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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 controls subsequent DNA damage signalling, cell cycle progression and apoptosis. (a) Experimental protocol. (b) DNA damage signalling in response to a prolonged mitotic arrest. Samples were analysed by immunoblotting using the antibodies indicated. Asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) DNA damage signalling in G1 following mitotic arrest. Samples were analysed by immunoblotting using specified antibodies (c), and cell cycle profiles were characterized by flow cytometry using propidium iodide as a marker of DNA content and phosphorylated Ser10 histone H3 as a marker of mitosis (d). (e) Induction of apoptosis during a mitotic arrest. Cells were incubated with a FAM-DEVD-fmk probe and analysed by flow cytometry. Percentages represent the amount of active cleaved caspase-3/7 positive cells. (f) γH2AX induction in cells arrested in mitosis is caspase-dependent. Cells were synchronized for 10 h in mitosis in the presence of z-VAD-fmk where indicated, cytospun and immunostained using an anti-γH2AX antibody. Representative microscopic fields are shown; Ap, apoptotic cells; scale bar, 50 µm.
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RSOB140156F1: The period of mitotic arrest controls subsequent DNA damage signalling, cell cycle progression and apoptosis. (a) Experimental protocol. (b) DNA damage signalling in response to a prolonged mitotic arrest. Samples were analysed by immunoblotting using the antibodies indicated. Asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) DNA damage signalling in G1 following mitotic arrest. Samples were analysed by immunoblotting using specified antibodies (c), and cell cycle profiles were characterized by flow cytometry using propidium iodide as a marker of DNA content and phosphorylated Ser10 histone H3 as a marker of mitosis (d). (e) Induction of apoptosis during a mitotic arrest. Cells were incubated with a FAM-DEVD-fmk probe and analysed by flow cytometry. Percentages represent the amount of active cleaved caspase-3/7 positive cells. (f) γH2AX induction in cells arrested in mitosis is caspase-dependent. Cells were synchronized for 10 h in mitosis in the presence of z-VAD-fmk where indicated, cytospun and immunostained using an anti-γH2AX antibody. Representative microscopic fields are shown; Ap, apoptotic cells; scale bar, 50 µm.

Mentions: To study cellular responses to mitotic arrest, we used a cell culture model in which rounded-up mitotic human osteosarcoma cells (U2OS) were collected after treatment of an asynchronous culture with nocodazole for 2 h (denoted N2M; figure 1a; electronic supplementary material, figure S1A). Mitotic cells were then replated in nocodazole for a further period and analysed by Western blotting (figure 1b). This procedure allows analysis of biochemical changes during the period of mitotic arrest and after subsequent entry into interphase without interference from cellular stresses generated by other methods of pre-synchronization. We found that a prolonged mitotic delay of up to 10 h followed by slippage into interphase (confirmed by flow cytometry; electronic supplementary material, figure S1B) activated components of the DNA damage response (DDR) [21] in a time-dependent manner (figure 1b). Ataxia–telangiectasia-mutated (ATM), a large phosphatidylinositol-3-kinase-related protein kinase (PIKK), became phosphorylated at Ser1981, a site associated with its activation and stabilization at sites of DNA damage [22]. The phosphorylation of ATM increased and persisted as cells slipped out of mitosis after 10 h (indicated by the loss of cyclin B1 and histone H3 Ser10 dephosphorylation), and correlated with activating phosphorylation of its targets, the protein kinase Chk2 and p53; there was also a transient activating phosphorylation of Chk1 at Ser345, a site targeted by ATR (ATM- and Rad3-related).Figure 1.


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 controls subsequent DNA damage signalling, cell cycle progression and apoptosis. (a) Experimental protocol. (b) DNA damage signalling in response to a prolonged mitotic arrest. Samples were analysed by immunoblotting using the antibodies indicated. Asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) DNA damage signalling in G1 following mitotic arrest. Samples were analysed by immunoblotting using specified antibodies (c), and cell cycle profiles were characterized by flow cytometry using propidium iodide as a marker of DNA content and phosphorylated Ser10 histone H3 as a marker of mitosis (d). (e) Induction of apoptosis during a mitotic arrest. Cells were incubated with a FAM-DEVD-fmk probe and analysed by flow cytometry. Percentages represent the amount of active cleaved caspase-3/7 positive cells. (f) γH2AX induction in cells arrested in mitosis is caspase-dependent. Cells were synchronized for 10 h in mitosis in the presence of z-VAD-fmk where indicated, cytospun and immunostained using an anti-γH2AX antibody. Representative microscopic fields are shown; Ap, apoptotic cells; scale bar, 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389791&req=5

RSOB140156F1: The period of mitotic arrest controls subsequent DNA damage signalling, cell cycle progression and apoptosis. (a) Experimental protocol. (b) DNA damage signalling in response to a prolonged mitotic arrest. Samples were analysed by immunoblotting using the antibodies indicated. Asterisk denotes a non-specific signal on pS15-p53 blot. (c,d) DNA damage signalling in G1 following mitotic arrest. Samples were analysed by immunoblotting using specified antibodies (c), and cell cycle profiles were characterized by flow cytometry using propidium iodide as a marker of DNA content and phosphorylated Ser10 histone H3 as a marker of mitosis (d). (e) Induction of apoptosis during a mitotic arrest. Cells were incubated with a FAM-DEVD-fmk probe and analysed by flow cytometry. Percentages represent the amount of active cleaved caspase-3/7 positive cells. (f) γH2AX induction in cells arrested in mitosis is caspase-dependent. Cells were synchronized for 10 h in mitosis in the presence of z-VAD-fmk where indicated, cytospun and immunostained using an anti-γH2AX antibody. Representative microscopic fields are shown; Ap, apoptotic cells; scale bar, 50 µm.
Mentions: To study cellular responses to mitotic arrest, we used a cell culture model in which rounded-up mitotic human osteosarcoma cells (U2OS) were collected after treatment of an asynchronous culture with nocodazole for 2 h (denoted N2M; figure 1a; electronic supplementary material, figure S1A). Mitotic cells were then replated in nocodazole for a further period and analysed by Western blotting (figure 1b). This procedure allows analysis of biochemical changes during the period of mitotic arrest and after subsequent entry into interphase without interference from cellular stresses generated by other methods of pre-synchronization. We found that a prolonged mitotic delay of up to 10 h followed by slippage into interphase (confirmed by flow cytometry; electronic supplementary material, figure S1B) activated components of the DNA damage response (DDR) [21] in a time-dependent manner (figure 1b). Ataxia–telangiectasia-mutated (ATM), a large phosphatidylinositol-3-kinase-related protein kinase (PIKK), became phosphorylated at Ser1981, a site associated with its activation and stabilization at sites of DNA damage [22]. The phosphorylation of ATM increased and persisted as cells slipped out of mitosis after 10 h (indicated by the loss of cyclin B1 and histone H3 Ser10 dephosphorylation), and correlated with activating phosphorylation of its targets, the protein kinase Chk2 and p53; there was also a transient activating phosphorylation of Chk1 at Ser345, a site targeted by ATR (ATM- and Rad3-related).Figure 1.

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