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.
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Abbreviations
- CL:
-
cytotoxic lymphocytes
- CTL:
-
cytotoxic T lymphocytes
- cyt c:
-
cytochrome c
- GraB:
-
granzyme B
- NK:
-
natural killer cells
- PI:
-
propidium iodide
- PS:
-
phosphatidylserine
- SMAC:
-
second mitochondrial activator of caspases
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Acknowledgements
We would like to thank Ilia Voskobonik (Peter MacCallum Cancer Centre) for helpful discussions, Herb Bull and Nancy Thornberry (MERCK research laboratories) for compound 20, Abbot Laboratories for ABT-737 and Sarah Ellis (Peter MacCallum Cancer Centre) for extensive support of the microscopy experiments. This work was supported by project grants to NJW (454506) and JO (454567) and program grants to JAT, RWJ and PIB from the NH&MRC Australia. NJW, JO and JS are RD Wright Fellows and JAT is a Senior Principal Research Fellow of the NH&MRC Australia. RWJ is a Pfizer Senior Research Fellow, Australia. We apologize to those whose papers were not cited due to editorial restrictions.
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Sedelies, K., Ciccone, A., Clarke, C. et al. Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity. Cell Death Differ 15, 708–717 (2008). https://doi.org/10.1038/sj.cdd.4402300
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DOI: https://doi.org/10.1038/sj.cdd.4402300
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