Induction of apoptosis by monastrol, an inhibitor ofthe mitotic kinesin Eg5, is independent of thespindle checkpoint
Gregory M. Chin1 and Ronald Herbst2
1DNAX Research Institute of Molecular and Cellular BiologyResearch Institute, Palo Alto, California and 2MedImmune, Inc.,Gaithersburg, Maryland
AbstractSpindle poisons such as paclitaxel are widely used ascancer therapeutics. By interfering with microtubuledynamics, paclitaxel induces mitotic arrest and apoptosis.Targeting the kinesin Eg5, which is required for theformation of a bipolar spindle, is a promising therapeuticalternative to drugs that interfere with microtubuledynamics. Recent data suggest that the spindle check-point can determine the response of tumor cells tomicrotubule poisons. The relationship between checkpointfunction and Eg5 inhibition, however, has not yet beenfully investigated. Here, we used time-lapse video micros-copy and biochemical analysis to study the effect ofspindle checkpoint abrogation on the response of HeLacells to monastrol, a selective Eg5 inhibitor. In HeLa cells,monastrol activated the spindle checkpoint, leading tomitotic arrest and apoptosis. Small interfering RNA–mediated depletion of the spindle checkpoint proteinsBubR1 or Mad2 significantly shortened drug-inducedarrest, causing premature mitotic exit without cell divi-sion. Time-lapse microscopy as well as analysis ofcaspase activation shows that these checkpoint-deficientcells initiate apoptosis after mitotic exit in response tomonastrol. Checkpoint-deficient cells treated with pacli-taxel, on the other hand, yielded a higher frequency ofcells with >4N DNA content and a decreased incidence ofapoptotic events, particularly in Mad2-depleted cells.These results indicate that the immediate fate of post-mitotic cells is influenced by both the nature of thecheckpoint defect and the type of drug used. Furthermore,these results show that inactivation of the kinesin Eg5 caninduce apoptosis in tumor cells in the absence of criticalspindle checkpoint components. [Mol Cancer Ther2006;5(10):2580–91]
IntroductionDrugs that target the mitotic spindle are among the mosteffective cancer therapeutics currently in use. Vincaalkaloids, which promote microtubule depolymerization,and taxanes (paclitaxel and Taxotere), which stabilizemicrotubules, inhibit spindle function by disrupting micro-tubule dynamics, leading to mitotic arrest and apoptosis(1, 2). Mitotic arrest is mediated by the spindle checkpoint,which is activated by microtubule-targeted drugs. Recently,inhibiting the mitotic kinesin Eg5 [also known as kinesinspindle protein (KSP)], which is required for the forma-tion of a bipolar spindle, has gained significant attentionas an alternative strategy to interfere with spindlefunction (3–5). Blockage of Eg5 function with selectiveinhibitors, such as monastrol, results in the characteristicmonoastral phenotype, mitotic arrest, and apoptosis invarious tumor cell lines (6–8). Similar to microtubulepoisons, inhibition of Eg5 leads to activation of the spindlecheckpoint (9).The spindle checkpoint prevents chromosome missegre-
gation and aneuploidy by ensuring the accurate segrega-tion of sister chromatids to the dividing daughter cellsduring mitosis (10–13). The spindle checkpoint remainsactive until all chromosome kinetochores are properlyattached to the bipolar spindle and chromosomes arealigned at the metaphase plate. Proper function of thespindle checkpoint requires the concerted action of severalcheckpoint proteins, which include BubR1, Bub1, Bub3,Mad1, and Mad2. Several of these components have beenshown to preferentially localize to unattached chromo-somes. The active checkpoint generates a ‘‘wait anaphasesignal’’ to inhibit the anaphase-promoting complex. Inhi-bition of the anaphase-promoting complex prevents thedegradation of several key mitotic proteins, which must bedegraded for anaphase initiation to occur. The presence ofunattached chromosomes or a lack of spindle tension that isnormally generated by bipolar chromosome attachmentresults in continued checkpoint activation, mitotic arrest,and eventually programmed cell death (14–17).Recent studies have shown a correlation between defects
in the spindle checkpoint and chromosomal instability,which is frequently observed in tumor cell lines (18–24). Inaddition to an association between defects in the spindlecheckpoint and chromosomal instability, spindle check-point defects are also associated with the susceptibility oftumor cells to induction of mitotic arrest and apoptosis bymicrotubule-targeted agents, such as paclitaxel and noco-dazole. Several studies have shown that impairment ofspindle checkpoint function leads to a reduction in the levelof mitotic arrest and apoptosis normally induced byantimicrotubule drugs (25–27). Other studies, however,
Received 4/11/06; revised 7/6/06; accepted 8/16/06.
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 toindicate this fact.
Requests for reprints: Ronald Herbst, MedImmune, Inc., Gaithersburg, MD.Phone: 301-398-5253. E-mail: [email protected]
Copyright C 2006 American Association for Cancer Research.
have concluded that inactivation of the spindle checkpointsensitizes cells to apoptosis induced by antimicrotubuledrugs (28, 29).The relationship between the spindle assembly check-
point and inhibition of the mitotic kinesin motor proteinEg5 is just beginning to be elucidated. In a recent study, Taoet al. (30) suggest that induction of apoptosis by an Eg5inhibitor requires sustained mitotic arrest, followed byadaptation and slippage into the next G1 phase. In theirstudy, cells refractory to slippage or cells with a weakenedmitotic checkpoint showed a diminished apoptotic re-sponse. The authors suggest that the presence of activatedcheckpoint components, specifically BubR1, may be re-quired for induction of apoptosis by Eg5-targeted drugsfollowing exit from mitosis.Here, we have sought to explore the relationship between
the spindle checkpoint and Eg5 inhibition in more detail.We used time-lapse video microscopy and time coursefluorescence-activated cell sorting (FACS) analysis tomonitor mitotic arrest and apoptosis of HeLa cells inresponse to the selective Eg5 inhibitor monastrol, as well asto the microtubule inhibitor paclitaxel. To analyze the effectof the spindle checkpoint on drug response, cells wererendered checkpoint deficient by small interfering RNA(siRNA)–mediated depletion of BubR1 or Mad2. Ourresults suggest that postmitotic events in checkpoint-deficient HeLa cells are dependent both on the type ofdrug used and the mode of checkpoint inhibition (BubR1versus Mad2 depletion). Furthermore, our results showthat a functional checkpoint is required for monastrol-induced mitotic arrest but not for apoptosis. In checkpoint-compromised HeLa cells, monastrol induced apoptosisfollowing mitotic exit into the next G1 phase, showing thatEg5 inhibition can lead to caspase activation and apoptosisin the absence of critical checkpoint components, such asBubR1 or Mad2.
Materials andMethodsCell CultureHeLa cells and HeLa cells constitutively expressing a
histone 2B-green fluorescent protein (31) were grown inDMEM supplemented with 10% fetal bovine serum and0.01 mol/L HEPES.
DrugTreatmentPaclitaxel (Sigma, St. Louis, MO) and monastrol (Tocris
Cookson, Inc., Ellisville, MS) were dissolved in DMSO andused at a final concentration of 100 nmol/L and 100 Amol/L,respectively. DMSO was added to mock-treated controls.
SiRNAsiRNA duplexes were used to knock down gene
expression in HeLa cells (32). All duplexes were synthe-sized by Dharmacon, Inc. (Lafayette, CO). The BubR1 targetsequence was 5¶-CUUCACUUGCGGAGAACAU-3¶ and theMad2 sequence was 5¶-GAGUCGGGACCACAGUUUA-3¶(33). A luciferase siRNA duplex (5¶-CAUUCUAUCCU-CUAGAGGAUG-3¶) was used as a control. Transfection ofsiRNA duplexes was done as described in Tanudji et al.
(34) using LipofectAMINE 2000 (Invitrogen, Grand Island,NY) as the transfection reagent.
ImmunoblottingBoth adherent and floating cells were collected in
radioimmunoprecipitation lysis buffer supplemented withprotease and phosphatase inhibitors. Following centrifuga-tion at 14,000 rpm for 10 minutes at 4jC, total proteinconcentration in the lysate was determined with theCoomassie Plus Protein Assay Reagent Kit (Pierce, Rock-ford, IL), and the lysate was resuspended in 4� NuPAGELDS sample buffer (Invitrogen). Ten micrograms of totalprotein lysate were run per lane on an SDS-PAGE gel andtransferred to nitrocellulose membrane following standardprocedures. Membranes were blocked with 4% milk inTBST buffer. Primary antibodies were used at the followingdilutions: anti-BubR1 (BD Transduction Labs, San Jose, CA)at 1:1,000, anti-actin (Sigma) at 1:10,000, anti-Mad2 (BDTransduction Labs) at 1:500, anti–poly(ADP-ribose)poly-merase (Cell Signaling, Danvers, MA) at 1:2,000, and anti–phospho-histone H3 (Upstate, Lake Placid, NY) at 1:2,000.Membranes were washed with TBST and horseradishperoxidase–conjugated secondary antibody (AmershamBiosciences, Piscataway, NJ) was added to 4% milk at1:5,000. Chemiluminesence was detected with Pierce Super-signal Westpico.
Flow CytometryAnalysisBoth adherent and floating cells were collected in PBS and
fixed in 4 mL of 70% ethanol overnight. Cells were washedonce in PBS with 1% fetal bovine serum and 0.03 mg/mLsaponin and resuspended in 100 AL of 10 Ag/AL anti-MPM2-FSE diluted in wash buffer. After a 2-hour incubation on ice,cells were washed twice with wash buffer and resuspendedin 0.4 mL of propidium iodide/RNase I solution. Toquantitate the number of cells with activated caspase, acarboxyfluorescein caspase detection kit (Biocarta, SanDiego, CA) was used according to the instructions of themanufacturer. Analysis was done on a FACScalibur (BectonDickson, San Diego, CA) machine using CellQuest software.
Time-LapseMicroscopyTime-lapse images were taken under a Zeiss Axiovert
S-100 inverted microscope in an environmental chamber(temperature and CO2 controlled). Images for each condi-tion were obtained in three separate fields of view with animage being taken every 10 to 15 minutes for 48 hours.Cells were pretreated with siRNA 24 hours before time-lapse analysis, and drug treatment was begun 1 to 2 hoursbefore the first time-lapse image acquisition. Mitoticprogression of individual cells was examined with Axiovi-sion V3.1 software (Zeiss, Thornwood, NY).
ResultsMitotic Arrest and Apoptosis in Monastrol-Treated
HeLa CellsThe potential fates of cells treated with antimitotic drugs
are either death in mitosis or adaptation and mitotic exit(slippage). Cells that manage to exit mitosis with 4N DNAcontent can either survive with continued rounds of DNA
replication, arrest in G1, or die via apoptosis (35). Theultimate outcome may be influenced by the spindlecheckpoint as well as other factors, such as p53. Toinvestigate the effect of the spindle checkpoint on theresponse to Eg5 inhibition, we used HeLa cells that arefrequently used for the study of mitosis as well for the studyof antimitotic drugs. HeLa cells have a functional spindlecheckpoint and show a durable mitotic arrest (no mitoticslippage) when treated with microtubule poisons atconcentrations that fully block microtubule dynamics(35, 36). To inhibit Eg5 ATPase activity, we made use ofmonastrol, a selective allosteric inhibitor of Eg5 (6, 37, 38).The mitotic arrest induced by monastrol is dose dependentand reaches a maximum level of arrest at a concentration of100 Amol/L in HeLa and other cell lines (9, 38, 39).At this concentration, centrosome separation is also maxi-mally inhibited (9). Therefore, we used monastrol at aconcentration of 100 Amol/L in all experiments. As acomparison, we also used the microtubule-stabilizing agentpaclitaxel. In contrast to monastrol, paclitaxel shows abiphasic dose response. At low nanomolar concentrations,paclitaxel results in aberrant mitotic exit, micronucleation,and eventual cell death. At high concentrations, however,paclitaxel induces a durable mitotic arrest in many cell lines(40). Here, we used paclitaxel at a concentration of 100nmol/L, which results in activation of the spindle check-point and maximal mitotic arrest (ref. 41; see also Fig. 1).First, we compared the kinetics of monastrol-induced
mitotic arrest with arrest induced by the microtubule-targeted drugs paclitaxel and nocodazole (Fig. 1A). Inasynchronously growing HeLa cells, maximal arrest wasobserved at 18 hours of drug treatment. The accumulationof cells in the mitotic phase confirms that all three drugsused activate the spindle checkpoint in HeLa cells becausecheckpoint-defective tumor cells do not show this responseto antimitotic drugs (42). Cells treated with monastrolarrested in mitosis with the characteristic rosette-likeconfiguration of condensed chromosomes (monoaster) asa result of unseparated centrosomes (Fig. 1A; ref. 6).Phenotypic analysis of DNA-stained cells further suggestedthat the decrease in mitotic index at later time points is dueto the induction of apoptosis (data not shown). Indeed, wefound that cleavage of poly(ADP-ribose)polymerase, a keyevent in the apoptotic signaling cascade, coincided witha decrease in the mitotic marker phospho-histone H3(Fig. 1B). To show on a molecular level that monastrolactivates the spindle checkpoint in HeLa cells, we tested fora phosphorylation-induced band-shift of BubR1 protein.This band-shift has previously been correlated withactivation of the spindle checkpoint (20, 24). Treatment ofHeLa cells with monastrol, paclitaxel, or nocodazoleresulted in hyperphosphorylation of BubR1, further con-firming activation of the spindle checkpoint (Fig. 1C).
