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Cell Cycle and Senescence

Suppression of Ser/Thr Phosphatase 4 (PP4C/PPP4C)Mimics a Novel Post-Mitotic Action of Fostriecin, ProducingMitotic Slippage Followed by Tetraploid Cell Death

Benjamin Theobald1, Kathy Bonness1, Alla Musiyenko1, Joel F. Andrews1,2, Gudrun Urban1, Xizhong Huang1,Nicholas M. Dean3, and Richard E. Honkanen1,2

AbstractFostriecin is a natural product purified from Sterptomyces extracts with antitumor activity sufficient to

warrant human clinical trials. Unfortunately, difficulties associated with supply and stable drug formulationstalled further development. At a molecular level, fostriecin is known to act as a catalytic inhibitor of four PPP-family phosphatases, and reports describing the design of molecules in this class suggest derivatives targetingenzymes within the fostriecin-sensitive subfamily can be successful. However, it is not clear if the tumor-selective cytotoxicity of fostriecin results from the inhibition of a specific phosphatase, multiple phosphatases,or a limited subset of fostriecin sensitive phosphatases. How the inhibition of sensitive phosphatasescontributes to tumor-selective cytotoxicity is also not clear. Here, high-content time-lapse imaging of livecells revealed novel insight into the cellular actions of fostriecin, showing that fostriecin-induced apoptosis isnot simply induced following a sustained mitotic arrest. Rather, apoptosis occurred in an apparent secondinterphase produced when tetraploid cells undergo mitotic slippage. Comparison of the actions of fostriecinand antisense-oligonucleotides specifically targeting human fostriecin-sensitive phosphatases revealed that thesuppression PP4C alone is sufficient to mimic many actions of fostriecin. Importantly, targeted suppression ofPP4C induced apoptosis, with death occurring in tetraploid cells following mitotic slippage. This effect was notobserved following the suppression of PP1C, PP2AC, or PP5C. These data clarify PP4C as a fostriecin-sensitive phosphatase and demonstrate that the suppression of PP4C triggers mitotic slippage/apoptosis.Implications: Future development of fostriecin class inhibitors should consider PP4C as a potentially important

target. Mol Cancer Res; 11(8); 845–55. �2013 AACR.

IntroductionFostriecin and structurally related phosphate mono-

esters produced by Streptomyces sp. (i.e., cytostatin) displaycytotoxicity and antitumor activity (for review, see refs. 1,2). Cytostatin has potent cytotoxic activity toward mel-anoma and leukemia cell lines and inhibits B16 melanomalung metastasis in a mouse model of cancer progression(3). The antitumor activity of fostriecin (also called

PD-110,161, CI-920 or NSC-339638) has been evaluat-ed extensively (4); for review, see refs. 1, 5). Fostriecinshows potent cytotoxicity against a number of cancer celllines and marked antitumor activity in animals (for review,see refs. 1, 5, 6). To evaluate its potential for use as a novelantitumor agent in humans, fostriecin entered humanclinical trials (7, 8). Although limited, the data obtainedfrom the phase I trials indicate that plasma levels offostriecin associated with antitumor activity in animals(9) can be achieved in humans (7, 8). Unfortunately,further development has been suspended for nearly adecade, due to early controversies regarding its mechanismof action, problems associated with the supply of fostriecinfrom natural sources, and difficulty associated with stabledrug formulation (8).Nowmethods for synthesis are known, and the molecular

targets of fostriecin are becoming clear. Fostriecin (1, 10–13), structurally related natural products [e.g. cytostatin(14, 15) phospholine, leustroducsin, and phoslactomycins(1, 16, 17)] and designed analogs used to explore thestructure–function properties and mechanisms of action ofmolecules in the class (11, 12, 15), all inhibit the catalyticactivity of a subset of PPP family serine/threonine protein

Authors' Affiliations: 1Department of Biochemistry and Molecular Biol-ogy, 2Mitchell Cancer Research Institute, University of South AlabamaCollege ofMedicine,Mobile, Alabama; and 3Department of Pharmacology,ISIS Pharmaceuticals, Carlsbad, California

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

B. Theobald and K. Bonness contributed equally to this work.

Corresponding Author: Richard E. Honkanen, Department of Biochem-istry and Molecular Biology, MSB 2362, University of South Alabama,Mobile, AL 36688. Phone: 251 460-6859; Fax: 251 460-6850; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-13-0032

�2013 American Association for Cancer Research.

MolecularCancer

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phosphatases. Fostriecin is a potent inhibitor of PP2AC[IC50�0.2 nmol/L (10, 11, 13)], a strong inhibitor of PP4C[IC50 � 4 nmol/L (18)], and a weak inhibitor of PP1C andPP5C (IC50, 72 and 60 mmol/L, respectively; refs. 10, 11).Structural studies have revealed that the fostriecin-sensitivephosphatases share a common catalytic mechanism (19).Structure–activity relationship (SAR) studies indicate thatselectivity for PP2AC is derived from the interaction of C3with a noncatalytic cysteine of PP2AC (C269), which iscontained in the b12–b13 loop that resides adjacent to thehighly conserved catalytic pocket (11, 12). This cysteine isnot conserved in PP1C or PP5 (12, 19), and the predictedcovalent adduct with PP2AC has been shown using a biotin-labeled derivative (20). The b12–b13 loops of PP4 and PP6contain a homologous cysteine, suggesting it serves as acommonality for strong inhibition (11, 12, 19). However, toour knowledge, these predicted actions on PP4C and PP6Chave not been tested experimentally. Fostriecin has noapparent affects on PP2B (calcineurin), PP7, or PPM familyphosphatases (for review, see refs. 1, 6). SAR studies have alsoprovided insight into other features needed for potency,selectivity, and stability, sparking renewed interest in thedevelopment of compounds in this class (11, 12, 15, 21, 22).The basis for the antitumor activity of fostriecin is not

clear. At the cellular level, fostriecin enters cells via folic acidtransporters (23, 24), and at concentrations sufficient toinhibit PP1C, PP2AC, PP4C and PP5C (>125 mmol/L), itkills both tumor and normal cells (13). Many of the toxiceffects are cell-cycle–independent and similar to the toxicactions produced by other natural compounds that act asstrong, nonselective inhibitors of PP1C, PP2AC, PP4C, andPP5 (e.g., microcystin-LR and calyculin A). Nonetheless,similar to early observations made with paclitaxel (taxol),preclinical studies indicate that fostriecin has antitumoractivity in animals at nontoxic concentrations (�50mmol/L; refs. 5, 8, 9, 25). At�50 mmol/L, fostriecin inducesan apoptotic response in many types of cancer cells (26).However, fostriecin-induced apoptosis is slow, requiringmore than 24 to 48 hours, and death is preceded by theaccumulation of cells with 4N DNA (13, 27, 28). Canthar-idin, a less selective natural inhibitor of PP1–PP5 that alsohas cytotoxic and antitumor activity, produces a similaraccumulation of cells with 4N DNA before the onset ofapoptosis. This suggested the antitumor activity of bothcompounds could arise from their ability to prolong mitosis,triggering mitotic/spindle checkpoint control mechanisms(29).To help clarify the relationship between tumor-selective

cytotoxicity and phosphatase inhibition, we developed 20-O-methyl phosphorothioate deoxyoligonucleotides capa-ble of specifically suppressing the expression of fostriecin-sensitive phosphatases (29–31). Previous studies haveshown that, like fostriecin, the suppression of PP2ACa,but not PP2ACb, PP1Cg1, or PP5C, induces a prolongedmitotic arrest (29). Further investigations revealed thatPP2AC forms a complex with a kinetochore-associatedprotein (Sgo1/MEI-S332) during metaphase (32–35).The PP2AC/Sgo1/kinetochore complex protects cohesin

residing at the kinetochore from phosphorylation-medi-ated dissociation before the onset of anaphase (32–35).Thus, when PP2AC is suppressed, the activation ofmitotic kinases (i.e., Plk1, Aurora A/B) results in thepremature dissociation of sister chromatids. This preventsbiorientation (the attachment of duplicated chromatidsvia their kinetochores to opposite poles within a micro-tubule-based mitotic spindle), which activates or main-tains the spindle assembly checkpoint controls that pre-vent progression into anaphase (32–35). These observa-tions lead to the widely accepted hypothesis that fostrie-cin-induced apoptosis was simply due to its ability toproduce a prolonged mitotic arrest via the inhibition ofPP2AC. However, the prolonged mitotic arrest inducedby the suppression of PP2AC alone did not triggerapoptosis (29), prompting us to re-examine the actionsof fostriecin. Here, high-content time lapse imaging of livecells reveals that fostriecin-induced apoptosis is not simplyproduced by the induction of a prolonged mitosis. Rather,death occurred in tetraploid cells produced followingmitotic slippage. This effect was mimicked by antisensetargeting PP4C (but not by antisense targeting PP1C,PP2AC, or PP5C), revealing a novel role for PP4C duringmitotic progression.

Materials and MethodsCell cultureA549 and HeLa cells were obtained from American Type

Culture Collection. A well-characterized stable HeLa cellline constitutively expressing a histone 2B-GFP fusionprotein (H2B-GFP; HeLa-H2B-GFP cells) was a generousgift from Dr. Kevin Sullivan (36). Both cell lines werecultured in Dulbecco'sModified Eagle's Medium (DMEM)supplementedwith 10%FBS and L-glutamine (4.0mmol/L)at 37�C in 5% CO2.

ChemicalsFostriecin was synthesized as described previously (11).

