c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release

Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.     

Redistribution of Cytochrome c Precedes the Caspase-Dependent Formation of Ultracondensed Mitochondria, with a Reduced Inner Membrane Potential, in Apoptotic Monocytes

Apoptosis was induced in human monocytic THP.1 cells by the use of chemicals with disparate mechanisms of action. Apoptotic cells were characterized by a reduced inner mitochondrial membrane potential, increased cytosolic cytochrome c, ultracondensed mitochondria, condensed chromatin, cytoplasmic inclusions of beta-actin, and fragmentation of the Golgi apparatus. All of these changes, except the release of cytochrome c, were prevented by caspase inhibition. Cells were separated into two populations, with either normal or low inner mitochondrial membrane potential, using fluorescence-activated cell sorting. Ultracondensed mitochondria were observed only in the cells with low inner mitochondrial membrane potential, whereas noncondensed mitochondria were found in the cells with a normal inner mitochondrial membrane potential. We have demonstrated a sequence of related biochemical and ultrastructural changes, commencing with the release of mitochondrial cytochrome c, followed by activation of caspases and a reduction of inner mitochondrial membrane potential. These changes involved the formation of ultracondensed but not swollen mitochondria. Thus the release of mitochondrial cytochrome c was not the result of the mitochondrial permeability transition, reduction of inner mitochondrial membrane potential, or rupture of the outer mitochondrial membrane. Discontinuities in the outer membrane of ultracondensed mitochondria may, however, facilitate the further release of caspase-activating proteins, thereby amplifying the apoptotic process.     

Release of mitochondrial cytochrome c and DNA fragmentation after cold injury-induced brain trauma in mice: possible role in neuronal apoptosis.

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. In this study, we examined the subcellular distribution of the cytochrome c protein after cold injury (CI), in which apoptosis is assumed to participate. Western blotting and immunohistochemistry showed cytosolic cytochrome c as early as 1 h after CI, and correspondingly, there was a reduction in mitochondrial cytochrome c after injury. Neuronal distribution of cytosolic cytochrome c was shown by double staining with a neuronal nuclear marker by immunohistochemistry. A significant amount of DNA laddering was detected 4 h after CI, and increased in a time-dependent manner. These data suggest that early cytochrome c release from mitochondria may contribute to apoptosis induction after traumatic brain injury.     

Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release,activation of multiple caspases,and virus release following coxsackievirus B3 infection

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release.     

Mitochondrial cytochrome c release in radiation-induced apoptosis of human peripheral T cells

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, using a microscopy-video system, mitochondrial membrane potential assay, annexin V, propidium iodide, and cytochrome c ELISA kit. After 5 Gy irradiation with 10 MV X-ray from a linear accelerator, the percentages of apoptotic T cells were estimated as approximately 5, 10, 20, 35, and 70%, at 0, 3, 6, 10, and 20 h after irradiation, respectively, as observed with the microscopy-video system. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, approximately half of the T cells showed dysfunction of mitochondrial membrane potential at 10 h after 5 Gy irradiation. With regard to annexin V and propidium iodide, approximately 40 and 5% of the human peripheral T cells showed positivity against annexin V and propidium iodide at that time, respectively. Mitochondrial cytochrome c release from the mitochondria to the cytosol was confirmed to start at 10 h and to reach a maximum at 20 h after 5 Gy of irradiation. These results demonstrated that mitochondrial cytochrome c release occurred following dysfunction of mitochondrial membrane potential in radiation-induced T cell apoptosis.     

The mitochondrial phase of the glucocorticoid-induced apoptotic response in thymocytes comprises sequential activation of adenine nucleotide transporter (ANT)-independent and ANT-dependent events

In thymocytes, dexamethasone initiates cytochrome c-dependent processing of caspase-9 and the activation of caspase-3 to trigger apoptotic damage. Using murine thymocytes or a thymocyte cell line WEHI 7.1, we show that this pathway is inhibited by dominant-negative caspase-9, the anti-apoptotic protein Bcl-2, or by blocking components of the mitochondrial permeability transition pore complex (PTPC). We use DIDS (dithiocyanatostilbene-2,2-disulfonic acid), a pharmacological modifier of VDAC (voltage-dependent anion channel) function or ectopic expression of hexokinase-II, to examine the role of the VDAC--a mitochondrial outer membrane protein--in this apoptotic pathway. This approach implicated the VDAC in dexamethasone-mediated cytochrome c release, processing of caspase-9 and caspase-3, the loss of mitochondrial transmembrane potential (Deltapsim), nuclear damage and cell lysis. Inhibiting the adenine nucleotide transporter (ANT), a protein on the mitochondrial inner membrane, also blocks dexamethasone-induced apoptosis, but the ANT regulates caspase-3 processing and nuclear damage but not the mitochondrial efflux of cytochrome c. Collectively, the data identify two separable, but connected events in dexamethasone-induced mitochondrial damage in thymocytes. The first event is an increase in permeability of the mitochondrial outer membrane leading to VDAC-regulated efflux of cytochrome c and initial processing of caspase-9 followed by ANT-dependent caspase-3 processing and apoptotic damage to cells.     

Apoptosis induction by interleukin-2-activated cytotoxic lymphocytes in a squamous cell carcinoma cell line and Daudi cells - involvement of reactive oxygen species-dependent cytochrome c and reactive oxygen species-independent apoptosis-inducing factors

Investigation of the induction of apoptosis by cytotoxic lymphocytes has mainly focused on the signalling associated with Fas and its adaptor proteins. The signal pathway via mitochondria, however, has not been sufficiently elucidated in cytotoxic lymphocyte-induced apoptosis. We examined the release of mitochondrial proapoptotic factors by lymphokine-activated killer (LAK) cells in two cell lines. LAK cell-induced DNA fragmentation of the target cells was suppressed to approximately 50% of control levels by the addition of neutralizing monoclonal antibody to Fas and a granzyme B inhibitor. When intracellular reactive oxygen species (ROS) were scavenged, the LAK cell-induced DNA fragmentation was decreased to approximately 60% of the non-treated cell level. Co-cultivation of Daudi cells with LAK cells increased cytosolic and mitochondrial ROS levels. Activation of procaspase-3 and apoptosis by treatment of oral squamous cell carcinoma cells (OSC) with LAK cells was partially inhibited by pretreatment of OSC cells with ROS scavengers and mitochondrial complex inhibitors. Furthermore, cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria by OSC cell treatment with supernatants of LAK cells. The supernatant-induced cytochrome c release was suppressed by mitochondrial complex inhibitors, but the inhibitors did not inhibit the release of AIF. These results indicate that LAK cells induce target cell apoptosis via not only the Fas/Fas ligand system and granzyme B, but also ROS-dependent cytochrome c and ROS-independent AIF release.     

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. We provide evidence that such coupling serves as a downstream effector function for Akt. First, Akt increases mitochondria-associated hexokinase activity. Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase. Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane.     

Immunoquantitation of cytochrome C in cardiac perfusate.

An indirect three step ELISA has been assessed in order to detect the possible release of cytochrome c, a mitochondrial protein, from isolated and perfused guinea-pig heart. The ELISA described in this study is sufficiently sensitive and accurate to measure extracellular cytochrome c.     

Fatty acid-induced apoptosis in neonatal cardiomyocytes: redox signaling

Exposure of neonatal rat cardiac myocytes to palmitate and glucose produces apoptosis as seen by cytochrome c release, caspase 3-like activation, DNA laddering, and poly(ADP-ribose) polymerase cleavage. The purpose of this study was to understand the role of reactive oxygen species in the initiation of programmed cell death by palmitate. We found that palmitate (but not oleate) produces inhibition of carnitine palmitoyltransferase I, accumulation of ceramide, and inhibition of electron transport complex III. These events are subsequent to cytochrome c release and loss of the mitochondrial membrane potential. No differences in H2O2 production or N-terminal c-Jun kinase phosphorylation were detected between myocytes incubated in palmitate and control myocytes (nonapoptotic) incubated in oleate. These results suggest that the palmitate-induced loss of the mitochondrial membrane potential is not associated with H2O2 synthesis and that a membrane potential is required to generate reactive oxygen species following ceramide inhibition of complex III.     

Antiapoptotic response to induced GSH depletion: involvement of heat shock proteins and NF-kappaB activation

Alteration of glutathione (GSH) homeostasis represents one of the earliest events during the commitment of stress-induced apoptosis. Extrusion of GSH into the extracellular milieu, in response to several oxidative stimuli, has been suggested as a molecular switch triggering apoptosis. However, chemical depletion of GSH does not induce cell death even though cytochrome c release from mitochondria has been observed. Here we report that U937 cells treated with buthionine sulfoximine (BSO) are able to survive and to inhibit the apoptotic program downstream of cytochrome c release. BSO treatment induces a highly significant decrease of GSH in both the cytosolic and mitochondrial fractions. The concomitant release of cytochrome c into the cytosol was associated with nuclear translocation of apoptosis-inducing factor. GSH depletion also resulted in reactive oxygen species production and in a specific increase of mitochondrial protein carbonyls. However, all these events were transiently present inside cells and efficiently counteracted by cell-repairing systems. We observed an increase in the proteasome activity and in the expression levels of heat shock protein 27 (Hsp27) and Hsp70. Moreover, nuclear factor-kappaB (NF-kappaB) was activated in our system as a survival cell response against the oxidative injury. Overall results suggest that activation of NF-kappaB and Hsp could allow cell adaptation and survival under exhaustive GSH depletion.     

Mycobacterium tuberculosis virulence correlates with mitochondrial cytochrome c release in infected macrophages

Mitochondria are at the centre of molecular events involved in energy production, cell survival and apoptosis. Mitochondrial membrane potential (Deltapsim) is maintained by cellular catabolic reactions and the electron transport chain of which cytochrome c is a constituent, whereas the proton leak pathway, ATP synthesis and turnover consume it. Mitochondrial alterations such as a drop in Deltapsim, swelling and cytochrome c release have been observed in apoptosis. However, there is a paucity of information concerning mitochondrial function in the course of intracellular infections, a process that must certainly induce stress on the host cell. This work analyses the effect that two strains of mycobacteria of opposing virulence have on the mitochondria of murine macrophages in the early stages of infection. It was found that infection of J774 cells with both Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv readily induced changes in Deltapsim as well as in mitochondrial morphology at the ultrastructural level. In addition, an increase in cytosolic ATP was found at 24 h post infection with both strains of M. tuberculosis. Interestingly, only M. tuberculosis H37Rv was able to induce cytochrome c release from mitochondria to the cytosol, thus suggesting the occurrence in M. tuberculosis H37Rv of a specific factor(s) capable of regulating cytochrome c translocation. The precise role of cytochrome c release in the context of a mycobacterial infection remains to be elucidated.     

Bid, a critical mediator for apoptosis induced by the activation of Fas/TNF-R1 death receptors in hepatocytes

The Bcl-2 family proteins consist of both antiapoptosis and pro-apoptosis members that regulate apoptosis typically at the mitochondrial level, 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 activation. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates the downstream caspases. This Bid-mediated pathway is critical in hepatocyte apoptosis induced by Fas/TNF-R1 engagement, where direct activation of cytosolic caspase cascade seems inefficient. The dependence on Bid, and thus on the mitochondrial cytochrome c release, of hepatocyte apoptosis induced by the death receptors also renders it sensitive to the inhibitory regulation by the anti-apoptosis members of the Bcl-2 family proteins, such as Bcl-2 and Bcl-xL. Moreover, the revealing of this death pathway in hepatocytes is important to the understanding of the pathogenesis of a number of hepatic diseases such as hepatitis or endotoxemia-related hepatic failure.     

Relocalization of cathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy

Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (delta psi(m)) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in delta psi(m) was detected using the delta psi(m)sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the delta psi(m). Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of delta psi(m) in cardiomyocytes exposed to naphthazarin.     

tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.     

