Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury Pothana Saikumar* ,1 , Zheng Dong 1 , Yogi Patel 1 , Kristi Hall 1 , Ulrich Hopfer 2 , Joel M Weinberg 3 and MA Venkatachalam 1 1 Department of Pathology, University of Texas Health Science Centre at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7750, USA; 2 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA; 3 Department of Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygena- tion. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omis- sion of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability. Keywords: hypoxia; reoxygenation; cell death; Bax; Bcl-2; cytochrome c Introduction Mechanisms of cell death resulting from ischemia and reperfusion are poorly understood but are thought to involve both apoptosis and necrosis in several cell types (Kato et al., 1997; Kajstura et al., 1998; Li et al., 1998a; Wiegele et al., 1998; Lieberthal and Levine, 1996; Noda et al., 1998). Injury during reperfusion is thought to be driven by processes that are related to reoxygenation (McCord, 1985; Cotran et al., 1998). On the other hand, cells may become damaged during the ischemic period itself, owing to energy declines caused by hypoxia and deprivation of metabolic substrates. These alterations can predispose previously hypoxic cells to undergo accelerated injury during reoxygena- tion (Cotran et al., 1998). Recent reports have suggested that hypoxic or ischemic cell death is prevented by anti-apoptotic members of Bcl-2 family (Shimizu et al., 1995; Jacobson and Ra, 1995; Martinou et al., 1994) and by inhibitors of the caspase family of cysteine proteases (Shimizu et al., 1996; Cheng et al., 1998; Yaoita et al., 1998). However, the relevant death mechanisms remain to be elucidated. Apoptotic processes set in motion by non-hypoxic stimuli involve at least two discrete mechanisms initiated by two distinct caspases, with downstream activation of common eector caspases which result in cellular disassembly. One mechanism, commonly associated with cytotoxic agents, radiation and growth factor withdrawal, involves the activation of initiator caspase-9 and requires the formation of a ternary complex with cytochrome c released from mitochon- dria and a cytosolic co-factor Apaf-1 in the presence of ATP or dATP (Li et al., 1997; Pan et al., 1998). Release of apoptogenic factors such as cytochrome c caused by increased mitochondrial permeability is a proximate trigger for this type of apoptosis (Yang et al., 1997; Kluck et al., 1997). Thus, by inhibiting cytochrome c release, anti-apoptotic members of the Bcl-2 family can abort the death cascade (Yang et al., 1997; Kluck et al., 1997). Caspase inhibitors have the power to prevent structural damage attributable to downstream enzymatic events, but need not, a priori, preserve mitochondrial integrity and therefore may only delay cell death (McCarthy et al., 1997; Miller et al., 1997; Xiang et al., 1996). The other mechanism underlies apoptosis caused by ligation of death receptors, which recruit and proteolytically activate the initiator caspase-8 (Ashke- nazi and Dixit, 1998) that in turn triggers eector caspases. This death cascade may also be ampli®ed by caspase-8-mediated mitochondrial damage and release of apoptogenic factors (Luo et al., 1998; Li et al., 1998b). Under these conditions caspase inhibitors oer complete protection against injury. Inhibition of the proximally located trigger caspase-8 not only aborts downstream, eector caspase related death events, but also ensures that caspase-8-mediated mitochondrial damage does not occur (Luo et al., 1998; Li et al., 1998b). Mitochondrial damage has long been considered to play a role in hypoxic and ischemic cell death, but the *Correspondence: P Saikumar The ®rst two authors have contributed equally to this work Received 3 November 1998; accepted 24 November 1998 Oncogene (1998) 17, 3401 ± 3415 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc