Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicat ive senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 mu M tert -butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells deve loped biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpressi on of p21(Waf-1/SDI-1/Cip1) , and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased af ter five stresses with 30 mu M t-BHP or a single stress under 450 mu M H2O2 . These corresponding genes include fibronectin, osteonectin, alpha 1(I)-pr ocollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at lat e CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxi dative stresses lead HDFs to a state close to replicative senescence. (C) 2 000 Elsevier Science Inc.