Long-term depression of the human masseter inhibitory reflex

Transient global ischemia reportedly results in glutamate receptor stimulation and harmful Ca(2+)-overloading, then activates some proteins involved in cell apoptosis in vivo and in vitro, but underlying mechanisms remain to be elucidated. Here we evaluated the role of N-methyl-D-aspartate (NMDA) receptor antagonist and L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist in mediating the release of cytochrome c and the expression of caspase-3 precursor protein (procaspase-3). Cytochrome c release from mitochondria is a critical step in the cell apoptotic process. We examined whether cytochrome c was translocated from mitochondria to the cytosol by Western blot in rat hippocampus after 15 min global ischemia. Released cytochrome c interacts with apoptotic protease activating factor-1 and caspase-9, both of which play important roles in the cytochrome c-dependent mitochondrial pathway of apoptosis by activating caspase-3. Our studies demonstrated that the inactive precursor and active cleaved subunits of caspase-3 protease increased dramatically with the extent of reperfusion time. Following pretreatment with ketamine (a non-competitive NMDA receptor antagonist) and nifedipine (L-VGCC antagonist), cytosolic cytochrome c and the expression of procaspase-3 dramatically decreased, which might result in less neuron damage after ischemia.     

Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes

Mitochondria play a central role in many apoptotic reactions. Although mitochondrial apoptotic changes and caspase activation have been demonstrated in the apoptotic thymocytes, cell death signal through mitochondria in TCR-stimulated thymocytes has not been fully understood. In this study, we show that TCR stimulation induced disruption of mitochondrial transmembrane potential (Delta Psi(m)), the cytochrome c release from mitochondira, capase-3 activation, and the cell death of thymocytes. Bongkrekic acid, an inhibitor of Delta Psi(m) disruption, blocked the cytochrome c release from mitochondria and the following caspase-3-mediated cell death. Furthermore, a pro-apoptotic Bcl-2 family protein, Bax, but not Bad or Bid, was translocated from cytosol to mitochondria in TCR-stimulated thymocytes. This translocation and the following apoptotic changes were inhibited by SB203580, a p38 kinase inhibitor, in a specific manner. These results suggest that activated p38 kinase pathway by TCR stimulation induces translocation of Bax to mitochondria, causing Delta Psi(m) disruption, and the release of cytochrome c, which finally induces caspase-3-mediated apoptosis in thymocytes.     

Ethanol withdrawal hastens the aging of cytochrome c oxidase

We investigated whether abrupt ethanol withdrawal (EW) age-specifically inhibits a key mitochondrial enzyme, cytochrome c oxidase (COX), and whether estrogen mitigates this problem. We also tested whether this possible effect of EW involves a substrate (cytochrome c) deficiency that is associated with proapoptotic Bcl2-associated X protein (BAX) and mitochondrial membrane swelling. Ovariectomized young, middle age, and older rats, with or without 17β-estradiol (E2) implantation, underwent repeated EW. Cerebelli were collected to measure COX activity and the mitochondrial membrane swelling using spectrophotometry and the mitochondrial levels of cytochrome c and BAX using an immunoblot method. The loss of COX activity and the mitochondrial membrane swelling occurred only in older rats under control diet conditions but occurred earlier, starting in the young rats under EW conditions. E2 treatment mitigated these EW effects. EW increased mitochondrial BAX particularly in middle age rats but did not alter cytochrome c. Collectively EW hastens but E2 delays the age-associated loss of COX activity. This EW effect is independent of cytochrome c but may involve the mitochondrial overload of BAX and membrane vulnerability.     

Early activation of the mitochondrial apoptotic pathway in Vesicular Stomatitis virus-infected cells

Vesicular Stomatitis Virus (VSV) has been shown to induce apoptosis in a caspase-dependent manner, but the precise apoptotic pathway remains unknown. We found that caspases 9 and 3, but not caspase 8, were activated during VSV-induced apoptosis in infected Vero cells. Since caspase 9 is related to the mitochondrial apoptotic pathway, we analyzed some mitochondrial events such as changes in the mitochondrial transmembrane potential (Deltapsim) and mitochondrial release of apoptogenic proteins such as cytochrome c and the apoptosis inducing factor (AIF). We found that VSV infection triggers the dissipation of the Deltapsim and the release of both cytochrome c and AIF from the mitochondrial intermembrane space very early in the VSV infection. These results indicate that the trigger of apoptosis in VSV-infected cells occurs through the early activation of the mitochondrial apoptotic pathway. On the other hand, intracellular levels of the anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, and the pro-apoptotic protein Bax, were assessed during viral infection. These analyses showed that as viral infection proceeded, the cellular level of Bcl-xL decreased, while the levels of Bax and Bcl-2 remained unaffected. The significance of the Bcl-xL modulation is also discussed.