cancer res 1983 mujagic 3591 7

1983;43:3591-3597.Cancer Res Hamza Mujagic, Shan-Shan Chen, Richard Geist, et al. 180 Cells

Sarcomaand Mitotic Accumulation in Asynchronously Growing Effects of Vincristine on Cell Survival, Cell Cycle Progression,

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[CANCER RESEARCH 43, 3591-3597, August 1983]

Effects of Vincristine on Cell Survival, Cell Cycle Progression, and Mitotic

Accumulation in Asynchronously Growing Sarcoma 180 Cells

Hamza Mujagic,1 Shan-Shan Chen, Richard Geist, Sandra J. Occhipinti, Bruce M. Conger, Charles A. Smith,

William H. Schuette, and Stanley E. Shackney

Section of Cell Kinetics, Clinical Pharmacology Branch, Division of Cancer Treatment, National Cancer Institute [H. M., S-S. C., R. G., S. J. 0., B. M. C., C. A. S.,S. E. S.], and Applied Clinical Engineering Section, BiomÃ©dical Engineering and Instrumentation Branch, Division of Research Services [W. H. S.], NIH,Bethesda, Maryland 20205

ABSTRACT

The effects of vincristine (VCR) on cell survival, cell cycleprogression, DMA synthesis, and metaphase accumulation werestudied in relation to drug concentration and drug exposureduration in Sarcoma 180 cells in vitro. VCR was found to affectcells in interphase, producing a transient G2 block at all drugconcentrations and drug exposure durations studied. VCR didnot affect DMA synthesis directly. Increases in the metaphaseindex were delayed and always peaked at approximately 8 hrafter drug removal, regardless of the duration of drug exposure.Increases in the metaphase index of sufficient magnitude to becommensurate with VCR lethality were observed only with prolonged drug exposure. VCR produced both nuclear fragmentation and polyploidy. The proportion of cells undergoing polyploidyincreased progressively with increasing drug exposure duration.Interference with cytokinesis during prolonged VCR exposuremay represent a lethal effect of VCR that is separate from itsshort-term effects. This could serve as the basis for the clinical

study of the antitumor effects of prolonged VCR infusions.

INTRODUCTION

VCR2 and vinblastine are cytotoxic tubulin-binding agents,

derived from the periwinkle (Vinca rosea), that have found theirplace as effective drugs in combination chemotherapy regimensfor the treatment of human leukemias, lymphomas, and a varietyof solid tumors.

Early experimental studies of the mode of action of the Vincaalkaloids focused on their disruptive effects on the mitotic spindle(6,10,13), and the lethality of these drugs was generally attributed to their effects on cells in mitosis (2, 6, 22). However, otherstudies have suggested that VCR exerts its lethal effects oninterphase cells (4, 7, 8,11,12, 20, 23).

Studies of the disposition of cells affected by the Vinca alkaloids have produced conflicting results. In some studies, metaphase arrest was found to be completely reversible (10, 13)while, in other studies, it was reported that arrested metaphasessubsequently became necrotic (4,6,11,12). Several authorshave emphasized that the reversibility of metaphase arrest wasdependent on drug concentration and drug exposure duration(5, 9, 24). Alabaster and Cassidy (1) demonstrated by means offlow cytometry that VCR produced polyploidy. These findings

1To whom requests for reprints should be addressed, at Building 10, Room

12C216, National Cancer Institute, Bethesda, Md. 20205.2The abbreviations used are: VCR, vincristine; HBSS, Hanks' balanced salt

solution; dThd, thymidine.Received October 22, 1982; accepted May 5, 1983.

