Oncogene (1999) 18, 7676 ± 7680 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase

Homayoun Vaziri1 and Samuel Benchimol*,2,3

1Stanford University School of Medicine, Department of Molecular Pharmacology, Edward's Building, 300 Pasteur Drive Stanford, California, CA 94305-5332, USA; 2Ontario Cancer Institute/Princess Margaret Hospital, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G-2M9; 3Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G-2M9

Telomere shortening may be one of several factors that A provocative and in¯uential model of cell contribute to the onset of senescence in human cells. The senescence and immortalization (Harley et al., 1992) p53 and pRb pathways are involved in the regulation of suggested that telomeres shorten in many proliferating cell cycle progression from G1 into S phase and somatic cells due to the end replication problem (the inactivation of these pathways leads to extension of life inability of the DNA replication machinery to span. Short dysfunctional telomeres may be perceived as completely copy chromosomal termini) (Olovnikov, damaged DNA and may activate these pathways, leading 1973; Watson, 1972). In this model, shortened to prolonged arrest in G1, typical of cells in senescence. telomeres signal the onset of senescence. Cells with Inactivation of the p53 and pRb pathways, however, does an extended life span bypass the senescence signal but not lead to cell immortalization. Cells that overcome continue to lose telomeric DNA until they reach crisis senescence and have an extended life span continue to and die. Overcoming crisis is associated with main- lose telomeric DNA and subsequently enter a second tenance of telomere length and reactivation of phase of growth arrest termed `crisis'. Forced expression telomerase (Counter et al., 1992). Although some of telomerase in human cells leads to the elongation of similarities exist between senescence and crisis, there telomeres and immortalization. The development of is now abundant evidence demonstrating that these human cancer is frequently associated with the inactiva- represent fundamentally di€erent cellular processes tion of the pRb and p53 pathways, attesting to the governed by distinct genetic regulatory components. importance of senescence in restricting the tumor- forming ability of human cells. Cancer cells must also maintain telomere length and, in the majority of cases, Involvement of the p53 and Rb pathways in senescence this is associated with expression of telomerase activity. The transforming proteins of DNA tumor viruses such Keywords: telomerase; hTERT; telomere; senescence; as SV40 large T antigen, adenovirus E1A/E1B or HPV aging; p53; pRb; TIEL; PARP E6/E7 can extend the life span of human cells in culture, but only a few, exceptional clones emerge as survivors following crisis. Extension of cellular life span is dependent on the ability of these viral proteins Two proliferative blockades: senescence and crisis to target the tumor suppressor proteins, p53 and pRb, and render them functionally inactive. In contrast to rodent cells, human cells rarely become pRb and p53 are important regulators of cell cycle immortal in culture. This di€erence may be attributed, progression and play key roles in controlling entry into at least partially, to the Hay¯ick barrier also termed S phase. pRb binds to members of the E2F family of `senescence'. In 1961, Hay¯ick showed that normal transcription factors. In the bound state, E2F-1 is human cells divide only a ®nite number of times in unable to activate E2F-1-responsive genes required for culture and undergo replicative senescence (Hay¯ick entry into S phase and for DNA synthesis. Phosphor- and Moorhead, 1961). Spontaneous escape from ylation of pRb by the G1 cyclin-dependent kinases senescence in human cells is an extremely rare event (CDKs) releases E2F-1 in a transcriptionally active but this can be facilitated through the action of certain form, allowing cells to progress from G1 into S phase. oncogenes or by exposure of primary cells to various The HPV E7 protein, as well as SV40 