doi: 10.1101/gad.13.11.1367.
c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release
Affiliations
- PMID: 10364155
- PMCID: PMC316765
- DOI: 10.1101/gad.13.11.1367
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c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release
Genes Dev.
.
Abstract
Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.
Figures
c-Myc induces cytochrome c release from mitochondria in serum-deprived fibroblasts. (A) Serum-deprived Rat-1/c-MycER fibroblasts were treated with OHT (100 nm ) in the presence or absence of IGF-1. IGF-1, if present, was added at the same time as OHT. At the indicated time points, subcellular fractionation was performed as described in Materials and Methods. The heavy membrane fraction (HMF; 10 μg of protein), containing crude mitochondria, and the postmitochondrial fraction (supernatant; S; 20 μg of protein) were analyzed for cytochrome c content by SDS-PAGE and immunoblotting with anti-cytochrome c antibody. (B) Rat-1/c-MycER fibroblasts were treated for 24 hr and subcellular fractionation was performed as in A. The heavy membrane (HMF; 10 μg of protein) and postmitochondrial fractions (supernatant; S; 20 μg of protein) were analyzed by SDS-PAGE analysis and immunoblotting for cytochrome c oxidase subunit IV. (C) Serum-deprived Rat-1/cMycER fibroblasts were treated as in A for 24 hr prior to fixation, immunostaining, and confocal-laser-scanning analysis as described in Materials and Methods. (Top left) Rat-1/c-MycER fibroblasts were left untreated and stained with anti-cytochrome c antibody; (top right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c antibody; (bottom left) Rat-1/c-MycER fibroblasts were treated with OHT and IGF-1 for 24 hr and stained with anti-cytochrome c antibody; (bottom right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c oxidase subunit IV antibody. Projections of 20 Z-sections (0.5-μm steps) ranging from the bottom to the top of the cells are shown. Bar = 20 μm. (D) Serum-deprived Rat-1/c-MycΔ(106–143)ER fibroblasts were treated with OHT. Subcellular fractionation and subsequent analysis of cytochrome c content in the heavy membrane fraction were performed as described in A at the indicated time points.
c-Myc induces cytochrome c release from mitochondria in serum-deprived fibroblasts. (A) Serum-deprived Rat-1/c-MycER fibroblasts were treated with OHT (100 nm ) in the presence or absence of IGF-1. IGF-1, if present, was added at the same time as OHT. At the indicated time points, subcellular fractionation was performed as described in Materials and Methods. The heavy membrane fraction (HMF; 10 μg of protein), containing crude mitochondria, and the postmitochondrial fraction (supernatant; S; 20 μg of protein) were analyzed for cytochrome c content by SDS-PAGE and immunoblotting with anti-cytochrome c antibody. (B) Rat-1/c-MycER fibroblasts were treated for 24 hr and subcellular fractionation was performed as in A. The heavy membrane (HMF; 10 μg of protein) and postmitochondrial fractions (supernatant; S; 20 μg of protein) were analyzed by SDS-PAGE analysis and immunoblotting for cytochrome c oxidase subunit IV. (C) Serum-deprived Rat-1/cMycER fibroblasts were treated as in A for 24 hr prior to fixation, immunostaining, and confocal-laser-scanning analysis as described in Materials and Methods. (Top left) Rat-1/c-MycER fibroblasts were left untreated and stained with anti-cytochrome c antibody; (top right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c antibody; (bottom left) Rat-1/c-MycER fibroblasts were treated with OHT and IGF-1 for 24 hr and stained with anti-cytochrome c antibody; (bottom right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c oxidase subunit IV antibody. Projections of 20 Z-sections (0.5-μm steps) ranging from the bottom to the top of the cells are shown. Bar = 20 μm. (D) Serum-deprived Rat-1/c-MycΔ(106–143)ER fibroblasts were treated with OHT. Subcellular fractionation and subsequent analysis of cytochrome c content in the heavy membrane fraction were performed as described in A at the indicated time points.
