Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent, caspases-3/-8-driven mitochondrial amplification loop Clarissa von Haefen 1,3 , Thomas Wieder 1,3 , Frank Essmann 1,3 , Klaus Schulze-Osthoff 2 , Bernd Do¨rken 1 and Peter T Daniel 1 1 Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charite´, Campus Berlin-Buch, Humboldt University of Berlin, Germany; 2 Institute for Molecular Medicine, University of Du¨sseldorf, Germany Caspase-8 is a key effector of death-receptor-triggered apoptosis. In a previous study, we demonstrated, however, that caspase-8 can also be activated in a death receptor- independent manner via the mitochondrial apoptosis pathway, downstream of caspase-3. Here, we show that caspases-3 and -8 mediate a mitochondrial amplification loop that is required for the optimal release of cytochrome c, mitochondrial permeability shift transition, and cell death during apoptosis induced by treatment with the microtubule-damaging agent paclitaxel (Taxol). In con- trast, Smac release from mitochondria followed a different pattern, and therefore seems to be regulated independently from cytochrome c release. Taxol-induced cell death was inhibited by the use of synthetic, cell- permeable caspase-3- (zDEVD-fmk) or caspase-8-specific (zIETD-fmk) inhibitors. Apoptosis signaling was not affected by a dominant-negative FADD mutant (FADD- DN), thereby excluding a role of death receptor signaling in the amplification loop and drug-induced apoptosis. The inhibitor experiments were corroborated by the use of BJAB cells overexpressing the natural serpin protease inhibitor, cytokine response modifier A. These data demonstrate that the complete activation of mitochondria, release of cytochrome c, and execution of drug-induced apoptosis require a mitochondrial amplification loop that depends on caspases-3 and -8 activation. In addition, this is the first report to demonstrate death receptor-indepen- dent caspase-8 autoprocessing in vivo. Oncogene (2003) 22, 2236–2247. doi:10.1038/sj.onc.1206280 Keywords: chemotherapy; apoptosis; mitochondria; amplification loop; BJAB cells; caspase-3; caspase-8; cytochrome c; Bcl-x L ; Taxol; paclitaxel Introduction Triggering of apoptotic cell death by different stimuli, such as death ligands, ionizing irradiation or chemother- apeutic drugs, leads to the activation of a family of cysteine proteases with an absolute requirement of aspartate residues at the substrate cleavage site which are thus called caspases (Nicholson, 1999). Caspases form the core of the apoptotic machinery and are involved both in initiation of receptor-mediated apop- totic signaling cascades as well as in the execution of the apoptotic program (Cohen, 1997; Thornberry and Lazebnik, 1998; Fadeel et al., 2000; Daniel et al., 2001). Depending on the death stimulus, one of these two major pathways is activated, that is, death receptor- dependent signaling and death receptor-independent signaling via the mitochondria (Los et al., 1999; Daniel, 2000; Daniel et al., 2001). In both cases, stimulation of the death pathway leads to processing and activation of initiator caspases, for example, caspases-8 or -9, which subsequently transmit the signal to downstream effector caspases, for example, caspases-3, -6, or -7. Interestingly, inactivation of apoptosis signaling pathways at the level of caspases might play a role in the development of cytotoxic drug resistance. Loss of caspase-3 impairs drug-induced apoptosis and reconsti- tution of caspase-3 reverts drug resistance in breast cancer cells with acquired drug resistance (Friedrich et al., 2001). We previously demonstrated that a severe disturbance of the mitochondrial apoptosis pathway occurs during relapse of childhood acute lymphoblastic leukemia (ALL). In this setting, chemoresistant relapse is associated with a decrease of the Bax/Bcl-2 ratio and a concomitant impaired caspase-3 processing (Prokop et al., 2000). Caspase-8 has been shown to act as initiator caspase in the signal transduction of death receptor-triggered apoptosis, that is, by CD95/Fas, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, and TNF-receptor-1 (for a review, see Daniel et al., 2001). Furthermore, it was suggested that cytotoxic drug-induced apoptosis is mediated by a CD95/Fas-dependent mechanism in T cells (Friesen et al., 1999; Nomura et al., 2000; Fulda et al., 2001b). Received 22 April 2002; revised 28 November 2002; accepted 28 November 2002 *Correspondence: PT Daniel, Molecular Hematology and Oncology, University Medical Center Charite´, Campus Berlin-Buch, Humboldt University of Berlin, Lindenberger We.g. 80, 13125 Berlin, Germany; E-mail: pdaniel@mdc-berlin.de 3 These authors contributed equally to the present study Oncogene (2003) 22, 2236–2247 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc