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.

Show MeSH

Related in: MedlinePlus

JNK Regulates the DNA Damage Response(A) U2OS cells were treated for 3 hr with 10 μM ABT-737, and phospho JNK1/2, ATM, and ATR were assessed by western blot. Ionizing radiation (2 Gy) was used as a positive control.(B) U2OS were treated with ABT-737 as in (A) in the presence or absence of Q-VD-OPh (10 μM) and immunoblotted for P-JNK1/2 and γH2A.X.(C) Representative images of P-JNK1/2 immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3); untreated mice (n = 3) were used as control.(D) HeLa cells were transiently transfected with siRNA for JNK1/2 and treated as in (A). Cell lysates were probed for total JNK1/2 and γH2A.X.(E) As in (D), except that siRNA oligos targeting JNK1, JNK2, or both JNK1 and JNK2 together were used.(F) U2OS cells were transfected with siRNA targeting CAD and treated with ABT-737. Cell lysates were probed for γH2A.X and P-JNK1/2.See also Figure S5.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352766&req=5

fig5: JNK Regulates the DNA Damage Response(A) U2OS cells were treated for 3 hr with 10 μM ABT-737, and phospho JNK1/2, ATM, and ATR were assessed by western blot. Ionizing radiation (2 Gy) was used as a positive control.(B) U2OS were treated with ABT-737 as in (A) in the presence or absence of Q-VD-OPh (10 μM) and immunoblotted for P-JNK1/2 and γH2A.X.(C) Representative images of P-JNK1/2 immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3); untreated mice (n = 3) were used as control.(D) HeLa cells were transiently transfected with siRNA for JNK1/2 and treated as in (A). Cell lysates were probed for total JNK1/2 and γH2A.X.(E) As in (D), except that siRNA oligos targeting JNK1, JNK2, or both JNK1 and JNK2 together were used.(F) U2OS cells were transfected with siRNA targeting CAD and treated with ABT-737. Cell lysates were probed for γH2A.X and P-JNK1/2.See also Figure S5.

Mentions: DNA damage-induced phosphorylation of H2A.X at S139 often occurs via the PI3K-related kinase family members ATM, ATR, and DNA-PK (Jackson and Bartek, 2009; Shiloh, 2003). However, we did not observe any significant increase in activated, phosphorylated ATM or ATR kinase following sub-lethal ABT-737 treatment (Figure 5A). Moreover, RNAi-mediated knockdown of ATM, ATR, or DNA-PK failed to affect BH3 mimetic-induced γH2A.X levels (Figures S5A and S5B). Besides ATM, ATR, and DNA-PK, c-Jun N-terminal kinase (JNK) has also been found to mediate H2A.X phosphorylation in some settings (Lu et al., 2006). Importantly, sub-lethal treatment with ABT-737 led to a caspase-dependent increase in JNK1/2 activation mirroring levels of γH2A.X (Figure 5B). JNK activation following ABT-737 administration was also detected in vivo in the small intestine (Figure 5C). To directly investigate the role of JNK in H2A.X phosphorylation, we used RNAi. Combined knockdown of JNK1/2 or selective knockdown of JNK2 effectively prevented ABT-737 induced γH2A.X implicating a direct role for JNK2 in H2A.X phosphorylation (Figures 5D and 5E). Accordingly, RNAi-mediated knockdown of CAD largely inhibited JNK1/2 phosphorylation (Figure 5F). These results identify JNK2 as a key player in the minority MOMP-induced DNA damage response.


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)

JNK Regulates the DNA Damage Response(A) U2OS cells were treated for 3 hr with 10 μM ABT-737, and phospho JNK1/2, ATM, and ATR were assessed by western blot. Ionizing radiation (2 Gy) was used as a positive control.(B) U2OS were treated with ABT-737 as in (A) in the presence or absence of Q-VD-OPh (10 μM) and immunoblotted for P-JNK1/2 and γH2A.X.(C) Representative images of P-JNK1/2 immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3); untreated mice (n = 3) were used as control.(D) HeLa cells were transiently transfected with siRNA for JNK1/2 and treated as in (A). Cell lysates were probed for total JNK1/2 and γH2A.X.(E) As in (D), except that siRNA oligos targeting JNK1, JNK2, or both JNK1 and JNK2 together were used.(F) U2OS cells were transfected with siRNA targeting CAD and treated with ABT-737. Cell lysates were probed for γH2A.X and P-JNK1/2.See also Figure S5.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352766&req=5

fig5: JNK Regulates the DNA Damage Response(A) U2OS cells were treated for 3 hr with 10 μM ABT-737, and phospho JNK1/2, ATM, and ATR were assessed by western blot. Ionizing radiation (2 Gy) was used as a positive control.(B) U2OS were treated with ABT-737 as in (A) in the presence or absence of Q-VD-OPh (10 μM) and immunoblotted for P-JNK1/2 and γH2A.X.(C) Representative images of P-JNK1/2 immunohistochemical staining in small intestine of mice treated with ABT-737 (75 mg/kg) for 1 day (n = 3); untreated mice (n = 3) were used as control.(D) HeLa cells were transiently transfected with siRNA for JNK1/2 and treated as in (A). Cell lysates were probed for total JNK1/2 and γH2A.X.(E) As in (D), except that siRNA oligos targeting JNK1, JNK2, or both JNK1 and JNK2 together were used.(F) U2OS cells were transfected with siRNA targeting CAD and treated with ABT-737. Cell lysates were probed for γH2A.X and P-JNK1/2.See also Figure S5.
Mentions: DNA damage-induced phosphorylation of H2A.X at S139 often occurs via the PI3K-related kinase family members ATM, ATR, and DNA-PK (Jackson and Bartek, 2009; Shiloh, 2003). However, we did not observe any significant increase in activated, phosphorylated ATM or ATR kinase following sub-lethal ABT-737 treatment (Figure 5A). Moreover, RNAi-mediated knockdown of ATM, ATR, or DNA-PK failed to affect BH3 mimetic-induced γH2A.X levels (Figures S5A and S5B). Besides ATM, ATR, and DNA-PK, c-Jun N-terminal kinase (JNK) has also been found to mediate H2A.X phosphorylation in some settings (Lu et al., 2006). Importantly, sub-lethal treatment with ABT-737 led to a caspase-dependent increase in JNK1/2 activation mirroring levels of γH2A.X (Figure 5B). JNK activation following ABT-737 administration was also detected in vivo in the small intestine (Figure 5C). To directly investigate the role of JNK in H2A.X phosphorylation, we used RNAi. Combined knockdown of JNK1/2 or selective knockdown of JNK2 effectively prevented ABT-737 induced γH2A.X implicating a direct role for JNK2 in H2A.X phosphorylation (Figures 5D and 5E). Accordingly, RNAi-mediated knockdown of CAD largely inhibited JNK1/2 phosphorylation (Figure 5F). These results identify JNK2 as a key player in the minority MOMP-induced DNA damage response.

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