Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne, Australia, 3800

Correspondence to: A Lawen, Department of Biochemistry and Molecular Biology, Monash University, PO Box 13D, Melbourne, Australia, 3800; E-mail: alfons.lawen@med.monash.edu.au

^aJD Ly and F Vaillant contributed equally to this publication.

^bCurrent address: Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK.

Permeability transition, and a subsequent drop in mitochondrial membrane potential (_m), have been suggested to be mechanisms by which cytochrome c is released from the mitochondria into the cytosol during apoptosis. Furthermore, a drop in _m has been suggested to be an obligate early step in the apoptotic pathway. Didemnin B, a branched cyclic peptolide described to have immunosuppressive, anti-tumour, and anti-viral properties, induces rapid apoptosis in a range of mammalian cell lines. Induction of apoptosis by didemnin B in cultured human pro-myeloid HL-60 cells is the fastest and most complete ever described with all cells being apoptotic after 3 h of treatment. By utilizing the system of didemnin B-induced apoptosis in HL-60 cells, and the potent inhibitors of mitochondrial permeability transition, cyclosporin A and bongkrekic acid, we show that permeability transition as determined by changes in _m and mitochondrial Ca²⁺ fluxing, is not a requirement for apoptosis or cytochrome c release. In this system, changes in mitochondrial membrane potential and cytochrome c release are shown to be dependent on caspase activation, and to occur concurrently with the release of caspase-9 from mitochondria, genomic DNA fragmentation and apoptotic body formation. Oncogene (2001) 20, 4085-4094.

didemnin B; apoptosis; mitochondria; cytochrome c; permeability transition; caspases

AIF, apoptosis inducing factor; CyA, cyclosporin A; BA, bongkrekic acid; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; PT, permeability transition; PTP, permeability transition pore; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DAPI, 4,6'-diamidino-2-phenylindole; mtDNA, mitochondrial DNA; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; Ac-DEVD-cho, acetyl-Asp-Glu-Val-L-Asp-aldehyde; Ac-YVAD-cmk, acetyl-Tyr-Val-Ala-Asp-chloromethylketone