inactivation of p53/pRb and expression of telomerase - Nature

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Oncogene (1999) 18, 7676 ± 7680 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 1

Stanford 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 contribute to the onset of senescence in human cells. The p53 and pRb pathways are involved in the regulation of cell cycle progression from G1 into S phase and inactivation of these pathways leads to extension of life span. Short dysfunctional telomeres may be perceived as damaged DNA and may activate these pathways, leading to prolonged arrest in G1, typical of cells in senescence. Inactivation of the p53 and pRb pathways, however, does not lead to cell immortalization. Cells that overcome senescence and have an extended life span continue to lose telomeric DNA and subsequently enter a second phase of growth arrest termed `crisis'. Forced expression of telomerase in human cells leads to the elongation of telomeres and immortalization. The development of human cancer is frequently associated with the inactivation of the pRb and p53 pathways, attesting to the importance of senescence in restricting the tumorforming ability of human cells. Cancer cells must also maintain telomere length and, in the majority of cases, this is associated with expression of telomerase activity. Keywords: telomerase; hTERT; telomere; senescence; aging; p53; pRb; TIEL; PARP

Two proliferative blockades: senescence and crisis In contrast to rodent cells, human cells rarely become immortal in culture. This di€erence may be attributed, at least partially, to the Hay¯ick barrier also termed `senescence'. In 1961, Hay¯ick showed that normal human cells divide only a ®nite number of times in culture and undergo replicative senescence (Hay¯ick and Moorhead, 1961). Spontaneous escape from senescence in human cells is an extremely rare event but this can be facilitated through the action of certain oncogenes or by exposure of primary cells to various mutagens. Cells that overcome senescence and have an extended life span subsequently enter a second phase of growth arrest termed `crisis'. Unlike senescent cells that are primarily arrested in the G1 phase of the cell cycle, cells at crisis undergo division and death. Escape from crisis occurs at very low frequency (361077) (Shay and Wright, 1989).

*Correspondence: S Benchimol

A provocative and in¯uential model of cell senescence and immortalization (Harley et al., 1992) suggested that telomeres shorten in many proliferating somatic cells due to the end replication problem (the inability of the DNA replication machinery to completely copy chromosomal termini) (Olovnikov, 1973; Watson, 1972). In this model, shortened telomeres signal the onset of senescence. Cells with an extended life span bypass the senescence signal but continue to lose telomeric DNA until they reach crisis and die. Overcoming crisis is associated with maintenance of telomere length and reactivation of telomerase (Counter et al., 1992). Although some similarities exist between senescence and crisis, there is now abundant evidence demonstrating that these represent fundamentally di€erent cellular processes governed by distinct genetic regulatory components. Involvement of the p53 and Rb pathways in senescence The transforming proteins of DNA tumor viruses such as SV40 large T antigen, adenovirus E1A/E1B or HPV 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 to target the tumor suppressor proteins, p53 and pRb, and render them functionally inactive. pRb and p53 are important regulators of cell cycle progression and play key roles in controlling entry into S phase. pRb binds to members of the E2F family of transcription factors. In the bound state, E2F-1 is unable to activate E2F-1-responsive genes required for entry into S phase and for DNA synthesis. Phosphorylation of pRb by the G1 cyclin-dependent kinases (CDKs) releases E2F-1 in a transcriptionally active form, allowing cells to progress from G1 into S phase. The HPV E7 protein, as well as SV40 large T antigen and adenovirus E1A, override this control circuit by binding to pRb and causing the inappropriate release of active E2F-1 in the absence of CDK-mediated phosphorylation of pRb (Weinberg, 1995). p53 promotes G1 cell cycle arrest or apoptosis in response to DNA damage and other forms of stress (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 in the p53 gene in human cancer eliminate the ability

