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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Minority MOMP Induces Caspase-Dependent DNA Damage(A) HeLa and U2OS cells were treated for 3 hr with indicated sub-lethal doses of ABT-737 or enantiomer (10 μM, ENA) in presence or absence of caspase inhibitor Q-VD-OPh (10 μM). Cell lysates were for probed by western blot for γH2A.X and actin (as loading control).(B) U2OS cells transiently expressing CytoGFP and MitoCherry were treated with ABT-737 (5 μM) for 3 hr or H2O2 (25 μM) for 10 min and immunostained for γH2A.X. Representative images are shown.(C) Quantification of γH2A.X foci in cells displaying minority MOMP (ABT-737-treated cells), control, and H2O2-treated cells. Data represent mean ± SEM of three independent experiments.(D) HeLa cells were treated as in (B) and subject to comet assay. Representative images are shown.(E) Quantification of comet tail moment following ABT-737 treatment. Data represent mean ± SEM of three independent experiments.(F–H) HeLa and HeLa overexpressing BCL-xL (F), wild-type MEF and MEF double knockout for Bax and Bak (G), or HeLa versus HeLa knockdown for APAF-1 (H) were treated as in (A) and western blotted for γH2A.X and actin.(I) A549 cells expressing caspase-9 fused to a FKBP dimerization domain were treated with indicated sub-lethal concentrations of homodimerizer (DM) for 3 hr to induce caspase-9 dimerization and activation. Cleavage of caspase-3 and γH2A.X was assessed by western blot.(J) Wild-type HeLa and U2OS cells and their Cad-deleted counterparts were treated and immunoblotted as in (A).(K) Wild-type and Cad-deleted HeLa cells were treated as in (D) and used to perform comet assay. Graph represents quantification of comet tail moment. Data represent mean ± SEM of four independent experiments.(L) Representative images of γH2A.X and TUNEL immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3).∗p < 0.05, compared versus control. See also Figure S3.
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fig3: Minority MOMP Induces Caspase-Dependent DNA Damage(A) HeLa and U2OS cells were treated for 3 hr with indicated sub-lethal doses of ABT-737 or enantiomer (10 μM, ENA) in presence or absence of caspase inhibitor Q-VD-OPh (10 μM). Cell lysates were for probed by western blot for γH2A.X and actin (as loading control).(B) U2OS cells transiently expressing CytoGFP and MitoCherry were treated with ABT-737 (5 μM) for 3 hr or H2O2 (25 μM) for 10 min and immunostained for γH2A.X. Representative images are shown.(C) Quantification of γH2A.X foci in cells displaying minority MOMP (ABT-737-treated cells), control, and H2O2-treated cells. Data represent mean ± SEM of three independent experiments.(D) HeLa cells were treated as in (B) and subject to comet assay. Representative images are shown.(E) Quantification of comet tail moment following ABT-737 treatment. Data represent mean ± SEM of three independent experiments.(F–H) HeLa and HeLa overexpressing BCL-xL (F), wild-type MEF and MEF double knockout for Bax and Bak (G), or HeLa versus HeLa knockdown for APAF-1 (H) were treated as in (A) and western blotted for γH2A.X and actin.(I) A549 cells expressing caspase-9 fused to a FKBP dimerization domain were treated with indicated sub-lethal concentrations of homodimerizer (DM) for 3 hr to induce caspase-9 dimerization and activation. Cleavage of caspase-3 and γH2A.X was assessed by western blot.(J) Wild-type HeLa and U2OS cells and their Cad-deleted counterparts were treated and immunoblotted as in (A).(K) Wild-type and Cad-deleted HeLa cells were treated as in (D) and used to perform comet assay. Graph represents quantification of comet tail moment. Data represent mean ± SEM of four independent experiments.(L) Representative images of γH2A.X and TUNEL immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3).∗p < 0.05, compared versus control. See also Figure S3.

Mentions: DNA fragmentation is a classical apoptotic hallmark mediated by caspase-activated DNase (CAD) (Sakahira et al., 1998). We hypothesized that limited caspase activity following minority MOMP might lead to low-level CAD activation, and, in turn, to induction of DNA damage in surviving cells. To test this, we treated HeLa and U2OS cells with ABT-737 in the presence or absence of Q-VD-OPh and analyzed for γH2A.X as readout for DNA damage. Importantly, in both cell lines, non-lethal treatment with BH3 mimetic led to caspase-dependent DNA damage as demonstrated by a caspase-dependent increase in γH2A.X (Figure 3A). Importantly, the extent of DNA-damage (measured by γH2A.X foci) correlated with minority MOMP, implicating a causal relationship between the two (Figures 3B and 3C). The ability of BH3 mimetics to engage DNA-damage in a caspase-dependent manner was also observed in other cell lines (Figure S3A). Further demonstrating caspase-dependent DNA damage, ABT-737 treatment also led to an increase in DNA breaks, measured by comet assay and Ser15 p53 phosphorylation dependent on caspase function, mirroring γH2A.X levels (Figures 3D, 3E, and S3B). Although p53 independent, induction of DNA damage depended on mitochondrial caspase activation, because overexpression of BCL-xL (HeLa), deletion of Bax (HCT-116) or Bax and Bak in murine embryonic fibroblasts (MEF), or knockdown of APAF-1 (HeLa) prevented BH3 mimetic-induced γH2A.X (Figures 3F–3H and S3C–S3E). Supporting these findings, direct, non-lethal activation of caspase-9 by chemical dimerization also led to DNA damage (Figures 3I and S3F).


