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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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Inhibition of DNA damage response kinases after release from mitotic arrest enhances the effects of microtubule poisons. (a) Experimental protocol. (b) Selective inhibition of PIKKs affects DNA damage signalling after a mitotic arrest. Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) Inhibition of PIKKs affects cell cycle progression following mitotic arrest. Cells arrested in mitosis for 2 h with nocodazole (c) or normal mitotic cells (d) were released as depicted in (a) and analysed using flow cytometry. The cumulative histograms show the percentage of cells in the different phases of the cell cycle, according to BrdU incorporation and DNA content. Data shown are from a representative experiment (n ≥ 3). (e,f) Inhibition of PIKKs reduces the viability and proliferation of cells following a mitotic arrest. (e) Cells treated as indicated in (a) were incubated with an FAM-DEVD-fmk probe to identify apoptotic cells and analysed by flow cytometry. The percentage of apoptotic cells is shown, values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001. (f) The relative number of viable, adherent cells was determined by crystal violet assay. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).
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RSOB140156F6: Inhibition of DNA damage response kinases after release from mitotic arrest enhances the effects of microtubule poisons. (a) Experimental protocol. (b) Selective inhibition of PIKKs affects DNA damage signalling after a mitotic arrest. Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) Inhibition of PIKKs affects cell cycle progression following mitotic arrest. Cells arrested in mitosis for 2 h with nocodazole (c) or normal mitotic cells (d) were released as depicted in (a) and analysed using flow cytometry. The cumulative histograms show the percentage of cells in the different phases of the cell cycle, according to BrdU incorporation and DNA content. Data shown are from a representative experiment (n ≥ 3). (e,f) Inhibition of PIKKs reduces the viability and proliferation of cells following a mitotic arrest. (e) Cells treated as indicated in (a) were incubated with an FAM-DEVD-fmk probe to identify apoptotic cells and analysed by flow cytometry. The percentage of apoptotic cells is shown, values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001. (f) The relative number of viable, adherent cells was determined by crystal violet assay. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).

Mentions: To address the significance of the DNA damage pathway activated in response to mitotic arrest, we analysed the effect of inhibiting ATM, ATR or DNA-PK after release from mitosis using the chemical inhibitors KU55933 [24], NU6027 [25] and NU7441 [26,27], respectively (for protocol, see figure 6a). Chemical inhibitors have an advantage over genetic approaches, because kinases can be targeted at specific points in the cell cycle; however, there remains the caveat that their cellular effects might be mediated by inhibition of other enzymes, especially other kinases (e.g. inhibition of CDK2 by NU6027 [25]). The relative selectivity of KU55933 and NU6027 was confirmed by their ability to block the phosphorylation of the ATM substrate Chk2 and the ATR substrate Chk1, respectively, as well as the selective inhibition of ATM autophosphorylation at S1981 by KU55933 but not NU6027 or NU7441 (figure 6b). We found that inhibition of ATM or DNA-PK in particular strongly suppressed the γH2AX signal generated immediately after release from mitosis, with a weaker effect by the ATR inhibitor. In addition, the ATM and ATR inhibitors both impaired p53 Ser15 phosphorylation and p21 induction at this time (figure 6b, left panels). We conclude that ATM, ATR and DNA-PK all contribute to DNA damage signalling after release from mitotic arrest.Figure 6.


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)

Inhibition of DNA damage response kinases after release from mitotic arrest enhances the effects of microtubule poisons. (a) Experimental protocol. (b) Selective inhibition of PIKKs affects DNA damage signalling after a mitotic arrest. Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) Inhibition of PIKKs affects cell cycle progression following mitotic arrest. Cells arrested in mitosis for 2 h with nocodazole (c) or normal mitotic cells (d) were released as depicted in (a) and analysed using flow cytometry. The cumulative histograms show the percentage of cells in the different phases of the cell cycle, according to BrdU incorporation and DNA content. Data shown are from a representative experiment (n ≥ 3). (e,f) Inhibition of PIKKs reduces the viability and proliferation of cells following a mitotic arrest. (e) Cells treated as indicated in (a) were incubated with an FAM-DEVD-fmk probe to identify apoptotic cells and analysed by flow cytometry. The percentage of apoptotic cells is shown, values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001. (f) The relative number of viable, adherent cells was determined by crystal violet assay. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389791&req=5

RSOB140156F6: Inhibition of DNA damage response kinases after release from mitotic arrest enhances the effects of microtubule poisons. (a) Experimental protocol. (b) Selective inhibition of PIKKs affects DNA damage signalling after a mitotic arrest. Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) Inhibition of PIKKs affects cell cycle progression following mitotic arrest. Cells arrested in mitosis for 2 h with nocodazole (c) or normal mitotic cells (d) were released as depicted in (a) and analysed using flow cytometry. The cumulative histograms show the percentage of cells in the different phases of the cell cycle, according to BrdU incorporation and DNA content. Data shown are from a representative experiment (n ≥ 3). (e,f) Inhibition of PIKKs reduces the viability and proliferation of cells following a mitotic arrest. (e) Cells treated as indicated in (a) were incubated with an FAM-DEVD-fmk probe to identify apoptotic cells and analysed by flow cytometry. The percentage of apoptotic cells is shown, values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001. (f) The relative number of viable, adherent cells was determined by crystal violet assay. Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).
Mentions: To address the significance of the DNA damage pathway activated in response to mitotic arrest, we analysed the effect of inhibiting ATM, ATR or DNA-PK after release from mitosis using the chemical inhibitors KU55933 [24], NU6027 [25] and NU7441 [26,27], respectively (for protocol, see figure 6a). Chemical inhibitors have an advantage over genetic approaches, because kinases can be targeted at specific points in the cell cycle; however, there remains the caveat that their cellular effects might be mediated by inhibition of other enzymes, especially other kinases (e.g. inhibition of CDK2 by NU6027 [25]). The relative selectivity of KU55933 and NU6027 was confirmed by their ability to block the phosphorylation of the ATM substrate Chk2 and the ATR substrate Chk1, respectively, as well as the selective inhibition of ATM autophosphorylation at S1981 by KU55933 but not NU6027 or NU7441 (figure 6b). We found that inhibition of ATM or DNA-PK in particular strongly suppressed the γH2AX signal generated immediately after release from mitosis, with a weaker effect by the ATR inhibitor. In addition, the ATM and ATR inhibitors both impaired p53 Ser15 phosphorylation and p21 induction at this time (figure 6b, left panels). We conclude that ATM, ATR and DNA-PK all contribute to DNA damage signalling after release from mitotic arrest.Figure 6.

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