone 3, lysine 9 and histone4, lysine 20 trimethylation. These chromatinmodications appear to be directed by mem-bers of the Rb family of proteins, which areable to bind and direct the actions of cer-tain chromatin-modifying enzymes: suppres-sor of variegation 39 homolog (SUV39H)and suppressor of variegation 420 homolog(SUV420H). Both proteins are histonemethyltransferases. Modulation of telomericchromatin leads to alteration in telomerelength regulation (Garcia-Cao et al. 2004).

TELOMERE-BINDINGPROTEINS: BEYOND THETELOMERE

As illustrated by the above examples, the earlyview that the telomere disguised or protectedthe ends of the chromosomes from DNA re-pair enzymes addresses only part of its func-tion. Thus, the DNA repair proteins andpathways are integral to normal telomere

homeostasis. It therefore should not be sur-prising that proteins previously thought tofunction exclusively at the telomeres exertfunctions that are unrelated to the mainte-nance of normal telomere function.

For example, as mentioned in the supple-mental text, tankyrase inhibits TRF1 bind-ing to telomeric DNA and promotes TRF1degradation, resulting in telomere elongation(Chang et al. 2003, Smith & de Lange 2000,Smith et al. 1998). A recent report showedthat tankyrase also plays a key role in sis-ter chromosome resolution (Dynek & Smith2004). Thus, RNAi-directed loss of tankyraseexpression resulted in cell cycle arrest and lossof sister chromosome resolution. And analysisof the arrested cells demonstrated that the sis-ter chromosomes lined up on the metaphaseplate but were unable to segregate becausethey remained attached via their telomeres.

TRF2 has been found to be localized todsDNA breaks immediately following theirformation. However, unlike proteins found inclassic DNA damage foci, such as H2A.X,localization of TRF2 was lost in a matter ofminutes (Bradshaw et al. 2005). A subsequentstudy demonstrated that the transient asso-ciation of TRF2 with sites of DNA damagerequires ATM activity (Tanaka et al. 2005).ATM phosphorylated TRF2 at an ATM con-sensus site in response to DNA damage, andinhibition of ATM activity resulted in loss ofTRF2 phosphorylation and DNA damage lo-calization following irradiation. Interestingly,the TRF2 found at sites of DNA damagedid not bind DNA, suggesting that TRF2was tethered indirectly to the DNA, osten-sibly through a protein-protein interaction.The precise role of TRF2 in DNA damageresponse/repair remains to be determined.Nevertheless, this observation highlights yetanother link between telomere-binding pro-teins, telomere homeostasis, and DNA dam-age response and repair. These observationssuggest that we need to reevaluate the re-lationship between DNA repair/replicationproteins and those responsible for telom-ere maintenance and that a number of these


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proteins likely play important roles in bothnormal cellular processes.

In addition to its apparent role in the DNAdamage response, TRF2 may be involved intumorigenesis. Thus, in a transgenic murinemodel, overexpression of TRF2 in the skinresulted in a severe phenotype in responseto light (Munoz et al. 2005). These animalsdisplayed hyperpigmentation, premature skindeterioration, and a condition that resem-bled xeroderma pigmentosum. The telom-eres had marked telomere shortening anddisplayed single-stranded telomeric DNAoverhang loss, which may reect the ability ofTRF2 to bind the XPF protein. This observa-tion is in contrast to what was observed whenTRF2 was overexpressed in human cells: in-creased telomere shortening with concomi-tant protection of the single-stranded over-hang (Karlseder et al. 2002). It is not clearwhy the human and mouse differ in regardto the effects of TRF2 on the single-strandedoverhang of the telomere. In addition, thetransgenic mice displayed increased telomereshortening when compared with age-matchedcontrols and increased genomic instability.Moreover, analyses of human basal and squa-mous cell carcinomas have revealed an in-crease in TRF2 expression levels in approx-imately 10% of cases (Munoz et al. 2005),consistent with a role for TRF2 in humancarcinogenesis.

Yet another study that examined humanmammary epithelial cell lines and cell linesderived from breast carcinomas also sug-gested that TRF2 plays a role in tumori-genesis. Analysis of TRF2 expression re-

vealed upregulation of the protein at theposttranscriptional level (Nijjar et al. 2005).In addition, upregulation of TRF2 proteinwas associated with altered TRF2 localiza-tion. TRF2 was found throughout the nu-cleus as well as at telomeres. Whether thisrelocalization represents the movement ofTRF2 to sites of DNA damage has not beendetermined.

CONCLUDING REMARKS ANDFUTURE DIRECTIONS

Is aging the price we pay for remaining can-cer free during our early life? And are telom-eres and replicative senescence at the center ofthis ongoing battle? Recent advances supportthe role of senescence in tumor suppressionand the loss of telomere integrity as a driv-ing force in the transformation process (Braiget al. 2005, Chen et al. 2005, Collado et al.2005; reviewed in Maser & DePinho 2002,Michaloglou et al. 2005). If we have learnedanything, it is that the telomere and its asso-ciated proteins play a critical role in genomicintegrity. Importantly, understanding how thetelomere interacts with the DNA repair ma-chinery in normal as well as pathological con-ditions will further our general understandingof telomere biology. In addition, it is now clearthat cell senescence is critical for tumor sup-pression, and accumulating data suggest thatthe former may also inuence many of thepathologies associated with aging. The com-ing years are sure to shed much light on telom-ere homeostasis and, in turn, on the roles ofthe telomere in human aging.

SUMMARY POINTS

1. Normal human somatic cells possess a limited replicative potential that is dictatedby the proper maintenance of the telomere and by their responses to certain cell-physiological stresses.

2. Senescence is often triggered by cell-physiological stresses that cells suffer in vitroand possibly in vivo.

3. Senescence functions as an important tumor suppressor mechanism.


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4. In the event that cell lineages circumvent the proliferative block imposed by senes-cence, the telomeres of these cells will continue to shorten, leading to crisis, in whichchromosomal fusions, genetic instability, and widespread cell death occur.

5. Telomere homeostasis, which is essential for the normal function of telomeres toprotect the ends of chromosomes, is dictated by both the conguration of telomericDNA and the functions of telomere-binding proteins.

6. DNA repair enzymes associate with telomeres and play critical roles in telomeremaintenance in still-unclear ways.

7. The two canonical tumor suppressor pathwaysinvolving the Rb and p53 tumorsuppressor proteinsimpose senescence in the event that telomeric DNA is disruptedin certain ways.

8. Cells can acquire replicative immortality either through the expression of telomeraseor through the activation of the alternative lengthening of telomeres (ALT) pathways.

FUTURE ISSUES

1. How is senescence activated at the molecular level, and what pathways govern activa-tion of the p53 and Rb tumor suppressor pathways, which impose a senescent growthstate?

2. What are the molecular mechanisms that enable the ALT pathway to maintain telom-eres, and will this pathway constitute an important escape pathway for cells subject toantitelomerase therapies?

3. Do telomere shortening and cell senescence contribute to human aging?

4. What is the precise relationship between DNA repair and telomere homeostasis?

ACKNOWLEDGMENTS

We would like to thank Abhishek Saharia for the telomere FISH and Lionel Guittat forhelp on several of the gures. S.A.S. is a Sidney Kimmel Foundation Scholar and an EdwardMallinckrodt, Jr. Foundation award recipient. R.A.W. is an American Cancer Society ResearchProfessor and a Daniel K. Ludwig Cancer Research Professor.

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