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A G2/M growth arrest response to low-dose intermittent H2O2 in normal uroepithelial cells
M Chien1, C Rinker-Schaeffer, W M Stadler
1Sections of Urology and Hematology/Oncology, Departments of Surgery and Medicine, Prostate Cancer Program, Cancer Research Center, University of Chicago, IL 60637, USA.
Abstract:
Studies in fibroblasts have shown that H2O2, as a model for oxidative damage, leads to a G1 growth arrest phenotypically similar to senescence. These observations as well as the observation that bladder cancer is associated with deletions of CDKN2, a gene important in normal senescence, led us to examine normal urothelial cell response to H2O2. We hypothesized that low dose H2O2 exposure would lead to p16 and/or p14arf mediated senescence. We show that H2O2 leads to endogenous beta-galactosidase expression similar to senescence, but instead of G1 arrest, it leads to G2/M growth arrest without induction of either p16 or p14arf. Lack of p21 induction and a similar G2/M growth arrest in E6 immortalized uroepithelial cells suggests that this response is independent of p53 as well. An increased level of cdc2 tyrosine-15 phosphorylation following H2O2 treatment suggests that the observed growth arrest is mediated by a G2 checkpoint mechanism.
Insights
Hydrogen peroxide (H2O2) induces senescence-like G2/M arrest in urothelial cells, independent of p53, p16, or p14arf. This oxidative damage response involves a G2 checkpoint mechanism.
Area of Science:
- Cell Biology
- Molecular Biology
- Urothelial Carcinogenesis
Background:
- Oxidative damage, modeled by hydrogen peroxide (H2O2), induces G1 arrest in fibroblasts, mimicking senescence.
- Bladder cancer is linked to deletions in CDKN2, a gene crucial for normal senescence.
- The response of normal urothelial cells to H2O2 requires investigation.
Purpose of the Study:
- To investigate the hypothesis that low-dose H2O2 exposure induces p16 and/or p14arf-mediated senescence in normal urothelial cells.
- To characterize the cellular response and underlying molecular mechanisms of H2O2-induced growth arrest in urothelial cells.
Main Methods:
- Exposure of normal urothelial cells and E6 immortalized uroepithelial cells to H2O2.
- Assessment of endogenous beta-galactosidase expression as a senescence marker.
- Cell cycle analysis to determine growth arrest phase (G1 vs. G2/M).
- Western blot analysis to detect levels of p16, p14arf, p21, and phosphorylated cdc2 (tyrosine-15).
Main Results:
- H2O2 exposure induced endogenous beta-galactosidase expression, indicative of senescence.
- Instead of G1 arrest, H2O2 caused a G2/M growth arrest.
- Neither p16 nor p14arf were induced by H2O2 treatment.
- p21 induction was absent, and G2/M arrest persisted in E6 immortalized cells, suggesting p53 independence.
- Increased cdc2 tyrosine-15 phosphorylation was observed, implicating a G2 checkpoint mechanism.
Conclusions:
- H2O2 induces a senescence-like phenotype in urothelial cells, characterized by G2/M arrest and beta-galactosidase expression.
- This response is independent of p16, p14arf, and p53.
- The G2/M growth arrest is mediated by a G2 checkpoint mechanism, likely involving cdc2 tyrosine-15 phosphorylation.