Implications for the role of microtubule dynamics in …Inhibition of mitosis by drugs 403 Table 1....

Journal of Cell Science 102, 401-416 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

401

Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles

Implications for the role of microtubule dynamics in mitosis

MARY ANN JORDAN*, DOUGLAS THROWER and LESLIE WILSON

Department of Biological Sciences, University of California, Santa Barbara, CA 93106, USA

*Author for correspondence

Summary

Inhibition of mitosis by many drugs that bind to tubulinhas been attributed to depolymerization of micro-tubules. However, we found previously that low concen-trations of vinblastine and vincristine blocked mitosis inHeLa cells with little or no depolymerization of spindlemicrotubules, and spindles appeared morphologicallynormal or nearly normal. In the present study, wecharacterized the effects of vinblastine, podophyllotoxinand nocodazole over broad concentration ranges onmitotic spindle organization in HeLa cells. These threedrugs are known to affect the dynamics of microtubulepolymerization in vitro and to depolymerize micro-tubules in cells. We wanted to probe further whethermitotic inhibition by these drugs is brought about by amore subtle effect on the microtubules than netmicrotubule depolymerization. We compared the effectsof vinblastine, podophyllotoxin and nocodazole on theorganization of spindle microtubules, chromosomes andcentrosomes, and on the total mass of microtubules.Spindle organization was examined by immunofluor-escence microscopy, and microtubule polymer mass was

assayed on isolated cytoskeletons by a quantitativeenzyme-linked immunoadsorbence assay for tubulin. Asthe drug concentration was increased, the organizationof mitotic spindles changed in the same way with allthree drugs. The changes were associated with mitoticarrest, but were not necessarily accompanied by netmicrotubule depolymerization. With podophyllotoxin,mitotic arrest was accompanied by microtubule depol-ymerization. In contrast, with vinblastine and nocod-azole, mitotic arrest occurred in the presence of a fullcomplement of spindle microtubules. All three drugsinduced a nearly identical rearrangement of spindlemicrotubules, an increasingly aberrant organization ofmetaphase chromosomes, and fragmentation of centro-somes. The data suggest that these anti-mitotic drugsblock mitosis primarily by inhibiting the dynamics ofspindle microtubules rather than by simply depolymer-izing the microtubules.

Key words: mitosis, microtubule dynamics, vinblastine,podophyllotoxin, nocodazole.

Introduction

Inhibition of mitosis by several anti-mitotic drugsincluding vinblastine, podophyllotoxin and nocodazole,that can depolymerize microtubules in vivo and in vitro,has been considered to occur by a mechanism involvingdepolymerization of microtubules (e.g. see Malawistaet al. 1968; Wilson and Bryan, 1974; DeBrabander et al.1976; Hoebeke et al. 1976; Zieve et al. 1980; reviewedby Dustin, 1984). However, we found that inhibition ofmitosis in HeLa cells by low concentrations of vincris-tine and vinblastine occurs with little or no depolym-erization of spindle microtubules (measured by en-zyme-linked immunoadsorbence assay (ELISA) oftubulin in isolated cytoskeletons; Jordan et al. 1991). Byimmunofluorescence microscopy with an antibody totubulin, blocked metaphase spindles appear morpho-logically normal or exhibit only slight abnormalities inmicrotubule organization. We also found that vinblast-

ine significantly inhibits the exchange of tubulin at theends of in vitro reassembled microtubules withoutexerting significant effects on the total mass of micro-tubule polymer (Jordan and Wilson, 1990). Theseresults indicated that vincristine and vinblastine inhibitmitosis, not by inducing microtubule depolymerization,but by stabilizing the dynamics of the spindle micro-tubules. We therefore wanted to determine whetherother microtubule-depolymerizing drugs inhibit mitosisby depolymerizing spindle microtubules or by a moresubtle action on the microtubules.

In the present study, we characterized further theeffects of vinblastine, and we characterized the effectsof podophyllotoxin and nocodazole on mitotic accumu-lation, the mass of cellular microtubules, and theorganization of the spindle microtubules, chromosomesand centrosomes in HeLa cells. We found that whileeach drug blocked mitosis with very different effects onmicrotubule polymer levels, all three drugs induced a

402 M. A. Jordan and others

nearly identical concentration-dependent series of re-arrangements of spindle microtubules, centrosomesand chromosomes. The series was characterized by adrug-concentration-dependent: (1) increase in thelength and number of astral microtubules; (2) decreasein the length of the central spindle; (3) increase in thenumber of chromosomes that were found near thespindle poles rather than in the metaphase plate; and(4) fragmentation of centrosomal material. With lowconcentrations of vinblastine and nocodazole, thesechanges occurred in the absence of any net depolym-erization of microtubules.

The observations reported in the present study,together with data on the effects of vinblastine,podophyllotoxin and nocodazole on the dynamics ofmicrotubules in vitro (see Discussion), suggest thatthese three antimitotic drugs, and perhaps others aswell, inhibit mitosis primarily by inhibiting microtubuledynamics. The data support the idea that the dynamicbehavior of microtubules is crucial to the progress ofmitosis and the cell cycle.

Materials and methods

Cell cultureHeLa S3 cells were provided by Dr. Jeannette Bulinski(Columbia University, New York, NY) or American TypeCulture Collection (Rockville, MD). Cells were grown inmonolayers at 37°C without antibiotics in 5% CO2 aspreviously described (Jordan et al. 1991). Subcultures of cellsfor assay of polymer mass were performed at a density of 1.5X 106 cells/ml in 15 ml. Approximately 20 h later freshmedium plus or minus drug was added. Cells were harvestedfor assay of polymerized and soluble tubulin 18-20 h after drugaddition. Subcultures of cells for immunofluorescence mi-croscopy and for assays of proliferation were plated at adensity of 4 x 104/2 ml in 35 mm dishes containing no. 1 glasscoverslips freshly coated with polylysine (50 /ig/ml, 2 h, 37°C,followed by a rinse with water and a rinse with medium).Approximately 40 h later, fresh medium containing drug (orno drug) was added. At this time, cells were scraped fromsome coverslips and counted by hemocytometer to determinecell number at the time of drug addition. Cell viability wasdetermined by exclusion of trypan blue. At 18-20 h after drugaddition samples of control and drug-treated cells werecounted to determine the increase in cell number. Two to fourindependent experiments were performed with each drug todetermine the concentration dependence for inhibition of celldivision. Simultaneously, parallel coverslips of cells werefixed and processed for immunofluorescence microscopy.Vinblastine was a gift from Eli Lilly and Co., Indianapolis,IN. Podophyllotoxin was obtained from Aldrich (Milwaukee,WI) and nocodazole from Janssen Pharmaceutical (Beerse,Belgium).

Immunofluorescence microscopy and determination ofconcentration dependence for metaphase arrest andspindle reorganizationCells grown on coverslips were prepared for immunofluor-escence microscopy as described previously; fixation was informalin followed by methanol (Jordan et al. 1991). Tubulinwas detected with a mouse monoclonal antibody (E7, IG^ agift from Dr. Michael Klymkowsky, University of Colorado,

Boulder, CO; Chu and Klymkowsky, 1989) that is specific for/3-tubulin in HeLa cell extracts (data not shown), centrosomeswere detected using antiserum 5051, a human autoimmuneanti-centrosomal antiserum (a gift from Dr. S. Doxsey,University of California, San Francisco, CA; Calarco-Gillamet al. 1983), and chromosomes were stained with DAPI (4,6-diamino-2-phenylindole; Sigma Chemical, St. Louis, MO).Second antibodies were fluorescein isothiocyanate-conju-gated goat anti-mouse IgG and rhodamine-conjugated goatanti-human IgG (Cappel, West Chester, PA). The percentageof cells arrested in metaphase was counted on preparationsdouble-stained with DAPI and for anti-tubulin immunofluor-escence; at least 400 cells were counted at each drugconcentration tested; two or three independent experimentswere performed for each drug. At drug concentrations thatwere just sufficient to induce metaphase arrest, metaphasewas clearly distinguishable from other stages of mitosis by thecharacteristic compact metaphase plate of chromosomes.However, at high drug concentrations associated with theformation of types 111 and IV mitotic spindles (see Results fordescription of types), cells with highly condensed masses ofchromatin were arbitrarily called "metaphase" as has beenthe convention in the literature. Sufficient numbers of mitoticcells were counted to acquire a minimum of 50 anaphaseand/or metaphase cells to determine the anaphase/metaphaseratio. Between 50 and several hundred metaphases werescored for each drug concentration to determine the fre-quencies of normal and types I-IV spindles.

The distance between the two poles was measured onmetaphase spindles that had been triply stained with DAPIand with anti-centrosomal and anti-tubulin antibodies.Measurement was done directly on the coverslip preparationusing a 40x Olympus oil immersion objective and an eyepiecereticle. Only spindles that had both centrosomes in the planeof focus were measured. A minimum of 22 spindles weremeasured for each drug concentration. Photo-micrographswere obtained using a Zeiss Photomicroscope III equippedwith an epi-fluorescence condenser and a 40x OlympusUVFL oil immersion objective of numerical aperture 1.3 asdescribed previously (Jordan et al. 1991).

