Caspase 3-mediated Cleavage of p21 WAF1/CIP1 Associated with the Cyclin A-Cyclin-dependent Kinase 2 Complex Is a Prerequisite for Apoptosis in SK-HEP-1 Cells*

Apoptosis of SK-HEP-1 human hepatoma cells induced by treatment with ginsenoside Rh2 (G-Rh2) is associated with rapid and selective activation of cyclin A-associated cyclin-dependent kinase 2 (Cdk2). Here, we show that in apoptotic cells, the Cdk inhibitory protein p21 WAF1/CIP1 , which is associated with the cyclin A-Cdk2 complex, undergoes selective proteolytic cleavage. In contrast, another Cdk inhibitory protein, p27 KIP1 , which is associated with cyclin A-Cdk2 and cyclin E-Cdk2 complexes, remained unaltered during apoptosis. Ectopic overexpression of p21 WAF1/CIP1 suppressed apoptosis as well as cyclin A-Cdk2 activity induced by treatment of SK-HEP-1 cells with G-Rh2. The suppressive effects of p21 WAF1/CIP1 were much higher in the cells transfected with p21D112N, an expression vector that encodes a p21 WAF1/CIP1 mutant resistant to caspase 3 cleavage. Overexpression of cyclin A in SK-HEP-1 cells dramatically up-regulated cyclin A-Cdk2 activity and accordingly enhances apoptosis induced by treatment with G-Rh2. These up-regulating effects were blocked by coexpression of a dominant negative allele of cdk2. Furthermore, olomoucine, a specific inhibitor of Cdks, also blocked G-Rh2-induced apoptosis. These data suggest that the induction of apoptosis in human hepatoma cells treated with G-Rh2 occurs by a mechanism that involves the activation of cyclin A-Cdk2 by caspase 3-mediated cleavage of p21 WAF1/CIP1 .