large T antigen mutagens. Cells that overcome senescence and have an and adenovirus E1A, override this control circuit by extended life span subsequently enter a second phase of binding to pRb and causing the inappropriate release growth arrest termed `crisis'. Unlike senescent cells that of active E2F-1 in the absence of CDK-mediated are primarily arrested in the G1 phase of the cell cycle, phosphorylation of pRb (Weinberg, 1995). cells at crisis undergo division and death. Escape from p53 promotes G1 cell cycle arrest or apoptosis in crisis occurs at very low frequency (361077) (Shay and response to DNA damage and other forms of stress Wright, 1989). (Levine, 1997). p53 protein functions as a transcription factor by binding to speci®c DNA sequences and regulating the transcription of target genes. This activity of p53 is considered to be important for its tumor suppressor function since most of the mutations *Correspondence: S Benchimol in the p53 gene in human cancer eliminate the ability Extension of cellular life span H Vaziri and S Benchimol 7677 of p53 protein to bind DNA or to promote cells (Vaziri et al., 1993) and endothelial cells (Chang transcription. p53 blocks cells in G1 primarily through and Harley, 1995). Several hypotheses have been its ability to induce expression of p21WAF1, a CDK proposed to explain how shortened telomeres might inhibitor (E1-Deiry et al., 1993). p21WAF1 provides a lead to senescence. We proposed that shortened link between the p53 and Rb regulatory pathways. telomeres per se are not the signal for senescence; p21WAF1 can act to limit the phosphorylation of pRb, rather that shortened telomeres compromise DNA thereby preventing cells from exiting the G1 phase of integrity and generate strand breaks that are sensed the cell cycle. The HPV E6 protein binds to p53 and by the DNA damage recognition machinery of the cell, promotes its degradation via the ubiquitin proteolytic leading to p53 activation and p21WAF1-mediated G1 pathway, while adenovirus E1B and SV40 large T arrest (Vaziri and Benchimol, 1996). In search of other antigen bind to p53 and block its transcriptional e€ector molecules in this pathway, we found that activity. In the absence of p53, pRb or p21WAF1, the inhibition of poly(ADP-ribosyl)ation in normal human cell cycle arrest pathway induced by DNA damage is ®broblasts extended cellular life span signi®cantly. lost (Kastan et al., 1992; Brugarolas et al., 1995; Deng Importantly, we demonstrated a physical and func- et al., 1995; Harrington et al., 1998). tional association between poly(ADP-ribose) polymer- Increased expression and/or activity of p53, p21 and ase and p53 in human cells (Vaziri et al., 1997). Hence another CDK inhibitor, p16INK4a, is detected in it is possible that short and dysfunctional telomeres senescent cells lending further support to the impor- signal growth arrest via poly(ADP-ribosyl)ation of tance of the p53 and Rb pathways in senescence critical e€ector molecules including p53. (Atadja et al., 1995; Noda et al., 1994; Kulju and Lehman, 1995; Vaziri et al., 1997; Alcorta et al., 1996; Hara et al., 1996). The mouse INK4a locus encodes the The telomerase complex CDK inhibitor, p16INK4a, as well as an unrelated protein, p19ARF which arises in large part from an Synthesis of telomeric repeats at the ends of chromo- alternative reading frame (Quelle et al., 1995). In somes is performed by the telomerase protein/RNA human cells, usage of an alternative reading frame in complex. Telomerase activity, measured by the ability of the INK4a locus similarly gives rise to the human the enzyme to add TTAGGG repeats to the end of homolog known as p14ARF. Ectopic overexpression of synthetic telomeric DNA, was originally discovered in p53, p21, p16INK4a,orp19ARF/p14ARF in proliferating the ciliate Tetrahymena (Greider and Blackburn, 1985) cells can induce G1 arrest. The ability of p16INK4a to and subsequently in the human immortal cell line HeLa induce cell cycle arrest is pRb dependent while p19ARF- (Morin, 1989), The ground breaking discovery of or p14ARF- induced cell cycle arrest