Effects of Vincristine on Cell Survival, Cell Cycle Progression, and Mitotic Accumulation in Asynchronously Growing Sarcoma 180 Cells

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

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

indicate that metaphase arrest and cell necrosis are not the only manifestations of VCR lethality. It has been suggested that The effects of vincristine (VCR) on cell survival, cell cycle polyploidy might also be a dose-dependent phenomenon (3), but progression, DMA synthesis, and metaphase accumulation were this has not been clearly established. studied in relation to drug concentration and drug exposure We have undertaken a systematic study of the effects of VCR duration in Sarcoma 180 cells in vitro. VCR was found to affect on cell survival, cell cycle progression, DNA synthesis, and cells in interphase, producing a transient G2 block at all drug metaphase accumulation in Sarcoma 180 in vitro in relation to concentrations and drug exposure durations studied. VCR did drug concentration and to drug exposure duration. The results not affect DMA synthesis directly. Increases in the metaphase of studies carried out on asynchronously growing cells are index were delayed and always peaked at approximately 8 hr described in this paper. The results of VCR studies carried out after drug removal, regardless of the duration of drug exposure. on recruited, partially synchronized cells are described in a Increases in the metaphase index of sufficient magnitude to be separate paper (14). The present studies demonstrated that VCR commensurate with VCR lethality were observed only with pro exerted its lethal effects on cells before they entered mitosis. longed drug exposure. VCR produced both nuclear fragmenta VCR did not inhibit DNA synthesis. However, it did produce a tion and polyploidy. The proportion of cells undergoing polyploidy transient accumulation of cells in G2. With regard to the disposi increased progressively with increasing drug exposure duration. tion of lethally damaged cells, both nuclear fragmentation and Interference with cytokinesis during prolonged VCR exposure polyploidy were observed, and the latter was found to be highly may represent a lethal effect of VCR that is separate from its dependent on drug concentration and drug exposure duration. short-term effects. This could serve as the basis for the clinical study of the antitumor effects of prolonged VCR infusions. MATERIALS AND METHODS ABSTRACT INTRODUCTION VCR2 and vinblastine are cytotoxic tubulin-binding agents,
All studies were carried out in Sarcoma 180 (Foley strain CCRF11 ; supplied by American Tissue Type Culture, Rockville, Md.) grown in vitro in Earle's Medium 199 (Flow Laboratories, Rockville, Md.), which was

derived from the periwinkle (Vinca rosea), that have found their supplemented with 10% fetal bovine serum, 2 rriM L-glutamine, 100 units place as effective drugs in combination chemotherapy regimens of penicillin per ml, and 100 ng of streptomycin per ml. Cultures were for the treatment of human leukemias, lymphomas, and a variety grown in monolayer in 250-ml plastic tissue culture flasks (growth surface area, 75 sq cm) (Costar, Cambridge, Mass.) containing 10 ml of medium. of solid tumors. Cells were plated at an initial concentration of 1 x 105 cells/ml. Medium Early experimental studies of the mode of action of the Vinca was changed on Days 2 and 4, and cells were subcultured on Day 5. alkaloids focused on their disruptive effects on the mitotic spindle Cell Survival Studies. Two-day-old log-phase cell cultures were in (6,10,13), and the lethality of these drugs was generally attrib cubated at 37°with VCR at final concentrations ranging from 0.01 to 10 uted to their effects on cells in mitosis (2, 6, 22). However, other /¿M drug exposure durations ranging from 1 to 24 hr. At the end of for studies have suggested that VCR exerts its lethal effects on the drug exposure period, the medium containing drug was removed, interphase cells (4, 7, 8,11,12, 20, 23). and the cells were rinsed 4 times with 5 ml of HBSS. Cells were harvested by incubation (37°)with 0.25% trypsin for 8 min. Controls were obtained Studies of the disposition of cells affected by the Vinca alka at each time point. Total cell counts were determined using a Coulter loids have produced conflicting results. In some studies, meta phase arrest was found to be completely reversible (10, 13) Counter (Coulter Electronics, Hialeah, Fla.). Cell suspensions were di luted with Medium 199, and known numbers of cells were cloned in soft while, in other studies, it was reported that arrested metaphases agar. Nine-day-old colonies were fixed, stained with Giemsa stain, and subsequently became necrotic (4,6,11,12). Several authors counted visually. Clonogenic cell yield, defined as the number of colonies have emphasized that the reversibility of metaphase arrest was enumerated divided by the number of cells plated, was averaged for 5 dependent on drug concentration and drug exposure duration replicate flasks at each concentration. The viable cell count per flask was (5, 9, 24). Alabaster and Cassidy (1) demonstrated by means of calculated as the total cell number per flask multiplied by the clonogenic flow cytometry that VCR produced polyploidy. These findings cell yield at a given time point. The surviving viable cell fraction following
drug exposure was calculated as the number of viable drug-treated
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.

