Beitr. Path. Vol. I61 , 13 I-14 I (I977)
Institute of Pathology I, University of Goteborg and Department of Pathology, Sundsvalls Hospital, Sundsvall, Sweden
In vitro Effects of Cytochalasin Bon TA3 Tumor Cells W. R YD and B. HAG MAR
Summary We studied the effect of Cytochalasin B (CB) on two lines of the ascites tumor TA3 by microcinematography and scanning electron microscopy. CB induces a dose dependent and reversible cell paralysis. One fJ,g CB/ml causes a pronounced bur incomplete paralysis. The cells retain their general shape but develop numerous blebs. Ten fJ,g CB/ml causes a complete cell paralysis and a retraction of pseudopods and microvilli. When CB is removed, the cells rapidly regain their original form and motility. The recove ry process starts within minutes aft er CB removal and is almost completed in 15 min.
In several cell types, Cytochalasin (CB) induces a reversible cell paralysis (Carter, I967; Wessells et al., I97I). The cell viability is apparantly unaffected, at least for the first hours of exposure (Carter, I967; Everhart and Rubin, I974). Effects of CB on cell mobility and morphology has mainly been studied on benign cells, on established tissue culture cell lines and on monocellular organisms (Carter, I967; Wessells et al., I971; Sanger and Holtzer, I972; Everhart and Rubin, I974; Boyde and Bailey, I974; Gershenbaum et al., I 974; Godman et al., I 97 5). There are few, if any, detailed reports about CB effects on transplantable malignant cells. In the present study, we used microcinematography and scanning electron microscopy (SEM) to find out how CB affects malignant cells from a transplantable mouse tumor. We were particularly interested in CB effects on the cell surface configuration and in the time-course of restitution, when CB is eliminated from the cells. We used two in vitro growing cell lines of the ascites tumor TA3. These lines have retained their malignancy and grow intraperitoneally from doses as small as the original ascites tumor (Ryd and Hagmar, unpubl.).
I
J2 . W. Ryd and B. Hagmar
Material and Methods We used the ascites tumor TA3, which has two sister lines, TA3-Ha and TA3-St (Friberg, 1972). We obtained both lines from the Karolinska Institute, Stockholm, by courtesy of dr George Klein. When received, TA3-Ha was in i.p. transfer generation 886 and TA3-St in generation 279. The tumors have been propagated in our laboratory since then by serial transplantation in inbred syngeneic A /Sn mice and as in vitro growing cell lines. The in vitro strain of TA3-Ha originates from i.p. transfer generation 891 and the TA3-S( strain from generation 281. Both lines grow in Eagle's MEM with 20°/ 0 calf serum and antibiotics (penicillin and streptomycin). Cytochalasin B (CB) (Calbiochem) was solubilized in dimethyl sulfoxide (DMSO) (Mallinckrodt) 10 mg/ml and diluted inMEM. Influence of CB on "wound" repair in confluent tumor cell cultures
TA3 cells were harvested from culture flasks and sown into plastic dishes with 6 cm diameter (Falcon). Each dish received approximately 2 X 10 5 cells. The dishes were incubated at 37° in 5°1o humified C02 atmosphere. At confluence, 6-8 days later, a 10 mm cell-free wound was created across the dish with a rubber policeman. The dishes were fed new culture medium containing I or 10 f..Ig CB/ml. In addition to control cultures in only MEM-serum we used cultures in MEM-serum containing 0.1°/0 DMSO, corresponding to the DMSO concentration in the 10 fAg CB medium. We filmed the cell growth into the wound with a Zeiss microcine-camera on an inverted phase contrast microscope. The temperature in the camera room was 37° and there was a continous flow of 5% C02 humified air across the dish. The filming started within 10 min after wounding and continued for 36 hours. The film speed was I frame/min. and we used an objective magnifying 10 times. The corresponding magnification on the film was 27 times. The width of the wound was measured from photo copies with a final magnification of 270. From TA3-St at least 4 films were taken at each CB concentration with appropriate controls. From TA3-Ha at least one film was taken in each type of medium. Effect of CB on membrane movements, cell mobility and cell shape
TA3 cells were grown for 1-3 days in a small glass chamber made for the camera and containing 5 ml medium. The bottom is a thin coverslip on which the cells grow. The chamber is fitted with tubes, which permit medium changes while the chamber is in the camera. We used an objective with 25 times magnification and a film speed of 4 frames per minute. The cell cultures were filmed before confluence. After two hours' filming, we injected CB into the chamber to achieve a final concentration of I or 10 flg/ ml. Control cells were exposed for 0.1% DMSO. After 60 min. CB and/or DMSO exposure, the chamber was perfused with fresh medium. Sixty millilitres were used for successive perfusion, which should reduce the 10 f..Ig/ml concentration to less than 0.01 flg/ml. The time for CB injection was 30-45 sec. and for medium restitution 2-4 min. During these periods the cells were out of focus in the camera, but otherwise they could be filmed continuously before, during and after CB treatment. The filming was continued for 2 hours after restitution. Scanning electron microscopic examination of CB treated cells
The in vitro growing sublines of TA3 were cultured on round coverslips (diam. 13 mm) in plastic dishes for 1-2 days. The medium was exchanged and new culture medium containing 10 or I flg CB/ml or 0.1% DMSO was added. Sixty minutes later the dishes
In vitro Effects of Cytochalasin B on TA3 Tumor Cells . 133 were washed three times with medium and fixed in 2.5% glutharaldehyde with 0.1 I M cacodylate buffer and 0.11 M succrose (pH 7.2). Additional dishes were washed 3 times with new medium and left with this CB-free medium for 5 or 15 min. The coverslips were prepared for SEM according to th e critical point method and covered with gold in a sputter equipment (Polaron ). The specimens were observed in a Jeol SM I scanning electron microscope.
