Journal of Cell Science 101, 79-91 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

79

Inhibition of mitosis by okadaic acid: possible involvement of a protein

phosphatase 2A in the transition from metaphase to anaphase

DALE D. VANDRE* and VICTORIA L. WILLS

Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, 4072 Graves Hall, 333 W. 10th Ave., Columbus, Ohio43210, USA

•Author for correspondence

Summary

The effects of the protein phosphatase inhibitor okadaicacid were examined using the pig kidney cell line LLC-PK. At relatively low concentrations of the inhibitor (8-40 nM), cells became blocked in a metaphase-like mitoticstate beginning 6-8 h after initial treatment. Spindlemicrotubules were present throughout the period of themitotic block, but were not stabilized since theyremained sensitive to nocodazole depolymerization.With increasing length of the mitotic block chromosomealignment at the metaphase plate was disrupted andmultipolar spindles developed. Cells continued to ac-cumulate in mitosis for at least 24 h, indicating that atthese low concentrations okadaic acid was not cytotoxic,but rather acted as a cytostatic agent. Upon release ofthe okadaic acid block, mitotic LLC-PK cells recoveredand completed anaphase. After extended periods oftreatment some cells were able to escape the okadaicacid-induced mitotic block. These cells were multi-nucleate and had undergone cytokinesis in the absence of

chromosome segregation. At higher concentrations ofokadaic acid (0.5-1.0 /iM), mitosis was blocked within30-60 min of treatment. However, within 90-120 mintreated cells rounded up and detached from themonolayer, regardless of whether they were in inter-phase or mitosis. Cytoplasmic microtubules were depol-ymerized in the detached cells, and these cells could notrecover from the cytotoxic effects of such high concen-trations of okadaic acid. Thus, differential effects of thephosphatase inhibitor could be demonstrated, depen-ding upon the concentration of okadaic acid applied tothe cultures. The okadaic acid-induced mitotic blockagewas probably due to the inhibition of a type 2A proteinphosphatase that is involved in the transition frommetaphase to anaphase.

Key words: mitotic block, okadaic acid, phosphataseinhibitor.

Introduction

The transition from interphase to mitosis is character-ized by the condensation of chromosomes, the break-down of the nuclear envelope, the disassembly of thecytoplasmic microtubule complex, and the formation ofthe mitotic spindle. These events appear to be initiatedby the specific phosphorylation of protein componentsassociated with each of these structures. These mitosis-specific phosphorylations correlate with the activationof a histone HI serine/threonine protein kinase activitythat has been identified as both the product of thefission yeast cell cycle control gene cdc2 (p34cdc2) and acomponent of maturation or mitotic promoting factor(MPF) (Arion et al. 1988; Dunphy et al. 1988; Gautieret al. 1988; Labb6 et al. 1988; Lohka et al. 1988).Mitotic protein substrates of the activated p34cdc2

kinase must subsequently be dephosphorylated uponexit from mitosis. These dephosphorylation eventsare temporally associated with the onset of anaphase

(Dore"e et al. 1983; Vandr6 and Borisy, 1989), andcorrelate with the inactivation of pM0**02 kinase.Therefore, a series of phosphorylation and dephos-phorylation reactions are intimately involved with theregulation of mitotic processes such as nuclear envelopebreakdown and re-formation (Newport, 1987; Dessevet al. 1991), and ultimately with the entry and exit ofcells from mitosis.

Okadaic acid (OA), a polyether derivative of a C^fatty acid, has been shown to be a potent tumor-promoting substance on mouse skin; however, OA doesnot bind to receptors of the phorbol ester class oftumor-promoting compounds or activate protein kinaseC (Suganuma et al. 1988). OA binds to and inhibitsprotein phosphatases present in the cell, specificallyprotein phosphatase 1(PP1) and protein phosphatase2A (PP2A) (reviewed by Cohen et al. 1990). ReportedID50 values for OA range from 0.04 to 1 nM against thePP2A catalytic subunit, and 12 to 500 nM against thecatalytic subunit of PP1 (Hescheler et al. 1988; Cohen et

80 D. D. Vandri and V. L. Wills

al. 1989). The resulting inhibition of phosphataseactivity leads to an increase in overall protein phos-phorylation in treated cells (Haystead et al. 1989).

When OA was microinjected into Xenopus or starfishoocytes, at a final intracellular concentration of 0.25 fjMand 1.2 pM, respectively, MPF activation and meioticmaturation were induced (Goris et al. 1989; Picard et al.1989). This suggests that OA-sensitive phosphatasesmaintain MPF in its inactive precursor form, pre-MPF.Activation of MPF in these oocyte systems leads to aburst of protein phosphorylation and germinal vesiclebreakdown (GVBD). Similarly, mouse oocytes treatedwith OA (25 nM-2.5 (M) also exhibit GVBD andchromosome condensation (Rim6 and Ozon, 1990;Gavin et al. 1991). In the first of these reports, spindleswere not detected in treated oocytes (Rime" and Ozon,1990); however, in approximately 50% of treatedoocytes in the second study spindles were present butthey showed an abnormal morphology (Gavin et al.1991). Spindle formation was also not observed inmicroinjected starfish oocytes (Picard et al. 1989).Therefore, either the cytoplasm of OA-treated oocyteswas not capable of organizing metaphase microtubulesin a normal fashion, or the phosphorylation state ofsome oocyte proteins must prevent the formation of orcause the depolymerization of spindle microtubules.

In Xenopus egg extracts, concentrations of OA thatinhibit PP2A but not PP1 were shown to transientlyactivate the p34cdc2 kinase ( Felix et al. 1990a), and theactivity of p34cdc2 kinase has been shown to influencethe dynamics of microtubule assembly in vitro usingXenopus extracts (Verde et al. 1990). This correlateswith the finding that a negative regulator of MPFactivity, INH, is an OA-sensitive PP2A (Lee et al.1991), and alteration in the activity of INH reduces thethreshold level of cyclin proteins, which are requiredfor MPF activation (Solomon et al. 1990).