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)

Minority MOMP Induces Caspase-Dependent DNA Damage(A) HeLa and U2OS cells were treated for 3 hr with indicated sub-lethal doses of ABT-737 or enantiomer (10 μM, ENA) in presence or absence of caspase inhibitor Q-VD-OPh (10 μM). Cell lysates were for probed by western blot for γH2A.X and actin (as loading control).(B) U2OS cells transiently expressing CytoGFP and MitoCherry were treated with ABT-737 (5 μM) for 3 hr or H2O2 (25 μM) for 10 min and immunostained for γH2A.X. Representative images are shown.(C) Quantification of γH2A.X foci in cells displaying minority MOMP (ABT-737-treated cells), control, and H2O2-treated cells. Data represent mean ± SEM of three independent experiments.(D) HeLa cells were treated as in (B) and subject to comet assay. Representative images are shown.(E) Quantification of comet tail moment following ABT-737 treatment. Data represent mean ± SEM of three independent experiments.(F–H) HeLa and HeLa overexpressing BCL-xL (F), wild-type MEF and MEF double knockout for Bax and Bak (G), or HeLa versus HeLa knockdown for APAF-1 (H) were treated as in (A) and western blotted for γH2A.X and actin.(I) A549 cells expressing caspase-9 fused to a FKBP dimerization domain were treated with indicated sub-lethal concentrations of homodimerizer (DM) for 3 hr to induce caspase-9 dimerization and activation. Cleavage of caspase-3 and γH2A.X was assessed by western blot.(J) Wild-type HeLa and U2OS cells and their Cad-deleted counterparts were treated and immunoblotted as in (A).(K) Wild-type and Cad-deleted HeLa cells were treated as in (D) and used to perform comet assay. Graph represents quantification of comet tail moment. Data represent mean ± SEM of four independent experiments.(L) Representative images of γH2A.X and TUNEL immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3).∗p < 0.05, compared versus control. See also Figure S3.
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fig3: Minority MOMP Induces Caspase-Dependent DNA Damage(A) HeLa and U2OS cells were treated for 3 hr with indicated sub-lethal doses of ABT-737 or enantiomer (10 μM, ENA) in presence or absence of caspase inhibitor Q-VD-OPh (10 μM). Cell lysates were for probed by western blot for γH2A.X and actin (as loading control).(B) U2OS cells transiently expressing CytoGFP and MitoCherry were treated with ABT-737 (5 μM) for 3 hr or H2O2 (25 μM) for 10 min and immunostained for γH2A.X. Representative images are shown.(C) Quantification of γH2A.X foci in cells displaying minority MOMP (ABT-737-treated cells), control, and H2O2-treated cells. Data represent mean ± SEM of three independent experiments.(D) HeLa cells were treated as in (B) and subject to comet assay. Representative images are shown.(E) Quantification of comet tail moment following ABT-737 treatment. Data represent mean ± SEM of three independent experiments.(F–H) HeLa and HeLa overexpressing BCL-xL (F), wild-type MEF and MEF double knockout for Bax and Bak (G), or HeLa versus HeLa knockdown for APAF-1 (H) were treated as in (A) and western blotted for γH2A.X and actin.(I) A549 cells expressing caspase-9 fused to a FKBP dimerization domain were treated with indicated sub-lethal concentrations of homodimerizer (DM) for 3 hr to induce caspase-9 dimerization and activation. Cleavage of caspase-3 and γH2A.X was assessed by western blot.(J) Wild-type HeLa and U2OS cells and their Cad-deleted counterparts were treated and immunoblotted as in (A).(K) Wild-type and Cad-deleted HeLa cells were treated as in (D) and used to perform comet assay. Graph represents quantification of comet tail moment. Data represent mean ± SEM of four independent experiments.(L) Representative images of γH2A.X and TUNEL immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3).∗p < 0.05, compared versus control. See also Figure S3.
Mentions: DNA fragmentation is a classical apoptotic hallmark mediated by caspase-activated DNase (CAD) (Sakahira et al., 1998). We hypothesized that limited caspase activity following minority MOMP might lead to low-level CAD activation, and, in turn, to induction of DNA damage in surviving cells. To test this, we treated HeLa and U2OS cells with ABT-737 in the presence or absence of Q-VD-OPh and analyzed for γH2A.X as readout for DNA damage. Importantly, in both cell lines, non-lethal treatment with BH3 mimetic led to caspase-dependent DNA damage as demonstrated by a caspase-dependent increase in γH2A.X (Figure 3A). Importantly, the extent of DNA-damage (measured by γH2A.X foci) correlated with minority MOMP, implicating a causal relationship between the two (Figures 3B and 3C). The ability of BH3 mimetics to engage DNA-damage in a caspase-dependent manner was also observed in other cell lines (Figure S3A). Further demonstrating caspase-dependent DNA damage, ABT-737 treatment also led to an increase in DNA breaks, measured by comet assay and Ser15 p53 phosphorylation dependent on caspase function, mirroring γH2A.X levels (Figures 3D, 3E, and S3B). Although p53 independent, induction of DNA damage depended on mitochondrial caspase activation, because overexpression of BCL-xL (HeLa), deletion of Bax (HCT-116) or Bax and Bak in murine embryonic fibroblasts (MEF), or knockdown of APAF-1 (HeLa) prevented BH3 mimetic-induced γH2A.X (Figures 3F–3H and S3C–S3E). Supporting these findings, direct, non-lethal activation of caspase-9 by chemical dimerization also led to DNA damage (Figures 3I and S3F).

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