Quantitation of tubulin in microtubulesCells were released from flasks by gentle scraping, collectedby centrifugation, and resuspended for counting and forcollection of stabilized microtubules in cytoskeletons asdescribed in detail previously (Thrower et al. 1991). Tubulinin microtubules was determined using an enzyme-linkedimmunoadsorbence assay (ELISA) (Thrower et al. 1991)using a monoclonal antibody to beta tubulin (1-1.1, IgM,kappa class; Ball et al. 1986). The tubulin standard was three-times-cycled microtubule-associated protein (MAP)-depletedbovine brain tubulin prepared as described by Farrell et al.(1987). Between 2 and 6 independent determinations ofmicrotubule polymer mass were made for each drug concen-tration.

Results

We incubated HeLa cells for a duration of one cell cyclewith a range of concentrations of vinblastine, podophyl-lotoxin and nocodazole. Cells were then fixed andprocessed for fluorescence microscopy to determine thearrangement of chromatin or chromosomes usingDAPI, the microtubules using anti-tubulin immunoflu-orescence, and the centrosomes using 5051, a human

Inhibition of mitosis by drugs 403

Table 1. Effects of vinblastine, podophyllotoxin andnocodazole on mitotic block, the mass of microtubule

polymer and cell division

0.01 0.1 1 10 100 1000 10000 100000

[ VinblastineJ (nM)

Fig. 1. Metaphase arrest and microtubule depolymerizationand spindle length after incubating HeLa cells withincreasing concentrations of vinblastine. (A) Percentage ofcells in metaphase (circles) and percentage decrease inmass of polymerized microtubules compared with controlcells (squares). At concentrations of 10 ,uM and 100 ,uMvinblastine, the polymer mass increased as a result offormation of vinblastine-tubulin paracrystals. The relativelylarge standard errors of polymer mass measurements at 10and 100 fiM vinblastine are probably due to the smallnumber of assays carried out at these concentrations and tocell death, which was prevalent in this vinblastineconcentration range (data not shown). (B) Percentagedecrease of interpolar distance of normal, type 1 and typeII spindles.

scleroderma autoimmune antiserum. The percentage ofcells in mitosis or in a mitotic-like stage after drugtreatment was determined from the stained prep-arations. In parallel, we isolated stabilized cytoskel-etons and determined the total mass of tubulin in theform of microtubules that remained after drug treat-ment as compared with control cells.

Effects of vinblastine on mitosisMitotic arrest and microtubule polymer mass

Incubation with vinblastine induced cells to accumulateat a stage resembling mitotic metaphase in a concen-tration-dependent manner (Fig. 1A, circles). A total of50% of the cells accumulated in metaphase (Kmet) at 0.8nM drug (Table 1), and a peak of maximal accumu-lation occurred at 6 nM vinblastine (Fig. 1A). Vinblast-ine inhibited cell division concomitant with metaphasearrest; half-maximal inhibition of cell division (Kdiv)occurred at 0.45 nM vinblastine (Table 1).

The ratio of the number of cells in anaphase to the

Drug"•met *^ana/met "-dep

(nM)

VinblastinePodophyllotoxinNocodazole

0.803054

0.405

12

1115

600

0.452022

140.5

11

Kmei, drug concentration that induced accumulation of 50% ofcells in metaphase after incubation for 18-20 h. From the data ofFigs 1A, 4A and 6A. Kima/mcx, drug concentration that induced a50% decrease in the ratio of the number of cells in anaphase tothe number of cells in metaphase after incubation for 18-20 h (datafor vinblastine is from Jordan et al. (1991). Kdcp, drugconcentration that induced a 50% decrease of microtubulepolymer mass as determined by quantitation of tubulin incytoskeletons isolated from cells 18-20 h after drug addition. Fromthe data of Figs 1A. 4A and 6A. ^Cdiv, drug concentration thatinduced inhibition of cell division by 50% after treatment ofexponentially growing cells for 18-20 h. The values weredetermined from plots of the percentage inhibition of increase innumber of cells after incubation for 18-20 h in the presence ofdrug vs the drug concentration (data not shown). For example, thevalue for vinblastine was derived from Fig. 1 of Jordan ct al.(1991).

number of cells in metaphase at each vinblastineconcentration was determined to ascertain whetheraccumulation of cells in metaphase by vinblastine wasdue to slowing of mitosis relative to the entire cell cycle,or whether it was due to a block of mitosis atmetaphase. An increase in the number of cells inmetaphase and a proportional increase in the number ofcells in anaphase would indicate that mitosis wasslowed, whereas an increase in the number of cells inmetaphase and a decrease in the number of cells inanaphase would indicate a metaphase block. Vinblast-ine induced a decrease in the ratio of cells in anaphaseto cells in metaphase in a concentration-dependentmanner, with 50% decrease in the ratio (Kana/met)occurring at 0.4 nM drug (Table 1). At 1.6 nMvinblastine, the anaphase/metaphase ratio was zero(data not shown). Thus, vinblastine blocked cells atmetaphase of mitosis.

Vinblastine affected the total mass of cellular micro-tubule polymer in a complex fashion with respect todrug concentration and with respect to metaphaseaccumulation (Fig. 1A, squares). Metaphase accumu-lation occurred without any reduction in total micro-tubule polymer mass at vinblastine concentrationsbetween 0.1 and 6 nM; maximal metaphase accumu-lation occurred in the concentration range in whichmicrotubule polymer mass was unaltered. Microtubulepolymer mass was reduced by 50% (Kdcp) at 11 nMvinblastine, 14 times the concentration required for50% accumulation of cells at metaphase (Kdep/Kmct,Table 1). Microtubules were completely depolymerizedat 100 nM vinblastine. Vinblastine at concentrationsgreater than 1 juM induced formation of vinblastine-tubulin paracrystals, resulting in increased polymer inisolated cytoskeletons (described further below).


Fig. 2. Microtubule, chromosome, and centrosomearrangement of two normal (control) cells in metaphase(the cell on the left and the cell on the right). Other cells(more dimly stained) are in interphase. (A) Indirectimmunofluorescence using anti-tubulin monoclonalantibody followed by a fluorescein isothiocyanate-conjugated second antibody on HeLa cells fixed andstained as described in Materials and methods, (a) DAPIstain of metaphase plates of chromosomes (arrows) in thesame cells, (a') Indirect immunofluorescence ofcentrosomes (arrows) using 5051, a human sclerodermaautoimmune antisemm, followed by a rhodamine-conjugated second antibody, in the same cells. Bar, 10 ixm.In this and other micrographs, staining of the same cell orgroup of cells using different antibodies and fluorochromesis designated by one alphabet character, as A, a or a'.

Effects of vinblastine on spindle organizationMetaphase spindles of control cells contained primarilykinetochore and interpolar microtubules; the few astralmicrotubules that were present were typically veryshort and were often barely detectable (Fig. 2A).Condensed chromosomes (Fig. 2a, arrows) were in acompact metaphase plate located midway between thetwo spindle poles. A single compact mass of centro-somal protein was located at each pole (Fig. 2a',arrows). The poles were separated by a distance of 7.9± 0.4 pan.

Fig. 3. Microtubule, chromosome and centrosomearrangement in HeLa cells after 18-20 h incubation invinblastine at the specified concentrations. (A,a,B) 0.8 nM;(C,c,c') 3.2 nM; (D,d,d') 6.4 nM; (E,e) 100 nM; (F) 1 (M;(G) 10 (M\ (H) 100 [M vinblastine. (A,C,D,E,F,G,H)Anti-tubulin immunofluorescence; (a,c,d,e) DAPI stain ofchromosomes; (B,c',d') anti-centrosomalimmunofluorescence. (A,a) Type I abnormal spindleinduced by incubation in 0.8 nM vinblastine, arrows pointto tufts of long astral microtubules and to chromosomeslocated near the spindle poles. (B) Fragmentedcentrosomal material induced by 0.8 nM vinblastine, themicrotubules and chromosomes of these three spindleswere in the normal metaphase configuration (not shown).C,c,c') Types I, II and III abnormal spindles (labelled I, IIand III) induced by 3.2 nM vinblastine. Arrows point tochromosomes located near the spindle poles. (D,d,d') TypeIII abnormal spindles with accompanying peripheralpunctate aggregates of tubulin (in D) and fragmentedcentrosomes induced by 6.4 nM vinblastine (in d').(E,e,F) Absence of microtubules and presence of punctateaggregates of tubulin induced by 100 nM and 1 fjMvinblastine, respectively; the chromosomes of these cells(type IV) were mitotic-like (condensed with no nuclearmembrane) except for the cell marked i which was ininterphase; each cell had one or two compact centrosomes(not shown). (G,H) Vinblastine-tubulin paracrystalsinduced by 10 JJM and 100 pM vinblastine, respectively; thenuclei of the cells in (G) were in interphase (not shown);the cells of (H) were judged to be dying from the pyknoticappearance of their chromatin and their inability to excludetrypan blue (not shown). Bars, 10 ^m; bar in a givesmagnification of A,a; bar in B gives magnification of allother panels.

