Caspase-3 Dependent Mitotic Checkpoint Inactivation by the … · B1 without mitotic checkpoint...

Preclinical Development

Caspase-3–Dependent Mitotic Checkpoint Inactivationby the Small-Molecule Inducers of Mitotic Slippage SU6656and Geraldol

Jenna L. Riffell1, Reiner U. J€anicke2, and Michel Roberge1

AbstractMicrotubule-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 termedmitotic 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.

Introduction

During cell division, genetic integrity is maintained byensuring that all chromosomes are attached to microtu-bules emanating from both poles of the mitotic spindlebefore segregation of sister chromatids begins (1). Thisprocess is monitored by the mitotic checkpoint, whichprevents initiation of anaphase until every kinetochore isattached and tension between kinetochores of pairedsister chromatids is sufficient, ensuring biorientation(2). To prevent aneuploidy and ensuing genetic defectsleading to cell death or tumorigenesis (3), the mitoticcheckpoint must be sufficiently sensitive to delay chro-

mosome separation when even 1 kinetochore is unat-tached. Exposure to drugs that interfere withmicrotubuledynamics, such as the taxanes (4) and the Vinca alkaloids(5), similarly activates the mitotic checkpoint and arrestscells at mitosis, effectively preventing further prolifera-tion.

The mitotic checkpoint acts through inhibition of theanaphase-promoting complex/cyclosome (APC/C; ref.6), the E3 ubiquitin ligase (7) that, when activated bycofactors 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 sisterchromatids, and exit from mitosis. The key componentsof the mitotic checkpoint are budding uninhibited bybenzimidazole related 1 (BubR1), budding uninhibitedby benzimidazole 3 (Bub3), and Cdc20, which form amitotic checkpoint complex (MCC; ref. 10). This complexis the main inhibitor of APC/C activity, along withmitotic arrest dependent 2 (Mad2), which initially bindsCdc20 (11) and catalyzes its binding to BubR1 and sub-sequent formation of theMCC (12). Cdc20 is an activatingcofactor of APC/C during mitosis (8); an active mitoticcheckpoint inhibits APC/C through APC/C-dependentpolyubiquitylation of Cdc20 and subsequent degradation

Authors' Affiliations: 1Department of Biochemistry and Molecular Biol-ogy, University of British Columbia, Vancouver, British Columbia, Canada;and 2Laboratory for Molecular Radiooncology, Clinic and Policlinic forRadiation Therapy and Radiooncology, Heinrich Heine Universit€atD€usseldorf, D€usseldorf, Germany

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

Corresponding Author: Michel Roberge, Department of Biochemistryand Molecular Biology, University of British Columbia, 2350 HealthSciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. Phone:604-822-2304; Fax: 604-822-5227. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0909

�2011 American Association for Cancer Research.

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by the proteasome (12). BubR1 binds to and inhibits bothCdc20 (13) and APC/C itself (14), acting as a pseudosub-strate inhibitor that, depending on acetylation status, canbe actively degraded by APC/CCdc20 (15). The role ofBub3 in the MCC is unclear, although in fission yeast it isinvolved in MCC localization (16). Other components ofthe mitotic checkpoint include the kinases Bub1, mono-polar spindle 1 (Mps1), and Aurora B (2).

Caspases have well-characterized apoptotic functions,but caspase-3 and caspase-7 have both recently beenobserved to play a role, yet to be defined, in mitoticprogression (17, 18). Their activities are tightly regulatedand must be restrained during mitotic stress to preventextensive cell death, most notably through survivin,which inhibits caspase activation during mitotic arrestand functions as part of themitotic checkpoint machinery(19).

Mitotic checkpoint activation during an unperturbedmitosis provides sufficient time for microtubule attach-ment, preventing aneuploidy (20) and increasing cellsurvival (21). However, long-term activation of themitotic checkpoint during exposure to antimitoticagents can be problematic because chromosome con-densation hinders RNA transcription (22). With time, animbalance between new protein production and proteindegradation may cause the levels of proteins essential tomaintain mitotic arrest to fall, triggering mitotic slip-page. Also termed mitotic checkpoint adaptation, mito-tic slippage occurs when cells exit mitosis withoutchromosome segregation or cell division (20, 23) andresults from slow APC/CCdc20- and proteasome-depen-dent degradation of cyclin B1 in the presence of anactive mitotic checkpoint (24, 25). Cells that have under-gone mitotic slippage enter a G1-like state with decon-densed chromosomes that form multiple micronuclei(23), allowing resumption of transcription and othercellular processes.

