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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 period of mitotic arrest determines subsequent cell fate. (a) The period of mitotic arrest controls subsequent DNA damage signalling. U2OS cells were arrested in mitosis for 2 (N2M) or 6 h (N6M) prior to release from mitosis for 2, 4 or 6 h and analysed by immunoblotting using the specified antibodies. Asynchronous cells (As), etoposide-treated cells (Et) and cells remaining adherent after 2 h treatment with nocodazole (N2A) were also analysed for comparison. (b) The period of mitotic arrest controls subsequent cell cycle progression. U2OS cells were treated as in (a) and untreated control mitotic cells were used as controls (M). BrdU incorporation to mark newly synthesized DNA was analysed by flow cytometry. Cumulative histograms show the percentages of cells in different phases of the cell cycle, according to the BrdU incorporation and DNA content. On top, percentages of BrdU positive cells (S phase cells) are given as means ± s.d. (n ≥ 3). (c,d) The period of mitotic arrest controls subsequent cell viability and proliferation. Cells treated as in (a) were collected at indicated times, incubated with an FAM-DEVD-fmk fluorescent probe for apoptotic caspase 3/7 activity and analysed by flow cytometry (c); values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05. The relative numbers of viable, adherent cells were determined by crystal violet assay at indicated times (d). Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).
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RSOB140156F2: The period of mitotic arrest determines subsequent cell fate. (a) The period of mitotic arrest controls subsequent DNA damage signalling. U2OS cells were arrested in mitosis for 2 (N2M) or 6 h (N6M) prior to release from mitosis for 2, 4 or 6 h and analysed by immunoblotting using the specified antibodies. Asynchronous cells (As), etoposide-treated cells (Et) and cells remaining adherent after 2 h treatment with nocodazole (N2A) were also analysed for comparison. (b) The period of mitotic arrest controls subsequent cell cycle progression. U2OS cells were treated as in (a) and untreated control mitotic cells were used as controls (M). BrdU incorporation to mark newly synthesized DNA was analysed by flow cytometry. Cumulative histograms show the percentages of cells in different phases of the cell cycle, according to the BrdU incorporation and DNA content. On top, percentages of BrdU positive cells (S phase cells) are given as means ± s.d. (n ≥ 3). (c,d) The period of mitotic arrest controls subsequent cell viability and proliferation. Cells treated as in (a) were collected at indicated times, incubated with an FAM-DEVD-fmk fluorescent probe for apoptotic caspase 3/7 activity and analysed by flow cytometry (c); values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05. The relative numbers of viable, adherent cells were determined by crystal violet assay at indicated times (d). Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).

Mentions: To test whether the period of mitotic arrest affects subsequent responses, we maintained cells for either 2 or 6 h in nocodazole-induced mitotic arrest, then released the cells by washing out the drug and analysed the DDR. Cells that had been arrested in mitosis for a total of 6 h exhibited stronger activating phosphorylation of ATM, Chk2 and p53 after release from mitosis and they were more likely to be delayed in G1 phase (BrdU negative adherent cells with 2N DNA content) than those that had been arrested in mitosis for only 2 h (figure 2a,b). The activating phosphorylation of Chk1 was also prolonged (figure 2a), consistent with a delay in S phase initiation (figure 2b). Similar results were obtained when cells were treated with taxol rather than treated with nocodazole (electronic supplementary material, figure S3A,B). Thus, the period of mitotic arrest determines the intensity of the DNA damage signalling and this correlates with restraint of subsequent cell cycle progression. Moreover, we found that U2OS cells arrested for longer in mitosis died more readily by apoptosis after release into interphase (figure 2c and electronic supplementary material, figure S3C). Together, these effects on cell cycle progression and apoptosis resulted in the strong inhibition of cell proliferation (figure 2d; electronic supplementary material, figure S3D).Figure 2.


