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

2000; Elsevier BV; Volume: 156; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)65082-1

1525-2191

Marco Tafani, Timothy Schneider, John G. Pastorino, John L. Farber,

Cell death mechanisms and regulation

The killing of L929 mouse fibroblasts by tumor necrosis factor-α (TNF-α) in the presence of 0.5 μg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-α alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-α causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-α can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8. The killing of L929 mouse fibroblasts by tumor necrosis factor-α (TNF-α) in the presence of 0.5 μg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-α alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-α causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-α can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8. The observation that cytochrome c can function as an activator of caspases1Zou H Henzel WJ Liu X Lutschg A Wang X Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3.Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar brought together two important aspects of the current interest in the mechanisms mediating programmed cell death (apoptosis). Considerable evidence implicates the caspases in this process.2Enari M Hug H Nagata S Involvement of an ICE-like protease in Fas-mediated apoptosis.Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar The caspases are cysteine proteases related to interleukin 1β-converting enzyme (ICE/caspase-1). At the same time, altered mitochondrial function is increasingly recognized as playing a critical role in apoptosis.3Reed JC Double identity for proteins of the Bcl-2 family.Nature. 1997; 387: 773-776Crossref PubMed Scopus (1391) Google Scholar, 4Orrenius S Burgess DH Hampton MB Zhivotovsky B Mitochondria as the focus of apoptosis research.Cell Death Differ. 1997; 4: 427-428Crossref PubMed Scopus (29) Google Scholar The mitochondrial permeability transition (MPT) is a well known alteration implicated as a mechanism of cell injury. The MPT refers to the regulated opening of a large, nonspecific pore in the inner mitochondrial membrane.5Gunter TE Gunter KK Sheu SS Gavin CE Mitochondrial calcium transport: physiological and pathological relevance.Am J Physiol. 1994; 267: C313-C339PubMed Google Scholar, 6Bernardi P Broekemeier KM Pfeiffer DR Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane.J Bioenerg Biomembr. 1994; 26: 509-517Crossref PubMed Scopus (528) Google Scholar, 7Zoratti M Szabò I The mitochondrial permeability transition.Biochim Biophys Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2199) Google Scholar Although the molecular elements that form this pore have not been definitively established, they are presumed to derive from well known membrane constituents, including the adenine nucleotide translocator, porin molecules, and the complex forming the peripheral benzodiazepine receptor.8McEnery MW Snowman AM Trifiletti RR Snyder SH Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier.Proc Natl Acad Sci USA. 1992; 89: 3170-3174Crossref PubMed Scopus (674) Google Scholar, 9McEnery MW The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel (VDAC).J Bioenerg Biomemr. 1992; 24: 63-69Crossref PubMed Scopus (68) Google Scholar The MPT is a critical event in the killing of cells that follows an inhibition of mitochondrial electron transport by anoxia.10Pastorino JG Snyder JW Serroni A Hoek JB Farber JL Cyclosporin and carnitine prevent the anoxic death of cultured hepatocytes by inhibiting the mitochondrial permeability transition.J Biol Chem. 1993; 268: 13791-13798Abstract Full Text PDF PubMed Google Scholar The MPT has also been implicated as a mechanism of mitochondrial dysfunction in apoptosis.11Zamzami N Hirsch T Dallaporta B Petit PX Kroemer G Mitochondrial implication in accidental and programmed cell death: apoptosis and necrosis.J Bioenerg Biomembr. 