Our group and others have identified chemicals thatstimulate mitotic slippage and observed that slippedcells typically undergo at least 1 round of DNA replica-tion without subsequent cell division but that, even-tually, all cells that undergo mitotic slippage die (26–30). Known chemical inducers of mitotic slippageinclude 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),G€o6976 (29), OM137 (27), and fisetin (30); Supplemen-tary Fig. S1]. We previously identified SU6656 andgeraldol as chemical inducers of mitotic slippage thatincreased cell killing after induction of mitotic arrest bymicrotubule-targeting agents. This study investigateshow chemicals can modulate mitotic slippage, revealsa mechanism for mitotic slippage that is different fromthat described for spontaneous mitotic slippage, andshows how interplay between pathways associated withmitosis and apoptosis can contribute to the outcome ofantimitotic cancer treatments.

Materials and Methods

Cell culture and chemicalsT98G cells, obtained from the American Type Culture

Collection (ATCC; characterized by short tandem repeatanalysis) and used within 6 months of resuscitation, weremaintained in Dulbecco’s Modified Eagle’s Medium(Invitrogen) supplemented with 10% FBS (Gibco).MCF-7 cell lines, obtained from the ATCC and stablytransfected with empty vector (pcDNA) or caspase-3(casp3), were maintained in RPMI (Invitrogen) supple-mented 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, andcell-permeable DEVD-CHO from Enzo Life Sciences.

Slippage induction assayT98G cells at 75% confluency were treated with 30

nmol/L paclitaxel, or MCF-7 cells were treated with 50nmol/L paclitaxel, for 20 hours at 37�C, and mitotic cellswere harvested by shake-off, counted using a hemacyt-ometer, seeded in a 96-well plate (PerkinElmer View-plate) at 5,000 cells perwell, and treatedwith chemicals asindicated for 4 hours at 37�C. Unattached mitotic cellswere then aspirated and discarded while attached,slipped cells were fixed in 3% paraformaldehyde(EMD) in PBS for 15 minutes at room temperature, andstained with Hoechst 33342 (Invitrogen) in PBS for 10minutes at room temperature. Five fields per well werecounted by a Cellomics ArrayScan VTI automated fluor-escence imager (ThermoFisher) by using a 10� objective.Individual nuclei of slipped cells were detected andcounted using the Cellomics Target Activation AnalysisProgram. In all figures, mitotic slippage was expressed asa percentage of the cells seeded in each well (26).

ImmunoblottingCells 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/LEDTA (Sigma), 1 mmol/L EGTA (Sigma), 1% TritonX-100 (LabChem Inc.), 2.5 mmol/L sodium pyropho-sphate (Fisher), 1 mmol/L b-glycerol phosphate (Sigma),1 mmol/L sodium orthovanadate (Sigma), and 1� pro-tease inhibitor cocktail (Roche). Lysates were spun at15,000 � g for 15 minutes, and supernatants wereremoved and assayed for protein concentration by usingthe Bradford assay (Sigma). Sample concentration wasequalized 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 toverify equal protein loading or transferred to a polyvi-nylidene difluoride membrane (Millipore Immobilon-P).The membrane was blocked in 5% milk (Nestle) in TBScontaining 0.1% Tween-20 (TBS-T; MP Biomedicals) for30 minutes and incubated overnight at 4�C with primaryantibody in 5% milk in TBS. Membranes were then

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Mol Cancer Ther; 10(5) May 2011 Molecular Cancer Therapeutics840




washed 2 � 10 minutes in TBS-T, incubated at roomtemperature with secondary antibody in 5% milk for 1hour, washed 3 � 10 minutes in TBS-T, and imaged bychemiluminescence (Millipore Immobilon Western).Antibodies used were mouse a-cyclin B1 (1:100; BDPharmingen), mouse a-BubR1 (1:1,000; BD Transduc-tion), mouse a-Mad2 (1:500; Santa Cruz), mousea-p55CDC/Cdc20 (1:1,000; Santa Cruz), mouse a-Mps1(1:500; Abcam), goat a-mouse horseradish peroxidase(1:10,000), and goat a-rabbit peroxidase conjugate(1:10,000).

