Limits...
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.

Show MeSH

Related in: MedlinePlus

Increased CCNG1 expression accompanies paclitaxel-induced SAC-mediated mitotic arrest in a p53-independent manner. Asynchronous U2OS, Cal51 and HCT116 cells were treated with 10 μM paclitaxel for 60 min. (a) Cells were harvested over 48 h and immunoblotted with anti-CCNG1 and anti-β-actin antibodies. β-actin is used as a loading control here and in other experiments. (b) Samples of the cells described in (a) were stained with mitotic phosphoprotein monoclonal antibody, MPM-2 and propidium iodide (PI) to measure DNA content, before analysis by flow cytometry. The percentage of viable cells with 4N DNA content, or positive for MPM-2 staining, is shown. (c) Paclitaxel-induced CCNG1 expression is independent of p53. Asynchronous HCT116 cells and their isogenic p53- counterparts (HCT116 p53−/−) were treated with paclitaxel as described before the analysis of CCNG1 and p53 expression by western blotting. (d) Samples of the cells described in (c) were fixed and stained with MPM-2 and PI as described in (b). Results shown are typical of three or more independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351588&req=5

fig1: Increased CCNG1 expression accompanies paclitaxel-induced SAC-mediated mitotic arrest in a p53-independent manner. Asynchronous U2OS, Cal51 and HCT116 cells were treated with 10 μM paclitaxel for 60 min. (a) Cells were harvested over 48 h and immunoblotted with anti-CCNG1 and anti-β-actin antibodies. β-actin is used as a loading control here and in other experiments. (b) Samples of the cells described in (a) were stained with mitotic phosphoprotein monoclonal antibody, MPM-2 and propidium iodide (PI) to measure DNA content, before analysis by flow cytometry. The percentage of viable cells with 4N DNA content, or positive for MPM-2 staining, is shown. (c) Paclitaxel-induced CCNG1 expression is independent of p53. Asynchronous HCT116 cells and their isogenic p53- counterparts (HCT116 p53−/−) were treated with paclitaxel as described before the analysis of CCNG1 and p53 expression by western blotting. (d) Samples of the cells described in (c) were fixed and stained with MPM-2 and PI as described in (b). Results shown are typical of three or more independent experiments.

Mentions: Asynchronous cultures of the cancer cell lines U2OS, Cal51 or HCT116 were exposed to 10 μM paclitaxel for 60 min before the drug was washed out (Sena et al., 1999; Michalakis et al., 2005). CCNG1 protein levels in cell lysates were determined by western blotting between 2 and 48 h after paclitaxel exposure (Figure 1a). These conditions of paclitaxel treatment induce an accumulation of cells in prometaphase, as marked by 4N DNA content and co-staining with the mitotic marker MPM-2 (Figure 1b). Pro-metaphase arrest was also confirmed through microscopic assessment of chromosome condensation visualized by 4′,6-diamidino-2-phenylindole staining (data not shown). MPM-2 staining increases after drug exposure in all the cell lines tested, peaking at >60% between 11 and 24 h after treatment, consistent with activation of the mitotic SAC. Although untreated cells express relatively low levels of the protein, CCNG1 protein levels increase sharply after paclitaxel exposure, exhibiting, for example, an approximately 100 × increase 16 h after exposure in the case of the HCT116 cells (Figure 1a). This increase coincides with the period of maximal mitotic arrest as determined by MPM-2 expression. CCNG1 protein levels decrease rapidly as cells undergo mitotic slippage and exit mitosis, and continue to decrease, but more slowly, over the following 48 h. Similar effects are elicited when HCT116 cells are treated with the inhibitors nocodazole or monastrol, which elicit mitotic arrest by mechanisms different from paclitaxel, suggesting that the changes in CCNG1 expression represent a general response to mitotic arrest (Supplementary Figure S1).


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)

Increased CCNG1 expression accompanies paclitaxel-induced SAC-mediated mitotic arrest in a p53-independent manner. Asynchronous U2OS, Cal51 and HCT116 cells were treated with 10 μM paclitaxel for 60 min. (a) Cells were harvested over 48 h and immunoblotted with anti-CCNG1 and anti-β-actin antibodies. β-actin is used as a loading control here and in other experiments. (b) Samples of the cells described in (a) were stained with mitotic phosphoprotein monoclonal antibody, MPM-2 and propidium iodide (PI) to measure DNA content, before analysis by flow cytometry. The percentage of viable cells with 4N DNA content, or positive for MPM-2 staining, is shown. (c) Paclitaxel-induced CCNG1 expression is independent of p53. Asynchronous HCT116 cells and their isogenic p53- counterparts (HCT116 p53−/−) were treated with paclitaxel as described before the analysis of CCNG1 and p53 expression by western blotting. (d) Samples of the cells described in (c) were fixed and stained with MPM-2 and PI as described in (b). Results shown are typical of three or more independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351588&req=5

fig1: Increased CCNG1 expression accompanies paclitaxel-induced SAC-mediated mitotic arrest in a p53-independent manner. Asynchronous U2OS, Cal51 and HCT116 cells were treated with 10 μM paclitaxel for 60 min. (a) Cells were harvested over 48 h and immunoblotted with anti-CCNG1 and anti-β-actin antibodies. β-actin is used as a loading control here and in other experiments. (b) Samples of the cells described in (a) were stained with mitotic phosphoprotein monoclonal antibody, MPM-2 and propidium iodide (PI) to measure DNA content, before analysis by flow cytometry. The percentage of viable cells with 4N DNA content, or positive for MPM-2 staining, is shown. (c) Paclitaxel-induced CCNG1 expression is independent of p53. Asynchronous HCT116 cells and their isogenic p53- counterparts (HCT116 p53−/−) were treated with paclitaxel as described before the analysis of CCNG1 and p53 expression by western blotting. (d) Samples of the cells described in (c) were fixed and stained with MPM-2 and PI as described in (b). Results shown are typical of three or more independent experiments.
Mentions: Asynchronous cultures of the cancer cell lines U2OS, Cal51 or HCT116 were exposed to 10 μM paclitaxel for 60 min before the drug was washed out (Sena et al., 1999; Michalakis et al., 2005). CCNG1 protein levels in cell lysates were determined by western blotting between 2 and 48 h after paclitaxel exposure (Figure 1a). These conditions of paclitaxel treatment induce an accumulation of cells in prometaphase, as marked by 4N DNA content and co-staining with the mitotic marker MPM-2 (Figure 1b). Pro-metaphase arrest was also confirmed through microscopic assessment of chromosome condensation visualized by 4′,6-diamidino-2-phenylindole staining (data not shown). MPM-2 staining increases after drug exposure in all the cell lines tested, peaking at >60% between 11 and 24 h after treatment, consistent with activation of the mitotic SAC. Although untreated cells express relatively low levels of the protein, CCNG1 protein levels increase sharply after paclitaxel exposure, exhibiting, for example, an approximately 100 × increase 16 h after exposure in the case of the HCT116 cells (Figure 1a). This increase coincides with the period of maximal mitotic arrest as determined by MPM-2 expression. CCNG1 protein levels decrease rapidly as cells undergo mitotic slippage and exit mitosis, and continue to decrease, but more slowly, over the following 48 h. Similar effects are elicited when HCT116 cells are treated with the inhibitors nocodazole or monastrol, which elicit mitotic arrest by mechanisms different from paclitaxel, suggesting that the changes in CCNG1 expression represent a general response to mitotic arrest (Supplementary Figure S1).

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