Early events in the anoikis program occur in the absence of caspase activation

Adhesion of many cell types to the extracellular matrix is essential to maintain their survival. In the absence of integrin-mediated signals, normal epithelial cells undergo a form of apoptosis termed anoikis. It has been proposed that the activation of initiator caspases is an early event in anoikis, resulting in Bid cleavage and cytochrome c release from mitochondria. We have previously demonstrated that the loss of integrin signaling in mammary epithelial cells results in apoptosis and that this is dependent upon translocation of Bax from the cytosol to the mitochondria. In this paper, we ask whether caspases are required for Bax activation and the associated changes within mitochondria. We show that Bax activation occurs extremely rapidly, within 15 min after loss of integrin-mediated adhesion to extracellular matrix. The conformational changes associated with Bax activation are independent of caspases including the initiator caspase-8. We also examined downstream events in the apoptosis program and found that cytochrome c release occurs after a delay of at least 1 h, with subsequent activation of the effector caspase-3. This delay is not due to a requirement for new protein synthesis, since cycloheximide has no effect on the kinetics of Bax activation, cytochrome c release, caspase-3 cleavage, or apoptosis. Together, our data indicate that the cellular decision for anoikis in mammary epithelial cells occurs in the absence of caspase activation. Moreover, although the conformational changes in Bax are rapid and synchronous, the subsequent events occur stochastically and with considerable delays.     

A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.     

Translocation of full-length Bid to mitochondria during anoikis

Epithelial cells require adhesion to the extracellular matrix for survival, and in the absence of adhesion they undergo apoptosis (anoikis). This is distinct from apoptosis induced by extracellular death ligands, such as tumor necrosis factor, which result in direct activation of caspase 8. Bid is a member of the BH3-only subfamily of the Bcl-2 proteins and is important for most cell types to apoptose in response to Fas and tumor necrosis factor receptor activation. Caspase 8 cleaves full-length Bid, resulting in truncated p15 tBid. p15 tBid is potently apoptotic and activates the multidomain Bcl-2 protein, Bax, resulting in release of cytochrome c from mitochondria. We have previously shown that Bax rapidly translocates from the cytosol to mitochondria following loss of adhesion and that this is required for anoikis. We have now examined the role of Bid in anoikis. Bid translocates to mitochondria with identical kinetics as Bax. Although Bid is required for anoikis, it does not require proteolytic cleavage by caspase 8. Furthermore, it does not require Bid to interact directly with other Bcl-2 family proteins, such as Bax. Our data indicate that Bid is important for regulating apoptosis via the intrinsic pathway and has implications for how Bid may fulfill that role.     

The N-terminal conformation of Bax regulates cell commitment to apoptosis

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.     

Subcellular localization and physiological consequences of introducing a mitochondrial matrix targeting signal sequence in bax and its mutants

Bax, a proapoptotic member of the Bcl-2 family of proteins, resides in the cytosol and translocates to the mitochondrial membrane upon induction of apoptosis. It has been proposed that Bax does not translocate to mitochondria under normal physiological conditions, due to interaction between amino (ART) and carboxy (TM) terminal domains. Here, we report the physiological consequences of introducing a matrix targeting mitochondrial signal sequence (Su9) at the amino terminus of Bax and its mutants lacking ART, TM, or both segments. In vitro mitochondrial protein import assays of the fusion proteins suggests localization to the mitochondrial matrix. When expressed in Cos-1 cells, Su9 could target Bax to mitochondria in the absence of an apoptotic stimulus. However, mitochondrial localization did not result in apoptosis. When ART, TM, or both segments of Bax were deleted, expression of fusion proteins containing Su9 resulted in apoptosis via cytochrome c release. Cell death was inhibited by the pan-caspase inhibitor zVAD-fmk. We thus demonstrate that an effective mitochondrial matrix targeting signal can override the inhibition of import of Bax to the organelle, presumed to arise as a result of interaction between ART and TM segments, in the absence of apoptotic stimulus. We also demonstrate the ability of truncated variants of Bax to cause apoptosis when targeted to mitochondria by cytochrome c release from an ectopic environment.     

