MILD HYPEROXIA SHORTENS TELOMERES AND INHIBITS PROLIFERATION OF FIBROBLASTS - A MODEL FOR SENESCENCE

Mild oxidative stress as exerted by culture of human WI-38 fibroblasts under 40% oxygen partial pressure blocks proliferation irreversibly a fter one to three population doublings. Hyperoxically blocked cells ar e similar to senescent ones in terms of general morphology and lipofus cin accumulation. Moreover, they, like senescent fibroblasts, are bloc ked preferentially in G1 as evident from DNA content measurements by h ow cytometry. Southern blotting of AluI- and HinfI-restricted genomic DNA shows an increase of the rate of telomere shortening from 90 bp pe r population doubling under normoxia to more than 500 bp per populatio n doubling under hyperoxia. In every case, proliferation is blocked if a telomere cutoff length of about 4 kb is arrived at. The fact that t elomere length correlates with the final inhibition of proliferation u nder conditions of varied oxidative stress, while the population doubl ing level does not, suggests that telomere shortening provides the sig nal for cell cycle exit in senescence. In postmitotic cells, no furthe r telomere shortening occurs. However, the sensitivity of terminal res triction fragments to S1 nuclease increases, indicating the accumulati on of single-strand breaks in telomeres of nondividing fibroblasts. Th is 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 importan t than incomplete replication per se. (C) 1995 Academic Press, Inc.