Treatment of tissue culture cells with OA hasdemonstrated morphological changes similar to those ofmitotic cells (Kipreos and Wang, 1990; Yamashita et al.1990). In each of these studies relatively high extracellu-lar concentrations of OA were added to the culturemedia, and cell rounding was observed within 60 min.Yamashita and coworkers (1990) showed in their studythat the rounded BHK cells detached from the growingsurface and entered a transient mitotic state character-ized by premature chromosome condensation. Spindleswere not observed in these cells while in the mitoticstate, and upon further incubation all microtubulesdepolymerized. On the other hand, the roundingobserved in NIH 3T3 cells by Kipreos and Wang (1990)was not associated with nuclear envelope breakdown orchromosome condensation. From these studies it wasnot clear whether the rounding of cells and alterationsin microtubule architecture were due solely to theinduction of a mitotic state. We, therefore, examinedthe effect of OA on cell growth, mitotic progression,and microtubule and spindle morphology, in LLC-PKcells. These results demonstrate that the effects of OAon mitotic progression and spindle morphology can bedistinguished from its effects on cell shape and

interphase microtubule arrays. Some of these resultshave appeared in preliminary form (Vandre", 1990).

Materials and methods

Cell cultureLLC-PK cells, derived from porcine kidney, were maintainedin monolayer culture at 37°C in a 5% CO2 atmosphere. Cellswere grown in DMEM media supplemented with 10% fetalbovine serum, penicillin (100 units ml"1), streptomycin (0.1mg ml"1), and 20 mM Hepes buffer (Sigma ChemicalCompany, St Louis, MO). Cells were subcultured 16-24 hprior to the addition of okadaic acid (OA) from a 100 ng ml"1

stock solution in dimethyl formamide (Moana Bioproducts,Honolulu, HI). Addition of equivalent amounts of dimethylformamide to the cultures had no effect on cell growth ormorphology. Toxicity of OA was determined by counting cellsthat remained attached to the monolayer growing surface ofthe flask after appropriate incubation time in OA. Attachedcells were collected by treatment with trypsin-EDTA, andcounted using a hemacytometer chamber. The effects ofvarying OA concentration and incubation time on the mitoticindex, mitotic stage and number of spindle poles present intreated cells were determined. Between 100 and 300 indi-vidual cells per coverslip were examined at each experimentalpoint. Cultures were grown on glass coverslips, incubatedwith OA, and then processed for immunofluorescencemicroscopy (see below). In some cases, cultures weresynchronized at the Gi/S boundary following exposure to 2mM hydroxyurea for 16 h prior to the addition of OA.

Immunofluorescence staining and microscopyCells remaining attached to coverslips were lysed in PHEMbuffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mMMgSO,t), pH 6.9, containing 0.5% Triton X-100 for 90 s.Following lysis, cells were rinsed in PHEM buffer and fixed inPHEM buffer containing 0.7% glutaraldehyde for 15 min.Fixative was aspirated and cells were rinsed in three changesof phosphate-buffered saline (PBS), pH 7.4. Unreactedaldehyde groups were reduced by two changes of NaBH* (1mg mP1 in Tris-buffered saline, pH 7.4) over 30 min.Coverslips were rinsed three times in PBS and incubated in4% normal goat serum for 30 min at 37°C, rinsed in PBS, andprocessed for tubulin immunofluorescence using the YL 1/2rat monoclonal anti-tubulin antibody (Accurate Chemical andScientific Corp., Westbury, NY). After three rinses in PBS,coverslips were incubated with fluorescein-conjugated anti-ratimmunoglobulin for 30 min at 37°C, and subsequently rinsedthree times in PBS. The next to last PBS rinse contained 5 jigml"1 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI,Polysciences, Inc., Warrington, PA). After a final rinse indistilled water coverslips were mounted in Mowiwol (Osbornand Weber, 1982) containing 1 mg ml"1 /wraphenylenediamine. For double-label immunofluorescence, cells wereincubated with a mixture of the MPM-2 mouse monoclonalantibody (Davis et al. 1983) and rabbit anti-Tyr-tubulinantibody (Gunderson et al. 1984). This was followed by anincubation in a mixture of rhodamine-conjugated goat anti-mouse and fluorescein-conjugated goat anti-rabbit immuno-globulins.

Media containing OA-treated cells that had rounded anddetached from the coverslip were collected, and cells weredeposited onto coverslips by cytocentrifugation using aCytospin 3 (Shandon Inc., Pittsburgh, PA). Cytocentrifugedcells were fixed in PHEM buffer containing 0.7% glutaralde-

Mitotic block induced by okadaic acid 81

hyde prior to their subsequent lysis in PHEM buffercontaining 0.5% Triton X-100. Lysed cells were processed forimmunofluorescence as described above.

Mounted coverslips were examined with a Zeiss IM-35microscope equipped with epifluorescence optics using aNikon x60 phase, 1.4 NA, planapochromat objective.Immunofluorescence micrographs were recorded on KodakTech-Pan 2415 film.

Results

Okadaic acid blocks LLC-PK cells in mitosisLLC-PK cells were treated with various concentrationsof okadaic acid (OA) to determine its effect on cellgrowth. OA was added to the culture media 24 h aftercells had been subcultured, and monolayers weremaintained for an additional 24-48 h, at which time thenumber of cells remaining in the monolayer wasdetermined (Fig. 1A). Cell growth was inhibited atconcentrations greater than 4.0 nM, and significantcytotoxicity was noted at concentrations of 62 nM orhigher. The LD50 for OA determined after 48 h ofexposure was approximately 10 nM (Fig. IB). Incomparison to control cultures, in drug-treated culturesa significantly greater number of cells remaining on themonolayer appeared to be in mitosis.

