THE RESPONSE OF HUMAN CULTURED LYMPHOCYTES TO CYTOCHALASIN B

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1 J. Cell Sci., (968) 595 Printed in Great Britain THE RESPONSE OF HUMAN CULTURED LYMPHOCYTES TO CYTOCHALASIN B M. A. C. RIDLER AND G. F. SMITH Kemtedy-Galton Centre, Harperbury Hospital, Near St Albans, Hertfordshire SUMMARY The cytochalasins are a new group of compounds which have been recently isolated from moulds. Some of their properties are of interest in the study of cytoplasmic and nuclear division since they have been shown to inhibit motility, produce nuclear extrusion and prevent cytoplasmic cleavage with the production of multinucleated cells. The present study is a report of the effects of cytochalasin B on cultured human lymphocytes. After exposure to cytochalasin B, a proportion of the cultured human lymphocytes became multinucleate. The magnitude of this effect was related to the concentration and the length of time of exposure to the compound. Cells with from one to eight nuclei were identified. Progressive variation in nuclear morphology became apparent as the number of nuclei in a cell increased. Examination of the chromosomes showed only diploid, tetraploid and a few octaploid cells. No obvious chromosomal abnormalities were detected, except in octaploid cells. It is suggested that in multinucleated cells there may be a failure of mitotic control leading to variations in size and shape of interphase nuclei. INTRODUCTION The cytochalasins are metabolites of moulds that have been recently isolated by Dr W. B. Turner, who was also one of the group that described the chemistry of these compounds (Aldridge, Armstrong, Speake & Turner, 967). Their special importance lies in their property of inhibiting cell division by blocking cytoplasmic cleavage. Such compounds are of value in the study of cellular function and these particular substances are unique in their ability to block cell division without preventing mitosis. Four cytochalasins have been isolated from culture filtrates: A and B from Helminthosporium dematioideum and C and D from Metarrhizium anisopliae. The substances are chemically similar but differ mainly in the tenfold increase of potency of C and D over A and B. Carter (967) has reported the inhibition of motility, nuclear extrusion and the prevention of cytoplasmic cleavage with the production of multinucleate cells by cytochalasin B in cultures of Earle's 'L' strain of mouse fibroblasts. Other interesting features are the low toxicity and reversibility of the action of the compound. Brief preliminary findings on the effect of cytochalasin B on suspension cultures of human lymphocytes have been recorded (Smith & Ridler, 967). In the present report, further observations are presented, including data on the chromosomes of lymphocytes exposed to cytochalasin B. 8 CeU Sci.

2 596 M. A. C. Ridler and G. F. Smith MATERIALS AND METHODS Cytochalasin B was obtained as a powder which was poorly soluble in water. It was made up as a o-i % stock solution in dimethyl sulphoxide (DMSO), divided into small portions and stored, frozen solid, at C. Blood, taken by venipuncture from normal adults, was treated with Phytohaemagglutinin (Burroughs Welcome), maintained in iced water for min and lightly centrifuged to obtain lymphocyte-rich plasma which was cultured with Tissue Culture Medium 99 by the modified method of Moorhead et al. (i96). The stock solution of cytochalasin was thawed and added at the commencement of the culture in concentrations ranging from -5 to /tg/ml. Cultures were incubated for up to 7 days. Aliquots were removed at intervals, rapidly fixed (glacial acetic acid: absolute ethyl alcohol, :) and spread by the air-drying method of Rothfels & Siminovitch (958) for microscopy. Slides were stained with Leishman's stain and mounted. Chromosome preparations were made from similar cultures to which colcemid was added in a final concentration of - mg/ml for 6 h. A prolonged exposure time to colcemid was thought desirable to accumulate metaphase cells during the critical time of multinucleated cell production. Cells were treated with hypotonic citrate, fixed, spread by the air-drying method and stained with Giemsa's stain. Control cultures without cytochalasin and with DMSO added alone were set up. RESULTS The first results obtained were extremely variable on account of growth failure in many cultures. The difficulty was apparently due to toxicity of DMSO at certain concentrations, and in some cultures it may have been due to deterioration of the cytochalasin after repeated freezing and thawing. The technique was later standardized. DMSO was never added in a concentration greater than o-i ml to ml of culture and cytochalasin was added from a small volume of stock solution that was thawed once only and discarded after use. Once standardized, the response of cultured lymphocytes to cytochalasin was found to vary slightly in different cultures but a general pattern was discernible. The most impressive response to cytochalasin B was the production of multinucleate cells. A few binucleate cells were found in the control cultures by 96 h but never more than %. A small proportion of binucleate cells was detected in the cytochalasin cultures at and 8 h but no marked response was obtained under 7 h incubation. At 7 h the number of binucleate cells rose to a peak when cytochalasin was used at a concentration of 6 /^g/ml. At 96 h the proportion of binucleate cells rose rapidly, reaching a peak level of about 5% with a cytochalasin concentration of 6 /ig/ml. Cells containing more than two nuclei showed a similar time/ dosage response. In a few cultures, incubation was continued for 7 days, by which time many of the cells appeared to be dead or dying, but in some of the cultures, a great number of multinucleate cells was found, reaching a maximum value of over 7 %. These results are illustrated in Fig.. Quantitative assessment of the proportion

3 Human lymphocytes and cytochalasin B 597 of multinucleated cells in a culture was hindered by the tendency of lymphocytes to form cell aggregates or clumps. These aggregates show considerable metabolic activity with poor cellular definition. The counts were, of necessity, made on separated cells or on very small clumps. Furthermore, the counts given are composite values from three of the most recent, standardized cultures. The values (Fig., Tables, ) are derived from varying numbers of observations at different times and dosages. The percentages of cells showing increased numbers of nuclei are given in Table. Binucleate cells were the most frequent, the proportion of multinucleated cells being inversely related to the number of nuclei per cell ^ u h 8 h 7 h 96 h 68 h Cytochalasin concentration (//g/ml) Fig.. Graphic representation of the proportion of cells with two nuclei (solid line) and total multinucleate cells (broken line) resulting from different exposures of lymphocyte cultures to various concentrations of cytochalasin B. The morphology of the cells underwent striking and progressive changes in response to cytochalasin. Up to 7 h only the usual blastic transformation was observed. Between 7 and 96 h some nuclei became slightly lobed, but in other cells separate nuclei of similar size were formed. After 7 h increasing numbers of cells with more than two nuclei were observed. Cells with from one to eight nuclei were counted with certainty. More than eight nuclei may have been present in some cells; however, overlapping and lobulation of nuclei made counting difficult. As the number of nuclei increased, so did over-all cell size, although the cytoplasmic volume did not appear to increase proportionately. In association with the increasing number of nuclei in a cell, there was a marked variation of size and shape of the nuclei within the same cell. Nuclei became lobulated, small remnants, or micronuclei, were formed and nuclear bridges of varying thickness, together with cytoplasmic bridges, were noted. Examination of mitotic figures showed all stages of mitosis with increasing complexity of the mitotic configurations. All the nuclei in any dividing cell appeared to be at the same stage of division though the mitotic figures were sometimes slightly out of phase with 8-

4 M. A. C. Ridler and G. F. Smith each other. Metaphase often showed bizarre chromosome arrangements with groups of chromosomes of varying size separated from the main groups. A series of cells showing variation in nuclear number and morphology are shown in Figs. -7 and a further series of cells in mitosis in Figs. 8-. Table. Percentage of cells (A)* with different numbers of nuclei (B) at various concentrations of cytochalasin (C) Time A in culture (h) i B C [ Control i) i- ' ' 5 6 > > 6 ; i v 5 6j 5 6 > 6, Figures based on counts of cells. /tg/ml /tg/ml. /tg/m /tg/ml 6/tg/ml 8 /tg/ml /tg/ml

5 Human lymphocytes and cytochalasin B 599 What appeared to be extrusion of cell nuclei was frequently seen, but this phenomenon needs to be observed in living cells because the effect may be an artefact due to spreading. Extrusion did seem to increase with cytochalasin concentration and was more marked than in the control cultures, but quantitative assessment proved impossible. Table. Chromosome counts in two lymphocyte cultures treated Chromosome count with cytochalasin Culture details < 6 6 > 6 < 9 9 Total cells. Cytochalasin 6 /tg/ml 6 8 Colcemid - mg/ml 6 h Terminated at 88 h. Cytochalasin /tg/ml 5 Colcemid mg/ml h Terminated at 96 h Table. Association of dividing nuclei containing various numbers of chromosomes* Chromosome complements of associated nuclei Two associated nuclei Number observed Three associated nuclei n + n + n Total Associated nuclei are likely to belong to the same parent cell. Enumeration of small, intermediate and large blasts and of the cells in mitosis revealed no effect of cytochalasin on the rate of transformation or growth, but we have not been able to investigate higher concentrations of the compound for toxic or lethal effects. Nor have we attempted, in this series of experiments, to observe the response of the cells to prolonged exposure to cytochalasin or to investigate the reversibility of its action. Chromosomes were examined in preparations from two cultures; counts and culture details are presented in Table. Diploid and tetraploid cells were most frequently found but octaploid cells were also present. In one culture, octaploid cells occurred in slightly less than % of the mitotic figures. In a culture exposed to /<g/ml of cytochalasin for 96 h, a third of the cells were tetraploid. In another culture, treated with cytochalasin at a concentration of 6 /ig/ml for 88 h, one half of the cells were tetraploid. After an extensive search, only cells with diploid, tetraploid and octaploid

6 M.A.C. Ridler and G. F. Smith chromosome complements were found. In some instances, however, two or three metaphase figures occurred in very close proximity to each other.

6 6 M.A.C. Ridler and G. F. Smith chromosome complements were found. In some instances, however, two or three metaphase figures occurred in very close proximity to each other. Some of these multiple metaphase figures showed fusion, but it was sometimes possible to define the components, the individual sets of chromosomes being differentially contracted. This effect may have been due to random confusion of nuclei with spreading, but it is probable that, in some instances, the confused metaphase figures were derived from the same parent cell. Counts of the proportion of different metaphase combinations are given in Table, and two representative chromosome configurations illustrated in Figs. 6 and 7. No morphological abnormalities of the chromosomes were detected in karyotypes prepared from representative diploid and tetraploid cells. Examples of these cells are shown in Figs. and 5. Karyotyping of octaploid cells was difficult; however, an occasional octaploid cell showed morphological peculiarities of some chromosomes (see Fig. 8). DISCUSSION It has been shown that cytochalasin inhibits motility, extrudes nuclei and prevents cytoplasmic cleavage, producing multinucleation in Earle's 'L' strain of mouse fibroblasts (Carter, 967). The present study of cultured human lymphocytes presents no data on motility, since the observations were made onfixed preparations. For the same reason, the nuclear extrusion reported here must be treated with reserve as a possible artefact, but our results confirm Carter's observation of cytoplasmic cleavage inhibition and multinucleation. The most obvious differences between the fibroblast and lymphocyte cultures were dosage effects and the production of variations in nuclear morphology. In the mouse cells, cleavage inhibition and multinucleation were manifest at a cytochalasin concentration of -5--/tg/ml, whereas very little response was evident in the lymphocytes below a concentration of 6-o /g/ml. The differences may represent a variation of sensitivity of different species to the metabolite or may be due to basic culture techniques, mouse fibroblasts being cultured as monolayers on glass while the lymphocytes are grown in suspension. Cells with one to eight nuclei were observed, which may lend some support to Carter's report of a progressive addition of one nucleus per division in the production of multinucleated cells. This is opposed to the usual synchronous mitosis where the number of nuclei would be expected to double at each division if cytoplasmic cleavage fails to occur. Carter suggests that the simplest explanation for the addition of only one nucleus per cycle is that a multinucleated cell may enter mitosis when only one of its nuclei is fully prepared and competent to divide. He has proposed the term ' pseudomitosis' for this process which appears as a synchronous mitosis, but which results in the division of only one nucleus, the remaining nuclei reverting unchanged to the interphase state. In the present study all the nuclei in dividing multinucleated cells appeared to be in the same stage of mitosis, although associated mitoses were sometimes slightly out of phase. No preparation gave evidence of cells containing a mitotic figure associated

7 Human lymphocytes and cytochalasin B 6 with an interphase nucleus. ' Pseudomitosis' may explain the production of series of nuclei of similar size and shape in Carter's cultures and in certain cells from our lymphocyte cultures, but fails to account for the multinucleated cells with nuclei of varied shape and size, and the occurrence of only diploid and tetraploid nuclei with a very occasional octaploid in our chromosome preparations. It would seem that any dividing nucleus may produce two similar daughter nuclei or a single polyploid nucleus. Furthermore, our preparations frequently produced a dividing cell, with more than a diploid set of chromosomes, in which the mitotic figures were of bizarre configuration, with large aggregates of chromosomes and small groups of only a few chromosomes (see Figs. -). The aggregates were sometimes completely separate and, at other times, joined by thick or thin chromosomal bridges (Fig. 8). The rounding up of these chromosome groups into interphase nuclei could explain our observation of variation of nuclear size and shape with incomplete separation, and nuclear bridging. Such cells appear to be inadequately equipped with mitotic apparatus to deal with the number of chromosomes present in the cell. It is possible that, in the production of a multinucleate cell, the first nuclear division can proceed normally but, thereafter, mitotic control of the chromosomes becomes disorganized. Mitosis and cleavage are known to be related and specific action of cytochalasin on the cell membrane may be responsible for all the observed anomalies. It is also possible that, in addition, the compound acts on the nuclear membrane or on some component of the mitotic apparatus. No such abnormality has been directly observed and, if present, may represent failure of replication of the mitotic apparatus resulting in inadequate control of the hyperdiploid set of chromosomes. One of the most important features of cytochalasin is its ability to inhibit cleavage without inhibiting mitosis, but much work remains to be done before its action is understood. The further investigation of this phenomenon may throw some light on the mechanisms of both normal and abnormal cellular division. The authors wish to thank Dr S. B. Carter for the cytochalasin B, Professor L. S. Penrose and Miss Helen Lang-Brown for guidance in the preparation of the manuscript, Miss Janet A. Faunch and Miss Madeleine J. Pendrey for technical assistance, and Mrs Helen Butcher for secretarial help. This work was supported by grants from The Joseph P. Kennedy Jr. Foundation, the Association for the Aid of Crippled Children and the National Society for Mentally Handicapped Children. REFERENCES ALDRIDGE, D. C, ARMSTRONG, J. J., SPEAKE, R. N. & TURNER, W. B. (967). The cytochalasins, a new class of biologically active mould metabolites. Chem. Commun., 6-7. CARTER, S. B. (967). Effects of cytochalasin on mammalian cells. Nature, Lond., 6-6. MOORHEAD, P. S., NOWELL, P. C, MELLMAN, W. J., BATTIPS, D. M. & HUNGERFORD, D. A. (i96). Chromosome preparations of leukocytes cultured from human peripheral blood. Expl Cell Res., ROTHFELS, K. H. & SIMINOVITCH, L. (958). An air-drying technique for flattening chromosomes in mammalian cells grown in vitro. Stain Technol., SMITH, G. F. & RIDLER, M. A. C. (967). The action of cytochalasin B on cultured human lymphocytes. Nature, Lond. Z6, -5. (Received 9 January 968)

8 6o M. A. C. Ridler and G. F. Smith Figs. -7. Variation in nuclear number and morphology in lymphocytes exposed to cytochalasin in culture.

9 Journal of Cell Science, Vol., No. t 5* M. A. C. RIDLER AND G. F. SMITH (Facing p. 6)

10 Figs. 8. Mitotic configurations of dividing lymphocytes from cultures exposed to cytochalasin.

11 Journal of Cell Science, Vol., No. V] M. A C. RIDLER AND G. F. SMITH

12 Fig.. Diploid cell in a chromosome preparation from a lymphocyte culture exposed to cytochalasin. Fig. 5. Tetraploid cell in a chromosome preparation from a lymphocyte culture exposed to cytochalasin. Fig. 6. Two associated diploid sets of chromosomes from a lymphocyte culture exposed to cytochalasin. Fig. 7. Associated diploid and tetraploid sets of chromosomes from a lymphocyte culture exposed to cytochalasin. Fig. 8. An octaploid cell with enlargement of some chromosomes showing structural peculiarities from a lymphocyte culture exposed to cytochalasin.

13 Journal of Cell Science, Vol., No. l 5 Sfi t ; 6 7-5/,] M\mV] M. A. C. RIDLER AND G. F. SMITH

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