Results Influence of CB on "wound" repair in confluent cultures Table 1 summarizes the results. The highest CB concentration, 10 .fAg/ml, causes a total cell paralysis. The cells round up and retract pseudopods. The small narrowing of the wound is not due to an active cell locomotion, but rather to mitoses, which take place in spite of the treatment during the first hours of CB exposure (cf Westermark, 1973). The monolayer also loosens up a little, presumably because of the cell retraction. With CB 1 'fAg/ml the wound diminishes, but the cell morphology is quite different from that of normal cells. The cells are rounded and show few cytoplasmic protrusions. Very few of the cells move actively. DMSO exposure alone does not appreciably inhibit wound closure for St cells. For Ha cells some retarding effect is seen, but the cell form and mode of growth is not altered.
Effect of CB on membrane movements, cell mobility and cell shape Before CB is added, the cells are well spread on the glass, with pseudopod extensions and ruffling activity of the cell membrane. Addition of CB 10fAg/ml induced an immediate retraction of pseudopods. The cells round up and the ruffling stops. The cells stay immobile and retracted during the whole exposure time. Within minutes after CB elimination, however, the cells begin to regain their flattened form and after 5-20 min they begin to move by pseudopods and membrane ruffling. Upon addition of CB 1 !!g/ml the cell membrane starts to ondulate, i.e. move quickly in a wave-like fashion. Enlarged pictures of such cells show numerous small blebs with a diameter of 0.1-2.5 'fAm on the cell surfaces. The protrusions are spherical and are evenly distributed on the upper cell surface. The blebbing continues during the entire exposure, but stops within
2 min after medium restitution. DMSO 0.10/0 does not noticeably change the cell behaviour.
134 . W. Ryd and B. Hagmar Table 1. Influence of CB on wound closure in confluent cultures of TA3-St and TA3Ha. Figures give the cell growth into the wound in mm X 380. The figures for TA3-St give the arithmetic mean of 4 films and for TA3-Ha the results from I film
Hours after wounding and medium change 4 12 18
Group
-
Control
20
DMSO 0.1 Ofo
15 8
CB
I
[tg/ml
CB 10 [tg/ml
Group
Control DMSO 0.1 Ofo CB 1 [tg/ml CB 10 [tg/ml
-----------
--~
80
50
70 60
21
25
26
9
II
II
Hours after wounding and medium change 12 18 4 0 0 0 0
30 20 15 0
40
20 20 0
70
24 55 25 25 0
Scanning electron microscopic examination of CB treated cells
The surface of TA3-St cells grown for two days is shown in fig. 1. Most cells are thin and flattened and they grow without signs of contact inhibition. Short microvilli are seen both on the upper surface and on the cell margins. Ruffling is common but only few blebs are seen. A minor part of the cell population is thicker and less well spread out. These cells usually show blebs and have longer microvilli. Some cells are spherical and covered with microvilli. They may represent cells in mitosis and early Gr phase (Porter et aI., r 973). The TA3-Ha cells, growing at the same density as St, have a more heterogenic appearance (Fig. 7). Some of the cells are large and flattened but most Ha cells are thick with fusiform, polygonal or rounded cell form. Blebs and microvilli are represented on all cell types but are generally fewer on the flattened cells. There is no accumulation of blebs at the cell margin as generally seen on benign cells (Harris, r 973). Spherical cells are often covered with microvilli.
In vitro Effects of Cytochalasin B on TA 3 Tumor Cells . 135
Fig.
I
Fig. 3
Fig.
l
Fig. -+
Treatment with CB 10 .f1g/ml for 60 min induces pronounced surface changes in both TA3-St (Fig. 2) and Ha cells (Fig. 8). The effect is most evident on flattened St cells, whose pseudopodia retract and only leave thin cytoplasmic remnants, "arborization", on the glass. In Ha, however, this process is not complete, for a few cells remain partly flattened. Ruffles are absent. The number of microvilli is reduced on both the flattened and
13 6 . W. Ryd and B. Hagmar
Fi . 5
Fig. 6
Fig. 7
Fig. 8
the thicker cell types but are not totally absent as reported for other CB treated cells (Everhart and Rubin, 1974). The CB effect disappears rapidly when the drug is removed. Tumor cells washed three times in MEM and fixed I min later (i.e. less than 2 min after CB elimination) still show pronounced changes but later during the restitution the number of microvilli, blebs and ruffles increase equaling that on control cells (Figs. 3, 9).