Monastrol-InducedMitotic Arrest Is ImpairedinSpin-dle Checkpoint ^Deficient CellsTo ensure the fidelity of chromosome segregation, the
spindle checkpoint monitors microtubule-kinetochoreattachment, as well as tension generated by a bipolar
Figure 1. Mitotic arrest and activation of the spindle checkpoint bymonastrol in HeLa cells. A, the mitotic index of monastrol- (100 Amol/L),paclitaxel- (Taxol; 100 nmol/L), nocodazole- (200 ng/mL), andDMSO-treated HeLa cells was determined using formaldehyde fix andHoechst DNA stain. HeLa cells were sampled at five time points aftereach drug was added to assess the level of mitotic arrest. For eachsample, >200 fixed cells were examined. The micrographs depict thetypical configuration of condensed chromosomes in mitotic cellsarrested with monastrol, paclitaxel, and nocodazole. Eg5 inhibitionleads to the characteristic rosette-like configuration of chromosomes(monoaster) as a result of unseparated centrosomes. B, decrease inmitotic index after drug-induced arrest coincides with induction ofapoptosis in HeLa cells. At the indicated time points, cell lysate wasimmunoblotted with anti –poly(ADP-ribose)polymerase (PARP ; to mon-itor apoptosis), anti –phospho-histone H3 (to monitor mitotic arrest),and anti-h-actin antibody (as a loading control). C, treatment ofHeLa cells with monastrol leads to BubR1 phosphorylation. Following16 h of drug treatment, a BubR1 band-shift was detected. Drug-induced mitotic arrest was verified by probing phospho-histone H3levels. Equal loading was shown by staining the membrane with amido-black (not shown).
spindle. Mad2 and BubR1 are integral components of thecheckpoint, which are required for checkpoint function (12).Because the mode of checkpoint inhibition may affect tumorcell response to antimitotic drugs, we established siRNAduplexes for both BubR1 and Mad2. As shown in Fig. 2A,transfection of HeLa cells with specific siRNA duplexesresulted in efficient depletion ofMad2 or BubR1. In repeatedexperiments, substantial knockdown of BubR1 and Mad2was observed as early as 16 hours after transfection andlasted for up to 96 hours (Fig. 2A, and data not shown).
Using these siRNA duplexes, we tested the effect ofspindle checkpoint abrogation on the mitotic arrest pheno-type induced by either monastrol or paclitaxel treatment.Monastrol and paclitaxel both efficiently induce mitoticarrest in control luciferase-transfected HeLa cells asevidenced by the round, detached cells that accumulatefollowing either drug treatment. In contrast, this response isnoticeably absent in phase-contrast images of drug-treatedHeLa cells that have been depleted of BubR1 protein(Fig. 2B). To better quantitate this difference, the accumu-lation of a mitotic marker, theMPM2 epitope found in manymitosis-specific proteins (43), was measured by FACSanalysis. In response to monastrol and paclitaxel treatment,24.0F 3.2% and 38.2F 4.5% of control luciferase-transfectedHeLa cells accumulated in mitosis, respectively. In contrastto the robust mitotic arrest seen in control luciferase-transfected cells, only 1.5 F 0.7% of monastrol-treated and4.6 F 0.3% of paclitaxel-treated BubR1-depleted HeLa cellswere MPM2 positive. This failure to accumulate MPM2-positive cells following either drug treatment indicates thatBubR1-depleted HeLa cells fail to maintain an efficientarrest in response to either monastrol or paclitaxel (Fig. 2C).Depletion of Mad2 similarly abrogated mitotic arrest inresponse to monastrol or paclitaxel. This mitotic arrestdefect was also corroborated by immunoblot analysis ofindependent mitotic markers, phopho-histone H3 andcyclin B1 (data not shown).To further explore the effect of spindle checkpoint
abrogation on drug-induced mitotic arrest, we employedtime-lapse video microscopy, which allowed us to followthe mitotic progression of individual cells. In luciferasesiRNA–transfected control cells, an average mitosis (mea-sured from the first sign of DNA condensation to thefirst sign of DNA decondensation) lasted 90 F 19 minutes(Fig. 3A). This average correlated well with the duration ofa normal HeLa cell mitosis described in previous reports(22, 36). Depletion of BubR1 resulted in a minor decrease inthe length of mitosis whereas depletion of Mad2 had amore pronounced effect with cells exiting mitosis after35.7 F 10 minutes (Fig. 3A). Control luciferase-transfectedHeLa cells treated with monastrol arrested in mitosis for229 F 19 minutes, which was followed by cell death asevidenced by hypercondensed and fragmented DNA indi-cative of apoptosis. In contrast, monastrol-induced arrestof BubR1- and Mad2-depleted HeLa cells was significantlyshortened to 42 F 5 and 22 F 7 minutes, respectively(Fig. 3B). Paclitaxel treatment yielded similar results(Fig. 3C). In addition to the obvious decrease in the dura-tion of mitotic arrest, the majority of spindle checkpoint–compromised cells treated with monastrol or paclitaxel alsoexited mitosis without division (see also below).
BubR1- and Mad2-Depleted Cells Abnormally ExitDrug-InducedMitotic ArrestMonastrol- and paclitaxel-induced mitotic arrest in
spindle checkpoint–competent HeLa cells leads to elimi-nation by apoptosis. In contrast, we found that abrogationof the spindle checkpoint allowed cells to rapidly exitmitosis without cell division. To determine the fate of
Figure 2. Loss of BubR1 or Mad2 function impairs mitotic arrest inresponse to monastrol and paclitaxel (Taxol). A, siRNA depletion of BubR1and Mad2 protein in HeLa cells. Efficient knockdown of BubR1 (top) andMad2 (bottom ) after siRNAi depletion is shown. h-Actin is included as aloading control. B, phase-contrast images of drug-treated HeLa cells. Cellswere transfected with siRNA and then treated with monastrol, paclitaxel,or DMSO 24 h later. Indicative of mitotic arrest, rounded control-transfected cells accumulate after 24 h of drug treatment. BubR1-depletedcells fail to show this arrest phenotype. C, quantitation of MPM2-positivecells by FACS analysis following drug treatment. The percentage ofMPM2-positive cells is severely reduced in BubR1-depleted cells treatedwith monastrol or paclitaxel (Taxol). Columns, average of three experi-ments; bars, SD.
postmitotic cells that lack either BubR1 or Mad2, we againemployed time-lapse microscopy to monitor cell fate.Figure 4 shows representative still images from time-lapsemovies of monastrol- and paclitaxel-treated HeLa cellsexpressing histone H2B-green fluorescent protein inthe presence or absence of a functional spindle checkpoint(see also Supplementary movies).3 Figure 1A shows thecontrols (drug treatment only and siRNA only) whereassiRNA-transfected and drug-treated cells are shown inFigure 1B and C. Quantitative data from several indepen-dent experiments are summarized in Table 1.Luciferase-transfected control HeLa cells mock treated
with DMSO efficiently completed mitosis, whereas control
cells treated with monastrol arrested in mitosis with thetypical monoastral configuration of chromosomes (Fig. 4A).After a period of mitotic arrest, cells die with hyper-condensated and fragmented chromosomal DNA, a hall-mark of apoptosis (last panel in row; ref. 44). Similarly,paclitaxel also induced mitotic arrest, which was followedby apoptosis in luciferase-transfected cells. During the48-hour period in which luciferase-transfected cells weremonitored by time-lapse microscopy, a total of 90.9%and 95.1% of monastrol- and paclitaxel-treated cells,respectively, underwent apoptosis (Table 1).No overt defects in cell morphology were observed in
BubR1-depleted HeLa cells, and mitosis was generallycompleted with relatively normal metaphase chromosomemorphology (Fig. 4A). However, consistent with recentreports (45), BubR1 knockdown also produced someexamples of unaligned metaphase chromosomes andlagging chromosomes during cell division (Fig. 4A; seealso Supplementary movies).3 The effect of Mad2 depletionwas more severe with cells undergoing a brief period ofchromosome hypercondensation that was neverthelessfollowed by chromosome segregation and cell division(Fig. 4A). Aligned metaphase chromosomes were neverobserved in Mad2-depleted cells, and lagging chromo-somes were identifiable in dividing cells. Using eitherduplex, only occasional apoptotic cells were observedwithin the 48-hour period of time-lapse analysis. Thus, atleast in the short term, loss of BubR1 or Mad2 function doesnot significantly affect the level of cell death.In both BubR1- and Mad2-depleted cells, monoastrol-
induced mitotic arrest was followed by rapid chromosomedecondensation and exit from mitosis (Fig. 4B; Supplemen-tary movies).3 BubR1-depleted cells treated with monastrol(70.7%) exited without division into a pseudo-G1 state.Interestingly, 26.8% of BubR1-depleted cells treated withmonastrol managed to exit with chromosome segregationand cell division despite the initial appearance of amonoastral spindle (Table 1). In contrast, the G1-like exitwas the only fate observed among monastrol-arrested cellsthat were depleted of Mad2; cell division was neverobserved (Fig. 4B). Following mitotic exit, we noticed thatHeLa cells depleted of either BubR1 or Mad2 remained ininterphase for varying lengths of time but nevertheless stillinitiated apoptosis in most cases. A measurement of thecumulative amount of cell death, which occurred duringthe 48 hours of monastrol treatment, revealed that 91.7% ofBubR1-depleted and 80.4% of Mad2-depleted HeLa cellsunderwent apoptosis (Table 1).Interestingly, treating checkpoint-deficient cells with
paclitaxel elicited different postmitotic fates than monastrol(Fig. 4C; Table 1; Supplementary movies).3 First, celldivision was never observed, among either BubR1- orMad2-depleted cells. Second, at least within the 48-hourduration of time-lapse analysis, the frequency of postmi-totic cell death was significantly reduced in paclitaxel-treated cells compared with monastrol-treated cells. Thecumulative cell death among BubR1- and Mad2-depletedcells treated with paclitaxel was 60.7% and 20.5%,
Figure 3. Duration of mitotic arrest in drug-treated cells. Individual cellsexpressing a histone 2B-green fluorescent protein fusion protein weremonitored by time-lapse microscopy (in a CO2- and temperature-controlledenvironment) to measure the duration of mitotic arrest. Images wereacquired as described in Materials and Methods. The line represents themedian duration of mitotic arrest (measured from the first sign of DNAcondensation to first sign of decondensation) of at least 34 DMSO- ordrug-treated cells. A, average duration of mitosis of DMSO-treated cellsused as reference. B, duration of mitosis in monastrol-treated cells. C,duration of mitosis in paclitaxel-treated cells (Taxol). Note that for cellstransfected with luciferase siRNA and treated with paclitaxel or monastrol,mitosis typically ends with the generation of apoptotic nuclei.
3 Supplementary material for this article is available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).
Figure 4. Analysis of mitotic and postmitotic events by time-lapse video microscopy. Drug-treated and control HeLa cells expressing histone H2B-greenfluorescent protein were monitored by time-lapse video microscopy in a CO2- and temperature-controlled environment. Still images of green fluorescentprotein– labeled chromosomes and phase-contrast cell morphology for a single cell are representative of several independent experiments. The number ofminutes that have elapsed at the time of image capture is indicated. A, images of controls treated with drugs or siRNA only. B and C, images ofcheckpoint-deficient cells treated with monastrol or paclitaxel (Taxol), respectively. Cells with hypercondensed and fragmented DNA were consideredapoptotic. To confirm cell death, cells were monitored for the full duration of the movie (images representing apoptotic cells are indicated with ‘‘+’’). A,DMSO-treated control cells show normal mitotic progression. Monastrol treatment elicits mitotic arrest, followed by apoptosis. Paclitaxel-treated cellsundergo programmed cell death following a prolonged mitotic arrest. Bar, 10 Am. B, BubR1 and Mad2 depletion affects mitotic progression. Mitoticchromosome congression is abbreviated in BubR1-depleted cells. Unaligned chromosomes and lagging chromosomes are sometimes observed. Mad2-depleted cells rapidly initiate anaphase and cell division following chromatin condensation, typically without a detectable metaphase alignment. Cellstransfected with luciferase siRNA and not drug treated showed normal mitotic progression similar to DMSO-treated cells described above (not shown). Bar,10 Am. B, loss of BubR1 or Mad2 function bypasses monastrol-induced mitotic arrest. BubR1- and Mad2-depleted cells predominantly escape mitoticarrest and exit without cell division (pictured). Apoptosis of cells depleted of either protein occurs after escape from mitotic arrest (+; final image ofthe sequence). Bar, 10 Am. C, BubR1- and Mad2-depleted cells respond differently to paclitaxel treatment. BubR1-depleted cells treated withpaclitaxel undergo abbreviated mitotic arrest and exit mitosis with micronucleation (image at 90 min). For the cell shown, apoptosis was observed at 40h (+; last image in sequence). In contrast, most MAD2-depleted cells treated with paclitaxel were still viable at the end of the 48-h movies. Mad2-depletedcells treated with paclitaxel often show repeated cycles of chromosome condensation and decondensation with progressive enlargement of the nuclearand cell body. Bar, 10 Am.
respectively (Table 1). Furthermore, examination of fluo-rescent and phase-contrast images of Mad2-depleted cellstreated with paclitaxel revealed successive rounds of DNAcondensation and cell body enlargement, which couldindicate DNA replication without subsequent cell division.Together, these results show that abrogation of the spindlecheckpoint allows cells to avoid monastrol- or paclitaxel-induced mitotic arrest. They also show that the majority ofcheckpoint-deficient cells treated with monastrol triggercell death following exit from mitosis. The differencesobserved between the type of drug treatment and the typeof checkpoint defect emphasize the idea that the ultimatefate of cells exiting mitosis is dependent on both the drugused and the underlying checkpoint defect.
DNA Synthesis in Paclitaxel- and Monastrol-TreatedCellsThat Are Spindle Checkpoint CompromisedThe results presented above indicate that Mad2-depleted
cells continue to synthesizeDNA following exit frommitosiswithout cell division, particularly when treated withpaclitaxel. Similarly, the majority of BubR1-compromisedcells that have been drug treated both escape apoptosis andfail to undergo nuclear division. Consequently, a highfraction of these cells would be expected to have abnormalDNA content. To test this directly, we quantitated the per-centage of cells with a >4N DNA content by FACS analysisof propidium iodide–stained cells. Figure 5A showsrepresentative FACS profiles of checkpoint-competent andcheckpoint-deficient cells following 24 hours of drugtreatment. Compared with monastrol treatment, paclitaxeltreatment resulted in a greater fraction of cells with >4NDNA content. This observation was further bolstered bymonitoring the increase of cells with >4N DNA content atmultiple time points (Fig. 5B). A distinct peak of cells with8N DNA content, however, was not observed at the timepoints analyzed,which is likely due to the eventual initiationof apoptosis following exit of checkpoint deficient cells intothe next G1 phase. Consistent with data shown in Fig. 4C andTable 1, DNA content of >4N was most prominent in Mad2-depleted cells treated with Taxol. Cells treated withmonastrol showed the lowest increase in the >4Npopulation
during the time course. In Mad2-depleted cells, 56.2 + 3.3%and 18.8 + 1.7% of cells had >4N DNA content following40 hours of treatment with paclitaxel or monastrol, respec-tively. The lower percentage of cells with >4N DNA contentin monastrol-treated cells is likely related to the higherincidence of apoptosis following exit from mitosis. ThisFACS data confirms that spindle checkpoint–deficientHeLacells treated with either monastrol or paclitaxel are compe-tent to exit mitotic arrest and enter another cell cycle,including a second round of DNA synthesis. In monastrol-treated cells, however, apoptosis was the most frequent cellfate following mitotic exit.
Abrogation of the Spindle Checkpoint DelaysMonas-trol- and Paclitaxel-Induced CaspaseActivationOur time-lapse experiments showed that, in the absence of
a functional spindle checkpoint, cells fail to trigger apoptosiswithin the mitotic phase in response to drug treatment.However, cell death was still observed at later time points,particularly in monastrol-treated cells. These observationssuggest that activation of the apoptotic machinery is noteliminated in the absence of an active spindle checkpointbut is merely delayed. Caspases play a central role in apop-totic cell death and are activated in response to a variety ofanticancer agents, including microtubule-targeted drugs(46). Using a quantitative FACS-based assay, we monitoredcaspase activation at several time points in control andBubR1-depleted cells treated with either monastrol orpaclitaxel. As shown in Fig. 6A, treatment of luciferase-transfected control cells with monastrol or paclitaxelresulted in a significant time-dependent increase in thecaspase-positive population. Caspase was also activated inBubR1-depleted cells by monastrol and paclitaxel, albeit to alesser degree. After 24 hours of monastrol or paclitaxeltreatment, activated caspases were detected in 35% and 30%of BubR1-depleted cells, respectively. In comparison, cas-pases were active in 45% of control-transfected cells at thistime point. After 40 hours of drug treatment, the caspase-positive population increased to f65% (monastrol andpaclitaxel) in checkpoint-deficient cells and to 75% (monas-trol) and 80% (paclitaxel) in luciferase-transfected cells.
Table 1. Loss of BubR1 or Mad2 function alters the post-arrest fate of monastrol- and paclitaxel-treated HeLa cells
*Cells were transfected with siRNA, drug treated as indicated, and monitored by time-lapse video microscopy for 48 hours. Individual cells were thenmonitored for the duration of the movie to score their fate. Each value represents the percent of the total number of cells scored that fell into each category (N ofat least 33).cCells that underwent apoptosis at any point during the time-lapse movie were identified, and a cumulative total of cell death was scored. The value takesinto account the change in cell number in a given field due to cell division. Each value represents the percent of cells that died in the time-lapse field (N of atleast 33).
To confirm activation of apoptotic effector caspases, wemonitored a caspase-3-dependent event, the cleavage ofpoly(ADP-ribose)polymerase from its 116-kDa pro-form toits 85-kDa active form (Fig. 6B). Compared with luciferase-transfected cells, poly(ADP-ribose)polymerase cleavageelicited by monastrol in BubR1-depleted cells was signif-icantly reduced. However, during the time course, agradual increase in the cleaved p85 form was neverthelessobserved. Similarly, the appearance of the cleaved p85 formof poly(ADP-ribose)polymerase in BubR1-depleted cellstreated with paclitaxel was delayed in comparison withcontrol cells. Similar results were observed in Mad2-
depleted cells (data not shown). Together with the datashown in Figs. 1 and 4, these results suggest that monastrolas well as paclitaxel leads to caspase activation during thearrest stage of spindle checkpoint–competent HeLa cells.In addition, the absence of a functional checkpoint delays,but does not prevent, the activation of the apoptotic caspasepathway.
DiscussionDisrupting the mitotic spindle is an effective strategy forcancer therapy. Microtubule-targeted drugs, which directlyinterfere with spindle dynamics, are widely used in theclinic. Other aspects of spindle function may also betargeted to interfere with mitosis. For example, histonedeacetylase inhibitors were recently shown to interferewith assembly of kinetochores, leading to activation of thespindle checkpoint and arrest of tumor cells at theprometaphase stage (47). Monastrol is the first example ofa new generation of antimitotic compounds that blockmitosis by inhibiting the kinesin motor protein Eg5. Thereis great interest in developing more potent Eg5 inhibitorsfor clinical applications due to the potential for improvedside effect profiles over taxanes. Recently, Muller et al. (48)described new analogues of monastrol with significantlyimproved activity. Structurally distinct Eg5 inhibitors areS-trityl-L-cystein and representatives of the dihydropyr-azole and quinazolinone compounds, which were alsoshown to mediate significant tumor growth inhibition in
Figure 6. Delayed activation of caspases by monastrol and paclitaxel(Taxol) in HeLa cells with a compromised spindle checkpoint. A,quantitation of apoptosis as measured by FACS analysis of caspaseactivity in live cells. Each condition was sampled 16, 24, and 40 h afteraddition of 100 Amol/L monastrol or 100 nmol/L paclitaxel. Columns,average of three time course experiments; bars, SD. B, initiation ofpoly(ADP-ribose)polymerase cleavage is delayed in BubR1-depleted HeLacells. Samples of protein lysate were collected 16, 24, and 40 h after drugtreatment. An antibody that recognizes both the uncleaved and cleavedforms of poly(ADP-ribose)polymerase was used to monitor the progressionof apoptotic activation.
Figure 5. Continued DNA synthesis in spindle checkpoint–compro-mised HeLa cells. DNA content of siRNA-transfected and drug-treatedcells was analyzed by propidium iodide staining and FACS analysis. A,representative FACS profiles of checkpoint-competent (luciferase siRNA)and checkpoint-deficient (BubR1 and MAD2 siRNA) cells after 24 h of drugtreatment. Top left, populations of cells with sub-G1, 2N, 4N, and >4NDNA content. B, time-dependent increase in cells with DNA content of>4N following siRNA transfection and treatment with DMSO, paclitaxel(Taxol), or monastrol. Knockdown of Mad2 or BubR1 alone (siRNA-transfected and DMSO-treated cells) does not lead to endoreduplication.Relative to monastrol, paclitaxel consistently resulted in higher levels ofendoreduplication, especially in the absence of Mad2. Columns, averageof three individual experiments; bars, SD.