1997; 29: 185-193Crossref PubMed Scopus (304) Google Scholar We have shown that the overexpression of Bax in Jurkat lymphocytes results in the killing of cells by a process that displays many of the features associated with apoptosis.12Pastorino JG Chen S-T Tafani M Snyder JW Farber JL The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition.J Biol Chem. 1998; 273: 7770-7775Crossref PubMed Scopus (535) Google Scholar In particular, the overexpression of Bax in the Jurkat cells induces the MPT, an event that is responsible for the loss of viability. The MPT is accompanied by the release of cytochrome c and, in turn, caspase-3 activation with the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) and the fragmentation of DNA. Inhibition of the MPT by cyclosporin A (CyA) prevented all manifestations of apoptosis, whereas caspase-3 inhibition prevented PARP cleavage and DNA fragmentation. The caspase-3 inhibitor was without effect on induction of the MPT and its functional consequences, namely cell death and the release of cytochrome c. Several important concerns were left unresolved by this earlier study. In particular, one needs to know how relevant the findings that occurred with Bax overexpression are to cell death occurring in other models. The killing of L929 mouse fibroblasts by tumor necrosis factor-α (TNF-α) also depends on induction of the MPT.13Pastorino JG Simbula G Yamamoto K Glascott Jr, PA Rothman RJ Farber JL The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.J Biol Chem. 1996; 271: 29792-29798Crossref PubMed Scopus (254) Google Scholar In this model, both the MPT and the cell killing induced by TNF-α are inhibited by CyA. Thus, the killing of L929 fibroblasts by TNF-α provides a very convenient system with which to further explore the nature and consequences of mitochondrial dysfunction in lethal cell injury. In the present study, we demonstrate that TNF-α causes a redistribution of cytochrome c from the mitochondria to the cytosol that is dependent on the induction of the MPT. In addition, the functional consequences of this redistribution of cytochrome c and their relationship to the loss of cell viability are defined. The L929 line of mouse fibroblasts (ATCC-CCL-1, American Type Culture Collection, Manassas, VA) was maintained in 25-cm2 polystyrene flasks (Corning Costar Corp., Oneonta, NY) with 5 ml of Dulbecco's modified Eagle's medium (DMEM. high glucose, without pyruvate; MediaTech), containing 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (complete DMEM) and incubated under an atmosphere of 95. air and 5% CO2. For all experiments, cells were plated at a density of 200,000/cm2 in complete DMEM. After overnight incubation the cells were washed twice with phosphate-buffered saline (PBS) and placed in DMEM without serum. In all experiments TNF-α (Sigma, St. Louis, MO) was added to a final concentration of 2 ng/ml (22 units/ng). TNF-α was dissolved in PBS and added to the cells in 0.2% volume. Act D (Sigma. was dissolved in dimethyl sulfoxide (DMSO), further diluted in PBS, and added in 0.2% volume to a final concentration of 0.5 μg/ml. Where indicated the cells were pretreated for 30 minutes with the following reagents before addition of TNF-α and Act D. Cyclosporin A (Biomol, Plymouth Meeting, PA) was dissolved in DMSO and added in a 0.2. volume to the cell culture media to give a final concentration of 5 μmol/L. Aristolochic acid (Biomol) was dissolved in PBS and added in a 0.2% volume to give a final concentration of 50 μmol/L. The cell permeable caspase-3 inhibitor (Z-Asp-Glu-Val-aspartic acid fluoromethylketone, Z-DEVD-FMK) and the cell permeable caspase-8 inhibitor (Z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone, Z-IETD-FMK. Kamyia Biomedical Co., Seattle, WA) were dissolved in DMSO and added in a 0.2% volume to give the concentrations indicated in the text. In all cases the vehicles used to prepare stock solutions of the reagents had no effect on the cells or the parameters measured at the concentrations used. Cell viability was determined by trypan blue exclusion. After treatment the cells were trypsinized and 10 μl of a 0.5% solution of trypan blue was added to 100 μl of treated cells. The suspension was then applied to a hemocytometer. Both viable and nonviable cells were counted. A minimum of 200 cells was counted for each data point in a total of eight microscopic fields. The assay is based on the ability of the active enzyme to cleave the chromophore pNA from the enzyme substrate DEVD-pNA. Extracts from treated cells were diluted 1:1 with 2× reaction buffer (10 mmol/L Tris, pH 7.4, 1 mmol/L dithiothreitol, 2 mmol/L EDTA, 0.1% (3-[3-cholamido- propyl]dimethylammonio)-1-propanesulfonate, 1 mmol/L PMSF, 10 μg/ml pepstatin, 10 μg/ml leupeptin). DEVD-pNA was added to a final concentration of 50 μmol/L, and the reaction was incubated for 1 hour at 37°C. The samples were then transferred to a 96-well plate, and the absorbance measurements were made with a 96-well plate reader at 405 nm. The activity of caspase-8 was measured with the Apo-Alert Caspase-8 Colorimetric Assay Kit (Clontech Laboratories Inc., Palo Alto, CA) according to the manufacturer's instructions. Absorbance measurements were made with a 96-well plate reader at 405 nm. Cells were harvested by trypsinization after treatment. Soybean trypsin inhibitor was used to neutralize trypsin after harvest. Cells were centrifuged at 750 × g for 10 minutes at 4°C. The pellets were washed with SHE-PI (10 mmol/L HEPES-KOH (pH 7.4), 1 mmol/L EDTA, protease inhibitor cocktail (PI), and 250 mmol/L sucrose). The cell pellets were resuspended in SHE-PI. The cell suspension was transferred to a dounce homogenizer and the cells broken open with 20 strokes of the pestle. The homogenate was transferred to a high speed centrifuge tube containing SHE-PI and 0.5% phenol red. HE-PI (10 mmol/L HEPES-KOH, pH 7.4, 1 mmol/L EDTA, protease inhibitor cocktail (PI) containing 750 mmol/L sucrose) was laid below the homogenate. Centrifugation was conducted at 10,000 × g for 30 minutes at 4°C. The resulting mitochondrial pellet was resuspended and centrifuged a second time into 750 mmol/L sucrose in HE-PI. The mitochondria were further concentrated in a Centricon (MW cutoff of 10,000) microconcentrator and the resulting retentate analyzed for cytochrome c. The supernatant from the 10,000 × g spin was centrifuged at 100,000 × g for preparation of cytosol. For the detection of cytochrome c, mitochondrial and cytosolic fractions were separated on 12. SDS-polyacrylamide electrophoresis gels with an equal amount of protein loaded onto each lane as determined by the bicinchoninic acid assay. Kaleidoscope prestained standards (Bio-Rad) were used to determine molecular weight. The gels were then electroblotted onto nitrocellulose membranes. Cytochrome c was detected by a monoclonal antibody (Pharmingen, San Diego, CA) at a dilution of 1:5000. Secondary goat-anti-mouse horseradish peroxidase-labeled antibody (1:2000) was detected by enhanced chemiluminescence. To detect Bid, the mitochondrial fraction was electrophoresed on 15. SDS-polyacrylamide gels. The gel was then electroblotted onto nitrocellulose membranes. Bid was detected with a polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500. The secondary anti-goat horseradish peroxidase-labeled antibody (1:20,000. was visualized by enhanced chemiluminescence. Cells (1.0 × 106) were trypsinized and collected by centrifugation at 750 × g for 10 minutes. The cell pellet was washed in PBS and then lysed in 200 μl of 10 mmol/L Tris, pH 8.0, 10 mmol/L EDTA, 0.5% Triton X-100. The lysate was centrifuged at 13,000 × g for 20 minutes at 4°C. RNase (0.2 mg/ml) was added, and the lysate was incubated for 30 minutes at 37°C. Proteinase K (0.1 mg/ml) and SDS (final concentration 1%) were added, followed by incubation at 50°C for 16 hours. DNA was extracted with phenol/chloroform and then with chloroform, precipitated with ethanol and sodium acetate, and electrophoresed on 1.2% agarose gels. Cells (1.0 × 105) were trypsinized and then pelleted at 750 × g, resuspended in 20 μl of SDS sample buffer, and boiled for 10 minutes. The samples were run on an 8% SDS-polyacrylamide electrophoresis gel. Protein content was determined by BCA, and molecular weight by external standards. The gels were electroblotted onto nitrocellulose membranes and probed with PARP rabbit antiserum (PA3–950, Affinity Bioreagents, Golden, CO) at 1:500 dilution. A secondary horseradish peroxidase-labeled goat-anti-rabbit antibody at 1:10,000 was detected using enhanced chemiluminesence. After treatment the cells were trypsinized and pelleted. The cells were then suspended and fixed in 2% gluteraldehyde with 1% tannic acid in 0.1 mol/L sodium cacodylate at pH 7.3 overnight at 4°C. The cells were rinsed three times in the sodium cacodylate buffer and then incubated in 2% osmium tetroxide in the same buffer for 2 hours at room temperature. The cells were rinsed three times in distilled water and then exposed to 1% uranyl acetate in water for 15 minutes at room temperature. The cells were then rinsed twice in distilled water, after which they were spun down into 3% agarose at 45°C and cooled to form blocks. The agarose blocks were dehydrated in graded steps of acetone and embedded in Spurr's low viscosity media. After polymerization overnight at 65°C, 80-nm sections were cut on a Reighert-Jung Ultra Cut E ultramicrotome and picked up on copper grids. The grids were poststained in uranyl acetate and bismuth subnitrate. The sections were observed in a Hitachi 7000 STEM and micrographs recorded on Kodak 4489-sheet film. MDH activity was measured in L929 cytosolic extracts according to the method of Ochoa.14Mehler AH Kornberg A Grisolia S Ochoa S Malic dehydrogenase from pig heart.J Biol Chem. 1948; 174: 961-966Abstract Full Text PDF PubMed Google Scholar Briefly, cytosolic extracts (2 mg of protein) were added to 300 μl of MDH buffer (259 mmol/L glycylglycine, pH 7.4, 1.5 mmol/L NADH). To this, 100 μl of 7.6 mmol/L oxalacetate was added to initiate the reaction. Readings were taken in a spectrophotometer at 340 nm and made against a blank containing all components except NADH. The decrease in the optical density between 30 and 45 seconds after the start of the reaction was used to calculate enzyme activity. The concentration of MDH in the cytosolic extracts was calculated by comparison to a standard curve. MDH and NADH were both from Sigma. TNF-α induces the mitochondrial permeability transition (MPT) in L929 fibroblasts and, in turn, the death of virtually all of the cells.13Pastorino JG Simbula G Yamamoto K Glascott Jr, PA Rothman RJ Farber JL The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.J Biol Chem. 1996; 271: 29792-29798Crossref PubMed Scopus (254) Google Scholar On treatment with TNF-α in the presence of either actinomycin D (ActD) or cycloheximide (CHX), the MPT (as measured by a loss of mitochondrial energization) is evident within 4 hours.13Pastorino JG Simbula G Yamamoto K Glascott Jr, PA Rothman RJ Farber JL The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.J Biol Chem. 1996; 271: 29792-29798Crossref PubMed Scopus (254) Google Scholar As shown in Figure 1, TNF-α produced over a similar time course an increase in caspase-3 activity in the cytosol of L929 fibroblasts. Within 4 hours caspase-3 activity increased by eightfold and 16-fold by 6 hours, a result that was entirely dependent on induction of the MTP. Cyclosporin A (CyA) together with the phospholipase A2 inhibitor aristolochic acid (ArA) prevents the development of the MPT in L929 cells treated with TNF-α.13Pastorino JG Simbula G Yamamoto K Glascott Jr, PA Rothman RJ Farber JL The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.J Biol Chem. 1996; 271: 29792-29798Crossref PubMed Scopus (254) Google Scholar Figure 2 shows that CyA + ArA inhibited the increase in caspase-3 activity induced by TNF-α. CyA + ArA lowered caspase-3 activity in TNF-α-treated cells to that in the control cells. Importantly, direct addition of CyA + ArA to cellular extracts of TNF-α-treated cells did not inhibit caspase-3 activity (data not shown), a result demonstrating that CyA + ArA does not directly inhibit caspase-3.Figure 2DEVD-FMK or CyA + ArA inhibit caspase-3 activation by TNF-α. L929 cells (5.0 × 106 cells) were pretreated for 30 minutes with either 5 μmol/L CyA and 50 μmol/L ArA or the indicated concentrations of DEVD-FMK. TNF-α 0.5 μg/ml ActD were then added. After 6 hours, cell extracts were prepared and caspase-3 activity assayed as described in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The caspase family of proteases differ with respect to the recognition sequence at which they cleave a given substrate. Caspase-3 cleaves at the amino acid sequence DEVDG, which in the case of the nuclear enzyme poly(ADP-ribose)polymerase (PARP) lies between Asp216 and Gly217 in the DNA binding domain of this caspase-3 substrate.15Nicholson DW Ali A Thornberry NA Vaillancourt JP Ding CK Gallant M Gareau Y Griffin PR Labelle M Lazebnik YA Munday NA Raju SM Smulson ME Yamin T-T Yu VL Miller DK Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar Z-DEVD-FMK is a specific, cell permeable and irreversible, tetrapeptide inhibitor of caspase-3.15Nicholson DW Ali A Thornberry NA Vaillancourt JP Ding CK Gallant M Gareau Y Griffin PR Labelle M Lazebnik YA Munday NA Raju SM Smulson ME Yamin T-T Yu VL Miller DK Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar L929 fibroblasts were pretreated with Z-DEVD-FMK for 30 minutes prior to the addition of TNF-α. As shown in Figure 2, Z-DEVD-FMK produced a dose-dependent inhibition of the increase in caspase-3 activity, with 100 μmol/L reducing caspase-3 activity to that of controls. Whereas the data in Figure 2 indicate that activation of caspase-3 is entirely a consequence of the MPT, there are potential, alternative mechanisms for caspase activation in response to TNF-α. In particular, caspase-8 can be activated as a direct consequence of the binding of TNF-α to the 55-kd receptor (TNFR-1) with recruitment of TRADD (TNFR-1-associated death domain protein) and, in turn, FADD (Fas-associated death domain protein).16Nagata S Apoptosis by death factor.Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar Caspase-8 can then activate downstream caspases, such as caspase-3.17Muzio M Stockwell BR Stennicke HR Salvesen GS Dixit VM An induced proximity model for caspase-8 activitation.J Biol Chem. 1998; 273: 2926-2930Crossref PubMed Scopus (885) Google Scholar, 18Wallach D Cell death induction by TNF: a matter of self control.Trends Biochem Sci. 1997; 22: 107-109Abstract Full Text PDF PubMed Scopus (209) Google Scholar Figure 3 details the activation of caspase-8 in L929 fibroblasts treated with TNF-α. Within 2 hours, TNF-α alone (no ActD) produced a threefold increase in caspase-8 activity. It deserves emphasis that there is no loss of viability with TNF-α alone.13Pastorino JG Simbula G Yamamoto K Glascott Jr, PA Rothman RJ Farber JL The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.J Biol Chem. 1996; 271: 29792-29798Crossref PubMed Scopus (254) Google Scholar In the presence of TNF-α and ActD, a 10-fold activation of caspase-8 occurred (Figure 3) CyA + ArA reduced the activation of caspase-8 by TNF-α and ActD to that with TNF-α alone (Figure 3) Thus, the additional activation of caspase-8 above that with TNF-α alone that occurred with TNF-α and ActD is a consequence of the MPT. Importantly, Z-IETD-FMK, a specific inhibitor of caspase-8,19Garcia-Calvo M Peterson EP Leiting B Ruel R Nicholson DW Thornberry NA Inhibition of human caspases by peptide-based and macromolecular inhibitors.J Biol Chem. 1998; 273: 32608-32613Crossref PubMed Scopus (849) Google Scholar prevented both components (that with TNF-α alone plus that with TNF-α and ActD) of the activation of this enzyme, thereby reducing its activity to the control level (Figure 3) Activation of Bid and its translocation to the mitochondria have been attributed to activation of caspase-8.20Li H Zhu H Xu C Yuan J Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 21Granville DJ Shaw JR Leong S Carthy CM Margaron P Hunt DW McManus BM Release of cytochrome c, Bax migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis.Am J Pathol. 1999; 155: 1021-1025Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Treatment of L929 fibroblasts with TNF-α and ActD caused the translocation of Bid to the mitochondria (Figure 4) Interestingly, despite the complete inhibition of caspase-8 activation by Z-IETD-FMK (Figure 3), the translocation of Bid is only partially prevented by this specific caspase inhibitor (Figure 4) This result is consistent with the previous report that, under conditions where the cell killing is not entirely dependent on caspase activation, Bid translocation is similarly not prevented by caspase inhibitors.22Desagher S Osen-Sand A Nichols A Eskes R Montessuit S Lauper S Maundrell K Antonsson B Martinou JC Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.J Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar Cleavage of PARP is a prominent manifestation of the activation of caspase-3 in many models of apoptosis.23Lazebnik YA Kaufmann SH Desnoyers S Poirier GG Earnshaw WC Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.Nature. 1994; 371: 346-347Crossref PubMed Scopus (2351) Google Scholar Similarly, the treatment of L929 fibroblasts (in the presence of ActD) causes a loss of the 116-kd PARP (Figure 5, land 2). This loss of PARP protein was not apparent with TNF-α in the absence of ActD (data not shown). Despite the fact that the antibody used here (PA3–590) recognizes the carboxyl terminal of mouse PARP, it was not possible to detect the 85-kd carboxyl terminal cleavage fragment of PARP in the L929 cell extracts. However, it could be demonstrated that the loss of the native PARP protein produced by TNF-α depended on caspase-3 activity. The TNF-α-induced PARP depletion was inhibited by pretreatment of the cells with Z-DEVD-FMK (Figure 5, lane 3). Importantly, the loss of PARP also depended on induction of the MPT as shown by the similar ability of CyA (in the presence of ArA) to maintain the control content of this enzyme (Figure 5, lane 4). The cleavage of DNA to oligonucleosome size fragments is another prominent feature of apoptosis. Treatment of L929 fibroblasts with TNF-α results in DNA fragmentation within 6 hours (Figure 6a). DNA fragmentation was not detected at the same times with TNF-α in the absence of ActD (data not shown). This DNA fragmentation induced by TNF-α is caspase-3-dependent. As shown in Figure 6b, the caspase-3 inhibitor Z-DEVD-FMK completely prevented the evidence of DNA fragmentation at 6 hours produced by TNF-α. As with the increase in caspase-3 activity itself and the depletion of PARP protein, DNA fragmentation was also dependent on the MPT. CyA + ArA completely prevented the DNA fragmentation evident 6 hours after treatment with TNF-α. The activation of caspases in some models of apoptosis has been attributed to the release and redistribution of cytochrome c from the mitochondria to the cytosol.24Yang J Liu X Bhalla K Kim CN Ibrado AM Cai J Peng TI Jones DP Wang X Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked.Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 25Kluck RM Bossy-Wetzel E Green DR Newmeyer DD The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar To determine the role of cytochrome c release in the activation of caspase-3 by TNF-α, cytosolic and mitochondrial fractions were derived from homogenates of TNF-α-treated L929 cells. Figure 7a shows that TNF-α (in the presence of ActD) produced a decrease in the mitochondrial content of cytochrome c that was evident after 4 hours and more prominent at 6 hours. This depletion of cytochrome c in the mitochondrial fraction was mirrored by a concomitant increase of the protein in the cytosolic fraction. There was no change in the cellular distribution of cytochrome c in cells treated with TNF-α in the absence of ActD (data not shown). The small amount of cytosolic cytochrome c evident at time zero was owing presumably to the trauma incurred by the mitochondria on homogenization and isolation of the respective subcellular fractions. The release of cytochrome c produced by TNF-α is dependent on induction of the MPT. CyA + ArA prevented the release and redistribution of cytochrome c from mitochondria to the cytosol at 6 hours (Figure 7b). By contrast, Z-DEVD-FMK had no effect on either the mitochondrial loss or cytosolic accumulation of cytochrome c in L929 cells treated TNF-α (Figure 7b). Similarly, Z-DEVD-FMK did not prevent induction of the MPT as measured by loss of mitochondrial membrane potential (data not shown). In keeping with the conclusion that the release of cytochrome c is a consequence of the MPT, Figure 8 shows that 20 μmol/L Z-IETD-FMK, a dose that prevents caspase-8 activation (Figure 3), is without effect on either the loss of cytochrome c from the mitochondria (Figure 8, top) or its accumulation in the cytosol (Figure 8, bottom). It has been hypothesized that the release of cytochrome c during apoptosis is the specific consequence of an as yet poorly characterized mechanism that disrupts the outer mitochondrial membrane without any loss of the mitochondrial membrane potential.25Kluck RM Bossy-Wetzel E Green DR Newmeyer DD The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar By contrast, induction of the MPT in isolated mitochondria results in the release of several proteins in addition to cytochrome c.26Igbavboa U Zwizinski CW Pfeiffer DR Release of mitochondrial matrix proteins through a Ca2+-requiring, cyclosporin-sensitive pathway.Biochem Biophys Res Commun. 1989; 161