In vitro Effects of Cytochalasin B on TA3 Tumor Cells . 137
f.ig.?
Fig.
10
Fig.
Fig.
12
II
Fig. 1-6. SEM photographs of TA3-St in culture. Fig. I, untreated cells. Fig. 2, cells exposed to CB 10 ltg/ml for 60 min. Fig. 3, I min. after and fig. 4, 15 min. after removal of CB. Fig. 5, cells exposed to I f-tg CB/ml for 60 min. Fig. 6, cells exposed to 0.1 % DMSO for 60 min. Fig. 7-12. SEM photographs of TA3-Ha in culture. Fig. 7, untreated cells. Fig. 8, cells exposed to CB 10 f-tg/ml for 60 min. Fig. 9, I min. after and fig. 10, 15 min. after removal of CB. Fig. I I, cells exposed to J.lg CB/ml for 60 min. Fig. 12, cells exposed to 0.1°/0 DMSO for 60 min.
138 . W. Ryd and B. Hagmar Fifteen minutes after CB removal most of the cells have regained a flattened form but are not as large as control cells. A few cells are still arborized (Figs. 4, 10). Tumor cells exposed to I /lg/ml CB retain their flattened form, but show numerous blebs, which are spread all over the cell surface (Figs. 5, I I). The blebs disappear as soon as CB is eliminated. DMSO does not affect the cell surface configuration of TA3-Ha and St cells (Figs. 6, I2).
Discussion The general effects of CB on our two ascites tumor lines are comparable to those on established cell lines in vitro (Carter, I967; Goldman, I972; Everhart and Rubin, I974; Godman et aI., I975). CB inhibits cell locomotion and reduces membrane movements such as ruffling and microvilli formation. If the CB concentration is adequate, in our case IO !lg/ ml, the cells round-up and are completely paralyzed during the entire period of treatment. There is obviously some variation among cell lines in the susceptibility to CB. Carter (I 967), using murine fibroblasts, obtained a complete cell paralysis with 0,5f-lg/ml. Everhart and Rubin (I974), on the other hand, found Chinese hamster cells morphologically unaffected by CB I .f-lg/ml for 2 hours. At a CB concentration of I f-lg/ml, our TA3 cells are not totally immobilized, nor do they retract all cytoplasmic protrusions. Numerous blebs appear all over the cell surface, however, and this phenomenon (zeiosis) is also seen, but to a lesser extent, at I of-lg/mI. Similar findings are described by Vesely and Boyde (I973), Gershenbaum et aI. (I974) and Godman et al. (1975). We do not interprete the blebbing as a devitalization phenomenon, for it is more pronounced at I f-lg/ ml CB than at 10 'f-lg/mI. It is also quickly reversible upon medium restitution and the cells retain their full transplantability (Hagmar and Ryd, I977). More probably CB disrupts the equilibrium at the cell periphery, a change which may be related to the cell cycle (Porter et aI., I 973; Rubin and Everhart, I 973; Boyde et aI., I 974; Gonda et aI., 1976). Everhart and Rubin (1974) found that CB caused blebs on synchronized cells in the Sand G2 phases, but not in the G1 phase. CB splits submembranously located micro filaments in different cell types (Wessells et aI., 1971; Gershenbaum et aI., 1974), either directly or by affecting the anchorage sites in the cell membrane (Parker et aI., I976).
In vitro Effects of Cytochalasin B on TA3 Tumor Cells· 139
When the micro filaments are disorganized by CB, the membrane weakens and allows herniations of the cytoplasm, which probably causes the blebbing (Godman et al., 1975). This process stops immediately upon restitution, indicating that the filaments are not destroyed but only disarranged. The quick recovery after complete cell paralysis may be interpreted in the same way. Our TA3 cells recovered within 1-2 min. after CB removal and the recovery was almost complete within 15 minutes. Thus, our malignant cells are similar in this respect to benign cells, such as those from hamster ovary (Everhart and Rubin, 1974). Little is known about the importance of an intact cell mobility for cell survival in vivo. For malignant cells, the ability to move and deform actively should be a prerequisite for an infiltrative behavior (Easty, 1967). But also in distant spread by lymph and blood vessels, the ability of the tumor cell to put out pseudopods and move actively should be important for vessel wall nidation and transgression (cf Weiss, 1967). TA3 cells, paralyzed by 10 flg CB/ml as above and injected i.v. give rise to more extrapulmonary metastases than untreated cells and cells treated with 1 'flg CB/ml (Hagmar and Ryd, 1977). In view of the present findings, we tend to ascribe the increase of extrapulmonary tumors to a favoured passage through lung vessels. Immobile rounded cells may more easily pass this first capillary filter. Perhaps the CB treated cells are also more deformable in a passive sense, a factor which may affect the metastasizibility beyond the first capillary bed (Sato and Suzuki,1975)· When CB is diluted and eliminated by the blood after injection, the cells, just as in vitro, probably regain their shape and mobility and can establish themselves as metastases in organs other than the lungs. Obviously, however, more studies are needed to elucidate the impact of cell paralysis on cell behavoir in vivo. The present in vitro studies show in summary, that CB causes various reversible morphologic changes in tumor cells. The changes are dose dependent and with 10 flg/ml we can achive a total, but still reversible, paralysis of TA3 cells. Acknowledgements The experiments were supported by grant No. 465-B75-o6X from the Swedish Cancer Society and by the Emil Anderson Fund for Medical Research. Our thanks are due
to
Mrs. Anne-Marie Malmberg, Miss Marianne Bremmer and Miss
Agnetha Jabosson for skillful technical assistance and
to
Miss Ann-Catrin Olsson for
typing the manuscript. Dr. Ulf Brunk is great fully acknowledged for his advice on electron microscopy.
14 0 . W. Ryd and B. Hagmar
References Boyde, A., Bailey, E., and Vesely, P.: SEM studies in the surface of various rat cell lines treated with cytochalasin B, colcemide and vinblastine. In: Johari and Corvin (Eds.), Scanning electron microscopy IITRI, pp. 597-604 (1974) Carter, S. B.: Effects of cytochalasins on mammalian cells. Nature 213, 261-264 (1967) Easty, G. c.: Invasion by cancer cells. In : Ambrose, E. ]., and Roe, F. ]. C. (Eds.) The biology of cancer, pp. 78-89. Van Nostrand, London (1967) Everhart, L. P., and Rubin, R. W.: Cyclic changes in the cell surface. II. The effect of cytochalasin B on the surface morphology of synchronized chinese hamster ovary cells. ]. Cell BioI. 60, 442-447 (1974) Friberg, jr., S.: Comparison of an immunoresistant and an immunosusceptible ascites subline from murine tumor TA3. I. Transplantability, morphology and some physicochemical characteristics. ]. Nat. Cancer Inst. 48, 1463-1476 (1972) Godman, G. c., Miranda, A. F., Deitch, A. D., and Tanenbaum, S. W.: Actions of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface. ]. Cell BioI. 64, 644-667 (1975) Goldman, R. D., Berg, G., Bushnell, A., Chang, C. M., Dickerman, L., Hopkins, N., Milles, M. L., Pollack, R., and Wang, E. P.: Fibrillar systems in cell mobility. In: Ciba Foundation Symposium 14, Locomotion of Tissue Cells, pp. 83-107. North Holland, Amsterdam (1973) Gonda, M. A., Aaronson, S. A., Ellmore, N., Zeve, V. H., and Nagashima, K.: Ultrastructural studies of surface features of human normal and tumor cells in tissue culture by scanning and transmission electron microscopy. ]. Nat. Cancer Inst. 56 245-263 (197 6 ) G ershenbaum, M. R., Shay, ]. W., and Porter, K. R.: The cffects of cytochalasin B on BALB /3T3 mammalian cells cultured in vitro as observed by scanning and high voltage electron microscopy. In: Johari and Corvin (Eds.), Scanning electron microscopy IITRI, pp. 589-59 6 (1974) Hagmar, B., and Ryd, W.: Tumor cell locomotion - a facror in metastasis formation? Influence of Cyrochalasin B on a tumor dissemination pattern. Int. ]. Cancer 19, 57 6-5 80 (1977) Harris, A. K.: Cell surface movements related to cell locomotion . In: Ciba Foundation Symposium 14, Locomotion of Tissue Cells, pp. 3-26. North Holland, Amsterdam (1973) Parker, C. W., Greene, W. c., and MacDonald, H. H.: Cyrochalasin binding in lymphocytes and polymorphonuclear leucocytes. Exp. Cell Res. 103,99-108 (1976) Porter, K., Prescott, D., and Frye, ]. : Changes in surface morphology of chinese hamster ovary cells during the cell cycle.]. Cell BioI. 57,815-836 (1973) Rubin, R. W., and Everhart, L. P . : The effect of cell-to-cell contact on the surface morphology of chinese hamster ovary cells. ]. Cell BioI. 57, 837-844 (1973) Sanger, ]. W., and Holtzer, H .: Cytochalasin B; effects on cell morphology, cell adhesion, and mucopolysaccharide synthesis. Proc. nat. Acad. Sci. (Wash.) 69, 253-257 (197 2) Sato, H., and Suzuki, M.: Studies on viability and deformability of tumor cells by transcapillary passage with special reference to metastasis in cancer. l!th Int. Cancer Congress, Abstracts, pp. 635-636 (1974) Vesely, P., and Boyde, A.: The significance of SEM evaluation of the cell surface for tumor cell biology. In: Johari and Corvin (Eds.), Scanning electron microscopy, IITRI pp. 69 0- 69 6 (1973)
In vitro Effects of Cytochalasin B on TA3 Tumor Cells . 14I Weiss, L.: The cell periphery, metastasis and other contact phenomena. North Holland Publishing Company, Amsterdam (I967) Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. 0., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M.: Microfilaments in cellular and developmental processes. Science 171, I35-I43 (I97I) Westermark, B.: Induction of a reversible GI block in human glia-like cells by Cytochalasin B. Exp. Cell Res. 82, 34I-350 (I973)
Received February 28, I977 . Accepted in revised form July 6, 1977
Key words: Ascites tumor - Cytochalasin B - DMSO - Microcinematography - Scanning electron microscopy Dr. W. Ryd, Institute of Pathology I, University of Goteborg, Goteborg, Sweden
10 Beitr. Path. Vol. 161
Institute of Pathology I, University of Goteborg and Department of Pathology, Sundsvalls Hospital, Sundsvall, Sweden
In vitro Effects of Cytochalasin Bon TA3 Tumor Cells W. R YD and B. HAG MAR