Oligonucleotide synthesis and assay for oligonucleotideinhibition of PP2ACa or PP4C expressionPhosphorothioate deoxyoligonucleotides and 20-O-meth-

yl phosphorothioate deoxyoligonucleotides were synthesizedand purified as described previously (37). The indicatedcells were plated in 60-mm dishes and cultured in DMEMcontaining 10% FBS. When about 50% confluent, thecells were treated with oligonucleotides as previouslydescribed (30, 38). Because phosphorothioate oligonu-cleotides act through RNAase H–dependent mRNA cleav-age mechanism in cells, the ability of each oligonucleotideto specifically inhibit the expression of PP4C was initiallydetermined by Northern blot analysis probing for levels ofPP4C mRNA (39). Antisense oligonucleotides targetingPP1C (g1), both isoforms of PP2Ac (PP2ACa, PP2ACb)and PP5 have been developed and characterized previously(29–31, 40). The characterization of antisense oligonu-cleotides targeting PP4C is provided in SupplementaryFig. S1.

Theobald et al.

Mol Cancer Res; 11(8) August 2013 Molecular Cancer Research846




Immunoblotting of PP2A, PP4C, PP5, and PP6Western blot analysis was conducted as described previ-

ously using polyclonal rabbit antibodies generated against asynthetic 15-amino acid peptide identical to unique regionsof PP2A, PP4C, PP5, and PP6 (29–31, 41). The antibodytargeting PP2A does not discriminate between PP2Aa andPP2Ab (29).

Caspase-3 activityCaspase-3 activity was measured using a CASP3C color-

imetric assay (optical density at 405 nm) according to theinstructions provided by the manufacture (Sigma). Allexperiments were repeated at least 3 times.

Flow cytometryA549 cells were harvested, stained with propidium iodide

(PI), and DNA content flow cytometry was conducted asdescribed previously (29).

Live cell imaging and time lapse video microscopyHeLa-H2B-GFP cells were cultured in 60-mm dishes and

incubated at 37�C in 5% CO2 using a Neuve live cellchamber fitted into an Eclipse Nikon TE 2000-U micro-scope (Nikon Instruments Inc.). Image acquisition wasachieved using a COOLSNAP ES monochrome cameraand processed using Elements (Nikon) and MetaMorphPremier software (Universal Imaging).

Analysis of live cell imagingTo study mitotic progression, we used live cell imaging

in combination with time lapse video microscopy using anestablished HeLa cell line (HeLa-H2B-GFP) expressinghistone 2B fused with GFP (29, 32, 36). Here, bothfluorescent and differential interference contrast (DIC)images of live cell cultures were taken at timed intervals(every 2–5 minutes) for the duration of experimentslasting from 12 to 72 hours. Short exposure times andneutral density filters were used to minimize UV exposureto cells, and control experiments using solvent or mis-matched antisense oligonucleotides were carried out toensure the observations made were not due to UV damageproduced during fluorescent imaging. In each experiment,images of more than 300 cells can be analyzed at everytime point for the duration of the experiment. H2B-GFPexpression in interphase cells is dispersed throughout thenucleus, and chromosome condensation (marking theonset of prophase) is easily distinguished from dispersednuclear fluorescence of interphase cells (Fig. 1A). Meta-phase alignment of sister chromatids, mitotic defects, andsister chromatid separation (indicating the onset of ana-phase) can also be easily distinguished. The nuclearenvelope breakdown and the onset of cytokinesis are easilydetected in corresponding DIC images. Therefore, anal-ysis of sequential images allows for quantitative assessmentof the time needed for cell-cycle progression from pro-phase to metaphase, anaphase, and cytokinesis. Analysis oflive cell images also allows us to provide a detaileddescription of mitotic defects induced by the various

treatments and to quantitate the frequency that thesedefects are produced.

ResultsQuantitation of cell-cycle progression in controlsTo further characterize the cellular effects of fostriecin, live

cell imaging, in combination with time lapse video micros-copy, was used using a HeLa cell line that stably expressesH2B fused with GFP (HeLa-H2B-GFP). This stable cellline is widely used because it allows high-resolution imagingof chromosomes without compromising nuclear or chro-mosomal structures (29, 32, 36), and it has proven useful as apowerful tool to study agents that affect chromosomecondensation, metaphase plate alignment of sister chroma-tids, and mitotic progression into anaphase (29, 32, 36). Ineach experiment, images of about 300 to 500 live cells weretaken at timed intervals (generally every 5 minutes) for 24 to72 hours, as indicated. Individual images were then exam-ined and analyzed with the aid of advanced imaging software(Elements and Metamorph). This procedure allows us tosimultaneously assess several aspects of cell-cycle progressionin about 50 to 300 individual cells per experiment. Then,following the fate of each cell, the duration required forprogression from nuclear envelope break down (NEBD) to(i) chromosome alignment at the metaphase plate, (ii) theonset of anaphase, (iii) chromosome decondensation, (iv)cleavage furrow formation, and (v) cytokinesis was quanti-fied. When abnormal behaviors were observed, the time ofonset (relative to NEBD) and the ensuing fate of the cellswere noted.Progression through mitosis in control cells is illustrated