indicate that metaphase arrest and cell necrosis are not the onlymanifestations of VCR lethality. It has been suggested thatpolyploidy might also be a dose-dependent phenomenon (3), but

this has not been clearly established.We have undertaken a systematic study of the effects of VCR

on cell survival, cell cycle progression, DNA synthesis, andmetaphase accumulation in Sarcoma 180 in vitro in relation todrug concentration and to drug exposure duration. The resultsof studies carried out on asynchronously growing cells aredescribed in this paper. The results of VCR studies carried outon recruited, partially synchronized cells are described in aseparate paper (14). The present studies demonstrated that VCRexerted its lethal effects on cells before they entered mitosis.VCR did not inhibit DNA synthesis. However, it did produce atransient accumulation of cells in G2. With regard to the disposition of lethally damaged cells, both nuclear fragmentation andpolyploidy were observed, and the latter was found to be highlydependent on drug concentration and drug exposure duration.

MATERIALS AND METHODS

All studies were carried out in Sarcoma 180 (Foley strain CCRF11 ;supplied by American Tissue Type Culture, Rockville, Md.) grown in vitroin Earle's Medium 199 (Flow Laboratories, Rockville, Md.), which was

supplemented with 10% fetal bovine serum, 2 rriM L-glutamine, 100 units

of penicillin per ml, and 100 ng of streptomycin per ml. Cultures weregrown in monolayer in 250-ml plastic tissue culture flasks (growth surface

area, 75 sq cm) (Costar, Cambridge, Mass.) containing 10 ml of medium.Cells were plated at an initial concentration of 1 x 105 cells/ml. Medium

was changed on Days 2 and 4, and cells were subcultured on Day 5.Cell Survival Studies. Two-day-old log-phase cell cultures were in

cubated at 37Â°with VCR at final concentrations ranging from 0.01 to 10

/Â¿Mfor drug exposure durations ranging from 1 to 24 hr. At the end ofthe drug exposure period, the medium containing drug was removed,and the cells were rinsed 4 times with 5 ml of HBSS. Cells were harvestedby incubation (37Â°)with 0.25% trypsin for 8 min. Controls were obtained

at each time point. Total cell counts were determined using a CoulterCounter (Coulter Electronics, Hialeah, Fla.). Cell suspensions were diluted with Medium 199, and known numbers of cells were cloned in softagar. Nine-day-old colonies were fixed, stained with Giemsa stain, and

counted visually. Clonogenic cell yield, defined as the number of coloniesenumerated divided by the number of cells plated, was averaged for 5replicate flasks at each concentration. The viable cell count per flask wascalculated as the total cell number per flask multiplied by the clonogeniccell yield at a given time point. The surviving viable cell fraction followingdrug exposure was calculated as the number of viable drug-treated cells

per flask divided by the number of viable untreated control cells per flask.Each experiment was performed at least 3 times, and the reportedresults represented the log means of replicate studies.

Metaphase Index Studies. Serial metaphase indices were obtainedat intervals during and after VCR exposure. Aliquots of cells were

AUGUST 1983 3591




VÃncrÃstineEffects in Sarcoma 180

(Chart 2A), there was no significant decrease in cell number, andthere was no delay in cell population growth. Exposure to drugfor 4 or 12 hr (Chart 2, B ana C) produced complete inhibition ofcell population growth throughout the drug exposure period atall drug concentrations studied and for 6 to 8 hr after drugremoval. Cells exposed to 0.1 Â¿tMVCR resumed growth within18 to 20 hr of the initiation of drug exposure, whether the drugwas present or not (in Chart 2, compare C and D). Exposure to0.5 or 2 Â¡IMVCR produced not only an inhibition of cell growth,but also a relative decrease in cell number of up to 10 to 25% insome experiments (Chart 2, B to D).

Metaphase Index Studies. The effects of VCR on the meta-phase index are shown in Chart 3. Incontrol cells, the metaphaseindex ranged from 0.01 to 0.02 throughout the period of observation in all 3 studies. After exposure to 0.1 Â¡MVCR for 1 hr,the metaphase index rose modestly to 0.03 at 4 hr, remained inthis range through 8 hr, and fen to control levels at 12 hr (Chart3A). After exposure to 0.5 Â¿<MVCR for 1 hr, the metaphase indexremained at the same level as that of controls at 2 hr, rose to0.08 at 8 hr, and fell at 12 hr. After exposure to 2.0 MMVCR for1 hr, the metaphase index remained at the same level as controlsthrough 4 hr, rose to 0.15 at 8 hr, and fell sharply at 12 hr.

