Apoptosis-inducing factor and apoptosis

Apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial intermembrane flavoprotein which has the ability to induce apoptosis via a caspase-independent pathway. AIF plays an important role in inducing nuclear chromatin condensation as well as large-scale DNA fragmentation (approximately 50 kb), and is essential for programmed cell death during cavitation of embryoid bodies. Two homologous proteins, AIF-homologous mitochondrion-associated inducer of death (AMID) and p53-responsive gene 3 (PRG3), also have apoptosis-inducing effects. Recent studies on mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans reveal that WAH-1(an AIF homolog in C. elegans) induced apoptosis is CED-3-dependent. AIF also interacts with cytochrome c and caspases during mammalian apoptosis processes, indicating that different apoptotic pathways may be mutually cross-regulated to activate an apoptotic program.     

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.     

Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL

A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochrome c, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochrome c plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL.     

Cytochrome c induces caspase-dependent apoptosis in intact hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-kinase-kinase, and malignant change

It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3-dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.     

Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions

Inhibition of the respiratory chain reaction by cyanide, rotenone or antimycin A (chemical hypoxia) induces necrotic cell death characterized by apparently intact chromatin, remarkable mitochondrial swelling with loss of crista structure, and loss of plasma membrane integrity. The treatments induce no apoptotic cell death, as defined by fragmented nuclei with condensed chromatin, fragmented or condensed cytoplasm. The anti-apoptotic proteins Bcl-2 and Bcl-xL effectively retard the chemical hypoxia-induced necrotic cell death. The necrotic cell death is also retarded by inhibitors of ICE(-like) proteases, including interleukin-1beta converting enzyme (ICE), which are common mediators of apoptosis. These results indicate that Bcl-2/Bcl-xL and ICE(-like) proteases modulate apoptotic and at least some forms of necrotic cell death. Both cell death pathways appear to involve some common mediators; however necrotic or apoptotic cell death signals might be transduced through multiple pathways, because Bcl-2/ Bcl-xL or inhibitors of ICE(-like) proteases are relatively less potent in blocking necrotic cell death than in preventing apoptosis.     

The central role of the mitochondrial megachannel in apoptosis: evidence obtained with intact cells, isolated mitochondria, and purified protein complexes

The mitochondrial megachannel (also called permeability transition pore) is a polyprotein complex formed in the contact site between the inner and the outer mitochondrial membranes and participates in the regulation of mitochondrial membrane permeability. We have obtained three independent lines of evidence suggesting the implication of the mitochondrial megachannel in apoptosis. First, in intact cells, apoptosis is accompanied by an early dissipation of the mitochondrial transmembrane potential (delta psi m). In several models of apoptosis, specific agents inhibiting the mitochondrial megachannels prevent this delta psi m dissipation and simultaneously abolish the manifestations of caspase- and endonuclease activation, indicating that megachannel opening is a critical event of the apoptotic process. Second, mitochondria are rate-limiting for caspase and nuclease activation in several cell-free systems of apoptosis. Isolated mitochondria release apoptogenic factors capable of activating pro-caspases or endonucleases upon opening of the mitochondrial megachannel in vitro. Third, opening of the purified megachannel reconstituted into liposomes is inhibited by recombinant Bcl-2 or Bcl-XL, two apoptosis-inhibitory proteins which also prevent megachannel opening in cells and isolated mitochondria. This indicates that the megachannel is under the direct regulatory control of anti-apoptotic members of the Bcl-2 family. Altogether, our results suggest that megachannel opening is sufficient and (mostly) necessary for triggering apoptosis.     

Functions of TGF-beta superfamily members in reproduction

We report here that BID, a BH3 domain-containing proapoptotic Bcl2 family member, is a specific proximal substrate of Casp8 in the Fas apoptotic signaling pathway. While full-length BID is localized in cytosol, truncated BID (tBID) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. tBID induces first the clustering of mitochondria around the nuclei and release of cytochrome c independent of caspase activity, and then the loss of mitochondrial membrane potential, cell shrinkage, and nuclear condensation in a caspase-dependent fashion. Coexpression of BclxL inhibits all the apoptotic changes induced by tBID. Our results indicate that BID is a mediator of mitochondrial damage induced by Casp8.     

Multi-step chromatin degradation in apoptosis. DNA breakdown in apoptosis

Elucidation of the mechanism and regulation of cell destruction in apoptosis requires knowledge of genome degradation. The cell genome, which encodes the fundamental cell programmes, is most likely to be the main sensitive target in cell apoptosis. Genome breakdown may be achieved by the generation and transduction of apoptogenic signal information to the specific chromatin regions of the nucleus, thus inactivating the basic cell programmes and inducing the endogenous pattern of chromatin degradation, which is determined by the genome organization of the eukaryotic nuclei. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation, thus leading to cell death of terminal differentiation or stress-induced apoptosis.     