In vitro kinase assaysSU6656 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 0.4mg/mL synthetic Src substrate (KVEKIGEGTYGVVYK)and 50 mmol/L 33P-ATP in kinase assay buffer containing25 mmol/L MOPS, pH 7.2, 12.5 mmol/L b-glycerolphosphate, 25 mmol/L MgCl2, 5 mmol/L EGTA, 2mmol/L EDTA, and 0.25 mmol/L DTT (Aurora kinases)or 25 mmol/L MOPS, pH 7.2, 12.5 mmol/L b-glycerolphosphate, 20 mmol/L MgCl2, 25 mmol/L MnCl2, 5mmol/L EGTA, 2 mmol/L EDTA, and 0.25 mmol/LDTT (Src). Ten microliters of this reaction mixture wasthen spotted on a phosphocelluloseMultiscreen plate andwashed 3 � 15 minutes in 1% phosphoric acid. Scintilla-tion fluid was added and the radioactivity on the platewas counted using a Trilux scintillation counter against acontrol incubated without substrate.

Results

Induction of mitotic slippage by SU6656 andgeraldol requires proteasomal activityA previous screening effort by our group identified

SU6656 and geraldol as chemicals capable of inducingmitotic slippage (26). When cells spontaneously slip outof mitotic arrest, as well as during normal exit frommitosis, proteasomal activity is required for the degrada-tion of cyclin B1 (24, 25). First, to determine whetherescape frommitotic arrest induced by SU6656 or geraldolsimilarly requires proteasome activity, mitotic slippagewas examined in the presence of the proteasome inhibitorMG-132. T98G cells at 75% confluency were arrested inmitosis by exposure to 30 nmol/L paclitaxel for 20 hours,harvested via shake-off, and seeded in 96-well plates. Thecells were exposed to the chemical inducers of mitoticslippage SU6656 (5 mmol/L) or geraldol (5 mmol/L) in thepresence of various concentrations of MG-132 for 4 hoursand in the continued presence of paclitaxel. Residualunattached mitotic cells were removed and the attached,slipped cells were fixed, stained with Hoechst 33342, andquantified using automated fluorescence microscopy(26). In the absence of MG-132, SU6656 and geraldolinduced 60% to 90% of mitotic cells to undergo mitoticslippage. MG-132 reduced the proportion of slipped cellsin a concentration-dependent manner (Fig. 1A), indicat-

ing that mitotic slippage induced by both chemicalsrequires proteasomal protein degradation. Both chemi-cals caused complete disappearance of cyclin B1, asdetermined by immunoblotting, and this degradationwas entirely prevented by coincubation with MG-132(Fig. 1B). Therefore, induction ofmitotic slippage by thesechemicals is similar to normal exit from mitosis andspontaneous slippage with respect to proteasome depen-dence and cyclin B1 degradation.

Induction of mitotic slippage involves proteasome-dependent degradation of mitotic checkpointproteins

Although spontaneous mitotic slippage occurs via thedegradation of cyclin B1 by APC/CCdc20, it does notinvolve inactivation of the mitotic checkpoint (24, 25).Using immunoblotting, the effects of SU6656 and geral-dol on the cellular levels of the main mitotic checkpointmediators Cdc20, BubR1, Mad2, and Mps1 were exam-ined. Levels of these proteins increased in cells arrested inmitosis (Fig. 1B), and subsequent exposure to SU6656 andgeraldol caused complete degradation of BubR1 andCdc20 and a sizeable reduction in Mps1 levels. Thedegradation of these 3 proteins was prevented by coin-cubation with MG-132 (Fig. 1B). Exposure to SU6656, butnot geraldol, decreased cellular levels of Mad2 and thisdepletion was not proteasome dependent (Fig. 1B).

In T98G cells, BubR1 is not degraded during comple-tion of mitosis (Supplementary Fig. S2), indicating that itsdegradation in cells induced to undergo mitotic slippageby SU6656 or geraldol is not simply a normal conse-quence of exiting mitosis. Depletion of BubR1 or Mps1has previously been shown to be sufficient to inactivatethe mitotic checkpoint (14, 35, 36). Therefore, our resultsimply that SU6656 and geraldol induce mitotic slippagethrough degradation of mitotic checkpoint proteins.

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

To determine whether SU6656 and geraldol induce theproteasome-dependent degradation of cyclin B1, BubR1,and Cdc20 in interphase and inmitotic cells, proliferatingT98G cells, which comprise 98% interphase cells, wereexposed to 5 mmol/l SU6656 or 5 mmol/L geraldol for 4hours and analyzed by immunoblotting. No decrease inthe levels of cyclin B1, BubR1, or Cdc20 was observed(Fig. 1C); rather, a slight increase in the levels of all 3proteins was observed. Simultaneous treatment with 20mmol/L MG-132 had no additional effect (Fig. 1C). Theseresults indicate that an active mitotic checkpoint isrequired for SU6656- and geraldol-induced selectivedegradation of mitotic checkpoint components.

