Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway

Even though retinoids are widely used as adjuvant in chemotherapeutic interventions to improve cancer cell death, their mechanism(s) of action involves multiple overlapping pathways that remain unclear. We have previously shown that vitamin A, the natural precursor of the retinoids, induces oxidative-dependent cytochrome c release from isolated mitochondria, suggesting a putative mechanism for apoptosis activation. Using Sertoli cells in culture, we show that retinol causes mitochondrial-dependent apoptosis, involving oxidative stress. Apoptosis was evaluated by nuclear morphology, DNA fragmentation, and caspase-3/7 activity. Retinol induced oxidant- and time-dependent imbalance of several mitochondrial parameters, cytochrome c release and caspase-3/7 activation, leading cells to commit apoptosis. All parameters tested were attenuated or blocked by trolox co-administration, suggesting that retinol induces apoptosis through oxidative damage, which mitochondria plays a pivotal role.     

Chelerythrine induces apoptosis through a Bax/Bak-independent mitochondrial mechanism

Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (DeltaPsi(m)) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.     

Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells

Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 μM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis

There are at least two distinct classes of caspases, initiators (e.g. caspases-8, -9, and -10) and effectors (e.g. caspase-3). Furthermore, it is believed that there are two distinct primary apoptotic signaling pathways, one of which is mediated by death receptors controlled by caspases-8/10, and the other by the release of cytochrome c and activation of a caspase-9/Apaf1/cytochrome c apoptosome. However, several recent reports have demonstrated that caspase-8, and its substrate Bid, are frequently activated in response to certain apoptotic stimuli in a death receptor-independent manner. These results suggest that significant cross-talk may exist between these two distinct signaling arms, allowing each to take advantage of elements unique to the other. Here we provide evidence that activation of caspase-8, and subsequent Bid cleavage, does indeed participate in cytochrome c-mediated apoptosis, at least in certain circumstances and cell types. Furthermore, the participation of activated caspase-3 is essential for activation of caspase-8 and Bid processing to occur. Although caspase-8 activation is not required for the execution of a cytochrome c-mediated death signal, we found that it greatly shortens the execution time. Thus, caspase-8 involvement in cytochrome c-mediated cell death may help to amplify weaker death signals and ensure that apoptosis occurs within a certain time frame.     

Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels

To define the responses of apoptotic regulatory proteins to different chemotherapeutic agents, we investigated the expression of Bcl-2 family gene products, the release of cytochrome c, and the activation of pro-caspase-3 during apoptosis induced by Taxol and Thiotepa, in the MCF-7 breast carcinoma and the HL-60 leukemia cell lines. The earliest event induced by drug exposure was increase in Bad protein levels, followed by Bcl-2 down-regulation, cytochrome c release, and Bcl-xL and Bax up-regulation. Bak accumulation was a late event. Activation of pro-caspase-3 and cleavage of Bcl-2 protein occurred in the HL-60 cells only, and followed the cytochrome c release. The overall responses were qualitatively similar in both cell types, but MCF-7 cells treated with Taxol showed a significant delay in apoptosis, correlating with early up-regulation of Bcl-2 and delayed release of cytochrome c. We conclude that Bad up-regulation is an early indicator of a cellular response that will lead to cell death, but may be modulated by survival mechanisms, which cumulatively govern the ultimate susceptibility to apoptosis.     

Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or exogenous reactive oxygen species-induced cell death

In this study, we have compared several features of cell death triggered by classical inducers of apoptotic pathways (etoposide and tumour necrosis factor (TNF)-α) versus exogenous reactive oxygen species (ROS; hydrogen peroxide (H?O?), tert-butyl hydroperoxide (t-BHP)) or a ROS generator (paraquat). Our aim was to characterize relationships that exist between ROS, mitochondrial perturbations, Bcl-2 and caspases, depending on source and identity of ROS. First, we have found that these five inducers trigger oxidative stress, mitochondrial membrane permeabilization (MMP), cytochrome c (cyt c) release from mitochondria and cell death. In each case, cell death could be inhibited by several antioxidants, showing that it is primarily ROS dependent. Second, we have highlighted that during etoposide or TNF-α treatments, intracellular ROS level, MMP and cell death are all regulated by caspases and Bcl-2, with caspases acting early in the process. Third, we have demonstrated that H?O?-induced cell death shares many of these characteristics with etoposide and TNF-α, whereas t-BHP induces both caspase-dependent and caspase-independent cell death. Surprisingly, paraquat-induced cell death, which harbours some characteristics of apoptosis such as cyt c release and caspase-3 activation, is not modulated by Bcl-2 and caspase inhibitors, suggesting that paraquat also triggers non-apoptotic cell death signals. On the one hand, these results show that endogenous or exogenous ROS can trigger multiple cell death pathways with Bcl-2 and caspases acting differentially. On the other hand, they suggest that H?O? could be an important mediator of etoposide and TNF-α-dependent cell death since these inducers trigger similar phenotypes.     

Release of cytochrome c from isolated mitochondria by etoposide

The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents.     

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.     

Osmostress-Induced Apoptosis in Xenopus Oocytes: Role of Stress Protein Kinases,Calpains and Smac/DIABLO

Hyperosmotic shock induces cytochrome c release and capase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.     

Hexokinase-I protection against apoptotic cell death is mediated via interaction with the voltage-dependent anion channel-1: mapping the site of binding

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents.     