in Fig. 1A. The data shown are typical of cell populations formore than 10 experiments, in which 50 to 100 mitotic cellsare analyzed in each experiment. Quantitation of the datarevealed that in untreated or solvent treated controls pro-gression from NEBD (designated as 0 minutes) to chromo-some decondensation and cytokinesis requires 1.3 � 0.82(mean � SD; n ¼ 300) hours (Fig. 2A). During this time,chromosome alignment at the metaphase plate (indictedwith an asterisk) is observed about 20 minutes after NEBDin >97%� 3% (mean � SD) of the cells scored (n¼ 500).Separation of sister chromatids, indicating the onset ofanaphase, occurs 49 � 25 minutes thereafter (mean � SD;n¼ 300) and is not observed until all of the chromosomes arealigned. In 96% of the cells analyzed, chromosome decon-densation and cytokinesis occur <2.5 hours after the onset ofNEBD. A delay during mitosis (defined as cells with con-densed chromosomes that fail to proceed into anaphase in<2.5 hours) is rarely observed in untreated or solvent con-trols (<4%, n ¼ 300) and is therefore considered anindication of "mitotic arrest."

Fostriecin prolongs mitosis and prevents cytokinesisFostriecin is known to induce apoptosis, and previous

studies using fluorescence-activated cell-sorting (FACS)analysis have shown that before death fostriecin producesa dose-dependent accumulation of cells with 4NDNA (27).Consistent with previous studies, in the HeLa-H2B-GFP

Suppression of PP4C Triggers Mitotic Slippage and Apoptosis

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Figure 1. Localization of H2B-GFP protein in HeLa cells. Fluorescent and corresponding DIC microscopic images of selected HeLa-H2B-GFP cellsrepresenting the typical behavior of the populations are shown at various phases of the cell cycle. A, representative images selected from time lapse live cellimaging of control cells typical of: interphase at the start of recording (i), the start of mitotic entry (set at 0 minutes), prometaphase (14 minutes),metaphase (45minutes), anaphase (1.2–1.4hours), chromosomedecondensation (2.2 hours), andafter the completion of cytokinesis (4.1 hours). Elapsed timefrom the start of mitotic entry is indicated below image pairs. B, images showing selected cells that represent the typical behavior of populations aftertreatment with 50 mmol/L fostriecin. No effects were observed before entry into mitosis (0 minutes). During a prolonged mitosis, condensed chromosomesfail to align properly at the metaphase plate (45 minutes; 4.6 hours). Subsequently, chromosomes segregated into unequal clusters (5.3 hours) andundergo decondensation (5.8 hours). During this period, more than one furrow appears (5.3, 5.8, and 7.7 hours). Then, the furrows undergo regression(shownhere at 10.9 hours). C, images showing selected cells that represent the typical behavior of populations after treatmentwith antisenseoligonucleotidestargeting PP4C. H2B-GFP expressing HeLa cells were treated with 400 mol/L ISIS 134947. After 24 hours, time lapse imaging studies were conductedfor 48 hours as above. The similarity to the effects produced by fostriecin is striking: PP4C-AS interferes with the integrity of the metaphase plate(55 minutes) prolongs mitosis and allows cleavage furrow regression following mitotic slippage (shown at 18.2 hours). The data shown are representative ofmore than 4 separate experiments in which 50 to 100 mitotic cells were scored in each experiment. Bars under figure pairs are colored to illustrate imagesrepresenting various stages of cell-cycle progression and are color matched to the quantitative data shown in Fig. 2. D, concentration-dependent increase in

Theobald et al.





model, fostriecin (<50 mmol/L) produced no notable effectson cell-cycle progression before NEBD (Fig. 1B; designatedas 0 minutes). Following chromosome condensation fos-triecin prevents the proper alignment of sister chromatids atthe metaphase plate (indicated at 45 minutes and 4.6 hoursby arrows). Cells that do not achieve metaphase alignmentarrest and do not progress into anaphase. Dose–responsestudies revealed a concentration-dependent increase in thefrequency of cells with aberrant metaphase alignment, withmaximal efficacy achieved with 50 mmol/L fostriecin (Fig.1D). Quantitation of the data revealed that fostriecin pro-longs mitosis [(5.4� 0.25 hours; mean� SE); compare redbars shown in Fig. 2A with Fig. 2B; Fig. 2E]. During thisperiod, the arrested cells remain rounded in appearance andthe chromosomes remain condensed. The sister chromatidsthat do align at the metaphase plate do not separate, and thechromosomes that fail to align move in an unorganizedmanner. The fostriecin-arrested cells then progress into anaberrant form of anaphase. At the onset of "anaphase," mostfostriecin-treated cells attempt chromosome separation in atri- or polypolar manner, which is associated with furrowingression at 3 or more sites (shown at 5.3 hours in Fig. 1B).In these cells, aberrant furrow ingression does not resultin abscission. Rather, after a delay the furrows regress,producing a single tetraploid cell. Some cells reform 3 ormore nuclei that are unequal in size (shown at 10.9 hours).Chromosome decondensation occurs before regression,and with time, the tetraploid cells become apoptotic. By48 hours, 50 mmol/L fostriecin killed more than 98% ofthe cells, and the IC50 for 72-hour cytotoxic activity offostriecin is about 5 to 7 mmol/L (11, 17, 25). Onlya small percentage (<4%) of the cells treated with50 mmol/L fostriecin become apoptotic prior the com-pletion of mitosis (defined here as chromosome decon-densation). Rather, in the majority of fostriecin-treatedcells, death occurred several hours after furrow regressionoccurred (Fig. 2B and D; black bars). The occasional cellthat eventually achieves metaphase alignment was notkilled and appeared to progress normally into anaphase.As reported previously for other cell types (13, 27),polyploidy above 4N was not observed in fostriecin-treated cells.

