Centmitor-1, a Novel Acridinyl-Acetohydrazide, Possesses Similar ... · Jenni H.E. M€aki-Jouppila...

Small Molecule Therapeutics

Centmitor-1, a Novel Acridinyl-Acetohydrazide, PossessesSimilar Molecular Interaction Field and Antimitotic CellularPhenotype as Rigosertib, ON 01910.Na

Jenni H.E. M€aki-Jouppila1,2,3,4, Leena J. Laine1,2, Jonathan Rehnberg1, Elli Narvi1,2, Pekka Tiikkainen1,Elvira Hukasova6, Pasi Halonen1, Arne Lindqvist6, Lila Kallio1, Antti Poso5, and Marko J. Kallio1,2

AbstractMitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic

inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and

electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of

phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were

tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohy-

drazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as

rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized

by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle

assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both

compoundsmodulatedmicrotubule plus-ends and reducedmicrotubule dynamics. Also,mitotic spindle forces

were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results

showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate

antimitotic effectswhen added to cells duringmitosis. Analysis of Plk1 activity in cells using a Forster resonance

energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken

together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic

processes without impairment of Plk1 kinase activity. Mol Cancer Ther; 13(5); 1054–66. �2014 AACR.

IntroductionMitosis has been a target of anticancer therapies for

decades. The earliest mitosis-perturbing drugs were anti-tubulin agents such as taxanes and vinca alkaloids, andtheir derivatives (1, 2). These drugs inhibit microtubuleassembly or disassembly dynamics by targeting tubulinsubunits, thebuildingblocksofmicrotubules.Microtubulesundergo major rearrangements during mitosis: within a

short period of time, interphase microtubules are disas-sembled and reassembled to form themitotic spindle appa-ratus that is needed for ordered chromosome segregationand exit from M phase. Tubulin-targeting drugs interferewith these processes and impair normal spindle functionand chromosome alignment at the metaphase plate (3, 4).This leads to persistent activity of the spindle assemblycheckpoint (SAC; ref. 5), typically resulting in a long-lastingmitotic arrest and ultimately cell death, a phenomenonthat possesses therapeutic value. Since the discovery oftubulin-targeting agents, several novel antimitotic com-pounds have been developed to specifically target mitotickinases andmotorproteins (for reviews see refs. 6, 7). Theseagents are expected to reduce side effects, such as neuro-toxicity, associated with antitubulin drugs (1, 2) and pro-vide new therapeutic opportunities for the treatment ofcancer pending on their successful clinical validation.

One interestinganticancercompound that is currently inphase II and III clinical trials for the treatment of bloodmalignancies and solid tumors (8–10) is rigosertib (ON01910.Na) developed by Onconova. Rigosertib functionsby inducing multipolarity, mitotic arrest, and subsequentcell death (11). The mechanism of action of rigosertib wasinitially reported to occur via the inhibition of polo-likekinase 1 (Plk1; ref. 11), an important mitotic regulator thatis necessary for spindle assembly, chromosomealignment,

Authors' Affiliations: 1VTT Health, VTT Technical Research Centre ofFinland; 2Centre for Biotechnology and 3Department of Pharmacology,Drug Development and Therapeutics, University of Turku, Turku; 4DrugResearch Doctoral Programme and FinPharma Doctoral Program DrugDiscovery; 5School of Pharmacy, University of Eastern Finland, Kuopio,Finland; and 6Department of Cell and Molecular Biology, Karolinska Insti-tutet, Stockholm, Sweden

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

J.H.E. M€aki-Jouppila and L.J. Laine contributed equally to this article.

Current address of P. Halonen: Division of Molecular Carcinogenesis, NKI,1066CX Amsterdam, the Netherlands.

CorrespondingAuthor:Marko J. Kallio, VTT Technical ResearchCentre ofFinland, It€ainen Pitk€akatu 4 C, 20521 Turku, Finland. Phone: 358-24788614; Fax: 358-207222840; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0685

�2014 American Association for Cancer Research.

MolecularCancer

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Mol Cancer Ther; 13(5) May 20141054

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and cytokinesis (12, 13). Because Plk1 is often overex-pressed in cancer cells and its inhibition lowers tumorcells’ viability (14), it is considered to be a promising drugtarget (15). In addition to targeting Plk1, rigosertib hasbeen reported to showactivity against phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase(MAPK) pathways (11, 16, 17). However, the mechanismof action of the compound is not completely understood.Here, we designed and executed a high-throughput

screen (HTS) to identify low molecular weight (LMW)compounds that possess similar steric and electrostaticfeatures but different chemical structure as rigosertib. Inthis study, we characterize a novel LMW compound, anacridinyl-acetohydrazide (C22H16BrN3O3), here termedasCentmitor-1 (Cent-1).We analyze and compare the effectsofCent-1 and rigosertib ondifferent aspects ofmitosis andmicrotubule dynamics.

Materials and MethodsChemicalsCent-1 (ChemBridge Corporation; 5676127)was used at

5 mmol/L concentration and rigosertib (ON 01910.Na;Selleck Chemicals; S1362) at 250 nmol/L concentrationunless stated otherwise. Nocodazole (Sigma; M1404) wasused at 0.5 to 3 mmol/L, taxol (Paclitaxel; Sigma; T7191) at0.1 to 0.6 mmol/L, thymidine (Sigma; T9250) at 2 mmol/L,insulin (Sigma; 19278) at 100 ng/mL, vinblastine (Sigma;V1377) at 3 mmol/L, staurosporine (Sigma;, S5921) at 1mmol/L, ZM447439 (Tocris Bioscience; 2458) at 5 mmol/L,MG132 (Sigma; C2211) at 10 to 20 mmol/L, monastrol(Sigma; M8515) at 100 mmol/L, dimethylenastron (AlexisBiochemicals; ALX-270-438) at 5 mmol/L, BI2536 (SelleckChemicals; S1109) at 200nmol/L, andwortmannin (TocrisBioscience; 1232) at 0.2 to 1 mmol/L concentration.

