A p21(waf1)-independent pathway for inhibitory phosphorylation of cyclin-dependent kinase p34(cdc2) and concomitant G(2)/M arrest by the chemopreventive flavonoid apigenin.

Apigenin, a nonmutagenic flavonoid, has been shown to inhibit ultraviolet light-induced skin tumorigenesis when topically applied to mouse skin. Our previous studies have shown that apigenin treatment of cultured mouse keratinocytes induces G(2)/M arrest accompanied by an increase in p53 protein stability and expression of p21(waf1). In this study, we determined whether the G(2)/M arrest induced by apigenin was dependent upon the presence of the cyclin dependent kinase inhibitor p21(waf1). We exposed WWT.8 (p21(waf1) wild-type) and WKO.16 (p21(waf1) null) mouse keratinocytes to various doses of apigenin for 24 h and observed G(2)/M arrest in both cell lines, thereby establishing that the apigenin-induced G(2)/M arrest was p21(waf1) independent. A 4-h treatment with apigenin induced increases in p53 protein level by sixfold and tenfold in the WWT.8 p21(waf1) wild-type cells and WKO.16 p21(waf1) null cells, respectively. After 24 h in WWT.8 cells, p21(waf1) protein also was induced in a dose-dependent manner, but it was not expressed in WKO.16 keratinocytes. We then measured the effect of apigenin treatment on the mammalian homologue of the yeast cdc2 gene (p34(cdc2)) cyclin-dependent kinase and cyclin B1 (cycB1), because these proteins complex to regulate G(2)/M progression. Apigenin treatment decreased the protein level of p34(cdc2), and p34(cdc2) kinase activity was inhibited in both p21(waf1)(+/+) and p21(waf1)(-/-) cell lines by approximately 40%. The inhibition of p34(cdc2) kinase activity by apigenin treatment correlated with increasing levels of p34(cdc2) phosphorylation at Tyr15, a site in the p34(cdc2) kinase that undergoes inhibitory phosphorylation by Wee1 kinase. Apigenin treatment also had no effect on the protein level or activity of the competing phosphatase, cdc25c, which dephosphorylates p34(cdc2) kinase at Tyr15. Apigenin had little effect on the accumulation of cycB1 protein. These results supported the conclusion that G(2)/M arrest induced by apigenin was accompanied by inhibition of the p34(cdc2) cyclin-dependent kinase protein level and activity in a p21(waf1)-independent manner.