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Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

Colin DJ, Hain KO, Allan LA, Clarke PR - Open Biol (2015)

Bottom Line: Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression.We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines.Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Research, Medical Research Institute, University of Dundee, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.

ABSTRACT
Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

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BH3 mimetics and inhibitors of the mitotic DNA damage response sensitize cancer cells synergistically to the effects of a microtubule poison. (a) Experimental protocol. (b,c) BH3 mimetics and inhibitors of the DNA damage response sensitize U2OS cells to taxol. U2OS cells in which p53 was stably depleted by shRNA (pRS-p53, confirmed by Western blot analysis) and control cells transfected with a scrambled shRNA (pRS-sc) were treated after release from taxol as indicated in (a). Apoptosis and proliferation of cells were followed in an IncuCyte. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3). (d) BH3 mimetics and inhibitors of the DNA damage response synergize with taxol. Indicated cell lines were treated as depicted in (a) first with taxol and then at least three different doses of the indicated inhibitor (see electronic supplementary material, experimental procedures) to determine their combination indexes (CIs) according to Chou [20]. The graphs show the CIs ± s.d. (n ≥ 3) obtained for 100 nM taxol treatments of U2OS and MDA-MB231 cells and 10 nM treatments of HCT116, SKOV-3 and OVCAR-3, 4, 5 cells. The CI values obtained at given concentrations depict an antagonism between the drugs when >1, an additive effect when equal to 1 and a synergism when <1.
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RSOB140156F7: BH3 mimetics and inhibitors of the mitotic DNA damage response sensitize cancer cells synergistically to the effects of a microtubule poison. (a) Experimental protocol. (b,c) BH3 mimetics and inhibitors of the DNA damage response sensitize U2OS cells to taxol. U2OS cells in which p53 was stably depleted by shRNA (pRS-p53, confirmed by Western blot analysis) and control cells transfected with a scrambled shRNA (pRS-sc) were treated after release from taxol as indicated in (a). Apoptosis and proliferation of cells were followed in an IncuCyte. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3). (d) BH3 mimetics and inhibitors of the DNA damage response synergize with taxol. Indicated cell lines were treated as depicted in (a) first with taxol and then at least three different doses of the indicated inhibitor (see electronic supplementary material, experimental procedures) to determine their combination indexes (CIs) according to Chou [20]. The graphs show the CIs ± s.d. (n ≥ 3) obtained for 100 nM taxol treatments of U2OS and MDA-MB231 cells and 10 nM treatments of HCT116, SKOV-3 and OVCAR-3, 4, 5 cells. The CI values obtained at given concentrations depict an antagonism between the drugs when >1, an additive effect when equal to 1 and a synergism when <1.

Mentions: We found that inhibitors of ATM, ATR and DNA-PK, including NVP-BEZ235, which also inhibits phosphatidyl-3-kinase and mTOR [28], as well as a Chk1 inhibitor (SB218078 [29]), all enhanced the induction of apoptosis in U2OS cells pre-treated for 6 h with taxol. So did Obatoclax or another BH3 mimetic Navitoclax (ABT-263) that selectively inhibits Bcl-2 and Bcl-xL [30] (figure 7a,b). Conversely, taxol strongly enhanced the induction of apoptosis by both kinase inhibitors and BH3 mimetics because these drugs caused only low levels of apoptosis in the absence of taxol. Each of the kinase inhibitors and BH3 mimetics decreased the total number of U2OS cells whether or not the cells expressed p53, although suppression of p53 reduced the inhibition of cell proliferation by taxol (figure 7c).Figure 7.


Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

Colin DJ, Hain KO, Allan LA, Clarke PR - Open Biol (2015)

BH3 mimetics and inhibitors of the mitotic DNA damage response sensitize cancer cells synergistically to the effects of a microtubule poison. (a) Experimental protocol. (b,c) BH3 mimetics and inhibitors of the DNA damage response sensitize U2OS cells to taxol. U2OS cells in which p53 was stably depleted by shRNA (pRS-p53, confirmed by Western blot analysis) and control cells transfected with a scrambled shRNA (pRS-sc) were treated after release from taxol as indicated in (a). Apoptosis and proliferation of cells were followed in an IncuCyte. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3). (d) BH3 mimetics and inhibitors of the DNA damage response synergize with taxol. Indicated cell lines were treated as depicted in (a) first with taxol and then at least three different doses of the indicated inhibitor (see electronic supplementary material, experimental procedures) to determine their combination indexes (CIs) according to Chou [20]. The graphs show the CIs ± s.d. (n ≥ 3) obtained for 100 nM taxol treatments of U2OS and MDA-MB231 cells and 10 nM treatments of HCT116, SKOV-3 and OVCAR-3, 4, 5 cells. The CI values obtained at given concentrations depict an antagonism between the drugs when >1, an additive effect when equal to 1 and a synergism when <1.
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RSOB140156F7: BH3 mimetics and inhibitors of the mitotic DNA damage response sensitize cancer cells synergistically to the effects of a microtubule poison. (a) Experimental protocol. (b,c) BH3 mimetics and inhibitors of the DNA damage response sensitize U2OS cells to taxol. U2OS cells in which p53 was stably depleted by shRNA (pRS-p53, confirmed by Western blot analysis) and control cells transfected with a scrambled shRNA (pRS-sc) were treated after release from taxol as indicated in (a). Apoptosis and proliferation of cells were followed in an IncuCyte. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3). (d) BH3 mimetics and inhibitors of the DNA damage response synergize with taxol. Indicated cell lines were treated as depicted in (a) first with taxol and then at least three different doses of the indicated inhibitor (see electronic supplementary material, experimental procedures) to determine their combination indexes (CIs) according to Chou [20]. The graphs show the CIs ± s.d. (n ≥ 3) obtained for 100 nM taxol treatments of U2OS and MDA-MB231 cells and 10 nM treatments of HCT116, SKOV-3 and OVCAR-3, 4, 5 cells. The CI values obtained at given concentrations depict an antagonism between the drugs when >1, an additive effect when equal to 1 and a synergism when <1.
Mentions: We found that inhibitors of ATM, ATR and DNA-PK, including NVP-BEZ235, which also inhibits phosphatidyl-3-kinase and mTOR [28], as well as a Chk1 inhibitor (SB218078 [29]), all enhanced the induction of apoptosis in U2OS cells pre-treated for 6 h with taxol. So did Obatoclax or another BH3 mimetic Navitoclax (ABT-263) that selectively inhibits Bcl-2 and Bcl-xL [30] (figure 7a,b). Conversely, taxol strongly enhanced the induction of apoptosis by both kinase inhibitors and BH3 mimetics because these drugs caused only low levels of apoptosis in the absence of taxol. Each of the kinase inhibitors and BH3 mimetics decreased the total number of U2OS cells whether or not the cells expressed p53, although suppression of p53 reduced the inhibition of cell proliferation by taxol (figure 7c).Figure 7.

Bottom Line: Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression.We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines.Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Research, Medical Research Institute, University of Dundee, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.

ABSTRACT
Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

Show MeSH
Related in: MedlinePlus