Cell cultureHeLa cells (ATCC CCL-2, obtained 2006) were grown in

Dulbecco’sModified EagleMedium (DMEM) supplemen-ted with 10% FBS, penicillin–streptomycin (0.1 mg/mL),L-glutamine (2 mmol/L), sodium pyruvate (1 mmol/L),HEPES (20 mmol/L), and nonessential amino acids(0.1 mmol/L). HeLa cells expressing H2B-GFP andmCherry tubulin were a kind gift from Stephan Geley’slaboratory (Medical University, Innsbruck, Austria), ob-tained in 2012. Their growth medium was supplied with250 mg/mL of G418. A549 cells (ATCC CCL-185, obtained2005) expressing GFP-tubulin were cultured in RPMI 1640(Gibco) supplemented with 10% FBS, penicillin–strepto-mycin (0.1 mg/mL), and L-glutamine (2 mmol/L). U2OScells stably expressing a Forster resonance energy transfer(FRET)-based Plk1 reporter (obtained in 2010 from thelaboratory of Ren�e Medema, the Netherlands CancerInstitute, Amsterdam, the Netherlands), described in 18,were cultured in DMEM with GlutaMAX (Invitrogen)supplemented with 6% heat-inactivated FBS and 1% pen-icillin–streptomycin. No authentication of the cell lineswas done by the authors.

For analyzing SAC activity by Western blot, HeLa cellswere synchronized with thymidine to G1–S border (cellswere incubated with 2 mmol/L thymidine for 19 hours,followedby8-hour release and second thymidine block for16 hours) and released into 5mmol/LCent-1 or 0.1mmol/Ltaxol for 12 hours. ZM447439 (20 mmol/L) was added tomitotic cells for 2 hours before cell harvest for Westernblotting. For analyzing PI3K/AKT pathway, HeLa cellswere starved in serum-free medium for 16 hours, treatedwith dimethyl sulfoxide (DMSO), 5 mmol/L Cent-1,250 nmol/L rigosertib or 1 mmol/Lwortmannin for a totalof 3 hours and 100 ng/mL insulin for 30 minutes.

Virtual HTSCompound structures for the virtual screen were col-

lected from four different vendors: Chembridge (Chem-Bridge Corporation), Chemical Diversity, Tripos (TriposInternational), and Micro Source. Altogether 65,000 com-pound structures were used in the virtual HTS. BrutusandAlmond softwareswere used for the superimpositionof the compound library against the template compoundrigosertib, and for similarity searches using three-dimen-sional (3D) structure and molecular interaction fields assearch criteria. All compounds were built using the ven-dor-provided sdf files and Sybyl ligand-preparation util-ities with default settings.

Cell-based screenHeLa cells were plated on 384-well plates with Multi-

drop Combi (Thermo Fisher Scientific) and screen com-poundswere added to the cells 24 hours after plating usingHamilton Microlab Star robotics (Hamilton) at 0.2 or 5mmol/L final concentrations. Eg5 inhibitor monastrol wasused as a positive control compound that induces mitoticarrest, and DMSO served as a negative control. Cells wereimaged live with phase contrast optics at 6 and 24 hoursafter treatment followed by fixation and DNA staining.

ImmunofluorescenceCells were fixed for 15 minutes with 2% paraformalde-

hyde in 60mmol/L Pipes, 25mmol/LHepes, 10 mmol/LEGTA, 4mmol/LMgSO4 (PHEM) containing 0.5%Triton-X-100. For imaging microtubules, 0.2% glutaraldehydewas included in the fix. For EB1 immunofluorescence,cells were fixed in ice-cold MeOH for 10 minutes, andrehydrated in PBS. Cells were washed with 10 mmol/LMOPS, 150 mmol/L NaCl, and 0.05% Tween 20 (MBST),blocked in MBST containing 20% boiled normal goatserum for 1 hour at room temperature, and stained withantibodies for 1 hour at room temperature. Primary anti-bodies includedmouse anti-Bub1 (Upstate; 05-899), rabbitanti-BubR1 (Proteinatlas), mouse anti-BubR1 (Abcam;ab4637), mouse anti-cenpA (Abcam; ab13939), mouseanti-CETN3 (Abnova; H00001070-M01), human autoim-mune serum (Crest; Antibodies Inc.), mouse-anti-EB1EA3 (kind gift from Prof. Gary Gorbsky, OklahomaMed-ical Research Foundation, Oklahoma City, OK), mouseanti-NuMA (kind gift from Prof. Markku Kallajoki,

Centmitor-1 Possesses Antimitotic Properties

www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1055




University of Jyvaskyla, Jyvaskyla, Finland), mouse anti-Plk1 (Abcam; 17057), rabbit anti-pericentrin (Abcam;ab4448), rat anti–a-tubulin YL-1/2 (Abcam; ab6160),mouse anti–a-tubulin DM1A (Abcam; ab7291), andmouse anti–g-tubulin (Abcam; ab11316). Secondary anti-bodies were Alexa Fluor 488, 555, and 647 dyes againstmouse, rabbit, rat, andhumanantigens (Invitrogen).DNAwas stained with DAPI (40,6-diamidino-2-phenylindole).Coverslips were washed with H2O and mounted onmicroscope slides with Vectashield mounting medium(Vector Laboratories; H-1000).

MicroscopyZeiss inverted 200M microscope (Zeiss GmbH)

equipped withMetaMorph software (Molecular Devices)was used for analyzing 384-well plates and fixed cells oncoverslips. Kinetochore intensities were quantified withMetamorph from maximum projections created from aZ-stack of images acquired every 0.5 mm. Zeiss Axiovert200M microscope equipped with spinning disk CSU22confocal scanner (Yokogawa) and SlideBook 5.0 software(Intelligent Imaging Innovations, Inc.)wasused formicro-tubule dynamics measurements and for the analysis ofkinetochore distances. ImageJ was used for image proces-sing and quantification of pole fragmentation. An areawasdrawnaroundeach centrosome so that all centrosomefragments that were seen adjacent to main centrosomeswere included inside the region of interest. Only bipolarcells were chosen for the analysis. The parameters mea-sured were area, aspect ratio (AR¼major axis divided byminor axis), and roundness (formulas available at http://rsbweb.nih.gov/ij/docs/menus/analyze.html).

In vitro tubulin polymerization assayFluorescence-based tubulin polymerization assay (Cyto-

skeleton Inc.; BK011P) was used to determine the effects ofCent-1 on tubulin polymerization in vitro. The assay wasperformed as described by themanufacturer. DMSO, taxol,and vinblastine were used as controls. Tubulin polymeri-

zationwas recorded everyminute for 60minutes by Victor1420 Multilabel HTS Counter (PerkinElmer).

Determination of microtubule dynamicityMicrotubule dynamicity measurements were per-

formed in live A549 cells stably expressing EGFP-a-tubu-lin (BD Biosciences; Clontech) using the spinning diskconfocal microscope with 100� oil objective. Measure-ments were performed 1 to 2 hours after compoundaddition on cells. Z-stacks containing four focus levelswith 0.3-mm step size were acquired every 10 secondsduring a filming session that lasted for 250 seconds. Theplus-ends of microtubules were digitally marked in therecorded time-lapsemovies to allowmeasurement of theirdynamicity. Dynamicity refers here to the combinedgrowth and shrinkage velocities of the microtubules,which were determined as a measurement of the distancethat themicrotubule plus-end traveled in a specific periodof time. Ten to 20 microtubules per cell and 5 to 8 cells persample were analyzed in each of three independentexperiments. Microtubule dynamics data were analyzedusing SlideBook 5.0 and Graph Pad Prism 4 (GraphPadSoftware, Inc.).

FRET for analysis of Plk1 kinase activityU2OS cells stably expressing a Plk1-responsible FRET-

based probe (18) were monitored on a DeltaVision Spec-tris Imaging system (Applied Precision), using a NA 0.7520� air objective. Cells were imaged at 37�C in Liebowitz-15 medium supplemented with 6% heat-inactivated FBSand 1% penicillin-streptomycin. Images were acquiredevery 20minutes andprocessedusing ImageJ (http://rsb.info.nih.gov/ij/). Imaging and quantification of FRETratio was performed as described previously (19).

ResultsDiscovery of Cent-1, an antimitotic LMW compound

To identify small-molecule analogs of rigosertib(Fig. 1A), we performed a ligand-based virtual HTS, in

Figure 1. Comparison of rigosertib and Cent-1 structures. A, structure of the template compound rigosertib (ON 01910.Na). B, hit compound N0-(3-bromo-4-hydroxybenzylidene)-2-(9-oxo-10(9H)-acridinyl)acetohydrazide named Cent-1. C, Brutus-aligned structures of rigosertib (blue ball-and-stick model) andCent-1 (gray/turquoise stick model) are shown. Brutus score of 1.62 indicates exceptionally high level of similarity in 3D structure and electrostatic potentialbetween the two compounds. Oxygens are colored red and sulfur yellow.

M€aki-Jouppila et al.

Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics1056




which 65,000 LMW compounds were screened for thosethat have similar interaction fields and 3D structures asthe template compound. Virtual screeningwas conductedusing Almond and Brutus (20–22), field-based molecularalignment, and virtual screening tools that take intoaccount both steric and electrostatic features of the mole-cules. One criterion was that the compounds should nothave similar chemical structures as the template com-pound. Therefore, compounds having similar backbonestructures as rigosertib were omitted from the hit list. The200 top-ranking compounds from each hit list were pur-chased and subjected to secondary cell-based screens, inwhich HeLa cells were treated with the compounds andanalyzed formitotic phenotypes. In total, 4 hit compoundsresulted in a notable increase in mitotic index and wereinvestigated further.One of the highest scoring compounds was an acridi-

nyl-acetohydrazide (C22H16BrN3O3) that we named asCent-1 (Fig. 1B) due to its effects on centrosome morphol-ogy. The field-based similarity score of Cent-1 in compar-ison with rigosertib was 1.62, which is very high (themaximum is 2.0, but less than0.01%of randomcompoundpairs will reach a value higher than 1.5 according to in-house tests), indicating that these molecules closelyresemble one another in their 3D structure and chargedistribution. The superimposition image of Cent-1 andrigosertib is shown in Fig. 1C. Besides the high similarityof Cent-1 to rigosertib in 3D comparisons, the compoundinduced a strong antimitotic phenotype that resembledthe cellular effects of rigosertib. For these reasons, Cent-1was selected for more detailed analyses.

Cent-1 induces a transient mitotic arrest followed byabnormal exit from M phase or cell deathTo analyze the impact of Cent-1 on cell-cycle progres-

sion, the compound (5 mmol/L) was applied on cyclingHeLa cells that were subsequently filmed with IncuCytelive-cell imager using 1-hour image capture interval(Fig. 2A). The fates of 60 individual mitotic cells weredetermined for each treatment condition (Fig. 2B). DMSOserved as a negative control, and nocodazole and taxol asmitotic arrest inducing positive controls. DMSO-treatedcells progressed normally through mitosis (the averageduration of mitosis was 1.5 � 1.8 hours). All nocodazole-and taxol-treated cells arrested inmitosis for several hoursbefore they underwent cell death (average time spent inmitosis before cell death was 14.3 � 5.4 and 16.7 � 5.9hours, respectively). Almost half of Cent-1–treated cells(48.3%) exhibited a long M-phase arrest followed by celldeath (average time spent in mitosis before cell death was14.3� 5.9 hours). Rest of the cells exhibited amitotic delaybefore they exitedmitosis with (38.3%) or without (13.3%)cytokinesis. Cells often died postmitotically soon after theaberrant exit from mitosis (result not shown). For com-parison,wedetermined the fate of rigosertib–treated cells.The majority of rigosertib-treated HeLa cells died inmitosis (76.7%), whereas a smaller proportion exited Mphase with (16.7%) or without (6.7%) cytokinesis (Sup-

plementary Fig. S1A).We alsodeterminedmitotic and celldeath indices from the same samples at 0-, 12-, 24-, and 36-hour timepoints after exposing the cells to the compounds(Fig. 2C). In Cent-1–treated populations, mitotic indexpeaked at 24 hours (49.4%) and cell death index was thehighest at 36 hours (50.8%). Rigosertib-treated cellscaused a similar accumulation of cells into mitosisfollowed by increase in cell death index (SupplementaryFig. S1B).

As rigosertib was earlier shown to cause apoptosis (11),we analyzed the potential apoptotic effects of Cent-1 onHeLa cells using Annexin V-FITC and propidium iodidestaining followed by flow cytometry, and Western blot-ting with an antibody recognizing cleaved PARP at 12-,24-, and 48-hour time points after adding the compound.Cent-1 caused the accumulation of cells to G2–M-phaseand an increase in the sub-G1 cell fraction in comparisonwith DMSO control (Supplementary Fig. S2A). After 48-hour Cent-1 treatment, 32.8% of cells were in early apo-ptosis and 14.3% in late apoptosis or necrosismeasuredbyAnnexin V-FITC and propidium iodide staining. In com-parison, after DMSO treatment, 10.4% of cells were earlyapoptotic and 8.6% late apoptotic or necrotic (Supplemen-tary Fig. S2B). Furthermore, Cent-1 caused a slightincrease in the level of cleaved PARP (SupplementaryFig. S2C).

Cent-1 induces multipolarity and centrosomefragmentation

Next, the mitotic effects of Cent-1 were investigatedusing a HeLa cell line stably expressing H2B-GFP andmCherry-tubulin. Cells were imaged live immediatelyafter Cent-1 or DMSO was added to the culture medium(Supplementary Movies S1 and S2). All Cent-1–treatedcells exhibited a long prometaphase delay that was fol-lowed by death or abnormal exit from mitosis. Typically,cells assembled multipolar spindles immediately uponnuclear envelope breakdown (NEB) and underwent mul-tipolar anaphase after spending several hours in prome-taphase (SupplementaryMovie S1). Alternatively, a num-ber of cells formed bipolar spindles through fusion ofmultiple poles during the prometaphase delay (Supple-mentaryMovie S1) or established a bipolar spindles uponentry into mitosis. Many chromosomes in these cellscongressed to themetaphase plate, indicating that spindlefibers and kinetochoreswere capable of establishing func-tional connections. However, cells always exhibited anumber of chromosomes that remained unaligned nearthe spindle poles (SupplementaryMovie S1). Control cellsestablished a bipolar spindle and divided normally (Sup-plementary Movie S2).

To confirm the live-cell imaging findings, DMSO- orCent-1–treatedHeLa cells were fixed and immunostainedwith various antibodies. Cent-1 treatment caused hetero-geneous anomalies that ranged from cells having bipolarspindles with a few unaligned chromosomes to cellshavingmultipolar spindles and fullymisaligned chromo-somes (Fig. 3A). The proportion of multipolar cells was






28.5% after 8-hour treatment with the drug (Fig. 3B) andincreased during longer incubation times (37.5% at 24hours and 44%at 48hours). Similarly, rigosertib treatmentresulted in a mixture of bipolar and multipolar cells(Supplementary Fig. S3).

Rigosertib was earlier shown to reduce the centrosome-associated pool of g-tubulin and cause centrosome frag-mentation (11). To evaluate the possible effects of Cent-1on centrosomes, cellswere stainedwith antibodies againstg-tubulin, pericentrin, and centrin after 6-hour Cent-1treatment. In these cells, g-tubulinwas greatly diminishedat spindle poles (Fig. 3C). Moreover, based on pericentrinstaining, the centrosomes were disintegrated (Fig. 3D).

Using morphometric analysis, we detected significantdifferences (P < 0.01–P < 0.001) in the following centro-someparameters: area, roundness, andARbetweenCent-1and DMSO, and between rigosertib and DMSO treat-ments (Fig. 3E). Furthermore, Cent-1–induced multipolarcells exhibited a-tubulin–positive foci that were oftennegative for pericentrin and centrin, suggesting that thesefoci lack normal centrosome constituents (Fig. 3F). Thesame observation was made in rigosertib–induced mul-tipolar cells (data not shown). In addition to havingcentrosome defects, Cent-1- and rigosertib-treated cellsexhibited shorter spindle length compared with control(Fig. 3G). In control cells treated with DMSO, the average

Figure 2. Cent-1 treatment inducesmitotic arrest followedby cell deathor forced mitotic exit. A,representative still images from thetime-lapse films of HeLa cellstreated with the indicatedcompounds. In DMSO, cellsundergo normal cell division. In thepresence of nocodazole (Noc) andtaxol, cells accumulate to mitosisand die after a long arrest. Cent-1–treated cells first arrest in mitosisfor several hours and theneither diefrom the arrest or undergo a forcedexit from M phase with or withoutcytokinesis. Cells were imagedwith IncuCyte after the addition ofDMSO, 5 mmol/L Cent-1, 3 mmol/LNoc, or 0.6 mmol/L taxol. Numbers,hours starting from compoundaddition. Arrows mark examplemitotic cells. Scale bar, 100 mm. B,graphs show the length of mitosisand fates of individual cells (n¼ 60)after the indicated treatments. C,graphs showmitotic and cell deathindices that were determined fromthe time-lapse films at differenttime points following the additionof the indicated compounds. Data,mean � SD from three film frames.






spindle lengthwas 10.4� 1.0 mm,whereasCent-1 reducedthe spindle length to 8.9� 1.4mm(P < 0.001) and rigosertibto 8.0 � 0.9 mm (P < 0.001).One mitotic protein contributing to the formation of

spindle poles and maintenance of normal spindle archi-

tecture is the nuclearmitotic apparatus protein 1 (NuMA;ref. 23). Interestingly, depletion of NuMA causes defectsin chromosome alignment (24), resembling the phenotypecaused by Cent-1 and rigosertib. Treatment of cells withCent-1 or rigosertib caused a prominent redistribution of

