Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells

Abstract

Human diploid fibroblast cells lose replicative potential after a certain number of population doublings. We use this experimental system to investigate the role of oxidative damage in cellular aging. Treating cells with H2O2 at < 300 microM did not affect the viability of the majority of cells when judged by morphology, trypan blue exclusion, and protein synthesis. However, the treatment caused a dose-dependent inhibition of DNA synthesis. After a 2-hr treatment with 200 microM H2O2, the cells failed to respond to a stimulus of serum, platelet-derived growth factor, basic fibroblast growth factor, or epidermal growth factor by synthesizing DNA, and the loss of response could not be recovered by 4 days. Subcultivation showed that, as in senescent cells, division of the treated cells was inhibited. The life-time cumulative growth curve showed that the loss of replication due to H2O2 treatment was cumulative and irreversible. The H2O2 treatment decreased the number of the population doublings in the rest of the life span by 35.3 +/- 10.3%. Enzymatic assays indicated that, like the cells in their senescent state, the treated cells were less able to activate ornithine decarboxylase and thymidine kinase. Furthermore, subcultivation after the H2O2 treatment showed that the cells developed the morphology of senescent cells. In conclusion, sublethal treatment of H2O2 "stunned" F65 cells and caused the cells to enter a state resembling senescence.