Advertisement
Preclinical Development

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

Jenna L. Riffell, Reiner U. Jänicke and Michel Roberge
DOI: 10.1158/1535-7163.MCT-10-0909 Published May 2011

Abstract

Microtubule-targeting cancer drugs such as paclitaxel block cell-cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells can spontaneously escape mitotic arrest and enter interphase without chromosome segregation by a process termed mitotic slippage that involves the degradation of cyclin B1 without mitotic checkpoint inactivation. Inducing mitotic slippage with chemicals causes cells to die after multiple rounds of DNA replication without cell division, which may enhance the antitumor activity of microtubule-targeting drugs. Here, we explore pathways leading to mitotic slippage by using SU6656 and geraldol, two recently identified chemical inducers of mitotic slippage. Mitotic slippage induced by SU6656 or geraldol was blocked by the proteasome inhibitor MG-132 and involved proteasome-dependent degradation of cyclin B1 and the mitotic checkpoint proteins budding uninhibited by benzimidazole related 1 (BubR1) and cell division cycle 20 (Cdc20) in T98G cells. Mitotic slippage and the degradation of BubR1 and Cdc20 were also inhibited by the caspase-3 and -7 inhibitor DEVD-CHO. MCF-7 cells lacking caspase-3 expression could not degrade BubR1 or undergo mitotic slippage in response to SU6656 or geraldol. Introduction of caspase-3 completely restored the ability of MCF-7 cells to degrade BubR1 and undergo mitotic slippage. However, lack of expression of caspase-3 did not affect cell death after exposure to paclitaxel, with or without mitotic slippage induction. The requirement for caspase-3 for chemically induced mitotic slippage reveals a new mechanism for mitotic exit and a link between mitosis and apoptosis that has implications for the outcome of cancer chemotherapy. Mol Cancer Ther; 10(5); 839–49. ©2011 AACR.

This article is featured in Highlights of This Issue, p. 709

Introduction

During cell division, genetic integrity is maintained by ensuring that all chromosomes are attached to microtubules emanating from both poles of the mitotic spindle before segregation of sister chromatids begins (1). This process is monitored by the mitotic checkpoint, which prevents initiation of anaphase until every kinetochore is attached and tension between kinetochores of paired sister chromatids is sufficient, ensuring biorientation (2). To prevent aneuploidy and ensuing genetic defects leading to cell death or tumorigenesis (3), the mitotic checkpoint must be sufficiently sensitive to delay chromosome separation when even 1 kinetochore is unattached. Exposure to drugs that interfere with microtubule dynamics, such as the taxanes (4) and the Vinca alkaloids (5), similarly activates the mitotic checkpoint and arrests cells at mitosis, effectively preventing further proliferation.

The mitotic checkpoint acts through inhibition of the anaphase-promoting complex/cyclosome (APC/C; ref. 6), the E3 ubiquitin ligase (7) that, when activated by cofactors cell division cycle 20 (Cdc20) or Cdh1 (8), polyubiquitylates the cyclin-dependent kinase 1 (Cdk1) cofactor cyclin B1 (7) and the separase regulator securin (9), targeting them for degradation by the proteasome. This results in inactivation of Cdk1, separation of sister chromatids, and exit from mitosis. The key components of the mitotic checkpoint are budding uninhibited by benzimidazole related 1 (BubR1), budding uninhibited by benzimidazole 3 (Bub3), and Cdc20, which form a mitotic checkpoint complex (MCC; ref. 10). This complex is the main inhibitor of APC/C activity, along with mitotic arrest dependent 2 (Mad2), which initially binds Cdc20 (11) and catalyzes its binding to BubR1 and subsequent formation of the MCC (12). Cdc20 is an activating cofactor of APC/C during mitosis (8); an active mitotic checkpoint inhibits APC/C through APC/C-dependent polyubiquitylation of Cdc20 and subsequent degradation by the proteasome (12). BubR1 binds to and inhibits both Cdc20 (13) and APC/C itself (14), acting as a pseudosubstrate inhibitor that, depending on acetylation status, can be actively degraded by APC/CCdc20 (15). The role of Bub3 in the MCC is unclear, although in fission yeast it is involved in MCC localization (16). Other components of the mitotic checkpoint include the kinases Bub1, monopolar spindle 1 (Mps1), and Aurora B (2).

Caspases have well-characterized apoptotic functions, but caspase-3 and caspase-7 have both recently been observed to play a role, yet to be defined, in mitotic progression (17, 18). Their activities are tightly regulated and must be restrained during mitotic stress to prevent extensive cell death, most notably through survivin, which inhibits caspase activation during mitotic arrest and functions as part of the mitotic checkpoint machinery (19).

Mitotic checkpoint activation during an unperturbed mitosis provides sufficient time for microtubule attachment, preventing aneuploidy (20) and increasing cell survival (21). However, long-term activation of the mitotic checkpoint during exposure to antimitotic agents can be problematic because chromosome condensation hinders RNA transcription (22). With time, an imbalance between new protein production and protein degradation may cause the levels of proteins essential to maintain mitotic arrest to fall, triggering mitotic slippage. Also termed mitotic checkpoint adaptation, mitotic slippage occurs when cells exit mitosis without chromosome segregation or cell division (20, 23) and results from slow APC/CCdc20- and proteasome-dependent degradation of cyclin B1 in the presence of an active mitotic checkpoint (24, 25). Cells that have undergone mitotic slippage enter a G1-like state with decondensed chromosomes that form multiple micronuclei (23), allowing resumption of transcription and other cellular processes.

