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© The Rockefeller University Press, 0021-9525/1997//469 $5.00
The Journal of Cell Biology, Volume 137, Number 2, , 1997 469-479

Article

Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells



Marion MacFarlane*, Kelvin Cain*, Xiao-Ming Sun*, Emad S. Alnemri{ddagger}, and Gerald M. Cohen*

* Medical Research Council Toxicology Unit, Centre for Mechanisms of Human Toxicity, University of Leicester, Leicester LE1 9HN, United Kingdom; and {ddagger} Centre for Apoptosis Research and Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107

Identification of the processing/activation of multiple interleukin-1β 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-1, 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-1, 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 (U170K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3{alpha} or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 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-1, 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-1, CPP32, Mch3{alpha}, and Mch2{alpha}. Together these observations demonstrate that processing/activation of Ich-1, 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.

Abbreviations used in this paper: ICE, interleukin-1β converting enzyme; PARP, poly(ADP-ribose) polymerase; TLCK, N-{alpha}-p-tosyl-L-lysine chloromethyl ketone; U1-70K, 70-kD protein component of U1 small nuclear ribonucleoprotein.

Please address all correspondence to Gerald M. Cohen, MRC Toxicology Unit, Centre for Mechanisms of Human Toxicity, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom. Tel.: (44) 116-252-5589/5600. Fax: (44) 116-252-5616. e-mail: gmc2{at}leicester.ac.uk


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