Activation of Caspases in p53‐induced Transactivation‐independent Apoptosis

Though p53‐induced apoptosis plays an important role in tumor suppression, the mechanism(s) by which p53 induces apoptosis is stillunclear. To elucidate the p53‐induced apoptotic pathway, we examined the role of p53 transactivation activity and caspase in J138V5C cells carrying a human temperature‐sensitive (ts) p53 mutant (138Ala→Val). The results showed that p53‐induced apoptosis was not blocked by cycloheximide, which effectively prevented the expression of p53 target genes, indicating that transactivation was not essential for p53‐induced apoptosis in this system. Western blotanalysis showed that PARP, CPP32 and ICH‐1 precursors were cleaved during apoptosis. The CPP32‐preferential tetrapeptide inhibitor Ac‐DEVD‐CHO blocked the cleavage of ICH‐1 and PARP precursors, suggesting that CPP32 or some other DEVD‐sensitive caspase(s) is the upstream activator of ICH‐1. We also examined the role of the Fas pathway by using Fas and Fas ligand‐neutralizing antibodies. Both antibodies failed to block p53‐induced apoptosis, suggesting that the Fas pathway was not essential for p53‐induced apoptosis in this system. Taken together, our results indicate that p53‐induced, transactivation‐independent apoptosis in Jurkat cells involves sequential activation of CPP32 or some other DEVD‐sensitive caspase(s) and ICH‐1, via a Fas‐independent pathway.