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Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

Ricci JE, Gottlieb RA, Green DR - J. Cell Biol. (2003)

Bottom Line: Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II.Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment.Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

ABSTRACT
During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

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Apoptotic cells maintain ΔΨm in the presence of TMPD/ascorbate but not in the presence of malate/palmitate or succinate as substrates. HeLa cells were treated with or without 0.3 μM actinomycin D (18 h), permeabilized with digitonin, and incubated with the indicated substrates. Oligomycin (10 μg/ml) was added as indicated. Cells were stained with TMRE and analyzed by flow cytometry. (A) Malate/palmitate; (B) rotenone and succinate; (C) antimycin A and TMPD/ascorbate. Concentrations for inhibitors and substrates were the same as for the experiment in Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.
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fig7: Apoptotic cells maintain ΔΨm in the presence of TMPD/ascorbate but not in the presence of malate/palmitate or succinate as substrates. HeLa cells were treated with or without 0.3 μM actinomycin D (18 h), permeabilized with digitonin, and incubated with the indicated substrates. Oligomycin (10 μg/ml) was added as indicated. Cells were stained with TMRE and analyzed by flow cytometry. (A) Malate/palmitate; (B) rotenone and succinate; (C) antimycin A and TMPD/ascorbate. Concentrations for inhibitors and substrates were the same as for the experiment in Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.

Mentions: Therefore, we sought to determine if complex I and II are targeted for caspase-mediated disruption during the process of apoptosis. HeLa cells were treated with actinomycin D to induce apoptosis, with and without zVAD-fmk to block caspase activation. Cells were then treated with digitonin in order to provide substrates with access to the mitochondria. Different substrates with or without exogenous cytochrome c were added, and ΔΨm was assessed. As shown in Fig. 7, ActD-treated cells lost ΔΨm driven by complex I, II, or IV substrates. In the absence of substrates, ΔΨm was minimal (unpublished data) as in Fig. 5 B. Addition of cytochrome c restored complex IV activity in this system (Fig. 7 C) but not the activities of complex I or II (Fig. 7, A and B). This is consistent with our results with caspase-treated mitochondria and permeabilized cells, supporting the idea that mitochondrial outer membrane permeabilization and caspase activation during apoptosis disrupted the function of complex I and II. Consistent with this idea, inhibition of caspase activation protected ΔΨm in each case.


Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

Ricci JE, Gottlieb RA, Green DR - J. Cell Biol. (2003)

Apoptotic cells maintain ΔΨm in the presence of TMPD/ascorbate but not in the presence of malate/palmitate or succinate as substrates. HeLa cells were treated with or without 0.3 μM actinomycin D (18 h), permeabilized with digitonin, and incubated with the indicated substrates. Oligomycin (10 μg/ml) was added as indicated. Cells were stained with TMRE and analyzed by flow cytometry. (A) Malate/palmitate; (B) rotenone and succinate; (C) antimycin A and TMPD/ascorbate. Concentrations for inhibitors and substrates were the same as for the experiment in Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.
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Related In: Results  -  Collection

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fig7: Apoptotic cells maintain ΔΨm in the presence of TMPD/ascorbate but not in the presence of malate/palmitate or succinate as substrates. HeLa cells were treated with or without 0.3 μM actinomycin D (18 h), permeabilized with digitonin, and incubated with the indicated substrates. Oligomycin (10 μg/ml) was added as indicated. Cells were stained with TMRE and analyzed by flow cytometry. (A) Malate/palmitate; (B) rotenone and succinate; (C) antimycin A and TMPD/ascorbate. Concentrations for inhibitors and substrates were the same as for the experiment in Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.
Mentions: Therefore, we sought to determine if complex I and II are targeted for caspase-mediated disruption during the process of apoptosis. HeLa cells were treated with actinomycin D to induce apoptosis, with and without zVAD-fmk to block caspase activation. Cells were then treated with digitonin in order to provide substrates with access to the mitochondria. Different substrates with or without exogenous cytochrome c were added, and ΔΨm was assessed. As shown in Fig. 7, ActD-treated cells lost ΔΨm driven by complex I, II, or IV substrates. In the absence of substrates, ΔΨm was minimal (unpublished data) as in Fig. 5 B. Addition of cytochrome c restored complex IV activity in this system (Fig. 7 C) but not the activities of complex I or II (Fig. 7, A and B). This is consistent with our results with caspase-treated mitochondria and permeabilized cells, supporting the idea that mitochondrial outer membrane permeabilization and caspase activation during apoptosis disrupted the function of complex I and II. Consistent with this idea, inhibition of caspase activation protected ΔΨm in each case.

Bottom Line: Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II.Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment.Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

ABSTRACT
During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

Show MeSH
Related in: MedlinePlus