The prevention of adhesions in surgery. A clinical review and experimental contribution

Induction of apoptosis in human monocytic THP.1 cells by etoposide or N-tosyl-L-phenylalanyl chloromethyl ketone resulted in release of mitochondrial cytochrome c, formation of ultracondensed mitochondria, development of outer mitochondrial membrane discontinuities and a reduction in mitochondrial membrane potential (delta psi m), as well as externalisation of phosphatidylserine, caspase-3 and -7 activation, proteolysis of poly(ADP-ribose) polymerase and lamin B1. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone inhibited all these ultrastructural and biochemical characteristics of apoptosis except for the release of cytochrome c. Release of mitochondrial cytochrome c was a late event in non-apoptotic cell death occurring after commitment to cell death and without caspase activation. Thus apoptosis is characterised by release of mitochondrial cytochrome c prior to formation of ultracondensed mitochondria and a reduction in delta psi m and by a mechanism independent of rupture of the outer mitochondrial membrane.     

Comparison of Mg2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system

Mitochondrial cytochrome c (cyt c) has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cyt c can initiate the activation cascade of caspases once it is released into the cytosol. The loss of a component of the mitochondrial electron transport chain also triggers the generation of superoxide. Although cyt c can be released independent of the mitochondrial permeability transition (MPT), the accompanying cellular redox change can trigger the MPT. Since another apoptotic protease, AIF, is released by MPT, the two separate pathways provide redundancy that ensures effective execution of the cell death program. Anti-apoptotic Bcl-2 family proteins function as gatekeepers to prevent the release of both cyt c and AIF. In spite of their stabilization effect on the mitochondrial outer membrane, Bcl-2 proteins may also be involved in the direct binding of Apaf molecules as regulatory elements further downstream from the mitochondrial apoptotic signals.     

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered delta psi(m) collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X(L) proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.     

Molecular quantum similarity measures tuned 3D QSAR: an antitumoral family validation study

It was recently reported that the mitochondrial protein cytochrome c is required for the induction of apoptosis, and that the overexpression of Bcl-2 caused increased retention of this apoptogenic factor by mitochondria. Several cellular toxins, including H2O2, tBOOH and Ca++, induce the Mitochondrial Permeability Transition (MPT); we tested the possibility that MPT is an intracellular sensor of toxicity that results in the release of cytochrome c. We observe that the release of cytochrome c from purified mitochondria is stimulated by the classical inducers of MPT, and is inhibited by the classical inhibitor of MPT, cyclosporin A (CsA). After induction of MPT, mitochondrial supernatants gained the activity to induce cleavage of caspase 3 (CPP32) in cytosolic extracts, and this gain of activity was inhibited by CsA pretreatment of mitochondria, and was cancelled by immunodepletion of cytochrome c from the supernatants. After induction of MPT, mitochondrial supernatants mixed with or without cytosolic extract gained the activity to ladder nuclei, and this gain of activity was inhibited by CsA pretreatment of mitochondria, and cancelled by immunodepletion of cytochrome c from the supernatants. These results demonstrate that the induction of MPT causes release of cytochrome c from mitochondria, which is required for the hallmarks of cytosolic and nuclear apoptosis, caspase 3 activation and nuclear laddering, and identify the MPT as a potential intracellular sensor of oxidants and other toxins, and as a target for the pharmacological inhibition of apoptosis.     

The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition

Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpression of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA-preventable loss of the mitochondrial membrane potential (DeltaPsim). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of DeltaPsim and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation.     

Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria

Apoptotic cell death involves a ritual series of morphological changes, presumably reflecting a conserved molecular pathway. We now report that the nuclear events typical of apoptosis can be reproduced in "apoptotic" Xenopus egg extracts. In this cell-free system, nuclear assembly and protein import are initially normal; after 2-4 hr, however, a process of nuclear destruction ensues involving chromatin condensation and the shrinkage and fragmentation of the nuclei. This apoptotic process, which also occurs in nuclei added exogenously, is blocked by the addition of baculovirus-expressed Bcl-2 protein. To block the disintegration of nuclei that are added later, Bcl-2 must be present during this latent period. "Apoptosis" in these extracts requires a dense organelle fraction enriched in mitochondria. The cell-free system described here provides a novel tool for understanding intracellular events in apoptosis and the inhibitory function of Bcl-2.     

Bax directly induces release of cytochrome c from isolated mitochondria

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.     

Drosophila grim induces apoptosis in mammalian cells

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.     

Bioavailability of phenytoin acid and phenytoin sodium with enteral feedings

In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207-6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J. G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529-535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-XL. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.     

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.     

Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.