Induction of p21CIP1/Waf1and Activation of p34cdc2Involved in Retinoic Acid-Induced Apoptosis in Human Hepatoma Hep3B Cells

Abstract

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21CIPI/Waf1and Bax proteins, as well as activation of p34cdc2kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27Kip1expression. Olomoucine, a potent p34cdc2and Cdk2 inhibitor, effectively blocked RA-mediated p34cdc2kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21CIP2/Waf1and p34cdc2mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21CIP2/Waf1overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34cdc2kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21CIP2/Waf1and p34cdc2is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.