Pinocytic loading of cytochrome c into intact cells specifically induces caspase-dependent permeabilization of mitochondria: evidence for a cytochrome c feedback loop

Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition.

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RESPIRATORY CARRIERS AND THE NATURE OF THE REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE OXIDASE SYSTEM IN XANTHOMONAS PHASEOLI

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o60-010 ◽

1960 ◽

Vol 38 (1) ◽

pp. 79-93 ◽

Cited By ~ 1

Author(s):

R. M. Hochster ◽

C. G. Nozzolillo

Keyword(s):

Cytochrome C ◽

Reductase Activity ◽

Inorganic Ions ◽

Product Formation ◽

Intact Cells ◽

Oxidase System ◽

Clear Cut ◽

Highly Active ◽

Inhibitor Study ◽

Terminal Oxidation

Intact cells and cell-free extracts of the phytopathogenic organism Xanthomonas phaseoli have been shown to contain flavoprotein and the respiratory carriers: cytochrome b1, cytochrome a1, and cytochrome a2. The reduced forms of these respiratory pigments are produced upon addition to a clear extract of substrate amounts of DPNH.The highly active DPNH oxidase system in extracts of this organism has been studied as to requirements for inorganic ions, optimum pH, product formation, distribution, and solubilization. Carbon monoxide inhibits the terminal oxidation system; this effect is reversed by bright light.An inhibitor study has shown members of the phenothiazine family of compounds to be most effective, followed by amytal, cyanide, BAL, atabrine, and pCMB. The most notable of the substances which did not inhibit were antimycin A, one of the quinoline-N-oxides, and azide.The possibility exists that H2O2may also be formed during the oxidation of DPNH although clear-cut evidence for its presence was difficult to obtain. X. phaseoli extracts do not contain a DPNH peroxidase. They exhibit, however, some DPNH – cytochrome c reductase activity which is believed to be quite independent of the DPNH oxidase system. The extracts are devoid of cytochrome c oxidase activity although they contain a respiratory system which readily oxidizes p-phenylenediamine.

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Spectroscopic studies of Rhodobacter capsulatus cytochrome c′ in the isolated state and in intact cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/0005-2728(92)90080-l ◽

1992 ◽

Vol 1100 (2) ◽

pp. 184-188 ◽

Cited By ~ 13

Author(s):

Fayez Monkara ◽

Stephen J. Bingham ◽

Fahmi H.A. Kadir ◽

Alastair G. McEwan ◽

Andrew J. Thomson ◽

...

Keyword(s):

Cytochrome C ◽

Rhodobacter Capsulatus ◽

Spectroscopic Studies ◽

Intact Cells

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Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release

Cell Death and Differentiation ◽

10.1038/sj.cdd.4400689 ◽

2000 ◽

Vol 7 (6) ◽

pp. 556-565 ◽

Cited By ~ 198

Author(s):

E A Slee ◽

S A Keogh ◽

S J Martin

Keyword(s):

Cytochrome C ◽

Uv Radiation ◽

Feedback Loop ◽

Caspase 3 ◽

Cytotoxic Drug ◽

Mitochondrial Cytochrome ◽

Cytochrome C Release ◽

Radiation Induced ◽

Induced Apoptosis ◽

Mitochondrial Cytochrome C

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Oxygen affinity of the respiratory chain of Acanthamoeba castellanii

Biochemical Journal ◽

10.1042/bj2140047 ◽

1983 ◽

Vol 214 (1) ◽

pp. 47-51 ◽

Cited By ~ 15

Author(s):

D Lloyd ◽

H Mellor ◽

J L Williams

Keyword(s):

Plasma Membrane ◽

Cytochrome C ◽

Cytochrome B ◽

Respiratory Chain ◽

Oxygen Affinity ◽

Acanthamoeba Castellanii ◽

Intact Cells ◽

Cytochrome Aa3 ◽

Isolated Mitochondria ◽

Soil Amoeba

Apparent Km values for O2 for the soil amoeba Acanthamoeba castellanii determined polarographically and by bioluminescence gave similar values (0.37 and 0.41 microM respectively). Mitochondria oxidizing succinate or NADH in the presence or absence of ADP gave values in the range 0.21-0.36 microM-O2. Oxidation of respiratory-chain components to 50% of the aerobic steady states in intact cells was observed at the following O2 concentrations: cytochrome aa3, 0.1-0.25 microM; cytochrome c, 0.3-0.6 microM; cytochrome b, 0.35-0.45 microM; flavoprotein, 2 microM. In isolated mitochondria corresponding values for a-, c- and b-type cytochromes were 0.007, 0.035-0.05 and 0.06-0.09 microM-O2. It is concluded that an O2 gradient exists between plasma membrane and mitochondria in A. castellanii.

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Aerobic and anaerobic respiratory systems in Campylobacter fetus subsp. jejuni grown in atmospheres containing hydrogen

Journal of Bacteriology ◽

10.1128/jb.152.1.306-314.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 306-314

Author(s):

G M Carlone ◽

J Lascelles

Keyword(s):

Cytochrome C ◽

Cytochrome B ◽

System Analysis ◽

Maximum Growth ◽

Oxidoreductase Activity ◽

Intact Cells ◽

Cytochrome O ◽

Campylobacter Fetus ◽

Fetus Subsp ◽

Carbon Monoxide Binding

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.

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KERNICTERUS

PEDIATRICS ◽

10.1542/peds.17.6.925 ◽

1956 ◽

Vol 17 (6) ◽

pp. 925-928

Author(s):

Richard Day

Keyword(s):

Cytochrome C ◽

Oxygen Uptake ◽

Intraperitoneal Injection ◽

Tetrahymena Pyriformis ◽

Newborn Rats ◽

Intact Cells ◽

Full Activity ◽

Mouse Diaphragm

Toxicity of heme pigments, in addition to that previously reported has been demonstrated as follows: By depression of oxygen uptake by bilirubin bathing relatively intact cells of yeast and diaphragm; by hematin bathing yeast, diaphragm and minced brain, and by mesobilirubin bathing mouse diaphragm. Oxidation cancels the toxicity of bilirubin. Cytochrome C cancels the toxicity of bilirubin and hematin. Mesobilirubin was not tested with cytochrome C. By immobilization of Tetrahymena pyriformis by bilirubin at a concentration as low as 15 mg./100 ml. Oxidation of the bilirubin or the addition of cytochrome C cancels the action of the bilirubin. Hematin does not immobilize the animals. Removal of the bilirubin restores the protozoa to full activity. Survival of newborn rats following intraperitoneal injection with bilirubin or hematin is impaired.

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Cytochrome c Induces Caspase-Dependent Apoptosis in Intact Hematopoietic Cells and Overrides Apoptosis Suppression Mediated by bcl-2, Growth Factor Signaling, MAP-Kinase-Kinase, and Malignant Change

Blood ◽

10.1182/blood.v92.4.1235.416k18_1235_1246 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1235-1246 ◽

Cited By ~ 2

Author(s):

John M. Garland ◽

Claudius Rudin

Keyword(s):

Growth Factor ◽

Cytochrome C ◽

Map Kinase ◽

Leukemic Cell ◽

Growth Factor Signaling ◽

Intact Cells ◽

Cell Extracts ◽

American Society ◽

Induced Apoptosis ◽

Map Kinase Kinase

It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)–dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3–dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control. © 1998 by The American Society of Hematology.

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Cytochrome c Induces Caspase-Dependent Apoptosis in Intact Hematopoietic Cells and Overrides Apoptosis Suppression Mediated by bcl-2, Growth Factor Signaling, MAP-Kinase-Kinase, and Malignant Change

Blood ◽

10.1182/blood.v92.4.1235 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1235-1246 ◽

Cited By ~ 43

Author(s):

John M. Garland ◽

Claudius Rudin

Keyword(s):

Growth Factor ◽

Cytochrome C ◽

Map Kinase ◽

Leukemic Cell ◽

Growth Factor Signaling ◽

Intact Cells ◽

Cell Extracts ◽

American Society ◽

Induced Apoptosis ◽

Map Kinase Kinase

Abstract It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)–dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3–dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control. © 1998 by The American Society of Hematology.

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Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10289-10299.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10289-10299 ◽

Cited By ~ 33

Author(s):

Paula B. Deming ◽

Zachary T. Schafer ◽

Jessica S. Tashker ◽

Malia B. Potts ◽

Mohanish Deshmukh ◽

...

Keyword(s):

Cell Death ◽

Cytochrome C ◽

Mitochondrial Cytochrome ◽

Caspase Activation ◽

Caspase 9 ◽

Cytochrome C Release ◽

Intact Cells ◽

Caspase Recruitment Domain ◽

Distinct Mechanism ◽

Mitochondrial Cytochrome C

ABSTRACT 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.

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