Down-regulation and decreased activity of cyclin-dependent kinase 2 in H2O2-induced premature senescence.
Premature senescence of human diploid fibroblasts (HDFs) induced by exposure to H2O2 at subcytotoxic concentration is characterized by many biomarkers of normal senescence such as irreversible growth arrest. Cyclin-dependent kinase inhibitor (CdKI) p21(Waf-1) is overexpressed in H2O2- and tert-butylhydroperoxide-induced premature senescence, likely explaining in part the hypophosphorylation of the retinoblastoma protein. p21(Waf-1) is known to inhibit the kinase activity of the cyclin-dependent kinase (CdK) 4 and 6 cyclin complexes. In this work, we investigated whether the kinase activity of the CdK4 and 6 cyclin complexes can be modulated by CdKI p16(Ink-4a), by changes in the protein level of CdKs and cyclins, or by changes in kinase activity of these CdKs not directly involving CdKIs. RNase protection assay, semi-quantitative RT-PCR, Western blot and kinase assay showed that the mRNA level, protein and kinase activity of CdK2 are decreased at 72h after H2O2 stress. These results suggest that the hypophosphorylation of the retinoblastoma protein is mediated in part by a decrease of the kinase activity of CdK2 not directly involving CdKIs. This CdK2-mediated effect should be considered in addition to the inhibition of cyclin D-CdK4 and 6 complexes by CdKI p21(Waf-1).
Frippiat
Christophe
C
Research Unit on Cellular Biology, University of Namur (FUNDP), 61 Rue de Bruxelles, B-5000, Namur, Belgium.
Remacle
José
J
Toussaint
Olivier
O
D042846:CDC2-CDC28 Kinases; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology*; D051357:Cyclin-Dependent Kinase 2; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000187:drug effects / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism
C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16; D006861:Hydrogen Peroxide; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases