Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase

Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.     

Reconstitution of Telomerase Activity Extends the Proliferative Life-span and Maintains the Neurogenetic Potential of Mesenchymal Stem Cells Derived from Human Fetal Muscle

目的 :通过重建端粒酶活性延长胎儿肌肉源间充质干细胞寿命 ,并对其成神经潜能进行研究 ,为组织工程神经修复提供种子细胞。方法 :将人端粒酶催化亚基 (hTERT)基因通过脂质体转染法导入胎儿肌肉源间充质干细胞 ,RT PCR检测hTERTmRNA的表达 ,TRAP PCR检测细胞端粒酶活性。用bFGF诱导已重建端粒酶活性的肌肉源间充质干细胞向神经细胞分化 ,免疫荧光及免疫印迹法检测分化情况。结果 :转染hTERT的胎儿肌肉源间充质干细胞能稳定表达端粒酶活性。转染后传 75代的细胞经bFGF诱导仍维持着自我更新及向神经细胞分化的潜能 ,且无恶性转化倾向。结论 :重建端粒酶活性可延长胎儿肌肉源间充质干细胞寿命并维持自我更新及成神经潜能 ,为建立组织工程标准细胞系提供了新的实验手段     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

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 Cre-expressing 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.