Suppression of PP4C prolongs mitosis and inducesfurrow regression before deathThe concentration of fostriecin that altered mitotic pro-

gression is sufficient to completely inhibit the activity ofPP2AC and PP4C, and the suppression of PP2AC did notproduced furrow regression or tetraploid cells (29). There-fore, we developed antisense that specifically suppressed

PP4C (Supplementary Fig. S1). Antisense oligonucleotidestargeting PP4C had no effect on the expression of PP2AC,PP5 or PP6C, and the level of these structurally relatedphosphatases do not increase to compensate for the lack ofPP4C (Fig. 3A). In the HeLa-H2B-GFP cell model, asobserved in other cell lines, following treatment with anti-sense oligonucleotides targeting PP4C (PP4C-AS) >30hours was required for PP4C protein levels to becomemaximally suppressed. Before about 50% suppression(�24 hours after treatment), cell division occurs normally.When PP4C levels are suppressed, PP4C-AS elicits aresponse that is strikingly similar to the response producedby fostriecin (compare Fig. 1B and C). First, the suppressionof PP4C produced no apparent differences from controlsbefore NEBD. Second, treatment with 400 nmol/L ISIS134947 prolonged mitotic progression in about 60% of thecells (4.33� 0.47 hour; mean� SE' Fig. 2) compared to lessthan 4% in cells treated MM controls. During this time,similar to the actions of fostriecin, cells arrested by PP4C-ASremain rounded in appearance and contained condensedchromosomes that fail to align properly at the metaphaseplate (shown in Fig. 1C at 55minutes and 2.5 hours). After aprolonged mitosis, multiple furrows initiate in the PP4-AS–treated cells (shown at15 hours). With time, the furrowsregress and chromosomes undergo decondensation, result-ing in a single tetraploid cell (shown at 18.2 hours). How-ever, it should be noted that when compared with fostriecin,both the average duration of mitosis and the time followingfurrow regression and proceeding apoptosis was highlyvariable in cells treated with PP4C-AS (compare bar lengthin Fig. 2B and C). This difference may reflect difficultiesassociated with the uniform delivery of antisense oligonu-cleotides to cell in culture or may reflect the combinedinhibition of both PP2AC and PP4C in the fostriecin-treated cells. Additional data comparing the actions offostriecin, PP4C-AS and PP2AC-AS is provided in Supple-mentary Figs. S2 and S3.

Suppression of PP4C expression induces apoptosis in theabsence of cellular stressInitial dose–response studies (Fig. 1E; Supplementary Fig.

S1) revealed that the suppression of PP4C kills HeLa cells inculture after 72 hours (similar effects were observed in otherlines of human cells). To determine whether death inducedby antisense oligonucleotides targeting PP4C is due toapoptosis, cells treated with ISIS 14376 or ISIS 134947were examined. Light microscopic examination of dyingcells revealed classic signs of apoptosis (i.e., membraneblebbing and condensed fragmented DNA visualized byDAPI staining). Electron microscopic analysis revealed

fostriecin-mediated failure of chromosomealignment at themetaphase plate. HeLa-H2B-GFP cells were treatedwith the indicated concentration of fostriecinor solvent (controls). After 24 hours, fluorescent images of random fields were taken, and 50 to 100 mitotic cells were examined for the presence ofcondensed chromosomes that failed to align properly at the metaphase plate. The data shown represent the mean � SD from 3 separate experiments.E, dose-dependent cell death following treatment with antisense targeting PP4C. HeLa cells were treated with the indicated concentration of ISIS14376, and cell viability was determined after 72 hours. For each experiment, random fields were examined and 100 cells were scored for by theability to exclude trypan blue. The data is plotted as death (%) as compared with death in cells treated with mismatched antisense oligonucleotides andrepresents the mean � SD (n ¼ 3).