c-Myc induces cytochrome c release from mitochondria in serum-deprived fibroblasts. (A) Serum-deprived Rat-1/c-MycER fibroblasts were treated with OHT (100 nm ) in the presence or absence of IGF-1. IGF-1, if present, was added at the same time as OHT. At the indicated time points, subcellular fractionation was performed as described in Materials and Methods. The heavy membrane fraction (HMF; 10 μg of protein), containing crude mitochondria, and the postmitochondrial fraction (supernatant; S; 20 μg of protein) were analyzed for cytochrome c content by SDS-PAGE and immunoblotting with anti-cytochrome c antibody. (B) Rat-1/c-MycER fibroblasts were treated for 24 hr and subcellular fractionation was performed as in A. The heavy membrane (HMF; 10 μg of protein) and postmitochondrial fractions (supernatant; S; 20 μg of protein) were analyzed by SDS-PAGE analysis and immunoblotting for cytochrome c oxidase subunit IV. (C) Serum-deprived Rat-1/cMycER fibroblasts were treated as in A for 24 hr prior to fixation, immunostaining, and confocal-laser-scanning analysis as described in Materials and Methods. (Top left) Rat-1/c-MycER fibroblasts were left untreated and stained with anti-cytochrome c antibody; (top right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c antibody; (bottom left) Rat-1/c-MycER fibroblasts were treated with OHT and IGF-1 for 24 hr and stained with anti-cytochrome c antibody; (bottom right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c oxidase subunit IV antibody. Projections of 20 Z-sections (0.5-μm steps) ranging from the bottom to the top of the cells are shown. Bar = 20 μm. (D) Serum-deprived Rat-1/c-MycΔ(106–143)ER fibroblasts were treated with OHT. Subcellular fractionation and subsequent analysis of cytochrome c content in the heavy membrane fraction were performed as described in A at the indicated time points.
c-Myc induces cytochrome c release from mitochondria in serum-deprived fibroblasts. (A) Serum-deprived Rat-1/c-MycER fibroblasts were treated with OHT (100 nm ) in the presence or absence of IGF-1. IGF-1, if present, was added at the same time as OHT. At the indicated time points, subcellular fractionation was performed as described in Materials and Methods. The heavy membrane fraction (HMF; 10 μg of protein), containing crude mitochondria, and the postmitochondrial fraction (supernatant; S; 20 μg of protein) were analyzed for cytochrome c content by SDS-PAGE and immunoblotting with anti-cytochrome c antibody. (B) Rat-1/c-MycER fibroblasts were treated for 24 hr and subcellular fractionation was performed as in A. The heavy membrane (HMF; 10 μg of protein) and postmitochondrial fractions (supernatant; S; 20 μg of protein) were analyzed by SDS-PAGE analysis and immunoblotting for cytochrome c oxidase subunit IV. (C) Serum-deprived Rat-1/cMycER fibroblasts were treated as in A for 24 hr prior to fixation, immunostaining, and confocal-laser-scanning analysis as described in Materials and Methods. (Top left) Rat-1/c-MycER fibroblasts were left untreated and stained with anti-cytochrome c antibody; (top right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c antibody; (bottom left) Rat-1/c-MycER fibroblasts were treated with OHT and IGF-1 for 24 hr and stained with anti-cytochrome c antibody; (bottom right) Rat-1/c-MycER fibroblasts were treated with OHT for 24 hr and stained with anti-cytochrome c oxidase subunit IV antibody. Projections of 20 Z-sections (0.5-μm steps) ranging from the bottom to the top of the cells are shown. Bar = 20 μm. (D) Serum-deprived Rat-1/c-MycΔ(106–143)ER fibroblasts were treated with OHT. Subcellular fractionation and subsequent analysis of cytochrome c content in the heavy membrane fraction were performed as described in A at the indicated time points.
Microinjected anti-cytochrome c antibodies inhibit c-Myc-induced cell death. Serum-deprived Rat-1/c-MycER fibroblasts were injected with either control or anti-cytochrome c antibodies (20 mg/ml) mixed with dextran-conjugated rhodamine dye. (□) Control antibodies; (█) anti-cytochrome c antibodies. Six hours later, the number of injected cells was evaluated, OHT (100 nm ) was added and the cells were then incubated at 37°C. At the indicated time points, cells were analyzed by fluorescence microscopy and the percentage of nonapoptotic cells was determined. Results are means ±s.e.m. of 4 (control antibody) and 6 (anti-cytochrome c antibody) independent experiments, each involving ∼70 cells.
