title: Pro- and anti-apoptotic effects of an inhibitor of chymotrypsin-like serine proteases.
abstract: The irreversible inhibitor of chymotrypsin-like serine proteases, N-tosyl-L-phenylalanine chloromethylketone (TPCK), was shown to prevent internucleosomal DNA cleavage caused by inducers of apoptosis. The pro-apoptotic properties of TPCK have been studied less thoroughly. The aim of the present study was to investigate the pro- and anti-apoptotic activities of TPCK on HL-60 cells and compare them with the actions of the mitochondrial electron transport inhibitor antimycin A (AMA). The results showed that TPCK alone caused activation of cell cycle checkpoints, mitochondrial cytochrome c release, caspase-3 activation, and chromatin condensation. Caspase-8 was not required for cytochrome c release but was crucial to caspase-3 activation. TPCK synergistically enhanced AMA-induced cytochrome c release and caspase-3 activation while completely blocking AMA-induced internucleosomal DNA fragmentation for at least 8 hours. Rather than blocking AMA-induced DNA fragmentation, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) actually enhanced it. The pro-apoptotic effect of TPCK may be due to activation of cell cycle checkpoints via inhibition of the proteasome. The apoptotic pathways activated by TPCK and AMA probably converge at the level of the mitochondria. The mode by which TPCK prevents internucleosomal DNA fragmentation is probably not through serine protease inhibition.
authors.affiliation: Department of Clinical Immunology, Pacific Medicine Laboratory Services, Royal North Shore Hospital, St. Leonards, Australia. making@doh.health.nsw.gov.au
authors.forename: Malcolm A
authors.initials: MA
authors.lastname: King
authors.forename: H Dorota
authors.initials: HD
authors.lastname: Halicka
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053181:Caspase 8; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus; D002918:Chymotrypsin / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013450:Sulfones / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology*
chemical_list: D061945:Caspase Inhibitors; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D014108:Tosylphenylalanyl Chloromethyl Ketone; D045304:Cytochromes c; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases