Regulation of apoptosis by respiration: cytochrome c release by respiratory substrates

Cytochrome c release from mitochondria is essential for apoptosis. Using human myelogenous leukemia ML-1a, its respiration-deficient and reconstituted cells, we demonstrated that respiratory function is essential for tumor necrosis factor-induced cytochrome c release. In a cell free system using mitochondrial fraction from ML-1a, initiation of respiration by substrates for complexes I, II, and III but not IV released cytochrome c, suggesting that reduction of coenzyme Q or complex III is essential for cytochrome c release. In the same system, disruption of mitochondrial outer membrane was neither enough nor the cause for cytochrome c release by succinate. These observations define an early pathway in which a change in respiration releases cytochrome c.     

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells

Cytochrome c is released from mitochondria into the cytosol in cells undergoing apoptosis. The temporal relationship between cytochrome c release and loss of mitochondrial membrane potential was monitored by laser-scanning confocal microscopy in single living pheochromocytoma-6 cells undergoing apoptosis induced by staurosporine. Mitochondrial membrane potential monitored by tetramethylrhodamine methyl ester decreased abruptly in individual cells from 2 to 7 h after treatment with staurosporine. Depolarization was accompanied by cytochrome c release documented by release of transfected green fluorescent protein-tagged cytochrome c in these cells. The results show that mitochondrial depolarization accompanies cytochrome c release in pheochromocytoma-6 cells undergoing apoptosis.     

Requirement of cytochrome c for apoptosis in human cells

During apoptosis, cytochrome c released from mitochondria activates Apaf-1, a cofactor of caspase-9. The evidence that cytochrome c can activate Apaf-1 is abundant, but the proof that cytochrome c is required for apoptosis is limited to two studies that used genetically modified mice. One of these studies concluded that in some tissues apoptosis may require Apaf-1 but not cytochrome c, which indicated the need to analyze the requirement of cytochrome c beyond the mouse models, and in human tumor cells in particular. In this study, we designed tools to silence cytochrome c expression in human cells and tested these tools in an experimental system of oncogenic transformation. We found that cytochrome c was required for apoptosis induced by both DNA damage and, unexpectedly, TNFalpha. Overall, this study established that cytochrome c is required for apoptosis in human cells and provided tools to dissect mechanisms of apoptosis in various experimental models.     

Translation factor eIF4E rescues cells from Myc-dependent apoptosis by inhibiting cytochrome c release

Eukaryotic translation initiation factor 4E (eIF4E) markedly reduces cellular susceptibility to apoptosis. However, the mechanism by which the translation apparatus operates on the cellular apoptotic machinery remains uncertain. Here we show that eIF4E-mediated rescue from Myc-dependent apoptosis is accompanied by inhibition of mitochondrial cytochrome c release. Experiments achieving gain and loss of function demonstrate that eIF4E-mediated rescue is governed by pretranslational and translational activation of bcl-x as well as by additional intermediates acting directly on, or upstream of, the mitochondria. Thus, our data trace a pathway controlling apoptotic susceptibility that begins with the activity state of the protein synthesis machinery and leads to interdiction of the apoptotic program at the mitochondrial checkpoint.     

Multiple pathways of cytochrome c release from mitochondria in apoptosis

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating permeabilization of the outer mitochondrial membrane and the release of intermembrane space proteins remain controversial. Here, we discuss possible scenarios of the outer membrane permeabilization. The mechanisms by which the intermembrane space proteins are released from mitochondria depend presumably on cell type and on the nature of the apoptotic stimulus. The variety of mechanisms that can lead to outer membrane permeabilization might explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c

We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with alpha-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-x(L) and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.     

Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells

Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of Parkinson's disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis.     

Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators.     