Summary We studied the effect of Cytochalasin B (CB) on two lines of the ascites tumor TA3 by microcinematography and scanning electron microscopy. CB induces a dose dependent and reversible cell paralysis. One fJ,g CB/ml causes a pronounced bur incomplete paralysis. The cells retain their general shape but develop numerous blebs. Ten fJ,g CB/ml causes a complete cell paralysis and a retraction of pseudopods and microvilli. When CB is removed, the cells rapidly regain their original form and motility. The recove ry process starts within minutes aft er CB removal and is almost completed in 15 min.
In several cell types, Cytochalasin (CB) induces a reversible cell paralysis (Carter, I967; Wessells et al., I97I). The cell viability is apparantly unaffected, at least for the first hours of exposure (Carter, I967; Everhart and Rubin, I974). Effects of CB on cell mobility and morphology has mainly been studied on benign cells, on established tissue culture cell lines and on monocellular organisms (Carter, I967; Wessells et al., I971; Sanger and Holtzer, I972; Everhart and Rubin, I974; Boyde and Bailey, I974; Gershenbaum et al., I 974; Godman et al., I 97 5). There are few, if any, detailed reports about CB effects on transplantable malignant cells. In the present study, we used microcinematography and scanning electron microscopy (SEM) to find out how CB affects malignant cells from a transplantable mouse tumor. We were particularly interested in CB effects on the cell surface configuration and in the time-course of restitution, when CB is eliminated from the cells. We used two in vitro growing cell lines of the ascites tumor TA3. These lines have retained their malignancy and grow intraperitoneally from doses as small as the original ascites tumor (Ryd and Hagmar, unpubl.).
I
J2 . W. Ryd and B. Hagmar
Material and Methods We used the ascites tumor TA3, which has two sister lines, TA3-Ha and TA3-St (Friberg, 1972). We obtained both lines from the Karolinska Institute, Stockholm, by courtesy of dr George Klein. When received, TA3-Ha was in i.p. transfer generation 886 and TA3-St in generation 279. The tumors have been propagated in our laboratory since then by serial transplantation in inbred syngeneic A /Sn mice and as in vitro growing cell lines. The in vitro strain of TA3-Ha originates from i.p. transfer generation 891 and the TA3-S( strain from generation 281. Both lines grow in Eagle's MEM with 20°/ 0 calf serum and antibiotics (penicillin and streptomycin). Cytochalasin B (CB) (Calbiochem) was solubilized in dimethyl sulfoxide (DMSO) (Mallinckrodt) 10 mg/ml and diluted inMEM. Influence of CB on "wound" repair in confluent tumor cell cultures
TA3 cells were harvested from culture flasks and sown into plastic dishes with 6 cm diameter (Falcon). Each dish received approximately 2 X 10 5 cells. The dishes were incubated at 37° in 5°1o humified C02 atmosphere. At confluence, 6-8 days later, a 10 mm cell-free wound was created across the dish with a rubber policeman. The dishes were fed new culture medium containing I or 10 f..Ig CB/ml. In addition to control cultures in only MEM-serum we used cultures in MEM-serum containing 0.1°/0 DMSO, corresponding to the DMSO concentration in the 10 fAg CB medium. We filmed the cell growth into the wound with a Zeiss microcine-camera on an inverted phase contrast microscope. The temperature in the camera room was 37° and there was a continous flow of 5% C02 humified air across the dish. The filming started within 10 min after wounding and continued for 36 hours. The film speed was I frame/min. and we used an objective magnifying 10 times. The corresponding magnification on the film was 27 times. The width of the wound was measured from photo copies with a final magnification of 270. From TA3-St at least 4 films were taken at each CB concentration with appropriate controls. From TA3-Ha at least one film was taken in each type of medium. Effect of CB on membrane movements, cell mobility and cell shape
TA3 cells were grown for 1-3 days in a small glass chamber made for the camera and containing 5 ml medium. The bottom is a thin coverslip on which the cells grow. The chamber is fitted with tubes, which permit medium changes while the chamber is in the camera. We used an objective with 25 times magnification and a film speed of 4 frames per minute. The cell cultures were filmed before confluence. After two hours' filming, we injected CB into the chamber to achieve a final concentration of I or 10 flg/ ml. Control cells were exposed for 0.1% DMSO. After 60 min. CB and/or DMSO exposure, the chamber was perfused with fresh medium. Sixty millilitres were used for successive perfusion, which should reduce the 10 f..Ig/ml concentration to less than 0.01 flg/ml. The time for CB injection was 30-45 sec. and for medium restitution 2-4 min. During these periods the cells were out of focus in the camera, but otherwise they could be filmed continuously before, during and after CB treatment. The filming was continued for 2 hours after restitution. Scanning electron microscopic examination of CB treated cells
