Involvement of different proteases in the execution and regulation of apoptosis in human monocytic THP.1 cells

Ced-3, a cysteine protease, is a key effector of apoptotic cell death in Caenorhabditis elegans. However the role of the Ced-3 mammalian homologues, caspases, and other classes of proteases in apoptosis has not been well understood. This study investigated the involvement of proteases in apoptosis induced by different mechanisms using human monocytic THP.1 cells as a model.;Apoptosis, as assessed by morphological and biochemical changes, including nuclear condensation and fragmentation, internucleosomal DNA cleavage and poly-(ADP-ribose) polymerase (PARP) degradation, was induced by cycloheximide (25 M), thapsigargin (100 nM), etoposide (25 M) and staurosporine (0.5 M). The induction of apoptosis by all these stimuli was enhanced by N-tosyl-L-lysinyl chloromethyl ketone (TLCK) (100 M), a trypsin-like protease inhibitor, except for etoposide, where apoptosis was inhibited. Staurosporine also induced necrotic cell death, which was prevented by TLCK.;N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) (50-75 M), a chymotrypsin-like protease inhibitor, induced all the characteristic apoptotic changes except the fully nuclear condensation and fragmentation, although it inhibited internucleosomal DNA cleavage induced by other stimuli.;Caspase-2, caspase-3, caspase-6, and caspase-7 were processed/activated during the induction of apoptosis. Caspase inhibitors either with low sensitivity or with higher selectivity for caspase-3 both proved to be potent in the inhibition of apoptosis. However staurosporine-induced necrosis was resistant to the inhibition of a caspase inhibitor.;This study demonstrated that apoptosis can be induced or modified by different protease inhibitors in a single cell line, implying tat proteases are involved in the regulation of apoptosis at multiple stages. TLCK and TPCK inhibitable proteases may control "upstream" events, whereas caspases have a fundamental role in the execution of apoptosis. This study also provides evidence that a apoptosis and necrosis involve different protease activities.