title: Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity.
abstract: Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.
authors.affiliation: Cancer Cell Death Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia.
authors.forename: K A
authors.initials: KA
authors.lastname: Sedelies
authors.forename: A
authors.initials: A
authors.lastname: Ciccone
authors.forename: C J P
authors.initials: CJ
authors.lastname: Clarke
authors.forename: J
authors.initials: J
authors.lastname: Oliaro
authors.forename: V R
authors.initials: VR
authors.lastname: Sutton
authors.forename: F L
authors.initials: FL
authors.lastname: Scott
authors.forename: J
authors.initials: J
authors.lastname: Silke
authors.forename: O
authors.initials: O
authors.lastname: Susanto
authors.forename: D R
authors.initials: DR
authors.lastname: Green
authors.forename: R W
authors.initials: RW
authors.lastname: Johnstone
authors.forename: P I
authors.initials: PI
authors.lastname: Bird
authors.forename: J A
authors.initials: JA
authors.lastname: Trapani
authors.forename: N J
authors.initials: NJ
authors.lastname: Waterhouse
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D053804:Granzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007694:Killer Cells, Natural / Q000187:drug effects / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D010539:Permeability; D011480:Protease Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000187:drug effects / Q000201:enzymology*; D013997:Time Factors; D014162:Transfection; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C028342:compound 20; D053804:Granzymes; D020169:Caspases