numerous cells with homogenous condensation of chroma-tin contained in one side or the periphery of the nucleus incells treated with antisense targeting PP4C, which is ahallmark characteristic of apoptosis (Fig. 3E). Analysis ofmore than 100 cells 48 hours after treatment revealedcondensed chromatin and other characteristics of apoptosisin 4%� 1% of the cells treated with lipofectin alone, 4%�2% of the cells treated with MM control oligonucleotides(500 nmol/L), 59%� 3% of the cells treated with PP4C-AS

at 300 nmol/L, and 75%� 8% of the cells treated PP4C-ASat 400 nmol/L. Antisense oligonucleotides targeting PP4also produced a dose-dependent increase in caspase-3 activ-ity (Fig. 3C). FACS analysis using an AnnexinV/FITC–labeled antibody also revealed an increase in apoptosis whenPP4C expression was suppressed (data not shown). Thesestudies indicate that antisense oligonucleotides capable ofsuppressing the expression of PP4C induce cell death via theinduction of apoptosis.

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Figure 2. Quantification of dataobtained from images taken duringexperiments, as shown in Fig. 1.The duration and fate of 50 to 100cells entering mitosis in eachexperiment was recorded. The plotdepicts the duration of mitosis andfate of the cells from a singlerepresentative experiment. Eachhorizontal bar represents a singlecell, and the color of the bardenotes the fate of the cell. Bluebars represent time spent ininterphase. Red bars represent thetime spent in mitosis (defined bythe time from NEBD to anaphaseonset), green bars indicate cells inwhich cytokinesis fails (producingtetraploid cells that then enter intoa second interphase). Black barsindicate death. The length of thebars (excluding black bars; death)denotes the duration of each fate,and the scale below the graphshows time in hours. A, controls.B, cells treated with 50 mmol/Lfostriecin. C, cells treated withPP4-AS as described in Fig. 1.D, bar graph representation of thepercentage of cells that: diefollowing mitosis (black), undergocleavage furrow regression (green);undergo mitotic slippage andreform >2 daughter nuclei ofunequal size (red); and arrest inmitosis for >2.5 hours (blue). ��, adifference from controls (P < 0.01)for bracketed valuesdeterminedby2-tailed t test. E, bar graphcomparing the average duration ofmitosis (red) and the average timespent in the second apparentinterphase (green; identified bycleavage furrow regression andchromosome decondensation)and preceding death. ��, adifference from controls (P < 0.01)according to independent t testsbetween means.

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Figure 3. Effects of antisense oligodoexynucleotides targeting PP4C. A, target-specific inhibition of PP4C protein levels. Cells were treated with the eitherantisense oligonucleotides targeting PP4C (ISIS 134947 illustrated) or mismatch control oligonucleotides (MM; ISIS 131370 or ISIS 131371). Proteinextracts were prepared at the times indicated and analyzed for structurally related phosphatases (PP2AC, PP4C, PP5, and PP6C) by Western blotanalysis asdescribed inMaterials andMethods [theantibody forPP2AC recognizes both theaandb isoformsofPP2AC (29)]. B, dose-dependent suppressionof PP4C mRNA levels by ISIS 134947 in HeLa cells. To ensure the uptake and efficacy of antisense oligonucleotides, for each experiment, replicateplates were treated with the oligonucleotides indicated and processed for Northern blot analysis after 24 hours, as described in Supplementary Fig. S1and Supplementary Methods. Representative data obtained using ISIS 134947 and MM (ISIS 131371) are shown. C, caspase-3 activity. A549 cellswere treated with nothing (1), lipofectin (lipid control; 2), 10 nmol/L TNFa (positive control; 3), 10 nmol/L okadaic acid (OA; second positive control; 4), or theindicated concentration of ISIS 14376 (PP4C-AS; 5, 6, 7). After 36 hours, cells were harvested and caspase-3 activity was measured as described inMaterials and Methods. The data is plotted as the mean OD405 � SE from 3 replicate plates and is representative of 3 separate experiments. D and E,representative electron micrographs of A549 cell populations after treatment with MM (control) or antisense oligonucleotides (ISIS 14376) targeting PP4C.After treatment (48 hours), cells were harvested, fixed, stained, and then viewed by electronmicroscopy using standard protocols. Controls (D) have an intactmembrane, organelles, and normal nuclearmorphology. ISIS 14376–treated cells (E) showhomogeneous chromatin condensationwithin the nucleus (arrow),membrane blebbing and other characteristics common to apoptotic cells.