Microinjected cytochrome c induces apoptosis. (A,B) Morphology of Rat-1/c-MycER fibroblasts following cytoplasmic microinjection of cytochrome c. Rat-1/c-MycER fibroblasts grown in 10% FCS in the absence of OHT were injected with either 25 μm pure cytochrome c (B) or water (A) and incubated for 2 hr at 37°C prior to microscopic analysis. Bar = 20 μm. (C) Cytochrome c-induced apoptosis is dose dependent. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with pure cytochrome c (hcC) at various concentrations mixed with dextran-conjugated rhodamine dye. (█) 2μm hcC; (●) 10 μm hcC; (▴) 25 μm hcC; (▵) 25μm hcC + zVad.fmk. Injected cells were then followed by time-lapse phase and fluorescence microscopy as described in Materials and Methods. The fates of 25 cells, picked randomly from a frame containing ∼50 injected cells, were analyzed. The onset of apoptosis was scored as the start of membrane blebbing and the end point of cell death was scored as the time of cell detachment from substratum. The time between these two events is represented for each individual cell death by the length of the horizontal line. These data are representative of at least three independent experiments. Where indicated, zVAD.fmk (100 μm ) was added 1 hr prior to microinjection. (D) Microinjected cytochrome c-induced apoptosis is independent of c-Myc activity. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with hcC (10 μm ) mixed with dextran-conjugated rhodamine dye. Where indicated (□), OHT (100 nm ) was added 2 hr prior to microinjection. (█) No OHT added. Microinjected cells were followed by time-lapse phase and fluorescence microscopy and scored for apoptosis as described in Materials and Methods. The number of cell deaths is expressed as a percentage of the total number of viable injected cells present in the entire frame at the beginning of the time lapse experiment (∼50 cells). Data shown are representative of at least three independent experiments. (E) IGF-1 does not protect against microinjected cytochrome c-induced apoptosis. Experiments were performed as described in D except that Rat-1/c-MycER fibroblasts serum deprived for 48 hr prior to microinjection were used. (█) IGF-1 (100 ng/ml) was added 2 hr prior to microinjection. (□) No IGF-1 added. Data shown are representative of at least three independent experiments.
Microinjected cytochrome c induces apoptosis. (A,B) Morphology of Rat-1/c-MycER fibroblasts following cytoplasmic microinjection of cytochrome c. Rat-1/c-MycER fibroblasts grown in 10% FCS in the absence of OHT were injected with either 25 μm pure cytochrome c (B) or water (A) and incubated for 2 hr at 37°C prior to microscopic analysis. Bar = 20 μm. (C) Cytochrome c-induced apoptosis is dose dependent. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with pure cytochrome c (hcC) at various concentrations mixed with dextran-conjugated rhodamine dye. (█) 2μm hcC; (●) 10 μm hcC; (▴) 25 μm hcC; (▵) 25μm hcC + zVad.fmk. Injected cells were then followed by time-lapse phase and fluorescence microscopy as described in Materials and Methods. The fates of 25 cells, picked randomly from a frame containing ∼50 injected cells, were analyzed. The onset of apoptosis was scored as the start of membrane blebbing and the end point of cell death was scored as the time of cell detachment from substratum. The time between these two events is represented for each individual cell death by the length of the horizontal line. These data are representative of at least three independent experiments. Where indicated, zVAD.fmk (100 μm ) was added 1 hr prior to microinjection. (D) Microinjected cytochrome c-induced apoptosis is independent of c-Myc activity. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with hcC (10 μm ) mixed with dextran-conjugated rhodamine dye. Where indicated (□), OHT (100 nm ) was added 2 hr prior to microinjection. (█) No OHT added. Microinjected cells were followed by time-lapse phase and fluorescence microscopy and scored for apoptosis as described in Materials and Methods. The number of cell deaths is expressed as a percentage of the total number of viable injected cells present in the entire frame at the beginning of the time lapse experiment (∼50 cells). Data shown are representative of at least three independent experiments. (E) IGF-1 does not protect against microinjected cytochrome c-induced apoptosis. Experiments were performed as described in D except that Rat-1/c-MycER fibroblasts serum deprived for 48 hr prior to microinjection were used. (█) IGF-1 (100 ng/ml) was added 2 hr prior to microinjection. (□) No IGF-1 added. Data shown are representative of at least three independent experiments.