is p53 dependent. Euplotes p123, the catalytic subunit of telomerase The human and mouse ARF proteins have been shown (Lingner et al., 1997), led to identi®cation of the human to stabilize and activate p53. This occurs, at least in telomerase catalytic subunit (hTERT) (Harrington et al., part, by the interaction of the ARF proteins with 1997b; Kilian et al., 1997; Meyerson et al., 1997; MDM2 resulting in abrogation of MDM2's ability to Nakamura et al., 1997). The human telomerase complex promote the degradation of p53 (Pomerantz et al., consists of an RNA subunit named hTR (Feng et al., 1998; Zhang et al., 1998; Kamijo et al., 1998). Primary 1995) and hTERT. Several other proteins have also been murine ®broblasts lacking p53 or p19ARF or both identi®ed that interact with the telomerase complex p16INK4a and p19ARF are easily established into including TEP1/TLP1 (Harrington et al., 1997a). immortal cell lines (Harvey et al., 1993; Serrano et Evidence from S. cerevisiae and Euplotes suggests that al., 1996; Kamijo et al., 1997). several other factors could also potentially interact and In human ®broblasts derived from patients with Li- be a part of the complex, including EST1, EST4 and p43 Fraumeni syndrome that are heterozygous for wild- (Nugent and Lundblad, 1998). type p53, senescence can be overcome (Bischo€ et al., 1990) through loss or mutation of the remaining wild- type p53 allele (Rogan et al., 1995). In addition, Telomerase and extension of life span dominant negative inhibition of p53 protein (Bond et al., 1994) or inactivation of p21WAF1 (Brown et al., Reconstitution of telomerase activity in vitro requires 1997) in human cells can also overcome senescence and the RNA template of human telomerase hTR and the lead to an extended life span. These cells, however, do catalytic subunit hTERT (Beattie et al., 1998; Weinrich not become immortal and, like those transformed by et al., 1997). Forced expression of hTERT in normal DNA tumor virus (Shay et al., 1991), enter crisis and human ®broblasts is sucient to fully reconstitute rarely emerge as immortal clones. An important telomerase activity, since the endogenous RNA question that arises in trying to relate these ®ndings template of human telomerase (hTR) is expressed in to the telomere shortening model of cell senescence is normal human cells (Feng et al., 1995). Reconstitution how the p53 and pRb pathways become activated in of telomerase activity leads to elongation of telomeres response to shortened telomeres to invoke the in hTERT expressing cells and is associated with the senescence program. extension of replicative life span (Bodnar et al., 1998; Vaziri and Benchimol, 1998). Forced overexpression of hTERT in post senescent human cells allows cells to Short telomeres may signal cell cycle exit via p53 protein proliferate beyond crisis (Counter et al., 1998; Halvorsen et al., 1999). These reports provide strong Shortening of telomeres as a function of in vitro age evidence in support of the model that telomere has been observed in variety of cell lineages including shortening is a mechanism that limits the replicative ®broblasts (Allsopp et al., 1992; Harley et al., 1990), T- capacity of human cells. Extension of cellular life span H Vaziri and S Benchimol 7678 In another study, reconstitution of telomerase This is further substantiated by the demonstration that activity by hTERT alone was unable to extend the functional p53 protein is expressed in these immortal life span of normal human keratinocytes or mammary cells (Vaziri et al., 1999). BJ-TIEL cells have an intact epithelial cells (HMECs) (Kiyono et al., 1998). Both p53-dependent G1 checkpoint in response to DNA pRb inactivation (through the use of HPV E7) and damage and are able to upregulate p53 protein in hTERT expression were required to immortalize these response to ionizing radiation (Vaziri et al., 1999) or human epithelial cells. These cells, unlike other human UV damage (Morales et al., 1999). Importantly, cells cell strains, undergo a telomere-independent, p16INK4a- immortalized by telomerase expression are non-