Extension of cellular life span H Vaziri and S Benchimol

of p53 protein to bind DNA or to promote transcription. p53 blocks cells in G1 primarily through its ability to induce expression of p21WAF1, a CDK inhibitor (E1-Deiry et al., 1993). p21WAF1 provides a link between the p53 and Rb regulatory pathways. p21WAF1 can act to limit the phosphorylation of pRb, thereby preventing cells from exiting the G1 phase of the cell cycle. The HPV E6 protein binds to p53 and promotes its degradation via the ubiquitin proteolytic pathway, while adenovirus E1B and SV40 large T antigen bind to p53 and block its transcriptional activity. In the absence of p53, pRb or p21WAF1, the cell cycle arrest pathway induced by DNA damage is lost (Kastan et al., 1992; Brugarolas et al., 1995; Deng et al., 1995; Harrington et al., 1998). Increased expression and/or activity of p53, p21 and another CDK inhibitor, p16INK4a, is detected in senescent cells lending further support to the importance of the p53 and Rb pathways in senescence (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 CDK inhibitor, p16INK4a, as well as an unrelated protein, p19ARF which arises in large part from an alternative reading frame (Quelle et al., 1995). In human cells, usage of an alternative reading frame in the INK4a locus similarly gives rise to the human homolog known as p14ARF. Ectopic overexpression of p53, p21, p16INK4a, or p19ARF/p14ARF in proliferating cells can induce G1 arrest. The ability of p16INK4a to induce cell cycle arrest is pRb dependent while p19ARFor p14ARF- induced cell cycle arrest is p53 dependent. The human and mouse ARF proteins have been shown to stabilize and activate p53. This occurs, at least in part, by the interaction of the ARF proteins with MDM2 resulting in abrogation of MDM2's ability to promote the degradation of p53 (Pomerantz et al., 1998; Zhang et al., 1998; Kamijo et al., 1998). Primary murine ®broblasts lacking p53 or p19ARF or both p16INK4a and p19ARF are easily established into immortal cell lines (Harvey et al., 1993; Serrano et al., 1996; Kamijo et al., 1997). In human ®broblasts derived from patients with LiFraumeni syndrome that are heterozygous for wildtype p53, senescence can be overcome (Bischo€ et al., 1990) through loss or mutation of the remaining wildtype p53 allele (Rogan et al., 1995). In addition, dominant negative inhibition of p53 protein (Bond et al., 1994) or inactivation of p21WAF1 (Brown et al., 1997) in human cells can also overcome senescence and lead to an extended life span. These cells, however, do not become immortal and, like those transformed by DNA tumor virus (Shay et al., 1991), enter crisis and rarely emerge as immortal clones. An important question that arises in trying to relate these ®ndings to the telomere shortening model of cell senescence is how the p53 and pRb pathways become activated in response to shortened telomeres to invoke the senescence program. Short telomeres may signal cell cycle exit via p53 protein Shortening of telomeres as a function of in vitro age has been observed in variety of cell lineages including ®broblasts (Allsopp et al., 1992; Harley et al., 1990), T-