Microinjected cytochrome c induces apoptosis. (A,B) Morphology of Rat-1/c-MycER fibroblasts following cytoplasmic microinjection of cytochrome c. Rat-1/c-MycER fibroblasts grown in 10% FCS in the absence of OHT were injected with either 25 μm pure cytochrome c (B) or water (A) and incubated for 2 hr at 37°C prior to microscopic analysis. Bar = 20 μm. (C) Cytochrome c-induced apoptosis is dose dependent. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with pure cytochrome c (hcC) at various concentrations mixed with dextran-conjugated rhodamine dye. (█) 2μm hcC; (●) 10 μm hcC; (▴) 25 μm hcC; (▵) 25μm hcC + zVad.fmk. Injected cells were then followed by time-lapse phase and fluorescence microscopy as described in Materials and Methods. The fates of 25 cells, picked randomly from a frame containing ∼50 injected cells, were analyzed. The onset of apoptosis was scored as the start of membrane blebbing and the end point of cell death was scored as the time of cell detachment from substratum. The time between these two events is represented for each individual cell death by the length of the horizontal line. These data are representative of at least three independent experiments. Where indicated, zVAD.fmk (100 μm ) was added 1 hr prior to microinjection. (D) Microinjected cytochrome c-induced apoptosis is independent of c-Myc activity. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with hcC (10 μm ) mixed with dextran-conjugated rhodamine dye. Where indicated (□), OHT (100 nm ) was added 2 hr prior to microinjection. (█) No OHT added. Microinjected cells were followed by time-lapse phase and fluorescence microscopy and scored for apoptosis as described in Materials and Methods. The number of cell deaths is expressed as a percentage of the total number of viable injected cells present in the entire frame at the beginning of the time lapse experiment (∼50 cells). Data shown are representative of at least three independent experiments. (E) IGF-1 does not protect against microinjected cytochrome c-induced apoptosis. Experiments were performed as described in D except that Rat-1/c-MycER fibroblasts serum deprived for 48 hr prior to microinjection were used. (█) IGF-1 (100 ng/ml) was added 2 hr prior to microinjection. (□) No IGF-1 added. Data shown are representative of at least three independent experiments.
Microinjected cytochrome c induces apoptosis. (A,B) Morphology of Rat-1/c-MycER fibroblasts following cytoplasmic microinjection of cytochrome c. Rat-1/c-MycER fibroblasts grown in 10% FCS in the absence of OHT were injected with either 25 μm pure cytochrome c (B) or water (A) and incubated for 2 hr at 37°C prior to microscopic analysis. Bar = 20 μm. (C) Cytochrome c-induced apoptosis is dose dependent. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with pure cytochrome c (hcC) at various concentrations mixed with dextran-conjugated rhodamine dye. (█) 2μm hcC; (●) 10 μm hcC; (▴) 25 μm hcC; (▵) 25μm hcC + zVad.fmk. Injected cells were then followed by time-lapse phase and fluorescence microscopy as described in Materials and Methods. The fates of 25 cells, picked randomly from a frame containing ∼50 injected cells, were analyzed. The onset of apoptosis was scored as the start of membrane blebbing and the end point of cell death was scored as the time of cell detachment from substratum. The time between these two events is represented for each individual cell death by the length of the horizontal line. These data are representative of at least three independent experiments. Where indicated, zVAD.fmk (100 μm ) was added 1 hr prior to microinjection. (D) Microinjected cytochrome c-induced apoptosis is independent of c-Myc activity. Rat-1/c-MycER fibroblasts grown in 10% FCS were microinjected with hcC (10 μm ) mixed with dextran-conjugated rhodamine dye. Where indicated (□), OHT (100 nm ) was added 2 hr prior to microinjection. (█) No OHT added. Microinjected cells were followed by time-lapse phase and fluorescence microscopy and scored for apoptosis as described in Materials and Methods. The number of cell deaths is expressed as a percentage of the total number of viable injected cells present in the entire frame at the beginning of the time lapse experiment (∼50 cells). Data shown are representative of at least three independent experiments. (E) IGF-1 does not protect against microinjected cytochrome c-induced apoptosis. Experiments were performed as described in D except that Rat-1/c-MycER fibroblasts serum deprived for 48 hr prior to microinjection were used. (█) IGF-1 (100 ng/ml) was added 2 hr prior to microinjection. (□) No IGF-1 added. Data shown are representative of at least three independent experiments.
