Processing/activation of at least four 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 substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-l, CPP32, and Mch3 alpha to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3 alpha but not of ICE. Fluorescence-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 (U1-70K), 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 morphologically normal cells, suggesting that cleavage of Ich-l may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-l, CPP32, Mch3 alpha, Mch2 alpha, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-l, CPP32, Mch3 alpha, and Mch2 alpha. Together these observations demonstrate that processing/activation of Ich-l, CPP32, Mch3 alpha, and Mch2 alpha accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.