PROCESSING ACTIVATION OF AT LEAST 4 INTERLEUKIN-1-BETA CONVERTING ENZYME-LIKE PROTEASES OCCURS DURING THE EXECUTION PHASE OF APOPTOSIS IN HUMAN MONOCYTIC TUMOR-CELLS/

Identification of the processing/activation of multiple interleukin-1 beta converting enzyme (ICE)-like proteases and their target substrate s in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at lea st four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by proces sing of Ich-l, CPP32, and Mch3 alpha to their catalytically active sub units, and lysates from these cells displayed a proteolytic activity w ith kinetics, characteristic of CPP32/Mch3 alpha but not of ICE. Fluor escence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-l, CPP32, and Mch3 alpha was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP) , the 70-kD protein component of U1 small nuclear ribonucleoprotein (U 1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3 alpha or the substrates was observed in normal eels. In cells exposed to an apoptotic stimulus, some processing of Ich-l was detected in morpholog ically normal cells, suggesting that cleavage of Ich-l may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxyc arbonyl-val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited a poptosis and cleavage of Ich-l, CPP32, Mch3 alpha, Mch2 alpha, PARP, U 1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apopt osis by inhibiting a key effector protease upstream of Ich-l, CPP32, M ch3 alpha, and Mch2 alpha. Together these observations demonstrate tha t processing/activation of Ich-l, CPP32, Mch3 alpha, and Mch2 alpha ac companies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like prote ases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.