After incubation with low concentrations of vinblast-ine (0.4-1.6 nM), between 12% and 69% of the cellswere blocked in metaphase (Fig. 1A). Yet, by immuno-fluorescence microscopy, many of the metaphasespindles were morphologically indistinguishable frommetaphase spindles of control cells. (The proportion ofall metaphase spindles that appeared normal was 69%after incubation with 0.4 nM vinblastine; the proportionthat appeared normal diminished to 9% of all meta-phase spindles with 1.6 nM vinblastine.) However,some spindles were clearly abnormal. The abnormalspindles could be described in terms of four types (I-IV), characterized by increasing degrees of disorganiz-ation (Jordan et al. 1991). Type I spindles were nearlynormal-looking bipolar spindles (Fig. 3A,a), exceptthat one or a few chromosomes were near one or bothspindle poles instead of being aligned with the majorityof the chromosomes at the metaphase plate (Fig. 3a,arrows). Also, the astral microtubules of type I spindleswere typically more numerous and somewhat longerthan those of normal spindles (Fig. 3A, arrows).

Type II abnormal spindles were bipolar (Fig. 3C,c,c')but the microtubules and chromosomes exhibited moreextensive rearrangement than those of type I spindles.More chromosomes were located at one or both poles intype II spindles than in type I spindles (Fig. 3c, arrows).Astral microtubules were prominent and often werevery long (Fig. 3C). The average the distance between


Fig. 3


the two poles in type I and type II spindles was 26%shorter than the distance between poles of normalspindles (Jordan et al. 1991). Thus in both type I andtype II spindles the astral microtubules were longer andthe kinetochore microtubules were shorter than nor-mal. The distinction between type I and II spindles wassubjective and was made primarily to emphasize thequalitative continuum of changes that occurred withincreasing drug concentration. Type III spindles (Fig.3C,c,c',D,d,d') were essentially monopolar and con-sisted of a ball of condensed chromatin (Fig. 3c,d), oneor more star-shaped aggregates of microtubules (Fig.3C,D), and one or more masses of centrosomal material(described further below) (Fig. 3c',d').

Between 0.2 nM and 1.6 nM vinblastine, the arrestedcell population contained a mixture of normal spindlesand abnormal types I, II and III spindles. For example,12% of the cells contained metaphase spindles at 0.4nM vinblastine; of those spindles 69% were normal,16% were types I and II, and 15% were type III. As thedrug concentration was increased from 0.2 nM to 1.6nM vinblastine, the frequency of normal and types I andII spindles diminished and the frequency of type IIIspindles increased. For example, 48% of the cellscontained metaphase spindles at 0.8 nM vinblastine; ofthose spindles 16% were normal, 12% were types I andII, and 72% were type III. Above 1.6 nM vinblastine, asfor example at 3 nM vinblastine, 82% of the cellscontained metaphase spindles, but there were nonormal-looking spindles; all spindles were types I, II orIII. At 6 nM vinblastine, 86% of the cells containedmetaphase spindles, but there were no bipolar spindlesand all spindles were type III (Fig. 3D,d,d').

Type IV mitotic figures (Fig. 3E,e,F) had nomicrotubules and contained nondescript aggregates ofcondensed chromosomes. Type IV mitotic figuresoccurred only upon incubation with vinblastine atconcentrations of 50 nM and higher; at these concen-trations no microtubule polymer was detected inisolated cytoskeletons (e.g. 100 nM vinblastine; Fig.1A, squares).

Small punctate aggregates of tubulin were observedby immunofluorescence microscopy at vinblastine con-centrations between 6 nM and 1000 nM. The aggregateswere present in very small numbers at vinblastineconcentrations of 6 nM (Fig. 3D); their numberincreased after incubation of cells with vinblastine atconcentrations that resulted in loss of measurablemicrotubule polymer. At 100 nM and 1000 nMvinblastine they were the only tubulin-containingstructures visible by immunofluorescence (Fig. 3E,F).The aggregates were located at the cell periphery incells displaying type III spindles (Fig. 3D) and werescattered throughout the cytoplasm in cells displayingtype IV mitotic figures (Fig. 3E,F).

The centrosomal material of cells incubated with verylow concentrations of vinblastine (0.4 nM or less)formed a single compact mass in interphase cells or twocompact masses in mitotic cells, one at each spindlepole (not shown). Between 0.8 and 13 nM vinblastine,centrosomal material in mitotic cells displaying normal

and types I, II and III spindles was fragmented intomultiple pieces that were located at or near the points ofconvergence of microtubule arrays. The fragmentationof centrosomal material induced by 0.8 nM vinblastineis shown in three cells in Fig. 3B. The location offragmented centrosomal material at the foci of micro-tubule arrays is shown in Fig 3C,c',D,d'. Above 50 nMvinblastine, mitotic centrosomes were compact (datanot shown); this occurred concomitant with totalmicrotubule depolymerization (Fig. 1 A). In contrast tothe effects of vinblastine on centrosomal material inmitotic cells, the centrosomal material of interphasecells was not fragmented at any vinblastine concen-tration examined (data not shown).

With increasing vinblastine concentration between0.1 nM and 6 nM, the distance between the spindlepoles decreased. Central spindles were half as long ascentral spindles in control cells after incubation with 3.6nM vinblastine (Fig. IB).

Vinblastine-tubulin paracrystal formationTubulin paracrystals formed only at high vinblastineconcentrations (> 1 fjM), concomitant with an increasein the mass of polymeric tubulin in cytoskeletal isolates(Fig. 1A, squares). At 10 /JM vinblastine, cellscontained several long, large, polygonal tubulin parac-rystals (Fig. 3G). The chromatin was either interphase-like (diffuse with an apparent nuclear membrane) ormitotic (condensed and aggregated with no apparentnuclear membrane) (not shown). At a vinblastineconcentration of 100 ^M, cells contained large numbersof small, globular paracrystals (Fig. 3H), and thechromatin often appeared extremely condensed (notshown).

Effects of podophyllotoxin on mitosisThe effects of podophyllotoxin on mitotic arrest andspindle organization appeared similar to the effects ofvinblastine in some ways, but there also were significantdifferences in the mode of action of the two drugs.

Mitotic arrest and microtubule polymer massLike vinblastine, podophyllotoxin induced cells toaccumulate in mitotic metaphase (Fig. 4A, circles). Atotal of 50% of the cells accumulated in metaphase(Kmet) at approximately 30 nM podophyllotoxin (Table1) and a peak of maximal metaphase accumulationoccurred at a podophyllotoxin concentration of 100 nM(Fig. 4A). Incubation of cells with podophyllotoxin,like incubation with vinblastine, decreased the ratio ofcells in anaphase to cells in metaphase in a concen-tration-dependent manner, with 50% decrease in theratio occurring at 5 nM podophyllotoxin (Kana/met,Table 1). The anaphase/metaphase ratio was zero at 33nM podophyllotoxin. Half-maximal inhibition of celldivision (Kdiv) occurred at 20 nM podophyllotoxin(Table 1).

In contrast to the action of low concentrations ofvinblastine, metaphase accumulation by low concen-trations of podophyllotoxin was associated with a highdegree of microtubule depolymerization (Fig. 4A,

10 100

[Podophyllotoxin] (nM)

1000 10000

Fig. 4. Podophyllotoxin concentration dependence ofmetaphase arrest and microtubule depolymerization (A)and of spindle and cell reorganization (B,C) in HeLa cellsafter treatment with podophyllotoxin at the statedconcentrations for the duration of one cell cycle.(A) Accumulation of cells in metaphase was concomitantwith depolymerization of microtubules. Percentage of cellsin metaphase (open circles) and percentage decrease inmass of polymerized microtubules (filled squares).(B) Percentage of metaphases that were types I or II (opentriangles) and types III and IV (filled triangles). Thepercentage of metaphase spindles that appeared normalequals 100% minus the percentages indicated by the filledand open triangles. (C) Percentage decrease of interpolardistance measured on bipolar spindles (normal, type I andtype II).

squares; Table 1, Kdcp/Kmcl=0.5). For example, 50%depolymerization of microtubules occurred at 15 nMpodophyllotoxin while only 8% of the cells werearrested in metaphase (Table 1, Kdcp; Fig. 4A, circles).

Spindle organizationBetween 3.3 nM and 18 nM podophyllotoxin, meta-phase spindles were frequently types I and II, exhibitingchromosomes located near the spindle poles and morenumerous, frequently longer, astral microtubules than


control spindles (Fig. 5A,a,b,C,c). The reorganizationof spindle microtubules, chromosomes and centro-somes induced by podophyllotoxin was qualitativelysimilar to that induced by incubation with vinblastine(see Fig. 3A,a,C,c). Type III spindles were prevalent at18 and 32 nM podophyllotoxin. Small star-shapedaggregates of microtubules were induced by podophyl-lotoxin (Fig. 5D,E), similar to but smaller than the star-shaped aggregates induced by vinblastine (Fig. 3C,D).Fig. 5b shows a particularly clear example of thearrangement of polar chromosomes in a type I spindle.Polar chromosomes were frequently distributedunequally at the two poles after incubation withvinblastine, podophyllotoxin or nocodazole (see be-low), suggesting that segregation of chromatids had notoccurred.

By contrast with vinblastine, podophyllotoxininduced significant distortions of spindle morphologywithout inducing accumulation of cells at metaphase orinhibiting cell division. For example, only 2.2% of thecells contained metaphase spindles at 6 nM podophyllo-toxin, but 22% of the spindles were types I and II (Fig.4B, open triangles). In addition, there was no netmicrotubule depolymerization at this concentration(Fig. 4A), and no inhibition of cell division. Even at 10nM podophyllotoxin, only 5% of the cells were inmetaphase (Fig. 4A, open circles), and there was nodetectable inhibition of cell proliferation (data notshown). However, spindle organization was signifi-cantly affected (Fig. 4B, 5C,c). Spindles were 23%shorter than control spindles (Fig. 4C) and were oftenabnormal (26% of the spindles were types I, II or III)(Fig. 4B). In addition, the anaphase/metaphase ratiowas reduced by 60% as compared with controls.

The observation that 10 nM podophyllotoxin induceda low frequency of cells in anaphase relative to cells inmetaphase and induced significant distortions of spindlemorphology without inhibiting cell division was initiallypuzzling. A plausible explanation, however, is that therate of progression through metaphase was slowed bylow concentrations of podophyllotoxin, but metaphasewas not blocked, and anaphase and cytokinesis oc-curred normally. These data suggest that the mechan-isms involved in proper construction of the metaphasespindle are more sensitive to podophyllotoxin than themechanisms responsible for progression from meta-phase to anaphase. The data also indicate that abnor-mal spindles do not necessarily result in metaphaseblockage, but that such spindles can proceed throughmetaphase and the cells can divide.

The extent of spindle damage and the effects of thedamage on mitotic accumulation increased dramaticallybetween 18 nM and 33 nM podophyllotoxin (Figs 4A,5D,d,E). At 18 nM podophyllotoxin, only 8% of thecells were in metaphase (Fig. 4A, open circles). Of cellsin metaphase, 32% had spindles with normal organiz-ation, 23% of the spindles were types I or II, and 45%were types III or IV (Fig. 4B). Bipolar spindles presentat this concentration were 63% shorter than controlspindles (Fig. 4C, open squares), and the mass ofmicrotubules was reduced by 52% as compared with


Fig. 5


Fig. 5. Microtubule, chromosome and centrosomearrangement in HeLa cells after 18-20 h incubation inpodophyllotoxin at the specified concentrations. (A,a) 3nM; (B,b) 18 nM; (C,c) 10 nM; (D,d) 18 nM; (E) 33 nM;(F) 37 nM; (G,g) 110 nM podophyllotoxin.(A,B,C,D,E,F,G) Anti-tubulin immunofluorescence;(a,b,c,g) DAPI staining of chromosomes; (d) anti-centrosomal immunofluorescence. (A,a) Type I abnormalspindle induced by 3 nM podophyllotoxin; arrows point totufts of long astral microtubules in (A) and chromosomeslocated near the spindle poles in a; (B,b) type I abnormalspindle induced by 18 nM podophyllotoxin; arrows in bpoint to chromosomes located near one pole, there werenone at the opposite pole; astral microtubules are notprominent in this particular spindle but there are a few atthe arrow in B. (C,c) Two type II spindles induced by 10nM podophyllotoxin; arrows point to long astralmicrotubules in C and to large numbers of chromosomeslocated near the spindle poles in c. (D,d) Three smallspindles induced by incubation with 18 nMpodophyllotoxin. All three spindles contain star-shapedaggregates of microtubules. The upper two spindles containfragmented centrosomal material. The chromosomes of thelower two spindles were condensed primarily into onespherical mass characteristic of type III spindles. Thechromosomes of the upper spindle retained a slightlyincreased density where one might expect the metaphaseplate, characteristic of a type II spindle (chromosomes notshown). (E) Star-shaped aggregates of microtubules in typeIII spindles (asterisks) and punctate aggregates of tubulinin type IV spindles (arrows) induced by 33 nMpodophyllotoxin. (F) Microtubules of interphase cellsinduced by incubation in 37 nM podophyllotoxin.

(G) Diffuse tubulin stain in both mitotic (labelled m) andinterphase (labelled i) cells induced by incubation with 110nM podophyllotoxin; and (g) parallel DAPI staining of thesame cells showing either masses of condensed mitoticchromatin or chromosomes (m) or diffuse interphasechromatin (i). Bars, 10 fim. Small bar in a showsmagnification of A,a and C-g; large bar in b showsmagnification of B and b.

control cells (Fig. 4A, squares). After incubation with33 nM podophyllotoxin, 65% of the cells were inmetaphase, and all of the mitotic figures were types IIIand IV (Fig. 4B, closed triangles). Mitotic cells lackedmicrotubules altogether (Fig. 5E, arrows), or containedvery small star-shaped aggregates of short microtubules(Fig. 5E, asterisks). Short microtubules remained insome interphase cells (Fig. 5F). Concomitantly, themicrotubule mass measured in isolated cytoskeletonswas reduced by 80% as compared with controls (Fig.4A, squares). At podophyllotoxin concentrationsgreater than or equal to 100 nM, no microtubules werepresent in interphase or mitotic cells (Fig. 4A, closedsquares; Fig. 5G), and all mitotic figures were type IV(Fig. 5G,g).

The centrosomal material was fragmented to somedegree at 10 nM podophyllotoxin and it was markedlyfragmented at 18 nM podophyllotoxin (Fig. 5d).However, the centrosomal material was compact (datanot shown) at high podophyllotoxin concentrations (>100 nM), which induced total microtubule depolymeriz-ation (Fig. 4A and Fig. 5G).

Effects of nocodazole on mitosisThe effects of very low concentrations of nocodazole onmitotic arrest and spindle organization were similar tothe effects of vinblastine in several significant ways.

Mitotic arrest and microtubule polymer massLike incubation of cells with vinblastine and podophyl-lotoxin, incubation of cells with nocodazole inducedmetaphase accumulation. A total of 50% of the cellshad accumulated in metaphase (/Cmet) at 54 nMnocodazole (Fig. 6A, circles; Table 1), and maximalaccumulation occurred at 100 nM nocodazole. Unlikevinblastine and podophyllotoxin, the percentage ac-

-20 10 100 1000 10000

[Nocodazole] (nM)

Fig. 6. Nocodazole concentration dependence of metaphasearrest and microtubule depolymerization (A) and ofspindle and cell reorganization (B,C) in HeLa cells aftertreatment with nocodazole at the stated concentrations forthe duration of one cell cycle. (A) Accumulation of cells inmetaphase was accompanied by little or nodepolymerization of microtubules. Percentage of cells inmetaphase (circles) and percentage decrease in mass ofpolymerized microtubules (squares). (B) Percentage ofmetaphases that were types I or II (open triangles) andtypes III and IV (filled triangles). The percentage ofmetaphase spindles that appeared normal equals 100%minus the percentages indicated by the filled and opentriangles. (C) Percentage decrease of interpolar distancemeasured on bipolar spindles (normal, type I and type II).


cumulation did not diminish at high concentrations ofnocodazole (Fig. 6A; compare with Figs 1A, 4A). Likevinblastine and podophyllotoxin, incubation withnocodazole resulted in a concentration-dependent de-crease in the ratio of cells in anaphase to cells inmetaphase, with 50% decrease in the ratio {Kana/met)occurring at 12 nM nocodazole (Table 1), and a ratio ofzero at 100 nM nocodazole. Half-maximal inhibition ofcell division (Kdiv) occurred at 22 nM nocodazole (Table1).

At concentrations of nocodazole (54 nM) that

induced 50% accumulation of cells in mitosis, there waslittle or no detectable microtubule depolymerization(Table 1, Fig. 6A). Some microtubule depolymerizationwas associated with high levels of metaphase arrest, butthe degree of depolymerization was slight comparedwith depolymerization associated with metaphase ar-rest induced by podophyllotoxin (Fig. 6A; comparewith Fig. 4A). For example, the microtubule polymerlevel was reduced by only 30% at 100 nM nocodazole, aconcentration that induced maximal mitotic accumu-lation. By comparison, maximal metaphase accumu-lation with podophyllotoxin was accompanied by 100%loss of polymer (Fig. 4A). An 11-fold higher concen-tration of nocodazole was required to depolymerize halfof the microtubule polymer than was required to induce50% accumulation of cells at metaphase (Kdep/Kmet,Table 1).

Spindle organizationBetween 3 nM and 100 nM nocodazole, metaphasespindles were frequently types I and II, exhibiting pole-associated chromosomes and lengthened, more numer-ous astral microtubules than control spindles (Fig.7A,a,a',B,b,b'). At 33 nM and higher nocodazoleconcentrations, spindles were frequently type III,consisting of star-shaped aggregates of microtubules,and type IV (Fig. 7B,b,b',C,c,c'). The reorganizationof spindle microtubules, chromosomes and centro-somes induced by nocodazole was qualitatively similarto that induced by incubation with vinblastine andpodophyllotoxin (see Figs 3 and 5).

The organization of a typical type I spindle is clearlyvisible in Fig. 7A,a,a'. It is evident that the chromo-somes that are not included in the metaphase plate arelocated at the ends of tufts of astral microtubules ratherthan strictly at the centrosomes. Chromosomes with apolar location were often located at the ends of astralmicrotubules after incubation of cells with all threedrugs, but the relationship is easier to visualize in themicroscope than in micrographs. Types I and II spindlesoccurred with relatively high frequency with nocod-azole as compared with the other drugs. A maximum of39% of all spindles were types I and II with nocodazole(Fig. 6B, open triangles); whereas the maximuminduced by podophyllotoxin was 23% (Fig. 4B) and themaximum induced by vinblastine was 17% (Jordan etal. 1991). However, as with vinblastine and podophyllo-toxin, some cells arrested in metaphase had normalspindles. For example, after incubation with 33 nMnocodazole, 22% of cells were arrested in metaphase.

Fig. 7. Microtubule, chromosome and centrosomearrangement in HeLa cells after incubation withnocodazole at the indicated concentrations for one cellcycle. (A,a,a') 33 nM; (B,b,b') 100 nM; (C,c,c') 330 nM;(D) 1 ,uM. (A,B,C,D) Anti-tubulin immunofluorescence;(a,b,c) DAP1 staining of chromatin; (a',b',c') anti-centrosomal immunofluorescence. (A,a,a') Two type Ispindles induced by incubation with 33 nM nocodazole.The spindles with long astral microtubules (arrows in A)and several chromosomes in the region of the poles(arrows in a). It is clear that many of these chromosomesare at the ends of long astral microtubules. Thecentrosomes (arrows in a') are fragmented. (B,b,b') Type I(I), FI (II) and III spindles (asterisks) induced byincubation with 100 nM nocodazole. The types I and IIspindles have some chromosomes located near the spindlepoles (arrows in b), whereas the chromosomes of the typeIII spindles are in spherical masses. Centrosomes arefragmented (arrows in b'). (C,c,c') Several type IIIspindles induced by 330 nM nocodazole. These differ fromthe type III spindles induced by lower concentrations ofnocodazole (B,b,b'); they contain multiple star-shapedaggregates of microtubules or other tubulin polymer (largearrows). Their centrosomes (arrows in c') are compact andlocated outside of the tubulin and chromosome-containingstructure. Centrosomes appear to have some tubulinassociated with them (compare centrosomal stain in c'(arrows) with tubulin stain at small arrows in C). There arealso a few punctate aggregates of tubulin at the cellperiphery in C. (D) Punctate aggregates of tubulin and afew short remnants of microtubules, 1 fiM nocodazole.Bars, 10 ,um. Large bar in A shows magnification ofA,a,a'; small bar in D shows magnification of B-D.

Of these, 20% had normal spindles, 39% were types Ior II, and 41% were type III (Fig. 4B).

The interpolar distance was decreased by nocodazoleconcentrations equal to or greater than 33 nM (Fig. 6C,squares). Nocodazole at these same concentrationsinduced formation of some monopolar spindles (typeIII) (Fig. 6B, filled triangles). Incubation with nocod-azole at concentrations that induced major rearrange-ments of spindle microtubules also induced fragmen-tation of centrosomal material. The centrosomalmaterial was, however, still located at the foci of themicrotubule arrays (Fig. 7A,a,B,b).

Mitotic cells at 330 nM nocodazole contained severalclusters of short, thick tubulin polymers (Fig. 7C). Toour knowledge, such structures have not been reportedpreviously with nocodazole. The tubulin polymers,which were enclosed within masses of condensedchromosomes (Fig. 7c), may be thick bundles ofmicrotubules or another polymeric form of tubulin. Incontrast to the fragmentation of centrosomes thatoccurred between approximately 33 and 100 nMnocodazole, the centrosomes were compact at 330 nMnocodazole. The centrosomes were often near theperiphery of the cells, some distance from the tubulinpolymer-chromosome array (Fig. 7C,c,c'). At 330 nMnocodazole, the cytoskeletal microtubule polymer masswas reduced by 40% as compared with the polymermass in control cells (Fig. 6A), consistent with theimpression by immunofluorescence microscopy that


Fig. 7


nocodazole induced significant microtubule depolym-erization.

By immunofluorescence microscopy, it was seen thathigh concentrations of nocodazole (> 1 //M) inducednearly complete depolymerization of microtubules.Punctate aggregates of tubulin were scattered through-out the cytoplasm of the cells, and only a few interphasecells contained short microtubule fragments (Fig. 7D).However, some microtubule polymer was present inisolated cytoskeletons (20-40% of controls; Fig. 6A).The presence of measurable cytoskeletal tubulinpolymer but the nearly complete absence of stainedmicrotubule polymer in the corresponding microscopicpreparations in this concentration range was the onlyinconsistency noted between the biochemical measure-ment of polymer mass and impressions of microtubulepolymer levels obtained by microscopy. The measur-able cytoskeletal tubulin polymer after incubation withnocodazole at concentrations ^ 1 [iM is probablyattributable to the tubulin aggregates and microtubulefragments present at these concentrations.

Discussion

Motility of cilia and flagella occurs by the interaction ofthe motor protein dynein with an array of stablemicrotubules (reviewed by Warner, 1979). Such stablemicrotubules clearly function as passive supports whoseassembly dynamics are not important in motility.Studies from many laboratories have also documentedthe probable importance of microtubule-associatedmotor proteins in mitosis (e.g. see Pfarr et al. 1990;Steuer et al. 1990; Hyman and Mitchison, 1991).However, mitotic spindle microtubules are highlydynamic, and it is reasonable to think that the dynamicsof the microtubules may be important in one or moreaspects of mitosis (e.g. see Saxton et al. 1984; Salmon etal. 1984; Mitchison, 1989).

Many drugs that inhibit mitosis are thought to act bydestroying spindle microtubules, and the actions ofthese drugs at high concentrations appear to result fromdepletion of microtubule polymer (Malawista et al.1968; Wilson and Bryan, 1974; De Brabander et al.1976; Sluder, 1979; Zieve et al. 1980). However, wefound that at low concentrations, vinblastine, podo-phyllotoxin, nocodazole, colchicine and taxol inhibitthe exchange of tubulin at microtubule ends in vitrowithout exerting significant effects on the total mass ofmicrotubule polymer (Jordan and Farrell, 1983; Wilsonet al. 1985; Wilson and Farrell, 1986; Jordan andWilson, 1990; R. Toso, M. A. Jordan and L. Wilson,unpublished results with nocodazole). Thus, it isconceivable that one or more of these drugs mightinhibit mitosis by affecting the dynamics of themicrotubules rather than merely by depolymerizing themicrotubules. In the present study, we found thatmitotic block by vinblastine and nocodazole in HeLacells occurred by a subtle effect on spindle micro-tubules, consistent with the idea that the drugs areacting by affecting the dynamics of spindle microtubulesrather than by depolymerizing the microtubules. Low

concentrations of vinblastine and nocodazole arrestedcells at metaphase of mitosis in the presence ofapparently correctly assembled spindles with a fullcomplement of microtubules. The mechanism by whichpodophyllotoxin induced mitotic arrest appeared toinvolve depolymerization of microtubules. However,podophyllotoxin also induced a reorganization of thespindle that was similar to that induced by vinblastineand nocodazole. It appears that podophyllotoxin in-duces alterations in spindle microtubule dynamics, butthat, at the lowest effective concentrations, the specificalterations are not sufficient to block the transition frommetaphase to anaphase.

Vinblastine, podophyllotoxin and nocodazole depol-ymerize microtubules in vitro at micromolar concen-trations (Wilson et al. 1976,1982; Hoebeke et al. 1976).However, at low concentrations, all three drugsstabilize microtubule ends in vitro. With in vitroreassembled MAP-rich bovine brain microtubules, themost sensitive effects of the three drugs on micro-tubules occur in the absence of significant changes in themass of assembled microtubules. For example, vinblast-ine inhibited exchange of tubulin at the ends ofmicrotubules half-maximally at 0.15 fiM, while thepolymer mass was reduced less than 3% as comparedwith controls (Jordan and Wilson, 1990). Similar resultswere obtained with podophyllotoxin (Jordan andFarrell, 1983) and with nocodazole (R. Toso, M. A.Jordan and L. Wilson, unpublished data). In addition,all three drugs suppress dynamic instability behavior ofMAP-free microtubules in vitro. Specifically, we foundthat vinblastine and nocodazole suppress rates ofgrowing and shortening at ends of individual micro-tubules and suppress transitions to phases of growingand shortening as determined by computer-enhancedvideo microscopy (R. Toso, K. W. Farrell, M. A.Jordan, B. Matsumoto and L. Wilson, unpublishedresults). Podophyllotoxin has been shown to inhibitdynamic instability in microtubule suspensions usingradiolabel exchange methodology and electron mi-croscopy (Schilstra et al. 1989). Thus, each of the drugscan inhibit tubulin exchange at microtubule ends invitro without significantly altering the mass of as-sembled microtubules. In other words, at low drugconcentrations, the association and dissociation rateconstants for tubulin addition to one or both micro-tubule ends are suppressed in ways that stabilizemicrotubule dynamics but do not alter the equilibriumbetween microtubule polymer and soluble tubulin. It islikely that the powerful capacity of vinblastine andnocodazole to inhibit microtubule dynamics and stabil-ize microtubule ends in vitro and a similar alteration invivo is responsible for their ability to block mitosis.With podophyllotoxin, inhibition of microtubule dy-namics in concert with net microtubule depolymeriz-ation appear to be responsible for mitotic block.

Effects of vinblastine, podophyllotoxin andnocodazole on mitosis

Effects on spindle organizationMetaphase accumulation by vinblastine, podophyllo-


toxin and nocodazole was associated with concen-tration-dependent rearrangements in the organizationof spindle microtubules and chromosomes that weresimilar for all three drugs. At the lowest effectiveconcentrations of all the drugs, many blocked spindleswere indistinguishable from normal (control) meta-phase spindles (Figs 4B, 6B). The mildest perturbationsof spindle organization in spindles that snowed abnor-malities were increasing numbers and lengths of astralmicrotubules, decreasing lengths of the centralspindles, the presence of one or a few chromosomeslocated near the spindle poles, and fragmentation ofcentrosomal material (Figs IB, 3, 4C, 5, 6C, 7) (types Iand II).

Types I and II abnormal spindles appeared to resultfrom a redistribution of microtubule polymer from thecentral spindle to the asters. The distance between thepoles decreased, and the number and length of astralmicrotubules increased. Thus, the interpolar andkinetochore microtubules must have shortened, whilethe astral microtubules got longer. Such length changescould occur by a common action of the three drugs on amechanism that controls the dynamics of tubulinaddition and loss at the ends of the microtubules. Forexample, prevention of catastrophic depolymerizationof astral microtubules could result in increased numbersand increased lengths of astral microtubules.

One of the most sensitive effects of the three drugswas on congression of chromosomes to form themetaphase plate. At the lowest effective concentrationsof the three drugs, most chromosomes congressednormally, while in some cells one or a few chromosomesappeared unable to congress. With increasing drugconcentrations, increasing numbers of chromosomeswere found at or near the spindle poles. Thus, thecorrect attachment of microtubules to both kineto-chores of a chromatid pair must be highly sensitive tothe drugs. During prometaphase, the astral micro-tubules repeatedly grow and shorten, apparentlyprobing the cytoplasm until kinetochore attachment isachieved (Rieder et al. 1990; Hayden et al. 1990). Onecan envision that interfering with growing and shorten-ing of the astral microtubules in prometaphase mightresult in the inability of the microtubules from one poleto reach the kinetochore of a chromosome located nearthe opposite pole. Alternatively, the attachment of themicrotubule to the kinetochore may be blocked by thepresence of the drug at the end of the microtubule.

Low concentrations of vinblastine, podophyllotoxinand nocodazole produced high proportions (16-39%) ofspindles that were bipolar but contained imperfectlycongressed chromosomes (types I and II). This obser-vation raises the question of whether a cell thateventually overcomes mitotic block (either duringincubation with drug or after drug removal) willundergo accurate chromosome segregation or not.Little information appears to exist concerning inductionof chromosome nondisjunction by these three drugs.However, other drugs (Colcemid, diethylstilbestrol)that affect microtubule polymerization dynamics havebeen found to cause aneuploidy (Kato and Yosida,

1970; Sharp and Parry, 1985; Wheeler et al., 1986). Theprevalence of abnormal bipolar spindles (types I and II)observed with vinblastine, podophyllotoxin, nocod-azole (Figs 4B, 6B), other vinca alkaloids (Jordan et al.1991), and colchicine and taxol (M. A. Jordan, D.Thrower and L. Wilson, unpublished results), suggeststhat any drug that affects microtubule dynamics mayhave the capacity to induce aneuploidy. Nicklas (1985)proposed, in words that presage the observationsreported here, that "a little explored but perhaps veryimportant source of aneuploidy stems from the delicatebalance that must be struck between a spindle that istoo stable for reorientation to occur and one that is sounstable that reorientation continues indefinitely".

At higher levels of metaphase accumulation with allthree drugs, the proportions of normal and type Ispindles decreased and types II and III became moreprevalent. Type III spindles were not bipolar. Therewas no distinguishable central plate of chromosomes;rather the chromosomes were arranged in a ballenclosing one or more star-shaped aggregates ofmicrotubules. In type III spindles there was noseparation of centrosomal material into two distinctmasses. Rather the centrosome consisted of multiplefragments that were generally located at the foci of themicrotubule array(s). The mechanism by which type IIImonopolar spindles form may involve inhibition ofspindle pole separation during prophase. By whatevermechanism they form, type III spindles appear torepresent an extreme in drug concentration-dependentredistribution of microtubule polymer from the centralspindle to the asters.

Sufficiently high concentrations of all three drugscaused microtubule depolymerization as determined byELISA (discussed below). The loss of microtubulepolymer in spindles was also clearly evident byimmunofluorescence microscopy (e.g. Figs 3F, 5G). Atsufficiently high concentrations of all three drugs, nomicrotubules were visible. Depolymerization resultedin the appearance of a type IV abnormal mitotic figureconsisting of a ball of condensed chromosomes and nomicrotubules; centrosomes were compact and locatedapparently randomly. Types III and IV spindles aretypical of the c-mitotic figures described by Levan(1938) and Ostergren (1944) in their early studies oncolchicine.

Effects on the mass of microtubule polymerAll three drugs induced accumulation of cells in ametaphase-like stage of mitosis. However, there was noclear relationship between the extent of mitotic ac-cumulation and microtubule depolymerization. Forexample, upon incubation of cells with concentrationsof vinblastine or nocodazole that induced 50% accumu-lation of cells in metaphase, there was little or nodetectable microtubule depolymerization (Figs 1A,6A). Thus, vinblastine and nocodazole appear to blockmitotic spindle function without changing the amountof microtubule polymer. In contrast, metaphase ac-cumulation with podophyllotoxin was accompanied bydepolymerization of microtubules. For example, at the


podophyllotoxin concentration that induced 50% ac-cumulation of cells at metaphase (30 nM), the druginduced an 85% loss of microtubule polymer (Fig. 4A).

A possible error in the foregoing analysis may ariseby comparing the microtubule polymer mass in culturesconsisting predominantly of mitotic cells after incu-bation with drugs with the microtubule mass in controlcultures that consist predominantly of interphase cells.It has not been previously determined whether mitoticcells have an inherently greater mass of microtubulesthan interphase cells. In the present study, we com-pared the microtubule content of interphase cells withthe microtubule content of a population of cells that wasenriched in mitotic stages but that had not beenincubated with any drug that induced metaphase block.We measured isolated cytoskeletons from HeLa cellsthat were synchronized in mitosis using a doublethymidine block (Rao and Engelberg, 1966) followedby mitotic shake-off (Robbins and Scharf, 1966). Themean mitotic index of the synchronized populations atthe time of cytoskeletal isolation was 45%, whereas themitotic index of the control populations was 2-3%.Tubulin in polymer form comprised 0.79 (± 0.24)% ofthe total protein in the synchronized mitotic popu-lations (w=5) compared with 1.03 (± 0.04)% forcontrol populations («=117). In addition, Y. Zhai andG.G. Borisy (University of Wisconsin, Madison, WI)measured the mass of microtubule polymer in indi-vidual LLC-PK cells after microinjection of fluorescenttubulin and found that, at 37°C, mitotic cells contained90% ± 5% of the microtubule polymer mass ofinterphase cells (personal communication). Thus, themass of microtubule polymer does not appear toincrease significantly, if at all, during mitosis.

In addition, two other lines of evidence suggest thatany mitosis-associated increase in microtubule polymermass must be small or nonexistent. First, Bulinski et al.(1980) found that the total tubulin concentration insynchronized HeLa cells in mitosis was the same as thetotal tubulin concentration in a mixed cell population.We found, after incubation with vinca alkaloids,podophyllotoxin and nocodazole over the range ofconcentrations used in this study, that the soluble(unpolymerized) tubulin concentration remained con-stant as the concentration of tubulin in polymer formwas altered (Jordan et al. 1991, and unpublished data).In other words, the soluble tubulin concentrationremained constant whether the cells were predomi-nantly in mitosis or whether they were predominantly ininterphase. Thus, mitotic cells do not have a signifi-cantly higher fraction of tubulin in polymer form thaninterphase cells, and from the results of Bulinski et al.(1980) we know that mitotic cells do not have more totaltubulin than interphase cells.

Second, incubation of cells with the drug taxolenhances microtubule polymerization (Schiff et al.1979; Schiff and Horwitz, 1980). However, in a separatestudy, we found that taxol at low concentrations caninduce approximately 33% mitotic accumulation inHeLa cells without increasing the microtubule polymermass as determined by ELISA of tubulin in isolated

cytoskeletons (M. A. Jordan, D. Thrower and L.Wilson, unpublished experiments). If mitotic cellscontained more microtubule polymer than interphasecells, one would expect to measure higher polymerlevels in such a population of cells blocked in mitosis bytaxol compared with control cells that were predomi-nantly in interphase.

Another possible error in the analysis of therelationship between mitotic accumulation and micro-tubule polymer mass with vinblastine, podophyllotoxinand nocodazole could arise if vinblastine or nocodazoleinduced the formation of a non-microtubule tubulinpolymer. However, only trace amounts of smallaggregates of tubulin were observed after incubation ofcells with 6.4 nM vinblastine (Fig. 3D) or 100 nMnocodazole (data not shown). These concentrationswere higher than the concentrations of vinblastine andnocodazole that induced mitotic accumulation (Figs1A, 6A). In addition, no paracrystals or macrotubules(Bensch and Malawista, 1969) were found after incu-bation of HeLa cells with 2 nM or 10 nM vinblastine, asdetermined by electron microscopy (K. Wendell, L.Wilson and M. A. Jordan, unpublished data). Afterincubation with 10 nM vinblastine the mean diameter ofmicrotubules was 24.9 nm. Some close alignment ofmicrotubules was observed by electron microscopy; thismay account for the thick appearance of the micro-tubule stain by immunofluorescence microscopy (Fig.3D).

Effects on the distance between spindle polesThe concentration-dependent decrease in pole-to-poledistance that occurred with vinblastine, podophyllo-toxin and nocodazole (Figs IB, 4C, 6C) indicates thatall three drugs diminish the forces holding the two polesapart. Suppression of dynamic instability or treadmill-ing of kinetochore microtubules (Mitchison et al. 1986;Hamaguchi et al. 1987; Mitchison, 1989) or sterichindrance of kinetochore/microtubule attachmentmight lead to a disruption of the balance of forces in thespindle and a resultant shortening of the spindle (for adiscussion of these forces, see Nicklas, 1988). It isconceivable that the poleward forces inherent intreadmilling or fluxing microtubules that are continuallyadding subunits at their (+) ends (kinetochore and/orinterpolar microtubules) are instrumental in maintain-ing centrosomal separation. In support of the idea thattreadmilling of kinetochore microtubules is responsiblefor pole-to-pole separation, Sawin and Mitchison(1991a,b) induced formation of asymmetric half-spindles in vitro composed of treadmilling or fluxingmicrotubules that extended between a centrosome anda mass of chromatin. Thus, interactions among thecomponents of a single half-spindle are sufficient tomaintain chromosome/centrosome separation and,therefore, in a whole spindle, centrosome/centrosomeseparation. Perhaps vinblastine, podophyllotoxin andnocodazole reduce the pole-to-pole distance by inhibit-ing the treadmilling dynamics that may be responsiblefor maintenance of the separation between the poles.


Effects on centrosomal organizationThe mitotic centrosome appears to play a significantrole in mitosis. One component (p34 2 protein kinase)of the mitotic control factor MPF is localized incentrosomes of mitotic HeLa cells (Bailly et al. 1989).Centrosomes nucleate microtubules (Kuriyama andBorisy, 1981), and a cyclical phosphorylation-and-dephosphorylation of centrosomal proteins parallelsthe increase in microtubule nucleating activity ofcentrosomes early in mitosis and the subsequentdecrease in nucleating activity at anaphase (Robbins etal. 1968; Kuriyama and Borisy, 1981; Vandre andBorisy, 1989). Using high concentrations of severalantimitotic drugs Kung et al. (1990) and Alfa et al.(1990) obtained evidence suggesting that intact micro-tubules play an essential role in the activation and /orinactivation of mitotic control proteins.

Sellitto and Kuriyama (1988) found that pericentrio-lar material was dispersed after treatment of CHO cellswith the antimitotic drug colcemid, but not aftertreatment with high concentrations of nocodazole(>330 nM). In agreement with their results, we foundthat centrosomes of HeLa cells were compact afterincubation with nocodazole at concentrations greaterthan 330 nM (Fig. 7c'). However, we found that, atlower concentrations of nocodazole, as well as withvinblastine and podophyllotoxin, the organization ofthe centrosomal material was altered in ways that weresimilar for all three drugs. Specifically, centrosomalmaterial was fragmented by concentrations of the threedrugs that affected the organization of spindle micro-tubules but did not totally destroy the microtubules.Fragmented centrosomal aggregates were alwayslocated at the foci of microtubule arrays (Figs3C,c',D,d', 5D,d, 7A,a',B,b'). Curiously, at drugconcentrations that induced complete microtubuledepolymerization, the centrosomal material of mitoticcells was compact, and the centrosomes of interphasecells were compact at all concentrations of the threedrugs. These results support the idea that organizationof the mitotic spindle microtubules by centrosomes isnot simply a one-way street, but that intact micro-tubules can alter the organization of the centrosome. Inaddition, they suggest that microtubule dynamics mayalso exert an important role in regulating the structure,and perhaps the function, of mitotic centrosomes.

Cell cycleWe have shown that vinblastine, podophyllotoxin andnocodazole can arrest the cell cycle at metaphase incells having apparently correctly assembled spindles.As the drug concentration is increased, the organizationof arrested metaphase spindles is altered in qualitativelythe same way with all three drugs. With vinblastine andnocodazole, metaphase arrest occurs in the presence ofa normal complement of microtubule polymer. Themetaphase/anaphase transition appears to be selec-tively sensitive to drug effects on microtubule-depen-dent functions. Microtubule-dependent processesrequired for progression of cells through interphase intomitosis were not inhibited by drug concentrations that

induced blockage of mitosis at the metaphase/anaphasetransition. Our results support the idea that not simplymicrotubules, but dynamic microtubules, are in someway crucial to mitosis. Spindle microtubules are knownto be considerably more dynamic than microtubules ininterphase cells (Saxton et al. 1984; Salmon et al. 1984).Murray and Kirschner (1989) postulated that a correctlyassembled spindle may be required for the degradationof cyclin and the exit from mitosis. It seems reasonableto think that the increased dynamics of spindlemicrotubules may be critical to the correct assembly ofthe spindle at a level that is not detectable by lightmicroscopy, or they may be critical for signalling thetransition from metaphase to anaphase.

We thank Dr. Roger Leslie and Dr. Teresa Burgess forstimulating discussions during the course of this work, Dr.Brian Matsumoto for the generous loan of his Olympusobjective, and Mr. Herb Miller for willing and patientassistance with computer software. We also thank Dr. TeresaBurgess, Mr. Rob Toso, and Ms. Kim Wendell for careful andthoughtful reading of the manuscript. This work wassupported by the American Cancer Society, grant CH 381.

References

Alfa, C. E., Ducommun, B., Beach, D. and Hyams, J. S. (1990).Distinct nuclear and spindle pole body populations of cyclin-cdc2 infission yeast. Nature 347, 680-682.

Bailly, E., Doree, M., Nurse, P. and Bornens, M. (1989). p34cdc2 islocated in both nucleus and cytoplasm; part is centrosomallyassociated at G2/M and enters vesicles at anaphase. EMBO J. 8,3985-3995.

Ball, R. L., Carney, D. H., Albrecht, T., Asai, D. J. and Thompson,W. C. (1986). A radiolabeled monoclonal antibody binding assayfor cytoskeletal tubulin in cultured cells. J. Cell Biol. 103, 1033-1041.

Bensch, K. G. and Malawista, S. E. (1969). Microtubular crystals inmammalian cells. J. Cell Biol. 40, 95-106.

Bulinski, J. C , Rodriguez, J. A. and Borisy, G. G. (1980). Test of fourpossible mechanisms for the temporal control of spindle andcytoplasmic microtubule assembly in HeLa cells. J. Biol. Chem.255, 1684-1688.

Calarco-Gillam, P. D., Siebert, M. C , Hubble, R., Mitchison, T. andKirschner, M. (1983). Centrosome development in early mouseembryos as defined by an autoantibody against pericentriolarmaterial. Cell 35, 621-629.

Chu, D. and Klymkowsky, M. W. (1989). Experimental analysis ofcytoskeletal function in early Xenopus lacvis embryos. In TheCytoskeleton in Differentiation and Development (ed. J. Arechagaand R. Maccioni ), pp. 331-333. Oxford, UK.: IRL Press.

De Brabander, M. J., Van De Veire, R. M. L., Aerts, F. E. M.,Borgers, M. and Janssen, P. A. J. (1976). The effects of methyl [5-(2-thienylcarbonyl)—lH-benzimidazol-2-yl] carbamate (R 17 943:NSC 238159), a new synthetic antitumoral drug interfering withmicrotubules, on mammalian cells cultured in vitro. Cancer Res.36, 905-916.

Dustin, P. (1984). Microtubules. Berlin: Springer-Verlag.Farrell, K. W., Jordan, M. A., Miller, H. and Wilson, L. (1987).

Phase dynamics at microtubule ends: the coexistence ofmicrotubule length changes and treadmilling. J. Cell Biol. 104,1035-1046.

Hamaguchi, T., Toriyama, M., Sakai, H. and Hiramoto, Y. (1987).Redistribution of fluorescently labeled tubulin in the mitoticapparatus of sand dollar eggs and the effects of taxol. Cell Struct.Fund. 12, 43-52.

Hayden, J. H., Bowser, S. S. and Rieder, C. L. (1990). Kinetochorescapture astral microtubules during chromosome attachment to the


mitotic spindle: direct visualization in live newt lung cells. J. CellBiol. I l l , 1039-1046.

Hoebeke, J., Van Nijen, G. and De Brabander, M. (1976). Interactionof oncodazole (R 17934), a new anti-tumoral drug, with rat braintubulin. Biochem. Biophys. Res. Commun. 69, 319-324.

Hyman, A. A. and Mitchison, T. (1991). Two different microtubule-based motor activities with opposite polarities in kinetochores.Nature 351, 206-211.

Jordan, M. A. and Farrell, K. W. (1983). Differential radiolabellingof opposite microtubule ends: methodology, equilibrium exchange-flux analysis, and drug poisoning. Anal. Biochem. 130, 41-53.

Jordan, M. A., Thrower, D. and Wilson, L. (1.991). Mechanism ofinhibition of cell proliferation by vinca alkaloids. Cancer Res. 51,2212-2222.

Jordan, M. A. and Wilson, L. (1990). Kinetic analysis of tubulinexchange at microtubule ends at low vinblastine concentrations.Biochemistry 29, 2730-2739.

Kato, H. and Yosida, T. H. (1970). Nondisjunction of chromosomesin a synchronized cell population initiated by reversal of colcemidinhibition. Exp. Cell Res 60, 459-464.

Kung, A. L., Sherwood, S. W. and Schimke, R. L. (1990). Cell line-specific differences in the control of cell cycle progression in theabsence of mitosis. Proc. Nat. Acad. Sci. USA 87, 9533-9557.

Kuriyama, R. and Borisy, G. G. (1981). Microtubule-nucleatingactivity of centrosomes in Chinese hamster ovary cells isindependent of the centriole cycle but coupled to the mitotic cycle.J. Cell Biol. 91, 822-826.

Levan, A. (1938). The effect of colchicine on root mitoses in Allium.Hereditas 24, 471-486.

Malawista, S. E., Bensch, K. G. and Sato, H. (1968). Vinblastine andgriseofulvin reversibly disrupt the living mitotic spindle. Science160, 770-772.

Mitchison, T. (1989). Polewards microtubule flux in the mitoticspindle: evidence from photoactivation of fluorescence. J. CellBiol. 109, 637-652.

Mitchison, T., Evans, L., Schulze, E. and Kirschner, M. (1986). Sitesof microtubule assembly and disassembly in the mitotic spindle.Cell 45, 515-527.

Murray, A. W. and Kirschner, M. W. (1989). Dominoes and clocks:the union of two views of the cell cycle. Science 246, 614-621.

Nicklas, R. B. (1985). Mitosis in eukaryotic cells: an overview ofchromosome distribution. In Aneuploidy, Etiology and Mechanism(ed. Dellarco, Voytek, Hollaender), p. 191. New York: Plenum.

Nicklas, R. B. (1988). The forces that move chromosomes in mitosis.Annu. Rev. Biophys. Biophys. Chem. 17, 431-449.

Ostergren, G. (1944). Colchicine mitosis, chromosome contraction,narcosis and protein chain folding. Hereditas 30, 429-467.

Pfarr, C. M., Coue, M., Grissom, P. M., Hays, T. S., Porter, M. E.and Mclntosh, J. R. (1990). Cytoplasmic dynein is localized tokinetochores during mitosis. Nature 345, 263-265.

Rao, P. N. and Engelberg, J. (1966). Effects of temperature on themitotic cycle of normal and synchronized mammalian cells. In CellSynchrony (cd. 1. L. Cameron and G. M. Padilla), pp. 332-352.New York: Academic Press.

Rieder, C. L., Alexander, S. P. and Rupp, G. (1990). Kinetochoresare transported poleward along a single astral microtubule duringchromosome attachment to the spindle in newt lung cells. J. CellBiol. 110, 81-95.

Robbins, E., Jentzsch, G. and Micali, A. (1968). The centriole cycle insynchronized HeLa cells. J. Cell Biol. 36, 329-339.

Robbins, E. and Scharff, M. (1966). Some macromolecularcharacteristics of synchronized HeLa cells. In Cell Synchrony (ed.I. L. Cameron and G. M. Padilla), pp. 353-374. New York:Academic Press.

Salmon, E. D., Leslie, R. J., Saxton, W. M., Karow, M. L. andMclntosh, J. R. (1984). Spindle microtubule dynamics in sea urchinembryos: analysis using a fluorescein-labeled tubulin and

measurements of fluorescence redistribution after laserphotobleaching. J. Cell Biol. 99, 2165-2174.

Sawin, K. and Mitchison, T. (1991a). Mitotic spindle assembly by twodifferent pathways in vitro. J. Cell Biol. 112, 925-940.

Sawin, K. and Mitchison, T. (1991b). Poleward microtubule flux inmitotic spindles assembled in vitro. J. Cell Biol. 112, 941-954.

Saxton, W. M., Stemple, D. L., Leslie, R. J., Salmon, E. D.,Zavortnik, M. and Mclntosh, J. R. (1984). Tubulin dynamics incultured mammalian cells. J. Cell Biol. 99, 2175-2186.

Schiff, P. B., Fant, J. and Horwitz, S. B. (1979). Promotion ofmicrotubule assembly in vitro by taxol. Nature 277, 665-667.

Schiff, P. B. and Horwitz, S. B. (1980). Taxol stabilizes microtubulesin mouse fibroblast cells. Proc. Nat. Acad. Sci. USA 77,1561-1565.

Schilstra, M. J., Martin, S. R. and Bayley, P. M. (1989). The effect ofpodophyllotoxin on microtubule dynamics. J. Biol. Chem. 264,8827-8834.

Sellitto, C. and Kuriyama, R. (1988). Distribution of pericentriolarmaterial in multipolar spindles induced by colcemid treatment inChinese hamster ovary cells. J. Cell Sci. 89, 57-65.

Sharp, D. C. and Parry, J. M. (1985). Diethylstilboestrol: Thebinding and effects of diethylstilboestrol upon the polymerisationand depolymerisation of purified microtubule protein in vitro.Carcinogenesis 6, 865-87.

Sluder, G. (1979). Role of spindle microtubules in the control of cellcycle timing. J. Cell Biol. 80, 674-691.

Steuer, E., Wordeman, L., Schroer, T. A. and Sheetz, M. P. (1990).Localization of cytoplasmic dynein to mitotic spindles andkinetochores. Nature 345, 266-268.

Thrower, D., Jordan, M. A. and Wilson, L. (1991). Quantitation ofcellular tubulin in microtubules and tubulin pools by a competitiveELISA. J. Immunol. Meth. 136, 45-51.

Vandre, D. D. and Borisy, G. G. (1989). Anaphase onset anddephosphorylation of mitotic phosphoproteins occurconcomitantly. J. Cell Sci. 94, 245-258.

Warner, F. D. (1979). Cilia and flagclla: microtubule sliding andregulated motion in microtubules. In Microtubules (ed. K. Robertsand J. S. Hyams), pp. 359-380. London: Academic Press.

Wheeler, W. J., Cherry, L. M., Downs, T. and Hsu, T. C. (1986).Mitotic inhibition and aneuploidy induction by naturally occurringand synthetic estrogens in Chinese hamster cells in vitro. Mutat.Res. 171, 31-41.

Wilson, L., Anderson, K. and Chin, D. (1976). Nonstoichiometricpoisoning of microtubule polymerization: a model for themechanism of action of the vinca alkaloids, podophyllotoxin, andcolchicine. In Cell Molility (ed. R. Goldman, T. Pollard and J.Rosenbaum), pp. 1051-1064. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY.

Wilson, L. W. and Bryan, J. (1974). Biochemical andpharmacological properties of microtubules. Advan. Cell Mol.Biol. 3, 21-72.

Wilson, L. and Farrell, K. W. (1986). Kinetics and steady statedynamics of tubulin addition and loss at opposite microtubule ends:the mechanism of action of colchicine. Ann. N.Y. Acad. Sci. 466,690-708.

Wilson, L., Jordan, M. A., Morse, A. and Margolis, R. L. (1982).Interaction of vinblastine with steady-state microtubules in vitro. J.Mol. Biol. 159, 125-149.

Wilson, L., Miller, H. P., Farrell, K. W., Snyder, K. B., Thompson,W. C. and Purich, D. L. (1985). Taxol stabilization of microtubulesin vitro: dynamics of tubulin addition and loss at opposite ends.Biochemistry 24, 5254-5262.

Zieve, G. W., Turnbull, D., Mullins, J. M. and Mclntosh, J. R.(1980). Production of large numbers of mitotic mammalian cells byuse of the reversible microtubule inhibitor nocodazole. Exp. CellRes. 126, 397-405.

(Received 15 January 1992 - Accepted 2 April 1992)

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Implications for the role of microtubule dynamics in …Inhibition of mitosis by drugs 403 Table 1....

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