The in vitro growing sublines of TA3 were cultured on round coverslips (diam. 13 mm) in plastic dishes for 1-2 days. The medium was exchanged and new culture medium containing 10 or I flg CB/ml or 0.1% DMSO was added. Sixty minutes later the dishes
In vitro Effects of Cytochalasin B on TA3 Tumor Cells . 133 were washed three times with medium and fixed in 2.5% glutharaldehyde with 0.1 I M cacodylate buffer and 0.11 M succrose (pH 7.2). Additional dishes were washed 3 times with new medium and left with this CB-free medium for 5 or 15 min. The coverslips were prepared for SEM according to th e critical point method and covered with gold in a sputter equipment (Polaron ). The specimens were observed in a Jeol SM I scanning electron microscope.
Results Influence of CB on "wound" repair in confluent cultures Table 1 summarizes the results. The highest CB concentration, 10 .fAg/ml, causes a total cell paralysis. The cells round up and retract pseudopods. The small narrowing of the wound is not due to an active cell locomotion, but rather to mitoses, which take place in spite of the treatment during the first hours of CB exposure (cf Westermark, 1973). The monolayer also loosens up a little, presumably because of the cell retraction. With CB 1 'fAg/ml the wound diminishes, but the cell morphology is quite different from that of normal cells. The cells are rounded and show few cytoplasmic protrusions. Very few of the cells move actively. DMSO exposure alone does not appreciably inhibit wound closure for St cells. For Ha cells some retarding effect is seen, but the cell form and mode of growth is not altered.
Effect of CB on membrane movements, cell mobility and cell shape Before CB is added, the cells are well spread on the glass, with pseudopod extensions and ruffling activity of the cell membrane. Addition of CB 10fAg/ml induced an immediate retraction of pseudopods. The cells round up and the ruffling stops. The cells stay immobile and retracted during the whole exposure time. Within minutes after CB elimination, however, the cells begin to regain their flattened form and after 5-20 min they begin to move by pseudopods and membrane ruffling. Upon addition of CB 1 !!g/ml the cell membrane starts to ondulate, i.e. move quickly in a wave-like fashion. Enlarged pictures of such cells show numerous small blebs with a diameter of 0.1-2.5 'fAm on the cell surfaces. The protrusions are spherical and are evenly distributed on the upper cell surface. The blebbing continues during the entire exposure, but stops within
2 min after medium restitution. DMSO 0.10/0 does not noticeably change the cell behaviour.
134 . W. Ryd and B. Hagmar Table 1. Influence of CB on wound closure in confluent cultures of TA3-St and TA3Ha. Figures give the cell growth into the wound in mm X 380. The figures for TA3-St give the arithmetic mean of 4 films and for TA3-Ha the results from I film
Hours after wounding and medium change 4 12 18
Group
-
Control
20
DMSO 0.1 Ofo
15 8
CB
I
[tg/ml
CB 10 [tg/ml
Group
Control DMSO 0.1 Ofo CB 1 [tg/ml CB 10 [tg/ml
-----------
--~
80
50
70 60
21
25
26
9
II
II
Hours after wounding and medium change 12 18 4 0 0 0 0
30 20 15 0
40
20 20 0
70
24 55 25 25 0
Scanning electron microscopic examination of CB treated cells
The surface of TA3-St cells grown for two days is shown in fig. 1. Most cells are thin and flattened and they grow without signs of contact inhibition. Short microvilli are seen both on the upper surface and on the cell margins. Ruffling is common but only few blebs are seen. A minor part of the cell population is thicker and less well spread out. These cells usually show blebs and have longer microvilli. Some cells are spherical and covered with microvilli. They may represent cells in mitosis and early Gr phase (Porter et aI., r 973). The TA3-Ha cells, growing at the same density as St, have a more heterogenic appearance (Fig. 7). Some of the cells are large and flattened but most Ha cells are thick with fusiform, polygonal or rounded cell form. Blebs and microvilli are represented on all cell types but are generally fewer on the flattened cells. There is no accumulation of blebs at the cell margin as generally seen on benign cells (Harris, r 973). Spherical cells are often covered with microvilli.
In vitro Effects of Cytochalasin B on TA 3 Tumor Cells . 135
Fig.