Figure 3. Cent-1 treatment causes chromosome alignment defects, multipolarity, and centrosome fragmentation. A, representative micrographs of DMSO-and Cent-1–treated HeLa cells showing bipolar and multipolar mitotic phenotype and abnormal chromosome orientations. B, graphs showing thequantification of spindle pole number (based ona-tubulin staining) at different time points after Cent-1 addition. DMSO treatment was for 8 hours. Data,mean� SD from two separate experiments. A total of 100 cells were analyzed per time point in both experiments. C and D, representative micrographs of DMSO-and Cent-1–treated HeLa cells after staining with g-tubulin or pericentrin antibodies showing reduced g-tubulin staining and spindle pole fragmentation.E, graph shows centrosome area, roundness, and AR according to the quantification of pericentrin-stained foci. Data, mean � SD of 40 cells from twoseparate experiments. F, micrographs show a representative multipolar cell after Cent-1 treatment stained with antibodies against a-tubulin, pericentrin, andcentrin. The arrow points to an a-tubulin–positive pole that is negative for pericentrin and centrin. G, the graph shows quantification of spindle lengthfrom bipolar cells stained for pericentrin after the indicated treatments. Data, mean� SD from two separate experiments (n¼ 40 cells). H, micrographs showNuMA localization in cells treated with DMSO, Cent-1, or rigosertib. Cells were incubated in the presence of DMSO, 5 mmol/L Cent-1, or 250 nmol/L rigosertibfor 6 hours unless stated otherwise. High-magnification views of one centrosome are shown in C, D, and F. Asterisks, statistically significant differencesbetween the controls and indicated compound treatments (��, P < 0.01; ��, P < 0.001). Scale bars, 10 mm.






NuMAfrom the spindlepoles to several newfoci through-out the cytosol in comparison with control (Fig. 3H).Collectively, these data show that Cent-1 and rigosertibinduce multipolarity and cause the disintegration of thecentrosomes.

Mitotic checkpoint is active in Cent-1–treated cellsTo investigate why cells arrest in mitosis after Cent-1

treatment, we analyzed the levels of proteins involvedin SAC signaling. The kinetochore occupancy of SACproteins BubR1 and Bub1 at the unaligned chromo-somes is a common marker for an active SAC (25,26). To analyze SAC activity, HeLa cells were incubatedin the presence of Cent-1, or control compounds (taxolor DMSO) for 6 hours. After fixation and immunoflu-orescence staining, BubR1 and Bub1 levels were quan-tified at the kinetochores of unaligned chromosomesnear the spindle poles. Only bipolar cells were chosenfor the analysis, because it was more feasible to cate-gorize aligned/unaligned chromosomes in comparisonwith multipolar cells. Similarly to control metaphasecells, BubR1 and Bub1 were mostly undetectable at thekinetochores of metaphase-aligned chromosomes inCent-1–treated cells (Fig. 4A and B). However, in thesame cells, the levels of both proteins remained elevatedat the kinetochores of unaligned chromosomes, indicat-ing continued SAC activity (Fig. 4C and D). Also inrigosertib-treated cells the kinetochores of unalignedchromosomes were positive for BubR1 and Bub1 (Sup-plementary Fig. S4).

In another approach, we used an Aurora B kinaseinhibitor ZM447439 that is known to cause prematureSAC inactivation, leading to forced exit fromMphase (27).Hela cells were accumulated into mitosis by treatmentwith Cent-1 or taxol, collected and further incubated withor without ZM447439 for 2 hours in the continued pres-ence of Cent-1 or taxol. If mitotic arrest is Aurora B andSAC-mediated, cells should exitmitosis within the 2-hourincubation period. This was the case for both Cent-1 andtaxol; the levels ofmitoticmarkers cyclin B1 andphospho-histone H3 were greatly reduced upon ZM447439 treat-ment (Fig. 4E).

Cent-1 and rigosertib modulate microtubuledynamics and microtubule plus-ends

It is well established that interference with microtu-bules leads to defects in spindle assembly, resulting in aSAC-mediated mitotic arrest (28). To test whether Cent-1impairs microtubule functions, we first investigatedwhether the compound directly affects tubulin polymer-ization in vitro. The polymerization rate of tubulin wasmonitored in the presence of various concentrations ofCent-1. Vinblastine that induces tubulin depolymeriza-tion and taxol that increases polymerization rate served ascontrols. Both reference drugs affected the rate of tubulinpolymerization as expected,whereas the additionofCent-1 at 1 or 5 mmol/L concentrations had no effect on thereaction (Fig. 5A). Higher concentrations (10 and 20

mmol/L) of Cent-1 resulted in a slight increase in tubulinpolymerization rate (Fig. 5A). It was previously reportedthat rigosertib does not affect the rate of tubulin polymer-ization in vitro althoughonly one concentration (5mmol/L)was tested (11).

Next, we investigated the effects of Cent-1, rigosertib,and control drugs nocodazole and taxol on the microtu-bule network of interphase cells using immunofluores-cence (Fig. 5B). HeLa cells were treated with the com-pounds for 6 hours, fixed and immunostained with anantibody against a-tubulin to visualize microtubules. Asexpected, nocodazole disrupted themicrotubule networkin comparison with DMSO, whereas taxol caused micro-tubule bundling (Fig. 5B). Cent-1 treatment caused veryminor, if any, changes compared with the DMSO control;the mass and shape of interphase cell microtubulesseemed normal (Fig. 5B). Only slight curving of micro-tubules was observed in some cells. Similarly, rigosertibincubation induced slight changes to the microtubulenetwork of interphase cells at 250 nmol/L concentration(Fig. 5B). It was previously published that at high con-centration (2.5 mmol/L), rigosertib depolymerizes micro-tubules (29) and our experiments confirmed this finding,most microtubules were lost after 6-hour incubation with2.5 mmol/L rigosertib (Supplementary Fig. S5).

Next, we fixed and immunostained Cent-1- or rigoser-tib-treated cells with an antibody against end-bindingprotein 1 (EB1), which is a highly conserved microtubuleplus-end protein that is needed for normal microtubuledynamics (30, 31). In DMSO-treated control cells, EB1showed a characteristic comet-like staining pattern at thetips of growingmicrotubuleplus-ends (Fig. 5C).AlthoughCent-1 and rigosertib did not have major effects on themicrotubule filaments in cells at low concentrations (Fig.5B), the same treatments caused notable changes in thelocalization of EB1 in interphase cells; EB1 comets weremuch shorter and seemed fragmented (Fig. 5C).