cells (Vaziri et al., 1993) and endothelial cells (Chang and Harley, 1995). Several hypotheses have been proposed to explain how shortened telomeres might lead to senescence. We proposed that shortened telomeres per se are not the signal for senescence; rather that shortened telomeres compromise DNA integrity and generate strand breaks that are sensed by the DNA damage recognition machinery of the cell, leading to p53 activation and p21WAF1-mediated G1 arrest (Vaziri and Benchimol, 1996). In search of other e€ector molecules in this pathway, we found that inhibition of poly(ADP-ribosyl)ation in normal human ®broblasts extended cellular life span signi®cantly. Importantly, we demonstrated a physical and functional association between poly(ADP-ribose) polymerase and p53 in human cells (Vaziri et al., 1997). Hence it is possible that short and dysfunctional telomeres signal growth arrest via poly(ADP-ribosyl)ation of critical e€ector molecules including p53. The telomerase complex Synthesis of telomeric repeats at the ends of chromosomes is performed by the telomerase protein/RNA complex. Telomerase activity, measured by the ability of the enzyme to add TTAGGG repeats to the end of synthetic telomeric DNA, was originally discovered in the ciliate Tetrahymena (Greider and Blackburn, 1985) and subsequently in the human immortal cell line HeLa (Morin, 1989), The ground breaking discovery of Euplotes p123, the catalytic subunit of telomerase (Lingner et al., 1997), led to identi®cation of the human telomerase catalytic subunit (hTERT) (Harrington et al., 1997b; Kilian et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997). The human telomerase complex consists of an RNA subunit named hTR (Feng et al., 1995) and hTERT. Several other proteins have also been identi®ed that interact with the telomerase complex including TEP1/TLP1 (Harrington et al., 1997a). Evidence from S. cerevisiae and Euplotes suggests that several other factors could also potentially interact and be a part of the complex, including EST1, EST4 and p43 (Nugent and Lundblad, 1998). Telomerase and extension of life span Reconstitution of telomerase activity in vitro requires the RNA template of human telomerase hTR and the catalytic subunit hTERT (Beattie et al., 1998; Weinrich et al., 1997). Forced expression of hTERT in normal human ®broblasts is sucient to fully reconstitute telomerase activity, since the endogenous RNA template of human telomerase (hTR) is expressed in normal human cells (Feng et al., 1995). Reconstitution of telomerase activity leads to elongation of telomeres in hTERT expressing cells and is associated with the 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 proliferate beyond crisis (Counter et al., 1998; Halvorsen et al., 1999). These reports provide strong evidence in support of the model that telomere shortening is a mechanism that limits the replicative capacity of human cells.

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In another study, reconstitution of telomerase activity by hTERT alone was unable to extend the life span of normal human keratinocytes or mammary epithelial cells (HMECs) (Kiyono et al., 1998). Both pRb inactivation (through the use of HPV E7) and hTERT expression were required to immortalize these human epithelial cells. These cells, unlike other human cell strains, undergo a telomere-independent, p16INK4adependent, growth arrest phase called M0 that can be overcome by E7 (Foster and Galloway, 1996; Foster et al., 1998). This important observation demonstrates that in certain cell strains, hTERT alone will not extend cellular life span. It should be recognized, however, that mammary epithelial cells undergo M0 after 7 ± 20 population doublings at a time when telomeres are still relatively long. Hence, it is unlikely that M0 occurs in response to telomere shortening and so it is not surprising that hTERT expression fails to 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, moreover, that telomerase reconstitution and telomere elongation in HMECs and human keratinocytes do not interfere with the di€erentiation programs of these cells. In contrast to these studies, other groups have been able to extend the life span of HMECs (Wang et al., 1998) and other epithelial cells (Bodnar et al., 1998). It is possible that there may be variability in the susceptibility of epithelial cells to di€erentiate in culture. Extension of cellular life span by telomerase is associated with maintenance of the p53/pRb pathways and maintenance of genomic integrity Extension of cellular life span beyond the Hay¯ick limit is associated with loss of genomic integrity and formation of aberrant chromosomes (Wolman et al., 1964). Senescence, therefore, may be viewed as a proliferative blockade to prevent the outgrowth of cells with genomic instability. As discussed thus far, cellular life span can be extended through, at least, two alternative pathways. The ®rst pathway involving inactivation of p53 and pRb is short-lived; cells acquire an extended life span but succumb to crisis. These post-senescence cells have lost G1 cell cycle control and as a result are prone to genetic rearrangements involved in tumor progression (Livingstone et al., 1992; Yin et al., 1992; Almasan et al., 1995). The second pathway involving telomerase expression and telomere maintenance leads to immortality. Are cells immortalized by telomerase genetically stable or unstable? On the basis of G-banding and spectral karyotyping, we reported previously that BJ-TIEL cells (BJ primary human ®broblasts immortalized by telomerase) displayed non clonal chromosomal aberrations initially after ectopic expression of telomerase (Vaziri et al., 1999). These aberrant structures were transient and subsequently disappeared in later cell passages. Morales et al. (1999) also reported no sign of chromosomal abnormalities in BJ-TIEL cells on the basis of G-banding analysis. It appears, therefore, that extension of cellular life span through telomerase reexpression does not compromise genomic integrity.