The effects of exposureto 0.1 //M VCR for 4 hr were similarin magnitudeto those observed after a 1-hr exposure (Chart

04812182404812182404812 2026048121824

Chart 2. Total eel number as a function of time during and after exposure toVCR. A, VCR exposure duration. 1 hr. e. VCR exposure duration. 4 hr. C. VCRexposure duration. 12 hr. D. VCR exposure duration. 24 hr. controls, â€¢0.1 JIMVCR. T: 0.5 UMVCR. A. 2 Â«MVCR. â€¢Bars, SE

SB).There was a modest rise in the metaphase index during thefirst 8 to 12 hr and a return to control values by 18 hr. Duringthe period of exposure to 0.5 and 2 Â¿>MVCR for 4 hr, there wasno rise m the metaphase index (Chart 35), and the metaphaseindex remained at control levels for 2 hr after drug removal. Thesubsequent behavior of the metaphase index after exposure to0.5 and 2 fitÂ»VCR for 4 hr was comparable to that observedafter drug exposure for 1 hr, except that the peaks in themetaphase index occurred at 12 hr rather than at 8 hr.

During a 12-hr exposure to 0.1 Â¿IMVCR, a modest but progressive rise in the metaphase index was observed from 4 to 14hr. Between 14 and 16 hr, the metaphase index doubled from0.05 to 0.1; it then feugradually to control values by 26 hr (Chart3C). In the presence of 0.5 and 2 /^MVCR, the metaphase indexdid not increase above control values until 8 hr and continued torise modestly through 14 hr to values of 0.08 to 0.12. Themetaphase index then rose sharply to values of 0.45 to 0.5 at20 hr, and then fell rapidly to control values by 26 hr.

In summary, for all 3 drug schedules. VCR produced modestincreases in the metaphase index during the period of drugexposure and for 2 to 4 hr thereafter. At the higher drug concentrations, the metaphase index rose sharply at 4 to 8 hr after thetermination of drug exposure, regardless of the duration of drugexposure.

Tubulin-bindingagentscan producea risein ine mitolicindexthat may be due in large part to selective loss of eels ininterphaserather than a true increasein mrtoticcete (11. 12,19). Wien the data in Charts 2 and 3 are consideredtogether,ittsdear thateventhemodest3-foldincreasesinthe metaphaseindex that were observedduringand after exposureto 0.1 KMVCR couid not be attributedto the selectivetossof interphasecells and reflectedtrue increasesin the absolute numbersasweMas the relativenumbersof mitoticceHs.

In general, the peak in the metaphase index varied directlywith drug concentrationand with drug exposure duration,butthese were not simple, linear relationships.Peak metaphaseindicesdidnotchangeasdrugexposuredurationincreasedfrom1 to 4 hr for all 3 drug concentrationsstudied(Chart 3, A and

0.5

Charta. Effects of VCR on the metaphaseindex. A, VCR exposure duration, 1 hr; B, VCflexposure duration. 4 hr; C. VCR exposure duration, 12 hr: controls. â€¢0.1 *M VCR, T; 05Â«cuVCR, A; 2 0*1 VCR. â€¢.Ban, SJE.

TIME, HR

AUGUST 1983 3593




H. Mujagic et al.

8); when drug exposure duration was extended to 12 hr, peakmetaphase indices increased 4- to 5-fold (Chart 3C). The peakmetaphase index of cells exposed to 0.5 MMVCR was one-half

that of cells exposed to 2 MMVCR for drug exposures of 1 or 4hr. After 12 hr of drug exposure, the peak metaphase index ofcells exposed to 0.5 MMVCR rose to 90% of that of cells exposedto 2 MM VCR. Thus, while both drug concentration and drugexposure duration are important, it would appear that prolongedduration of drug exposure is the dominant factor in producingmetaphase arrest.

Parallel changes in the peak metaphase index and in clono-

genic cell killing as a function of drug concentration and drugexposure duration might suggest a simple causal relation between the two. However, when Charts 1 and 3 are consideredtogether, it is clear that this is not the case. Following exposureto 2 MMVCR for 12 hr, 90% of the clonogenic cells were killed(Chart 1). Allowing that 70% of all of the cells in the populationwere clonogenic, then the overall fraction of the cell populationthat was lethally damaged by the drug was 0.63 (0.9 x 0.7). Thepeak metaphase index of 0.5 after exposure to 2 UMVCR for 12hr (Chart 3C) is in reasonably good agreement with this value.By comparison, following exposure to 2 MM VCR for only 4 hr,over 80% of the clonogenic cells were killed (Chart 1). Again,allowing for the fact that 70% of all the cells were clonogenic,one can estimate that the overall fraction of lethally damagedcells was 0.56 (0.8 x 0.7). However, the peak metaphase indexof 0.16 (Chart 38) was too low to account for all of these lethallydamaged cells. Thus, while delayed arrest in metaphase mightbe taken as a prominent feature of lethally damaged cells following continuous exposure to high concentrations of VCR for 12hr, it would appear that most clonogenic cells that were killedafter only 4 hr of drug exposure did not exhibit this feature.

Flow Cytometry Studies. Flow cytometry studies were carriedout in parallel with the metaphase index studies on separatealiquots of cells from the same flasks. Representative drug-

induced changes in the DMA histogram are illustrated by thedata obtained after a 12-hr exposure to 0.5 tiM VCR (Chart 4).

After 12 hr of drug exposure, many cells had progressed fromthe GÃ¬and early S regions to the late S and G2-M regions of the

DMA histogram (Chart 46). At 16 and 20 hr, the majority of cellsprogressed to the G2-M region of the histogram and accumulated

there (Chart 4, C and D); by 20 hr, some cells had gone on todivide, resulting in a slight increase in the height of the d peak(Chart 40). By 26 hr, the height of the G2-M peak decreased

A CONTROL B 12 HR C 16HRPREÂ«,0, S G.MPOSTUMi

i ji\.i

I : 'â€¢;\

Ã±M1

20HR

>.

26 HR 32 HR

DNA CONTENT

Chart 4. Series of DNA histograms obtained at various times after exposure to0.5 UM VCR for 12 hr. Histogram is divided into PRE-G,, G,, S, G2-M, and POST-Gi-M regions, as shown in A; G, and G2-M regions, shaded panels.

considerably (Chart 4E). The fate of cells that had accumulatedearlier in the G2-M region could be traced through one of 3 paths.Many of these cells appeared as nuclear fragments in the pre-Gi region. The presence of micronuclei was confirmed histolog-ically in Feulgen-stained smears of these cells. Some of the cellsthat had been blocked in the G2-M region went on to divide,appearing in the d and early-S regions of the histogram (com

pare G, regions of Chart 4, D and Â£).Cells that persisted in theG2-M region of the histogram at 26 hr went on to endoreduplicateand were found to proceed in a broad wave through the post-G2-M region at 32 hr (Chart 4F). Feulgen-stained smears of cells

obtained at this time point contained many large cells withmultilobed nuclei or multiple micronuclei.

Systematic changes in the fractions of cells in different regionsof the DNA histogram in relation to VCR concentration and drugexposure duration are described quantitatively in Chart 5.

The most striking change that was observed with all drugschedules was the accumulation of cells in the G2-M region

during and after exposure to VCR (Chart 5, A4 to D4). In general,the accumulation of cells was greater and more prolonged withincreasing drug concentration and increasing drug exposureduration. However, the patterns were quite different from thoseof the metaphase index data (Chart 3). Let us first consider theG2-M accumulation during and after exposure to 2 UM VCR

(Chart 5, A4 to D4, uppermost curves). The earliest drug effectwas observed within 2 hr of the onset of drug exposure. Peakaccumulation in the G2-M region always occurred at 8 hr after

the onset of drug exposure, regardless of the duration of drugexposure, and involved 70 to 90% of the cells with drug exposuredurations of 4 hr or longer. The peak G2-M accumulation was

not sustained, even in the continued presence of the drug (Chart5, C4 and D4). When the relative numbers of cells and the timesof peak accumulations in the flow cytometry data (Chart 5, At toC4)are compared with those of the metaphase index data (Chart3, A to C), it is clear that the peaks in the flow cytometry dataconsisted predominantly of premitotic cells. For example, duringa 12-hr exposure to 2 MMVCR, 75% of the cells accumulated inthe G2-M region of the histogram at 8 hr (Chart 5 C4); however,

at 8 hr, the metaphase index was only 0.05 (Chart 3C). Thus,approximately 70% of the cells in the G2-M region of the DNA

histogram were in G2 and not in M at this time. The same canbe said for peak accumulations of the cells in the G2-M region at

8 hr during exposure to 0.5 MM VCR (compare Chart 5C4 withChart 3C). The fractions of cells in the G2-M region following

exposure to 0.5 and 2 MM VCR fell at 20 hr to 0.4 and 0.5,respectively (Chart 5C4); these values corresponded to the peakvalues of the metaphase index at 20 hr at the 2 respective drugconcentrations (Chart 3C).

Of the cells that accumulated in G2 and were killed by thedrug, some may have been permanently arrested in G2 or M.However, it is apparent from Chart 5 that the cells that accumulated transiently in G2 during and after drug exposure underwent one of several subsequent fates:

1. Decreases in the fractions of cells in the G2-M region of theDNA histogram were accompanied by increases of 10 to 20% inthe fractions of cells in the pre-d region of the histogram. The

magnitude of these increases was modest and only weaklydependent on drug concentration and drug exposure duration(Chart 5, A, to Di).

2. With the decrease in the peak fraction of cells in the G2-M

3594 CANCER RESEARCH VOL. 43




VCR 1 HR 4HR 12 HR

VincristineEffects in Sarcoma 180

24 HR

PRE-G

0 4 8 12 18 24 0 4 8 12 18 24 0 4 8 12 20 26 0 4 8 12 18 24

TIME,HRCharts. Changes in the fractions of cells in different regions of the DMA exposure duration are taken from one experiment. Drug exposure duration periods,

histograms in relation to VCR concentration and drug exposure duration. Data for shaded panels. Controls, â€¢;0.1 tÂ¡uVCR, T; 0.5 UNI VCR, A; 2 >Â»MVCR, â€¢.1-hr, 4-hr, and 12-hr exposure durations, means of 3 experiments; Hata for 24-hr Changes are shown in pre-Gi, GÃ¬,S, Gz-M, and post-G2-M regions. Bars, S.E.

region of the histogram, there also occurred a concomitant 5A2) and became less pronounced at higher drug concentrationsincrease in the fraction of cells in the GÃ¬region of the histogram and longer drug exposure durations (Chart 5, A2 to D2).Wheneverthat was greatest after a 1-hr exposure to 0.1 /Â¿MVCR (Chart a wave of cells was observed to enter and move through the GÃ¬

AUGUST 1983 3595




H. Mujagic et al.

8 12 16 20 24

Time,hrChart 6. ['HJdThd tf'H/dTft) incorporation during continuous exposure to VCR.

Controls, â€¢ â€¢;0.1 Â«MVCR, â€¢ â€¢;0.5 /*MVCR, â€¢â€”â€¢;2 MMVCR,â€¢ Â».Bars.S.E.

region of the histogram (Chart 5, A2 to C2), the wave was seento be propagated through the S region of the histogram 6 to 8hr later (Chart 5, A3 to C3).

3. Another fate of G^blocked cells was the development ofpolyploidy. White this effect was drug concentration-dependent,

it was most striking with prolonged drug exposure durations(Chart 5, As to D5). In these respects, polyploidy is similar in itsbehavior to that of the metaphase index, and the fractions ofpolyploid cells at 26 hr after the onset of a 12-hr drug exposure

(Chart 5C5) were comparable to corresponding peak values ofthe metaphase index at 20 hr (Chart 3C).

[3H]dThd Incorporation during Continuous Exposure toVCR. The patterns of [3H]dThd incorporation during continuous

exposure to VCR concentrations of 0.1,0.5, and 2 pM are shownin Chart 6. There was a gradual, progressive decline in [3H]dThdincorporation for up to 12 hr. There followed an increase in [3H]-

dThd incorporation, with a return toward control values in thepresence of 0.1 UM VCR, and a 2.5- to 3-fdd overshoot above

controls in the presence of 0.5 and 2 //M VCR. The overshootcan be attributed both to the fact that there is twice as muchDMA being synthesized in polyploid cells, and to the partialsynchronization of the polyploid cells following transient metaphase accumulation (Chart 4F).

DISCUSSION

The present studies indicate that VCR affects cells in inter-phase and that metaphase arrest is a late consequence of VCR-induced cell damage. The earliest effect of VCR was manifestwithin 1 to 2 hr of drug administration as a progressive accumulation of cells in G2 that was observed at all drug concentrations and drug exposure durations studied (Chart 5, At to D4).The accumulation of cells in G2 was transient; the fraction ofcells in the G2 region of the histogram peaked at 4 to 8 hr afterthe onset of drug administration and decreased thereafter, evenin the continued presence of the drug (Chart 5, A4 to D4). A blockin G2 following VCR administration has also been reported byothers (24). Although the cause of this G2 block could not be

determined directly from our studies, it would seem reasonableto suppose that it might be due to the disruption or inhibition offormation of microtubular structures other than the mitotic spindle that might be required for the initiation of mitosis.

We have examined the susceptibility of interphase cells to thelethal effects of VCR in greater detail in a separate study insynchronized Sarcoma 180 cells (14). In brief, cells were foundto become more susceptible to the lethal effects of VCR as theyprogressed through late S and G2, suggesting an underlyingmechanism of action that was independent of DMA synthesisbut involved a cell constituent, presumably tubulin, whose synthesis overlapped with that of DMA.

It is clear from the present study that VCR did not inhibit DNAsynthesis directly. Cells that were in the d and S regions of theDNA histogram at the onset of drug administration succeeded intraversing the S region during continuous exposure to drug for24 hr and accumulated transiently in the G2-M region (Chart 5D4).Thus, the gradual decrease in [3H]dThd incorporation that was

observed during the first 12 hr of VCR exposure (Chart 6)reflected the failure of cells to divide and replenish the d and Sregions of the DNA histogram (Chart 5, D2 and D3) but did notrepresent a direct inhibitory effect on DNA synthesis itself. Thiswas confirmed by the overshoot in [3H]dThd incorporation in the

presence of VCR (Chart 6), corresponding to the wave of polyploid cells traversing S (Chart 5D5).

In the present studies, the metaphase index peaked at approximately 4 to 8 hr after drug removal, regardless of theduration of the preceding drug exposure period (Chart 3). Thisdelay was attributable at least in part to the prior transient blockin G2 (compare Chart 5, /44 to C4, with Chart 3). In any event, itis clear that the accumulation of cells in metaphase was theresult of drug-induced damage that was sustained earlier in the

cell cycle. It is also apparent from a comparison of Chart 5, A4to C4, with Chart 3 that, following brief exposure to high concentrations of VCR, not all cells that were blocked transiently in G2subsequently accumulated in metaphase. Furthermore, the degree of metaphase accumulation following exposure to highconcentrations of VCR for 1 to 4 hr was not sufficient to accountfor the magnitude of the cell kill that was observed (compareChart 3 with Chart 1). Metaphase accumulation was pronouncedonly with prolonged drug exposure (Chart 3C), and only then didthe peak metaphase index correspond with drug-induced cell

kill. Thus, it would appear that VCR may have multiple effectson cells and that some of these effects may become moreprominent with prolonged drug exposure duration.

The fate of arrested metaphases has been studied by anumber of investigators (4-6, 8, 10-13, 24). In some early

studies, metaphase arrest was considered to be reversible (10,13). However, the reversibility of metaphase arrest was found tobe dependent on drug concentration and drug exposure duration(5,10, 24). Following exposure to cytotoxic drug concentrations,arrested metaphases were found commonly to undergo necrosis,multipolar divisions, and/or dissolution (4-6, 8,11,12, 24). Our

findings are consistent with the fragmentation of some mitoticcells, particularly after relatively brief exposure to high VCRconcentrations. The evidence for this was the appearance of cellfragments in the pre-d region of the histogram (Chart 5, A, to

Ci), usually peaking at 4 to 6 hr after peak metaphase accumulation (compare with Chart 3). However, most of the metaphasesthat accumulated after 12 hr of exposure to 0.5 and 2 ^M VCR

3596 CANCER RESEARCH VOL. 43




Vincristine Effects in Sarcoma 180

could not be accounted for either by fragmentation (compareChart 3C with Chart 5C,) or by dissolution (compare Chart 3Cwith Chart 2C). On the other hand, both the magnitude andtiming of the rise in the polyploid cell fraction are consistent withthe progression of most of the arrested metaphases to polyploidy(compare Chart 3C with Chart 5C5).

The development of polyploidy is an effect of VCR that becameincreasingly more pronounced with the prolongation of drugexposure duration. Indeed, as drug exposure duration was increased, this effect was demonstrated at progressively lowerVCR concentrations (Chart 5, /45 to D5). The development ofpolyploidy following VCR administration in vivo has been reported by Alabaster and Cassidy (1). Camplejohn (3) suggestedthat this finding may have been due to the very high doses ofVCR that were used in that study. However, the maintenance ofeffective drug levels for prolonged periods after administrationof high doses in vivo may be an alternative explanation.

The mechanism underlying the development of polyploidyfollowing prolonged drug exposure is of special interest. Thelethal effects of the Vinca alkaloids have been attributed to theirbinding to tubulin subunits and the inhibition of polymerization oftubulin subunits into microtubules (15-17). However, microtu-

bules participate in a wide variety of cellular processes, and themitotic sequence itself involves several different classes of microtubules. Early studies focused on the inhibition or disruptionof the mitotic spindle by tubulin-binding agents and the resultant

prevention of centriole separation during mitosis (6, 21). Thedevelopment of polyploidy calls attention to another microtubule-

dependent mitotic function that may be inhibited by VCR, namely,cytokinesis. The Vinca alkaloids are known to inhibit the development of the cleavage furrow of sea urchin eggs (18) andinterfere with the normal separation of mammalian cells in telo-

phase (10, 11, 24). Thus, interference with cytokinesis duringprolonged exposure to VCR would represent a lethal effect ofthe drug that may be distinct from its short-term effects, and

which could account for the development of polyploidy duringand after prolonged drug exposure. This could serve as the basisfor clinical studies of the antitumor effects of prolonged VCRinfusions.

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15. Oweilen. R. J., Donigian. O. W., Hartke, C. A.. Oickerson. R. M.. and Kuhar.M. J. The binding of vinblastine to tubulin and to paniculate fractions ofmammalainbrain. Cancer Res., 34: 3180-3186,1974.

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