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.
Rubio
Miguel A
MA
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Kim
Sahn-Ho
SH
Campisi
Judith
J
D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D016922:Cellular Senescence; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics; D011839:Radiation, Ionizing; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000096:biosynthesis*; D016615:Telomere / Q000502:physiology* / Q000648:ultrastructure; D013997:Time Factors; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D019098:Telomerase