title: Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases.
abstract: The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl) -fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly(ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.
authors.affiliation: Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4955, USA.
authors.forename: T
authors.initials: T
authors.lastname: Shimizu
authors.forename: Y
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases; D002467:Cell Nucleus / Q000187:drug effects; D002474:Cell-Free System; D003374:Coumarins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation; D017353:Gene Deletion; D016158:Genes, p53; D018922:HL-60 Cells / Q000502:physiology*; D006801:Humans; D049934:Isocoumarins; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D011355:Prodrugs; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D049934:Isocoumarins; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D011355:Prodrugs; D015842:Serine Proteinase Inhibitors; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011064:Poly Adenosine Diphosphate Ribose; C046235:3,4-dichloroisocoumarin; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D002166:Camptothecin