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

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 μM 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 μM 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.