Our group and others have identified chemicals that stimulate mitotic slippage and observed that slipped cells typically undergo at least 1 round of DNA replication without subsequent cell division but that, eventually, all cells that undergo mitotic slippage die (26–30). Known chemical inducers of mitotic slippage include CDK1 inhibitors (roscovitine, RO3066; ref. 28), histone deacetylase complex inhibitors (SBHA, SAHA, sodium butyrate, trichostatin A; refs. 31, 32), and Aurora inhibitors [ZM447439 (33), MLN8054 (34), Gö6976 (29), OM137 (27), and fisetin (30); Supplementary Fig. S1]. We previously identified SU6656 and geraldol as chemical inducers of mitotic slippage that increased cell killing after induction of mitotic arrest by microtubule-targeting agents. This study investigates how chemicals can modulate mitotic slippage, reveals a mechanism for mitotic slippage that is different from that described for spontaneous mitotic slippage, and shows how interplay between pathways associated with mitosis and apoptosis can contribute to the outcome of antimitotic cancer treatments.

Materials and Methods

Cell culture and chemicals

T98G cells, obtained from the American Type Culture Collection (ATCC; characterized by short tandem repeat analysis) and used within 6 months of resuscitation, were maintained in Dulbecco's Modified Eagle's Medium (Invitrogen) supplemented with 10% FBS (Gibco). MCF-7 cell lines, obtained from the ATCC and stably transfected with empty vector (pcDNA) or caspase-3 (casp3), were maintained in RPMI (Invitrogen) supplemented with 10% FBS and 10 mmol/L HEPES, pH 7.3 (Invitrogen). Paclitaxel was obtained from USB, SU6656, and MG-132 from Sigma, geraldol from Chromadex, and cell-permeable DEVD-CHO from Enzo Life Sciences.

Slippage induction assay

T98G cells at 75% confluency were treated with 30 nmol/L paclitaxel, or MCF-7 cells were treated with 50 nmol/L paclitaxel, for 20 hours at 37°C, and mitotic cells were harvested by shake-off, counted using a hemacytometer, seeded in a 96-well plate (PerkinElmer Viewplate) at 5,000 cells per well, and treated with chemicals as indicated for 4 hours at 37°C. Unattached mitotic cells were then aspirated and discarded while attached, slipped cells were fixed in 3% paraformaldehyde (EMD) in PBS for 15 minutes at room temperature, and stained with Hoechst 33342 (Invitrogen) in PBS for 10 minutes at room temperature. Five fields per well were counted by a Cellomics ArrayScan VTI automated fluorescence imager (ThermoFisher) by using a 10× objective. Individual nuclei of slipped cells were detected and counted using the Cellomics Target Activation Analysis Program. In all figures, mitotic slippage was expressed as a percentage of the cells seeded in each well (26).

Immunoblotting

Cells were washed in PBS and lysed for 5 minutes on ice in lysis buffer containing 20 mmol/L Tris-HCl (Fisher), pH 7.5, 150 mmol/L NaCl (Fisher), 1 mmol/L EDTA (Sigma), 1 mmol/L EGTA (Sigma), 1% Triton X-100 (LabChem Inc.), 2.5 mmol/L sodium pyrophosphate (Fisher), 1 mmol/L β-glycerol phosphate (Sigma), 1 mmol/L sodium orthovanadate (Sigma), and 1× protease inhibitor cocktail (Roche). Lysates were spun at 15,000 × g for 15 minutes, and supernatants were removed and assayed for protein concentration by using the Bradford assay (Sigma). Sample concentration was equalized and diluted in 50 mmol/L Tris-HCl (Fisher), pH 6.8, 2% SDS (Fisher), 0.1% bromophenol blue (Sigma), and 10% glycerol (Fisher), run on a 12% acrylamide (Bio-Rad) gel, and stained with Coommassie Brilliant Blue to verify equal protein loading or transferred to a polyvinylidene difluoride membrane (Millipore Immobilon-P). The membrane was blocked in 5% milk (Nestle) in TBS containing 0.1% Tween-20 (TBS-T; MP Biomedicals) for 30 minutes and incubated overnight at 4°C with primary antibody in 5% milk in TBS. Membranes were then washed 2 × 10 minutes in TBS-T, incubated at room temperature with secondary antibody in 5% milk for 1 hour, washed 3 × 10 minutes in TBS-T, and imaged by chemiluminescence (Millipore Immobilon Western). Antibodies used were mouse α-cyclin B1 (1:100; BD Pharmingen), mouse α-BubR1 (1:1,000; BD Transduction), mouse α-Mad2 (1:500; Santa Cruz), mouse α-p55CDC/Cdc20 (1:1,000; Santa Cruz), mouse α-Mps1 (1:500; Abcam), goat α-mouse horseradish peroxidase (1:10,000), and goat α-rabbit peroxidase conjugate (1:10,000).

In vitro kinase assays

SU6656 or geraldol were incubated for 20 to 30 minutes at room temperature with 20 to 40 nmol/L active kinase, 0.2 mg/mL myelin basic protein (Aurora kinases), or 0.4 mg/mL synthetic Src substrate (KVEKIGEGTYGVVYK) and 50 μmol/L 33P-ATP in kinase assay buffer containing 25 mmol/L MOPS, pH 7.2, 12.5 mmol/L β-glycerol phosphate, 25 mmol/L MgCl2, 5 mmol/L EGTA, 2 mmol/L EDTA, and 0.25 mmol/L DTT (Aurora kinases) or 25 mmol/L MOPS, pH 7.2, 12.5 mmol/L β-glycerol phosphate, 20 mmol/L MgCl2, 25 mmol/L MnCl2, 5 mmol/L EGTA, 2 mmol/L EDTA, and 0.25 mmol/L DTT (Src). Ten microliters of this reaction mixture was then spotted on a phosphocellulose Multiscreen plate and washed 3 × 15 minutes in 1% phosphoric acid. Scintillation fluid was added and the radioactivity on the plate was counted using a Trilux scintillation counter against a control incubated without substrate.

