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.     

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.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria.     

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.     

Mitochondrial cytochrome c release precedes transmembrane depolarisation and caspase-3 activation during ceramide-induced apoptosis of Jurkat T cells

Whilst the role of ceramide, a second messenger of the sphingolipid family, in the initiation of receptor-mediated apoptosis is controversial, a growing body of evidence is emerging for a role of ceramide in the amplification of apoptosis via mitochondrial perturbations that culminate in the activation of execution caspases. Treatment of Jurkat T cells with the cell-permeable analog, C₂-ceramide, resulted in the rapid onset of apoptosis as evidenced by Annexin V-FITC staining of externalised phosphatidylserine residues. Cells bearing this early apoptotic marker had a reduced mitochondrial transmembrane potential (m) that was preceded by the release of cytochrome c from mitochondria. Subsequent activation of caspase-3 provides the link between these ceramide-induced mitochondrial changes and execution caspases that ultimately result in the physical destruction of the cell. Collectively these results demonstrate that ceramide signalling results in caspase-mediated apoptosis via mitochondrial cytochrome c release and are further supportive of the role of ceramide in the amplification of apoptosis.     

Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.     

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.     

Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.     

Caspase-mediated Bak activation and cytochrome c release during intrinsic apoptotic cell death in Jurkat cells

Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.     

A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.

The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.     

Ethyl-nitrosourea transformed astrocytes exhibit mitochondrial membrane hyperpolarization and constrained apoptosis

Recent studies have shown the mitochondria and mitochondrial DNA are altered in gliomas, studied either as primary tissues or in culture. Few studies have been performed which evaluate the mitochondria during the development of glial malignancy. We used an ethyl-nitrosourea (ENU) in vitro model to assess the changes in mitochondrial parameters with progression to astrocyte transformation. When compared to the untreated control cells mitochondrial mass of the ENU treated cells significantly decreased ontologically early with concurrent increase in mitochondrial membrane potential, resulting in hyperpolarization of the mitochondrial membrane. At successive divisions, the degree of spontaneous apoptosis during astrocyte transformation was significantly diminished in the ENU treated cells. With 24 h pre- and co-treatment of ENU cells with citrate, an allosteric inhibitor of phosphofructokinase, the astrocytes still became immortal, but did not manifest any of the mitochondrial changes nor acquire the transformed properties of the ENU treated cells without the inhibition. Indeed, the degree of apoptosis noted in these dually treated cells was increased, associated with a loss of anchorage independence and low density growth. Transformed subclones exposed to citrate after the development of malignant properties also exhibited increased apoptosis, and did not form colonies in low density plating conditions. These results suggest that the development of transformed properties in an ENU model is associated with marked hyperpolarization of mitochondrial membrane potential and diminished spontaneous apoptosis. Exposure to citrate attenuated these mitochondrial changes and in vitro growth properties, with increases in apoptosis. The development of transformed astrocytes involve constraints on apoptosis related to alterations in mitochondrial membrane potential and mass.     

Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release

Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (Deltapsi(m)) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of Deltapsi(m) and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca(2+), atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca(2+) sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.     

Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.     

p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.     

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.     

A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis

Treatment of HL-60 cells with staurosporine (STS) induced mitochondrial cytochrome c efflux into the cytosol, which was followed by caspase-3 activation and apoptosis. Consistent with these observations, in vitro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required for caspase-3 activation. In contrast, treatment of HCW-2 cells (an apoptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, and apoptosis. In vitro assays strongly suggested that a lack of cytochrome c in the cytosol was the primary limiting factor for caspase-3 activation in HCW-2 cells. To explore the mechanism which regulates mitochondrial cytochrome c efflux, we developed an in vitro assay which showed that cytosolic extracts from STS-treated, but not untreated, HL-60 cells contained an activity, which we designated 'CIF' (cytochrome c-efflux inducing factor), which rapidly induced cytochrome c efflux from HL-60 mitochondria. In contrast, there was no detectable CIF activity in STS-treated HCW-2 cells although the mitochondria from HCW-2 cells were responsive to the CIF activity from STS-treated HL-60 cells. These experiments have identified a novel activity, CIF, which is required for cytochrome c efflux and they indicate that the absence of CIF is the biochemical explanation for the impaired ability of HCW-2 cells to activate caspase-3 and undergo apoptosis.     

Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia.     

Cardiolipin deficiency releases cytochrome c from the inner mitochondrial membrane and accelerates stimuli-elicited apoptosis

Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.