title: Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.
abstract: Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Deltapsi). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Deltapsi and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Deltapsi suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Deltapsi suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.
authors.affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9.
authors.forename: G
authors.initials: G
authors.lastname: MacDonald
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: C
authors.initials: C
authors.lastname: Vande Velde
authors.forename: J
authors.initials: J
authors.lastname: Lieberman
authors.forename: A H
authors.initials: AH
authors.lastname: Greenberg
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D053818:Chymases; D003574:Cytochrome c Group / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D019254:Genes, bcl-2 / Q000235:genetics; D053804:Granzymes; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D053802:Tryptases
chemical_list: D003574:Cytochrome c Group; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D017382:Reactive Oxygen Species; D015842:Serine Proteinase Inhibitors; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; C509215:Gzmb protein, rat; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509223:Tpsab1 protein, rat; C509221:Tpsb2 protein, rat; D053802:Tryptases; D020169:Caspases