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 Promotes Transformation and Tumorigenesis(A and B) Primary mouse embryonic fibroblasts (MEF) were treated daily for seven passages with the indicated concentrations of ABT-737 and transduced with E1A- and KRAS-expressing retrovirus. Representative images are shown in (A), and quantitation of the number of transformed colonies is depicted in (B). Data represent mean ± SEM of triplicate samples for a representative experiment (out of three independent experiments).(C) Primary p19Arf  MEFs or p19Arf  MEFs transduced with empty vector or BCL-xL-expressing retrovirus were treated for ten passages with ABT-737 (10 μM) in the presence or absence of Q-VD-OPh (10 μM), enantiomer (10 μM), or DMSO (Ctrl), and their anchorage-independent growth was assessed by soft agar assay. Representative images for each condition are shown.(D) Quantification of triplicate samples (mean ± SEM) from one representative soft agar assay (out of three independent experiments).(E) Primary p19Arf  MEF were treated with ABT-737 (10 μM) or enantiomer (ENA, 10 μM) for ten passages. CD1-Nude female mice were injected subcutaneously with treated MEF and tumor growth was measured over time. Data are plotted as mean with error bars representing 95% CI (n = 15 for both treatments). Applying Welch’s t test to the area-under-the-curve data produces a p value of 3.48 × 10−7 for the tumor growth rates.See also Figure S7.
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fig7: Minority MOMP Promotes Transformation and Tumorigenesis(A and B) Primary mouse embryonic fibroblasts (MEF) were treated daily for seven passages with the indicated concentrations of ABT-737 and transduced with E1A- and KRAS-expressing retrovirus. Representative images are shown in (A), and quantitation of the number of transformed colonies is depicted in (B). Data represent mean ± SEM of triplicate samples for a representative experiment (out of three independent experiments).(C) Primary p19Arf MEFs or p19Arf MEFs transduced with empty vector or BCL-xL-expressing retrovirus were treated for ten passages with ABT-737 (10 μM) in the presence or absence of Q-VD-OPh (10 μM), enantiomer (10 μM), or DMSO (Ctrl), and their anchorage-independent growth was assessed by soft agar assay. Representative images for each condition are shown.(D) Quantification of triplicate samples (mean ± SEM) from one representative soft agar assay (out of three independent experiments).(E) Primary p19Arf MEF were treated with ABT-737 (10 μM) or enantiomer (ENA, 10 μM) for ten passages. CD1-Nude female mice were injected subcutaneously with treated MEF and tumor growth was measured over time. Data are plotted as mean with error bars representing 95% CI (n = 15 for both treatments). Applying Welch’s t test to the area-under-the-curve data produces a p value of 3.48 × 10−7 for the tumor growth rates.See also Figure S7.

Mentions: Genomic instability can promote oncogenic transformation. Therefore, we investigated if minority MOMP-induced DNA damage leading to genomic instability could potentiate E1A/KRAS-induced transformation in primary mouse embryonic fibroblasts (MEF). Primary MEFs were treated with sub-lethal doses of ABT-737 prior to transformation with E1A/KRAS. Remarkably, BH3-only stress was able to potentiate the transforming ability of E1A/KRAS in primary cells in a dose-dependent manner (Figures 7A and 7B). We next tested if minority MOMP could promote transformation in a different setting. Primary MEF deficient in the tumor suppressor p19Arf were treated with enantiomer or ABT-737 in the presence or absence of caspase-inhibitor Q-VD-OPh (Figure S7A). Additionally, primary p19Arf deficient MEF expressing BCL-xL or empty vector were treated with ABT-737 (Figures S7B and S7C). Following treatment, cellular transformation was assessed by anchorage-independent growth in soft agar. Strikingly, ABT-737 treatment led to transformation that was completely prevented by inhibiting caspases (with Q-VD-OPh) or by inhibiting MOMP through BCL-xL expression (Figures 7C and 7D). We next compared ABT-737 or enantiomer treated p19Arf MEF for their tumorigenic potential in vivo following subcutaneous injection in CD1-Nude mice. Significantly, ABT-737 treated MEF formed tumors much more rapidly than enantiomer-treated MEF (Figure 7E). Collectively, these results demonstrate that minority MOMP promotes both cellular transformation and tumorigenesis.


