H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

Abstract To defend against the potential damages induced by reactive oxygen species, proliferating cells enter a transient cell cycle arrest. We treated mouse fibroblasts with H2O2 and found that sublethal doses of H2O2 induced a transient multi-phase cell cycle arrest at the G1, S, and G2 phases but not the M phase. Western blot analysis demonstrated that this transient cell cycle arrest is associated with the down-regulation of cyclins D1 and D3 and up-regulation of the CKI p21Cip1 expression. We also demonstrate that the induction in p21Cip1 expression by H2O2 is at least partially mediated at the transcriptional level and can occur in the absence of p53 function. Further immunoprecipitation kinase and immunodepletion assays indicated that in response to H2O2 treatment, the down-regulation of cyclin Ds expression are associated with repression of cyclin D-CDK4, whereas the accumulation of p21Cip1 is responsible for the inhibition of cyclin E and A-CDK2 activity and associated with the down-regulation of cyclin B-CDC2 activity. These data could account for the cell cycle arrest at the G1, S, and G2 phases following H2O2stimulation. Deletion of p21Cip1, restoration of cyclin D expression, or overexpression of cyclin E alone is insufficient to effectively overcome the cell cycle arrest caused by sublethal doses of H2O2. By contrast, overexpression of the humanHerpesvirus 8 K cyclin, which can mimic the function of cyclin D and E, is enough to override this transient cell cycle arrest. On the basis of our findings, we propose a model in which moderate levels of H2O2 induce a transient multi-phase cell cycle arrest at least partially through up-regulation of p21Cip1 and down-regulation of cyclin D expression.