This prompted us to investigate whether microtubuledynamics was affected by the compounds in cells. Themodel used was a human A549 lung carcinoma cell linestably expressing EGFP-a-tubulin that we have earliervalidated for the measurement of microtubule dynamics(32). In A549 EGFP-a-tubulin cells, 6-hour treatment with5 mmol/L Cent-1 caused a similar phenotype as in HeLacells; the cells showed chromosome misalignment andmultipolarity, and exhibited a long mitotic delay (Sup-plementary Fig. S6A and S6B). Moreover, 5 mmol/L Cent-1 had only minor effects on interphase cell microtubules(Supplementary Fig. S6C). Interestingly, these cells weremuch more sensitive to rigosertib treatment than HeLacells, and treatment with 250 nmol/L concentration ofrigosertib completely abolished microtubules (data notshown), whereas 50 nmol/L concentration did not elim-inate microtubules but was sufficient to cause mitoticarrest (Supplementary Fig. S6A–S6C). For determiningmicrotubule dynamics, A549 EGFP-a-tubulin cells weretreated with DMSO, 5 mmol/L Cent-1, or 50 nmol/Lrigosertib for 1 to 2 hours. The cells were then imaged






live and microtubule plus-ends were tracked manuallyto determine average microtubule dynamicity (the dis-tance a microtubule plus-end travels in a set time) foreach treatment. In control cells (n ¼ 21), the averagemicrotubule dynamicity was 0.096� 0.036 mm/swhereasin Cent-1 (n ¼ 20) and rigosertib-treated cells (n ¼ 18)overall microtubule dynamicity was reduced to 0.051 �0.019 (P < 0.001) and 0.039 � 0.014 (P < 0.001) mm/s,respectively (Fig. 5D). Representative time-lapse moviesof Cent-1-, rigosertib-, and DMSO-treated cells are avail-able as Supplementary material (Supplementary MoviesS3–S5). On the basis of these results, Cent-1 and rigosertibmodulate microtubule plus-ends and microtubule dyna-micity in interphase cells.

Cent-1 and rigosertib decrease interkinetochoretension by targeting mitotic substrates other thanPlk1To extend the analysis of Cent-1 and rigosertib effects

on microtubules to mitotic cells, we measured interkine-

tochore distances in HeLa cells using Cenp-A as an innerkinetochore marker (33). Generation of tension acrosssister kinetochores stabilizes microtubule–kinetochoreinteractions and is required for SAC inactivation andnormal mitotic progression (5). Cells were treated withCent-1, rigosertib, or controls (DMSO, nocodazole, ortaxol) for 6 hours. Interkinetochore distances were mea-sured separately for chromosomes that had aligned at themetaphase plate and chromosomes that remainedunaligned near the spindle poles in Cent-1- and rigoser-tib-treated cells. Only bipolar cells were selected for theanalysis. Results show that the average interkinetochoredistance of control cells atmetaphase platewas 1.51� 0.09mm, whereas treatment with Cent-1 and rigosertibreduced the distance to 1.07 � 0.09 mm (P < 0.001) and1.06� 0.04mm(P< 0.001), respectively (Fig. 6AandB). Theaverage interkinetochore distances of unaligned chromo-someswere at similar range in control prometaphase cells(0.89 � 0.07 mm), in Cent-1–treated cells (0.85 � 0.02 mm),and in rigosertib-treated cells (0.85 � 0.04 mm). As

Figure 4. Mitotic checkpoint isactive in Cent-1–treated cells. Aand B, representative micrographsshowing BubR1 and Bub1 stainingin control prometaphase andmetaphase cells, and incompound-treated cells. HeLacells were treated with DMSO, 0.1mmol/L taxol, or 5mmol/LCent-1 for6 hours before fixation andimmunostaining. Scale bars, 10mm.C andD,micrographs showingquantification of kinetochore signalintensities of BubR1 and Bub1immunostainings normalizedagainst the Crest signals in cellstreated as indicated. Data, mean�SD from three separateexperiments. Twenty kinetochoresper cell were quantified from 15cells in one experiment, except inCent-1–treated cells, in whichkinetochores of all unalignedchromosomes (4–20 kinetochoresper cell) were quantified. E,Western blot showing cyclin B,phospho-histone H3, andglyceraldehyde-3-phosphatedehydrogenase levels in taxol- orCent-1–treated cell extracts beforeand after the addition of Aurora Binhibitor ZM447439 that causes anoverride of SAC-mediated mitoticarrest.






anticipated, the shortest distances were measured for thecontrol drugs nocodazole (0.77� 0.05 mm) and taxol (0.78� 0.05 mm) that diminish tension to minimum by inter-fering directly with tubulin polymerization. These resultsindicate that although many chromosomes were able tomove to the metaphase plate in Cent-1- and rigosertib-treated cells, the kinetochores were not under normaltension, suggesting that Cent-1 and rigosertib influencemitotic spindle forces.

To study whether Cent-1 and rigosertib elicit their anti-mitotic effects through targeting a protein that is operatingduring M phase, we applied the compounds on mitoticpost-NEB HeLa cells and monitored their fate using Incu-Cyte at 15-minute intervals for 24 hours. Proteasome

inhibitor MG132 and kinesin Eg5 inhibitors monastrol(34) and dimethylenastron were used as controls. Asexpected, MG132 induced amitotic arrest followed by celldeath due to the inhibition of protein degradation essent-ial for anaphase onset and ordered exit fromMphase (35).In agreement with the known early mitotic function ofEg5 in centrosome separation, monastrol and dimethyle-nastron had no effect when applied to post-NEB mitoticcells (34, 36). In sharp contrast, both Cent-1 and rigosertibcaused an immediate effect in mitotic cells; most cellsarrested to M phase for several hours before they died(Fig. 6C). On the basis of these results, the target protein ofCent-1 and rigosertib is present in mitotic cells in which itcontributes to microtubule-mediated processes.