Cytochrome c release induced by c-Myc is neither blocked by dominant-negative mutants of p53 or FADD nor by the caspase inhibitor zVAD.fmk (A) Effect of p53min on apoptosis induced by c-Myc. (▵) Rat-1/cMycER and (○) Rat-1/c-MycER/p53min cells were serum deprived for 48 hr and c-Myc activated by the addition of OHT. The fate of the cells was followed by time-lapse videomicroscopy at one frame every 3 min and the results are expressed as cumulative cell deaths against time. (B) p53min does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/p53min fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the post-mitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A. (C) zVAD.fmk does not inhibit c-Myc-induced cytochrome c release. Subcellular fractionation and subsequent immunoblot analysis of serum-deprived Rat-1/c-MycER fibroblasts treated with OHT was performed as described in Fig. 1A. Where indicated, zVAD.fmk (100 μm ) was added at the same time as OHT. (D) Inhibition of c-Myc-induced apoptosis in Rat-1 fibroblasts by a dominant-negative mutant of FADD. (○) Rat-1/c-MycER − OHT, (●) Rat-1/c-MycER + OHT, (▴) Rat-1/c-MycER/FADD DN − OHT, or (▵) Rat-1/c-MycER-FADD DN + OHT fibroblasts were serum-deprived and cell-death analyzed following activation of c-Myc with 100 nm OHT by phase-contrast time-lapse microscopy. (E) A dominant-negative mutant of FADD does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/FADD DN fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the postmitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A.
Cytochrome c release induced by c-Myc is neither blocked by dominant-negative mutants of p53 or FADD nor by the caspase inhibitor zVAD.fmk (A) Effect of p53min on apoptosis induced by c-Myc. (▵) Rat-1/cMycER and (○) Rat-1/c-MycER/p53min cells were serum deprived for 48 hr and c-Myc activated by the addition of OHT. The fate of the cells was followed by time-lapse videomicroscopy at one frame every 3 min and the results are expressed as cumulative cell deaths against time. (B) p53min does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/p53min fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the post-mitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A. (C) zVAD.fmk does not inhibit c-Myc-induced cytochrome c release. Subcellular fractionation and subsequent immunoblot analysis of serum-deprived Rat-1/c-MycER fibroblasts treated with OHT was performed as described in Fig. 1A. Where indicated, zVAD.fmk (100 μm ) was added at the same time as OHT. (D) Inhibition of c-Myc-induced apoptosis in Rat-1 fibroblasts by a dominant-negative mutant of FADD. (○) Rat-1/c-MycER − OHT, (●) Rat-1/c-MycER + OHT, (▴) Rat-1/c-MycER/FADD DN − OHT, or (▵) Rat-1/c-MycER-FADD DN + OHT fibroblasts were serum-deprived and cell-death analyzed following activation of c-Myc with 100 nm OHT by phase-contrast time-lapse microscopy. (E) A dominant-negative mutant of FADD does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/FADD DN fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the postmitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A.
Cytochrome c release induced by c-Myc is neither blocked by dominant-negative mutants of p53 or FADD nor by the caspase inhibitor zVAD.fmk (A) Effect of p53min on apoptosis induced by c-Myc. (▵) Rat-1/cMycER and (○) Rat-1/c-MycER/p53min cells were serum deprived for 48 hr and c-Myc activated by the addition of OHT. The fate of the cells was followed by time-lapse videomicroscopy at one frame every 3 min and the results are expressed as cumulative cell deaths against time. (B) p53min does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/p53min fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the post-mitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A. (C) zVAD.fmk does not inhibit c-Myc-induced cytochrome c release. Subcellular fractionation and subsequent immunoblot analysis of serum-deprived Rat-1/c-MycER fibroblasts treated with OHT was performed as described in Fig. 1A. Where indicated, zVAD.fmk (100 μm ) was added at the same time as OHT. (D) Inhibition of c-Myc-induced apoptosis in Rat-1 fibroblasts by a dominant-negative mutant of FADD. (○) Rat-1/c-MycER − OHT, (●) Rat-1/c-MycER + OHT, (▴) Rat-1/c-MycER/FADD DN − OHT, or (▵) Rat-1/c-MycER-FADD DN + OHT fibroblasts were serum-deprived and cell-death analyzed following activation of c-Myc with 100 nm OHT by phase-contrast time-lapse microscopy. (E) A dominant-negative mutant of FADD does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/FADD DN fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the postmitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A.
