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Cyclin G1 regulates the outcome of taxane-induced mitotic checkpoint arrest.

Russell P, Hennessy BT, Li J, Carey MS, Bast RC, Freeman T, Venkitaraman AR - Oncogene (2011)

Bottom Line: However, the mechanisms that determine these outcomes remain unclear.Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds.Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Oncology and The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, UK.

ABSTRACT
Anti-mitotic chemotherapeutic agents such as taxanes activate the spindle assembly checkpoint (SAC) to arrest anaphase onset, but taxane-exposed cells eventually undergo slippage to exit mitosis. The therapeutic efficacy of taxanes depends on whether slippage after SAC arrest culminates in continued cell survival, or in death by apoptosis. However, the mechanisms that determine these outcomes remain unclear. Here, we identify a novel role for cyclin G1 (CCNG1), an atypical cyclin. Increased CCNG1 expression accompanies paclitaxel-induced, SAC-mediated mitotic arrest, independent of p53 integrity or signaling through the SAC component, BUBR1. CCNG1 overexpression promotes cell survival after paclitaxel exposure. Conversely, CCNG1 depletion by RNA interference delays slippage and enhances paclitaxel-induced apoptosis. Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds. Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy.

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Signaling through BUBR1 is dispensable for increased CCNG1 expression during mitosis. (a) Asynchronous U2OS cells were exposed to BUBR1 or non-targeting (NT) control siRNA for 24 h before treatment with 10 μM paclitaxel for 60 min. Individual cells were visualized by bright-field microscopy every 3–5 min from entry into mitotic prophase, indicated by characteristic changes in cell morphology, until anaphase, indicated by the onset of furrowing. The median duration for this interval, and the inter-quartile range are indicated. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (b) Asynchronous U2OS cells were exposed to BUBR1 or NT control siRNA before paclitaxel treatment, and released into 5 μM MG132. Cells were harvested by MSO 90 min afterwards, and stained with anti-MPM-2 antibody for analysis by flow cytometry. The percentage of intact cells positive for MPM-2 staining is shown. (c) Extracts prepared from samples of the cells described in (b) were immunoblotted with anti-BUBR1, anti-CCNG1 and anti-β-actin antibodies. (d) Asynchronous Cal51 cells were treated with 10 μM paclitaxel, for 60 min, released and after 12 h, treated with 2 μM ZM 447439 (ZM). Cells were harvested 90 and 180 min after ZM addition for analysis by MPM-2 staining using flow cytometry. The percentage of viable cells positive for MPM-2 staining is shown. (e) Extracts from samples of the cells described in (d) were immunoblotted as described above. Results shown are typical of at least two independent experiments.
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fig3: Signaling through BUBR1 is dispensable for increased CCNG1 expression during mitosis. (a) Asynchronous U2OS cells were exposed to BUBR1 or non-targeting (NT) control siRNA for 24 h before treatment with 10 μM paclitaxel for 60 min. Individual cells were visualized by bright-field microscopy every 3–5 min from entry into mitotic prophase, indicated by characteristic changes in cell morphology, until anaphase, indicated by the onset of furrowing. The median duration for this interval, and the inter-quartile range are indicated. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (b) Asynchronous U2OS cells were exposed to BUBR1 or NT control siRNA before paclitaxel treatment, and released into 5 μM MG132. Cells were harvested by MSO 90 min afterwards, and stained with anti-MPM-2 antibody for analysis by flow cytometry. The percentage of intact cells positive for MPM-2 staining is shown. (c) Extracts prepared from samples of the cells described in (b) were immunoblotted with anti-BUBR1, anti-CCNG1 and anti-β-actin antibodies. (d) Asynchronous Cal51 cells were treated with 10 μM paclitaxel, for 60 min, released and after 12 h, treated with 2 μM ZM 447439 (ZM). Cells were harvested 90 and 180 min after ZM addition for analysis by MPM-2 staining using flow cytometry. The percentage of viable cells positive for MPM-2 staining is shown. (e) Extracts from samples of the cells described in (d) were immunoblotted as described above. Results shown are typical of at least two independent experiments.

Mentions: To further verify that paclitaxel-induced CCNG1 expression is independent of SAC signaling, we depleted the essential SAC component, BUBR1(Meraldi et al., 2004), from U2OS cells using siRNA, before exposure to paclitaxel as before (Figure 3a). Serial time-lapse imaging confirms that BUBR1 depletion suppresses the mitotic arrest normally triggered by paclitaxel, allowing anaphase entry and rapid exit from mitosis. Therefore, to prevent mitotic exit, BUBR1-depleted, paclitaxel-treated cells were exposed to 5 μM MG132 (Brito and Rieder, 2006), as were controls, before mitotic cells were harvested by MSO (Figure 3b). We find no detectable difference in paclitaxel-induced CCNG1 expression after BUBR1 depletion, suggesting that CCNG1 induction during mitosis is independent of signaling by the SAC (Figure 3c).