animal models (8, 49). Interestingly, these more potentstructures seem to target the same binding site in Eg5 asmonastrol (49–51). Several of these structures may provideclinical leads, and the first quinazoline-based Eg5 inhibitorshave entered clinical trials (52).The effects of microtubule poisons on tumor cells have
been studied in considerable detail. In light of potentialclinical implications, the relationship between the spindlecheckpoint and the response of tumor cells to microtubule-targeted drugs has been of significant interest in recentyears. However, the effect of Eg5 inhibitors on tumor cellsand the possible implications of the spindle checkpoint inmitotic progression and induction of apoptosis have not yetbeen studied in detail. Using spindle checkpoint–compe-tent and checkpoint-deficient HeLa cells, we show here thata functional checkpoint is required for Eg5 inhibitor–induced mitotic arrest but is not required for induction ofapoptosis.Several studies have previously argued in favor of the
need for an active spindle checkpoint in achieving efficientarrest and cell death by microtubule-targeted drugs.Masuda et al. (27) found a correlation between humancancer cell lines that are spindle checkpoint impaired andresistant to apoptosis induced by antimicrotubule agents.In that study, cell lines that failed to proficiently inducemitotic arrest when challenged with an antimicrotubuleagent, such as nocodazole, produced a much lower level ofapoptosis compared with spindle checkpoint–competentcells (27). Similarly, another study showed that MCF-7 cellsdepleted of either BubR1 or Mad2 showed an increasedresistance to paclitaxel-induced apoptosis (26).However, in contrast to the aforementioned reports,
recently a few studies have provided evidence thatinactivation of the spindle checkpoint sensitizes cells tothe effects of antimicrotubule drugs. Sihn et al. (29) showedthat a truncated form of hCDC20, the anaphase-promotingcomplex target of BubR1 and Mad2, could bypassmicrotubule drug-induced mitotic arrest and induce anincrease in apoptosis when compared with similarlytreated control cells. Another study has shown thatdepletion of BubR1 protein in HeLa cells by RNAinterference increased HeLa cell sensitivity to both pacli-taxel and nocodazole (28). Finally, Kienitz et al. (53) haveshown that partial down-regulation of Mad1 inactivatedthe spindle checkpoint but did not confer resistance toeither paclitaxel- or monastrol-induced apoptosis.There are several factors that may have contributed to the
alternative results produced by these two sets of studies.One major difference between them is the duration of drugtreatment that occurred before apoptosis was measured. Ascells need to complete a cell cycle and traverse from G2 intoM phase to be inhibited by antimitotic drugs, the cell cyclelength or doubling time of the chosen cell system alsoneeds to be considered. For example, the doubling time ofMCF-7 cells used by Sudo et al. (26) is significantly longerthan that of HeLa cells (54). Here, a time point beyond48 hours may have revealed more substantial (or delayed)apoptosis in checkpoint compromised cells. In addition, drug
concentration and the type of drug used varied betweenstudies and could have affected the outcome becausemicrotubule-stabilizing and microtubule-destabilizingdrugs can produce different mitotic and postmitotic effectsin tumor cells (55). Paclitaxel is known to activate a varietyof signaling pathways, including the mitogen-activatedprotein kinase pathway, in various cancer cell lines. Theseeffects have also been linked to the mitotic and apoptoticeffects of paclitaxel (refs. 56, 57 and references therein).Interestingly, monastrol, which does not directly interferewith microtubules, does not stimulate activity of extracel-lular signal–regulated kinase or p38 kinases in several celllines tested.4 The ability or inability of antimitotic drugs tomodulate certain signaling pathways may also account forsome of the differences noted in this study betweenpaclitaxel and monastrol. Differences in the expression ofproapoptotic and antiapoptotic factors, such as Bcl2 familymembers, also need to be considered when comparingdifferent cell lines (see also below). For example, MCF-7cells do not express caspase-3 and contain high levels ofantiapoptotic Bcl2 (refs. 58, 59 and references therein).Whereas MCF-7 cells respond to paclitaxel (26), we foundthese cells relatively resistant to Eg5 inhibitor–induced celldeath.5
Finally, as noted by Kienitz et al. (53) and our study,altering the functional status of different spindle check-point components may produce varying effects on thesensitivity of a cell to antimitotic drugs (see below). Thus, atleast in the case of antimicrotubule drugs, the requirementof a functional spindle checkpoint for drug-inducedapoptosis may be dependent on a variety of differentfactors.The relationship between mitotic arrest and apoptosis
induced by Eg5 inhibition and the spindle checkpoint isjust beginning to be elucidated. Using the Eg5 inhibitorKSP-IA, Tao et al. (30) observed that abrogation of thespindle checkpoint significantly reduced the apoptoticresponse. These results led them to propose that inductionof apoptosis in response to Eg5 inhibition requires mitoticslippage into the next G1 phase in the presence of anactivated spindle checkpoint. The authors concluded thatactivated checkpoint components like BubR1, together withthe inactivation of antiapoptotic factors like survivin, arelikely required for initiation of cell death following mitoticslippage.Here, we have shown that the predominant outcome of
treating HeLa cells with monastrol is apoptosis, regardlessof checkpoint function. HeLa cells show a durable arrestand do not undergo mitotic slippage in response tomoderate concentrations of paclitaxel and other mitoticdrugs (35). In the present study, we used monastrol andpaclitaxel at concentrations that trigger a durable mitoticarrest (Figs. 1A and 2C). The absence of mitotic slippage is
4 Vijapurkar and Herbst, unpublished data.5 Bookstein and Herbst, unpublished data.
further confirmed by our time-lapse analysis of monastrol-and paclitaxel-treated HeLa cells (Table 1). Our time-lapseanalysis also shows that drug-treated HeLa cells canefficiently undergo apoptosis during the mitotic arrestphase. Chromosome decondensation, which would indi-cate mitotic slippage into the next G1 phase, was neverobserved before the appearance of apoptotic nuclei. Thesetime-lapse observations are also supported by an increasein caspase activation and poly(ADP-ribose)polymerasecleavage, which coincides with a drop in mitotic index indrug-treated cells (Fig. 1). HeLa cells with an intact spindlecheckpoint showed these responses whether they weretreated with the Eg5 inhibitor monastrol or the microtubulepoison paclitaxel. Although checkpoint-compromisedHeLa cells were able to avoid drug-induced cell deathduring mitosis, apoptosis nevertheless occurred followingmitotic exit in most cases. Careful observation of individualHeLa cells by time-lapse video microscopy not onlyconfirmed that the apoptotic pathway was, in fact, stillcapable of activation but also revealed that death occurredin a postmitotic stage. Although both paclitaxel andmonastrol elicited postmitotic cell death, we found thatEg5 inhibition induced a higher level of apoptosis incheckpoint-deficient cells. Quantitative time-lapse analysisalso showed that, relative to controls, the duration of themitotic phase in checkpoint-deficient cells is significantlyshortened, especially in Mad2-depleted cells. However,despite the decreased mitotic phase, we found thatcheckpoint-compromised cells initiated cell death laterthan their checkpoint-competent counterparts. Thesetime-lapse observations correlate with a measurable delayin caspase activation and poly(ADP-ribose)polymerasecleavage in checkpoint-compromised cells treated witheither monastrol or paclitaxel. Interestingly, the resultsreported by Tao et al. (30) may also indicate a delayedapoptotic response in checkpoint-compromised cells, al-though the overall level of cell death achieved after 48hours of drug treatment is significantly lower than reportedhere. This may, at least in part, be due to the differentmethods used to assess apoptosis and/or the differentapproaches to override the spindle checkpoint. The resultspresented here show that, following exit from mitosis,apoptosis can be induced by Eg5 inhibitors, even in theabsence of critical spindle checkpoint components, suchas BubR1 and Mad2. Whereas paclitaxel can trigger celldeath through multiple mechanisms (40), the intrinsicmitochondrial death pathway seems to be critical for Eg5inhibitor– induced apoptosis (60).4 Thus, the integrity of themitochondrial apoptotic pathway, rather than the integrityof the spindle checkpoint, may be a major requirement forEg5 inhibitor– induced apoptosis.The results obtained with paclitaxel further emphasize
the importance of individual checkpoint components in thefate of drug-treated cells. Similar to monastrol, the majorityof BubR1-depleted cells treated with paclitaxel undergoapoptosis within 48 hours (the duration of our time-lapseanalysis). In contrast, most Mad2-depleted cells treatedwith paclitaxel remained viable at this point. Cumulative
cell death amounted to 60.7% and 20.5% of the BubR1- andMad2-depleted cell populations, respectively. This discrep-ancy in apoptotic response may be explained by theincreased level of cells with >4N DNA content observedamong Mad2-depleted cells.Thus, whereas depletion of either BubR1 or Mad2 will
abrogate spindle checkpoint function, these approachesmay not be functionally equivalent (61). In accordance withseveral studies (22, 62–64), we found that the timespanning the first sign of chromatin condensation at theonset of mitosis to the first sign of chromatin decondensa-tion during telophase is significantly reduced in cellsdepleted of either BubR1 or Mad2 protein. Meraldi et al.(63) proposed that BubR1 and Mad2 not associated with thekinetochore may be responsible for the control of mitotictiming occurring between the checkpoint function of Emi1in early prometaphase and the kinetochore spindle check-point function during mitosis. The significant differences intiming of the two checkpoint-depleted cell types observedin our studies further suggest that Mad2 and BubR1 maymonitor different aspects of mitotic progression. Theprometaphase chromatin morphology of Mad2-defectivecells appeared disorganized, and condensed metaphaseand anaphase chromosomes were never observed. Instead,the chromatin seemed to decondense rapidly following anabnormal prometaphase. In contrast, DNA condensationoccurred normally in BubR1-depleted HeLa cells despite adecrease in mitotic duration. In addition to the requirementfor Mad2 in checkpoint function, these observations maysuggest additional roles in the regulation of mitotic events,such as chromatin condensation, and could account for thedifferent outcomes induced by paclitaxel in BubR1- andMad2-depleted cells. These observations may also help toreconcile at least some of the differences in the earlierstudies addressing the effect of the spindle checkpoint onthe response to antimicrotubule drugs.Inhibitors of the mitotic kinesin Eg5 are promising new
alternatives to antimicrotubule drugs, such as the taxanesand Vinca alkaloids. Similar to microtubule-targeted drugs,Eg5 inhibition leads to mitotic arrest and apoptosis.However, due to the specific requirement of this kinesinfor mitosis, this approach is expected to have a better sideeffect profile in the clinic (5, 7, 8). Furthermore, Eg5inhibitors have proved to be effective in paclitaxel-resistanttumor cells (65). The results presented here have expandedour understanding of the antiproliferative and proapop-totic effects of monastrol and also revealed both similaritiesand significant differences between Eg5 inhibition and themicrotubule-targeted drug paclitaxel. Finally, our resultssuggest that Eg5-targeted therapeutics should be effectivein spindle checkpoint–compromised tumor cells, irrespec-tive of the underlying checkpoint defect.
