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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 depletion prolongs paclitaxel-induced mitotic arrest and increases drug-induced cell death. (a) Asynchronous U2OS and Cal51 cells exposed to CCNG1 or NT control siRNA were treated with paclitaxel, and harvested 12 h afterwards. Relative CCNG1 mRNA expression was quantitated by semi-quantitative RT–PCR. The graph shows the mean±s.e.m. from triplicate experiments. (b) Extracts from samples of the cells described in (a) were immunoblotted with anti-CCNG1 and anti-β-Actin antibodies. (c) Individual, asynchronous U2OS cells, treated as in (a), were monitored by bright-field microscopy as described in Figure 3 to determine the interval from prophase to anaphase. Cumulative frequency plots of the time taken are shown. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (d) The median time taken from prophase to anaphase onset after CCNG1 depletion using two distinct siRNAs is increased in a statistically significant manner (CCNG1 siRNA_1 () (*P=0.0294) and CCNG1 siRNA_2 () (***P<0.0001) when compared with NT control () using a two-tailed Mann–Whitney test). The median and the inter-quartile range for each group are indicated. (e) The effect of CCNG1 depletion on cell viability was assessed utilizing the CellTiter-Blue Cell Viability Assay (Promega) over a 3-day period following paclitaxel treatment. Cell viability is expressed relative to the controls. Data points represent the mean of triplicate observations with the error bars representing a single s.d. from the mean.
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fig4: CCNG1 depletion prolongs paclitaxel-induced mitotic arrest and increases drug-induced cell death. (a) Asynchronous U2OS and Cal51 cells exposed to CCNG1 or NT control siRNA were treated with paclitaxel, and harvested 12 h afterwards. Relative CCNG1 mRNA expression was quantitated by semi-quantitative RT–PCR. The graph shows the mean±s.e.m. from triplicate experiments. (b) Extracts from samples of the cells described in (a) were immunoblotted with anti-CCNG1 and anti-β-Actin antibodies. (c) Individual, asynchronous U2OS cells, treated as in (a), were monitored by bright-field microscopy as described in Figure 3 to determine the interval from prophase to anaphase. Cumulative frequency plots of the time taken are shown. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (d) The median time taken from prophase to anaphase onset after CCNG1 depletion using two distinct siRNAs is increased in a statistically significant manner (CCNG1 siRNA_1 () (*P=0.0294) and CCNG1 siRNA_2 () (***P<0.0001) when compared with NT control () using a two-tailed Mann–Whitney test). The median and the inter-quartile range for each group are indicated. (e) The effect of CCNG1 depletion on cell viability was assessed utilizing the CellTiter-Blue Cell Viability Assay (Promega) over a 3-day period following paclitaxel treatment. Cell viability is expressed relative to the controls. 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 regulates paclitaxel-induced mitotic arrest, using serial time-lapse imaging to precisely assess mitotic progression in CCNG1-depleted or control U2OS cells (Figures 4a and b), before and after drug exposure. Images were taken every 3 min from the start of cell rounding that starts in late prophase until the formation of a cleavage furrow in anaphase A, visualizing at least 25 individual cells per sample. The cumulative frequency of cells progressing from prophase to anaphase was then plotted against time, providing a sensitive measure of SAC enforcement. Consistent with expectation, the prophase–anaphase interval is greatly prolonged from a median of 30 min to 270 min when control cells are exposed to paclitaxel (Figure 4c). Conversely, and also as expected (Meraldi et al., 2004; Sudo et al., 2004), the depletion of BUBR1, an essential SAC component, reduces the prophase–anaphase interval after paclitaxel treatment to a median of 21 min. Interestingly, the depletion of CCNG1 using either of the two sequence-independent siRNAs significantly increased the mitotic delay induced by paclitaxel, prolonging the prophase–anaphase interval to a median of 381 min (P=0.0294) and 474 min (P<0.0001) (Figure 4d). However, CCNG1 depletion did not alter the prophase–anaphase interval in unchallenged cells, suggesting that it is dispensable for normal mitotic timing. From these results we infer that CCNG1 is not an essential component of the SAC machinery per se, but instead, may have a role in promoting slippage and mitotic exit after SAC activation.


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 depletion prolongs paclitaxel-induced mitotic arrest and increases drug-induced cell death. (a) Asynchronous U2OS and Cal51 cells exposed to CCNG1 or NT control siRNA were treated with paclitaxel, and harvested 12 h afterwards. Relative CCNG1 mRNA expression was quantitated by semi-quantitative RT–PCR. The graph shows the mean±s.e.m. from triplicate experiments. (b) Extracts from samples of the cells described in (a) were immunoblotted with anti-CCNG1 and anti-β-Actin antibodies. (c) Individual, asynchronous U2OS cells, treated as in (a), were monitored by bright-field microscopy as described in Figure 3 to determine the interval from prophase to anaphase. Cumulative frequency plots of the time taken are shown. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (d) The median time taken from prophase to anaphase onset after CCNG1 depletion using two distinct siRNAs is increased in a statistically significant manner (CCNG1 siRNA_1 () (*P=0.0294) and CCNG1 siRNA_2 () (***P<0.0001) when compared with NT control () using a two-tailed Mann–Whitney test). The median and the inter-quartile range for each group are indicated. (e) The effect of CCNG1 depletion on cell viability was assessed utilizing the CellTiter-Blue Cell Viability Assay (Promega) over a 3-day period following paclitaxel treatment. Cell viability is expressed relative to the controls. Data points represent the mean of triplicate observations with the error bars representing a single s.d. from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig4: CCNG1 depletion prolongs paclitaxel-induced mitotic arrest and increases drug-induced cell death. (a) Asynchronous U2OS and Cal51 cells exposed to CCNG1 or NT control siRNA were treated with paclitaxel, and harvested 12 h afterwards. Relative CCNG1 mRNA expression was quantitated by semi-quantitative RT–PCR. The graph shows the mean±s.e.m. from triplicate experiments. (b) Extracts from samples of the cells described in (a) were immunoblotted with anti-CCNG1 and anti-β-Actin antibodies. (c) Individual, asynchronous U2OS cells, treated as in (a), were monitored by bright-field microscopy as described in Figure 3 to determine the interval from prophase to anaphase. Cumulative frequency plots of the time taken are shown. Results shown are typical of at least two independent experiments in which 25 mitoses were followed for each experimental group. (d) The median time taken from prophase to anaphase onset after CCNG1 depletion using two distinct siRNAs is increased in a statistically significant manner (CCNG1 siRNA_1 () (*P=0.0294) and CCNG1 siRNA_2 () (***P<0.0001) when compared with NT control () using a two-tailed Mann–Whitney test). The median and the inter-quartile range for each group are indicated. (e) The effect of CCNG1 depletion on cell viability was assessed utilizing the CellTiter-Blue Cell Viability Assay (Promega) over a 3-day period following paclitaxel treatment. Cell viability is expressed relative to the controls. 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 regulates paclitaxel-induced mitotic arrest, using serial time-lapse imaging to precisely assess mitotic progression in CCNG1-depleted or control U2OS cells (Figures 4a and b), before and after drug exposure. Images were taken every 3 min from the start of cell rounding that starts in late prophase until the formation of a cleavage furrow in anaphase A, visualizing at least 25 individual cells per sample. The cumulative frequency of cells progressing from prophase to anaphase was then plotted against time, providing a sensitive measure of SAC enforcement. Consistent with expectation, the prophase–anaphase interval is greatly prolonged from a median of 30 min to 270 min when control cells are exposed to paclitaxel (Figure 4c). Conversely, and also as expected (Meraldi et al., 2004; Sudo et al., 2004), the depletion of BUBR1, an essential SAC component, reduces the prophase–anaphase interval after paclitaxel treatment to a median of 21 min. Interestingly, the depletion of CCNG1 using either of the two sequence-independent siRNAs significantly increased the mitotic delay induced by paclitaxel, prolonging the prophase–anaphase interval to a median of 381 min (P=0.0294) and 474 min (P<0.0001) (Figure 4d). However, CCNG1 depletion did not alter the prophase–anaphase interval in unchallenged cells, suggesting that it is dispensable for normal mitotic timing. From these results we infer that CCNG1 is not an essential component of the SAC machinery per se, but instead, may have a role in promoting slippage and mitotic exit after SAC activation.

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