cells

per flask divided by the number of viable untreated control cells per flask. Each experiment was performed at least 3 times, and the reported results represented the log means of replicate studies. Metaphase Index Studies. Serial metaphase indices were obtained at intervals during and after VCR exposure. Aliquots of cells were

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VÃ-ncrÃ-stine Effects in Sarcoma 180 (Chart 2A), there was no significant decrease in cell number, and there was no delay in cell population growth. Exposure to drug for 4 or 12 hr (Chart 2, B ana C) produced complete inhibition of cell population growth throughout the drug exposure period at all drug concentrations studied and for 6 to 8 hr after drug removal. Cells exposed to 0.1 ¿tMCR resumed growth within V 18 to 20 hr of the initiation of drug exposure, whether the drug was present or not (in Chart 2, compare C and D). Exposure to 0.5 or 2 ¡IM VCR produced not only an inhibition of cell growth, but also a relative decrease in cell number of up to 10 to 25% in some experiments (Chart 2, B to D). Metaphase Index Studies. The effects of VCR on the metaphase index are shown in Chart 3. In control cells, the metaphase index ranged from 0.01 to 0.02 throughout the period of obser vation in all 3 studies. After exposure to 0.1 ¡MVCR for 1 hr, the metaphase index rose modestly to 0.03 at 4 hr, remained in this range through 8 hr, and fen to control levels at 12 hr (Chart 3A). After exposure to 0.5 ¿<M for 1 hr, the metaphase index VCR remained at the same level as that of controls at 2 hr, rose to 0.08 at 8 hr, and fell at 12 hr. After exposure to 2.0 MMVCR for 1 hr, the metaphase index remained at the same level as controls through 4 hr, rose to 0.15 at 8 hr, and fell sharply at 12 hr. The effects of exposure to 0.1 //M VCR for 4 hr were similar in magnitudeto those observed after a 1-hr exposure (Chart SB). There was a modest rise in the metaphase index during the first 8 to 12 hr and a return to control values by 18 hr. During the period of exposure to 0.5 and 2 ¿>M for 4 hr, there was VCR no rise m the metaphase index (Chart 35), and the metaphase index remained at control levels for 2 hr after drug removal. The subsequent behavior of the metaphase index after exposure to 0.5 and 2 fit» VCR for 4 hr was comparable to that observed after drug exposure for 1 hr, except that the peaks in the metaphase index occurred at 12 hr rather than at 8 hr. During a 12-hr exposure to 0.1 ¿IM VCR, a modest but pro gressive rise in the metaphase index was observed from 4 to 14 hr. Between 14 and 16 hr, the metaphase index doubled from 0.05 to 0.1; it then feugradually to control values by 26 hr (Chart 3C). In the presence of 0.5 and 2 /^MVCR, the metaphase index did not increase above control values until 8 hr and continued to rise modestly through 14 hr to values of 0.08 to 0.12. The metaphase index then rose sharply to values of 0.45 to 0.5 at 20 hr, and then fell rapidly to control values by 26 hr. In summary, for all 3 drug schedules. VCR produced modest increases in the metaphase index during the period of drug exposure and for 2 to 4 hr thereafter. At the higher drug concen trations, the metaphase index rose sharply at 4 to 8 hr after the termination of drug exposure, regardless of the duration of drug exposure. Tubulin-binding agentscan producea rise in ine mitolicindex that may be due in large part to selective loss of eels in interphaserather than a true increasein mrtoticcete (11. 12, 19). Wien the data in Charts 2 and 3 are consideredtogether, ittsdear that eventhe modest3-foldincreasesinthe metaphase index that were observedduringand after exposureto 0.1 KM VCR couid not be attributedto the selectivetossof interphase cells and reflected true increasesin the absolute numbers as weMas the relativenumbersof mitoticceHs. In general, the peak in the metaphase index varied directly with drug concentrationand with drug exposure duration,but these were not simple, linear relationships.Peak metaphase indicesdidnotchangeas drugexposuredurationincreasedfrom 1 to 4 hr for all 3 drug concentrationsstudied(Chart 3, A and

