Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis

Abstract

Various molecules such as cytokines and anticancer drugs, as well as factor deprivation, rapidly induce apoptosis (programmed cell death), which is morphologically characterized by cell shrinkage and the blebbing of plasma membranes and by nuclear condensation. Caspases, particularly caspase 3, are proteases that are activated during apoptosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin. Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA. In the accompanying Article, we have identified and molecularly cloned a caspase-activated 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-expressing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation. The ICAD transformants were also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase. Our results indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, and that ICAD works as an inhibitor of this process.

Perforin‐dependent activation‐induced cell death acts through caspase 3 but not through caspases 8 or 9

“…Caspase 3 -/-cells increased from only 5% to 6% hypodiploid with recrosslinking (p X 0.001). Given that hypodiploid counts are dependent upon DNA fragmentation and subsequent loss of fragments, the low hypodiploid counts for caspase 3-deficient cells are consistent with reports that caspase 3 is responsible for the release of a caspase-activated DNase required for DNA fragmentation [26,27].…”

“…Other apoptotic events act through compromise of mitochondrial activity and lead to activation of caspase 9 (reviewed in [21,22]). Both receptor-mediated and mitochondrial pathways converge upon caspase 3 [23,24], which causes DNA fragmentation through degradation of a DNase inhibitor (ICAD) and release of a caspase-activated DNase [25][26][27].…”

Dexamethasone protects primary cultured hepatocytes from death receptor-mediated apoptosis by upregulation of cFLIP

“…Activated caspase-9 subsequently activates the executioner proteases including caspase-3, which in turn liberates a DNase termed CAD (caspase-activated DNase) from an inhibitor of CAD (ICAD/ DFF-45) by cleaving the ICAD protein. 4 This process leads to DNA degradation, a hallmark event in apoptosis.…”

DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells

“…DNA fragmentation is one of characteristic features of apoptosis (Raff, 1992) and triggered by a heterodimeric DNA fragmentation factor (DFF), which is composed of DFF40/CAD/CPAN nuclease and its inhibitor DFF45/ICAD. Upon apoptotic stimuli, DFF45 is cleaved by caspase-3 into three fragments and dissociates from DFF40, resulting in the activation of DFF40 (Liu et al, 1997(Liu et al, , 1998Enari et al, 1998;Halenbeck et al, 1998;Mukae et al, 1998;Sakahira et al, 1998). DFF45 also acts as a folding chaperone required to produce active DFF40 Sakahira et al, 1998).…”

“…Upon apoptotic stimuli, DFF45 is cleaved by caspase-3 into three fragments and dissociates from DFF40, resulting in the activation of DFF40 (Liu et al, 1997(Liu et al, , 1998Enari et al, 1998;Halenbeck et al, 1998;Mukae et al, 1998;Sakahira et al, 1998). DFF45 also acts as a folding chaperone required to produce active DFF40 Sakahira et al, 1998). In this study, we examined a role(s) of DFF45 in the regulation of NBL cell death.…”