1. ¹Cancer Cell Death, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia
2. ²Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA
3. ³Gene Regulation Laboratories, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia
4. ⁴Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia

Correspondence: NJ Waterhouse, Cancer Cell Death, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia. Tel: +61 –3 –9656 3725; Fax: +61 –3 –9656 1411; E-mail: nigel.waterhouse@petermac.org

Received 28 December 2004; Revised 15 July 2005; Accepted 5 August 2005; Published online 16 September 2005.

Abstract

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage – upstream or downstream of caspases – remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (m) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their m. Loss of m was not required for rapid granzyme B-induced apoptosis and regeneration of m following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of m demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.