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Proc Natl Acad Sci U S A. Oct 1, 2002; 99(20): 12520–12522.
Published online Sep 23, 2002. doi:  10.1073/pnas.212514699
PMCID: PMC130491

Telomerase extracurricular activities

The extended growth potential of cancer cells is critically dependent upon the maintenance of functional telomeres, G-rich repeat sequences that cap the ends of most eukaryotic chromosomes and serve to protect natural DNA ends from being recognized as double-stranded breaks (reviewed in ref. 1). The inability of DNA polymerase to synthesize fully the terminal ends of the lagging strand leads to progressive telomere shortening with each round of cell division. The gradual erosion of telomeres to a critically short length elicits the successive cellular responses of senescence and crisis; each has been shown to represent formidable barriers to continued cell growth in culture (2, 3). These cell culture-based studies also seeded the view that long-term cancer cell growth and survival requires activation of one of two known mechanisms of telomere maintenance. The first and most common mechanism involves reactivation of the enzyme telomerase (4), a specialized ribonucleoprotein complex that contains a complementary RNA template (TERC) and a reverse transcriptase catalytic subunit (TERT). In telomerase reactivation, transcriptional up-regulation of the TERT gene is often the limiting event (5, 6), although TERT activity can be controlled on multiple posttranscriptional and posttranslational levels (7). The second telomere maintenance mechanism, encountered in only a minority of cancer cells, involves a telomerase-independent process termed ALT (for alternative lengthening of telomeres), which is, perhaps, mediated by the homologous recombination pathway (8, 9). The consistent presence of either mechanism in advanced human cancers has supported the assumption that the key, and perhaps only, factor in the promotion of full malignant transformation is adequate telomere reserves, and that the particular telomere maintenance mechanism used was less relevant. In this issue of PNAS, Stewart et al. (10) force a re-evaluation of this commonly held view with a provocative set of experiments showing that the actions of TERT in tumor progression extend beyond the singular role of telomere maintenance, and that TERT-mediated vs. ALT-mediated telomere maintenance are not functionally equivalent in promoting tumorigenesis.

The actions of TERT in tumor progression extend beyond the singular role of telomere maintenance.

A large body of work in human cell culture models has documented the biological and genomic consequences of telomere attrition and their relationship to the suppression or promotion of cancer. Replicative senescence (also termed the Hayflick limit or Mortality Stage I) is the first cellular response elicited by passage-induced telomere attrition, and its induction requires intact p53 and RB tumor-suppressor pathways (11, 12). Inactivation of these key tumor-suppressor pathways permits extended replicative potential but also continued telomere erosion and eventual loss of telomere capping function. Uncapped telomeres are highly recombinogenic, resulting in the formation of dicentric chromosomes and breakage at the time of cell division; they also fuel high degrees of genomic instability and loss of cell viability, a period aptly termed “crisis” (2). It is well established that only rare (1 × 10−7 to 1 × 10−5) cultured cells emerge from crisis (13, 14), and that enforced hTERT expression and, hence, telomerase activity, can avert both senescence and crisis in primary culture cells (15, 16). Importantly, hTERT expression enables full malignant transformation of primary human cells by small and large T antigen and activated H-RAS (17). Together, these studies have underscored the essential role of telomere maintenance in sustaining the proliferation of normal and neoplastic cells.

A smaller fraction of human tumors employs ALT to maintain telomere lengths during neoplastic growth (8, 18). These ALT+ tumors most often derive from mesenchymal tissues and rarely derive from epithelial compartments, which, instead, show near-exclusive activation of telomerase (8). In addition, ALT appears to be exceedingly rare in hematological malignancies; this may be related, in part, to the more ready activation of telomerase in normal lymphoid cells and tissues (19). It is tempting to speculate that this dichotomy in telomere maintenance mechanisms and tissues of origin might reflect tissue-specific differences in the ability to derepress the TERT promoter during tumorigenesis and/or inherent functional differences in ALT vs. telomerase and cell type-specific responses to these differences. ALT tumor cells are characterized by very heterogeneous telomere lengths and the presence of ALT-associated promyelocytic leukemia (PML) bodies, nuclear structures containing telomeric DNA and proteins involved in DNA recombination and replication (20). Although the molecular mechanisms underlying ALT continue to be a focus of investigation, emerging data in mammalian cell culture systems have implicated interchromosomal recombination mechanisms as the means to maintain telomeres above a threshold length that elicits crisis (9).