Suppression of PP4C is associated with aberrant mitoticspindlesTo explore mechanisms by which PP4C-AS may prolong

mitosis, we examined the effects of ISIS 134947 on micro-tubule behavior. Immunostaining with fluorescent-taggedanti-a-tubulin antibodies revealed that PP4C-AS has noapparent affect on microtubules in nondividing cells (Sup-plementary Fig. S4), whereas in mitotic cells, marked differ-ences were observed between the mismatch control andPP4C-AS treatment. At mitosis, more than 98% of thecontrol cells contain a typical array of anti-parallel micro-tubules characteristic of a normal bipolar mitotic spindle. Incontrast, in PP4C-AS–treated cells, the spindle apparatus

was aberrant, often composed of multiple "poles" (Fig. 4;indicated by arrows). On the basis of fluorescent immunos-taining of fixed mitotic cells, it appears that PP4C-AS doesnot prevent the polymerization of microtubule structures ingeneral. Rather, PP4C-AS disrupts the organization of thespindles.

DiscussionMicrotubule binding drugs that disrupt mitotic progres-

sion (e.g., Vinca alkaloids and taxanes) have shown efficacyin the treatment of different types of cancer. Accordingly,intense efforts have been devoted to further the understand-ing of how cancer cells responded to known antimitotic

CBA

Figure 4. Mitotic abnormalitiesassociated with the suppression ofPP4C. Representative confocalmicroscopic images of serial Zsections through a HeLa celltreated with antisense targetingPP4C. Cells were treated with 400nmol/L ISIS 134947 and fixed oncoverslips 30 hours later. A,microtubules (red) were thenvisualized by immunofluorescencefollowing treatment with anti-a-tubulin and Alexa Fluor 594–labeled antibodies as described inMaterials and Methods. B, DNA(blue) was visualized by stainingwith Hoechst 33342. C, computer-assisted merge of images A and B.Similar results were obtained with�3 independent experiments.

Theobald et al.





drugs and to the development of agents that perturb mitoticprogression via other, less toxic mechanisms. Indeed, agentstargeting mitotic kinases [i.e., Polo-like kinase-1 (Plk1) andAurora kinases] and microtubule-associated motor proteins(e.g., Eg5/Kinesin-5/KIF11 and CENP-E) are already inpre- or early clinical development. The studies describedhere add support to the hypothesis that the inhibition offostriecin-sensitive phosphatases can also disrupt normalmitotic progression (Fig. 1) without directly targetingmicro-tubules, mitotic kinases, or motor proteins (10, 27). Ourdata also provide new insight into the cytotoxic actions offostriecin and reveal, for thefirst time, that the suppression ofa little-studied fostriecin-sensitive phosphatase (PP4C) issufficient to prolong mitosis, alter cleavage furrow position/ingression, and induce apoptosis in the tetraploid cellsproduced following mitotic slippage.Fostriecin is known to act as a strong inhibitor of PP2AC

(10–12, 27), and fostriecin induces the accumulation of cellswith 4NDNA that have aberrant mitotic spindles before theonset of apoptosis (10, 13, 27). Therefore, when studies ofPP2AC action during mitotic progression revealed thatPP2AC (in a complex with PR61/B' and Sgo1 at thekinetochore) functions to protect centromeric cohesionfrom phosphorylation-mediated dissociation by the activa-tion of mitotic kinases (i.e., Plk-1 or Aurora B; refs. 32,33, 35), a simple hypothesis for the antitumor activity offostriecin emerged. That is, the inhibition of PP2AC mayallow the premature dissociation of centromeric cohesion(32–35), which prevents biorientation and triggers/main-tains mitotic spindle assembly checkpoint controls. Studieswith antisense oligonucleotides targeting PP2AC supportthis hypothesis, in the respect that the specific suppression ofPP2ACa is sufficient to arrest cells in mitosis, and thePP2AC-AS–arrested cells have lagging chromosomes thatfailed to align at the metaphase plate (29). Nonetheless,PP2AC can dephosphorylate a number of cyclin B–Cdk1substrates (42, 43), suggesting that the inhibition of PP2ACcould also prolong mitosis via numerous other mechanisms.A key novel observation reported here is that although

fostriecin treatment does prolong mitosis, apoptosis onlyoccurs rarely during mitotic arrest (Figs. 1 and 2). Thisargues that fostriecin does not kill cells by simply prolongingmitosis. Further characterization revealed that the fostriecin-treated cells exit mitosis (defined here as chromosomedecondensation) but then fail to complete cytokinesis.Death then occurs after mitotic slippage in the tetraploidcells that are produced, during what appears to be a secondinterphase. These novel actions of fostriecin are not observedfollowing the suppression of PP1Cg1, PP2ACa, PP2ACb,or PP5 (29–31) but are faithfully mimicked by the suppres-sion of PP4C (Figs. 1C and 2C).Careful examination of the data reveals that there is great

variation in the timing of cell death after treatment withfostriecin or antisense targeting PP4C. In general the actionsof fostriecin were more uniform, which may reflect difficul-ties associated with various aspects of using antisense oligo-nucleotides in cultured cells (44) or differences produced bythe rapid inhibition of PP4C activity by fostriecin compared

with the gradual loss of PP4C protein following treatmentwith PP4-AS. Nonetheless, after treatment with eitherfostriecin or PP4-AS, we observed no clear correlationbetween the duration of mitotic delay and cell fate, suggest-ing that in this cell model, the time a cell spends in mitosisdoes not dictate whether it dies in mitosis or slips frommitotic arrest and dies later after cleavage furrow regression.Studies of PP4C function in human cells have revealed

that it plays an important role in the recovery of cell-cycleprogression after DNA damage–mediated S-phase arrest(45), whichwas not addressed by our experiments.However,investigations of PP4C function in lower organisms mayprovide insight into some of the other affects produced byantisense oligonucleotides targeting PP4C in HeLa cells. InDrosophila embryos, the PP4C ortholog (91% identical tohuman PP4C) is required for the organization of micro-tubules at the centrosomes (18), and siRNA-mediatedsuppression in Drosophila affects polar division in neurons(46). The association of PP4C at themitotic centrosomes hasalso been observed in higher organisms and is required forproper centrosome organization of microtubules (47, 48).Our studies show that the mitotic spindles in the PP4C-AS–treated cells are aberrant (Fig. 4), whereas the microtubulesin nonmitotic cells appeared normal (Supplementary Fig.S4). Although we cannot rule out the possibility that PP4Cplays an independent and nonredundant role to PP2AC inthe regulation of centromeric cohesion, the appearance ofmisaligned and Y-shaped metaphase alignment of sisterchromatids, produced by PP4C-AS or fostriecin (but notPP2AC-AS), would be an expected consequence of agentsthat disrupt normal microtubule attachment at the centro-somes. Presumably disruption of centrosome/microtubuleinteractions would also interfere with biorentation andprolong mitosis, which was observed after treatment withantisense specifically targeting PP4C.Studies into the molecular mechanisms associated with

cytokinetic abscission and signaling mechanism by whichfailures in cytokinesis can lead to regression and tetraploidysuggest reversible phosphorylation plays many regulatoryroles. For example, in HeLa cells, chromosome bridgessustain Aurora B activity, and Aurora B has been shown toprotect missegregating cells against cleavage furrow regres-sion via a mechanism in which Aurora B–mediated phos-phorylation of Mklp1 stabilizes Mklp1 at the intracellularcanal (49). Chemical genetic studies of Plk1 function duringmitosis indicate that the partial suppression of Plk1 can alsoresult in cleavage furrow regression, leading to the formationof tetraploid cells (50). Phosphorylation at specific sites hasbeen shown to activate Aurora B (Thr-232), Mlkp1 (Ser911), and Plk1 (Thr-210). However, our data are notconsistent with PP4C acting to directly dephosphorylatekey activating sites on these kinases because the inhibition ofAurora B, Plk1, or PP4C lead to furrow regression and theformation of tetraploid cells. Our data appears to be con-sistent with PP4C playing an upstream role in which PP4Cactivity is needed to activate or maintain the activation ofAurora B or Plk1. Alternatively, PP4C could have indirectactions that affect a poorly characterized aspect of these






known pathways. Clearly future studies will be required todetermine the molecular mechanisms by which the suppres-sion of PP4C produces mitotic slippage and tetraploid celldeath.

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

Authors' ContributionsConception and design: B. Theobald, K. Bonness, R.E. HonkanenDevelopment of methodology: B. Theobald, K. Bonness, G. Urban, A. Huang, R.E.HonkanenAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): B. Theobald, K. Bonness, A. Musiyenko, G. Urban, A. HuangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): B. Theobald, K. Bonness, A. Musiyenko, J.F. Andrews, G. Urban,A. Huang, R.E. HonkanenWriting, review, and/or revision of the manuscript: B. Theobald, K. Bonness,N.M. Dean, R.E. Honkanen

Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): K. Bonness, A. Musiyenko, R.E. HonkanenStudy supervision: K. Bonness, R.E. Honkanen

Grant SupportThis work was supported in part by the University of South Alabama Cancer

Research Fund, an intramural grant program funded by the USA Mitchell CancerInstitute, the NIH (NCI grant CA-60750 to R.E. Honkanen) and the NationalScience Foundation (NSF 0751684). This investigation use equipment purchasedwith funds from a shared instrument grant (S10RR027535) and was conductedin a facility constructed with support from Research Facilities ImprovementProgram Grant (C06 RR11174) from the National Center for ResearchResources.

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

Received January 21, 2013; revised April 11, 2013; accepted April 30, 2013;published OnlineFirst May 13, 2013.

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2013;11:845-855. Published OnlineFirst May 13, 2013.Mol Cancer Res Benjamin Theobald, Kathy Bonness, Alla Musiyenko, et al. Followed by Tetraploid Cell DeathNovel Post-Mitotic Action of Fostriecin, Producing Mitotic Slippage

) Mimics aPPP4CSuppression of Ser/Thr Phosphatase 4 (PP4C/

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