Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells

The biological activity of retinoic acid (RA) was examined in human hepatom a Hep3B cells, Under serum-deprived conditions, RA induced S/M-phase elevat ion and mitotic index increase within 24 h, followed by apoptosis, This RA- induced apoptosis was accompanied by p53-independent up-regulation of endog enous p21(CIPI/Waf1) proteins, as well as activation of p34(cdc2) kinase, a nd increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-X-L; Bcl-X-S; cyclins A, B, D1, D3, o r E; or Rbl expression but markedly downmodulated Cdk2 kinase activity and reduced Cdk4 expression, RA also slightly delayed p27(Kip1) expression. Olo moucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-medi ated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthe rmore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cd c2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necess ary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment le ads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincid ent with an aberrant mitotic progression, followed by appearance of abnorma l nucleus. This aberrant cell cycle progression appeared requisite for RA-i nduced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with indu ction of Bar and involved in cell death with apoptosis when Hep3B cells are exposed to RA. (C) 1999 Academic Press.