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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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CCNG1 overexpressing cells escape paclitaxel-induced apoptosis independent of p53. (a) Asynchronous HCT116 and HCT116 p53−/− cells were transiently transfected with a construct encoding an EGFP-CCNG1 fusion protein or the empty vector. Cells were treated with paclitaxel 24 h after transfection, and harvested at the indicated times for analysis by immunoblotting or flow cytometry. (b) Extracts from cells harvested at 0, 18 and 44 h were immunoblotted with anti-CCNG1 and anti-β-actin antibodies. (d) The percentage of HCT116 (c) or HCT116 p53−/− cells expressing EGFP was determined by flow cytometry. Data points represent the mean of triplicate observations with the error bars representing a single s.d. from the mean.
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fig5: CCNG1 overexpressing cells escape paclitaxel-induced apoptosis independent of p53. (a) Asynchronous HCT116 and HCT116 p53−/− cells were transiently transfected with a construct encoding an EGFP-CCNG1 fusion protein or the empty vector. Cells were treated with paclitaxel 24 h after transfection, and harvested at the indicated times for analysis by immunoblotting or flow cytometry. (b) Extracts from cells harvested at 0, 18 and 44 h were immunoblotted with anti-CCNG1 and anti-β-actin antibodies. (d) The percentage of HCT116 (c) or HCT116 p53−/− cells expressing EGFP was determined by flow cytometry. Data points represent the mean of triplicate observations with the error bars representing a single s.d. from the mean.

Mentions: We therefore tested whether CCNG1 overexpression could conversely enhance cell survival after exposure to anti-mitotic drugs. To this end, we used a method we have previously established (Lee et al., 1999) to test whether cells overexpressing an EGFP-CCNG1 fusion protein have a survival advantage after paclitaxel exposure when compared with untransfected cells in the same culture. Accordingly, HCT116 cells were transfected (Figure 5a) with either EGFP-CCNG1 or EGFP, such that no more than 10–30% in each culture received the fluorophore-expressing construct, whereas the remaining cells were untransfected. Cultures were exposed to 10 μM paclitaxel for 60 min, before harvesting at different times over the following 72 h (Figure 5b), and enumeration by flow cytometry of the ratio of viable EGFP-positive to EGFP-negative cells (Figure 5c). Viable cells that express EGFP-CCNG1 are ∼2fold increased over those expressing EGFP alone within 24 h after drug treatment. This advantage was not manifest in untreated cultures confirming that EGFP-CCNG1 promotes survival only after paclitaxel exposure. Similar results were obtained in p53−/− HCT116 cells (Figure 5d), demonstrating that EGFP-CCNG1 expression promotes cell survival after SAC activation independent of p53 integrity.


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)

CCNG1 overexpressing cells escape paclitaxel-induced apoptosis independent of p53. (a) Asynchronous HCT116 and HCT116 p53−/− cells were transiently transfected with a construct encoding an EGFP-CCNG1 fusion protein or the empty vector. Cells were treated with paclitaxel 24 h after transfection, and harvested at the indicated times for analysis by immunoblotting or flow cytometry. (b) Extracts from cells harvested at 0, 18 and 44 h were immunoblotted with anti-CCNG1 and anti-β-actin antibodies. (d) The percentage of HCT116 (c) or HCT116 p53−/− cells expressing EGFP was determined by flow cytometry. Data points represent the mean of triplicate observations with the error bars representing a single s.d. from the mean.
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fig5: CCNG1 overexpressing cells escape paclitaxel-induced apoptosis independent of p53. (a) Asynchronous HCT116 and HCT116 p53−/− cells were transiently transfected with a construct encoding an EGFP-CCNG1 fusion protein or the empty vector. Cells were treated with paclitaxel 24 h after transfection, and harvested at the indicated times for analysis by immunoblotting or flow cytometry. (b) Extracts from cells harvested at 0, 18 and 44 h were immunoblotted with anti-CCNG1 and anti-β-actin antibodies. (d) The percentage of HCT116 (c) or HCT116 p53−/− cells expressing EGFP was determined by flow cytometry. Data points represent the mean of triplicate observations with the error bars representing a single s.d. from the mean.
Mentions: We therefore tested whether CCNG1 overexpression could conversely enhance cell survival after exposure to anti-mitotic drugs. To this end, we used a method we have previously established (Lee et al., 1999) to test whether cells overexpressing an EGFP-CCNG1 fusion protein have a survival advantage after paclitaxel exposure when compared with untransfected cells in the same culture. Accordingly, HCT116 cells were transfected (Figure 5a) with either EGFP-CCNG1 or EGFP, such that no more than 10–30% in each culture received the fluorophore-expressing construct, whereas the remaining cells were untransfected. Cultures were exposed to 10 μM paclitaxel for 60 min, before harvesting at different times over the following 72 h (Figure 5b), and enumeration by flow cytometry of the ratio of viable EGFP-positive to EGFP-negative cells (Figure 5c). Viable cells that express EGFP-CCNG1 are ∼2fold increased over those expressing EGFP alone within 24 h after drug treatment. This advantage was not manifest in untreated cultures confirming that EGFP-CCNG1 promotes survival only after paclitaxel exposure. Similar results were obtained in p53−/− HCT116 cells (Figure 5d), demonstrating that EGFP-CCNG1 expression promotes cell survival after SAC activation independent of p53 integrity.

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