Results

Induction of mitotic slippage by SU6656 and geraldol requires proteasomal activity

A previous screening effort by our group identified SU6656 and geraldol as chemicals capable of inducing mitotic slippage (26). When cells spontaneously slip out of mitotic arrest, as well as during normal exit from mitosis, proteasomal activity is required for the degradation of cyclin B1 (24, 25). First, to determine whether escape from mitotic arrest induced by SU6656 or geraldol similarly requires proteasome activity, mitotic slippage was examined in the presence of the proteasome inhibitor MG-132. T98G cells at 75% confluency were arrested in mitosis by exposure to 30 nmol/L paclitaxel for 20 hours, harvested via shake-off, and seeded in 96-well plates. The cells were exposed to the chemical inducers of mitotic slippage SU6656 (5 μmol/L) or geraldol (5 μmol/L) in the presence of various concentrations of MG-132 for 4 hours and in the continued presence of paclitaxel. Residual unattached mitotic cells were removed and the attached, slipped cells were fixed, stained with Hoechst 33342, and quantified using automated fluorescence microscopy (26). In the absence of MG-132, SU6656 and geraldol induced 60% to 90% of mitotic cells to undergo mitotic slippage. MG-132 reduced the proportion of slipped cells in a concentration-dependent manner (Fig. 1A), indicating that mitotic slippage induced by both chemicals requires proteasomal protein degradation. Both chemicals caused complete disappearance of cyclin B1, as determined by immunoblotting, and this degradation was entirely prevented by coincubation with MG-132 (Fig. 1B). Therefore, induction of mitotic slippage by these chemicals is similar to normal exit from mitosis and spontaneous slippage with respect to proteasome dependence and cyclin B1 degradation.

Figure 1.
Figure 1.

Mitotic slippage occurs through proteasome-dependent degradation of mitotic checkpoint proteins. A, T98G cells arrested in mitosis by 30 nmol/L paclitaxel were harvested by shake-off, seeded in 96-well plates, and exposed to 5 μmol/L SU6656 or 5 μmol/L geraldol simultaneously with 0 to 75 μmol/L MG-132. After 4 hours, the attached, slipped cells were fixed, stained, and quantified using an automated fluorescence imager. Error bars represent 95% CIs. B, mitotic T98G cells were harvested by shake-off and exposed to 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. C, cycling T98G cells were exposed to 5 μmol/L SU6656 or 5 μmol/L geraldol for 4 hours without or with 20 μmol/L MG-132, lysed, and immunoblotted for the indicated proteins.

Induction of mitotic slippage involves proteasome-dependent degradation of mitotic checkpoint proteins

Although spontaneous mitotic slippage occurs via the degradation of cyclin B1 by APC/CCdc20, it does not involve inactivation of the mitotic checkpoint (24, 25). Using immunoblotting, the effects of SU6656 and geraldol on the cellular levels of the main mitotic checkpoint mediators Cdc20, BubR1, Mad2, and Mps1 were examined. Levels of these proteins increased in cells arrested in mitosis (Fig. 1B), and subsequent exposure to SU6656 and geraldol caused complete degradation of BubR1 and Cdc20 and a sizeable reduction in Mps1 levels. The degradation of these 3 proteins was prevented by coincubation with MG-132 (Fig. 1B). Exposure to SU6656, but not geraldol, decreased cellular levels of Mad2 and this depletion was not proteasome dependent (Fig. 1B).

In T98G cells, BubR1 is not degraded during completion of mitosis (Supplementary Fig. S2), indicating that its degradation in cells induced to undergo mitotic slippage by SU6656 or geraldol is not simply a normal consequence of exiting mitosis. Depletion of BubR1 or Mps1 has previously been shown to be sufficient to inactivate the mitotic checkpoint (14, 35, 36). Therefore, our results imply that SU6656 and geraldol induce mitotic slippage through degradation of mitotic checkpoint proteins.

SU6656 and geraldol do not induce the degradation of cyclin B1, BubR1, or Cdc20 in interphase cells

To determine whether SU6656 and geraldol induce the proteasome-dependent degradation of cyclin B1, BubR1, and Cdc20 in interphase and in mitotic cells, proliferating T98G cells, which comprise 98% interphase cells, were exposed to 5 μmol/l SU6656 or 5 μmol/L geraldol for 4 hours and analyzed by immunoblotting. No decrease in the levels of cyclin B1, BubR1, or Cdc20 was observed (Fig. 1C); rather, a slight increase in the levels of all 3 proteins was observed. Simultaneous treatment with 20 μmol/L MG-132 had no additional effect (Fig. 1C). These results indicate that an active mitotic checkpoint is required for SU6656- and geraldol-induced selective degradation of mitotic checkpoint components.

Mitotic checkpoint inactivation and slippage induction by SU6656 and geraldol require caspase-3

Caspases have been implicated in mitotic progression (18), and, in particular, BubR1 is reportedly degraded by the effector caspase-3 during exit from mitosis (17), although we did not observe this effect in T98G cells (Supplementary Fig. S2). To determine whether mitotic slippage induced by SU6656 and geraldol involves caspase-3, paclitaxel-arrested T98G cells harvested via shake-off were exposed to 5 μmol/L SU6656 or 5 μmol/L geraldol concurrently with cell-permeable DEVD-CHO, an inhibitor of caspases-3 and -7, for 4 hours. DEVD-CHO prevented induction of mitotic slippage at 50 to 100 μmol/L (Fig. 2A), indicating a requirement for caspase activity in mitotic slippage induction by SU6656 and geraldol. Immunoblotting of paclitaxel-arrested T98G cells exposed to SU6656 or geraldol together with 50 μmol/L DEVD-CHO revealed that degradation of BubR1 and Cdc20 is caspase dependent (Fig. 2B), in addition to being proteasome dependent (Fig. 1B). In contrast, cyclin B1 degradation during induction of mitotic slippage is not dependent on caspase-3 or caspase-7 (Fig. 2B), as cotreatment with DEVD-CHO did not prevent cyclin B1 degradation in response to SU6656 or geraldol.