Mitotic checkpoint inactivation and slippageinduction by SU6656 and geraldol require caspase-3

Caspases have been implicated in mitotic progression(18), and, in particular, BubR1 is reportedly degraded bythe effector caspase-3 during exit from mitosis (17),

Caspase-3–Dependent Chemical Induction of Mitotic Slippage

www.aacrjournals.org Mol Cancer Ther; 10(5) May 2011 841




although we did not observe this effect in T98G cells(Supplementary Fig. S2). To determine whether mitoticslippage induced by SU6656 and geraldol involves cas-pase-3, paclitaxel-arrestedT98Gcells harvested via shake-off were exposed to 5 mmol/L SU6656 or 5 mmol/Lgeraldol 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 to100mmol/L (Fig. 2A), indicating a requirement for caspaseactivity in mitotic slippage induction by SU6656 andgeraldol. Immunoblotting of paclitaxel-arrested T98Gcells exposed to SU6656 or geraldol together with 50mmol/L DEVD-CHO revealed that degradation of BubR1and Cdc20 is caspase dependent (Fig. 2B), in addition tobeing proteasome dependent (Fig. 1B). In contrast, cyclinB1 degradation during induction ofmitotic slippage is notdependent on caspase-3 or caspase-7 (Fig. 2B), as cotreat-ment with DEVD-CHO did not prevent cyclin B1 degra-dation in response to SU6656 or geraldol.

We previously observed that MCF-7 cells do notundergo mitotic slippage, spontaneous (26) or inducedby SU6656 or geraldol (not shown). MCF-7 cells do notexpress caspase-3 because of a deletion within exon 3 ofthe CASP-3 gene that results in the introduction of apremature stop codon that completely abrogates transla-tion of the CASP-3 mRNA (37). This observation wasused to determine whether caspase-3 is required formitotic slippage induction by SU6656 and geraldol; theinability 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 mitoticslippage in MCF-7 cells stably transfected with an emptyexpression vector (MCF-7pcDNA; Fig. 2C). However,stable transfection of CASP-3 cDNA into MCF-7 cells(MCF-7casp3), which results in expression of procas-pase-3 (37), was sufficient to enable cells to undergorobust mitotic slippage in the presence of SU6656 orgeraldol (Fig. 2C). Therefore, caspase-3 is required forinduction of mitotic slippage by these chemicals.

We next asked whether there were differences in thedegradation of cyclin B1, BubR1, and Cdc20 in MCF-7pcDNA and MCF-7casp3 cells during exposure toSU6656 and geraldol. Paclitaxel-arrested MCF-7pcDNAand MCF-7casp3 cells were harvested via shake-off,exposed to 5 mmol/L SU6656 or 5 mmol/L geraldol for4 hours, and analyzed by immunoblotting (Fig. 2D).Cyclin B1 was completely degraded in both cell lineson exposure to either SU6656 or geraldol. Because MCF-7pcDNA cells do not undergo mitotic slippage underthese conditions whereas MCF-7casp3 cells do, this resultimplies that cyclin B1 degradation is not sufficient toinduce mitotic slippage. Cdc20 was degraded in bothMCF-7pcDNA and MCF-7casp3 cells. Interestingly,BubR1 was degraded only in MCF-7casp3 cells(Fig. 2D), implying that BubR1 is degraded in a cas-pase-3–dependent manner during mitotic slippage andthat its degradation is required for mitotic slippageinduction. The observation by Kim and colleagues that

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Figure 1. Mitotic slippage occursthrough proteasome-dependentdegradation of mitotic checkpointproteins. A, T98G cells arrested inmitosis by 30 nmol/L paclitaxelwere harvested by shake-off,seeded in 96-well plates, andexposed to 5 mmol/L SU6656 or 5mmol/L geraldol simultaneouslywith 0 to 75 mmol/L MG-132. After4 hours, the attached, slippedcells were fixed, stained, andquantified using an automatedfluorescence imager. Error barsrepresent 95% CIs. B, mitoticT98G cells were harvestedby shake-off and exposed to5 mmol/L SU6656 or 5 mmol/Lgeraldol without or with 20 mmol/LMG-132 for 4 hours. Lysates wereimmunoblotted for the indicatedproteins. C, cycling T98G cellswere exposed to 5 mmol/L SU6656or 5 mmol/L geraldol for 4 hourswithout or with 20 mmol/L MG-132, lysed, and immunoblotted forthe indicated proteins.

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caspase-3 can directly cleave BubR1 during mitotic exit(17) suggests that caspase-3 may degrade BubR1 directlyduring 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-7casp3cells was not inhibited by MG-132 (Fig. 2D), furtherindicating that it is caspase-3 and not the proteasomethat is required for the degradation of BubR1 duringinduction of mitotic slippage. Cdc20 degradation was

proteasome dependent, but the degradation of cyclinB1 was not (Fig. 2D). Taken together, these results indi-cate that SU6656 and geraldol stimulate the degradationof BubR1 by caspase-3, inactivating the mitotic check-point and resulting in mitotic slippage.

Approximately 20% of MCF-7pcDNA cells underwentmitotic slippage in the absence of SU6656 and geraldol(Fig. 2C), indicating that spontaneous mitotic slippagedoes not require caspase-3. To extend this observation,mitotic arrest and slippage in MCF-7pcDNA and

Figure 2. Mitotic checkpointinactivation but not cyclin B1degradation occurs throughcaspase-3–dependent cleavageof BubR1. A, T98G cells werearrested in mitosis by 30 nmol/Lpaclitaxel, harvested by shake-off,and seeded in 96-well plates. Afterexposure to 5 mmol/L SU6656 or 5mmol/L geraldol simultaneouslywith 0 to 100 mmol/L Ac-DEVD-CHO for 4 hours, the slipped cellswere stained with Hoechst 33342and quantified by automatedfluorescence microscopy. Errorbars represent 95%CIs. B, mitoticT98G cells were harvested byshake-off and incubated with 5mmol/L SU6656 or 5 mmol/Lgeraldol without or with 50 mmol/LDEVD-CHO for 4 hours. Lysateswere immunoblotted for theindicated proteins. C, MCF-7 cellsstably transfected with emptyvector (MCF-7pcDNA) orcaspase-3 (MCF-7casp3) werearrested in mitosis by 50 nmol/Lpaclitaxel, harvested by shake-off,and seeded in 96-well plates. Thecells were exposed to 0 to15 mmol/L SU6656 or geraldol for4 hours, stained with Hoechst33342, and quantified using anautomated fluorescence imager.Error bars represent 95% CIs. D,mitotic MCF-7pcDNA and MCF-7casp3 cells were harvested byshake-off and incubated with 5mmol/L SU6656 or 5 mmol/Lgeraldol without or with 20 mmol/LMG-132 for 4 hours. Lysates wereimmunoblotted for the indicatedproteins. E, MCF-7pcDNA orMCF-7casp3 cells were exposedto 100 nmol/L paclitaxel for up to28 hours, and nuclei were fixedand stained with Hoechst 33342.The total number of cells wasquantified using automatedfluorescence microscopy, and theimages were visually inspected todetermine the proportion ofslipped and mitotic cells at eachtime.

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MCF-7casp3 cells were examined during exposure topaclitaxel. Cells were exposed to 100 nmol/L paclitaxelfor up to 28 hours and the proportion of interphase,mitotic, and slipped cells was determined (Fig. 2E). Inboth cell lines, mitotic and slipped cells accumulated overtime as the proportion of interphase cells declined(Fig. 2E). After 24 hours, the proportion of slipped cellsbecame greater than that of mitotic cells. The kinetics ofaccumulation of mitotic cells and slipped cells were verysimilar in MCF-7pcDNA and MCF-7casp3 cells (Fig. 2E),confirming that caspase-3 is not required for mitoticarrest or spontaneous slippage in response to paclitaxel.Therefore, although caspase-3 is not required for sponta-neous mitotic slippage in response to antimitotic agents,it is absolutely required for mitotic slippage induction bySU6656 and geraldol.

Mitotic slippage correlates temporally withdegradation of BubR1 and Cdc20

To examine the timing of chemically induced exit frommitosis, paclitaxel-arrested mitotic cells were harvestedby shake-off, seeded in 96-well plates, and exposed to

geraldol for up to 3 hours while cell attachment and TG3fluorescence were measured (Fig. 3A). TG3 recognizesnucleolin phosphorylated by Cdk1/cyclin B1 and is amarker for mitosis (38). A significant proportion of cellsbegan to attach after 2 hours of exposure to geraldol, andthe proportion of attached cells continued to increaseover time (Fig. 3A, left). TG3 fluorescence decreasedappreciably within 60 minutes of exposure to geraldoland continued to decrease over time (Fig. 3A, right). TG3fluorescence during exposure to SU6656 could not bemeasured because of autofluorescence of the compound.The timing of degradation of cyclin B1, BubR1, and Cdc20during mitotic slippage was also examined. Paclitaxel-arrested cells were exposed to 5 mmol/L SU6656 or 5mmol/L geraldol for 15 minutes to 3 hours. Cyclin B1disappeared completely within 30 minutes (Fig. 3B).BubR1 and Cdc20 were partially degraded within 15minutes of exposure and almost completely degradedafter about 2 hours (Fig. 3B), around the time when cellsbegan to attach and lose nucleolin phosphorylation, con-sistent with a requirement for degradation of BubR1 andCdc20 for mitotic slippage.

