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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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Cellular responses to microtubule poisons are determined by caspase-dependent DNA damage signalling initiated during delayed mitosis. During normal mitosis, release of cytochrome c from mitochondria is inhibited by the action of Bcl-2, Bcl-xL and Mcl-1; downstream caspase activation is restrained by the inhibitory phosphorylation of caspase-9. Increasing phosphorylation of Bcl-2 and Bcl-xL, however, reduces their activity, whereas phosphorylation of Mcl-1 by CDK1–cyclin B initiates its proteolytic destruction. After a prolonged mitotic arrest, this results in the partial release of cytochrome c from mitochondria and the subapoptotic activation of caspase-3/7 when coupled with a slow decline in CDK1–cyclin B kinase activity and dephosphorylation of caspase-9. Caspase-3/7 activity results in localized DNA damage and activation of ATM, ATR and DNA-PK. The mitotic DNA damage response is amplified as cells slip out of mitosis, resulting in the phosphorylation of p53 and induction of p21, which inhibits CDK activity required for S-phase. ATM and ATR also activate Chk2 and Chk1, respectively, which inhibit subsequent cell cycle progression. ATM, ATR and DNA-PK also have functions in recovery from telomere damage and restrain apoptosis.
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RSOB140156F8: Cellular responses to microtubule poisons are determined by caspase-dependent DNA damage signalling initiated during delayed mitosis. During normal mitosis, release of cytochrome c from mitochondria is inhibited by the action of Bcl-2, Bcl-xL and Mcl-1; downstream caspase activation is restrained by the inhibitory phosphorylation of caspase-9. Increasing phosphorylation of Bcl-2 and Bcl-xL, however, reduces their activity, whereas phosphorylation of Mcl-1 by CDK1–cyclin B initiates its proteolytic destruction. After a prolonged mitotic arrest, this results in the partial release of cytochrome c from mitochondria and the subapoptotic activation of caspase-3/7 when coupled with a slow decline in CDK1–cyclin B kinase activity and dephosphorylation of caspase-9. Caspase-3/7 activity results in localized DNA damage and activation of ATM, ATR and DNA-PK. The mitotic DNA damage response is amplified as cells slip out of mitosis, resulting in the phosphorylation of p53 and induction of p21, which inhibits CDK activity required for S-phase. ATM and ATR also activate Chk2 and Chk1, respectively, which inhibit subsequent cell cycle progression. ATM, ATR and DNA-PK also have functions in recovery from telomere damage and restrain apoptosis.

Mentions: Regulation of the mitotic stress response by Bcl-2 family proteins suggests a mechanism for temporal control of the initiation of the response (figure 8). Cells in mitosis are restricted in their ability to induce new transcription and changes during mitosis are mediated primarily through post-translational mechanisms, notably protein phosphorylation and protein degradation by the ubiquitin–proteasome system. Mcl-1 is destroyed after a prolonged mitotic arrest [11], and loss of Mcl-1 is likely to be a key step in the initiation of the response. Although Bcl-2 and Bcl-xL are stable during mitotic arrest, they are highly phosphorylated, and this phosphorylation has been associated with the inhibition of their function [13]. Nevertheless, cells arrested in mitosis are exquisitely sensitive to Navitoclax [31] (D.J.C., K.O.H., L.A.A. & P.R.C. 2015, unpublished data), showing that Bcl-2 and/or Bcl-xL are critical to prevent the induction of mitotic stress when Mcl-1 has been degraded. It seems, therefore, that the threshold to induce caspase activation is reduced in mitotically arrested cells through both the destruction of Mcl-1 and the partial inhibition of Bcl-2/Bcl-xL.Figure 8.


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)

Cellular responses to microtubule poisons are determined by caspase-dependent DNA damage signalling initiated during delayed mitosis. During normal mitosis, release of cytochrome c from mitochondria is inhibited by the action of Bcl-2, Bcl-xL and Mcl-1; downstream caspase activation is restrained by the inhibitory phosphorylation of caspase-9. Increasing phosphorylation of Bcl-2 and Bcl-xL, however, reduces their activity, whereas phosphorylation of Mcl-1 by CDK1–cyclin B initiates its proteolytic destruction. After a prolonged mitotic arrest, this results in the partial release of cytochrome c from mitochondria and the subapoptotic activation of caspase-3/7 when coupled with a slow decline in CDK1–cyclin B kinase activity and dephosphorylation of caspase-9. Caspase-3/7 activity results in localized DNA damage and activation of ATM, ATR and DNA-PK. The mitotic DNA damage response is amplified as cells slip out of mitosis, resulting in the phosphorylation of p53 and induction of p21, which inhibits CDK activity required for S-phase. ATM and ATR also activate Chk2 and Chk1, respectively, which inhibit subsequent cell cycle progression. ATM, ATR and DNA-PK also have functions in recovery from telomere damage and restrain apoptosis.
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RSOB140156F8: Cellular responses to microtubule poisons are determined by caspase-dependent DNA damage signalling initiated during delayed mitosis. During normal mitosis, release of cytochrome c from mitochondria is inhibited by the action of Bcl-2, Bcl-xL and Mcl-1; downstream caspase activation is restrained by the inhibitory phosphorylation of caspase-9. Increasing phosphorylation of Bcl-2 and Bcl-xL, however, reduces their activity, whereas phosphorylation of Mcl-1 by CDK1–cyclin B initiates its proteolytic destruction. After a prolonged mitotic arrest, this results in the partial release of cytochrome c from mitochondria and the subapoptotic activation of caspase-3/7 when coupled with a slow decline in CDK1–cyclin B kinase activity and dephosphorylation of caspase-9. Caspase-3/7 activity results in localized DNA damage and activation of ATM, ATR and DNA-PK. The mitotic DNA damage response is amplified as cells slip out of mitosis, resulting in the phosphorylation of p53 and induction of p21, which inhibits CDK activity required for S-phase. ATM and ATR also activate Chk2 and Chk1, respectively, which inhibit subsequent cell cycle progression. ATM, ATR and DNA-PK also have functions in recovery from telomere damage and restrain apoptosis.
Mentions: Regulation of the mitotic stress response by Bcl-2 family proteins suggests a mechanism for temporal control of the initiation of the response (figure 8). Cells in mitosis are restricted in their ability to induce new transcription and changes during mitosis are mediated primarily through post-translational mechanisms, notably protein phosphorylation and protein degradation by the ubiquitin–proteasome system. Mcl-1 is destroyed after a prolonged mitotic arrest [11], and loss of Mcl-1 is likely to be a key step in the initiation of the response. Although Bcl-2 and Bcl-xL are stable during mitotic arrest, they are highly phosphorylated, and this phosphorylation has been associated with the inhibition of their function [13]. Nevertheless, cells arrested in mitosis are exquisitely sensitive to Navitoclax [31] (D.J.C., K.O.H., L.A.A. & P.R.C. 2015, unpublished data), showing that Bcl-2 and/or Bcl-xL are critical to prevent the induction of mitotic stress when Mcl-1 has been degraded. It seems, therefore, that the threshold to induce caspase activation is reduced in mitotically arrested cells through both the destruction of Mcl-1 and the partial inhibition of Bcl-2/Bcl-xL.Figure 8.

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