Abstract
Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging. To investigate the role of cellular senescence in aging it is necessary to define the time-dependent molecular events by which it is characterized. Here we investigated changes in levels of key proteins involved in cell cycle regulation, DNA replication, and stress resistance in senescing human fibroblasts following oxidative stress. An immediate response in stressed cells was dephosphorylation of retinoblastoma (Rb) and cessation of DNA synthesis. This was followed by sequential induction of p53, p21, and p16. Increase in hypophosphorylated Rb and induction of p53 and p21 by a single stress treatment was transient, whereas sustained induction or dephosphorylation were achieved by a second stress. Down-regulation of the critical DNA replication initiation factor Cdc6 occurred early after stress concurring with p53 induction, and was followed by a decrease in Mcm2 levels. A late event in the stress-induced molecular sequence was the induction of SOD1, catalase, and HSP27 coinciding with development of the fully senescent phenotype. Our data suggest that loss of proliferative capacity in oxidatively stressed cells is a multistep process regulated by time-dependent molecular events that may play differential roles in induction and maintenance of cellular senescence.
Highlights
Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging
Cessation of DNA Replication and Induction of Senescence in the H2O2-treated Cells—To understand molecular mechanisms underlying aging, we investigated key cell cycle regulatory proteins and stress-resistant factors in normal human diploid fibroblasts that were subjected to oxidative stress
Molecular Events in Replicatively Senescing Cells—To confirm that molecular events induced by oxidative stress are similar to those in replicatively senescing cells, we examined changes in p53, p21, p16, and Mcm2 in cells when they were in early population doubling (PD30), in a near senescent state (PD57), and in the fully established replicative senescent state (4 weeks into replicative senescence)
Summary
The inset is a representative BrdUrd incorporation assay of control cells. Data represent the mean Ϯ S.E. of three independent experiments. The inset is a representative SA--gal assay of H2O2-induced senescent cells. The mean Ϯ S.E. of three independent experiments are shown. Senescence in human fibroblasts by well defined sequential molecular events
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