The mitotic index of treated cultures was determinedby examining cells fixed and then stained with anti-tubulin antibodies to detect microtubule patterns andDAPI to detect DNA. A dramatic increase in mitoticindex was observed after incubation in OA. Thisincrease was dependent upon both the concentration ofOA applied (Fig. 2A) and the length of exposure todrug (Fig. 2B). At these low concentrations of OA,there was a significant lag period prior to the accumu-lation of mitotic cells that was highly concentrationdependent. At 22 nM OA, the mitotic index did notincrease significantly until nearly 8 h after treatment.This lag period was longer at lower concentrations. Incomparing Fig. 2A and B it is apparent that at 15 nMOA mitotic cells began to accumulate only after 14 h ofexposure, while the mitotic block was readily apparentafter 12 h with 22 nM. Further incubation at 15 nM OAshowed that cells continued to accumulate in mitosis(data not presented). The lag time prior to the mitoticblock was significantly reduced at higher concentrationsof OA (see below).

Treated cells were examined to determine the mitoticstage blocked by OA. At concentrations of OA that hadan effect on mitotic index (15-31 nM; Fig. 2A), cellsaccumulated in a prometaphase-like state, as defined bythe absence of an intact nuclear envelope, the presenceof spindle microtubules and the lack of metaphasealignment of condensed chromosomes. Almost nometaphase, anaphase or telophase cells were present inthe cultures treated overnight with 31 nM OA.However, little change in the percentage of mitotic cellsin prophase was detected (data not presented), sugges-ting that cells were continuing to enter mitosis, but wereblocked in a mitotic state prior to anaphase onset.When cells were first treated overnight with hydroxy-

1 10OA concn (nM)

Fig. 1. Effect of OA on the growth of LLC-PK cells grownin monolayer culture. (A) Cultures were incubated withvarious concentrations of OA and the cell numbersdetermined after 24 and 48 h: control (O O); 4 nM(T T); 8 nM (• D); 15 nM (A A); 31 nM(O O); 62 nM ( • • ) ; 124 nM (V V) OA. (B)Percentage of cells surviving treatment with OA for 48 h incomparison to control populations; LD5o = 10 nM.

urea followed by low concentrations of OA in thecontinued presence of hydroxyurea, no mitotic cellswere detected even after extended periods of incu-bation. Mitotic cells did accumulate if the hydroxyureablock was released at the time of OA addition (data notpresented). Together, these results indicate that duringthe first few hours of exposure to OA other stages of cellcycle progression were not inhibited.

The okadaic acid-induced mitotic block ischaracterized by the presence of an abnormal mitoticspindleVarious spindle morphologies were represented in theblocked mitotic cells following overnight treatment with22 nM OA acid; the most common types are shown in

82 D. D. Vandrt and V. L. Wills

0 4 8 15OA concn (nM)

0 4 8 12 24Time (h) (22 nM OA)

Fig. 2. Effect of OA on the mitotic index of LLC-PK cells.(A) Cultures were incubated for 14 h with variousconcentrations of OA, and the mitotic index of thepopulation was determined. Only a slight effect on mitoticindex was observed at 15 nM OA but the mitotic index wasnearly 6 times control levels at 31 nM OA. (B) The mitoticindex of treated cultures was determined following incubation with 22 nM OA for various periods of time. Anincrease in mitotic index was not observed until afternearly 8 h of treatment, and the mitotic index continued toincrease with longer exposures to the OA.

Fig. 3. Some cells contained a normal bipolar spindlewith chromosomes aligned on the metaphase plate (Fig.3A and B). However, some of the bipolar spindles witha typical microtubule pattern showed a displacement ofsome chromosomes off the metaphase plate. Thesedisplaced chromosomes were usually oriented near oneof the spindle poles (Fig. 3C and D). Many cellscontained bipolar spindles that had a much greaterpole-to-pole separation compared to typical bipolarspindles (compare Fig. 3A with Fig. 4E and G).Chromosomes were no longer aligned at the metaphaseplate but were distributed in a fairly random fashionbetween the spindle poles (Fig. 3F). In a few examples,the spindle fibers appeared to collapse, giving theimpression of an extended narrow spindle (Fig. 3G).The chromosomes in this particular cell appear to be inthree groups, one group associated with each spindle

pole and one group in a metaphase position (Fig. 3H).Multipolar cells were also fairly common, with thechromosomes often randomly distributed between thespindle poles (Fig. 31 and J). The last major category ofspindle morphology present in blocked cells wasrepresented by what appeared to be a disruption in theassociation between the two half-spindles (Fig. 3K andL). Interzonal microtubules connecting the half-spindles were absent, and chromosomes were indepen-dently associated with each half-spindle. In eachexample of aberrant spindle morphology the sisterchromatids remained paired.

Like the increase in mitotic index (Fig. 2), thefrequency with which multipolar spindles were ob-served increased with the concentration of OA (Fig.4A), and length of exposure to OA (Fig. 4B). Theincrease in multipolar cells appeared to proceed in atemporal sequence, with the development of tripolarcells preceding an increase in cells that contained fouror more spindle poles.

LLC-PK cells arrested in mitosis with many drugs,such as colcemid or nocodazole, lack spindle micro-tubules; however, cells blocked in mitosis with taxol, amicrotubule stabilizing agent, retain polymerizedmicrotubules. Spindle microtubules were present inOA-blocked cells after 36 h of treatment, whichsuggested that the OA may, in part, block mitosis bystabilizing microtubules. To test this possibility OA-blocked cells were treated with nocodazole at concen-trations that resulted in net depolymerization ofmicrotubules in untreated mitotic cells. OA-blockedcells showed the same sensitivity to nocodazole as didcontrol cells (data not presented). Thus, OA treatmentdid not appear to stabilize spindle microtubules;therefore, stabilization cannot account for the persist-ence of spindle microtubules in blocked cells.