Figure 2.
Figure 2.

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.

We previously observed that MCF-7 cells do not undergo mitotic slippage, spontaneous (26) or induced by SU6656 or geraldol (not shown). MCF-7 cells do not express caspase-3 because of a deletion within exon 3 of the CASP-3 gene that results in the introduction of a premature stop codon that completely abrogates translation of the CASP-3 mRNA (37). This observation was used to determine whether caspase-3 is required for mitotic slippage induction by SU6656 and geraldol; the inability of these chemicals to stimulate mitotic slippage in MCF-7 cells could be due to lack of caspase-3 activity. Indeed, SU6656 and geraldol did not induce mitotic slippage in MCF-7 cells stably transfected with an empty expression vector (MCF-7pcDNA; Fig. 2C). However, stable transfection of CASP-3 cDNA into MCF-7 cells (MCF-7casp3), which results in expression of procaspase-3 (37), was sufficient to enable cells to undergo robust mitotic slippage in the presence of SU6656 or geraldol (Fig. 2C). Therefore, caspase-3 is required for induction of mitotic slippage by these chemicals.

We next asked whether there were differences in the degradation of cyclin B1, BubR1, and Cdc20 in MCF-7pcDNA and MCF-7casp3 cells during exposure to SU6656 and geraldol. Paclitaxel-arrested MCF-7pcDNA and MCF-7casp3 cells were harvested via shake-off, exposed to 5 μmol/L SU6656 or 5 μmol/L geraldol for 4 hours, and analyzed by immunoblotting (Fig. 2D). Cyclin B1 was completely degraded in both cell lines on exposure to either SU6656 or geraldol. Because MCF-7pcDNA cells do not undergo mitotic slippage under these conditions whereas MCF-7casp3 cells do, this result implies that cyclin B1 degradation is not sufficient to induce mitotic slippage. Cdc20 was degraded in both MCF-7pcDNA and MCF-7casp3 cells. Interestingly, BubR1 was degraded only in MCF-7casp3 cells (Fig. 2D), implying that BubR1 is degraded in a caspase-3–dependent manner during mitotic slippage and that its degradation is required for mitotic slippage induction. The observation by Kim and colleagues that caspase-3 can directly cleave BubR1 during mitotic exit (17) suggests that caspase-3 may degrade BubR1 directly during chemically induced mitotic slippage.

Although mitotic slippage induction by SU6656 and geraldol in MCF-7casp3 cells is proteasome dependent (data not shown), BubR1 degradation in MCF-7casp3 cells was not inhibited by MG-132 (Fig. 2D), further indicating that it is caspase-3 and not the proteasome that is required for the degradation of BubR1 during induction of mitotic slippage. Cdc20 degradation was proteasome dependent, but the degradation of cyclin B1 was not (Fig. 2D). Taken together, these results indicate that SU6656 and geraldol stimulate the degradation of BubR1 by caspase-3, inactivating the mitotic checkpoint and resulting in mitotic slippage.

Approximately 20% of MCF-7pcDNA cells underwent mitotic slippage in the absence of SU6656 and geraldol (Fig. 2C), indicating that spontaneous mitotic slippage does not require caspase-3. To extend this observation, mitotic arrest and slippage in MCF-7pcDNA and MCF-7casp3 cells were examined during exposure to paclitaxel. Cells were exposed to 100 nmol/L paclitaxel for up to 28 hours and the proportion of interphase, mitotic, and slipped cells was determined (Fig. 2E). In both cell lines, mitotic and slipped cells accumulated over time as the proportion of interphase cells declined (Fig. 2E). After 24 hours, the proportion of slipped cells became greater than that of mitotic cells. The kinetics of accumulation of mitotic cells and slipped cells were very similar in MCF-7pcDNA and MCF-7casp3 cells (Fig. 2E), confirming that caspase-3 is not required for mitotic arrest or spontaneous slippage in response to paclitaxel. Therefore, although caspase-3 is not required for spontaneous mitotic slippage in response to antimitotic agents, it is absolutely required for mitotic slippage induction by SU6656 and geraldol.

Mitotic slippage correlates temporally with degradation of BubR1 and Cdc20

To examine the timing of chemically induced exit from mitosis, paclitaxel-arrested mitotic cells were harvested by shake-off, seeded in 96-well plates, and exposed to geraldol for up to 3 hours while cell attachment and TG3 fluorescence were measured (Fig. 3A). TG3 recognizes nucleolin phosphorylated by Cdk1/cyclin B1 and is a marker for mitosis (38). A significant proportion of cells began to attach after 2 hours of exposure to geraldol, and the proportion of attached cells continued to increase over time (Fig. 3A, left). TG3 fluorescence decreased appreciably within 60 minutes of exposure to geraldol and continued to decrease over time (Fig. 3A, right). TG3 fluorescence during exposure to SU6656 could not be measured because of autofluorescence of the compound. The timing of degradation of cyclin B1, BubR1, and Cdc20 during mitotic slippage was also examined. Paclitaxel-arrested cells were exposed to 5 μmol/L SU6656 or 5 μmol/L geraldol for 15 minutes to 3 hours. Cyclin B1 disappeared completely within 30 minutes (Fig. 3B). BubR1 and Cdc20 were partially degraded within 15 minutes of exposure and almost completely degraded after about 2 hours (Fig. 3B), around the time when cells began to attach and lose nucleolin phosphorylation, consistent with a requirement for degradation of BubR1 and Cdc20 for mitotic slippage.