Acknowledgments
We thank Emma Lees, Wolfgang Seghezzi, Xiaomin Schebye, MarcelTanudji, Jerelyn Wong, Ulka Vijapurkar, and Wei Wang for helpfuldiscussions and critical reading of the manuscript. DNAX ResearchInstitute of Molecular and Cellular Biology Research is a subsidiary of theSchering-Plough Corporation.
1. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs.Nat Rev Cancer 2004;4:253–65.
2. Wood KW, Cornwell WD, Jackson JR. Past and future of the mitoticspindle as an oncology target. Curr Opin Pharmacol 2001;1:370–7.
3. Sawin KE, LeGuellec K, Philippe M, Mitchison TJ. Mitotic spindleorganization by a plus-end-directed microtubule motor. Nature 1992;359:540–3.
4. Blangy A, Lane HA, d’Herin P, Harper M, Kress M, Nigg EA.Phosphorylation by p34cdc2 regulates spindle association of humanEg5, a kinesin-related motor essential for bipolar spindle formation in vivo .Cell 1995;83:1159–69.
5. Mandelkow E, Mandelkow EM. Kinesin motors and disease. Trends CellBiol 2002;12:585–91.
6. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, MitchisonTJ. Small molecule inhibitor of mitotic spindle bipolarity identified in aphenotype-based screen. Science 1999;286:971–4.
7. DeBonis S, Skoufias DA, Lebeau L, et al. In vitro screening forinhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumoractivities. Mol Cancer Ther 2004;3:1079–90.
8. Sakowicz R, Finer JT, Beraud C, et al. Antitumor activity of a kinesininhibitor. Cancer Res 2004;64:3276–80.
9. Kapoor TM, Mayer TU, Coughlin ML, Mitchison TJ. Probing spindleassembly mechanisms with monastrol, a small molecule inhibitor of themitotic kinesin, Eg5. J Cell Biol 2000;150:975–88.
10. Compton DA. Spindle assembly in animal cells. Annu Rev Biochem2000;69:95–114.
11. Karsenti E, Vernos I. The mitotic spindle: a self-made machine.Science 2001;294:543–7.
12. Musacchio A, Hardwick KG. The spindle checkpoint: structuralinsights into dynamic signalling. Nat Rev Mol Cell Biol 2002;3:731–41.
13. Amon A. The spindle checkpoint. Curr Opin Genet Dev 1999;9:69–75.
14. Chan GK, Jablonski SA, Sudakin V, Hittle JC, Yen TJ. HumanBUBR1 is a mitotic checkpoint kinase that monitors CENP-E functionsat kinetochores and binds the cyclosome/APC. J Cell Biol 1999;146:941–54.
15. Fang G. Checkpoint protein BubR1 acts synergistically with Mad2 toinhibit anaphase-promoting complex. Mol Biol Cell 2002;13:755–66.
16. Sudakin V, Chan GK, Yen TJ. Checkpoint inhibition of the APC/C inHeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.J Cell Biol 2001;154:925–36.
17. Wu H, Lan Z, Li W, et al. p55CDC/hCDC20 is associated with BUBR1and may be a downstream target of the spindle checkpoint kinase.Oncogene 2000;19:4557–62.
18. Baker DJ, Jeganathan KB, Cameron JD, et al. BubR1 insufficiencycauses early onset of aging-associated phenotypes and infertility in mice.Nat Genet 2004;36:744–9.
19. Cahill DP, Lengauer C, Yu J, et al. Mutations of mitotic checkpointgenes in human cancers. Nature 1998;392:300–3.
20. Dai W, Wang Q, Liu T, et al. Slippage of mitotic arrest and enhancedtumor development in mice with BubR1 haploinsufficiency. Cancer Res2004;64:440–5.
21. Hanks S, Coleman K, Reid S, et al. Constitutional aneuploidy andcancer predisposition caused by biallelic mutations in BUB1B. Nat Genet2004;36:1159–61.
22. Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty VV,Benezra R. Complete loss of the tumor suppressor MAD2 causespremature cyclin B degradation and mitotic failure in human somatic cells.Proc Natl Acad Sci U S A 2004;101:4459–64.
23. Saeki A, Tamura S, Ito N, et al. Frequent impairment of the spindleassembly checkpoint in hepatocellular carcinoma. Cancer 2002;94:2047–54.
24. Shin HJ, Baek KH, Jeon AH, et al. Dual roles of human BubR1,a mitotic checkpoint kinase, in the monitoring of chromosomal instability.Cancer Cell 2003;4:483–97.
25. Kasai T, Iwanaga Y, Iha H, Jeang KT. Prevalent loss of mitotic spindlecheckpoint in adult T-cell leukemia confers resistance to microtubuleinhibitors. J Biol Chem 2002;277:5187–93.
26. Sudo T, Nitta M, Saya H, Ueno NT. Dependence of paclitaxel
sensitivity on a functional spindle assembly checkpoint. Cancer Res 2004;64:2502–8.
27. Masuda A, Maeno K, Nakagawa T, Saito H, Takahashi T. Associationbetween mitotic spindle checkpoint impairment and susceptibility to theinduction of apoptosis by anti-microtubule agents in human lung cancers.Am J Pathol 2003;163:1109–16.
