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Arsenite slows S phase progression via inhibition of cdc25A dual specificity phosphatase gene transcription
Geniece M Lehmann1, Michael J McCabe
1Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, 575 Elmwood Avenue, Rochester, New York 14642, USA.
Abstract:
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.
Insights
Sodium arsenite inhibits cell cycle progression by reducing cdc25A protein levels. This occurs through decreased gene transcription, not protein degradation or altered mRNA stability, impacting S phase entry.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Cancer Research
Background:
- Arsenic compounds exhibit dual roles as toxicants and chemotherapeutics, with unclear mechanisms.
- Sodium arsenite at 5 microM inhibits cell cycle progression, including S phase, in U937 cells.
- Cdc25A phosphatase is crucial for S phase entry and progression by regulating cyclin E-cdk2 activity.
Purpose of the Study:
- To investigate the mechanism by which sodium arsenite inhibits cell cycle progression.
- To determine the effect of arsenite on cdc25A protein levels and activity.
- To elucidate the impact of arsenite on cdc25A gene expression and mRNA stability.
Main Methods:
- Immunoblotting to assess cdc25A protein levels.
- Coimmunoprecipitation and kinase activity assays to evaluate cyclin E-cdk2 phosphorylation and activity.
- Cycloheximide and actinomycin D half-life assays to study protein and mRNA stability.
- Real-time RT-PCR to quantify cdc25A mRNA levels.
- Sucrose density gradient polysomal analysis to assess mRNA translation.
Main Results:
- Arsenite treatment significantly decreased cdc25A protein levels in U937 cells.
- Arsenite treatment led to increased phosphorylation of cyclin E-cdk2 at inhibitory sites (Thr14 and Tyr15) and reduced kinase activity.
- Contrary to known checkpoints, arsenite stabilized cdc25A protein.
- Real-time RT-PCR showed a substantial decrease in cdc25A mRNA levels, while mRNA stability remained unaffected.
- Reduced mRNA translation was ruled out as the cause for decreased cdc25A protein.
Conclusions:
- Arsenite-induced depletion of cdc25A protein likely contributes to S phase inhibition.
- The reduction in cdc25A protein is primarily due to decreased gene transcription, not enhanced proteasomal degradation or altered mRNA stability.
- Further studies are needed to fully define the transcriptional regulation mechanism of cdc25A by arsenite.
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