Figure 3.
Figure 3.

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 the Aurora kinases by SU6656 and geraldol

The Aurora kinases play complex roles in metaphase arrest and anaphase initiation, including chromosome congression and interkinetochore tension sensing (39, 40). Inhibition of Aurora A or Aurora B in mitotic cells results in mitotic slippage (33, 34). SU6656 was designed as a Src family kinase inhibitor (41) but has since been reported to inhibit Aurora B in vitro (42, 43). Geraldol has no known biological activity, but fisetin, a closely structurally related flavonoid that induces mitotic slippage less potently than geraldol (Supplementary Fig. S3), has also been reported to inhibit Aurora B (30). Geraldol was assayed for in vitro inhibition of a panel of kinases including Src, Aurora A, and Aurora B (Supplementary Table S1) and showed significant inhibition of Aurora A and Aurora B but not Src. SU6656 and geraldol were then assayed at 0.1 to 10 μmol/L for inhibition of Aurora A and Aurora B kinase activity (Fig. 4A). Both compounds inhibited Aurora A and Aurora B, although Aurora B was inhibited more potently. The intracellular effects of SU6656 and geraldol were compared with those of ZM447439, a well-characterized Aurora B inhibitor that induces mitotic slippage (33). ZM447439 induces 50% to 60% of mitotic MCF-7 cells to undergo mitotic slippage (Fig. 4B), whereas SU6656 and geraldol require the introduction of caspase-3 to induce mitotic slippage in MCF-7 cells (Fig. 2C). Thus, mitotic slippage induction through inhibition of Aurora B does not seem to require caspase-3 activation. Therefore, although SU6656 and geraldol may stimulate mitotic slippage in part by inhibition of Aurora B, these compounds probably have additional activities.

Figure 4.
Figure 4.

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.

Cell survival after mitotic slippage is not affected by caspase-3

Given the role that caspase-3 plays in apoptosis and in mitosis (44, 45), induction of mitotic slippage may affect the survival of cells lacking and expressing caspase-3 differently. MCF-pcDNA and MCF-7casp3 cells arrested at mitosis with paclitaxel were harvested via shake-off and exposed to dimethyl sulfoxide (DMSO), SU6656, or geraldol for 4 hours. The unattached mitotic cells were removed and the attached, slipped cells were cultured in the absence of any drugs for up to 14 days while cell numbers were determined by automated fluorescence microscopy (Fig. 5A). Extensive cell death occurred in both cell lines such that 14 days after mitotic slippage, less than 20% of the initial number of mitotic cells remained (Fig. 5A). Therefore, caspase-3 expression does not seem to play a major role in cell survival after mitotic slippage.

Figure 5.
Figure 5.

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.

The fate of the entire cell population after exposure to paclitaxel and SU6656 or geraldol was also examined. MCF-7pcDNA and MCF-7casp3 cells were exposed to 50 nmol/L paclitaxel for 20 hours and 0.1% DMSO, 5 μmol/L SU6656, or 5 μmol/L geraldol was added for a further 4 hours before both drugs were washed away. The cells were then allowed to grow in fresh cell culture medium for up to 10 days before staining and quantification. Initially, a small increase in cell number was observed, indicating that some cells recovered and were able to divide (Fig. 5B). However, extensive cell death began to occur 5 days following drug treatment and the majority of cells died before day 10 (Fig. 5B). Caspase-3 expression did not alter this response.

We previously reported that, after undergoing mitotic slippage, cells remained metabolically active for up to several days and underwent 1 or more rounds of DNA replication without cell division before undergoing apoptosis (26). MCF-7pcDNA and MCF-7casp3 cells were treated as before and metabolic activity was examined using the MTT assay. The metabolic activity of treated cells in both cell lines increased during the first 3 days to roughly the same extent as untreated cells (Fig. 5C), although untreated cells proliferated rapidly during that time and there was a minimal increase in the number of treated cells (Fig. 5B). Metabolic activity reached a plateau after 3 days and decreased considerably after 7 days, a response not altered by caspase-3 expression. This result indicates that, for 3 days after treatment with paclitaxel without or with SU6656 or geraldol, little or no cell proliferation or death took place, but the cells continued to grow in size. Cell growth was arrested between 3 and 7 days before extensive cell death took place after day 7.

Discussion

This study aimed to better understand pathways leading to mitotic slippage through the use of chemicals. SU6656 and geraldol, 2 compounds found to stimulate mitotic slippage in cells exposed to a microtubule-targeting agent (26), induce the proteasome-dependent degradation of cyclin B1 as occurs during exit from mitosis (Fig. 1B; ref. 46). However, these chemicals inactivate the mitotic checkpoint through the proteasome-dependent degradation of BubR1 (Fig. 1B) that is sufficient to compromise the mitotic checkpoint (35, 36, 47). This effect occurs only in mitotic cells (Fig. 1C), and BubR1 is not degraded during completion of mitosis in T98G cells (Supplementary Fig. S2). These results suggest that, rather than accelerating spontaneous mitotic slippage, SU6656 and geraldol activate an alternate pathway leading to mitotic slippage through BubR1 degradation.