I
Fig. 3
Fig.
l
Fig. -+
Treatment with CB 10 .f1g/ml for 60 min induces pronounced surface changes in both TA3-St (Fig. 2) and Ha cells (Fig. 8). The effect is most evident on flattened St cells, whose pseudopodia retract and only leave thin cytoplasmic remnants, "arborization", on the glass. In Ha, however, this process is not complete, for a few cells remain partly flattened. Ruffles are absent. The number of microvilli is reduced on both the flattened and
13 6 . W. Ryd and B. Hagmar
Fi . 5
Fig. 6
Fig. 7
Fig. 8
the thicker cell types but are not totally absent as reported for other CB treated cells (Everhart and Rubin, 1974). The CB effect disappears rapidly when the drug is removed. Tumor cells washed three times in MEM and fixed I min later (i.e. less than 2 min after CB elimination) still show pronounced changes but later during the restitution the number of microvilli, blebs and ruffles increase equaling that on control cells (Figs. 3, 9).
In vitro Effects of Cytochalasin B on TA3 Tumor Cells . 137
f.ig.?
Fig.
10
Fig.
Fig.
12
II
Fig. 1-6. SEM photographs of TA3-St in culture. Fig. I, untreated cells. Fig. 2, cells exposed to CB 10 ltg/ml for 60 min. Fig. 3, I min. after and fig. 4, 15 min. after removal of CB. Fig. 5, cells exposed to I f-tg CB/ml for 60 min. Fig. 6, cells exposed to 0.1 % DMSO for 60 min. Fig. 7-12. SEM photographs of TA3-Ha in culture. Fig. 7, untreated cells. Fig. 8, cells exposed to CB 10 f-tg/ml for 60 min. Fig. 9, I min. after and fig. 10, 15 min. after removal of CB. Fig. I I, cells exposed to J.lg CB/ml for 60 min. Fig. 12, cells exposed to 0.1°/0 DMSO for 60 min.
138 . W. Ryd and B. Hagmar Fifteen minutes after CB removal most of the cells have regained a flattened form but are not as large as control cells. A few cells are still arborized (Figs. 4, 10). Tumor cells exposed to I /lg/ml CB retain their flattened form, but show numerous blebs, which are spread all over the cell surface (Figs. 5, I I). The blebs disappear as soon as CB is eliminated. DMSO does not affect the cell surface configuration of TA3-Ha and St cells (Figs. 6, I2).
Discussion The general effects of CB on our two ascites tumor lines are comparable to those on established cell lines in vitro (Carter, I967; Goldman, I972; Everhart and Rubin, I974; Godman et aI., I975). CB inhibits cell locomotion and reduces membrane movements such as ruffling and microvilli formation. If the CB concentration is adequate, in our case IO !lg/ ml, the cells round-up and are completely paralyzed during the entire period of treatment. There is obviously some variation among cell lines in the susceptibility to CB. Carter (I 967), using murine fibroblasts, obtained a complete cell paralysis with 0,5f-lg/ml. Everhart and Rubin (I974), on the other hand, found Chinese hamster cells morphologically unaffected by CB I .f-lg/ml for 2 hours. At a CB concentration of I f-lg/ml, our TA3 cells are not totally immobilized, nor do they retract all cytoplasmic protrusions. Numerous blebs appear all over the cell surface, however, and this phenomenon (zeiosis) is also seen, but to a lesser extent, at I of-lg/mI. Similar findings are described by Vesely and Boyde (I973), Gershenbaum et aI. (I974) and Godman et al. (1975). We do not interprete the blebbing as a devitalization phenomenon, for it is more pronounced at I f-lg/ ml CB than at 10 'f-lg/mI. It is also quickly reversible upon medium restitution and the cells retain their full transplantability (Hagmar and Ryd, I977). More probably CB disrupts the equilibrium at the cell periphery, a change which may be related to the cell cycle (Porter et aI., I 973; Rubin and Everhart, I 973; Boyde et aI., I 974; Gonda et aI., 1976). Everhart and Rubin (1974) found that CB caused blebs on synchronized cells in the Sand G2 phases, but not in the G1 phase. CB splits submembranously located micro filaments in different cell types (Wessells et aI., 1971; Gershenbaum et aI., 1974), either directly or by affecting the anchorage sites in the cell membrane (Parker et aI., I976).
