Consequences of Telomerase Loss In Vivo The Telomerase Knockout Mouse

Telomeric repeats in human cells are typically between 10 and 15 kb in length. Furthermore, telomerase activity is stringently regulated in human tissues, i.e., unde-tectable in many somatic tissues but present in germ cells, activated leukocytes, and stem cells from a variety of organs (Harley et al. 1994, Wright et al. 1996, Newbold 1997, Weng et al. 1997). This regulation is achieved almost entirely through stringent downregulation of hTert (Meyerson et al. 1997). In contrast to humans, the laboratory mouse species Mus musculus possesses much longer telomeres, ranging in size from 40 to 80 kb, although some chromosomes harbor significantly shorter telomeres (Blasco et al. 1997, Zijlmans et al. 1997). In addition, telomerase activity and gene expression of mouse Tert (mTert) appear to be less stringently regulated in murine somatic cells (Kipling and Cooke 1990, Prowse et al. 1993, Prowse and Greider 1995, Greenberg et al. 1998, Martin-Rivera et al. 1998). Despite these species-specific differences, mTert expression appears to be the critical determinant of telomerase activity in mouse cells, since mTert expression correlates well with the distribution of telomerase activity in mouse tissues and cells (Greenberg et al. 1998). Although murine telomerase is regulated less stringently, murine telomeres do exhibit telomere shortening during development and somatic growth in some tissues, implying that the low levels of telomerase present may be inadequate or that other factors allowing telomere access may be limiting (Prowse and Greider 1995, Rudolph et al. 1999).

Work from a number of laboratories has shown that the mouse is an excellent system for studying telomere biology (reviewed in Chang et al. 2001, Goytisolo and Blasco 2002). The telomerase-deficient mouse lacks the critical RNA subunit mTerc, is viable, fertile, and has no significant morphological abnormalities (Blasco et al. 1997). Telomere length measurements revealed that, as in yeast and human cells lacking telomerase, telomeres shortened by approximately 120 bp per cell division. Given the long telomeres in mice, successive generations of telomerase-deficient mice were produced to achieve sufficient telomere attrition. By the sixth generation (G6), defects were revealed in multiple tissue compartments with high proliferative histories, including hematopoietic and reproductive systems. Defects in the hematopoietic system were manifested by a marked decline in hematopoietic precursor cell numbers, reduced proliferation of T and B lymphocytes upon mitogenic stimulation, and splenic atrophy, with a reduction in germinal center function (Lee et al. 1998, Herrera et al. 2000). Decreased fecundity was first noted in G4 mice, culminating in sterility in late-generation (G6) animals. Both male and female reproductive functions were severely compromised at this point, with the males displaying marked testicular atrophy accompanied by germ cell depletion, leading to a complete absence of spermatogenesis. In the females, a decrease in the number of oocytes upon ovulation was observed (Lee et al. 1998). Importantly, reconstitution of telomerase activity in late-generation mTerc-/- mice increased tel-omere lengths preferentially at the shortest telomeres and rescued many of the pathologies associated with telomere dysfunction (Hemann et al. 2001, Samper et al. 2001). Taken together, these results strongly support the view that the long-term renewal of germ and hematopoietic cells is dependent upon telomere function, and that telomerase expression is vital in long-term cellular survival and organ homeostasis (see also Zimmermann and Martens, this volume, Gutierrez and Ju, this volume).

Owing to its anti-proliferative effects, replicative senescence has long been postulated to be a potent tumor suppression mechanism in vivo (reviewed by Campisi 2005; see also Rudolph, this volume, and Blasco, this volume). Mouse models bearing dysfunctional telomeres have been generated to address this hypothesis. The INK4a~'~ mouse lacks both p16INK4a and p19ARF, which have important roles in both the pRb and p53 pathways. Importantly, the p53-dependent DDR is intact in the INK4a~'~ mouse. Treatment of early-generation mTerc1' INK4a~'~ mice with DMBA and UVB revealed that these mice are highly cancer prone (Greenberg et al. 1999).

However, similar treatment of late-generation mTerc~'~INK4a~'~ mice with short dysfunctional telomeres yielded a reduction in tumor incidence and much longer survival. This result suggests that in the setting of telomere dysfunction and an intact p53 checkpoint, tumor formation is impaired. A similar finding was observed in a skin carcinogenesis model, in which late-generation mTerc~'~ mice produced significantly fewer skin tumors upon chemical carcinogenesis of the skin compared to wild-type controls with long telomeres (Gonzalez-Suarez et al. 2000). Upregulation of p53 was detected in late-generation mTerc~'~ papillomas, most likely due to dysfunctional tel-omeres being sensed as DSBs, resulting in induction of p53 levels.

However, given that the aforementioned mouse models all possess wild-type p53, it is not clear how dysfunctional telomeres limit neoplastic growth in vivo. Which function of p53 (apoptosis versus cell cycle arrest/senescence) is more important for telomere-dependent tumor suppression? The generation of mTerc~'~ mice with dysfunctional telomeres in the setting of a p53 knock-in mutation (p53R172P allele) that is incapable of initiating a p53-dependent apoptosis but is competent to execute p53-mediated cell cycle arrest/replicative senescence allowed this question to be addressed directly (Cosme-Blanco et al. 2007 ). In this system, dysfunctional telomeres induce p53-mediated replicative senescence to suppress spontaneous tumorigenesis, while p53-dependent apoptosis appears dispensable for tumor suppression. Dysfunctional telomere-induced senescence was accompanied by robust increase in p53, p21, and SA-P-gal activity in all tissue compartments examined, suggesting that a telomere-dependent DDR is activated in vivo. These observations parallel a recent study indicating that p53 is dispensable for tumor suppression induced by radiation damage (a process mediated mainly through p53's apoptotic functions), while suppression of radiation-induced lymphomagenesis requires an intact p19 pathway (presumably to initiate oncogene-mediated cellular senescence) (Christophorou et al. 2006). Since the DDR is activated at the earliest stages in many human carcinomas (Bartkova et al. 2005), these results predict that activation of an intact DDR pathway by dysfunctional telomeres would promote p53-dependent senescence, suppressing further tumor progression. The increased genome instability in nascent tumor cells that stochastically inactivate the DDR and/or the p53-p21 dependent senescence pathways would promote tumor progression. However, recent studies in mTERC~'~p21~'~ mice revealed that deletion of p21 improved stem cell function, organ maintenance, and life span of mice possessing dysfunctional telomeres without accelerating tumor formation (Choudhury et al. 2007; see also Rudolph, this volume). The impact of cell cycle checkpoints and apoptosis for tumor suppression in response to telomere shortening thus needs to be better defined in future studies.

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