Cytochrome c release induced by c-Myc is neither blocked by dominant-negative mutants of p53 or FADD nor by the caspase inhibitor zVAD.fmk (A) Effect of p53min on apoptosis induced by c-Myc. (▵) Rat-1/cMycER and (○) Rat-1/c-MycER/p53min cells were serum deprived for 48 hr and c-Myc activated by the addition of OHT. The fate of the cells was followed by time-lapse videomicroscopy at one frame every 3 min and the results are expressed as cumulative cell deaths against time. (B) p53min does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/p53min fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the post-mitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A. (C) zVAD.fmk does not inhibit c-Myc-induced cytochrome c release. Subcellular fractionation and subsequent immunoblot analysis of serum-deprived Rat-1/c-MycER fibroblasts treated with OHT was performed as described in Fig. 1A. Where indicated, zVAD.fmk (100 μm ) was added at the same time as OHT. (D) Inhibition of c-Myc-induced apoptosis in Rat-1 fibroblasts by a dominant-negative mutant of FADD. (○) Rat-1/c-MycER − OHT, (●) Rat-1/c-MycER + OHT, (▴) Rat-1/c-MycER/FADD DN − OHT, or (▵) Rat-1/c-MycER-FADD DN + OHT fibroblasts were serum-deprived and cell-death analyzed following activation of c-Myc with 100 nm OHT by phase-contrast time-lapse microscopy. (E) A dominant-negative mutant of FADD does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/FADD DN fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the postmitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A.
Cytochrome c release induced by c-Myc is neither blocked by dominant-negative mutants of p53 or FADD nor by the caspase inhibitor zVAD.fmk (A) Effect of p53min on apoptosis induced by c-Myc. (▵) Rat-1/cMycER and (○) Rat-1/c-MycER/p53min cells were serum deprived for 48 hr and c-Myc activated by the addition of OHT. The fate of the cells was followed by time-lapse videomicroscopy at one frame every 3 min and the results are expressed as cumulative cell deaths against time. (B) p53min does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/p53min fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the post-mitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A. (C) zVAD.fmk does not inhibit c-Myc-induced cytochrome c release. Subcellular fractionation and subsequent immunoblot analysis of serum-deprived Rat-1/c-MycER fibroblasts treated with OHT was performed as described in Fig. 1A. Where indicated, zVAD.fmk (100 μm ) was added at the same time as OHT. (D) Inhibition of c-Myc-induced apoptosis in Rat-1 fibroblasts by a dominant-negative mutant of FADD. (○) Rat-1/c-MycER − OHT, (●) Rat-1/c-MycER + OHT, (▴) Rat-1/c-MycER/FADD DN − OHT, or (▵) Rat-1/c-MycER-FADD DN + OHT fibroblasts were serum-deprived and cell-death analyzed following activation of c-Myc with 100 nm OHT by phase-contrast time-lapse microscopy. (E) A dominant-negative mutant of FADD does not inhibit c-Myc-induced cytochrome c release. Serum-deprived Rat-1/c-MycER/FADD DN fibroblasts were treated with OHT for the indicated time. Subcellular fractionation and immunoblot analysis of cytochrome c content in the heavy membrane fraction (HMF) and the postmitochondrial fraction (supernatant; S) were then performed as described in Fig. 1A.
A dominant-negative mutant of FADD confers increased resistance to microinjected cytochrome c and inhibits c-Myc-induced caspase activation. (A) (●) Rat-1/c-MycER and (○) Rat-1/c-MycER/FADD DN fibroblasts grown in 10% FCS were microinjected with 25 μm pure hcC mixed with dextran-coupled rhodamine dye and the number of injected cells then evaluated by fluorescence microscopy. Cells were incubated at 37°C and, at the indicated time points, the number of nonapoptotic cells was evaluated and expressed as percentage of the initial number of injected cells. Results presented are the mean ±s.e.m. of five independent experiments, each of ∼100 injected cells. (B) Rat-1/c-MycER and Rat-1/c-MycER/FADD DN fibroblasts were serum-deprived prior to addition of OHT (100 nm ). Where indicated, IGF-1 (100 ng/ml) was added simultaneously. Twenty-four hours later, postmitochondrial fractions were prepared and assayed for DEVD cleaving activity as described in Materials and Methods. Results presented are the mean ±s.e.m. of at least three independent experiments.
