Mild Hyperoxia Shortens Telomeres and Inhibits Proliferation of Fibroblasts: A Model for Senescence?

Abstract

Mild oxidative stress as exerted by culture of human WI-38 fibroblasts under 40% oxygen partial pressure blocks proliferation irreversibly after one to three population doublings. Hyperoxically blocked cells are similar to senescent ones in terms of general morphology and lipofuscin accumulation. Moreover, they, like senescent fibroblasts, are blocked preferentially in G1 as evident from DNA content measurements by flow cytometry. Southern blotting of AluI- and HinfI-restricted genomic DNA shows an increase of the rate of telomere shortening from 90 bp per population doubling under normoxia to more than 500 bp per population doubling under hyperoxia. In every case, proliferation is blocked if a telomere cutoff length of about 4 kb is arrived at. The fact that telomere length correlates with the final inhibition of proliferation under conditions of varied oxidative stress, while the population doubling level does not, suggests that telomere shortening provides the signal for cell cycle exit in senescence. In postmitotic cells, no further telomere shortening occurs. However, the sensitivity of terminal restriction fragments to S1 nuclease increases, indicating the accumulation of single-strand breaks in telomeres of nondividing fibroblasts. This effect is found both under normoxic and hyperoxic culture, although it is more pronounced under conditions of higher oxidative stress. It might be speculated that accumulation of single-strand breaks and the resultant loss of distal single-stranded fragments during replication could be a major cause of telomere shortening, possibly more important than incomplete replication per se.