Reversible Manipulation of Telomerase Expression and Telomere Length

Abstract

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Creexpressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.

Effects of hTERT on metal ion-induced genomic instability

“…BJ normal human foreskin fibroblasts (which normally senesce at approximately PD 80) were infected at PD 50 with pLXSNhTERT retroviruses (as in Rubio et al, 2002). The plasmid was created by introducing hTERT from pBABE-PUROhTERT (Rubio et al, 2002) in the pLXSN retroviral vector (Clontech).…”

“…The plasmid was created by introducing hTERT from pBABE-PUROhTERT (Rubio et al, 2002) in the pLXSN retroviral vector (Clontech). Cells were selected with G418, and the population (hTERT þ ) expanded before being frozen, before use, after PD 55.…”

Death receptor signaling regulatory function for telomerase: hTERT abolishes TRAIL-induced apoptosis, independently of telomere maintenance

“…Note that the sensitivity of all cell lines to cisplatin, or etoposide was not modified by ATRA pretreatment (data not shown), suggesting that the increased sensitivity of the NB4-LR1/hTERT-GFP cells to cisplatin or etoposide represents likely an intrinsic property of this cell line independent of telomerase expression. There are apparently contradictory reports regarding the effect of hTERT on cisplatin-induced apoptosis and in general in DNA-damaged induced cell death (Kondo et al, 1998;Folini et al, 2000;Lu et al, 2001;Ludwig et al, 2001;Gorbunova et al, 2002;Yuan and Mei, 2002;Rubio et al, 2002Rubio et al, , 2004. These controversial results obtained in these studies, including ours, could be tentatively explained by the fact that the cell response to genotoxic stress may depend on the combination of stress of distinct nature, the presence of telomeric proteins which can interfere with some types of DNA repair and on the length of the shortest telomere and the ability of telomerase to elongate short telomeres, as recently demonstrated (Rubio et al, 2002(Rubio et al, , 2004.…”

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

“…To provide further functional validation of the CHK2-specific ZFP TF repressor, we used retroviruses to constitutively express ZFP-5475 in untransformed human fibroblasts that were immortalized by hTERT, the catalytic subunit of telomerase (23). Several independent single-cell-derived clones were obtained in which ZFP TF-driven CHK2 repression was evident by immunoblot analysis (Fig.…”

“…Human fibroblasts (strain 82-6) were obtained, cultured, and immortalized with an hTERT-expressing retrovirus, as described (22,23). The ZFP-5475 cDNA was subcloned into the pLXSN retroviral vector, infectious virus was produced, and hTERT-expressing cells were infected and selected as mass cultures.…”