Caspase-3 activity and carbonyl cyanide m-chlorophenylhydrazone-induced apoptosis in HL-60 cells

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone ( CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Tr eatment of HL-60 cells with micromolar concentrations of CCCP resulted in c ell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a disti nct increase in caspase-3 activity. The results, however, indicated that fu ll caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp- Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, no r did it affect the manifestation of apoptotic hallmarks, including apoptot ic bodies formation and nucleosomal DNA fragmentation. The only distinct ef fect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma m embrane. We therefore assume that caspase-3 activity itself is not essentia l for the manifestation of apoptotic features mentioned above. Similarly, t he pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromet hyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-F MK completely prevented DNA cleavage and apoptotic body formation, but it f ailed to completely counteract chromatin condensation. Thus, in the presenc e of Z-VAD-FMK, application of CCCP concentrations that otherwise induced a poptosis, resulted in the appearance of two morphologically different group s of dead cells with intact DNA. The first group included cells with necrot ic-like nuclear morphology, and therefore could be taken as being "truly" n ecrotic in nature, because they had intact DNA. The cells of the second gro up formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as "truly" necrotic cells. It is evident that in the experimental system, caspase proteases play an esse ntial role in the formation of apoptotic bodies and in the cleavage of nucl eosomal DNA, but not in the condensation of chromatin. Therefore, it is lik ely that the choice between cell death modalities is not solely a matter of the caspase proteases present.