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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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The role of p53 in the fate of cells following a mitotic arrest. (a) Characterization of p53-depleted U2OS cells. Stably transfected U2OS cells were treated as in figure 1a and analysed by immunoblotting using the specified antibodies. (b) Role of p53 in cell cycle progression following a mitotic arrest. Stably transfected U2OS cells treated as indicated were analysed by 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 repeated three times. (c,d) Role of p53 in the viability and proliferation of cells following a mitotic arrest. (c) Cells treated as depicted in (a) were incubated with an FAM-DEVD-fmk probe and analysed by flow cytometry. The percentage of apoptotic cells with active caspase 3/7 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. (d) The relative number of viable, adherent cells was determined by following cell proliferation using IncuCyte-based cell density analyses. Values are means ± s.d. from quadruplicate wells of a representative experiment repeated three times.
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RSOB140156F5: The role of p53 in the fate of cells following a mitotic arrest. (a) Characterization of p53-depleted U2OS cells. Stably transfected U2OS cells were treated as in figure 1a and analysed by immunoblotting using the specified antibodies. (b) Role of p53 in cell cycle progression following a mitotic arrest. Stably transfected U2OS cells treated as indicated were analysed by 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 repeated three times. (c,d) Role of p53 in the viability and proliferation of cells following a mitotic arrest. (c) Cells treated as depicted in (a) were incubated with an FAM-DEVD-fmk probe and analysed by flow cytometry. The percentage of apoptotic cells with active caspase 3/7 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. (d) The relative number of viable, adherent cells was determined by following cell proliferation using IncuCyte-based cell density analyses. Values are means ± s.d. from quadruplicate wells of a representative experiment repeated three times.

Mentions: Because mitotic delay resulted in the activation of p53 downstream of DNA damage signalling, we tested the role of p53 in the cellular response to a synchronized mitotic delay (figure 5). We examined the effects of mitotic arrest on a cell line derived from U2OS cells [18] in which p53 was stably depleted by expression of an shRNA (pRS-p53); cells expressing a scrambled shRNA (pRS-sc) were used for comparison. Depletion of p53 inhibited the induction of p21 following arrest in mitosis by nocodazole for 2 h (figure 5a) and prevented the accumulation of cells in the subsequent G1 phase (figure 5b). By contrast, loss of p53 did not affect the normal cell cycle progression of mitotic cells collected without nocodazole treatment. Interestingly, the rate of progression into S-phase (2N-4N BrdU positive cells) was apparently enhanced in p53-depleted cells by prior mitotic delay as compared with those cells that had progressed from a normal mitosis (figure 5b). These results demonstrate that p53 plays a key role in restraining cell cycle progression following mitotic stress.Figure 5.


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)

The role of p53 in the fate of cells following a mitotic arrest. (a) Characterization of p53-depleted U2OS cells. Stably transfected U2OS cells were treated as in figure 1a and analysed by immunoblotting using the specified antibodies. (b) Role of p53 in cell cycle progression following a mitotic arrest. Stably transfected U2OS cells treated as indicated were analysed by 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 repeated three times. (c,d) Role of p53 in the viability and proliferation of cells following a mitotic arrest. (c) Cells treated as depicted in (a) were incubated with an FAM-DEVD-fmk probe and analysed by flow cytometry. The percentage of apoptotic cells with active caspase 3/7 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. (d) The relative number of viable, adherent cells was determined by following cell proliferation using IncuCyte-based cell density analyses. Values are means ± s.d. from quadruplicate wells of a representative experiment repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389791&req=5

RSOB140156F5: The role of p53 in the fate of cells following a mitotic arrest. (a) Characterization of p53-depleted U2OS cells. Stably transfected U2OS cells were treated as in figure 1a and analysed by immunoblotting using the specified antibodies. (b) Role of p53 in cell cycle progression following a mitotic arrest. Stably transfected U2OS cells treated as indicated were analysed by 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 repeated three times. (c,d) Role of p53 in the viability and proliferation of cells following a mitotic arrest. (c) Cells treated as depicted in (a) were incubated with an FAM-DEVD-fmk probe and analysed by flow cytometry. The percentage of apoptotic cells with active caspase 3/7 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. (d) The relative number of viable, adherent cells was determined by following cell proliferation using IncuCyte-based cell density analyses. Values are means ± s.d. from quadruplicate wells of a representative experiment repeated three times.
Mentions: Because mitotic delay resulted in the activation of p53 downstream of DNA damage signalling, we tested the role of p53 in the cellular response to a synchronized mitotic delay (figure 5). We examined the effects of mitotic arrest on a cell line derived from U2OS cells [18] in which p53 was stably depleted by expression of an shRNA (pRS-p53); cells expressing a scrambled shRNA (pRS-sc) were used for comparison. Depletion of p53 inhibited the induction of p21 following arrest in mitosis by nocodazole for 2 h (figure 5a) and prevented the accumulation of cells in the subsequent G1 phase (figure 5b). By contrast, loss of p53 did not affect the normal cell cycle progression of mitotic cells collected without nocodazole treatment. Interestingly, the rate of progression into S-phase (2N-4N BrdU positive cells) was apparently enhanced in p53-depleted cells by prior mitotic delay as compared with those cells that had progressed from a normal mitosis (figure 5b). These results demonstrate that p53 plays a key role in restraining cell cycle progression following mitotic stress.Figure 5.

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