Induction of apoptosis in AK-5 tumor cells by a serum factor from tumor rejecting animals: cytochrome c release independent of Bcl-2 and caspases

The ability to selectively induce apoptosis in tumor cells is the prime goal in cancer immunotherapy and aims at identifying potential molecular targets, regulating this process. Here we show that the sera from the animals which had spontaneously rejected the AK-5 tumor (a rat histiocytoma) had an effective and potent ability to counteract and kill tumor cells by inducing apoptosis, with a high degree of specificity. Apoptosis induced by the serum factor involved the activation of caspases and cytochrome c release to the cytosol. A reduction in mitochondrial transmembrane potential (Delta psi(m)) occurred considerably later than cytochrome c translocation. The anti-apoptotic protein Bcl-2 and the pancaspase inhibitor zVAD-fmk did not prevent cytochrome c release, but completely blocked the reduction in Delta psi(m), DNA fragmentation and apoptosis. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT) pore had no effect on cytochrome c release and apoptosis mediated by serum factor in AK-5 cells, suggesting that apoptosis was independent of MPT. Taken together these results suggest that the serum factor in conjunction with the immune cells may be participating in the efficient rejection of the tumor in syngeneic hosts and Delta psi(m) disruption but not cytochrome c release, is a critical and decisive event to trigger apoptotic cell death induced by the serum factor in AK-5 tumor cells.     

The Role of Cytochrome c on Apoptosis Induced by Anagrapha falcifera Multiple Nuclear Polyhedrosis Virus in Insect Spodoptera litura Cells

There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell- free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.     

Characterization of 4-O-methyl-ascochlorin-induced apoptosis in comparison with typical apoptotic inducers in human leukemia cell lines

Apoptosis can be induced by various stimuli such as the ligands of death receptors, chemotherapeutic drugs and irradiation. It is generally believed that chemotherapeutic drugs induce mitochondrial damage, cytochrome c release and activation of caspase-9, leading to apoptosis. Here, we found that an isoprenoid antibiotic, 4-O-methyl ascochlorin, significantly induces typical apoptotic events in Jurkat cells including the degradation of poly (ADP-ribose) polymerase, DNA fragmentation, activation of caspase-3, -9 and -8, and cytochrome c release from mitochondria. Similar to Fas stimulation, 4-O-methyl ascochlorin but not staurosporine, cycloheximide and actinomycin D, induced apoptosis in SKW6.4 cells, in which apoptosis is strongly dependent on death-inducing signaling-complex. Bcl-2 overexpression in Jurkat cells completely suppressed the apoptosis, but procaspase-9 processing was partially induced. A caspase-8 inhibitor, IETD-fmk, effectively suppressed poly (ADP-ribose) polymerase cleavage and cytochrome c release. However, 4-O-methyl ascochlorin induced apoptosis in Jurkat cells deficient of caspase-8 or Fas-associated death domain protein. These results suggest that 4-O-methyl ascochlorin induces apoptosis through the mechanism distinct from conventional apoptosis inducers.     

The molecular mechanism of Noxa-induced mitochondrial dysfunction in p53-mediated cell death

Genotoxic stresses stabilize the p53 tumor suppressor protein which, in turn, transactivates target genes to cause apoptosis. Although Noxa, a "BH3-only" member of the Bcl-2 family, was shown to be a target of p53-mediated transactivation and to function as a mediator of p53-dependent apoptosis through mitochondrial dysfunction, the molecular mechanism by which Noxa causes mitochondrial dysfunction is largely unknown. Here we show that two domains (BH3 domain and mitochondrial targeting domain) in Noxa are essential for the release of cytochrome c from mitochondria. Noxa-induced cytochrome c release is inhibited by permeability transition pore inhibitors such as CsA or MgCl2, and Noxa induces an ultra-structural change of mitochondria yielding "swollen" mitochondria that are unlike changes induced by tBid. This indicates that Noxa may activate the permeability transition-related pore to release cytochrome c from mitochondria into cytosol. Moreover, Bak-oligomerization, which is an essential event for tBid-induced cytochrome c release in the extrinsic death signaling pathway, is not associated with Noxa-induced cytochrome c release. This finding suggests that the pathway of Noxa-induced mitochondrial dysfunction is distinct from the one of tBid-induced mitochondrial dysfunction. Thus, we propose that there are at least two different pathways of mitochondrial dysfunction; one mediated through Noxa in response to genotoxic stresses and the other through tBid in response to death ligands.     

Distinct pathways for stimulation of cytochrome c release by etoposide

Induction of apoptosis by DNA-damaging agents, such as etoposide, is known to involve the release of mitochondrial cytochrome c, although the mechanism responsible for this event is unclear. In the present study, using Jurkat T-lymphocytes, a reconstituted cell-free system, or isolated liver mitochondria, we demonstrate the ability of etoposide to induce cytochrome c release via two distinct pathways. Caspase inhibition by either benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) or benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk) attenuates cytochrome c release triggered by a low dose of etoposide via an apparent inhibition of nuclear events involving the release of protein factor(s) that is (are) able to interact with mitochondria. In contrast, caspase inhibition has no effect on cytochrome c release induced by a higher dose of etoposide. Moreover, the higher dose of etoposide heightens the sensitivity of Ca(2+)-loaded isolated mitochondria to mitochondrial permeability transition, an effect that is completely abolished by cyclosporin A. Interestingly, cyclosporin A is ineffective at preventing similar mitochondrial damage in Jurkat cells treated with etoposide. We propose that lower doses of etoposide predominantly target the nucleus and stimulate the release of caspase-sensitive protein factor(s) that interact with mitochondria to trigger cytochrome c release, whereas higher doses of the drug impart a more direct effect on mitochondria and thus are not mitigated by caspase inhibition.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.     

Role of cytochrome C in apoptosis: increased sensitivity to tumor necrosis factor alpha is associated with respiratory defects but not with lack of cytochrome C release

Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-alpha-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways.     

Myc Potentiates Apoptosis by Stimulating Bax Activity at the Mitochondria

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.