APOPTOSIS IN HUMAN MONOCYTIC THP.1 CELLS INVOLVES SEVERAL DISTINCT TARGETS OF N-TOSYL-L-PHENYLALANYL CHLOROMETHYL KETONE (TPCK)

N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK), a chymotrypsin-like serine protease inhibitor, affected apoptosis in human monocytic THP. 1 cells differently dependent on both the concentration used and the a poptotic stimulus. TPCK (50-75 mu M) induced both biochemical and ultr astructural changes characteristic of apoptosis, including proteolysis of poly (ADP ribose) polymerase (PARP) and lamins together with forma tion of large kilobase pair fragments of DNA, particularly of 30-50 an d 200-300 kilobase pairs in length but without internucleosomal cleava ge of DNA. The induction of apoptosis by TPCK also involved the proces sing of CPP32 and Mch 3 to their catalytically active subunits. Benzyl oxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), an ICE- like protease inhibitor, completely prevented all the biochemical and morphological changes induced by TPCK demonstrating the involvement of ICE-like proteases in the execution phase of apoptosis. Lower concent rations of TPCK (5-20 mu M) prevented internucleosomal cleavage of DNA induced by other apoptotic stimuli. TPCK (10 mu M) inhibited cell dea th induced by etoposide but potentiated that induced by cycloheximide demonstrating that it differentially affected apoptosis in THP.1 cells dependent on the stimulus used. These results are consistent with at least three distinct TPCK targets, one being important for cell surviv al, the second in facilitating internucleosomal cleavage of DNA and th e third in the modulation of apoptosis induced by different apoptotic stimuli.