Limits...
Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence of cell death.

Ichim G, Lopez J, Ahmed SU, Muthalagu N, Giampazolias E, Delgado ME, Haller M, Riley JS, Mason SM, Athineos D, Parsons MJ, van de Kooij B, Bouchier-Hayes L, Chalmers AJ, Rooswinkel RW, Oberst A, Blyth K, Rehm M, Murphy DJ, Tait SW - Mol. Cell (2015)

Bottom Line: Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis.Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event.Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.

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Sub-Lethal BH3-Only Protein and Apoptotic Stress Induces Minority MOMP and DNA Damage(A) MelJuSo tBID tetON or wild-type cells were treated for 12 hr with 1 μg/ml of doxycycline (DOX), and cell lysates were probed for tBID.(B) MelJuSo tBID tetON cells were treated for 6 hr with DOX and cell viability was assessed by Annexin V-based flow cytometry. Data represent mean ± SEM of three independent experiments. ∗p < 0.05, compared to control.(C) Cytosolic fractions from MelJuSo tetON tBID cells treated as in (B) were probed for cytochrome c. To induce apoptosis, 1 μg/ml DOX was used as a positive control. WCE, whole-cell extract.(D) MelJuSo tBID tetON expressing CytoGFP/MitoCherry were treated with DOX as in (A) and imaged by confocal microscopy. Arrows denote mitochondria undergoing permeabilization.(E) Quantification of cells undergoing minority MOMP. Data represent mean ± SEM of three independent experiments.(F) MelJuSo tBID tetON were treated with DOX as in (A) and cell lysates were probed by western blot for γH2A.X, PARP, and tBID.(G) U2OS cells stably expressing empty vector or BCL-xL were treated for 3 hr with FAS ligand (10 ng/ml) and CHX (1 μg/ml) and scored for the presence of minority MOMP. Data represent mean ± SEM of three independent experiments.(H) U2OS cells were treated for 3 hr with the indicated concentrations of FAS ligand and CHX (1 μg/ml), and western blot was performed for γH2A.X.(I) PDAC cells were treated with MG132 (2.5 μM) for 3 hr, and minority MOMP was quantified. Data represent mean ± SEM of three independent experiments.∗p < 0.05, compared versus control. See also Figure S4.
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fig4: Sub-Lethal BH3-Only Protein and Apoptotic Stress Induces Minority MOMP and DNA Damage(A) MelJuSo tBID tetON or wild-type cells were treated for 12 hr with 1 μg/ml of doxycycline (DOX), and cell lysates were probed for tBID.(B) MelJuSo tBID tetON cells were treated for 6 hr with DOX and cell viability was assessed by Annexin V-based flow cytometry. Data represent mean ± SEM of three independent experiments. ∗p < 0.05, compared to control.(C) Cytosolic fractions from MelJuSo tetON tBID cells treated as in (B) were probed for cytochrome c. To induce apoptosis, 1 μg/ml DOX was used as a positive control. WCE, whole-cell extract.(D) MelJuSo tBID tetON expressing CytoGFP/MitoCherry were treated with DOX as in (A) and imaged by confocal microscopy. Arrows denote mitochondria undergoing permeabilization.(E) Quantification of cells undergoing minority MOMP. Data represent mean ± SEM of three independent experiments.(F) MelJuSo tBID tetON were treated with DOX as in (A) and cell lysates were probed by western blot for γH2A.X, PARP, and tBID.(G) U2OS cells stably expressing empty vector or BCL-xL were treated for 3 hr with FAS ligand (10 ng/ml) and CHX (1 μg/ml) and scored for the presence of minority MOMP. Data represent mean ± SEM of three independent experiments.(H) U2OS cells were treated for 3 hr with the indicated concentrations of FAS ligand and CHX (1 μg/ml), and western blot was performed for γH2A.X.(I) PDAC cells were treated with MG132 (2.5 μM) for 3 hr, and minority MOMP was quantified. Data represent mean ± SEM of three independent experiments.∗p < 0.05, compared versus control. See also Figure S4.

Mentions: Although our data demonstrate that the BH3 mimetic drug ABT-737 triggers minority MOMP and DNA damage, we sought to demonstrate that these effects also occur following MOMP triggered through other means. BH3-only proteins are the endogenous inducers of MOMP through their ability to activate Bax and Bak. Therefore, we next asked whether BH3-only proteins themselves could also engage minority MOMP. For this purpose, we generated a MelJuSo cell line expressing the BH3-only protein tBID under a doxycycline inducible promoter. Whereas doxycycline addition at 1 μg/ml led to robust tBID expression and apoptotic cell death, we were able to titrate doxycycline down to induce non-lethal tBID expression (Figures 4A, 4B, and S4A–S4D). We assessed whether expression of tBID at non-lethal levels could also trigger minority MOMP. Importantly, sub-lethal amounts of tBID (induced by 2.5 and 1 ng/ml doxycycline) led to minority MOMP as detected by the presence of cytochrome c in the cytosol (Figure 4C). We aimed to validate these findings using our method to detect MOMP via GFP re-localization. Non-lethal levels of tBID expression led to a clear increase in cells displaying minority MOMP in a manner that could be prevented through co-expression of BCL-xL (Figures 4D and 4E). Importantly, as was observed for BH3-mimetic treatment, the caspase inhibitor Q-VD-OPh also prevented H2A.X phosphorylation upon induction of sub-lethal levels of tBID (Figure 4F). We next addressed if a physiological apoptotic stimulus could also trigger minority MOMP and DNA-damage. Accordingly, sub-lethal treatment of U2OS cells with FAS ligand triggered minority MOMP and DNA-damage in a BCL-xL and caspase-inhibitable manner (Figures 4G, 4H, and S4E). Similarly, sub-lethal treatment of cells with the proteasome inhibitor MG132 also induced minority MOMP (Figure 4I). Collectively, these data demonstrate that similar to BH3-mimetics, apoptotic stimuli and pro-apoptotic BH3-only proteins also induce minority MOMP and DNA damage.


Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence of cell death.

Ichim G, Lopez J, Ahmed SU, Muthalagu N, Giampazolias E, Delgado ME, Haller M, Riley JS, Mason SM, Athineos D, Parsons MJ, van de Kooij B, Bouchier-Hayes L, Chalmers AJ, Rooswinkel RW, Oberst A, Blyth K, Rehm M, Murphy DJ, Tait SW - Mol. Cell (2015)