dependent, growth arrest phase called M0 that can be tumorigenic and do not have changes typically overcome by E7 (Foster and Galloway, 1996; Foster et associated with the malignant phenotype (Vaziri et al., 1998). This important observation demonstrates al., 1999; Morales et al., 1999; Jiang et al., 1999). that in certain cell strains, hTERT alone will not Maintenance of telomere length by telomerase is extend cellular life span. It should be recognized, probably critical for preventing chromosomal aberra-

however, that mammary epithelial cells undergo M0 tions associated with eroded telomeres. after 7 ± 20 population doublings at a time when Together, these ®ndings further substantiate the idea telomeres are still relatively long. Hence, it is unlikely that extension of cellular life span by telomerase is

that M0 occurs in response to telomere shortening and fundamentally di€erent from that induced by inactiva- so it is not surprising that hTERT expression fails to tion of the p53 and pRb pathways.

bypass M0. This is consistent with M0 being part of a di€erentiation program in certain human cell lineages. The study by Kiyono et al. (1998) demonstrates, p53, pRb, telomeres and cancer moreover, that telomerase reconstitution and telomere elongation in HMECs and human keratinocytes do not The development of human cancers is frequently interfere with the di€erentiation programs of these associated with the inactivation of the pRb and p53 cells. In contrast to these studies, other groups have pathways, attesting to the importance of senescence in been able to extend the life span of HMECs (Wang et restricting the tumor-forming ability of human cells. al., 1998) and other epithelial cells (Bodnar et al., Cancer cells maintain telomere length and in the 1998). It is possible that there may be variability in the majority of cases this is associated with expression of susceptibility of epithelial cells to di€erentiate in telomerase activity, although alternative mechanisms culture. for telomere maintenance in the absence of telomerase must also exist (Colgin and Reddel, 1999). An important question that arises from these ®ndings is Extension of cellular life span by telomerase is whether there is a connection between the p53/pRb associated with maintenance of the p53/pRb pathways and telomerase expression in immortal pathways and maintenance of genomic integrity cancer cells. Cells with an extended life span in which the p53 Extension of cellular life span beyond the Hay¯ick and pRb pathways have been inactivated continue to limit is associated with loss of genomic integrity and lose telomeric DNA until crisis. Telomerase activity is formation of aberrant chromosomes (Wolman et al., not detectable in these cells. Furthermore, telomerase 1964). Senescence, therefore, may be viewed as a activity in cell lines is independent of their p53 status proliferative blockade to prevent the outgrowth of (Milas et al., 1998) and activation of telomerase in cells with genomic instability. As discussed thus far, keratinocytes by HPV16-E6 is independent of the cellular life span can be extended through, at least, two ability of E6 to compromise p53 function (Klingel- alternative pathways. The ®rst pathway involving hutz et al., 1996). Together, these ®ndings suggest that inactivation of p53 and pRb is short-lived; cells loss of p53 gene function is not by itself sucient for acquire an extended life span but succumb to crisis. telomerase activation, telomere elongation and cellular These post-senescence cells have lost G1 cell cycle immortality. Little is known about the regulation of control and as a result are prone to genetic hTERT gene expression and how this gene becomes rearrangements involved in tumor progression (Living- activated in cells emerging from crisis. It is possible stone et al., 1992; Yin et al., 1992; Almasan et al., that the genetic instability resulting from the inactiva- 1995). The second pathway involving telomerase tion of the p53 and pRb pathways facilitates events expression and telomere maintenance leads to immor- that lead to telomerase expression. tality. Are cells immortalized by telomerase genetically stable or unstable? On the basis of G-banding and spectral karyotyping, Telomerase inhibition: hitting the Achilles heel of the we reported previously that BJ-TIEL cells (BJ primary cancer cell or causing further genomic instability? human ®broblasts immortalized by telomerase) dis- played non clonal chromosomal aberrations initially There is currently much interest in the development of after ectopic expression of telomerase (Vaziri et al., telomerase inhibitors as anti-cancer agents. Inhibition 1999). These aberrant structures were transient and of telomerase activity by anti-sense inhibition of hTR subsequently disappeared in later cell passages. has shown some limited success in inducing cell death Morales et al. (1999) also reported no sign of (Feng et al., 1995). Telomerase-de®cient mice experi- chromosomal abnormalities in BJ-TIEL cells on the ence telomere shortening and accompanying genetic basis of G-banding analysis. It appears, therefore, that instability including aneuploidy and chromosome extension of cellular life span through telomerase re- abnormalities (Blasco et al., 1997). If telomerase expression does not compromise genomic integrity. activity is required for immortality and subsequent Extension of cellular life span H Vaziri and S Benchimol 7679 maintenance of genomic stability, telomerase inhibition normal, self-renewing stem cells would be harmful should destabilize the genome by interfering with (Lee et al., 1998). This points out the necessity of telomere function. Tumor cells treated in vivo with understanding the mechanism underlying the survival anti-telomerase drugs might be expected to become of telomerase-negative cells. Eventual inhibition of highly unstable especially in the absence of functional tumor growth may rely on inhibition of both the p53 and pRb. Although telomerase inhibition in the telomerase-dependent and telomerase-independent short term may be bene®cial for eradication of tumor pathways of telomere maintenance. cells, long term inhibition of the enzyme may cause further genomic instability and eventual emergence of drug resistant telomerase-negative tumor cells. Emer- gence of immortal, telomerase-negative cells both in Acknowledgments vitro (Bryan et al., 1995) and in vivo (Bryan et al., 1997) This work was supported by grants from the Medical attest to this possibility. In addition, drugs that Research Council of Canada and the National Cancer interfere with the function of telomerase-positive, Institute of Canada.

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