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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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Mcl-1 controls caspase-dependent DNA damage signalling and subsequent fate of cells following mitotic arrest. (a) Experimental protocol. (b) Regulation of caspase activity affects DNA damage signalling in response to mitotic arrest. Where indicated, mitotically arrested U2OS cells were co-treated with z-VAD-fmk (left panel) or Obatoclax (right panel). U2OS cells expressing exogenous Mcl-1 were compared with mock-transfected controls (middle panel). Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c) Regulation of caspase activity affects cell cycle progression following a mitotic arrest. Cells were treated as indicated in (a) and analysed by flow cytometry. The cumulative histograms show the percentages of cells in the different phases of the cell cycle, according to BrdU incorporation into newly synthesized DNA and DNA content by propidium iodide staining. Percentages shown are from a representative experiment (n ≥ 3).
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RSOB140156F4: Mcl-1 controls caspase-dependent DNA damage signalling and subsequent fate of cells following mitotic arrest. (a) Experimental protocol. (b) Regulation of caspase activity affects DNA damage signalling in response to mitotic arrest. Where indicated, mitotically arrested U2OS cells were co-treated with z-VAD-fmk (left panel) or Obatoclax (right panel). U2OS cells expressing exogenous Mcl-1 were compared with mock-transfected controls (middle panel). Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c) Regulation of caspase activity affects cell cycle progression following a mitotic arrest. Cells were treated as indicated in (a) and analysed by flow cytometry. The cumulative histograms show the percentages of cells in the different phases of the cell cycle, according to BrdU incorporation into newly synthesized DNA and DNA content by propidium iodide staining. Percentages shown are from a representative experiment (n ≥ 3).

Mentions: Analysis of the DDR in interphase cells following release from mitotic arrest (figure 4a) showed that the activating phosphorylation of ATM, Chk1, Chk2 and p53, as well as the induction of p21, were strongly inhibited by addition of zVAD-fmk during the arrest (figure 4b, left panel), and were therefore dependent on upstream caspase activity. This is consistent with a mechanism in which the response to caspase-dependent DNA damage foci generated during a delayed mitosis is enhanced following release into interphase. By contrast, the increase in total p53 protein following mitotic delay was not affected by zVAD-fmk, indicative of a caspase-independent mechanism for p53 induction. Caspase-dependent cleavage of poly(ADP ribose) polymerase (PARP) was not observed until 10 h of incubation (N10M), consistent with flow cytometry data (figure 1e) showing that, until this time, few cells were undergoing apoptosis. Remarkably, however, treatment with zVAD-fmk reduced the delay in G1 of non-apoptotic cells released from 2 h mitotic arrest and promoted their entry into S phase (figure 4c, left panel).Figure 4.


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)

Mcl-1 controls caspase-dependent DNA damage signalling and subsequent fate of cells following mitotic arrest. (a) Experimental protocol. (b) Regulation of caspase activity affects DNA damage signalling in response to mitotic arrest. Where indicated, mitotically arrested U2OS cells were co-treated with z-VAD-fmk (left panel) or Obatoclax (right panel). U2OS cells expressing exogenous Mcl-1 were compared with mock-transfected controls (middle panel). Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c) Regulation of caspase activity affects cell cycle progression following a mitotic arrest. Cells were treated as indicated in (a) and analysed by flow cytometry. The cumulative histograms show the percentages of cells in the different phases of the cell cycle, according to BrdU incorporation into newly synthesized DNA and DNA content by propidium iodide staining. Percentages shown are from a representative experiment (n ≥ 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140156F4: Mcl-1 controls caspase-dependent DNA damage signalling and subsequent fate of cells following mitotic arrest. (a) Experimental protocol. (b) Regulation of caspase activity affects DNA damage signalling in response to mitotic arrest. Where indicated, mitotically arrested U2OS cells were co-treated with z-VAD-fmk (left panel) or Obatoclax (right panel). U2OS cells expressing exogenous Mcl-1 were compared with mock-transfected controls (middle panel). Samples were analysed by immunoblotting using the specified antibodies; asterisk denotes a non-specific signal on pS15-p53 blot. (c) Regulation of caspase activity affects cell cycle progression following a mitotic arrest. Cells were treated as indicated in (a) and analysed by flow cytometry. The cumulative histograms show the percentages of cells in the different phases of the cell cycle, according to BrdU incorporation into newly synthesized DNA and DNA content by propidium iodide staining. Percentages shown are from a representative experiment (n ≥ 3).
Mentions: Analysis of the DDR in interphase cells following release from mitotic arrest (figure 4a) showed that the activating phosphorylation of ATM, Chk1, Chk2 and p53, as well as the induction of p21, were strongly inhibited by addition of zVAD-fmk during the arrest (figure 4b, left panel), and were therefore dependent on upstream caspase activity. This is consistent with a mechanism in which the response to caspase-dependent DNA damage foci generated during a delayed mitosis is enhanced following release into interphase. By contrast, the increase in total p53 protein following mitotic delay was not affected by zVAD-fmk, indicative of a caspase-independent mechanism for p53 induction. Caspase-dependent cleavage of poly(ADP ribose) polymerase (PARP) was not observed until 10 h of incubation (N10M), consistent with flow cytometry data (figure 1e) showing that, until this time, few cells were undergoing apoptosis. Remarkably, however, treatment with zVAD-fmk reduced the delay in G1 of non-apoptotic cells released from 2 h mitotic arrest and promoted their entry into S phase (figure 4c, left panel).Figure 4.

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