Suppression of cytochrome c release and apoptosis in testes with heat stress by minocycline

Spermatogenic cells are susceptible to heat stress and undergo apoptosis. Although a variety of factors appear to be involved in the apoptotic process, the nature of the intracellular signaling pathway is ambiguous. To clarify the process, we chose a simple model in which testes of mice were exposed to mild heating by immersion in hot water at 42 degrees C for 15 min. In situ DNA fragmentation was detected by a TUNEL method. The release of cytochrome c into the cytoplasm was observed by Western blotting both in heat-treated testis and in isolated spermatogenic cells that had been incubated at 42 degrees C for 1h, but not in Sertoli cells. Minocycline, a semisynthetic tetracycline, is known to reach the brain by permeating the blood-brain barrier and suppresses apoptosis in neuronal cells. Since the testis also has a similar barrier, minocycline was examined as a possible agent to inhibit heat stress-induced apoptosis. The results indicate that minocycline suppressed the release of cytochrome c from mitochondria both in vivo and in vitro and significantly decreased the number of TUNEL-positive cells. These findings suggest that heat stress of testes triggers the release of cytochrome c from mitochondria in spermatogenic cells, leading to the activation of an apoptotic pathway.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.     

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

A Ca2+ signal is found upstream of cytochrome c release during apoptosis in HeLa cells

We showed previously that a cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in HeLa cells. In this study, we found evidence that this Ca(2+) signal occurs upstream of the release of cytochrome c from mitochondria. First, when we abolished [Ca(2+)](i) increases by injecting BAPTA or heparin into UV-treated HeLa cells, cytochrome c release was either blocked or severely delayed. Second, using a living cell imaging technique, we observed a series of transient [Ca(2+)](i) increases (typically lasting about 40-60s) in many apoptotic cells induced by either UV- or TNFalpha-treatment. Third, using GFP-tagged cytochrome c, we found that the Ca(2+) spikes appear in a time window before cytochrome c was released. Finally, by fixing the TNFalpha-treated cell at the time when it started to display Ca(2+) spikes, we examined the distribution of its endogenous cytochrome c using immunostaining. We found that cytochrome c was not yet released from mitochondria. These findings suggest the existence of certain apoptotic pathways, in which an early Ca(2+) signal is activated upstream of cytochrome c release.     

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.     

Different levels of reactive endogenous cytochrome c in mitochondria rich cells revealed by diaminobenzidine staining

Light microscopic examination of rat and mouse tissues incubated in a medium containing 3,3'-diaminobenzidine (DAB) and catalase revealed that cells known to possess abundant mitochondria (hepatocytes, cardiomyocytes, renal proximal and distal tubular cells, parietal cells of gastric mucosa, and retinal photoreceptor cells) were stained with different intensity: from moderate (parietal cells, cardiomyocytes, renal distal tubular cells) to weak (hepatocytes, renal proximal tubular cells) or even negative (photoreceptors). When exogenous cytochrome c was added to the incubation medium, all these cells displayed quite uniform, strong staining, indicating a comparable activity of cytochrome oxidase. Since DAB is oxidized directly by cytochrome c which in turn undergoes reoxidation by cytochrome oxidase, the observed differences of staining intensity in the absence of exogenous cytochrome c are postulated to result from different content of reactive endogenous cytochrome c in mitochondria of the investigated cells.     

A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c

Multiple apoptotic pathways release cytochrome c from the mitochondrial intermembrane space, resulting in the activation of downstream caspases. In vivo activation of Fas (CD95) resulted in increased permeability of the mitochondrial outer membrane and depletion of cytochrome c stores. Serial measurements of oxygen consumption, NADH redox state and membrane potential revealed a loss of respiratory state transitions. This tBID-induced respiratory failure did not require any caspase activity. At early time points, re-addition of exogenous cytochrome c markedly restored respiratory functions. Over time, however, mitochondria showed increasing irreversible respiratory dysfunction as well as diminished calcium buffering. Electron microscopy and tomographic reconstruction revealed asymmetric mitochondria with blebs of herniated matrix, distended inner membrane and partial loss of cristae structure. Thus, apoptogenic redistribution of cytochrome c is responsible for a distinct program of mitochondrial respiratory dysfunction, in addition to the activation of downstream caspases.