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 period of mitotic arrest determines subsequent cell fate. (a) The period of mitotic arrest controls subsequent DNA damage signalling. U2OS cells were arrested in mitosis for 2 (N2M) or 6 h (N6M) prior to release from mitosis for 2, 4 or 6 h and analysed by immunoblotting using the specified antibodies. Asynchronous cells (As), etoposide-treated cells (Et) and cells remaining adherent after 2 h treatment with nocodazole (N2A) were also analysed for comparison. (b) The period of mitotic arrest controls subsequent cell cycle progression. U2OS cells were treated as in (a) and untreated control mitotic cells were used as controls (M). BrdU incorporation to mark newly synthesized DNA was analysed by flow cytometry. Cumulative histograms show the percentages of cells in different phases of the cell cycle, according to the BrdU incorporation and DNA content. On top, percentages of BrdU positive cells (S phase cells) are given as means ± s.d. (n ≥ 3). (c,d) The period of mitotic arrest controls subsequent cell viability and proliferation. Cells treated as in (a) were collected at indicated times, incubated with an FAM-DEVD-fmk fluorescent probe for apoptotic caspase 3/7 activity and analysed by flow cytometry (c); values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05. The relative numbers of viable, adherent cells were determined by crystal violet assay at indicated times (d). 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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Show All Figures
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RSOB140156F2: The period of mitotic arrest determines subsequent cell fate. (a) The period of mitotic arrest controls subsequent DNA damage signalling. U2OS cells were arrested in mitosis for 2 (N2M) or 6 h (N6M) prior to release from mitosis for 2, 4 or 6 h and analysed by immunoblotting using the specified antibodies. Asynchronous cells (As), etoposide-treated cells (Et) and cells remaining adherent after 2 h treatment with nocodazole (N2A) were also analysed for comparison. (b) The period of mitotic arrest controls subsequent cell cycle progression. U2OS cells were treated as in (a) and untreated control mitotic cells were used as controls (M). BrdU incorporation to mark newly synthesized DNA was analysed by flow cytometry. Cumulative histograms show the percentages of cells in different phases of the cell cycle, according to the BrdU incorporation and DNA content. On top, percentages of BrdU positive cells (S phase cells) are given as means ± s.d. (n ≥ 3). (c,d) The period of mitotic arrest controls subsequent cell viability and proliferation. Cells treated as in (a) were collected at indicated times, incubated with an FAM-DEVD-fmk fluorescent probe for apoptotic caspase 3/7 activity and analysed by flow cytometry (c); values are means ± s.d. (n ≥ 3). Statistical differences were analysed with the Mann–Whitney test; n.s., non-significant, *p < 0.05. The relative numbers of viable, adherent cells were determined by crystal violet assay at indicated times (d). Values are means ± s.d. from quadruplicate wells of a representative experiment (n ≥ 3).
Mentions: To test whether the period of mitotic arrest affects subsequent responses, we maintained cells for either 2 or 6 h in nocodazole-induced mitotic arrest, then released the cells by washing out the drug and analysed the DDR. Cells that had been arrested in mitosis for a total of 6 h exhibited stronger activating phosphorylation of ATM, Chk2 and p53 after release from mitosis and they were more likely to be delayed in G1 phase (BrdU negative adherent cells with 2N DNA content) than those that had been arrested in mitosis for only 2 h (figure 2a,b). The activating phosphorylation of Chk1 was also prolonged (figure 2a), consistent with a delay in S phase initiation (figure 2b). Similar results were obtained when cells were treated with taxol rather than treated with nocodazole (electronic supplementary material, figure S3A,B). Thus, the period of mitotic arrest determines the intensity of the DNA damage signalling and this correlates with restraint of subsequent cell cycle progression. Moreover, we found that U2OS cells arrested for longer in mitosis died more readily by apoptosis after release into interphase (figure 2c and electronic supplementary material, figure S3C). Together, these effects on cell cycle progression and apoptosis resulted in the strong inhibition of cell proliferation (figure 2d; electronic supplementary material, figure S3D).Figure 2.

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