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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 mmol/L geraldol for 15 minutes to 3 hours. Slipped cells were quantified after staining withHoechst (left) or with mouse TG3 antibody against mitotically phosphorylated nucleolin (right). The proportion of slipped cells was lower than usuallyobserved 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 incubatedwith 5 mmol/L SU6656 or 5 mmol/L geraldol for 15 minutes to 3 hours and lysates were immunoblotted for the indicated proteins.

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Inhibition of the Aurora kinases by SU6656 andgeraldolThe Aurora kinases play complex roles in metaphase

arrest and anaphase initiation, including chromosomecongression and interkinetochore tension sensing (39,40). Inhibition of Aurora A or Aurora B in mitotic cellsresults in mitotic slippage (33, 34). SU6656 was designedas a Src family kinase inhibitor (41) but has since beenreported to inhibit Aurora B in vitro (42, 43). Geraldol hasno known biological activity, but fisetin, a closely struc-turally related flavonoid that induces mitotic slippageless potently than geraldol (Supplementary Fig. S3), hasalso been reported to inhibit Aurora B (30). Geraldol wasassayed for in vitro inhibition of a panel of kinasesincluding Src, Aurora A, and Aurora B (SupplementaryTable S1) and showed significant inhibition of Aurora Aand Aurora B but not Src. SU6656 and geraldol were thenassayed at 0.1 to 10 mmol/L for inhibition of Aurora Aand Aurora B kinase activity (Fig. 4A). Both compoundsinhibited Aurora A andAurora B, althoughAurora Bwas

inhibited more potently. The intracellular effects ofSU6656 and geraldol were compared with those ofZM447439, a well-characterized Aurora B inhibitor thatinduces mitotic slippage (33). ZM447439 induces 50% to60% of mitotic MCF-7 cells to undergo mitotic slippage(Fig. 4B), whereas SU6656 and geraldol require the intro-duction of caspase-3 to induce mitotic slippage in MCF-7cells (Fig. 2C). Thus, mitotic slippage induction throughinhibition of Aurora B does not seem to require caspase-3activation. Therefore, although SU6656 and geraldol maystimulate mitotic slippage in part by inhibition of AuroraB, these compounds probably have additional activities.

Cell survival after mitotic slippage is not affected bycaspase-3

Given the role that caspase-3 plays in apoptosis and inmitosis (44, 45), induction of mitotic slippage may affectthe survival of cells lacking and expressing caspase-3differently. MCF-pcDNA and MCF-7casp3 cellsarrested at mitosis with paclitaxel were harvested via

Figure 4. Inhibition of Aurora Aand Aurora B by SU6656 andgeraldol. A, SU6656 and geraldolwere assayed for in vitro inhibitionof Aurora A and Aurora B asdescribed in Materials andMethods. B, MCF-7 cells werearrested in mitosis by 50 nmol/Lpaclitaxel for 20 hours, harvestedby shake-off, seeded in 96-wellplates, and incubated with 0.1 to20 mmol/L ZM447439 for 4 hours.Cells were fixed, stained withHoechst 33342, and imaged byautomated fluorescencemicroscopy. Error bars represent95% CIs.

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shake-off and exposed to dimethyl sulfoxide (DMSO),SU6656, or geraldol for 4 hours. The unattached mitoticcells were removed and the attached, slipped cells werecultured in the absence of any drugs for up to 14 dayswhile cell numbers were determined by automatedfluorescence microscopy (Fig. 5A). Extensive cell deathoccurred in both cell lines such that 14 days after mitoticslippage, less than 20% of the initial number of mitoticcells remained (Fig. 5A). Therefore, caspase-3 expres-sion does not seem to play a major role in cell survivalafter mitotic slippage.