Figure 5. Cent-1 and rigosertib reducemicrotubule dynamicity and causemislocalization of EB1 in cells. A, the graph showing in vitro tubulin polymerization inthe presence of Cent-1 or indicated control compounds. B, representative micrographs of drug-treated interphase HeLa cells immunostained with a-tubulinantibody.Cent-1 and rigosertib inducemild effects on themicrotubule network, whereas the positive control drugs induce strongmicrotubule depolymerizing(Noc) and stabilizing (taxol) effects. C, representative micrographs of drug-treated interphase cells stained with EB1 antibody. HeLa cells were treatedwith DMSO, 5 mmol/L Cent-1, 250 nmol/L rigosertib, 0.5 mmol/L nocodazole (Noc), or 0.1 mmol/L taxol for 6 hours. The morphology of the EB1 comets wasnotably altered by all compound treatments compared with the DMSO control. Scale bars, 10 mm. D, the graph showing changes in microtubule dynamicityupon Cent-1 and rigosertib treatments compared with DMSO. Microtubule dynamicity (the distance a microtubule plus-end travels in a set time) wasmeasured in A549 cells expressing EGFP-a-tubulin after 1-hour incubation with DMSO, 5 mmol/L Cent-1, or 50 nmol/L rigosertib. Each dot in the graphrepresents the averagemicrotubule plus-end velocity per analyzed cell (total number of cells analyzed per treatment was 18–21 from three experiments). Foreach analyzed cell, we recorded 10 to 20 microtubules. Line, the mean microtubule dynamicity in each treatment category. Asterisks, statistically significantdifferences between the controls and indicated treatments (��, P < 0.001).






Figure 6. Cent-1 and rigosertib cause a reduction in interkinetochore distance, and have immediate antimitotic effects without influencing Plk1 kinase activity.A, representative micrographs of control- and drug-treated mitotic cells stained for inner kinetochore marker CenpA (red) and centromere marker Crest(green). HeLa cells were incubated with DMSO, 5 mmol/L Cent-1, 250 nmol/L rigosertib, 0.5 mmol/L nocodazole (Noc), or 0.1 mmol/L taxol for 6 hours beforefixation and immunocytochemistry. Images aremaximumZ-projections of 5 to 7 layers acquired at 0.5-mmstep interval. Insets, higher-magnification views ofselected sister kinetochore pairs. Scale bar, 10 mm. B, the graph shows quantification of interkinetochore distances from control- and drug-treatedcells. Data, mean � SD from three experiments. A total of 100 kinetochore pairs were measured from 10 cells in each experiment, except for unalignedkinetochore pairs in Cent-1 and rigosertib treatments (for these, 1–10 kinetochores were measured per cell). Asterisks, statistically significant differencesbetween the controls and indicated treatments (��,P < 0.001). C, mitotic HeLa cells respond to Cent-1 and rigosertib treatments. Cells in mitosis (n¼ 22–36)were treated with 5 mmol/L Cent-1, 250 nmol/L rigosertib, 10 mmol/L MG132, 100 mmol/L monastrol (Mon), or 5 mmol/L dimethylenastron (DMA) andtheir fate was recorded using time-lapse filming at 15-minute intervals. Graph, the percentage of mitotic cells exhibiting the indicated fates. D and E,U2OScells expressing a FRET-based sensor formeasuring Plk1 activitywere treatedwith 200 nmol/LBI2536, 5 or 10mmol/LCent-1, or 500 nmol/L rigosertib.Each dot represents one cell, n¼ 20 cells/condition. D, duration ofmitosis. Note thatmany of the BI2536-treated cells were still inmitosis when filming ended,and therefore the data for BI2536 underrepresent the actual duration of mitosis. E, quantification of YFP–YFP/CFP–YFP emission ratio of mitotic cellsin D. Plk1 activity is absent in interphase and peaks in mitosis.






Plk1 localization at the kinetochores of individual chro-mosomes changes upon their congression to the cellequator; the protein accumulates to unaligned kineto-chores but is removed upon chromosome alignment tothe metaphase plate (37). It has been reported that failureto remove active Plk1 from the kinetochores of chromo-somes at the metaphase plate leads to decreased inter-kinetochore tension (38). To determine whether Plk1 isaffected by Cent-1 or rigosertib, we analyzed Plk1 local-ization in compound-treated cells. Microscopic analysisdid not reveal any changes from the normal dynamicbehavior of Plk1 at mitotic kinetochores (SupplementaryFig. S7A).

We next measured the effects of Cent-1 and rigosertibon Plk1 kinase activity using U2OS cells stably expressinga FRET-based probe (18). Cent-1 and rigosertib caused amitotic delay in U2OS cells (Fig. 6D). As expected, Plk1activity increased when cells progressed from interphaseto mitosis. The mitotic increase in Plk1 activity was effi-ciently blocked byBI2536 (Fig. 6E), a knownPlk1 inhibitor(13). However, in cells treated with Cent-1 or rigosertib,Plk1 activity stayed at the normal mitotic level (Fig. 6E).Taken together, our results suggest that Cent-1 and ribo-sertib do not act through Plk1 but instead target anothermitotic protein(s).

Finally, we analyzed whether Cent-1 and rigosertibaffect PI3K activity in cells as rigosertib has beenreported to inhibit PI3K in vitro (17). HeLa cells werestarved in serum-free medium and the PI3K/AKT path-way was activated by the addition of insulin (39). On thebasis of the analysis of AKT phosphorylation at Ser473,a marker of PI3K activity (40), the addition of insulinactivated the kinase in the presence of DMSO, Cent-1,and rigosertib, whereas a known PI3K inhibitor wort-mannin completely abolished AKT phosphorylation(Supplementary Fig. S7B and S7C). This result suggeststhat Cent-1 and rigosertib do not function by directlyinhibiting PI3K in HeLa cells.

DiscussionIn this study, we described the identification and char-

acterization of Cent-1, a novel LMW compound thatpossesses similar steric and electrostatic features andantimitotic phenotype as the template compound rigo-sertib, which is a multikinase inhibitor with claimedactivity against PI3K and Plk1 pathways (11, 16, 17). Thetemplate compoundhas showncytotoxic effects inhumantumor cell lines and tumor growth suppression in xeno-graft models (11). At the moment, rigosertib is clinicallyevaluated for the treatment of hematopoietic malignan-cies and solid tumors (9, 10, 41).We analyzed the effects ofCent-1 in cells side by side with rigosertib and relevantcontrol compounds. Cent-1 was found to cause multipo-larity and chromosome misalignment (Fig. 3) that trig-gered a SAC-mediatedM-phase arrest (Fig. 4) followedbymitotic cell deathor aberrant exit fromcelldivision (Fig. 2).Multipolarity was often associated with the formation ofacentrosomal poles that were negative for pericentrin and

centrin (Fig. 3). Furthermore, we detected other spindleand microtubule defects such as reduced microtubuledynamics in interphase cells (Fig. 5) and reduced inter-kinetochore tension in mitotic cells (Fig. 6). Treatment ofcells with rigosertib induced a similar pleiotropic antimi-totic phenotype.

