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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 Promotes Genomic Instability(A) HeLa cells were treated daily with ABT-737 or enantiomer (10 μM, Ctrl) at the indicated concentrations for either 5 (P5) or 10 (P10) passages and then scored for micronuclei. Data represent mean ± SEM of three independent experiments.(B) U2OS cells stably expressing BCL-xL (U2OS BCL-xL) or empty vector (U2OS LZRS) were treated as in (A) and assessed for micronuclei. Data represent mean ± SEM of three independent experiments.(C) MelJuSo tetON or wild-type MelJuSo cells were treated daily for ten passages with the indicated concentration of doxycycline (DOX) in the absence or presence of Q-VD-OPh, and micronuclei were scored. Data represent mean ± SEM of three independent experiments.(D) PDAC and 3T3-SA cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM). Data represent mean ± SEM of three independent experiments.(E) Genomic DNA was extracted from PALA-resistant PDAC, 3T3-SA, and WEHI-S clones, and Cad gene levels were quantified by qPCR. Data represent the mean ± SD from triplicate samples from a representative experiment carried out twice independently.(F) Representative images of PALA-resistant colonies from PDAC cells stably expressing BCL-xL (PDAC BCL-xL) or empty vector (PDAC LZRS). Cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM).(G) Quantification of PALA-resistance clonogenic survival in PDAC BCL-xL versus PDAC LZRS cells. Data represent mean ± SEM of three independent experiments.(H) Cad expression in PDAC BCL-xL and PDAC-LZRS PALA resistant colonies. Data represent the mean from a representative experiment carried out twice independently.Where stated, ∗p < 0.05, compared versus control. See also Figure S6.
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fig6: Minority MOMP Promotes Genomic Instability(A) HeLa cells were treated daily with ABT-737 or enantiomer (10 μM, Ctrl) at the indicated concentrations for either 5 (P5) or 10 (P10) passages and then scored for micronuclei. Data represent mean ± SEM of three independent experiments.(B) U2OS cells stably expressing BCL-xL (U2OS BCL-xL) or empty vector (U2OS LZRS) were treated as in (A) and assessed for micronuclei. Data represent mean ± SEM of three independent experiments.(C) MelJuSo tetON or wild-type MelJuSo cells were treated daily for ten passages with the indicated concentration of doxycycline (DOX) in the absence or presence of Q-VD-OPh, and micronuclei were scored. Data represent mean ± SEM of three independent experiments.(D) PDAC and 3T3-SA cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM). Data represent mean ± SEM of three independent experiments.(E) Genomic DNA was extracted from PALA-resistant PDAC, 3T3-SA, and WEHI-S clones, and Cad gene levels were quantified by qPCR. Data represent the mean ± SD from triplicate samples from a representative experiment carried out twice independently.(F) Representative images of PALA-resistant colonies from PDAC cells stably expressing BCL-xL (PDAC BCL-xL) or empty vector (PDAC LZRS). Cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM).(G) Quantification of PALA-resistance clonogenic survival in PDAC BCL-xL versus PDAC LZRS cells. Data represent mean ± SEM of three independent experiments.(H) Cad expression in PDAC BCL-xL and PDAC-LZRS PALA resistant colonies. Data represent the mean from a representative experiment carried out twice independently.Where stated, ∗p < 0.05, compared versus control. See also Figure S6.

Mentions: Based on our results, we hypothesized that by causing DNA damage, minority MOMP may promote genomic instability and transformation. To test this possibility, we repeatedly treated HeLa and U2OS cells with sub-lethal doses of ABT-737 for five (P5) or ten passages (P10). Following blockage of cytokinesis, we then quantified the number of cells with micronuclei, a well-established marker for chromosomal damage (Figure S6A) (Fenech, 2007). Strikingly, U2OS and HeLa cells displayed a significant increase in micronuclei number following ABT-737 treatment in a dose-dependent manner (Figures 6A and S6B). Ectopic BCL-xL expression inhibited micronuclei accumulation in U2OS cells, confirming that the observed genomic instability required mitochondrial permeabilization (Figure 6B). In an analogous manner, induction of sub-lethal levels of the BH3-only protein tBID in MelJuSo cells also promoted micronuclei accumulation in a dose-dependent manner (Figure 6C). Micronuclei accumulation following tBID expression was reduced to control levels by the caspase inhibitor Q-VD-OPh, demonstrating a requirement for caspases in minority MOMP-induced chromosomal damage (Figure 6C).