A dominant-negative mutant of FADD confers increased resistance to microinjected cytochrome c and inhibits c-Myc-induced caspase activation. (A) (●) Rat-1/c-MycER and (○) Rat-1/c-MycER/FADD DN fibroblasts grown in 10% FCS were microinjected with 25 μm pure hcC mixed with dextran-coupled rhodamine dye and the number of injected cells then evaluated by fluorescence microscopy. Cells were incubated at 37°C and, at the indicated time points, the number of nonapoptotic cells was evaluated and expressed as percentage of the initial number of injected cells. Results presented are the mean ±s.e.m. of five independent experiments, each of ∼100 injected cells. (B) Rat-1/c-MycER and Rat-1/c-MycER/FADD DN fibroblasts were serum-deprived prior to addition of OHT (100 nm ). Where indicated, IGF-1 (100 ng/ml) was added simultaneously. Twenty-four hours later, postmitochondrial fractions were prepared and assayed for DEVD cleaving activity as described in Materials and Methods. Results presented are the mean ±s.e.m. of at least three independent experiments.
Microinjected cytochrome c cooperates with CD95 stimulation and X-irradiation. (A) Dose-dependent sensitization to CD95 ligation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with either 2 or 10 μm pure hcC mixed with dextran-conjugated rhodamine dye. Where indicated, CD95Ls (50 ng/ml) was added immediately after injection. (▵) 10 μm hcC + CD95Ls, (▴) 10 μm hcC − CD95Ls, (○) 2 μm hcC + CD95Ls, (●) 2 μm hcC − CD95Ls. Cell death was scored as described in Fig. 3D. Data shown are representative of four (10 μm hcC ± CD95Ls) and three (2 μm hcC ± CD95Ls) independent experiments, each involving ∼50 injected cells. (B) Sensitization to X-irradiation by c-Myc. Rat-1/c-MycER fibroblasts were grown in 2% FCS for 48 hr. X-ray irradiation was then performed as described in Materials and Methods. (○) OHT (100 nm ) added just after irradiation; (●) OHT not added. (C) Sensitization to X-irradiation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with 10 μm pure hcC mixed with dextran-conjugated rhodamine dye or with the dye alone. Where indicated, cells were exposed to X-rays, as described in Materials and Methods, immediately following microinjection. (▵) 10 μm hcC, (○) no hcC + 10 Gy, (●) 10 μm hcC + 10 Gy. Cells were then incubated at 37°C and, at the indicated time points after irradiation, the number of apoptotic cells was evaluated and expressed as percentage of the initial number of injected cells. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (D) Sensitization to X-irradiation by microinjected cytochrome c is inhibited by p53min. Rat-1/c-MycER/p53 min fibroblasts were injected with 10 μm and exposed to X-rays as described in C. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (▵) 10 μm hcC, (●) 10 μm hcC + 10 Gy.
Microinjected cytochrome c cooperates with CD95 stimulation and X-irradiation. (A) Dose-dependent sensitization to CD95 ligation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with either 2 or 10 μm pure hcC mixed with dextran-conjugated rhodamine dye. Where indicated, CD95Ls (50 ng/ml) was added immediately after injection. (▵) 10 μm hcC + CD95Ls, (▴) 10 μm hcC − CD95Ls, (○) 2 μm hcC + CD95Ls, (●) 2 μm hcC − CD95Ls. Cell death was scored as described in Fig. 3D. Data shown are representative of four (10 μm hcC ± CD95Ls) and three (2 μm hcC ± CD95Ls) independent experiments, each involving ∼50 injected cells. (B) Sensitization to X-irradiation by c-Myc. Rat-1/c-MycER fibroblasts were grown in 2% FCS for 48 hr. X-ray irradiation was then performed as described in Materials and Methods. (○) OHT (100 nm ) added just after irradiation; (●) OHT not added. (C) Sensitization to X-irradiation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with 10 μm pure hcC mixed with dextran-conjugated rhodamine dye or with the dye alone. Where indicated, cells were exposed to X-rays, as described in Materials and Methods, immediately following microinjection. (▵) 10 μm hcC, (○) no hcC + 10 Gy, (●) 10 μm hcC + 10 Gy. Cells were then incubated at 37°C and, at the indicated time points after irradiation, the number of apoptotic cells was evaluated and expressed as percentage of the initial number of injected cells. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (D) Sensitization to X-irradiation by microinjected cytochrome c is inhibited by p53min. Rat-1/c-MycER/p53 min fibroblasts were injected with 10 μm and exposed to X-rays as described in C. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (▵) 10 μm hcC, (●) 10 μm hcC + 10 Gy.