Bax interacts with the voltage-dependent anion channel and mediates ethanol-induced apoptosis in rat hepatocytes

Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis.     

Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.     

Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.     

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.     

Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells

Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochrome c-conducting channel in the outer mitochondrial membrane.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.     

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.     

Natural diterpenoid compound elevates expression of Bim protein, which interacts with antiapoptotic protein Bcl-2, converting it to proapoptotic Bax-like molecule

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.     

Fragmented mitochondria are sensitized to Bax insertion and activation during apoptosis

Recent studies have shown mitochondrial fragmentation during cell stress and have suggested a role for the morphological change in mitochondrial injury and ensuing apoptosis. However, the underlying mechanism remains elusive. Here we demonstrate that mitochondrial fragmentation facilitates Bax insertion and activation in mitochondria, resulting in the release of apoptogenic factors. In HeLa cells, overexpression of mitofusins attenuated mitochondrial fragmentation during cisplatin- and azide-induced cell injury, which was accompanied by less apoptosis and less cytochrome c release from mitochondria. Similar effects were shown by inhibiting the mitochondrial fission protein Drp1 with a dominant negative mutant (dn-Drp1). Mitofusins and dn-Drp1 did not seem to significantly affect Bax translocation/accumulation to mitochondria; however, they blocked Bax insertion and activation in mitochondrial membrane. Consistently, in rat kidney proximal tubular cells, small interfering RNA knockdown of Drp1 prevented mitochondrial fragmentation during azide-induced ATP depletion, which was accompanied by less Bax activation, insertion, and oligomerization in mitochondria. These cells released less cytochrome c and AIF from mitochondria and showed significantly lower apoptosis. Finally, mitofusin-null mouse embryonic fibroblasts (MEF) had fragmented mitochondria. These MEFs were more sensitive to cisplatin-induced Bax activation, release of cytochrome c, and apoptosis. Together, this study provides further support for a role of mitochondrial fragmentation in mitochondrial injury and apoptosis. Mechanistically, mitochondrial fragmentation may sensitize the cells to Bax insertion and activation in mitochondria, facilitating the release of apoptogenic factors and consequent apoptosis.     

Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis

During etoposide-induced apoptosis in HL-60 cells, cytochrome c release was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition was also observed; however, it was not the primary cause of mitochondrial swelling. Potassium uptake and swelling of mitochondria were blocked by bcl-2 overexpression. As a result, cytochrome c release was reduced, and apoptosis delayed. Residual cytochrome c release in the absence of swelling in bcl-2 expressing cells could be due to observed Bax translocation into mitochondria. This study suggests several novel aspects of apoptotic signaling: (1) potassium related swelling of mitochondria; (2) inhibition of mitochondrial potassium uptake by bcl-2; (3) co-existence within one system of multiple mechanisms of cytochrome c release: mitochondrial swelling and swelling-independent permeabilization of the outer mitochondrial membrane.     

Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria

Bax is a potent pro-apoptotic member of the Bcl-2 protein family that localizes to the mitochondrial membrane during apoptosis. Tauroursodeoxycholic acid (TUDCA) modulates the apoptotic threshold, in part, by preventing Bax translocation both in vitro and in vivo. The mechanisms by which Bax induces and TUDCA inhibits release of cytochrome c are unclear. We show here that recombinant Bax protein induced cytochrome c release in isolated mitochondria without detectable swelling. Co-incubation with TUDCA prevented efflux of mitochondrial factors and proteolytic processing of caspases in cytosolic extracts. Spectroscopic analyses of mitochondria exposed to Bax revealed increased polarity and fluidity of the membrane lipid core as well as altered protein order, indicative of Bax binding, together with loss of spin-label paramagnetism, characteristic of oxidative damage. TUDCA markedly abrogated the Bax-induced membrane perturbation. In conclusion, our results indicate that Bax protein directly induces cytochrome c release from mitochondria through a mechanism that does not require the permeability transition. Rather, it is accompanied by changes in the organization of membrane lipids and proteins. TUDCA is a potent inhibitor of Bax association with mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation through pathways that are also independent of the mitochondrial permeability transition.     

Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.