CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis.

DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic chromatin condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of chromatin condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic chromatin condensation.

Activation of multiple interleukin-1beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis.

Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1. 5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.