Mitosis-specific phosphorylations are maintained inblocked cellsThe MPM-2 antibodies (Davis et al. 1983) recognize aset of mitotic phosphoproteins some of which arelocalized to the centrosome (Vandre' et al. 1984). Thesephosphoproteins are a marker for the mitotic state andare lost or dephosphorylated only after anaphase onset(Vandr6 and Borisy, 1989). Yamashita and coworkers(1990) have reported that MPM-specific staining wasinitially detected, but subsequently lost from BHK cellsafter a premature mitotic state was induced byrelatively high concentrations of OA. Therefore, LLC-PK cells blocked in mitosis with OA were examinedwith the MPM-2 antibody to determine if MPM-reactive phosphoproteins characteristic of mitosis weremaintained throughout the length of the blockage.

There was no increase in the MPM staining levels inany interphase cells, but MPM-reactive material wasassociated with spindle poles and kinetochores in allcells blocked in mitosis regardless of the length of OAtreatment (Fig. 5). Typically, two of the spindle poles ormicrotubule nucleating centers in multipolar cellsreacted more intensely with the MPM-2 antibody (Fig.

Mitotic block induced by okadaic acid 83

Fig. 3. Indirect immunofluorescence staining of tubulin in OA-treated LLC-PK cells. Cells grown on coverslips weretreated with 22 nM OA for 18 h, at which time the cells fixed and processed for anti-tubulin indirect immunofluorescencestaining (A, C, E, G, I and K). DNA was stained with DAPI (B, D, F, H, J and L). Various spindle morphologies arepresent in OA-blocked cells, and representative examples are presented. These morphologies range from normal bipolarspindles with chromosomes aligned on the metaphase plate (A and B), to spindles with a few chromosomes displaced fromthe metaphase plate (C and D), bipolar spindles with a dispersed set of chromosomes (E and F), compacted narrowbipolar spindles (G and H), multipolar spindles (I and J), and disrupted spindles in which the half-spindles are no longerassociated (K and L). Bar, 10 im\.

5B and D), which suggested that these were thecentriole-containing structures. The staining intensityof other poles varied greatly, some reacted fairlystrongly while others could not be detected. Thegeneration of secondary poles may result from thefragmentation of pericentriolar material from theoriginal centrosomes, which may account for the widerange of staining intensity observed with the MPM

antibody. The generation of new microtubule organiz-ing centers in these treated cells was probably not dueto the separation of existing centrioles or to theformation of new centrioles, since sufficient time tocomplete an additional centriole cycle is not requiredprior to the appearance of new nucleating sites. Thesepossibilities will be resolved only by following ultra-structural analysis, however.

84 D. D. Vandri and V. L. Wills

100

60

40

20

4 8 15OA concn (nM)

31

100

2 *o

40 -

20 -

B1 1 1 1

I 11 11J

-

-

4 8 12Time (h) (22 nM OA)

24

Fig. 4. Effect of OA on the spindle pole number in cellsblocked in mitosis. The percentages of mitotic cells thatwere bipolar (•), tripolar (•) and multipolar (more thanthree poles) (ffl), were determined in cells labeled forindirect immunofluorescence with antibodies to tubulin.(A) The change in spindle pole number in relation to theconcentration of OA after treatment of 16 h. Higherconcentrations of OA produce a greater percentage ofmultipolar spindles in treated cells. (B) The length ofexposure to OA effects spindle pole number in treatedcells. An increase in the percentage of multipolar cellscorresponds to the length of exposure to OA. Tripolar cellsappear prior to multipolar cells in response to both theconcentration and the time of exposure to OA.

Okadaic acid-treated cells can recover from the mitoticblockTo determine whether the mitotic block induced by OA

Fig. 5. MPM-reactive mitotic phosphoproteins are presentin OA-blocked mitotic cells. LLC-PK cells were treatedwith 22 nM OA (A and B) or 31 nM OA (C and D) for 16h and examined by double-label immunofluorescencemicroscopy for tubulin staining (A and C) and MPM-2staining (B and D). Multipolar spindles were shown tohave MPM-reactive material concentrated at the foci ofmicrotubule-organizing centers. Two of these MPM-reactive centers stained more intensely (large arrowheadsin D), and probably correlate with the position of thecentriole-containing centrosomes. Other foci showedvariable, but less intense, levels of MPM staining (smallarrowheads in D). The number of microtubules nucleatedfrom an individual organizing center varied, and the mostprominent nucleating sites did not necessarily correspondto the position of the two major MPM-reactive centers.Bar, 10 jiM.

(22 nM) was reversible, treated cultures were washedfree of the drug and incubated in fresh media. Sampleswere examined at hourly intervals following drugrelease, for the appearance of anaphase or telophasecells. As in the initial mitotic block, a significant lagperiod, in this case 4-5 h, was required before there wasan increase in these later mitotic stages. Spindles inrecovering cells were generally bipolar, but occasionalmultipolar anaphase cells were observed (data notpresented). The number of multipolar spindles wassignificantly lower than the number detected prior torelease, which suggested that a bipolar spindle was re-established during recovery. Occasionally one or bothof the centrosomes appeared to be detached from thespindle in recovering cells, but normal anaphaseseparation of chromosomes appeared to take place(data not presented). After 14 h of release nearly all

Mitotic block induced by okadaic acid 85

Fig. 6. Multinucleated cells that had escaped the OA-induced mitotic block were observed after extended incubation.Phase-contrast (A, D and G), DNA staining by DAPI (B, E and H), and anti-tubulin staining (C, F and I) ofmultinucleated LLC-PK cells present in cultures treated with 22 nM OA for 30 h. The 1-2% of the cells in the culture thatescaped the OA-induced mitotic block were characterized by multiple micronuclei and a midbody. These morphologiessuggested that chromosome segregation did not occur, but also indicated that the cells underwent cytokinesis. Thiscytokinesis resulted in some cells that appeared to cleave off only a small anuclear piece of cytoplasm (A-C), while in mostcleavage occurred near the center of the cell (D-I). This cleavage often resulted in a micronucleus being trapped in thecleavage furrow (D-F). Bars: 10 ^m (A-F); 20 [M (G-I).

cells in the treated population recovered from the OA-induced mitotic block.