The template compound rigosertib was originally pro-posed to target Plk1 via an allosteric mechanism of action(11), but that notion has been challenged by others(29, 42, 43). Our results showing that cells exhibit strongantimitotic phenotypes upon treatment with Cent-1 orrigosertib without any inhibition of Plk1 activity (Fig. 6)support the view that Plk1 inhibition is not themechanismby which these compounds function. Moreover, whereasCent-1 and rigosertib treatments lead to multipolarity,specific Plk1 inhibition with small molecules causesmonopolarity (13). More recently, rigosertib was shownto inhibit the PI3K/AKT pathway (16, 17). In our analysis,we did not observe significant changes in the phosphor-ylation status ofAKTSer473, a downstream target epitopeof PI3K (40) uponCent-1 or rigosertib treatment in insulin-stimulated cells (Supplementary Fig. S7). This does not,however, exclude the possibility that the compoundscould perturb downstream elements of the PI3K/AKTpathway, or that differences in the experimental setupcould explain the discrepancy between the results.Importantly, because both compounds induced imme-diate M-phase arrest when applied to early mitotic cells,at least one of their target proteins must be present inmitotic cells.

We made three particularly interesting observations inmitotic cells thatmay shed some light to themechanismofaction of Cent-1 and rigosertib duringMphase. First, bothcompounds caused centrosome fragmentation andreduced the amount of centrosome-associated g-tubulin(Fig. 3). Second, they significantly retarded microtubuledynamics in interphase cells (Fig. 5), shortened spindlelength in mitosis (Fig. 3), and decreased tension acrosssister kinetochores inmitotic chromosomes (Fig. 6). Third,the compounds caused delocalization of NuMA (Fig. 3)andEB1 (Fig. 5) inmitotic cells. Together, these data implythat Cent-1 and rigosertib impair microtubule-mediatedprocesses during M phase. It is noteworthy that acentro-somal spindlepoles (44), reduced interkinetochore tension(45), mislocalization of NuMA and EB1 (46–48), and chro-mosome misalignment (45) are all consequences of treat-ment of cells with low doses of microtubule drugs, whichstrengthens the notion that the compoundsmaymodulatemicrotubuledynamics. The compoundsdonot apparentlyperturb microtubule polymerization in vitro at low-rangeconcentrations (Fig. 5; ref. 11).Also, the current data donotallow to concludewhether their impact onmicrotubules incells is direct or indirect. Cent-1 and rigosertibmay impairmicrotubule functions in cells by having an impact onmicrotubule-associatedproteins.At themoment, the iden-tity of suchproteins remainsunknownbut it is noteworthythat depletion of EB1 has been reported to slow downmicrotubule plus-end dynamics and reduce spindle






length (30, 49, 50). It is therefore plausible to hypothesizethat EB1 mislocalization by Cent-1 and rigosertib cancontribute to the observed microtubule defects in cells.In summary, our results provide evidence that ligand-

based in silico HTS is a feasible method for the identifica-tion of novel antimitotic compounds when a knowninhibitor is used as a template. Cent-1 is a new mitoticinhibitor that possesses similar steric and electrostaticfeatures as the template compound rigosertib, whichshows clinical anticancer potency. In cells, both com-pounds induced various defects in mitotic processes thatinvolve dynamic microtubules. However, the precisemechanism of action of Cent-1 and rigosertib remains tobe clarified.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.H.E. M€aki-Jouppila, L.J. Laine, J. Rehnberg,P. Tiikkainen, M.J. KallioDevelopment of methodology: J. Rehnberg, E. Narvi, P. Tiikkainen,P. HalonenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.H.E. M€aki-Jouppila, J. Rehnberg, E. Narvi,E. Hukasova, P. HalonenAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): J.H.E. M€aki-Jouppila, L.J. Laine,J. Rehnberg, E. Narvi, P. Tiikkainen, E. Hukasova, P. Halonen, A. Poso,M.J. Kallio

Writing, review, and/or revision of themanuscript: J.H.E.M€aki-Jouppila,L.J. Laine, E. Narvi, A. Poso, M.J. KallioAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.H.E. M€aki-JouppilaStudy supervision: L.J. Laine, A. Lindqvist, L. Kallio, M.J. Kallio

AcknowledgmentsThe authors thank Jouko Sandholm for the help with flow cytometry

that was performed at the Cell Imaging Core, Turku Centre for Biotech-nology. They also thank Stephan Geley for providing the HeLa H2B-GFPmCherry-tubulin cell line, and professors Markku Kallajoki and GaryGorbsky for providing antibodies.

Grant SupportThis studywas supported byAcademyof Finlandgrants 120804 (toM.J.

Kallio) and 134272 (to L.J. Laine), Marie Curie EXT grant 002697 (to M.J.Kallio and J. Rehnberg), the Academy of Finland Centre of Excellence forTranslational Genome-Scale Biology funding (for M.J. Kallio, P. Tiikkai-nen, P.Halonen, E. Narvi, and L. Kallio), the Finnish CancerOrganisationsgrants (to M.J. Kallio and J.H.E. M€aki-Jouppila), FinPharma DoctoralProgram funding (for J.H.E. M€aki-Jouppila), and Finnish Cultural Foun-dation grant (to J.H.E. M€aki-Jouppila). A. Poso was funded by the Uni-versity of Eastern Finland, School of Pharmacy. E. Hukasova and A.Lindqvist were funded by the Swedish Research Council, the SwedishCancer Society, and the Swedish Foundation for Strategic Research.

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 August 16, 2013; revised February 5, 2014; accepted February25, 2014; published OnlineFirst April 18, 2014.

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2014;13:1054-1066. Published OnlineFirst April 18, 2014.Mol Cancer Ther Jenni H.E. Mäki-Jouppila, Leena J. Laine, Jonathan Rehnberg, et al. Rigosertib, ON 01910.NaMolecular Interaction Field and Antimitotic Cellular Phenotype as Centmitor-1, a Novel Acridinyl-Acetohydrazide, Possesses Similar

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