This is further substantiated by the demonstration that functional p53 protein is expressed in these immortal cells (Vaziri et al., 1999). BJ-TIEL cells have an intact p53-dependent G1 checkpoint in response to DNA damage and are able to upregulate p53 protein in response to ionizing radiation (Vaziri et al., 1999) or UV damage (Morales et al., 1999). Importantly, cells immortalized by telomerase expression are nontumorigenic and do not have changes typically associated with the malignant phenotype (Vaziri et al., 1999; Morales et al., 1999; Jiang et al., 1999). Maintenance of telomere length by telomerase is probably critical for preventing chromosomal aberrations associated with eroded telomeres. Together, these ®ndings further substantiate the idea that extension of cellular life span by telomerase is fundamentally di€erent from that induced by inactivation of the p53 and pRb pathways. p53, pRb, telomeres and cancer The development of human cancers is frequently associated with the inactivation of the pRb and p53 pathways, attesting to the importance of senescence in restricting the tumor-forming ability of human cells. Cancer cells maintain telomere length and in the majority of cases this is associated with expression of telomerase activity, although alternative mechanisms for telomere maintenance in the absence of telomerase must also exist (Colgin and Reddel, 1999). An important question that arises from these ®ndings is whether there is a connection between the p53/pRb pathways and telomerase expression in immortal cancer cells. Cells with an extended life span in which the p53 and pRb pathways have been inactivated continue to lose telomeric DNA until crisis. Telomerase activity is not detectable in these cells. Furthermore, telomerase activity in cell lines is independent of their p53 status (Milas et al., 1998) and activation of telomerase in keratinocytes by HPV16-E6 is independent of the ability of E6 to compromise p53 function (Klingelhutz et al., 1996). Together, these ®ndings suggest that loss of p53 gene function is not by itself sucient for telomerase activation, telomere elongation and cellular immortality. Little is known about the regulation of hTERT gene expression and how this gene becomes activated in cells emerging from crisis. It is possible that the genetic instability resulting from the inactivation of the p53 and pRb pathways facilitates events that lead to telomerase expression. Telomerase inhibition: hitting the Achilles heel of the cancer cell or causing further genomic instability? There is currently much interest in the development of telomerase inhibitors as anti-cancer agents. Inhibition of telomerase activity by anti-sense inhibition of hTR has shown some limited success in inducing cell death (Feng et al., 1995). Telomerase-de®cient mice experience telomere shortening and accompanying genetic instability including aneuploidy and chromosome abnormalities (Blasco et al., 1997). If telomerase activity is required for immortality and subsequent

Extension of cellular life span H Vaziri and S Benchimol

maintenance of genomic stability, telomerase inhibition should destabilize the genome by interfering with telomere function. Tumor cells treated in vivo with anti-telomerase drugs might be expected to become highly unstable especially in the absence of functional p53 and pRb. Although telomerase inhibition in the short term may be bene®cial for eradication of tumor cells, long term inhibition of the enzyme may cause further genomic instability and eventual emergence of drug resistant telomerase-negative tumor cells. Emergence of immortal, telomerase-negative cells both in vitro (Bryan et al., 1995) and in vivo (Bryan et al., 1997) attest to this possibility. In addition, drugs that interfere with the function of telomerase-positive,

normal, self-renewing stem cells would be harmful (Lee et al., 1998). This points out the necessity of understanding the mechanism underlying the survival of telomerase-negative cells. Eventual inhibition of tumor growth may rely on inhibition of both the telomerase-dependent and telomerase-independent pathways of telomere maintenance.

Acknowledgments This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.

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