title: Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis.
abstract: A detailed kinetic analysis of three extranuclear end points of apoptosis, phosphatidylserine exposure, alpha-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentation and the activation of the interleukin-1beta-converting enzyme (ICE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas monoclonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine exposure was quantitated by plasma clotting time, as well as annexin V-fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited ICE-like activity in both cell types studied, whereas the calpain inhibitor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurkat cells stimulated by anti-Fas monoclonal antibody, indicating that ICE-like proteases play an important role in the regulation of this apoptotic system. Calpain inhibitors were ineffective in this system. TNF-induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Jurkat cells. This suggests that ICE-like enzymes predominate in anti-Fas monoclonal antibody-stimulated Jurkat cells, whereas proteases affected by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells. Importantly, the two apoptotic systems seem to be regulated by different proteases.
authors.affiliation: Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden.
authors.forename: D M
authors.initials: DM
authors.lastname: Vanags
authors.forename: M I
authors.initials: MI
authors.lastname: Pörn-Ares
authors.forename: S
authors.initials: S
authors.lastname: Coppola
authors.forename: D H
authors.initials: DH
authors.lastname: Burgess
authors.forename: S
authors.initials: S
authors.lastname: Orrenius
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism*; D020170:Caspase 1; D003374:Coumarins / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D006801:Humans; D019169:Jurkat Cells; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019014:fas Receptor / Q000276:immunology; D013049:Spectrin
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D020170:Caspase 1; D003374:Coumarins; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D010718:Phosphatidylserines; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D013049:Spectrin; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin