Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P10415 (Bcl-2)
 33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent reports substantiating the role of cytochrome c in the induction of apoptosis led us to examine the kinetics and mechanisms involved in this process as an extension of our ongoing studies of cell injury and cell death. Microinjection of cytochrome c into NRK-52E kidney cells produced rapid apoptosis, which usually began within 30 minutes and reached a maximum of 60-70% by 3 hours. The changes that occurred included four phases: an initial shrinkage phase, an active phase, a spherical phase, and a necrotic phase. For morphological purposes, the progressive changes were followed by phase-contrast and fluorescence microscopy, transmission and scanning electron microscopy, and time-lapse video microscopy. Cells first showed shrinkage, then displayed multiple pseudopods, which rapidly extended and retracted, giving the cells a bosselated appearance. During this active phase there was chromatin condensation, mitochondria were swollen but retained membrane potential, and the endoplasmic reticulum was dilated. Within 2-4 hours, active-phase cells became spherical and smooth-surfaced but were still alive, the nuclei showed chromatin clumping, the mitochondria underwent high-amplitude swelling but retained membrane potential, the endoplasmic reticulum was highly dilated, and many large apical vacuoles were present. Elevation of [Ca(2+)](i) was seen at the late spherical phase, shortly before cell death. Pretreatment with the caspase 3 inhibitor (Ac-DEVD-CHO) prevented apoptosis, whereas overexpression of Bcl-2 did not. Depletion of cellular ATP by cyanide inhibition of energy metabolism prevented cytochrome c from inducing the active and later phases of apoptosis. The results clearly indicate that cytochrome c-induced apoptosis is a dynamic and energy-requiring process that has a distinct active and spherical phase before cell death.
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PMID:Studies on the mechanisms and kinetics of apoptosis induced by microinjection of cytochrome c in rat kidney tubule epithelial cells (NRK-52E). 1066 93

Cytochrome c (cyto c) release from mitochondria is a critical event in apoptosis. By investigating the ordering of molecular events during genotoxic stress-induced apoptosis, we found that ionizing radiation (IR) and etoposide induced the release of cyto c from mitochondria in two distinct stages. The early release of low levels of cyto c into the cytosol preceded the activation of caspase 9 and 3, but had no effect on ATP levels or mitochrondrial transmembrane potential (Deltapsim). In contrast, the late stage cyto c release resulted in a drastic loss of mitochondrial cyto c and was associated with reduction of ATP levels and Deltapsim. Moreover, caspases contributed to the late cyto c release since the caspase inhibitor zVAD prevented only the late but not the early-stage cyto c release. Recombinant caspase 3 induced cyto c release from isolated mitochondria in the absence of cytosolic factors. Bcl-2 but not Bid was cleaved during apoptosis after caspase activation. This suggests that Bcl-2 cleavage might contribute to the late cyto c release, which results in mitochondrial dysfunction manifested by the decrease of ATP and Deltapsim. zVAD prevented the reduction of ATP, Deltapsim, and nuclear condensation when added up to 8 h after IR, at the time the caspases were highly activated but when the majority of cyto c was still maintained in the mitochondria. These findings link the feedback loop control of caspase-induced cyto c release with mitochondrial dysfunction manifested by ATP and Deltapsim decline.
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PMID:Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. 1071 37

Previously we reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondria/cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. We also established a cell-free Bax degradation assay in which an in vitro-translated, (35)S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. Our studies strongly suggest that ubiquitin/proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.
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PMID:Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. 1072

Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein. As well as effluxing xenotoxins, functional P-gp can confer resistance to caspase-dependent apoptosis induced by a range of different stimuli, including Fas ligand, tumor necrosis factor, UV irradiation, and serum starvation. However, P-gp-positive cells remain sensitive to caspase-independent death induced by cytotoxic T-cell granule proteins, perforin, and granzyme B. It is, therefore, possible that agents that induce cell death in a caspase-independent manner might circumvent P-gp-mediated MDR. We demonstrated here that hexamethylene bisacetamide (HMBA) induced equivalent caspase-independent cell death in both P-gp-positive and -negative cell lines at concentrations of 10 mmol/L and above. The HMBA-induced death pathway was marked by release of cytochrome c from the mitochondria and reduction of Bcl-2 protein levels. In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected. These studies greatly enhance our understanding of the molecular cell death events that can be regulated by functional P-gp and highlight the potential clinical use of drugs that function via a caspase-independent pathway for the treatment of MDR tumors.
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PMID:HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance. 1073 10

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.
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PMID:Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. 1074 29

The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.
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PMID:Role of oxidative phosphorylation in Bax toxicity. 1077 48

Coupled cellular respiration requires that ATP and ADP be efficiently exchanged between the cytosol and the mitochondrial matrix. When growth factors are withdrawn from dependent cells, metabolism is disrupted by a defect in ATP/ADP exchange across the mitochondrial membranes. Unexpectedly, we find that this defect results from loss of outer mitochondrial membrane permeability to metabolic anions. This decrease in anion permeability correlates with the changes in conductance properties that accompany closure of the voltage-dependent anion channel (also known as mitochondrial porin). Loss of outer membrane permeability (i) results in the accumulation of stored metabolic energy within the intermembrane space in the form of creatine phosphate, (ii) is prevented by the outer mitochondrial membrane proteins Bcl-x(L) and Bcl-2, and (iii) can be reversed by growth factor readdition. If outer membrane impermeability persists, the disruption of mitochondrial homeostasis culminates in loss of outer mitochondrial membrane integrity, cytochrome c redistribution, and apoptosis. The recognition that outer membrane permeability is regulated under physiological conditions has important implications for the understanding of bioenergetics and cell survival.
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PMID:Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. 1078 Oct 72

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.
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PMID:Mitochondria and cell death. 1081 21

We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O-*2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.
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PMID:The role of a mitochondrial pathway in the induction of apoptosis by chemicals extracted from diesel exhaust particles. 1094 1

Exposure to 1,3-dinitrobenzene (DNB) is associated with neuropathologic changes in specific brainstem nuclei, mediated by oxidative stress and mitochondrial dysfunction. The expression of Bcl-2-family proteins as a function of sensitivity to 1, 3-dinitrobenzene (DNB)-induced mitochondrial permeability transition (MPT) was examined in C6 glioma and SY5Y neuroblastoma cells. Neuroblastoma cells were 10-fold more sensitive than glioma cells to DNB-induced decreases in mitochondrial reducing potential, measured by reduction of the tetrazolium compound, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The IC(50) values for DNB-related inhibition of MTT reduction were 107+/-25 microM in SY5Y cells and 1047+/-101 microM in C6 cells. Levels of reactive oxygen species (ROS) were increased in both SY5Y and C6 cells following DNB exposure by 4.6- and 6.0-fold above control, respectively. DNB caused abrupt depolarization of mitochondria in both neuroblastoma and glioma cells that was inhibited by trifluoperazine. The first order rate constants for mitochondrial depolarization were: C6, k=0.31+/-0.02 min(-1); SY5Y, k=0.14+/-0.01 min(-1). Onset of MPT occurred at 10-fold lower concentration of DNB in SY5Y cells than in C6 cells. The antioxidants, deferoxamine and alpha-tocopherol, effectively prevented DNB-induced MPT in C6 and SY5Y cells, suggesting involvement of ROS in the initiation of MPT. Exposure to DNB resulted in decreased cellular ATP content in SY5Y cells and efflux of mitochondrial calcium in both SY5Y and C6 cells, concurrent with onset of MPT. The expression of Bcl-2, Bcl-X(L), and Bax was evaluated in both cell types by Western blot analysis. C6 glioma cells strongly expressed Bcl-X(L) and only weakly expressed Bcl-2 and Bax, whereas SY5Y neuroblastoma cells expressed lower levels of Bcl-X(L) and higher levels of both Bcl-2 and Bax. Collectively, these results suggest that higher constitutive expression of Bcl-X(L), rather than Bcl-2, correlates with resistance to DNB-induced MPT in SY5Y and C6 cells and that differential regulation of the permeability transition pore may underlie the cell-specific neurotoxicity of DNB.
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PMID:Differential cellular regulation of the mitochondrial permeability transition in an in vitro model of 1,3-dinitrobenzene-induced encephalopathy. 1096 Jun 1


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