Caspase-3–Dependent Mitotic Checkpoint Inactivation by the Small-Molecule Inducers of Mitotic Slippage SU6656 and Geraldol

Mitotic checkpoint inactivation but not cyclin B1 degradation occurs through caspase-3–dependent cleavage of BubR1. A, T98G cells were arrested in mitosis by 30 nmol/L paclitaxel, harvested by shake-off, and seeded in 96-well plates. After exposure to 5 μmol/L SU6656 or 5 μmol/L geraldol simultaneously with 0 to 100 μmol/L Ac-DEVD-CHO for 4 hours, the slipped cells were stained with Hoechst 33342 and quantified by automated fluorescence microscopy. Error bars represent 95% CIs. B, mitotic T98G cells were harvested by shake-off and incubated with 5 μmol/L SU6656 or 5 μmol/L geraldol without or with 50 μmol/L DEVD-CHO for 4 hours. Lysates were immunoblotted for the indicated proteins. C, MCF-7 cells stably transfected with empty vector (MCF-7pcDNA) or caspase-3 (MCF-7casp3) were arrested in mitosis by 50 nmol/L paclitaxel, harvested by shake-off, and seeded in 96-well plates. The cells were exposed to 0 to 15 μmol/L SU6656 or geraldol for 4 hours, stained with Hoechst 33342, and quantified using an automated fluorescence imager. Error bars represent 95% CIs. D, mitotic MCF-7pcDNA and MCF-7casp3 cells were harvested by shake-off and incubated with 5 μmol/L SU6656 or 5 μmol/L geraldol without or with 20 μmol/L MG-132 for 4 hours. Lysates were immunoblotted for the indicated proteins. E, MCF-7pcDNA or MCF-7casp3 cells were exposed to 100 nmol/L paclitaxel for up to 28 hours, and nuclei were fixed and stained with Hoechst 33342. The total number of cells was quantified using automated fluorescence microscopy, and the images were visually inspected to determine the proportion of slipped and mitotic cells at each time.

Timeline of mitotic checkpoint inactivation and slippage. A, T98G cells arrested in mitosis by 30 nmol/L paclitaxel were harvested by shake-off, seeded in 96-well plates, and incubated with DMSO or 5 μmol/L geraldol for 15 minutes to 3 hours. Slipped cells were quantified after staining with Hoechst (left) or with mouse TG3 antibody against mitotically phosphorylated nucleolin (right). The proportion of slipped cells was lower than usually observed because of the numerous washes during immunofluorescent staining that removed many attached, slipped cells. Error bars represent 95% CIs. B, cycling T98G cells (U) were arrested in mitosis by exposure to 30 nmol/L paclitaxel and harvested by shake-off. Mitotic cells (M) were incubated with 5 μmol/L SU6656 or 5 μmol/L geraldol for 15 minutes to 3 hours and lysates were immunoblotted for the indicated proteins.

Inhibition of Aurora A and Aurora B by SU6656 and geraldol. A, SU6656 and geraldol were assayed for in vitro inhibition of Aurora A and Aurora B as described in Materials and Methods. B, MCF-7 cells were arrested in mitosis by 50 nmol/L paclitaxel for 20 hours, harvested by shake-off, seeded in 96-well plates, and incubated with 0.1 to 20 μmol/L ZM447439 for 4 hours. Cells were fixed, stained with Hoechst 33342, and imaged by automated fluorescence microscopy. Error bars represent 95% CIs.

Dependence of the outcome of spontaneous and induced mitotic slippage on caspase-3. A, slipped cells, MCF-7pcDNA and MCF-7casp3 cells were exposed to 50 nmol/L paclitaxel for 20 hours, harvested by shake-off, and seeded in 96-well plates. After exposure to 0.1% DMSO, 5 μmol/L SU6656, or 5 μmol/L geraldol for 4 hours, unattached (mitotic) cells were removed and adherent (slipped) cells were allowed to grow in fresh culture medium for up to 14 days before staining with Hoechst and quantification as a proportion of mitotic cells by automated fluorescence microscopy. B and C, all cells and viable cells, MCF-7pcDNA or MCF-7casp3 cells in 96-well plates were exposed to 0.1% DMSO or 50 nmol/L paclitaxel for 20 hours and then 0.1% DMSO, 5 μmol/L SU6656, or 5 μmol/L geraldol for a further 4 hours. Drugs were washed away and the cells were allowed to grow in fresh culture medium for up to 14 day before staining with Hoechst and quantification (all cells) or analysis of cell viability by the MTT assay (viable cells). Error bars represent 95% CIs.