Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by the expression of a dominant-negative hTERT mutant.

As activation of telomerase represents a key step in themalignant transformation process, experimental modelsto develop anti-telomerase drugs provide a rational basisfor anticancer strategies. We analysed the short and long-term e†cacy of a stably expressed dominant-negativemutant (DN) of the telomerase catalytic unit (hTERT) inUT-7 and U937 human leukemia cell lines by using anIRES-e-GFP retrovirus. As expected, telomerase inacti-vation resulted in drastic telomere shortening, cytogeneticinstability and cell growth inhibition in all e-GFP positiveDN clones after 15–35 days of culture. However, despitethis initial response, 50% of e-GFP positive DN cloneswith short telomeres escaped from crisis after 35 days ofculture and recovered a proliferation rate similar to thecontrol cells. This rescue was associated with atelomerase reactivation inducing telomere lengthening.We identified two pathways, one involving the loss of theDN transgene expression and the other the transcrip-tional up-regulation of endogenous hTERT withpersistence of the DN transgene expression. Althoughthis second mechanism appears to be a very rare event(one clone), these findings suggest that genomic instabilityinduced by short telomeres after telomerase inhibitionmight enhance the probability of activation or selection oftelomere maintenance mechanisms dependent on hTERTtranscription.Oncogene (2002) 21, 8262–8271. doi:10.1038/sj.onc.1206054Keywords: telomere; telomerase; leukemia; retrovirus;cell death; therapyIntroductionTelomeres are dynamic DNA-protein complexes(Blackburn, 2001) that cap the ends of linearchromosomes consisting of TTAGGG repeats (deLange et al., 1990) and telomere-binding proteins(Broccoli et al., 1997; Chong et al., 1995). Telomeresprotect the chromosome ends from degradation,recombination and DNA repair activities (Bailey etal., 1999; d’Adda di Fagagna et al., 1999; Samper etal., 2000). In addition, telomere length is progressivelyreduced with cell divisions (Blasco et al., 1997; Harleyet al., 1990), due to the ‘end-replication problem’(Hastie et al., 1990) and the putative exonucleaseactivity in the CA-rich strand (Makarov et al., 1997).Telomere shortening in aging cells induces replicativesenescence (Morin, 1997). When telomeres reach acritical size, chromosomes become unstable and under-go end-to-end fusions, DNA fragmentation, andmutations (Blasco et al., 1997; Gisselsson et al.,2001). Conversely, telomere length is stable in cellswith long-lived replicative life spans, such as germ lineand embryonic cells, and in immortalized and tumorcells (Wright et al., 1996).Telomerase activation is the major mechanism thatmaintains telomere integrity (Morin, 1989). Thehuman telomerase reverse transcriptase (hTERT) isan RNA-dependent DNA polymerase which synthe-sizes telomeric DNA using the human telomeraseRNA (hTR) as a template (Colgin and Reddel, 1999;Feng et al., 1995; Morin, 1989; Nugent andLundblad, 1998). Unlike germline and embryoniccells, somatic cells do not have telomerase activity,with the exception of regenerative tissues such ashematopoietic stem cells (Chiu et al., 1996; Morrisonet al., 1996) or lymphocytes (Hiyama et al., 1995). Incontrast, most malignant cells are characterized by anincreased telomerase activity (Meyerson et al., 1997;Raymond et al., 1996). Enhanced telomerase activityrepresents an important step in the transformationprocess of human cells, as the combined expression ofhTERT, SV40 large T antigen and H-Ras results indirect conversion from normal to tumor cells (Hahnet al., 1999a). In addition, acquisition of telomeraseactivity may be associated with escape from senes-cence (Bodnar et al., 1998; Counter et al., 1998a;Pendino et al., 2001). These findings validate