Gnidimacrin (NSC252940) shows significant antiproliferating activity against human tumor cell lines. This compound binds to and directly activates protein kinase C (PKC). Human hepatoma HLE cells, which lose p53 function and retinoblastoma protein (Rb) expression, are resistant to gnidimacrin. However, PKC betaII gene-transfected HLE (HLE/PKC betaII) cells became sensitive to gnidimacrin, through which cdc2 inhibition and G(2)-phase arrest was caused. p21(WAF1/Cip1) induction and cdc2 reduction were observed and this reduction was abolished through the suppression of p21(WAF1/Cip1) induction by the MEK1/2 inhibitor U0126. Translocation of E2F-4 to the nucleus was also observed in the cells but not in parental HLE cells. Consequently gnidimacrin inhibited cell growth through G(2)-phase arrest not only by the p21(WAF1/Cip1)-dependent suppression of cdc2 activity, but also by subsequent transcriptional suppression of cdc2 itself. In addition, involvement of E2F-4 in cdc2 suppression through a long-lasting induction of p21(WAF1/Cip1) by gnidimacrin is suggested in HLE/PKC betaII cells.