Sub-Lethal BH3-Only Protein and Apoptotic Stress Induces Minority MOMP and DNA Damage(A) MelJuSo tBID tetON or wild-type cells were treated for 12 hr with 1 μg/ml of doxycycline (DOX), and cell lysates were probed for tBID.(B) MelJuSo tBID tetON cells were treated for 6 hr with DOX and cell viability was assessed by Annexin V-based flow cytometry. Data represent mean ± SEM of three independent experiments. ∗p < 0.05, compared to control.(C) Cytosolic fractions from MelJuSo tetON tBID cells treated as in (B) were probed for cytochrome c. To induce apoptosis, 1 μg/ml DOX was used as a positive control. WCE, whole-cell extract.(D) MelJuSo tBID tetON expressing CytoGFP/MitoCherry were treated with DOX as in (A) and imaged by confocal microscopy. Arrows denote mitochondria undergoing permeabilization.(E) Quantification of cells undergoing minority MOMP. Data represent mean ± SEM of three independent experiments.(F) MelJuSo tBID tetON were treated with DOX as in (A) and cell lysates were probed by western blot for γH2A.X, PARP, and tBID.(G) U2OS cells stably expressing empty vector or BCL-xL were treated for 3 hr with FAS ligand (10 ng/ml) and CHX (1 μg/ml) and scored for the presence of minority MOMP. Data represent mean ± SEM of three independent experiments.(H) U2OS cells were treated for 3 hr with the indicated concentrations of FAS ligand and CHX (1 μg/ml), and western blot was performed for γH2A.X.(I) PDAC cells were treated with MG132 (2.5 μM) for 3 hr, and minority MOMP was quantified. Data represent mean ± SEM of three independent experiments.∗p < 0.05, compared versus control. See also Figure S4.
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fig4: Sub-Lethal BH3-Only Protein and Apoptotic Stress Induces Minority MOMP and DNA Damage(A) MelJuSo tBID tetON or wild-type cells were treated for 12 hr with 1 μg/ml of doxycycline (DOX), and cell lysates were probed for tBID.(B) MelJuSo tBID tetON cells were treated for 6 hr with DOX and cell viability was assessed by Annexin V-based flow cytometry. Data represent mean ± SEM of three independent experiments. ∗p < 0.05, compared to control.(C) Cytosolic fractions from MelJuSo tetON tBID cells treated as in (B) were probed for cytochrome c. To induce apoptosis, 1 μg/ml DOX was used as a positive control. WCE, whole-cell extract.(D) MelJuSo tBID tetON expressing CytoGFP/MitoCherry were treated with DOX as in (A) and imaged by confocal microscopy. Arrows denote mitochondria undergoing permeabilization.(E) Quantification of cells undergoing minority MOMP. Data represent mean ± SEM of three independent experiments.(F) MelJuSo tBID tetON were treated with DOX as in (A) and cell lysates were probed by western blot for γH2A.X, PARP, and tBID.(G) U2OS cells stably expressing empty vector or BCL-xL were treated for 3 hr with FAS ligand (10 ng/ml) and CHX (1 μg/ml) and scored for the presence of minority MOMP. Data represent mean ± SEM of three independent experiments.(H) U2OS cells were treated for 3 hr with the indicated concentrations of FAS ligand and CHX (1 μg/ml), and western blot was performed for γH2A.X.(I) PDAC cells were treated with MG132 (2.5 μM) for 3 hr, and minority MOMP was quantified. Data represent mean ± SEM of three independent experiments.∗p < 0.05, compared versus control. See also Figure S4.
Mentions: Although our data demonstrate that the BH3 mimetic drug ABT-737 triggers minority MOMP and DNA damage, we sought to demonstrate that these effects also occur following MOMP triggered through other means. BH3-only proteins are the endogenous inducers of MOMP through their ability to activate Bax and Bak. Therefore, we next asked whether BH3-only proteins themselves could also engage minority MOMP. For this purpose, we generated a MelJuSo cell line expressing the BH3-only protein tBID under a doxycycline inducible promoter. Whereas doxycycline addition at 1 μg/ml led to robust tBID expression and apoptotic cell death, we were able to titrate doxycycline down to induce non-lethal tBID expression (Figures 4A, 4B, and S4A–S4D). We assessed whether expression of tBID at non-lethal levels could also trigger minority MOMP. Importantly, sub-lethal amounts of tBID (induced by 2.5 and 1 ng/ml doxycycline) led to minority MOMP as detected by the presence of cytochrome c in the cytosol (Figure 4C). We aimed to validate these findings using our method to detect MOMP via GFP re-localization. Non-lethal levels of tBID expression led to a clear increase in cells displaying minority MOMP in a manner that could be prevented through co-expression of BCL-xL (Figures 4D and 4E). Importantly, as was observed for BH3-mimetic treatment, the caspase inhibitor Q-VD-OPh also prevented H2A.X phosphorylation upon induction of sub-lethal levels of tBID (Figure 4F). We next addressed if a physiological apoptotic stimulus could also trigger minority MOMP and DNA-damage. Accordingly, sub-lethal treatment of U2OS cells with FAS ligand triggered minority MOMP and DNA-damage in a BCL-xL and caspase-inhibitable manner (Figures 4G, 4H, and S4E). Similarly, sub-lethal treatment of cells with the proteasome inhibitor MG132 also induced minority MOMP (Figure 4I). Collectively, these data demonstrate that similar to BH3-mimetics, apoptotic stimuli and pro-apoptotic BH3-only proteins also induce minority MOMP and DNA damage.

Bottom Line: Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis.Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event.Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.

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Related in: MedlinePlus