ACTIVATION OF MULTIPLE INTERLEUKIN-1-BETA CONVERTING-ENZYME HOMOLOGS IN CYTOSOL AND NUCLEI OF HL-60 CELLS DURING ETOPOSIDE-INDUCED APOPTOSIS

Recent genetic and biochemical studies have implicated cysteine-depend ent aspartate-directed proteases (caspases) in the active phase of apo ptosis. In the present study, three complementary techniques were util ized to follow caspase activation during the course of etoposide-induc ed apoptosis in HL-60 human leukemia cells. Immunoblotting revealed th at levels of procaspase-2 did not change during etoposide-induced apop tosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities t hat cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoum arin increased 100- and 20-fold, respectively; but there was only a 1. 5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity . Affinity labeling with enzyloxycarbonylglutamyl-N-epsilon-biotinylly syl)- aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone indicated that multiple active caspase species sequentially appeared in the cyt osol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated wi th caspase-6 and three comigrated with active caspase-3 species, sugge sting that several splice or modification variants of these enzymes ar e active during apoptosis. Polypeptides that comigrate with the cytoso lic caspases were also labeled in nuclei of apoptotic HL-60 cells. The se 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.