Arecoline-induced phosphorylated p53 and p21WAF1protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells

Abstract

Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21^WAF1 protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21^WAF1 and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21^WAF1 protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21^WAF1 at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30–60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21^WAF1, but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21^WAF1, at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21^WAF1 gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21^WAF1 and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21^WAF1 protein expression is dependent on ATM and PI3K. Moreover, p21^WAF1 gene is transcriptionally induced by arecoline-activated ATM. J. Cell. Biochem. 107: 408–417, 2009.