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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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Apoptosis, loss of ΔΨm, and ROS production are caspase dependent. HeLa cells were treated with actinomycin D or UV (as indicated) in the presence or absence of 50 μM of zVAD-fmk, harvested, and stained with annexin V–FITC and PI to assess cell death, TMRE to measure ΔΨm, and 2-HE to measure ROS production, and then analyzed by flow cytometry.
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fig1: Apoptosis, loss of ΔΨm, and ROS production are caspase dependent. HeLa cells were treated with actinomycin D or UV (as indicated) in the presence or absence of 50 μM of zVAD-fmk, harvested, and stained with annexin V–FITC and PI to assess cell death, TMRE to measure ΔΨm, and 2-HE to measure ROS production, and then analyzed by flow cytometry.

Mentions: To examine the role of caspases in mitochondrial functions during apoptosis, we first examined the effects of their inhibition on two important parameters, ΔΨm (using tetramethylrhodamine ethyl ester [TMRE] [Farkas et al., 1989]) and the generation of ROS (using dihydroethidium [2-HE] [Heibein et al., 1999]). HeLa cells were treated with actinomycin D (ActD) or UV to induce apoptosis, which was assessed by annexin V–FITC/propidium iodide (PI) staining (Fig. 1). Cell death, loss of ΔΨm, and production of ROS showed a close correspondence in each case. Addition of the caspase inhibitor N-benzoylcarbanyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk) delayed both cell death and loss of ΔΨm as observed previously (Waterhouse et al., 2001b). Interestingly, inhibition of caspase activation also blocked ROS production. Therefore, caspase functions appeared to be required for the rapid onset of these events during apoptosis.


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

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

Apoptosis, loss of ΔΨm, and ROS production are caspase dependent. HeLa cells were treated with actinomycin D or UV (as indicated) in the presence or absence of 50 μM of zVAD-fmk, harvested, and stained with annexin V–FITC and PI to assess cell death, TMRE to measure ΔΨm, and 2-HE to measure ROS production, and then analyzed by flow cytometry.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172744&req=5

fig1: Apoptosis, loss of ΔΨm, and ROS production are caspase dependent. HeLa cells were treated with actinomycin D or UV (as indicated) in the presence or absence of 50 μM of zVAD-fmk, harvested, and stained with annexin V–FITC and PI to assess cell death, TMRE to measure ΔΨm, and 2-HE to measure ROS production, and then analyzed by flow cytometry.
Mentions: To examine the role of caspases in mitochondrial functions during apoptosis, we first examined the effects of their inhibition on two important parameters, ΔΨm (using tetramethylrhodamine ethyl ester [TMRE] [Farkas et al., 1989]) and the generation of ROS (using dihydroethidium [2-HE] [Heibein et al., 1999]). HeLa cells were treated with actinomycin D (ActD) or UV to induce apoptosis, which was assessed by annexin V–FITC/propidium iodide (PI) staining (Fig. 1). Cell death, loss of ΔΨm, and production of ROS showed a close correspondence in each case. Addition of the caspase inhibitor N-benzoylcarbanyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk) delayed both cell death and loss of ΔΨm as observed previously (Waterhouse et al., 2001b). Interestingly, inhibition of caspase activation also blocked ROS production. Therefore, caspase functions appeared to be required for the rapid onset of these events during apoptosis.

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