CLEAVAGE OF CAD INHIBITOR IN CAD ACTIVATION AND DNA-DEGRADATION DURING APOPTOSIS

Various molecules such as cytokines and anticancer drugs, as well as f actor deprivation, rapidly induce apoptosis (programmed cell death)(1, 2), which is morphologically characterized by cell shrinkage and the b lebbing of plasma membranes and by nuclear condensation(3,4). Caspases , particularly caspase 3, are proteases that are activated during apop tosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin(5,6). Apoptosis is also accompanied by the i nternucleosomal degradation of chromosomal DNA(7-9). In the accompanyi ng Article(10), we have identified and molecularly cloned a caspase-ac tivated deoxyribonuclease (CAD) and its inhibitor (ICAD). Here we show that caspase 3 cleaves ICAD and inactivates its CAD-inhibitory effect , We identified two caspase-3 cleavage sites in ICAD by site-directed mutagenesis. When human Jurkat cells were transformed with ICAD-expres sing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation, The ICAD transformants w ere also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase. Our result s indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, an d that ICAD works as an inhibitor of this process.