Cyclin G1 regulates the outcome of taxane-induced mitotic checkpoint arrest.

Russell P, Hennessy BT, Li J, Carey MS, Bast RC, Freeman T, Venkitaraman AR - Oncogene (2011)

Signaling through BUBR1 is dispensable for increased CCNG1 expression during mitosis. (a) Asynchronous U2OS cells were exposed to BUBR1 or non-targeting (NT) control siRNA for 24 h before treatment with 10 μM paclitaxel for 60 min. Individual cells were visualized by bright-field microscopy every 3–5 min from entry into mitotic prophase, indicated by characteristic changes in cell morphology, until anaphase, indicated by the onset of furrowing. The median duration for this interval, and the inter-quartile range are indicated. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (b) Asynchronous U2OS cells were exposed to BUBR1 or NT control siRNA before paclitaxel treatment, and released into 5 μM MG132. Cells were harvested by MSO 90 min afterwards, and stained with anti-MPM-2 antibody for analysis by flow cytometry. The percentage of intact cells positive for MPM-2 staining is shown. (c) Extracts prepared from samples of the cells described in (b) were immunoblotted with anti-BUBR1, anti-CCNG1 and anti-β-actin antibodies. (d) Asynchronous Cal51 cells were treated with 10 μM paclitaxel, for 60 min, released and after 12 h, treated with 2 μM ZM 447439 (ZM). Cells were harvested 90 and 180 min after ZM addition for analysis by MPM-2 staining using flow cytometry. The percentage of viable cells positive for MPM-2 staining is shown. (e) Extracts from samples of the cells described in (d) were immunoblotted as described above. Results shown are typical of at least two independent experiments.
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fig3: Signaling through BUBR1 is dispensable for increased CCNG1 expression during mitosis. (a) Asynchronous U2OS cells were exposed to BUBR1 or non-targeting (NT) control siRNA for 24 h before treatment with 10 μM paclitaxel for 60 min. Individual cells were visualized by bright-field microscopy every 3–5 min from entry into mitotic prophase, indicated by characteristic changes in cell morphology, until anaphase, indicated by the onset of furrowing. The median duration for this interval, and the inter-quartile range are indicated. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (b) Asynchronous U2OS cells were exposed to BUBR1 or NT control siRNA before paclitaxel treatment, and released into 5 μM MG132. Cells were harvested by MSO 90 min afterwards, and stained with anti-MPM-2 antibody for analysis by flow cytometry. The percentage of intact cells positive for MPM-2 staining is shown. (c) Extracts prepared from samples of the cells described in (b) were immunoblotted with anti-BUBR1, anti-CCNG1 and anti-β-actin antibodies. (d) Asynchronous Cal51 cells were treated with 10 μM paclitaxel, for 60 min, released and after 12 h, treated with 2 μM ZM 447439 (ZM). Cells were harvested 90 and 180 min after ZM addition for analysis by MPM-2 staining using flow cytometry. The percentage of viable cells positive for MPM-2 staining is shown. (e) Extracts from samples of the cells described in (d) were immunoblotted as described above. Results shown are typical of at least two independent experiments.
Mentions: To further verify that paclitaxel-induced CCNG1 expression is independent of SAC signaling, we depleted the essential SAC component, BUBR1(Meraldi et al., 2004), from U2OS cells using siRNA, before exposure to paclitaxel as before (Figure 3a). Serial time-lapse imaging confirms that BUBR1 depletion suppresses the mitotic arrest normally triggered by paclitaxel, allowing anaphase entry and rapid exit from mitosis. Therefore, to prevent mitotic exit, BUBR1-depleted, paclitaxel-treated cells were exposed to 5 μM MG132 (Brito and Rieder, 2006), as were controls, before mitotic cells were harvested by MSO (Figure 3b). We find no detectable difference in paclitaxel-induced CCNG1 expression after BUBR1 depletion, suggesting that CCNG1 induction during mitosis is independent of signaling by the SAC (Figure 3c).

Bottom Line: However, the mechanisms that determine these outcomes remain unclear.Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds.Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Oncology and The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, UK.

ABSTRACT
Anti-mitotic chemotherapeutic agents such as taxanes activate the spindle assembly checkpoint (SAC) to arrest anaphase onset, but taxane-exposed cells eventually undergo slippage to exit mitosis. The therapeutic efficacy of taxanes depends on whether slippage after SAC arrest culminates in continued cell survival, or in death by apoptosis. However, the mechanisms that determine these outcomes remain unclear. Here, we identify a novel role for cyclin G1 (CCNG1), an atypical cyclin. Increased CCNG1 expression accompanies paclitaxel-induced, SAC-mediated mitotic arrest, independent of p53 integrity or signaling through the SAC component, BUBR1. CCNG1 overexpression promotes cell survival after paclitaxel exposure. Conversely, CCNG1 depletion by RNA interference delays slippage and enhances paclitaxel-induced apoptosis. Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds. Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy.

Show MeSH
Related in: MedlinePlus