Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK(-) cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK(-) osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK(-) cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.     

A comparison of three flow cytometry methods for evaluating mitochondrial damage during staurosporine-induced apoptosis in Jurkat cells.

Measuring cytochrome c release during apoptosis provides valuable information about the nature and extent of apoptosis. Several years ago a flow cytometric method (based on selective permeabilization of the plasma membrane with digitonin) was developed that has advantages over other techniques. These experiments describe a comprehensive evaluation of that method. Apoptosis was triggered in Jurkat cells with staurosporine and then flow cytometry was used to measure three aspects of mitochondrial damage: (1) cytochrome c release (with the digitonin assay and a commercially available kit based on the same principle), using a DNA-binding dye to define cell cycle stage; (2) loss of mitochondrial cardiolipin, assessed by a decrease in 10 N-nonyl acridine orange (NAO) binding; and (3) loss of mitochondrial membrane potential, assessed by a decrease in tetramethylrhodamineethylester (TMRE) binding. The results from these three assays were compared with an antibody-based assay for cleaved caspase 3. The digitonin assay and the commercially available kit gave comparable results, showing that staurosporine caused cytochrome c release in all phases of the cell cycle and clearly defining those cells that had lost DNA due to internucleosomal DNA fragmentation. The pattern of fluorescence demonstrated that the mitochondrial apoptotic pathway was either the sole or the predominant pathway to be activated and that cytochrome c release in an individual cell was all-or-nothing. However, comparison with the other assays showed that the cytochrome c release assay underestimated the true extent of apoptosis. This was caused by the selective loss of some digitonin-treated apoptotic cells. The flow cytometry assay for cytochrome c release provides valuable information but it underestimates the percentage of apoptotic cells.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

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.     

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane.

BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.     

Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.     

Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.     

Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential

Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.     

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.     

Bioenergetic remodeling during cellular differentiation: changes in cytochrome c oxidase regulation do not affect the metabolic phenotype.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%-40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%-20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells

Mitochondrial respiration, the key process behind cellular energy production, is critical for cell proliferation, growth and survival. However, the regulation of mitochondrial respiratory function in tumor cells is not well understood. In this study, we propose a model whereby tumor cells possess the capacity to fine-tune the balance between energy demands and mitochondrial reactive oxygen species (ROS) status, to maintain a milieu optimal for survival. This is achieved through the moderation of mitochondrial respiration, depending on the ROS context within the organelle, with the main players being Bcl-2 and cytochrome c oxidase (COX). We report a higher level of COX activity, oxygen consumption and mitochondrial respiration in tumor cells overexpressing Bcl-2. Transient overexpression, gene silencing and pharmacological inhibition of Bcl-2 corroborate these findings. Interestingly, Bcl-2 is also able to regulate mitochondrial respiration and COX activity in the face of mounting ROS levels, triggered by mitochondrial complex inhibitors. In this respect, it is plausible to suggest that Bcl-2 may be able to create an environment, most suited for survival by adjusting mitochondrial respiration accordingly to meet energy requirements, without incurring an overwhelming, detrimental increase in intracellular ROS.     

Mitochondrial function in glucocorticoid triggered T-ALL cells with transgenic bcl-2 expression

Independent of apoptosis, dexamethasone induced and a decrease of respiration and citrate synthase activity per cell in cells with and without transgenic Bcl-2 expression. The reduction of respiration, however, was slightly, but statistically more pronounced in apoptotic cells compared to non-apoptotic Bcl-2 over-expressing cells. A slight cytochrome c release was detected in apoptotic cells only. Importantly, the stimulatory effect of FCCP was maintained, indicating that oxidative phosphorylation remained coupled in active mitochondria. Coupled and uncoupled respiration were reduced to almost identical degrees as the activities of the marker enzymes citrate synthase (matrix) and cytochrome c oxidase (respiratory chain). Therefore, the reduction of cellular respiration was mainly caused by a decrease in mitochondrial content per cell. The functional integrity of mitochondria was preserved, apart from the slight degree of cytochrome c release, either through a pore formed by the oligomerisation of BAK in coupled mitochondria or by permeability transition of a small fraction of injured mitochondria.     

Dissociation of cytochrome c from the inner mitochondrial membrane during cardiac ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.     

Estrogens prevent calcium-induced release of cytochrome c from heart mitochondria

We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium-induced release of cytochrome c from mitochondria. 10 nM-10 microM 17beta-estradiol or 4-hydroxytamoxifen did not affect mitochondrial respiration rate and membrane potential in state 3 and state 4. Higher concentrations of both agents decreased state 3 respiration rate and membrane potential. 100 nM 17beta-estradiol and 4-hydroxytamoxifen blocked high calcium-induced cytochrome c release from mitochondria but not mitochondrial swelling. Thus, at physiological concentrations estrogens do not affect mitochondrial respiratory functions but protect heart mitochondria from high calcium-induced release of cytochrome c.     

Cytochrome c is released in a single step during apoptosis

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).