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 Transformation and Tumorigenesis(A and B) Primary mouse embryonic fibroblasts (MEF) were treated daily for seven passages with the indicated concentrations of ABT-737 and transduced with E1A- and KRAS-expressing retrovirus. Representative images are shown in (A), and quantitation of the number of transformed colonies is depicted in (B). Data represent mean ± SEM of triplicate samples for a representative experiment (out of three independent experiments).(C) Primary p19Arf  MEFs or p19Arf  MEFs transduced with empty vector or BCL-xL-expressing retrovirus were treated for ten passages with ABT-737 (10 μM) in the presence or absence of Q-VD-OPh (10 μM), enantiomer (10 μM), or DMSO (Ctrl), and their anchorage-independent growth was assessed by soft agar assay. Representative images for each condition are shown.(D) Quantification of triplicate samples (mean ± SEM) from one representative soft agar assay (out of three independent experiments).(E) Primary p19Arf  MEF were treated with ABT-737 (10 μM) or enantiomer (ENA, 10 μM) for ten passages. CD1-Nude female mice were injected subcutaneously with treated MEF and tumor growth was measured over time. Data are plotted as mean with error bars representing 95% CI (n = 15 for both treatments). Applying Welch’s t test to the area-under-the-curve data produces a p value of 3.48 × 10−7 for the tumor growth rates.See also Figure S7.
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fig7: Minority MOMP Promotes Transformation and Tumorigenesis(A and B) Primary mouse embryonic fibroblasts (MEF) were treated daily for seven passages with the indicated concentrations of ABT-737 and transduced with E1A- and KRAS-expressing retrovirus. Representative images are shown in (A), and quantitation of the number of transformed colonies is depicted in (B). Data represent mean ± SEM of triplicate samples for a representative experiment (out of three independent experiments).(C) Primary p19Arf MEFs or p19Arf MEFs transduced with empty vector or BCL-xL-expressing retrovirus were treated for ten passages with ABT-737 (10 μM) in the presence or absence of Q-VD-OPh (10 μM), enantiomer (10 μM), or DMSO (Ctrl), and their anchorage-independent growth was assessed by soft agar assay. Representative images for each condition are shown.(D) Quantification of triplicate samples (mean ± SEM) from one representative soft agar assay (out of three independent experiments).(E) Primary p19Arf MEF were treated with ABT-737 (10 μM) or enantiomer (ENA, 10 μM) for ten passages. CD1-Nude female mice were injected subcutaneously with treated MEF and tumor growth was measured over time. Data are plotted as mean with error bars representing 95% CI (n = 15 for both treatments). Applying Welch’s t test to the area-under-the-curve data produces a p value of 3.48 × 10−7 for the tumor growth rates.See also Figure S7.
Mentions: Genomic instability can promote oncogenic transformation. Therefore, we investigated if minority MOMP-induced DNA damage leading to genomic instability could potentiate E1A/KRAS-induced transformation in primary mouse embryonic fibroblasts (MEF). Primary MEFs were treated with sub-lethal doses of ABT-737 prior to transformation with E1A/KRAS. Remarkably, BH3-only stress was able to potentiate the transforming ability of E1A/KRAS in primary cells in a dose-dependent manner (Figures 7A and 7B). We next tested if minority MOMP could promote transformation in a different setting. Primary MEF deficient in the tumor suppressor p19Arf were treated with enantiomer or ABT-737 in the presence or absence of caspase-inhibitor Q-VD-OPh (Figure S7A). Additionally, primary p19Arf deficient MEF expressing BCL-xL or empty vector were treated with ABT-737 (Figures S7B and S7C). Following treatment, cellular transformation was assessed by anchorage-independent growth in soft agar. Strikingly, ABT-737 treatment led to transformation that was completely prevented by inhibiting caspases (with Q-VD-OPh) or by inhibiting MOMP through BCL-xL expression (Figures 7C and 7D). We next compared ABT-737 or enantiomer treated p19Arf MEF for their tumorigenic potential in vivo following subcutaneous injection in CD1-Nude mice. Significantly, ABT-737 treated MEF formed tumors much more rapidly than enantiomer-treated MEF (Figure 7E). Collectively, these results demonstrate that minority MOMP promotes both cellular transformation and tumorigenesis.

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.

Show MeSH
Related in: MedlinePlus