Consequences of Telomerase Inhibition in HSCs

When telomeres shorten until they are significantly eroded, a DNA damage cascade is activated, and cells usually undergo replicative senescence or apoptosis (Harley et al. 1994, Maser and DePinho 2002). Because the majority of tumor cells is reliant on telomerase for telomere stabilization, inhibition of this enzyme represents an attractive concept for cancer therapy (reviewed in Zimmermann 2007). Proof of the underlying principle for such strategies was given by studies in which telomerase was abolished by overexpression of DN-hTERT in immortal cancer cell lines, resulting in telomere erosion and induction of senescence or apoptosis (Hahn et al. 1999, Zhang et al. 1999). In addition to genetic approaches, different synthetic telomerase inhibitors are tested at present with promising results for specific killing of tumor cells (Asai et al. 2003, Damm et al. 2001, El-Daly et al. 2005, Herbert et al. 1999) (reviewed in Zimmermann 2007), some of which progress significantly toward clinical application (Dikmen et al. 2005, Djojosubroto et al. 2005, Gellert et al. 2006). The main portion of normal human cells considered as telomerase-negative should not be affected by telomerase inhibition, but potential side effects by such treatment modalities concern telomerase-positive stem cells such as HSCs, which most likely turn out less dramatically in light of the low estimated HSC replication rates in vivo. As seen above, the exact role of telomerase in these cells is not completely understood, yet there is clear evidence that telomerase activity is required for health and viability within the entire life span (Mitchell et al. 1999, Vulliamy et al. 2001). A particular model to study the role of telomerase in vivo is the human disease Dyskeratosis congenita (DKC), which is characterized by anemia, immune deficiency, skin and nail lesions, chromosomal instability, and cancer (reviewed in Collins and Mitchell 2002; see also Du et al., this volume). Two genes encoding proteins of the telomerase complex were found to be mutated, resulting in a partial telomerase inhibition; abnormally short telomeres, and a dramatically limited proliferative capacity of hematopoietic and epithelial tissues in DKC patients (Mitchell et al. 1999). Autosomal-dominant forms of the disease are caused by mutations in the core RNA component of telomerase hTERC (Goldman et al. 2005, Vulliamy et al. 2001) (reviewed in Vulliamy and Dokal 2003). The unusual appearance of two distinct mutations in both hTERC alleles in a DKC patient was reported recently (Ly et al. 2005b), whereby haploinsufficiency of hTERC is most likely resulting in an anticipation of the autosomal-dominant DKC disease forms correlated with an increase in telomere shortening in successive generations of families with this disease (Vulliamy et al. 2004). The hematological abnormalities developed in most DKC patients clarify that highly proliferative cell lineages like the hematopoietic progenitor cells would suffer most in the course of telomerase inhibition.

Another human disease possibly associated with telomerase is the bone marrow failure syndrome aplastic anemia (AA), which is characterized by pancytopenia related to reduced or absent immature hematopoietic cells (Maciejewski et al. 1994, Marsh et al. 1990, Scopes et al. 1994). The higher turnover of HSCs is here expressed in significantly shorter telomeres in mature blood cells like peripheral lymphocytes and granulocytes than those in age-adjusted healthy controls (Ball et al. 1998, Brummendorf et al. 2001a, Lee et al. 2001). As in DKC, mutations in telomerase hTERC (Vulliamy et al. 2002) may be responsible for the compromised telomerase activity resulting in the observed phenotypes. Most recently, evidence for a direct link between hTERC sequence variants found in AA patients and abolished telom-erase activity was described (Ly et al. 2005a). Analogously, mutations in hTERT were recently identified among AA patients, again associated with short telomeres and low telomerase enzymatic activity (Yamaguchi et al. 2005). Bone marrow failure of variable severity due to DKC may be present in otherwise phenotypic normal adults and may masquerade as AA, whereas common mutations in hTERC are linking the two diseases (Dokal and Vulliamy 2003, Fogarty et al. 2003, Marrone et al. 2004). Interestingly, hTERC haploinsufficiency in autosomal-dominant DKC cases, which is associated with a modest reduction of telomerase activation of around 50%, is sufficient to induce the severe phenotypes of the disease described above. Overall, this indicates the need of a tight control of telomerase levels throughout life in human HSCs (reviewed in Collins and Mitchell 2002).

Recent studies suggest that even normal human cells like primary fibroblasts harbor some previously undetected telomerase activity (Masutomi et al. 2003). These basal telomerase levels seem to be important for the maintenance of the 3' overhang, cell proliferation, and cellular life span without any consequences on the overall telomere loss (Masutomi et al. 2003). It would be interesting to demonstrate a similar function of telomerase in the stem cell compartment. We found that telomerase ablation in CB AC133+ cells by DN-hTERT overexpression is accompanied by a reduction in clonogenic growth without changing the mean telomere length, supporting the hypothesis above that telomerase even in HSCs may have additional functions beyond simple telomere lengthening (Zimmermann et al. 2004). In addition, we observed that high concentrations of the small molecule telomerase inhibitor BIBR1532 specifically kill tumor cells of the hematopoietic system and do not harm proliferation and clonogenic growth of normal CD34+ cells, reflecting potential differences between genetic and pharmacological approaches of telomerase inhibition (El-Daly et al. 2005).

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