04812182404812182404812

2026048121824

Chart 2. Total eel number as a function of time during and after exposure to VCR. A, VCR exposure duration. 1 hr. e. VCR exposure duration. 4 hr. C. VCR exposure duration. 12 hr. D. VCR exposure duration. 24 hr. controls, •0.1 JIM VCR. T: 0.5 UMVCR. A. 2 «M VCR. • Bars, SE

0.5

Charta. Effects of VCR on the metaphase index. A, VCR exposure duration, 1 hr; B, VCfl exposure duration. 4 hr; C. VCR exposure du ration, 12 hr: controls. •0.1 *M VCR, T; 05 «cu VCR, A; 2 0*1 VCR. •. Ban, SJE.

TIME,

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H. Mujagic et al. considerably (Chart 4E). The fate of cells that had accumulated earlier in the G2-M region could be traced through one of 3 paths. Many of these cells appeared as nuclear fragments in the prethat of cells exposed to 2 MMVCR for drug exposures of 1 or 4 Gi region. The presence of micronuclei was confirmed histologhr. After 12 hr of drug exposure, the peak metaphase index of ically in Feulgen-stained smears of these cells. Some of the cells cells exposed to 0.5 MMVCR rose to 90% of that of cells exposed that had been blocked in the G2-M region went on to divide, to 2 MM VCR. Thus, while both drug concentration and drug appearing in the d and early-S regions of the histogram (com exposure duration are important, it would appear that prolonged pare G, regions of Chart 4, D and £).Cells that persisted in the duration of drug exposure is the dominant factor in producing G2-M region of the histogram at 26 hr went on to endoreduplicate and were found to proceed in a broad wave through the postmetaphase arrest. Parallel changes in the peak metaphase index and in clono- G2-M region at 32 hr (Chart 4F). Feulgen-stained smears of cells genic cell killing as a function of drug concentration and drug obtained at this time point contained many large cells with exposure duration might suggest a simple causal relation be multilobed nuclei or multiple micronuclei. Systematic changes in the fractions of cells in different regions tween the two. However, when Charts 1 and 3 are considered together, it is clear that this is not the case. Following exposure of the DNA histogram in relation to VCR concentration and drug to 2 MM VCR for 12 hr, 90% of the clonogenic cells were killed exposure duration are described quantitatively in Chart 5. (Chart 1). Allowing that 70% of all of the cells in the population The most striking change that was observed with all drug were clonogenic, then the overall fraction of the cell population schedules was the accumulation of cells in the G2-M region that was lethally damaged by the drug was 0.63 (0.9 x 0.7). The during and after exposure to VCR (Chart 5, A4 to D4). In general, peak metaphase index of 0.5 after exposure to 2 UM VCR for 12 the accumulation of cells was greater and more prolonged with hr (Chart 3C) is in reasonably good agreement with this value. increasing drug concentration and increasing drug exposure By comparison, following exposure to 2 MM VCR for only 4 hr, duration. However, the patterns were quite different from those over 80% of the clonogenic cells were killed (Chart 1). Again, of the metaphase index data (Chart 3). Let us first consider the allowing for the fact that 70% of all the cells were clonogenic, G2-M accumulation during and after exposure to 2 UM VCR one can estimate that the overall fraction of lethally damaged (Chart 5, A4 to D4, uppermost curves). The earliest drug effect cells was 0.56 (0.8 x 0.7). However, the peak metaphase index was observed within 2 hr of the onset of drug exposure. Peak of 0.16 (Chart 38) was too low to account for all of these lethally accumulation in the G2-M region always occurred at 8 hr after damaged cells. Thus, while delayed arrest in metaphase might the onset of drug exposure, regardless of the duration of drug be taken as a prominent feature of lethally damaged cells follow exposure, and involved 70 to 90% of the cells with drug exposure ing continuous exposure to high concentrations of VCR for 12 durations of 4 hr or longer. The peak G2-M accumulation was hr, it would appear that most clonogenic cells that were killed not sustained, even in the continued presence of the drug (Chart after only 4 hr of drug exposure did not exhibit this feature. 5, C4 and D4). When the relative numbers of cells and the times Flow Cytometry Studies. Flow cytometry studies were carried of peak accumulations in the flow cytometry data (Chart 5, At to out in parallel with the metaphase index studies on separate C4) are compared with those of the metaphase index data (Chart aliquots of cells from the same flasks. Representative drug- 3, A to C), it is clear that the peaks in the flow cytometry data induced changes in the DMA histogram are illustrated by the consisted predominantly of premitotic cells. For example, during data obtained after a 12-hr exposure to 0.5 tiM VCR (Chart 4). a 12-hr exposure to 2 MMVCR, 75% of the cells accumulated in After 12 hr of drug exposure, many cells had progressed from the G2-M region of the histogram at 8 hr (Chart 5 C4); however, the Gì nd early S regions to the late S and G2-M regions of the at 8 hr, the metaphase index was only 0.05 (Chart 3C). Thus, a DMA histogram (Chart 46). At 16 and 20 hr, the majority of cells approximately 70% of the cells in the G2-M region of the DNA progressed to the G2-M region of the histogram and accumulated histogram were in G2 and not in M at this time. The same can there (Chart 4, C and D); by 20 hr, some cells had gone on to be said for peak accumulations of the cells in the G2-M region at divide, resulting in a slight increase in the height of the d peak 8 hr during exposure to 0.5 MM VCR (compare Chart 5C4 with (Chart 40). By 26 hr, the height of the G2-M peak decreased Chart 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, A HRPRE«, CONTROL B 12 HR C 16 respectively (Chart 5C4); these values corresponded to the peak POSTUMi 0, S G.M ;\ : '• I values of the metaphase index at 20 hr at the 2 respective drug i ji\.i concentrations (Chart 3C). Of the cells that accumulated in G2 and were killed by the drug, some may have been permanently arrested in G2 or M. 1 However, it is apparent from Chart 5 that the cells that accu mulated transiently in G2 during and after drug exposure under 32 HR 26 HR 20HR went one of several subsequent fates: >. 1. Decreases in the fractions of cells in the G2-M region of the DNA histogram were accompanied by increases of 10 to 20% in the fractions of cells in the pre-d region of the histogram. The magnitude of these increases was modest and only weakly DNA CONTENT dependent on drug concentration and drug exposure duration Chart 4. Series of DNA histograms obtained at various times after exposure to 0.5 UM VCR for 12 hr. Histogram is divided into PRE-G,, G,, S, G2-M, and POST- (Chart 5, A, to Di). Gi-M regions, as shown in A; G, and G2-M regions, shaded panels. 2. With the decrease in the peak fraction of cells in the G2-M 8); when drug exposure duration was extended to 12 hr, peak metaphase indices increased 4- to 5-fold (Chart 3C). The peak metaphase index of cells exposed to 0.5 MMVCR was one-half

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Vincristine Effects in Sarcoma 180

VCR 1 HR

4HR

12 HR

24 HR

PRE-G

0 4 8 12

18

24 0 4

8 12

18

24 0 4 8 12 TIME, HR

20

26 0 4

8 12

18 24

Charts. Changes in the fractions of cells in different regions of the DMA histograms in relation to VCR concentration and drug exposure duration. Data for 1-hr, 4-hr, and 12-hr exposure durations, means of 3 experiments; Hata for 24-hr

exposure duration are taken from one experiment. Drug exposure duration periods, shaded panels. Controls, •;0.1 t¡u VCR, T; 0.5 UNI VCR, A; 2 >»M VCR, •. Changes are shown in pre-Gi, Gì, Gz-M, and post-G2-M regions. Bars, S.E. S,

region of the histogram, there also occurred a concomitant increase in the fraction of cells in the Gìegion of the histogram r that was greatest after a 1-hr exposure to 0.1 /¿M VCR (Chart

5A2) and became less pronounced at higher drug concentrations and longer drug exposure durations (Chart 5, A2 to D2). Whenever a wave of cells was observed to enter and move through the Gì

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H. Mujagic et al. determined directly from our studies, it would seem reasonable to suppose that it might be due to the disruption or inhibition of formation of microtubular structures other than the mitotic spin dle that might be required for the initiation of mitosis. We have examined the susceptibility of interphase cells to the lethal effects of VCR in greater detail in a separate study in synchronized Sarcoma 180 cells (14). In brief, cells were found to become more susceptible to the lethal effects of VCR as they progressed through late S and G2, suggesting an underlying mechanism of action that was independent of DMA synthesis but involved a cell constituent, presumably tubulin, whose syn thesis overlapped with that of DMA. It is clear from the present study that VCR did not inhibit DNA synthesis directly. Cells that were in the d and S regions of the DNA histogram at the onset of drug administration succeeded in traversing the S region during continuous exposure to drug for 24 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 S regions of the DNA histogram (Chart 5, D2 and D3) but did not represent a direct inhibitory effect on DNA synthesis itself. This was confirmed by the overshoot in [3H]dThd incorporation in the presence of VCR (Chart 6), corresponding to the wave of poly ploid cells traversing S (Chart 5D5). In the present studies, the metaphase index peaked at ap proximately 4 to 8 hr after drug removal, regardless of the duration of the preceding drug exposure period (Chart 3). This delay was attributable at least in part to the prior transient block in G2 (compare Chart 5, /44 to C4, with Chart 3). In any event, it is clear that the accumulation of cells in metaphase was the result of drug-induced damage that was sustained earlier in the cell cycle. It is also apparent from a comparison of Chart 5, A4 to C4, with Chart 3 that, following brief exposure to high concen trations of VCR, not all cells that were blocked transiently in G2 subsequently accumulated in metaphase. Furthermore, the de gree of metaphase accumulation following exposure to high concentrations of VCR for 1 to 4 hr was not sufficient to account for the magnitude of the cell kill that was observed (compare Chart 3 with Chart 1). Metaphase accumulation was pronounced only with prolonged drug exposure (Chart 3C), and only then did the peak metaphase index correspond with drug-induced cell kill. Thus, it would appear that VCR may have multiple effects on cells and that some of these effects may become more prominent with prolonged drug exposure duration. The fate of arrested metaphases has been studied by a number 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 to be 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 mitotic cells, particularly after relatively brief exposure to high VCR concentrations. The evidence for this was the appearance of cell fragments in the pre-d region of the histogram (Chart 5, A, to Ci), usually peaking at 4 to 6 hr after peak metaphase accumu lation (compare with Chart 3). However, most of the metaphases that accumulated after 12 hr of exposure to 0.5 and 2 ^M VCR

8

12

16

20

24

Time, hr Chart 6. ['HJdThd tf'H/dTft) incorporation during continuous exposure to VCR. Controls, • •; «M 0.1 VCR, • •; /*M VCR, •—•; 0.5 2 MMVCR, • ».Bars.S.E.

region of the histogram (Chart 5, A2 to C2), the wave was seen to be propagated through the S region of the histogram 6 to 8 hr later (Chart 5, A3 to C3). 3. Another fate of G^blocked cells was the development of polyploidy. 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 its behavior to that of the metaphase index, and the fractions of polyploid cells at 26 hr after the onset of a 12-hr drug exposure (Chart 5C5) were comparable to corresponding peak values of the metaphase index at 20 hr (Chart 3C). [3H]dThd Incorporation during Continuous Exposure to VCR. The patterns of [3H]dThd incorporation during continuous exposure to VCR concentrations of 0.1,0.5, and 2 pM are shown in Chart 6. There was a gradual, progressive decline in [3H]dThd incorporation for up to 12 hr. There followed an increase in [3H]dThd incorporation, with a return toward control values in the presence 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 overshoot can be attributed both to the fact that there is twice as much DMA being synthesized in polyploid cells, and to the partial synchronization of the polyploid cells following transient meta phase accumulation (Chart 4F). DISCUSSION The present studies indicate that VCR affects cells in interphase and that metaphase arrest is a late consequence of VCRinduced cell damage. The earliest effect of VCR was manifest within 1 to 2 hr of drug administration as a progressive accu mulation of cells in G2 that was observed at all drug concentra tions and drug exposure durations studied (Chart 5, At to D4). The accumulation of cells in G2 was transient; the fraction of cells in the G2 region of the histogram peaked at 4 to 8 hr after the onset of drug administration and decreased thereafter, even in the continued presence of the drug (Chart 5, A4 to D4). A block in G2 following VCR administration has also been reported by others (24). Although the cause of this G2 block could not be

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Vincristine Effects in Sarcoma 180 could not be accounted for either by fragmentation (compare Chart 3C with Chart 5C,) or by dissolution (compare Chart 3C with Chart 2C). On the other hand, both the magnitude and timing of the rise in the polyploid cell fraction are consistent with the 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 became increasingly more pronounced with the prolongation of drug exposure duration. Indeed, as drug exposure duration was in creased, this effect was demonstrated at progressively lower VCR concentrations (Chart 5, /45 to D5). The development of polyploidy following VCR administration in vivo has been re ported by Alabaster and Cassidy (1). Camplejohn (3) suggested that this finding may have been due to the very high doses of VCR that were used in that study. However, the maintenance of effective drug levels for prolonged periods after administration of high doses in vivo may be an alternative explanation. The mechanism underlying the development of polyploidy following prolonged drug exposure is of special interest. The lethal effects of the Vinca alkaloids have been attributed to their binding to tubulin subunits and the inhibition of polymerization of tubulin subunits into microtubules (15-17). However, microtubules participate in a wide variety of cellular processes, and the mitotic sequence itself involves several different classes of mi crotubules. Early studies focused on the inhibition or disruption of the mitotic spindle by tubulin-binding agents and the resultant prevention of centriole separation during mitosis (6, 21). The development of polyploidy calls attention to another microtubuledependent mitotic function that may be inhibited by VCR, namely, cytokinesis. The Vinca alkaloids are known to inhibit the devel opment of the cleavage furrow of sea urchin eggs (18) and interfere with the normal separation of mammalian cells in telophase (10, 11, 24). Thus, interference with cytokinesis during prolonged exposure to VCR would represent a lethal effect of the drug that may be distinct from its short-term effects, and which could account for the development of polyploidy during and after prolonged drug exposure. This could serve as the basis for clinical studies of the antitumor effects of prolonged VCR infusions.
3. Camplejohn, R. S. A critical review of the use of vincristine (VCR) as a tumour cell synchronizing agent in cancer therapy. Cefi Tissue Kinet., 13: 327-335, 1980. 4. Camplejohn. R. S., Schultze. B., and Maurer, W. An in vivo double-labeling study with the JB-1 mouse ascites tumour of the subsequent fate of cells arrested in metaphase by vincristine. Cell Tissue Kinet., 13:239-250,1980. 5. Dahl, W. N., Oftebro, R., Petterson, E. 0., and Brustad. T. Inhibitory and cytotoxic effects of oncovm (vincristine sulfate) on cells of human line NHIK 3025. Cancer Res., 36: 3101-3105,1976. 6. George, P., Journey, L J., and Goldstein. M. N. Effect of vincristine on the fine structure of HeLa cells during mitosis. J. Nati. Cancer Inst.. 35: 355-375, 1965. 7. Hill, B. T., and Whelan, R. D. H. Comparative cell killing and kinetic effects of vincristine or vindesinein mammaliancell lines. J. Nati. Cancer Inst., 67:437443, 1981. 8. Jelinghaus, W., Schultze, B.. and Maurer. W. The effect of vincristine on mouse jejuna! crypt cells of differing cell age: double labeling autoradiographic studes using "H- and "C-TdR. Cell Tissue Kinet., 10:147-156,1977. 9. Klein, H. O. Cell kinetic alterations in normal and neoplastic cell populations in vitro and in vivo following vincristine. Cell Tissue Kinet., 13:425-434,1980. 10. Krishan. A. Time-lapse and ultrastructure studies on the reversal of mitotic arrest induced by vinblastine sulfate in Earie's L cells. J. Nati. Cancer Inst., 41: 581-595.1968. 11. Lengsfeld. A. M., Schultze, B., and Maurer. W. Tune-lapse studies on the effect of vincristine on HeLa cells. Eur. J. Cancer, 17: 307-319, 1981. 12. Madoc-Jones,H., and Mauro, F. Interphaseaction of vinblastineand vincristine: differencesin their lethal action through the mitotic cycle of cultured mammalian cells. J. Cell Physiol., 72:185-196.1968. 13. Malawista, S E.. Sato, H., and Bensch, K. G. VnUastine and gnseofuiv/n reversibly disrupt the living mitotic spindle. Science (Wash. D. C.), 760: 770772.1968. 14. Mujagic, H., Conger, B. M., Smith, C. A., Occhipinti,S. J., Schiette, W. H., and Shackney.S. E. Scheduledependenceof vincristinelethalityin sarcoma 180 cells followingpartialsynchronization with hydroxyurea CancerRes., 43. 3598-3603, 1983. 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 of mammalainbrain. Cancer Res., 34: 3180-3186,1974. 16. Oweilen. R. J., Hartke. C. A.. Oickerson. R. M.. and Hans. F. O. Inhibition of !ubulin-microtjbule polymerizationby drugs of the Vincaalkaloid class. Cancer Res.. 36.:1499-1502,1976. 17. Oweten, R. J.. Owens. A. H., Jr., and Donigian.D.W. Thebinding of vincristine, vinblastine and colchicine to tubufci. Bnchem. Biophys. Res. Commun., 47: 685-691.1972. 18. Remfflard,S., and Rebhun, L. I. Antimitotic activity of the potent tumor inhibitor maytansine. Science (Wash. 0. C.). 789; 1002-1005.1975. 19. Shackney, S. E., Bum, P. A and Ford. S. S. The effects of cofcemd on mouse bone marrow. Cell Tissue Kinet., 9: 363-369,1976. 20. Stryckmans, P. A., Lurie, P. M., Manaster, J., and Vamecq. G. Mode of action of chemotherapy in vivo on acute leukemia. II. Vincristine. Eur. J. Cancer, 9 613-620.1973. 21. Stubblefield. E. Centriole replication in mammalian cells. In: The Proliferation and Spread of Neoplastic Cells, pp. 175-193. Baltimore. Md.: The Williams & WilkmsCo.. 1968. 22. Tucker, R. W., Oweilen, R. J.. and Harris, S. B. Correlation of cytotoxoty and mitotoic spindle dissolution by vmbiastine in mammalian cells. Cancer Res., 37:4346-4351,1977. 23. Wibe, E. Age-dependent ce««activation vincristine alone or in combination  by with 1-propargyl-5-chloropyrimidin-2-one.Cancer Res., 40: 2069-2073,1980. 24. Wibe, E., Oftebro, R., Christensen, T., Laland, S. G., Pettersen, E. O., and Lindmo. T. Inhibitory effects of the new mitotic inhibitor 5<hloropyrimkin-2one and of vincristine on human cete in vitro. Cancer Res.. 38: 560-565. 1978.

REFERENCES
1. Alabaster, O., and Cassidy, M. Flow rricrofluorimetric analysisof P388 murine leukemia after administration of vincristine and maytansine m vivo. J. Nati. Cancer Inst.. 60: 649-652,1978. 2. Bruchovsky, N., Owen, A. A., Becker, A. J., and Till, J. E. Effects of vinolastine on the proliferate capacity of L-cells and their progress through the division cycle. Cancer Res., 25. 1232-1237, 1965.

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