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H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1
Karin Barnouin1, Marlène L Dubuisson, Emma S Child
1CRC Laboratories and the Section of Cancer Cell Biology, Imperial College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom.
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 H(2)O(2) and found that sublethal doses of H(2)O(2) induced a transient multi-phase cell cycle arrest at the G(1), S, and G(2) 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 p21(Cip1) expression. We also demonstrate that the induction in p21(Cip1) expression by H(2)O(2) 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 H(2)O(2) treatment, the down-regulation of cyclin Ds expression are associated with repression of cyclin D-CDK4, whereas the accumulation of p21(Cip1) 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 G(1), S, and G(2) phases following H(2)O(2) stimulation. Deletion of p21(Cip1), 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 H(2)O(2). By contrast, overexpression of the human Herpesvirus 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 H(2)O(2) induce a transient multi-phase cell cycle arrest at least partially through up-regulation of p21(Cip1) and down-regulation of cyclin D expression.
Insights
Sublethal hydrogen peroxide (H2O2) induces a transient, multi-phase cell cycle arrest in fibroblasts. This arrest is linked to altered cyclin and cyclin-dependent kinase inhibitor (CKI) expression, specifically p21(Cip1).
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Proliferating cells utilize cell cycle arrest to mitigate damage from reactive oxygen species.
- Hydrogen peroxide (H2O2) is a key reactive oxygen species implicated in cellular stress responses.
Purpose of the Study:
- To investigate the molecular mechanisms underlying H2O2-induced transient cell cycle arrest in mouse fibroblasts.
- To elucidate the roles of specific cell cycle regulatory proteins, including cyclins and cyclin-dependent kinase inhibitors (CKIs), in this response.
Main Methods:
- Treatment of mouse fibroblasts with sublethal doses of H2O2.
- Cell cycle analysis using flow cytometry.
- Western blot analysis to assess protein expression levels (cyclins, CKIs).
- Immunoprecipitation kinase assays and immunodepletion assays to determine protein interactions and functions.
Main Results:
- Sublethal H2O2 induced a transient cell cycle arrest at G1, S, and G2 phases, but not M phase.
- This arrest correlated with decreased cyclin D1/D3 and increased p21(Cip1) expression, partly via transcriptional regulation independent of p53.
- H2O2 treatment led to cyclin D-CDK4 repression and p21(Cip1)-mediated inhibition of cyclin E/A-CDK2 and cyclin B-CDC2 activities.
Conclusions:
- Moderate H2O2 levels trigger a multi-phase cell cycle arrest primarily through p21(Cip1) upregulation and cyclin D downregulation.
- Overcoming this arrest requires more than single-gene interventions; viral cyclins that mimic cyclin D/E functions can override it.
- The findings propose a model for H2O2-mediated cell cycle regulation involving key cell cycle machinery.