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 Promotes Genomic Instability(A) HeLa cells were treated daily with ABT-737 or enantiomer (10 μM, Ctrl) at the indicated concentrations for either 5 (P5) or 10 (P10) passages and then scored for micronuclei. Data represent mean ± SEM of three independent experiments.(B) U2OS cells stably expressing BCL-xL (U2OS BCL-xL) or empty vector (U2OS LZRS) were treated as in (A) and assessed for micronuclei. Data represent mean ± SEM of three independent experiments.(C) MelJuSo tetON or wild-type MelJuSo cells were treated daily for ten passages with the indicated concentration of doxycycline (DOX) in the absence or presence of Q-VD-OPh, and micronuclei were scored. Data represent mean ± SEM of three independent experiments.(D) PDAC and 3T3-SA cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM). Data represent mean ± SEM of three independent experiments.(E) Genomic DNA was extracted from PALA-resistant PDAC, 3T3-SA, and WEHI-S clones, and Cad gene levels were quantified by qPCR. Data represent the mean ± SD from triplicate samples from a representative experiment carried out twice independently.(F) Representative images of PALA-resistant colonies from PDAC cells stably expressing BCL-xL (PDAC BCL-xL) or empty vector (PDAC LZRS). Cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM).(G) Quantification of PALA-resistance clonogenic survival in PDAC BCL-xL versus PDAC LZRS cells. Data represent mean ± SEM of three independent experiments.(H) Cad expression in PDAC BCL-xL and PDAC-LZRS PALA resistant colonies. Data represent the mean from a representative experiment carried out twice independently.Where stated, ∗p < 0.05, compared versus control. See also Figure S6.
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fig6: Minority MOMP Promotes Genomic Instability(A) HeLa cells were treated daily with ABT-737 or enantiomer (10 μM, Ctrl) at the indicated concentrations for either 5 (P5) or 10 (P10) passages and then scored for micronuclei. Data represent mean ± SEM of three independent experiments.(B) U2OS cells stably expressing BCL-xL (U2OS BCL-xL) or empty vector (U2OS LZRS) were treated as in (A) and assessed for micronuclei. Data represent mean ± SEM of three independent experiments.(C) MelJuSo tetON or wild-type MelJuSo cells were treated daily for ten passages with the indicated concentration of doxycycline (DOX) in the absence or presence of Q-VD-OPh, and micronuclei were scored. Data represent mean ± SEM of three independent experiments.(D) PDAC and 3T3-SA cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM). Data represent mean ± SEM of three independent experiments.(E) Genomic DNA was extracted from PALA-resistant PDAC, 3T3-SA, and WEHI-S clones, and Cad gene levels were quantified by qPCR. Data represent the mean ± SD from triplicate samples from a representative experiment carried out twice independently.(F) Representative images of PALA-resistant colonies from PDAC cells stably expressing BCL-xL (PDAC BCL-xL) or empty vector (PDAC LZRS). Cells were treated daily for five passages with ABT-737 at the indicated concentrations, and clonogenic survival assay was performed in media containing PALA (100 μM).(G) Quantification of PALA-resistance clonogenic survival in PDAC BCL-xL versus PDAC LZRS cells. Data represent mean ± SEM of three independent experiments.(H) Cad expression in PDAC BCL-xL and PDAC-LZRS PALA resistant colonies. Data represent the mean from a representative experiment carried out twice independently.Where stated, ∗p < 0.05, compared versus control. See also Figure S6.
Mentions: Based on our results, we hypothesized that by causing DNA damage, minority MOMP may promote genomic instability and transformation. To test this possibility, we repeatedly treated HeLa and U2OS cells with sub-lethal doses of ABT-737 for five (P5) or ten passages (P10). Following blockage of cytokinesis, we then quantified the number of cells with micronuclei, a well-established marker for chromosomal damage (Figure S6A) (Fenech, 2007). Strikingly, U2OS and HeLa cells displayed a significant increase in micronuclei number following ABT-737 treatment in a dose-dependent manner (Figures 6A and S6B). Ectopic BCL-xL expression inhibited micronuclei accumulation in U2OS cells, confirming that the observed genomic instability required mitochondrial permeabilization (Figure 6B). In an analogous manner, induction of sub-lethal levels of the BH3-only protein tBID in MelJuSo cells also promoted micronuclei accumulation in a dose-dependent manner (Figure 6C). Micronuclei accumulation following tBID expression was reduced to control levels by the caspase inhibitor Q-VD-OPh, demonstrating a requirement for caspases in minority MOMP-induced chromosomal damage (Figure 6C).

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