Arsenite slows S phase progression via inhibition of cdc25A dual specificity phosphatase gene transcription.
Arsenic acts as a toxicant, a carcinogen, and an effective chemotherapeutic agent, but its mechanisms of action are unclear. We have previously shown that treatment of U937 cells with 5 microM sodium arsenite inhibits cell cycle progression through each cell cycle phase, including S phase. Cdc25A dual specificity phosphatase controls entry into and progression through S phase by dephosphorylating sites of inhibitory phosphorylation on cyclin E-cdk2 (Thr14 and Tyr15). Immunoblotting reveals that a 3-h treatment of U937 cells with 5 microM sodium arsenite results in a dramatic decrease in cdc25A protein levels. Coimmunoprecipitation experiments confirm that cyclin E-cdk2 is more phosphorylated at Thr14 and Tyr15 in the presence of arsenite, and kinase activity assays reveal a decrease in cyclin E-associated cdk2 activity. Therefore, arsenite-dependent cdc25A depletion could contribute to S phase inhibition. There exists an S phase checkpoint known to be mediated by proteasomal cdc25A degradation. However, cycloheximide half-life assay reveals that cdc25A is actually stabilized in arsenite-treated cells. Real-time RT-PCR shows that cdc25A mRNA levels are substantially decreased with arsenite treatment, and actinomycin D half-life assay reveals no change in message stability. Decreased cdc25A message translation is shown by sucrose density gradient polysomal analysis to be an unlikely cause for the profound arsenite-dependent reduction in cdc25A protein levels. Studies are ongoing to establish the mechanism by which 5 microM arsenite decreases cdc25A message abundance, but we surmise that, given the lack of effect on mRNA stability, an inhibition of gene transcription is likely involved.
Lehmann
Geniece M
GM
Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, 575 Elmwood Avenue, Rochester, New York 14642, USA.
McCabe
Michael J
MJ
D018053:Arsenites / Q000633:toxicity*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004338:Drug Combinations; D004791:Enzyme Inhibitors / Q000633:toxicity*; D006801:Humans; D009000:Monocytes / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D016196:S Phase / Q000187:drug effects*; D017670:Sodium Compounds / Q000633:toxicity*; D013439:Sulfhydryl Reagents / Q000633:toxicity*; D014158:Transcription, Genetic / Q000187:drug effects*; D020298:U937 Cells; D020687:cdc25 Phosphatases / Q000235:genetics* / Q000378:metabolism
D018053:Arsenites; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D017670:Sodium Compounds; D013439:Sulfhydryl Reagents; D003609:Dactinomycin; C017947:sodium arsenite; D003513:Cycloheximide; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases