ToxSci Advance Access originally published online on June 1, 2007
Toxicological Sciences 2007 99(1):70-78; doi:10.1093/toxsci/kfm142
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Arsenite Slows S Phase Progression via Inhibition of cdc25A Dual Specificity Phosphatase Gene Transcription
Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, 575 Elmwood Avenue, Rochester, New York 14642
1 To whom correspondence should be addressed. Fax: (585) 256-2591. E-mail: michael_mccabe{at}urmc.rochester.edu.
Received April 2, 2007; accepted May 28, 2007
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Abstract |
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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 5µM 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 5µM 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 5µM 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.
Key Words: arsenite; S phase inhibition; cell cycle; cdc25A; U937.