Microinjected cytochrome c cooperates with CD95 stimulation and X-irradiation. (A) Dose-dependent sensitization to CD95 ligation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with either 2 or 10 μm pure hcC mixed with dextran-conjugated rhodamine dye. Where indicated, CD95Ls (50 ng/ml) was added immediately after injection. (▵) 10 μm hcC + CD95Ls, (▴) 10 μm hcC − CD95Ls, (○) 2 μm hcC + CD95Ls, (●) 2 μm hcC − CD95Ls. Cell death was scored as described in Fig. 3D. Data shown are representative of four (10 μm hcC ± CD95Ls) and three (2 μm hcC ± CD95Ls) independent experiments, each involving ∼50 injected cells. (B) Sensitization to X-irradiation by c-Myc. Rat-1/c-MycER fibroblasts were grown in 2% FCS for 48 hr. X-ray irradiation was then performed as described in Materials and Methods. (○) OHT (100 nm ) added just after irradiation; (●) OHT not added. (C) Sensitization to X-irradiation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with 10 μm pure hcC mixed with dextran-conjugated rhodamine dye or with the dye alone. Where indicated, cells were exposed to X-rays, as described in Materials and Methods, immediately following microinjection. (▵) 10 μm hcC, (○) no hcC + 10 Gy, (●) 10 μm hcC + 10 Gy. Cells were then incubated at 37°C and, at the indicated time points after irradiation, the number of apoptotic cells was evaluated and expressed as percentage of the initial number of injected cells. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (D) Sensitization to X-irradiation by microinjected cytochrome c is inhibited by p53min. Rat-1/c-MycER/p53 min fibroblasts were injected with 10 μm and exposed to X-rays as described in C. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (▵) 10 μm hcC, (●) 10 μm hcC + 10 Gy.
Microinjected cytochrome c cooperates with CD95 stimulation and X-irradiation. (A) Dose-dependent sensitization to CD95 ligation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with either 2 or 10 μm pure hcC mixed with dextran-conjugated rhodamine dye. Where indicated, CD95Ls (50 ng/ml) was added immediately after injection. (▵) 10 μm hcC + CD95Ls, (▴) 10 μm hcC − CD95Ls, (○) 2 μm hcC + CD95Ls, (●) 2 μm hcC − CD95Ls. Cell death was scored as described in Fig. 3D. Data shown are representative of four (10 μm hcC ± CD95Ls) and three (2 μm hcC ± CD95Ls) independent experiments, each involving ∼50 injected cells. (B) Sensitization to X-irradiation by c-Myc. Rat-1/c-MycER fibroblasts were grown in 2% FCS for 48 hr. X-ray irradiation was then performed as described in Materials and Methods. (○) OHT (100 nm ) added just after irradiation; (●) OHT not added. (C) Sensitization to X-irradiation by microinjected cytochrome c. Rat-1/c-MycER fibroblasts were injected with 10 μm pure hcC mixed with dextran-conjugated rhodamine dye or with the dye alone. Where indicated, cells were exposed to X-rays, as described in Materials and Methods, immediately following microinjection. (▵) 10 μm hcC, (○) no hcC + 10 Gy, (●) 10 μm hcC + 10 Gy. Cells were then incubated at 37°C and, at the indicated time points after irradiation, the number of apoptotic cells was evaluated and expressed as percentage of the initial number of injected cells. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (D) Sensitization to X-irradiation by microinjected cytochrome c is inhibited by p53min. Rat-1/c-MycER/p53 min fibroblasts were injected with 10 μm and exposed to X-rays as described in C. Results presented are representative of at least five independent experiments, each involving ∼100 injected cells. (▵) 10 μm hcC, (●) 10 μm hcC + 10 Gy.
Mechanism of c-Myc-induced sensitization to apoptosis. c-Myc activation triggers release of cytochrome c into the cytosol. This release is blocked by IGF-1. Cells with increased cytosolic cytochrome c are sensitized to distinct death pathways such as that mediated by CD95 and p53, and possibly others too. Neither FADD DN nor p53min blocks c-Myc-induced cytochrome c release and so neither affects the sensitized state of a cell expressing c-Myc. See text for details.
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