H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression.
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.
Barnouin
Karin
K
CRC Laboratories and the Section of Cancer Cell Biology, Imperial College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom.
Dubuisson
Marlène L
ML
Child
Emma S
ES
Fernandez de Mattos
Silvia
S
Glassford
Janet
J
Medema
René H
RH
Mann
David J
DJ
Lam
Eric W-F
EW
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D056741:Cyclin D; D019927:Cyclin E / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D011233:Precipitin Tests; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D015854:Up-Regulation
C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D056741:Cyclin D; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D007544:Isopropyl Thiogalactoside; D006861:Hydrogen Peroxide; D010100:Oxygen