28. Lee EA, Keutmann MK, Dowling ML, Harris E, Chan G, Kao GD.Inactivation of the mitotic checkpoint as a determinant of the efficacy ofmicrotubule-targeted drugs in killing human cancer cells. Mol Cancer Ther2004;3:661–9.
29. Sihn CR, Suh EJ, Lee KH, Kim TY, Kim SH. p55CDC/hCDC20 mutantinduces mitotic catastrophe by inhibiting the MAD2-dependent spindlecheckpoint activity in tumor cells. Cancer Lett 2003;201:203–10.
30. Tao W, South VJ, Zhang Y, et al. Induction of apoptosis by aninhibitor of the mitotic kinesin KSP requires both activation of thespindle assembly checkpoint and mitotic slippage. Cancer Cell 2005;8:49–59.
31. Kanda T, Sullivan KF, Wahl GM. Histone-GFP fusion protein enablessensitive analysis of chromosome dynamics in living mammalian cells.Curr Biol 1998;8:377–85.
32. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T.Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells. Nature 2001;411:494–8.
33. Martin-Lluesma S, Stucke VM, Nigg EA. Role of Hec1 in spindlecheckpoint signaling and kinetochore recruitment of Mad1/Mad2. Science2002;297:2267–70.
34. Tanudji M, Shoemaker J, L’Italien L, Russell L, Chin G, Schebye XM.Gene silencing of CENP-E by small interfering RNA in HeLa cells leads tomissegregation of chromosomes after a mitotic delay. Mol Biol Cell 2004;15:3771–81.
35. Rieder CL, Maiato H. Stuck in division or passing through: whathappens when cells cannot satisfy the spindle assembly checkpoint. DevCell 2004;7:637–51.
36. Bomont P, Maddox P, Shah JV, Desai AB, Cleveland DW. Unstablemicrotubule capture at the centromere-associated protein CENP-F. EMBOJ 2005;24:3927–39.
37. Cochran JC, Gatial JE, III, Kapoor TM, Gilbert SP. Monastrol inhibitionof the mitotic kinesin Eg5. J Biol Chem 2005;280:12658–67.
38. DeBonis S, Simorre JP, Crevel I, et al. Interaction of the mitoticinhibitor monastrol with human kinesin Eg5. Biochemistry 2003;42:338–49.
39. DeBonis S, Skoufias DA, Lebeau L, et al. In vitro screening forinhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumoractivities. Mol Cancer Ther 2004;3:1079–90.
40. Wang TH, Wang HS, Soong YK. Paclitaxel-induced cell death. Cancer2000;88:2619–28.
41. Giannakakou P, Robey R, Fojo T, Blagosklonny M. Low concen-trations of paclitaxel induce cell type-dependent p53, p21 and G1/G2
arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity. Oncogene 2001;20:3806–13.
42. Chill DP, Lengauer C, Yu J, et al. Mutations of mitotic checkpointgenes in human cancers. Nature 1998;392:300–3.
43. Davis FM, Tsao TY, Fowler SK, Rao PN. Monoclonal antibodies tomitotic cells. Proc Natl Acad Sci U S A 1983;80:2926–30.
44. Jaattela M. Multiple cell death pathways as regulators of tumourinitiation and progression. Oncogene 2004;23:2746–56.
45. Kops GJ, Foltz DR, Cleveland DW. Lethality to human cancer cellsthrough massive chromosome loss by inhibition of the mitotic checkpoint.Proc Natl Acad Sci U S A 2004;101:8699–704.
46. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485–95.
47. Robbins AR, Jablonski SA, Yen TJ, et al. Inhibitors of histonedeacetylases alter kinetochore assembly by disrupting pericentromericheterochromatin. Cell Cycle 2005;4:717–26.
48. Muller C, Gross D, Sarli V, et al. Inhibitors of kinesin Eg5:antiproliferative activity of monastrol analogues against human glioblas-toma cells. Cancer Chemother Pharmacol. Epub 2006 May 16.
49. Cox CD, Breslin MJ, Mariano BJ, et al. Kinesin spindle protein (KSP)inhibitors: Part 1. The discovery of 3,5-diaryl-4,5-dihidropyrazoles aspotent and selective inhibitors of the mitotic kinesin KSP. Bioorg MedChem Lett 2005;15:2041–5.
50. Brier S, Lemaire D, Debonis S, Forest E, Kozielski F. Moleculardissection of the inhibitor binding pocket of the mitotic kinesin Eg5reveals mutants that confer resistance to antimitotic agents. J Mol Biol2006; epub.
51. Brier S, Lemaire D, Debonis S, Forest E, Kozielski F. Identification ofthe protein binding region of S-trityl-L-cystein, a new potent inhibitor ofthe mitotic kinesin Eg5. Biochemistry 2004;43:13072–82.
52. Bergnes G, Brejc K, Belmont L. Mitotic kinesins: prospects forantimitotic drug discovery. Curr Top Med Chem 2005;5:127–45.
53. Kienitz A, Vogel C, Morales I, Muller R, Bastians H. Partial down-regulation of MAD1 causes spindle checkpoint inactivation and aneuploi-dy, but does not confer resistance towards Taxol. Oncogene 2005;24:4301–10.
54. Descamps S, Lebourhis X, Delehedde M, Boilly B, Hondermarck H.Nerve growth factor is mitogenic for cancerous but not normal humanbreast epithelial cells. J Biol Chem 1998;273:16659–62.
55. Chen J-G, Horwitz SB. Differential mitotic responses to microtubule-stabilizing and -destabilizing drugs. Cancer Res 2002;62:1935–8.
57. McDaid HM, Lopez-Barcons L, Grossman A, et al. Enhancement of thetherapeutic efficacy of paclitaxel by the mitogen-activated protein kinaseinhibitor CI-1040 in nude mice bearing human heterotransplants. CancerRes 2005;65:2854–60.
58. Boldt S, Weidle UH, Kolch W. The role of MAPk pathways in theaction of chemotherapeutic drugs. Carcinogenesis 2002;23:1831–8.
59. Morse DL, Gray H, Payne CM, Gillies RJ. Docetaxel induces cell deaththrough mitotic catastrophe in human breast cancer cells. Mol Cancer Ther2005;4:1495–504.
60. Leizerman I, Avunie-Masala R, Elkabets M, Fich A, Gheber L.Differential effects of monastrol in two human cell lines. Cell Mol LifeSci 2004;61:2060–70.
61. Skoufias DA, Andreassen PR, Lacroix FB, Wilson L, Margolis RL.Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachmentand kinetochore-tension checkpoints. Proc Natl Acad Sci U S A 2001;98:4492–7.
62. Gorbsky GJ, Chen RH, Murray AW. Microinjection of antibody toMad2 protein into mammalian cells in mitosis induces prematureanaphase. J Cell Biol 1998;141:1193–205.
63. Meraldi P, Draviam VM, Sorger PK. Timing and checkpoints in theregulation of mitotic progression. Dev Cell 2004;7:45–60.
64. Canman JC, Sharma N, Straight A, Shannon KB, Fang G, Salmon ED.Anaphase onset does not require the microtubule-dependent depletion ofkinetochore and centromere-binding proteins. J Cell Sci 2002;115:3787–95.
65. Marcus AI, Peters U, Thomas SL, et al. Mitotic kinesin inhibitorsinduce mitotic arrest and cell death in Taxol-resistant and -sensitive cancercells. J Biol Chem 2005;280:11569–77.