The fate of the entire cell population after exposure topaclitaxel and SU6656 or geraldol was also examined.MCF-7pcDNA and MCF-7casp3 cells were exposed to50 nmol/L paclitaxel for 20 hours and 0.1% DMSO,5 mmol/L SU6656, or 5 mmol/L geraldol was added fora further 4 hours before both drugs were washed away.The cells were then allowed to grow in fresh cell culturemedium for up to 10 days before staining and quantifica-tion. Initially, a small increase in cell number wasobserved, indicating that some cells recovered and wereable to divide (Fig. 5B). However, extensive cell deathbegan to occur 5 days following drug treatment and themajority of cells died before day 10 (Fig. 5B). Caspase-3expression did not alter this response.

We previously reported that, after undergoing mitoticslippage, cells remained metabolically active for up toseveral days and underwent 1 or more rounds of DNAreplication without cell division before undergoing apop-tosis (26). MCF-7pcDNA and MCF-7casp3 cells weretreated as before and metabolic activity was examinedusing the MTT assay. The metabolic activity of treatedcells in both cell lines increased during the first 3 days toroughly the same extent as untreated cells (Fig. 5C),although untreated cells proliferated rapidly during thattime and there was a minimal increase in the number oftreated cells (Fig. 5B). Metabolic activity reached a pla-teau after 3 days and decreased considerably after 7 days,a response not altered by caspase-3 expression. Thisresult indicates that, for 3 days after treatment withpaclitaxel without or with SU6656 or geraldol, little orno cell proliferation or death took place, but the cellscontinued to grow in size. Cell growth was arrestedbetween 3 and 7 days before extensive cell death tookplace after day 7.

Discussion

This study aimed to better understand pathways lead-ing to mitotic slippage through the use of chemicals.

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DMSOSU6656Geraldol

DMSOPaclitaxel + DMSOPaclitaxel + SU6656Paclitaxel + geraldol

DMSOPaclitaxel + DMSOPaclitaxel + SU6656Paclitaxel + geraldol

Figure 5. Dependence of theoutcome of spontaneous andinduced mitotic slippage oncaspase-3. A, slipped cells, MCF-7pcDNA and MCF-7casp3 cellswere exposed to 50 nmol/Lpaclitaxel for 20 hours, harvestedby shake-off, and seeded in 96-well plates. After exposure to0.1%DMSO, 5 mmol/L SU6656, or5 mmol/L geraldol for 4 hours,unattached (mitotic) cells wereremoved and adherent (slipped)cells were allowed to grow in freshculture medium for up to 14 daysbefore staining with Hoechst andquantification as a proportion ofmitotic cells by automatedfluorescence microscopy. B andC, all cells and viable cells, MCF-7pcDNA or MCF-7casp3 cells in96-well plates were exposed to0.1% DMSO or 50 nmol/Lpaclitaxel for 20 hours and then0.1%DMSO, 5 mmol/L SU6656, or5 mmol/L geraldol for a further 4hours. Drugs were washed awayand the cells were allowed to growin fresh culture medium for up to14 day before staining withHoechst and quantification (allcells) or analysis of cell viability bythe MTT assay (viable cells). Errorbars represent 95% CIs.

Riffell et al.





SU6656 and geraldol, 2 compounds found to stimulatemitotic slippage in cells exposed to a microtubule-target-ing agent (26), induce the proteasome-dependent degra-dation of cyclin B1 as occurs during exit from mitosis(Fig. 1B; ref. 46). However, these chemicals inactivate themitotic checkpoint through the proteasome-dependentdegradation of BubR1 (Fig. 1B) that is sufficient to com-promise the mitotic checkpoint (35, 36, 47). This effectoccurs only in mitotic cells (Fig. 1C), and BubR1 is notdegraded 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 lead-ing to mitotic slippage through BubR1 degradation.Examination of the timing of mitotic checkpoint inac-

tivation and slippage revealed that mitotic slippage,defined in this experiment by cell attachment and lossof the mitotic phosphoepitope recognized by the TG3antibody, begins to occur 2 hours following exposure togeraldol whereas cyclin B1 degradation is complete 30minutes after drug treatment (Fig. 3A and B). BubR1 andCdc20 degradation occurs more slowly (Fig. 3B) andapproximately correlate with mitotic slippage, indicatinga possible requirement for checkpoint inactivation priortomitotic slippage, even in the absence of Cdk1/cyclin B1activity. In agreement with this observation, cyclin B1 isalso completely degraded in MCF-7 cells in response toSU6656 and geraldol, althoughmitotic slippage cannot beinduced (Fig. 2B). It is not known whether the degrada-tion of other APC/C substrates or dephosphorylation ofmitotic checkpoint kinase substrates might be involved inmitotic slippage.Caspase-3 is upregulated during mitosis (18) and has

been shown to increase the formation of micronuclei inresponse to antimitotic agents (48), but a role for caspase-3 in mitosis remains undefined. This study reveals anovel role for caspase-3: mitotic slippage induced bySU6656 and geraldol is caspase-3 dependent. Cotreat-ment of mitotic cells with SU6656 or geraldol and thecaspase-3 and -7 inhibitor DEVD-CHO prevented mitoticslippage and checkpoint inactivation (Fig. 2A and B).Although introduction of caspase-3 intoMCF-7 cells doesnot affect the frequency of spontaneous mitotic slippagein response to paclitaxel (Fig. 2E), mitotic slippage can beinduced by SU6656 and geraldol in MCF-7 cells onlywhen exogenous caspase-3 is expressed (Fig. 2C), indi-cating that caspase-3 is absolutely required for mitoticslippage induction by SU6656 and geraldol.This requirement for caspase-3 is likely due to its role in

inactivation of the mitotic checkpoint; mitotic slippageand BubR1 degradation occur in MCF-7 cells with activecaspase-3 but not in MCF-7 cells lacking caspase-3(Fig. 2C and D). BubR1 degradation is sufficient to inac-tivate themitotic checkpoint (14, 35, 36), and furthermore,depletion of BubR1 by mutation (47), gene knockdown,or, recently, in response to chemicals (36) has been impli-cated in the development of polyploidy. Several factorscan influence the degradation of BubR1 during mitosis.

Choi and colleagues observed that BubR1 deacetylationat metaphase results in abrogation of its anaphase inhibi-tion effects and in its degradation by APC/CCdc20 (15).Cotreatment of mitotic cells with SU6656 or geraldol andthe deacetylase inhibitor trichostatin A did not preventchemical induction of mitotic slippage (data not shown),indicating that SU6656 and geraldol do not induce pre-mature deacetylation of BubR1. Although a small protea-some-dependent decrease in BubR1 was observed inMCF-7pcDNA cells in response to SU6656 and geraldol(Fig. 2D), this is not sufficient for extensive mitotic slip-page to occur and is probably due to some deacetylationand proteasome-dependent degradation of BubR1. Kimand colleagues observed cleavage of BubR1 by caspase-3during 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), indicatingthat caspase-3 initiates mitotic slippage through cleavageof BubR1.

However, caspase-3 does not seem to be required forcell death following paclitaxel treatment (Fig. 5).Although caspase-3 is required for DNA fragmentationduring apoptosis (37), cells that lack caspase-3 can never-theless undergo apoptosis. Other cell death pathwaysmay also be involved in the fate of cells following expo-sure to an antimitotic agent: for instance, necrosis andautophagy have both been implicated in cell death fol-lowing antimitotic therapy (49, 50).

Spontaneous mitotic slippage has been describedto occur through slow ubiquitylation of cyclin B1 byAPC/CCdc20 and subsequent proteasome-dependentdegradation 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

Cyclin B1

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Figure 6. Model for spontaneous and induced mitotic slippage.






depletion, combined with phosphatase activity, results inloss of the mitosis-specific Cdk1/cyclin B1 inhibitoryphosphorylation on caspase-9 (45). Active caspase-9may then cleave procaspase-3, and activated caspase-3can cleave BubR1, resulting in inactivation of the mitoticcheckpoint and activation of APC/CCdc20. This eventwould lead to further ubiquitylation and degradationof cyclin B1, combining to force exit frommitosis throughmitotic slippage.

In summary, these results show that the chemicalinducers of mitotic slippage SU6656 and geraldol causeproteasome- and caspase-dependent inactivation of themitotic checkpoint, in contrast to the accepted model forspontaneous mitotic slippage. Caspase-3 is required formitotic slippage induction and checkpoint inactivationthrough degradation of BubR1, although not for celldeath in response to antimitotic agents. We propose amodel for induced mitotic slippage that includes animportant role for caspases in modulation of mitoticarrest. In response to the cellular stress presented by aprolonged mitotic arrest, caspases may contribute to the

outcome of antimitotic cancer therapy both throughmitotic slippage and through apoptosis.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

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

Grant Support

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

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 30, 2010; revised February 1, 2011; acceptedMarch11, 2011; published OnlineFirst March 25, 2011.

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2011;10:839-849. Published OnlineFirst March 25, 2011.Mol Cancer Ther Jenna L. Riffell, Reiner U. Jänicke and Michel Roberge GeraldolSmall-Molecule Inducers of Mitotic Slippage SU6656 and

Dependent Mitotic Checkpoint Inactivation by the−Caspase-3

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