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Glen MacDonald^*, Lianfa Shi^*, Christine Vande Velde^*, Judy Lieberman, and Arnold H. Greenberg^*

From the ^* Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9; and the Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115

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 (). 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 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, 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-X_L suppressed caspase activation, mitochondrial cytochrome c release, 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.

Key Words: granzyme B • apoptosis • mitochondria • cytochrome c • caspase

Abbreviations used: BA, bongkrekic acid; CsA, cyclosporine acid; , mitochondrial transmembrane potential; DEVD-CHO, Ac-Asp-Glu-Val-Asp-acid aldehyde; FA-FMK, Cbz-Phe-Ala-fluoromethyl ketone; GraB, granzyme B; GraK, granzyme K; PT, permeability transition; Rh123, Rhodamine 123; ROS, reactive oxygen species; YVAD-CHO, Ac-Tyr-Val-Ala-Asp-acid aldehyde; zVAD-FMK, Cbz-Val-Ala-Asp-fluoromethyl ketone.

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