Examination of the timing of mitotic checkpoint inactivation and slippage revealed that mitotic slippage, defined in this experiment by cell attachment and loss of the mitotic phosphoepitope recognized by the TG3 antibody, begins to occur 2 hours following exposure to geraldol whereas cyclin B1 degradation is complete 30 minutes after drug treatment (Fig. 3A and B). BubR1 and Cdc20 degradation occurs more slowly (Fig. 3B) and approximately correlate with mitotic slippage, indicating a possible requirement for checkpoint inactivation prior to mitotic slippage, even in the absence of Cdk1/cyclin B1 activity. In agreement with this observation, cyclin B1 is also completely degraded in MCF-7 cells in response to SU6656 and geraldol, although mitotic slippage cannot be induced (Fig. 2B). It is not known whether the degradation of other APC/C substrates or dephosphorylation of mitotic checkpoint kinase substrates might be involved in mitotic slippage.

Caspase-3 is upregulated during mitosis (18) and has been shown to increase the formation of micronuclei in response to antimitotic agents (48), but a role for caspase-3 in mitosis remains undefined. This study reveals a novel role for caspase-3: mitotic slippage induced by SU6656 and geraldol is caspase-3 dependent. Cotreatment of mitotic cells with SU6656 or geraldol and the caspase-3 and -7 inhibitor DEVD-CHO prevented mitotic slippage and checkpoint inactivation (Fig. 2A and B). Although introduction of caspase-3 into MCF-7 cells does not affect the frequency of spontaneous mitotic slippage in response to paclitaxel (Fig. 2E), mitotic slippage can be induced by SU6656 and geraldol in MCF-7 cells only when exogenous caspase-3 is expressed (Fig. 2C), indicating that caspase-3 is absolutely required for mitotic slippage induction by SU6656 and geraldol.

This requirement for caspase-3 is likely due to its role in inactivation of the mitotic checkpoint; mitotic slippage and BubR1 degradation occur in MCF-7 cells with active caspase-3 but not in MCF-7 cells lacking caspase-3 (Fig. 2C and D). BubR1 degradation is sufficient to inactivate the mitotic checkpoint (14, 35, 36), and furthermore, depletion of BubR1 by mutation (47), gene knockdown, or, recently, in response to chemicals (36) has been implicated in the development of polyploidy. Several factors can influence the degradation of BubR1 during mitosis. Choi and colleagues observed that BubR1 deacetylation at metaphase results in abrogation of its anaphase inhibition effects and in its degradation by APC/CCdc20 (15). Cotreatment of mitotic cells with SU6656 or geraldol and the deacetylase inhibitor trichostatin A did not prevent chemical induction of mitotic slippage (data not shown), indicating that SU6656 and geraldol do not induce premature deacetylation of BubR1. Although a small proteasome-dependent decrease in BubR1 was observed in MCF-7pcDNA cells in response to SU6656 and geraldol (Fig. 2D), this is not sufficient for extensive mitotic slippage to occur and is probably due to some deacetylation and proteasome-dependent degradation of BubR1. Kim and colleagues observed cleavage of BubR1 by caspase-3 during mitosis, which also led to exit from mitosis (17). BubR1 is degraded in a caspase-3- but not proteasome-dependent manner in MCF-7 cells (Fig. 2D), indicating that caspase-3 initiates mitotic slippage through cleavage of BubR1.

However, caspase-3 does not seem to be required for cell death following paclitaxel treatment (Fig. 5). Although caspase-3 is required for DNA fragmentation during apoptosis (37), cells that lack caspase-3 can nevertheless undergo apoptosis. Other cell death pathways may also be involved in the fate of cells following exposure to an antimitotic agent: for instance, necrosis and autophagy have both been implicated in cell death following antimitotic therapy (49, 50).

Spontaneous mitotic slippage has been described to occur through slow ubiquitylation of cyclin B1 by APC/CCdc20 and subsequent proteasome-dependent degradation despite mitotic checkpoint activity (24, 25). We propose a model (Fig. 6) for induced mitotic slippage, wherein decreased Cdk1 activity due to slow cyclin B1 depletion, combined with phosphatase activity, results in loss of the mitosis-specific Cdk1/cyclin B1 inhibitory phosphorylation on caspase-9 (45). Active caspase-9 may then cleave procaspase-3, and activated caspase-3 can cleave BubR1, resulting in inactivation of the mitotic checkpoint and activation of APC/CCdc20. This event would lead to further ubiquitylation and degradation of cyclin B1, combining to force exit from mitosis through mitotic slippage.

Figure 6.
Figure 6.

Model for spontaneous and induced mitotic slippage.

In summary, these results show that the chemical inducers of mitotic slippage SU6656 and geraldol cause proteasome- and caspase-dependent inactivation of the mitotic checkpoint, in contrast to the accepted model for spontaneous mitotic slippage. Caspase-3 is required for mitotic slippage induction and checkpoint inactivation through degradation of BubR1, although not for cell death in response to antimitotic agents. We propose a model for induced mitotic slippage that includes an important role for caspases in modulation of mitotic arrest. In response to the cellular stress presented by a prolonged mitotic arrest, caspases may contribute to the outcome of antimitotic cancer therapy both through mitotic slippage and through apoptosis.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by Canadian Breast Cancer Foundation grant (M. Roberge), Michael Smith Foundation for Health Research Junior Graduate Scholarship (J.L. Riffell), and Deutsche Forschungsgemeinschaft (SFB 728/B1) grant (R.U. Jänicke).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

We thank Connie Kim for her help in characterizing analogues of geraldol and Peter Davies for the TG3 antibody.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Received September 30, 2010.
  • Revision received February 1, 2011.
  • Accepted March 11, 2011.

