The Journal of Experimental Medicine
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Published 5 November 2001. doi:10.1084/jem.194.9.1325
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© Rockefeller University Press, 0022-1007/2001/11/1325/ $5.00
The Journal of Experimental Medicine, Volume 194, Number 9, November 5, 2001 1325-1338

Original Article

Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

Gui-Qiang Wang1, Eva Wieckowski1, Leslie A. Goldstein1, Brian R. Gastman2, Asaf Rabinovitz1, Andrea Gambotto3, Shuchen Li1, Bingliang Fang5, Xiao-Ming Yin1 and Hannah Rabinowich1,4

1 Department of Pathology, The University of Pittsburgh School of Medicine
2 Department of Otolaryngology, The University of Pittsburgh School of Medicine
3 Department of Surgery, The University of Pittsburgh School of Medicine
4 The University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213
5 Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center Houston, Texas 77030

Address correspondence to Hannah Rabinowich, University of Pittsburgh Cancer Institute, W952 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Phone: 412-624-0289; Fax: 412-624-7737; E-mail: rabinow{at}pitt.edu

Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

Key Words: apoptosis • Bak • Bid • cytochrome c • granzyme B • mitochondria


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