At the low concentrations used to generate themitotic block, OA was not cytotoxic, as shown by therecovery of cells when the drug was removed. Inaddition, occasional multinucleated cells (approxi-mately 1-2% of the mitotic cells) were observed incultures blocked with OA longer than 24 h (Fig. 6). Thissuggested that some treated cells escaped the mitoticblock. Closer examination of the multinucleated cellsindicated that they had undergone cytokinesis, as inevery example a midbody was present (Fig. 6). Thedistribution of nuclei appeared random, as some cellscleaved off only a small portion of cytoplasm lackingnuclei or DNA (Fig. 6A-C) while others showedvarying numbers of nuclei in the two daughter cells(Fig. 6D-I). In some cases it was apparent that re-forming nuclei had been trapped between the daughtercells as the cleavage furrow progressed (Fig. 6D-E).The level of MPM staining in these multinucleated cellswas also at interphase levels (data not presented).

Thus, at the concentrations that resulted in a mitoticblockage OA did not inhibit cytokinesis.

These results suggested that in cells escaping the OA-induced mitotic block chromosome segregation did nottake place but, rather, multiple nuclei formed aroundthe chromosomes that were randomly dispersed on thespindle (see Fig. 3). A cleavage plane was established,however, since a spindle was present in the OA-blockedcells. Cells escaping the block could complete cytokin-esis in the absence of chromosome separation. Thedephosphorylation events associated with anaphaseoccurred as the cells escaped the mitotic block. It hasnot been determined whether sister chromatid disjunc-tion occurred in these cells prior to nuclear envelopebreakdown.

High concentrations of okadaic acid block mitosisrapidly, but are also cytotoxicAt high concentrations of OA (0.5-1.0 /xM), inhibitionof mitosis is observed within 30-90 min; however, therewas no evidence that cells entered a premature mitotic

86 D. D. Vandrd and V. L. Wills

V

Fig. 7. Mitosis was inhibited rapidly at higher concentrations of OA. Treated LLC-PK cells were examined by phase-contrast (A,D and G) and double-label immunofluorescence staining with anti-tubulin (B, E and H) and MPM-2 antibodies(C, F and I). Normal bipolar metaphase spindles were still present in cells treated with 0.5 fjM OA for 60 min (A-C). At1.0 fjM OA, treatment for 60 minutes resulted in an inhibition of mitosis (D-I). Chromosomes were displaced from themetaphase plate to varying degrees, but remained associated with spindle microtubules. Spindle microtubules at this higherconcentration of OA were typically curved or wavy in appearance (E). MPM-2 antibodies clearly stained the spindle polesand kinetochores in these treated mitotic cells (C, F and I). Bar, 10 /an.

state. Normal metaphase spindles were present in cellsexposed to 0.5 ;uM OA for 60 min (Fig. 7A-C), but wereno longer observed after 90 min. At concentrations of1.0 fjM OA, bipolar spindles were present after a 60 minincubation, but they were structurally aberrant (Fig.7D-I). Chromosomes alignment at the metaphase platewas lost, the spindles were generally larger than normalmetaphase spindles, and half-spindles were oftencomposed of wavy or curved microtubules (Fig. 7E).MPM staining was typical for mitotic cells, and waslocalized to the spindle poles and kinetochores, andalong spindle fibers. Further incubation led to thedetachment of mitotic cells from the monolayer, andloss of spindle microtubules (data not presented).

The MPM staining of interphase cells at these higherOA concentrations was not increased, even thoughcontinued incubation at these elevated concentrations

resulted in the rounding and detachment of cells fromthe monolayer. Interphase cells having typical mor-phology were still apparent after 60 min at 0.5 /iM OA(Fig. 8A-C), but the majority of cells were rounded andfloating in the culture media after 120 min of treatment.Most interphase cells were rounded after incubationwith 1.0 /JM OA for 60 min (Fig. 8D-F), and almost allwere detached and floating in the culture media after120 min (Fig. 8G-I). Only a few cytoplasmic micro-tubules remained in rounded cells (Fig. 8E), and in thedetached cells what few microtubules remained wereextremely short and were only associated with thecentrosomes (Fig. 8H). Although in many cell types cellrounding is characteristic of mitotic cells, LLC-PK cellsdo not normally round up appreciably in mitosis.Rounding of cells following OA treatment was also notassociated with induction of mitosis in the LLC-PK cells

Mitotic block induced by okadaic acid 87

Fig. 8. The morphology of interphase cells was altered following treatment with high concentrations of OA. Treated LLC-PK cells were examined by phase-contrast (A,D and G) and double-label immunofluorescence staining with anti-tubulin(B, E and H) and MPM-2 antibodies (C, F and I). Typical interphase morphology, microtubule arrays and MPM-stainingwere still present in cells treated with 0.5 /iM OA for 60 min (A-C). Interphase cells in cultures treated with 1.0 /*M OAfor 60 min had rounded up (D), and this was associated with a loss of many of the cytoplasmic microtubules (E). TheMPM staining intensity remained at low levels typical of interphase cells. After 2 h in the presence of either 0.5 /.iM or 1.0/iM OA almost all cells in treated cultures had detached from the monolayer. The nuclear envelope remained intact (G),and the levels of MPM staining remained low in the majority of the detached cells (I). The cytoplasmic microtubulespresent were short, few in number, and radiated from the centrosome, giving the appearance of an aster in some cells (H).In other detached cells only a few very short microtubules were present (arrowhead, H). The rounding of cells and aster-like appearance was not associated with a premature mitosis in the LLC-PK cells.

as determined by both the presence of intact interphasenuclei and the low levels of MPM staining that arecharacteristic of interphase cells. Cells that detachedfrom the monolayer did not survive subculturing intomedia lacking OA (data not presented). Thus, themorphological changes associated with high OA con-centrations were not due to the induction of a mitoticstate, but more closely reflected the cytotoxicity of thedrug at these concentrations.

When cells were treated with high OA concentrationsafter first being synchronized at G]/S phase withhydroxyurea, rounding and detachment of the hydroxy-urea-blocked cells was observed. There was no evi-dence in these rounded cells for chromosome conden-sation or nuclear envelope breakdown (data not

presented), indicating that the rounding of cells wasmore likely to be the result of additional drug effectsthat were not related to the mitotic blockage observedat lower concentrations of OA.

Discussion

Treatment of LLC-PK cells with low concentrations ofOA (8-40 nM) results in a unique metaphase-likemitotic block that is characterized by the presence of anintact mitotic spindle. Deleterious effects of OA atthese concentrations were not apparent, since mitoticcells continued to accumulate for at least 24 h and themitotic block was reversible. This is in contrast to the

88 D. D. Vandri and V. L. Wills

effects of OA at higher concentrations (0.5-1.0which resulted in cell rounding and detachment fromthe monolayer followed by loss of cell viability. Spindlemorphology in OA-blocked cells demonstrated pro-gressive alterations, which were dependent upon boththe length of time an individual cell remained blockedin mitosis and the concentration of drug. The variousspindle morphologies represented in treated cell popu-lations suggested a temporal sequence for the develop-ment of the abnormal spindle structures. Treated cellsentered prophase at a fairly constant rate even after OAtreatment. As prophase was completed a bipolarspindle was formed with chromosomes aligned on themetaphase plate in a fairly normal fashion. At this pointthe transition from metaphase to anaphase was blockedand there was no further mitotic progression. Chromo-some displacement from the metaphase plate followed.Initially only a few chomosomes were affected, butcontinued loss of chromosomes from their metaphasealignment accompanied the further disruption ofspindle structure. Multipolar spindles or dissociatedhalf-spindles developed after an extended period ofblockage in mitosis.

Chromosomes in blocked cells appear to remainattached to spindle microtubules via an interaction atthe kinetochore even after extended periods in themitotic block; however, they appear to lose the abilityto position themselves properly. This may reflect adisruption in the normal function or regulation of motormolecules required for proper movement of chromo-somes, a change in the dynamics of spindle micro-tubules, or a combination of these events as a result ofthe elevated state of phosphorylation that is maintainedin the blocked cells. Although in some blocked cellsgroups of chromosomes would appear to be clusteredaround the spindle poles, there was no evidence ofsister chromatid separation. Similar morphologies havebeen observed following microinjection of OA intoprometaphase PtKi cells (Vandr6 and Borisy, unpub-lished observations). Cells that escaped the mitoticblock exhibited a unique morphology; they underwentcytokinesis but in the absence of chromosome segre-gation resulting in daughter cells with a variablenumber of micronuclei.

Cells blocked in mitosis also showed the presence ofMPM-reactive mitosis-specific phosphoproteins (Daviset al. 1983; Vandre' et al. 1984). These phosphoproteinswere only detected in treated cells that entered mitosis,and the level of MPM-reactive material associated withcentrosomes and kinetochores was maintained through-out the mitotic block. Generally, only two major MPM-reactive sites were typically detected in multipolar cells.This major MPM staining indirectly reflects the numberof centriole-containing MTOCs as indicated by ourpreliminary ultrastructural analysis of OA-treated cells(Vandr6, unpublished observations). Therefore, theincrease in MTOCs either reflects the fragmentation ofpericentriolar material from existing centrosomes or theaggregation of additional microtubule-nucleating ma-terial from other extracentrosomal locations within thecell rather than the splitting of existing centriole pairs

and/or the generation of new centrioles and centro-somes.

There is a significant lag period between addition ofOA to the culture medium and the initial appearance ofcells blocked in mitosis. This lag period is concentrationdependent, with an effect on mitosis being detectedwithin 30 min at 1 /*M OA, whereas several hours wererequired to observe an initial mitotic block at 22 nMOA. In general, higher concentrations of OA (0.25-15juM) have been used to elicit rapid responses in intactcells. For example, OA has been shown to activate akinase activity that phosphorylates microtubule-associ-ated protein in quiescent fibroblasts with a maximaleffect at 10-20 fM. following a 15 min incubation (Gotohet al. 1990) and a 10-fold stimulation of myelin basicprotein kinase activity has been reported in adipocytesfollowing treatment with 10 fjM OA for 20 min(Haystead et al. 1990). At these high OA concen-trations no apparent toxic effects are observed in short-term incubations (Cohen et al. 1990). While higherconcentrations of OA are required to generate aresponse in intact cells measured in terms of minutesrather than hours, these higher concentrations are alsocytotoxic. Kim et al. (1990) reported the stimulation ofc-fos expression in the A-549 human lung adenocarci-noma cell line by OA after exposure for 24-48 h atconcentrations around 50 nM, but these authors notedOA cytotoxicity at 125 nM against the A-549 cells and60 nM against human synoviocytes in similar assays.OA toxicity was also shown to be high against 3T3 cells,ranging from 10 to 20 nM (Herschman et al. 1989). Incontrast to intact cells, in cell extracts lower concen-trations of OA may be used to observe rapid inhibitionof phosphatase activity (Cohen et al. 1989).

To observe a more rapid response to OA, LLC-PKcells were treated with high concentrations of OA (1/zM) for 30-60 min. At this concentration of drug,however, nearly all cells in the population also roundedup and subsequently detached from the surface of theculture flask. Rounded cells, either before or afterdetachment from the monolayer, exhibited no apparentinduction of a mitotic state, since both the nuclearenvelope remained intact and the level of MPMantibody staining remained at interphase levels. De-tachment was followed by a generalized depolymeriz-ation of microtubules, which ultimately resulted in atotal loss of microtubules. Microtubule dynamics can beinfluenced by phosphorylation events regulated byp34cdc2 kinase as has been demonstrated in Xenopusoocyte extracts (Verde et al. 1990), which may bemediated through a MAP kinase (Gotoh et al. 1991).Phosphorylation of microtubule-associated proteins(MAPs) has also been shown to influence the stability ofmicrotubules (Jameson et al. 1980), and the phos-phorylation state of several MAPs has been shown to bemodulated through the cell cycle (Gard and Kirschner,1987; Vandr6 et al. 1991). It is likely that the depolym-erization of interphase microtubules results from theinhibition of phosphatase activities in addition to thesephosphatase activities responsible for the mitotic blockin LLC-PK cells. Thus, cell rounding that resulted from

Mitotic block induced by okadaic acid 89

OA treatment is not an indicator of entry into a mitotic-like state, but may reflect a generalized increase in thephosphorylation state of MAPs and other proteinsinvolved in maintaining cell shape. A similar disruptionof the interphase microtubule array was not observed atconcentrations of OA that blocked mitosis but did notcause cell rounding and detachment.

Thus, differential effects of OA on interphase andmitotic microtubules could be distinguished by usingdifferent drug concentrations. These effects couldreflect the differential specificity of OA for the catalyticsubunits of PP1 and PP2A. The low concentrations ofOA necessary to generate a mitotic block suggest that aspecific, highly sensitive phosphatase activity, perhaps aform of PP2A, is involved in processes that trigger theonset of anaphase. The phosphatase activity involved inanaphase onset has yet to be identified, but this couldbe a specific isoform of PP2A or a unique phosphatase.Multiple isozymes of PP2A have been cloned as well asseveral novel protein phosphatases related to PP1 andPP2A (Cohen and Cohen, 1989). Recently, eight to tendifferent protein serine/threonine phosphatases havebeen shown to be expressed in a single cell type(Wadzinski et al. 1990), suggesting a multiplicity ofregulatory roles for various phosphatases. Most re-cently, a unique OA-sensitive phosphatase designatedPP3 has been described that may be involved in mitoticprogression (Honkanen et al. 1991). The initial effectsof OA, such as an inhibition of mitosis, are observedmore rapidly at higher concentrations of the drug.However, as the intracellular concentration of OAcontinues to increase, there is also the potential for therapid inhibition of additional PP2A activities and/orPP1 activities. The inhibition of these additionalphosphatase activities could be involved in the re-arrangement of microtubules and cell rounding that areobserved at higher OA concentrations.

Recently, Yamashita and coworkers (1990) reportedon the treatment of BHK cells with 0.5 /zM OA. Cellssynchronized in early S phase by isoleucine deprivationand hydroxyurea treatment rounded and detached fromthe monolayer after exposure to OA, similar to thebehavior of LLC-PK cells at equivalent drug concen-trations. Unlike LLC-PK cells, many of the detachedBHK cells exhibited a transient premature mitotic statecharacterized by nuclear envelope breakdown, prema-ture chromosome condensation, and an increase inMPM antibody staining. A spindle, however, was notformed during this induced mitotic state. Roundedcellular morphology suggesting a mitotic phenotype hasalso been observed in 3T3 cells following treatmentwith a high concentration of OA (Kipreos and Wang,1990). However, like the LLC-PK cells, the rounding of3T3 fibroblasts was not associated with nuclear envel-ope breakdown or chromosome condensation. How canthe apparent differences in the cellular response to OAbe reconciled? Activation of p34cdc2 kinase, whichregulates entry into mitosis, requires both the accumu-lation of the cyclin protein A and/or B to a criticalthreshold level, and the subsequent post-translationalmodification of p34cdc2. These modification events

involve both the phosphorylation and dephosphoryl-ation of the protein. Levels of OA that specificallyinhibit PP2A activities have been shown to transientlyactivate p34cdc2 in Xenopus oocyte extracts (Felix et al.1990a). Subsequent studies have demonstrated that OAinhibits a negative regulator of p34cdc2 activity termedINH (Solomon et al. 1990), and the INH has beenshown to be a PP2A (Lee et al. 1991). If INH activity isinhibited by OA, the threshold level of cyclin B isreduced, and once threshold levels are achieved thedelay in cyclin B activation of p34cdc2 is eliminated(Solomon et al. 1990). In BHK cells, cyclin B is presentin early S phase and continues to accumulate through-out S phase (Yamashita et al. 1990). In conjunction withthe accumulation of cyclin B, if threshold levels ofcyclin B were lowered by OA treatment, prematureentry into mitosis would occur in many of the treatedBHK cells. This correlation between cyclin B levels andpremature mitosis has recently been established inBHK cells (Steinmann et al. 1991). The transient natureof the premature mitotic state reported by Yamashita etal. (1990) may be a result of cytotoxic effects from thehigh concentrations of OA used in these studies. CyclinB does not accumulate in S phase HeLa cells, however,but accumulates later in G2 phase (Pines and Hunter,1989). This correlates with the inability to inducechemically premature mitosis in human cells with OA(Steinmann et al. 1991). Therefore, if cyclin levels inLLC-PK and 3T3 cells more closely resemble those ofHeLa cells, few cells in an unsynchronized populationwould enter a premature mitosis following exposure toOA. It will be of interest to determine if lowerconcentrations of OA would be able to induce a morestable premature mitotic state in BHK cells in theabsence of cell rounding and detachment from themonolayer.

Owing to the low concentrations of OA required, themitotic block obtained with LLC-PK cells suggests theinvolvement of a PP2A-like phosphatase in the regu-lation of anaphase onset. A role for PP2A, in the formof INH, has been clearly established in mitoticregulation (Solomon et al. 1990; Lee et al. 1991).However, the regulation of mitotic progression mayalso involve the activity of PP1. Mutations in genes withsequence homology to mammalian PP1 like those thathave been identified in Aspergillus nidulans, the bimGgene (Doonan and Morris, 1989); the fission yeastSchizosaccharomyces pombe, the dis2 gene (Ohkura etal. 1989); and Drosophila, the PP1 87B gene (Axton etal. 1990), support this concept. Each of these mutationsappears to block stages of mitosis subsequent tometaphase. The defects in spindle morphology ob-served in Drosophila PP1 mutants are strikingly similarto spindle abnormalities observed in LLC-PK cellsblocked in mitosis with concentrations of OA thatwould not be expected to affect PP1 activity. It ispossible that PP1 is involved in the dephosphorylationof mitotic phosphoproteins, and mutations or inhibitionof PP1 would mimic the effects of stabilized p34cdc2. Ineach case elevated levels of mitosis-specific phosphoryl-ation would be maintained.

90 D. D. Vandrd and V. L. Wills

In LLC-PK cells blocked in mitosis with eithernocodazole (Vandr6 and Borisy, 1989) or low concen-trations of OA (this study), MPM-reactive mitosis-specific phosphorylations are maintained for extendedperiods, suggesting that p34cdc2 kinase activity isstabilized under these conditions. MPF kinase activityalso remains elevated in nocodazole-blocked cells(Morla et al. 1989; Yamashita et al. 1990), and incytostatic factor (CSF)-blocked oocytes (Newport andKirschner, 1984; Sagata et al. 1989). Microinjection ofOA in to starfish oocytes also induces a stable mitoticblock (Picard et al. 1989). Loss of mitotic activityrequires the inactivation of pM"102 kinase, and loss ofkinase activity coincides with the proteolytic degra-dation of cyclin (Murray et al. 1989; Draetta et al. 1989;Felix et al. 1990b). A proteolysis-resistant mutant cyclinmolecule that contains a truncated N terminus has beenshown to prevent exit from mitosis and loss of MPFkinase activity (Murray et al. 1989). In a similar fashion,the activity of cytostatic factor (CSF) is responsible formaintaining MPF activity and the meiotic block typicalof oocytes (Newport and Kirschner, 1984). The activecomponent of CSF has recently been identified as thec-mos proto-oncogene product (Sagata et al. 1989),which is a serine/threonine kinase that is capable ofphosphorylating cyclin B (Roy et al. 1990). Theseresults suggest that phosphorylated cyclin is moreresistant to proteolytic degradation, and that dephos-phorylation of the cyclin is involved in the cyclindegradation pathway. The results obtained with theLLC-PK cells suggest that a phosphatase responsiblefor the dephosphorylation of cyclin was inhibited at lowconcentrations of OA. Thus, an OA-sensitive phospha-tase could be involved in triggering the metaphase toanaphase transition by inhibiting the signal regulatingcyclin degradation. The result of this inhibition wouldbe an extended metaphase. While consistent with thepresent results, this hypothesis requires the measure-ment of p34cdc2 kinase activity, cyclin levels andphosphatase activities in cells blocked in mitosis withOA.

Phosphatase inhibitors must be used under carefullycontrolled conditions to establish which phosphatasesare directly involved in regulating specific events suchas mitosis. In general, phosphatases have a broadsubstrate specificity, and inhibition of some phospha-tases may have pleiotropic effects. While OA demon-strates a higher specificity for PP2A than PP1, thisdifference is influenced by the concentrations of thephosphatases within the cell. It is not clear whetherindividual phosphatase isozymes within each of theseclasses would be more or less sensitive to OAinhibition, but this may also be influenced by intracellu-lar concentrations of the individual isozymes. We havedemonstrated, however, that the effects of OA on cellcycle progression and cell morphology can be dis-tinguished at specific drug concentrations. The mitoticblock observed in LLC-PK cells following treatmentwith low levels of OA appears to be a generalphenomenon, as similar mitotic effects have beenobserved in CHO and HeLa cells (Vandr6, unpublished

observations), and most recently in human leukemiaK562 cells (Zheng et al. 1991). These results indicatethat the transition from metaphase to anaphase mayinvolve the activity of a specific phosphatase, andinhibition of this phosphatase activity blocks cells in anextended metaphase-like state. This phosphatase ac-tivity is distinct from other phosphatase activities thatmay be involved in the maintenance of microtubulesand cell shape. Whether the phosphatase involved inregulating the metaphase to anaphase transition isrelated to the PP2A phosphatase ENH, which isinvolved in the activation of p34cdc2 kinase, remains tobe determined.

The authors thank April Smart for her technical assistancein the early phases of this work and Dr John Robinson for hiscomments regarding the manuscript. This work was sup-ported by a National Science Foundation grant DCB-8902338,and American Cancer Society grant IRG-16-30.

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{Received 30 May 1991 - Accepted, in revised form,19 September 1991)