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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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Mitotic arrest elicits a localized caspase-dependent DNA damage response under the control of Bcl-2 family proteins. Cells were synchronized in prolonged mitosis for different times (N2M, N6M, N10M) and compared with untreated mitotic cells (M). Some cells were co-treated with the pan-caspase inhibitor z-VAD-fmk (20 µM) or the Mcl-1/Bcl-2/Bcl-xL inhibitor Obatoclax (500 nM). U2OS cells over-expressing Mcl-1 were prepared by transient transfection. Cells were cytospun and immunostained using an anti-γH2AX antibody. (a) Representative microscopic fields are shown; scale bar, 40 µm. (b,c) The histograms show the numbers of γH2AX foci per mitotic cell treated as indicated in (a) or in which Mcl-1 was depleted by siRNA (si Mcl-1) compared with control cells in which an irrelevant luciferase siRNA was transfected (si Luc); values are means ± s.d. (n ≥ 3). Statistical differences were analysed using one-way ANOVA statistical tests; *p < 0.05, **p < 0.01 and ***p < 0.001.
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RSOB140156F3: Mitotic arrest elicits a localized caspase-dependent DNA damage response under the control of Bcl-2 family proteins. Cells were synchronized in prolonged mitosis for different times (N2M, N6M, N10M) and compared with untreated mitotic cells (M). Some cells were co-treated with the pan-caspase inhibitor z-VAD-fmk (20 µM) or the Mcl-1/Bcl-2/Bcl-xL inhibitor Obatoclax (500 nM). U2OS cells over-expressing Mcl-1 were prepared by transient transfection. Cells were cytospun and immunostained using an anti-γH2AX antibody. (a) Representative microscopic fields are shown; scale bar, 40 µm. (b,c) The histograms show the numbers of γH2AX foci per mitotic cell treated as indicated in (a) or in which Mcl-1 was depleted by siRNA (si Mcl-1) compared with control cells in which an irrelevant luciferase siRNA was transfected (si Luc); values are means ± s.d. (n ≥ 3). Statistical differences were analysed using one-way ANOVA statistical tests; *p < 0.05, **p < 0.01 and ***p < 0.001.

Mentions: In agreement with previous descriptions [15,16], we found that most cells arrested in mitosis for 2 or more hours exhibited localized γH2AX foci (figure 3). Counting the number of γH2AX foci, we found that untreated mitotic cells exhibited only occasional foci (figure 3a) with a mean of 2.2 foci per cell (figure 3b). By contrast, cells arrested in mitosis had a distribution of up to 20 foci per cell (electronic supplementary material, figure S4), with a mean of 5.4 in cells arrested for 2 h. The mean number of foci per cell increased when the period of the arrest was prolonged, showing that it is time-dependent (figure 3b; electronic supplementary material, figure S4). Importantly, we found that the formation of these foci was inhibited by the caspase inhibitor zVAD-fmk (figure 3). By contrast, γH2AX foci induced in mitotic cells by the topoisomerase II poison etoposide, which causes widespread DNA double-strand breaks, were not dependent on caspase activity (electronic supplementary material, figure S5).Figure 3.


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)

Mitotic arrest elicits a localized caspase-dependent DNA damage response under the control of Bcl-2 family proteins. Cells were synchronized in prolonged mitosis for different times (N2M, N6M, N10M) and compared with untreated mitotic cells (M). Some cells were co-treated with the pan-caspase inhibitor z-VAD-fmk (20 µM) or the Mcl-1/Bcl-2/Bcl-xL inhibitor Obatoclax (500 nM). U2OS cells over-expressing Mcl-1 were prepared by transient transfection. Cells were cytospun and immunostained using an anti-γH2AX antibody. (a) Representative microscopic fields are shown; scale bar, 40 µm. (b,c) The histograms show the numbers of γH2AX foci per mitotic cell treated as indicated in (a) or in which Mcl-1 was depleted by siRNA (si Mcl-1) compared with control cells in which an irrelevant luciferase siRNA was transfected (si Luc); values are means ± s.d. (n ≥ 3). Statistical differences were analysed using one-way ANOVA statistical tests; *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4389791&req=5

RSOB140156F3: Mitotic arrest elicits a localized caspase-dependent DNA damage response under the control of Bcl-2 family proteins. Cells were synchronized in prolonged mitosis for different times (N2M, N6M, N10M) and compared with untreated mitotic cells (M). Some cells were co-treated with the pan-caspase inhibitor z-VAD-fmk (20 µM) or the Mcl-1/Bcl-2/Bcl-xL inhibitor Obatoclax (500 nM). U2OS cells over-expressing Mcl-1 were prepared by transient transfection. Cells were cytospun and immunostained using an anti-γH2AX antibody. (a) Representative microscopic fields are shown; scale bar, 40 µm. (b,c) The histograms show the numbers of γH2AX foci per mitotic cell treated as indicated in (a) or in which Mcl-1 was depleted by siRNA (si Mcl-1) compared with control cells in which an irrelevant luciferase siRNA was transfected (si Luc); values are means ± s.d. (n ≥ 3). Statistical differences were analysed using one-way ANOVA statistical tests; *p < 0.05, **p < 0.01 and ***p < 0.001.
Mentions: In agreement with previous descriptions [15,16], we found that most cells arrested in mitosis for 2 or more hours exhibited localized γH2AX foci (figure 3). Counting the number of γH2AX foci, we found that untreated mitotic cells exhibited only occasional foci (figure 3a) with a mean of 2.2 foci per cell (figure 3b). By contrast, cells arrested in mitosis had a distribution of up to 20 foci per cell (electronic supplementary material, figure S4), with a mean of 5.4 in cells arrested for 2 h. The mean number of foci per cell increased when the period of the arrest was prolonged, showing that it is time-dependent (figure 3b; electronic supplementary material, figure S4). Importantly, we found that the formation of these foci was inhibited by the caspase inhibitor zVAD-fmk (figure 3). By contrast, γH2AX foci induced in mitotic cells by the topoisomerase II poison etoposide, which causes widespread DNA double-strand breaks, were not dependent on caspase activity (electronic supplementary material, figure S5).Figure 3.

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