Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after gamma-irradiation by stabilization of the protein

Expression of the cyclin kinase inhibitor, p21, is regulated both transcrip tionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p2 1 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppresse d the ubiquitination of p21, In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cell s resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY 294002 pretreatment of cells. However, the levels of p21 mRNA were not affe cted by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21 . In addition, we examined the involvement of DNA-PK, whose activity is inh ibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibro blast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY394002, was evaluated. ATM deficient cells ind uced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclu de that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor AT M gene product for activity. (C) 2000 Elsevier Science B.V. All rights rese rved.