The Mitochondrial Permeability Transition Is Required for Tumor Necrosis Factor Alpha-Mediated Apoptosis and Cytochrome c Release

ABSTRACT This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IκB superrepressor, tumor necrosis factor alpha (TNFα) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFα induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFα treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFα treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFα-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing ΔFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFα, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFα-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.

Download Full-text

Critical involvement of transmembrane tumor necrosis factor-alpha in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin

Blood ◽

10.1182/blood.v86.11.4184.bloodjournal86114184 ◽

1995 ◽

Vol 86 (11) ◽

pp. 4184-4193 ◽

Cited By ~ 129

Author(s):

G Eissner ◽

F Kohlhuber ◽

M Grell ◽

M Ueffing ◽

P Scheurich ◽

...

Keyword(s):

Cell Death ◽

Tumor Necrosis Factor ◽

Programmed Cell Death ◽

Ionizing Radiation ◽

Tumor Necrosis Factor Alpha ◽

Tumor Necrosis ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Induced Apoptosis ◽

Necrosis Factor

In this report, we show that ionizing radiation (IR) at a clinically relevant dose (4 Gy) causes apoptosis in macrovascular and microvascular human endothelial cells. Treatment of irradiated cells with a low dose of bacterial endotoxin (LPS), similar to the levels observed in serum during endotoxemia, enhanced the rate of apoptosis, although LPS alone was unable to induce programmed cell death. The cytokine and endotoxin antagonist interleukin-10 (IL-10) reduced the rate of LPS + IR-induced apoptosis to levels obtained with irradiation alone. Using neutralizing antibodies against tumor necrosis factor- alpha (TNF), we could show crucial involvement of TNF in the LPS- mediated enhancement of IR-induced apoptosis, but not in the IR-induced apoptosis per se. However, further analysis strongly suggested the transmembrane form of TNF (mTNF), but not soluble TNF, to be accountable for the LPS-mediated cytotoxic effects. Studies with anatagonistic receptor specific antibodies clearly showed that TNF receptor type I (TR60) is essential and sufficient to elicit this effect. These findings are of potential clinical importance because they may disclose a relevant mechanism that leads to endothelial damage after radiotherapy or total body irradiation used for conditioning in bone marrow transplantation and that may thus contribute to transplant related complications, especially in association with endotoxemia or related inflammatory states.

Download Full-text

Adenine Nucleotide (ADP/ATP) Translocase 3 Participates in the Tumor Necrosis Factor–induced Apoptosis of MCF-7 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-12-1161 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4681-4689 ◽

Cited By ~ 22

Author(s):

Ziqiang Yang ◽

Wei Cheng ◽

Lixin Hong ◽

Wanze Chen ◽

Yanhai Wang ◽

...

Keyword(s):

Cell Death ◽

Mitochondrial Permeability Transition ◽

Adenine Nucleotide ◽

Permeability Transition ◽

Mitochondrial Permeability ◽

Tnf Α ◽

Induced Apoptosis ◽

Necrosis Factor ◽

Mcf 7

Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor α (TNF-α)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion–mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-α–induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress–induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-α–induced apoptosis observed in ANT3 mutant (ANT3mut) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type– and stimulus-dependent.

Download Full-text

Arachidonic Acid Causes Cell Death through the Mitochondrial Permeability Transition

Journal of Biological Chemistry ◽

10.1074/jbc.m010603200 ◽

2000 ◽

Vol 276 (15) ◽

pp. 12035-12040 ◽

Cited By ~ 214

Author(s):

Luca Scorrano ◽

Daniele Penzo ◽

Valeria Petronilli ◽

Francesco Pagano ◽

Paolo Bernardi

Keyword(s):

Cell Death ◽

Arachidonic Acid ◽

Tumor Necrosis ◽

Aristolochic Acid ◽

Mitochondrial Permeability Transition ◽

Permeability Transition ◽

Mitochondrial Permeability ◽

Factor Α ◽

Necrosis Factor

We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent depolarization or respiratory inhibition, suggesting a specific effect on the PT pore. When added to intact MH1C1 cells, arachidonic acid but not palmitic acid caused a mitochondrial PTin situthat was accompanied by cytochromecrelease and rapidly followed by cell death. All these effects of arachidonic acid could be prevented by cyclosporin A but not by the phospholipase A2inhibitor aristolochic acid. In contrast, tumor necrosis factor α caused phospholipid hydrolysis, induction of the PT, cytochromecrelease, and cell death that could be inhibited by both cyclosporin A and aristolochic acid. These findings suggest that arachidonic acid produced by cytosolic phospholipase A2may be a mediator of tumor necrosis factor α cytotoxicityin situthrough induction of the mitochondrial PT.

Download Full-text

Cytochrome c-Dependent Activation of Caspase-3 by Tumor Necrosis Factor Requires Induction of the Mitochondrial Permeability Transition

American Journal Of Pathology ◽

10.1016/s0002-9440(10)65082-1 ◽

2000 ◽

Vol 156 (6) ◽

pp. 2111-2121 ◽

Cited By ~ 62

Author(s):

Marco Tafani ◽

Timothy G. Schneider ◽

John G. Pastorino ◽

John L. Farber

Keyword(s):

Tumor Necrosis Factor ◽

Cytochrome C ◽

Tumor Necrosis ◽

Caspase 3 ◽

Mitochondrial Permeability Transition ◽

Permeability Transition ◽

Mitochondrial Permeability ◽

Necrosis Factor

Download Full-text

Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death.

Journal of Clinical Investigation ◽

10.1172/jci119114 ◽

1996 ◽

Vol 98 (12) ◽

pp. 2854-2865 ◽

Cited By ~ 506

Author(s):

K A Krown ◽

M T Page ◽

C Nguyen ◽

D Zechner ◽

V Gutierrez ◽

...

Keyword(s):

Cell Death ◽

Tumor Necrosis Factor ◽

Tumor Necrosis Factor Alpha ◽

Tumor Necrosis ◽

Signaling Cascade ◽

Cardiac Cell ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Induced Apoptosis ◽

Necrosis Factor

Download Full-text

Transcription Factor AP-2α Is Preferentially Cleaved by Caspase 6 and Degraded by Proteasome during Tumor Necrosis Factor Alpha-Induced Apoptosis in Breast Cancer Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.4856-4867.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 4856-4867 ◽

Cited By ~ 32

Author(s):

Okot Nyormoi ◽

Zhi Wang ◽

Dao Doan ◽

Maribelis Ruiz ◽

David McConkey ◽

...

Keyword(s):

Breast Cancer ◽

Breast Cancer Cells ◽

Tumor Necrosis ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Factor Alpha ◽

Caspase 6 ◽

Induced Apoptosis ◽

Necrosis Factor

ABSTRACT Several reports have linked activating protein 2α (AP-2α) to apoptosis, leading us to hypothesize that AP-2α is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-α) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF-α downregulates AP-2α and AP-2γ expression posttranscriptionally during TNF-α-induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF-α-induced apoptosis and AP-2α downregulation. In vivo tests showed that AP-2α was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2α preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF-α-treated cells, thus confirming its involvement in AP-2α cleavage. All three caspases cleaved AP-2α at asp19 of the sequence asp-arg-his-asp (DRHD19). Mutating D19 to A19abrogated AP-2α cleavage by all three caspases. TNF-α-induced cleavage of AP-2α in vivo led to AP-2α degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2α degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2α is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2α are resistant to TNF-α-induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2α and apoptosis. This is the first report to demonstrate that degradation of AP-2α is a critical event in TNF-α-induced apoptosis. Since the DRHD sequence in vertebrate AP-2 is widely conserved, its cleavage by caspases may represent an important mechanism for regulating cell survival, proliferation, differentiation, and apoptosis.

Download Full-text