References

  1. 1.↵
    1. O'Connell CB,
    2. Khodjakov AL
    . Cooperative mechanisms of mitotic spindle formation. J Cell Sci 2007;120:1717–22.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Musacchio A,
    2. Salmon ED
    . The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 2007;8:379–93.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Ganem NJ,
    2. Storchova Z,
    3. Pellman D
    . Tetraploidy, aneuploidy and cancer. Curr Opin Genet Dev 2007;17:157–62.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Schiff PB,
    2. Fant J,
    3. Horwitz SB
    . Promotion of microtubule assembly in vitro by taxol. Nature 1979;277:665–7.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Jordan MA,
    2. Thrower D,
    3. Wilson L
    . Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res 1991;51:2212–22.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Fang G,
    2. Yu H,
    3. Kirschner MW
    . Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol Cell 1998;2:163–71.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. King RW,
    2. Peters JM,
    3. Tugendreich S,
    4. Rolfe M,
    5. Hieter P,
    6. Kirschner MW
    . A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 1995;81:279–88.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Visintin R,
    2. Prinz S,
    3. Amon A
    . CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 1997;278:460–3.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Cohen-Fix O,
    2. Peters JM,
    3. Kirschner MW,
    4. Koshland D
    . Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 1996;10:3081–93.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Sudakin V,
    2. Chan GK,
    3. Yen TJ
    . Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J Cell Biol 2001;154:925–36.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Kallio M,
    2. Weinstein J,
    3. Daum JR,
    4. Burke DJ,
    5. Gorbsky GJ
    . Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J Cell Biol 1998;141:1393–406.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Nilsson J,
    2. Yekezare M,
    3. Minshull J,
    4. Pines J
    . The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat Cell Biol 2008;10:1411–20.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Wu H,
    2. Lan Z,
    3. Li W,
    4. Wu S,
    5. Weinstein J,
    6. Sakamoto KM,
    7. et al.
    p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase. Oncogene 2000;19:4557–62.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Chan GK,
    2. Jablonski SA,
    3. Sudakin V,
    4. Hittle JC,
    5. Yen TJ
    . Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC. J Cell Biol 1999;146:941–54.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Choi E,
    2. Choe H,
    3. Min J,
    4. Choi JY,
    5. Kim J,
    6. Lee H
    . BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis. EMBO J 2009;28:2077–89.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Vanoosthuyse V,
    2. Meadows JC,
    3. Van Der Sar SJ,
    4. Millar JB,
    5. Hardwick KG
    . Bub3p facilitates spindle checkpoint silencing in fission yeast. Mol Biol Cell 2009;20:5096–105.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Kim M,
    2. Murphy K,
    3. Liu F,
    4. Parker SE,
    5. Dowling ML,
    6. Baff W,
    7. et al.
    Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression. Mol Cell Biol 2005;25:9232–48.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Hashimoto T,
    2. Yamauchi L,
    3. Hunter T,
    4. Kikkawa U,
    5. Kamada S
    . Possible involvement of caspase-7 in cell cycle progression at mitosis. Genes Cells 2008;13:609–21.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Peterson D,
    2. Lee J,
    3. Lei XC,
    4. Forrest WF,
    5. Davis DP,
    6. Jackson PK,
    7. et al.
    A chemosensitization screen identifies TP53RK, a kinase that restrains apoptosis after mitotic stress. Cancer Res 2010;70:6325–35.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Andreassen PR,
    2. Martineau SN,
    3. Margolis RL
    . Chemical induction of mitotic checkpoint override in mammalian cells results in aneuploidy following a transient tetraploid state. Mutat Res 1996;372:181–94.
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Jin L,
    2. Williamson A,
    3. Banerjee S,
    4. Philipp I,
    5. Rape M
    . Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 2008;133:653–65.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Blagosklonny MV
    . Mitotic arrest and cell fate: why and how mitotic inhibition of transcription drives mutually exclusive events. Cell Cycle 2007;6:70–4.
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Elhajouji A,
    2. Cunha M,
    3. Kirsch-Volders M
    . Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay. Mutagenesis 1998;13:193–8.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Brito DA,
    2. Rieder CL
    . Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr Biol 2006;16:1194–200.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Lee J,
    2. Kim JA,
    3. Margolis RL,
    4. Fotedar R
    . Substrate degradation by the anaphase promoting complex occurs during mitotic slippage. Cell Cycle 2010;9:1792–801.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Riffell JL,
    2. Zimmerman C,
    3. Khong A,
    4. McHardy LM,
    5. Roberge M
    . Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation. Cell Cycle 2009;8:3025–38.
    OpenUrlPubMed
  27. 27.↵
    1. DeMoe JH,
    2. Santaguida S,
    3. Daum JR,
    4. Musacchio A,
    5. Gorbsky GJ
    . A high throughput, whole cell screen for small molecule inhibitors of the mitotic spindle checkpoint identifies OM137, a novel Aurora kinase inhibitor. Cancer Res 2009;69:1509–16.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Chan YW,
    2. Ma HT,
    3. Wong W,
    4. Ho CC,
    5. On KF,
    6. Poon RY
