Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells.
Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.
Bilodeau
J F
JF
Unit of Health and Environment, Laval University Medical Research Centre, CHUQ, Department of Medicine, Laval University, Ste-Foy, Québec, Canada.
Faure
R
R
Piedboeuf
B
B
Mirault
M E
ME
D015152:Blotting, Northern; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology*; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D016196:S Phase / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
C493956:CDKN1A protein, human; D019925:Cyclin A; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D014443:Tyrosine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D010100:Oxygen