In the current study, Stewart et al. (10) asked whether an immortal telomerase-negative fibroblast cell line (GM 847) that exhibits the ALT-mediated telomere maintenance phenotype would provide a permissive cellular context for the full transforming effects of activated H-RAS. Activated H-RAS expression in this nontumorigenic cell line was shown to confer anchorage-independent growth in vitro, yet failed to promote a tumorigenic phenotype as assayed by s.c. implantation in immunocompromised mice. This finding led, in turn, to the experiment showing that the coexpression of H-RAS and hTERT in these ALT-positive cultures readily produced a robust tumorigenic phenotype, implying that telomere maintenance by ALT- and hTERT-mediated mechanisms are not biologically equivalent in promoting tumorigenesis and/or that the pro-tumorigenic actions of hTERT include non-telomere functions. To distinguish between these possibilities, these investigators made elegant use of a hemaglutanin (HA) epitope-tagged hTERT mutant protein (hTERT-HA) that is defective in maintaining telomeres in vivo because of the presence of the epitope tag (21). Strikingly, despite this telomere maintenance defect, hTERT-HA was able to cooperate with H-RAS coexpression to effect a highly penetrant tumorigenic phenotype. The elemental conclusion is that hTERT possesses protumorigenic activities that extend beyond its classical role in telomerase-mediated telomere synthesis.

The first clues that TERT may have roles other than telomere-length maintenance came from studies implicating a possible role for TERT in neuroprotection (22). Inhibition or overexpression of TERT in neuronal model systems was found to modulate the apoptotic threshold in the setting of the neurotoxic protein amyloid β-peptide, a protein believed to promote neuronal degeneration in Alzheimer's disease. Indeed, Stewart et al. demonstrated that under conditions of physiological stress (0.4% oxygen, 2.5 mM glucose, and 0.1% serum), hTERT expression in the GM847 ALT cells conferred a selective growth advantage over cells expressing a control vector. This observation is in line with data suggesting that TERT expression also can enhance cell survival in the face of proapoptotic cellular stress.

More recently, a complementary series of transgenic mouse studies have been conducted to assess the pathophysiological impact of high-level mTERT expression in various tissues. Laboratory mice possess long telomeres and express telomerase in somatic tissues; thus, cellular growth and transformation are not normally limited by their telomere reserve. However, despite the ample telomere reserves in mouse cells, telomerase is up-regulated in a spectrum of developing tumor types (2325), suggesting that telomerase activation may confer enhanced proliferative and survival potential in tumorigenesis, independent of its role in telomere maintenance. The long telomeres in mice and the lack of critical telomere shortening in mouse tissues thus provide a model system in which to evaluate in vivo the impact of enforced TERT expression in a setting where telomere length is not limiting.

Targeted expression of telomerase in epidermal basal keratinocytes resulted in high levels of telomerase activity and normal telomere length (26). The epidermis of these animals showed increased wound healing rates and an increased incidence of carcinogen-induced skin tumors compared with littermate controls—findings in line with a growth-promoting role for TERT in vivo. Furthermore, expression of telomerase under the control of a ubiquitously expressed promoter resulted in expression in a broad range of tissues, including mammary glands, splenocytes, and skin (27). Interestingly, whereas telomerase expression was without discernable effects in most tissues (perhaps relating, in part, to low levels of mTERC), aged mTERT transgenic mice developed a highly penetrant ductal carcinoma in situ phenotype as well as spontaneous mammary tumors. Nontransgenic age-matched controls remained breast cancer-free. mTERT-induced breast cancers were shown to be invasive and exhibited malignant histological features, including nuclear pleiomorphism and high mitotic activity (27). This result indicates that, even in a setting where telomeres are not limiting, enforced mTERT expression can promote neoplastic lesions in mammary glands to enable progression to fully invasive lethal tumors in aged animals. These mouse studies are intriguing in the light of previous human cell culture studies (28) assessing the impact of ectopic hTERT expression in human mammary cells. Enforced hTERT expression in cultures that have epigenetically silenced p16INK4a during adaptation to culture resulted in increased resistance to growth arrest mediated by transforming growth factor β, suggesting that TERT expression can cooperate with p16INK4a inactivation to influence cancer-relevant signaling pathways (28). Taken together, these mouse and human model systems support the existence of telomere-independent functions for TERT in mediating cellular proliferation, survival, and tumorigenesis.

Although it is intriguing to assign previously uncharacterized functions to TERT, it is also possible that the end result of telomere maintenance via telomerase may be fundamentally different from that mediated by ALT. The telomere length heterogeneity characteristic of ALT cells renders a population of chromosome ends with very long telomeres, whereas other termini may have denuded telomeres (29, 30). “Marked” telomeres in ALT cells have been shown to cycle from gradual shortening in culture to rapid increases in length to a resumption of gradual attrition rates (31). This cyclical nature of telomere dynamics in ALT cells raises the possibility that there exists to some degree ongoing telomere dysfunction in the population, possibly activating checkpoint responses and limiting cellular growth and survival, hence quelling tumorigenic potential. Indeed, cytogenetic evidence suggests that some ALT cells possess telomere-free chromosomal ends that give rise to chromosomal fusions and complex translocations, which are all features of dysfunctional telomeres (ref. 32; and S.C. and R.A.D., unpublished work). In contrast, telomerase has been shown to be effective (and perhaps preferential) in the repair of the shortest telomeres and the rescue of the cellular defects associated with telomere dysfunction (33, 34). Examination of ALT cells reconstituted with telomerase activity by PNA-fluorescence in situ hybridization (FISH) suggests that telomerase also targeted the shortest telomeres in this system (30, 35). In the study by Stewart et al. (10), the use of the defective HA-TERT argues against the rescue of short telomeres as the sole advantage conferred by TERT in processes of tumorigenesis. A future use of quantitative PNA-FISH analysis of telomere lengths in these cells pre- and post-hTERT reconstitution should serve to reinforce these important conclusions.

The findings by Stewart et al. (10) also may provide a rational explanation for why cancer cells overwhelmingly favor telomerase activation over the ALT mechanism in maintaining telomere function. Their data, along with the aforementioned studies, suggest that the ALT mechanism(s) is not functionally equivalent to telomerase in supporting full malignant transformation. This hypothesis gains added support from the analysis of telomerase-deficient tumor cell lines with critically short dysfunctional telomeres (S.C. and R.A.D., unpublished work). Although these cells form tumors upon s.c. implantation, they fail to metastasize to the lungs after tail-vein injection. Expectedly, metastatic potential can be acquired by mTerc reconstitution, which restores telomerase activity and telomere function. Intriguingly, in the absence of reconstitution, extended passage of these transformed mTerc−/− mouse cells in vivo yields sublines that exhibit an ALT phenotype of long heterogeneous telomeres. These ALT cells, although exhibiting robust tumor growth upon s.c. implantation, are completely unable to metastasize to the lung. Thus, despite the increased overall telomere lengths generated by ALT, these transformed cells were radically distinct from those with mTERC-reconstituted telomerase activity. These results further underscore the potential benefits of telomerase-mediated telomere maintenance in advanced stages of cancer.

The nonequivalence of telomerase and ALT in tumorigenesis has implications for the use of telomerase inhibitors as anti-tumor agents in human cancers. An understanding of the signaling pathways engaged by TERT may provide opportunities for the development of compounds that sever this link, thereby leading to synergy with the adverse cellular consequences of telomere erosion. In addition, despite concerns for activation of ALT in the course of anti-telomerase treatment, the study by Stewart et al. suggests that ALT cells may not be as biologically robust as telomerase-positive cancer cells and may impair advanced stages of malignancy despite activation of this pathway.

Footnotes

See companion article on page 12606.

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