    . CDK1 inhibitors antagonize the immediate apoptosis triggered by spindle disruption but promote apoptosis following the subsequent rereplication and abnormal mitosis. Cell Cycle 2008;7:1449–61.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Stolz A,
    2. Vogel C,
    3. Schneider V,
    4. Ertych N,
    5. Kienitz A,
    6. Yu H,
    7. et al.
    Pharmacologic abrogation of the mitotic spindle checkpoint by an indolocarbazole discovered by cellular screening efficiently kills cancer cells. Cancer Res 2009;69:3874–83.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Salmela AL,
    2. Pouwels J,
    3. Varis A,
    4. Kukkonen AM,
    5. Toivonen P,
    6. Halonen PK,
    7. et al.
    Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint. Carcinogenesis 2009;30:1032–40.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Stevens FE,
    2. Beamish H,
    3. Warrener R,
    4. Gabrielli B
    . Histone deacetylase inhibitors induce mitotic slippage. Oncogene 2008;27:1345–54.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Noh EJ,
    2. Lim DS,
    3. Jeong G,
    4. Lee JS
    . An HDAC inhibitor, trichostatin A, induces a delay at G2/M transition, slippage of spindle checkpoint, and cell death in a transcription-dependent manner. Biochem Biophys Res Commun 2009;378:326–31.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Ditchfield C,
    2. Johnson VL,
    3. Tighe A,
    4. Ellston R,
    5. Haworth C,
    6. Johnson T,
    7. et al.
    Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 2003;161:267–80.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Wysong DR,
    2. Chakravarty A,
    3. Hoar K,
    4. Ecsedy JA
    . The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents. Cell Cycle 2009;8:876–88.
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Dai W,
    2. Wang Q,
    3. Liu T,
    4. Swamy M,
    5. Fang Y,
    6. Xie S,
    7. et al.
    Slippage of mitotic arrest and enhanced tumor development in mice with BubR1 haploinsufficiency. Cancer Res 2004;64:440–5.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Tovar C,
    2. Higgins B,
    3. Deo D,
    4. Kolinsky K,
    5. Liu JJ,
    6. Heimbrook DC,
    7. et al.
    Small-molecule inducer of cancer cell polyploidy promotes apoptosis or senescence: implications for therapy. Cell Cycle 2010;9:3364–75.
    OpenUrlCrossRefPubMed
  37. 37.↵
    1. Janicke RU,
    2. Sprengart ML,
    3. Wati MR,
    4. Porter AG
    . Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 1998;273:9357–60.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    1. Anderson HJ,
    2. de Jong G,
    3. Vincent I,
    4. Roberge M
    . Flow cytometry of mitotic cells. Exp Cell Res 1998;238:498–502.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Kim Y,
    2. Holland AJ,
    3. Lan W,
    4. Cleveland DW
    . Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E. Cell 2010;142:444–55.
    OpenUrlCrossRefPubMed
  40. 40.↵
    1. Liu D,
    2. Vader G,
    3. Vromans MJ,
    4. Lampson MA,
    5. Lens SM
    . Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates. Science 2009;323:1350–3.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Blake RA,
    2. Broome MA,
    3. Liu X,
    4. Wu J,
    5. Gishizky M,
    6. Sun L,
    7. et al.
    SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol Cell Biol 2000;20:9018–27.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Bain J,
    2. McLauchlan H,
    3. Elliott M,
    4. Cohen P
    . The specificities of protein kinase inhibitors: an update. Biochem J 2003;371:199–204.
    OpenUrlCrossRefPubMed
  43. 43.↵
    1. Bain J,
    2. Plater L,
    3. Elliott M,
    4. Shpiro N,
    5. Hastie CJ,
    6. McLauchlan H,
    7. et al.
    The selectivity of protein kinase inhibitors: a further update. Biochem J 2007;408:297–315.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Tao W,
    2. South VJ,
    3. Zhang Y,
    4. Davide JP,
    5. Farrell L,
    6. Kohl NE,
    7. et al.
    Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell 2005;8:49–59.
    OpenUrlCrossRefPubMed
  45. 45.↵
    1. Allan LA,
    2. Clarke PR
    . Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis. Mol Cell 2007;26:301–10.
    OpenUrlCrossRefPubMed
  46. 46.↵
    1. Gascoigne KE,
    2. Taylor SS
    . Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs. Cancer Cell 2008;14:111–22.
    OpenUrlCrossRefPubMed
  47. 47.↵
    1. Shin HJ,
    2. Baek KH,
    3. Jeon AH,
    4. Park MT,
    5. Lee SJ,
    6. Kang CM,
    7. et al.
    Dual roles of human BubR1, a mitotic checkpoint kinase, in the monitoring of chromosomal instability. Cancer Cell 2003;4:483–97.
    OpenUrlCrossRefPubMed
  48. 48.↵
    1. Decordier I,
    2. Cundari E,
    3. Kirsch-Volders M
    . Survival of aneuploid, micronucleated and/or polyploid cells: crosstalk between ploidy control and apoptosis. Mutat Res 2008;651:30–9.
    OpenUrlPubMed
  49. 49.↵
    1. Yeung TK,
    2. Germond C,
    3. Chen X,
    4. Wang Z
    . The mode of action of taxol: apoptosis at low concentration and necrosis at high concentration. Biochem Biophys Res Commun 1999;263:398–404.
    OpenUrlCrossRefPubMed
  50. 50.↵
    1. Gorka M,
    2. Daniewski WM,
    3. Gajkowska B,
    4. Lusakowska E,
    5. Godlewski MM,
    6. Motyl T
    . Autophagy is the dominant type of programmed cell death in breast cancer MCF-7 cells exposed to AGS 115 and EFDAC, new sesquiterpene analogs of paclitaxel. Anticancer Drugs 2005;16:777–88.
    OpenUrlCrossRefPubMed
Back to top
Molecular Cancer Therapeutics: 10 (5)
May 2011
Volume 10, Issue 5

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Email Article
Citation Tools
Caspase-3–Dependent Mitotic Checkpoint Inactivation by the Small-Molecule Inducers of Mitotic Slippage SU6656 and Geraldol
Jenna L. Riffell, Reiner U. Jänicke and Michel Roberge
Mol Cancer Ther May 1 2011 (10) (5) 839-849; DOI: 10.1158/1535-7163.MCT-10-0909
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement