Proteasome-Dependent Downregulation of p21 Waf1/Cip1 Induced by Reactive Oxygen Species

Abstract

After hydrogen peroxide (H₂O₂) treatment, the p21 (p21^Waf1/Cip1) protein level in GM00637 fibroblast cells was rapidly decreased, reaching its nadir around 3 h. However, it rebounded within 5 hours to a level higher than that before treatment. Fluorescence microscopic analyses revealed that nuclear p21 was downregulated during the initial oxidative stress. H₂O₂-induced downregulation of p21 protein was accompanied by a gradual increase in p21 mRNA levels. Other inducers of genotoxic stress, such as treatment with adriamycin, a DNA damage compound, did not cause a significant decrease in p21 protein levels. Pretreatment of GM00637 cells with the proteasome inhibitors, lactacystin or MG132, completely blocked H₂O₂-induced p21 downregulation, suggesting that H₂O₂ treatment accelerated p21 degradation. Conversely, cotreatment of cells with a protein synthesis inhibitor, cycloheximide, and H₂O₂ drastically shortened the half-life of p21. Moreover, p21 mRNA levels were not downregulated by treatment with proteasome or protein synthesis inhibitors. Taken together, our studies indicate that oxidative stress induces rapid, but reversible, downregulation of functional p21 by accelerating its protein turnover.