In vitro Effects of Cytochalasin B on TA3 Tumor Cells· 139
When the micro filaments are disorganized by CB, the membrane weakens and allows herniations of the cytoplasm, which probably causes the blebbing (Godman et al., 1975). This process stops immediately upon restitution, indicating that the filaments are not destroyed but only disarranged. The quick recovery after complete cell paralysis may be interpreted in the same way. Our TA3 cells recovered within 1-2 min. after CB removal and the recovery was almost complete within 15 minutes. Thus, our malignant cells are similar in this respect to benign cells, such as those from hamster ovary (Everhart and Rubin, 1974). Little is known about the importance of an intact cell mobility for cell survival in vivo. For malignant cells, the ability to move and deform actively should be a prerequisite for an infiltrative behavior (Easty, 1967). But also in distant spread by lymph and blood vessels, the ability of the tumor cell to put out pseudopods and move actively should be important for vessel wall nidation and transgression (cf Weiss, 1967). TA3 cells, paralyzed by 10 flg CB/ml as above and injected i.v. give rise to more extrapulmonary metastases than untreated cells and cells treated with 1 'flg CB/ml (Hagmar and Ryd, 1977). In view of the present findings, we tend to ascribe the increase of extrapulmonary tumors to a favoured passage through lung vessels. Immobile rounded cells may more easily pass this first capillary filter. Perhaps the CB treated cells are also more deformable in a passive sense, a factor which may affect the metastasizibility beyond the first capillary bed (Sato and Suzuki,1975)· When CB is diluted and eliminated by the blood after injection, the cells, just as in vitro, probably regain their shape and mobility and can establish themselves as metastases in organs other than the lungs. Obviously, however, more studies are needed to elucidate the impact of cell paralysis on cell behavoir in vivo. The present in vitro studies show in summary, that CB causes various reversible morphologic changes in tumor cells. The changes are dose dependent and with 10 flg/ml we can achive a total, but still reversible, paralysis of TA3 cells. Acknowledgements The experiments were supported by grant No. 465-B75-o6X from the Swedish Cancer Society and by the Emil Anderson Fund for Medical Research. Our thanks are due
to
Mrs. Anne-Marie Malmberg, Miss Marianne Bremmer and Miss
Agnetha Jabosson for skillful technical assistance and
to
Miss Ann-Catrin Olsson for
typing the manuscript. Dr. Ulf Brunk is great fully acknowledged for his advice on electron microscopy.
14 0 . W. Ryd and B. Hagmar
References Boyde, A., Bailey, E., and Vesely, P.: SEM studies in the surface of various rat cell lines treated with cytochalasin B, colcemide and vinblastine. In: Johari and Corvin (Eds.), Scanning electron microscopy IITRI, pp. 597-604 (1974) Carter, S. B.: Effects of cytochalasins on mammalian cells. Nature 213, 261-264 (1967) Easty, G. c.: Invasion by cancer cells. In : Ambrose, E. ]., and Roe, F. ]. C. (Eds.) The biology of cancer, pp. 78-89. Van Nostrand, London (1967) Everhart, L. P., and Rubin, R. W.: Cyclic changes in the cell surface. II. The effect of cytochalasin B on the surface morphology of synchronized chinese hamster ovary cells. ]. Cell BioI. 60, 442-447 (1974) Friberg, jr., S.: Comparison of an immunoresistant and an immunosusceptible ascites subline from murine tumor TA3. I. Transplantability, morphology and some physicochemical characteristics. ]. Nat. Cancer Inst. 48, 1463-1476 (1972) Godman, G. c., Miranda, A. F., Deitch, A. D., and Tanenbaum, S. W.: Actions of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface. ]. Cell BioI. 64, 644-667 (1975) Goldman, R. D., Berg, G., Bushnell, A., Chang, C. M., Dickerman, L., Hopkins, N., Milles, M. L., Pollack, R., and Wang, E. P.: Fibrillar systems in cell mobility. In: Ciba Foundation Symposium 14, Locomotion of Tissue Cells, pp. 83-107. North Holland, Amsterdam (1973) Gonda, M. A., Aaronson, S. A., Ellmore, N., Zeve, V. H., and Nagashima, K.: Ultrastructural studies of surface features of human normal and tumor cells in tissue culture by scanning and transmission electron microscopy. ]. Nat. Cancer Inst. 56 245-263 (197 6 ) G ershenbaum, M. R., Shay, ]. W., and Porter, K. R.: The cffects of cytochalasin B on BALB /3T3 mammalian cells cultured in vitro as observed by scanning and high voltage electron microscopy. In: Johari and Corvin (Eds.), Scanning electron microscopy IITRI, pp. 589-59 6 (1974) Hagmar, B., and Ryd, W.: Tumor cell locomotion - a facror in metastasis formation? Influence of Cyrochalasin B on a tumor dissemination pattern. Int. ]. Cancer 19, 57 6-5 80 (1977) Harris, A. K.: Cell surface movements related to cell locomotion . In: Ciba Foundation Symposium 14, Locomotion of Tissue Cells, pp. 3-26. North Holland, Amsterdam (1973) Parker, C. W., Greene, W. c., and MacDonald, H. H.: Cyrochalasin binding in lymphocytes and polymorphonuclear leucocytes. Exp. Cell Res. 103,99-108 (1976) Porter, K., Prescott, D., and Frye, ]. : Changes in surface morphology of chinese hamster ovary cells during the cell cycle.]. Cell BioI. 57,815-836 (1973) Rubin, R. W., and Everhart, L. P . : The effect of cell-to-cell contact on the surface morphology of chinese hamster ovary cells. ]. Cell BioI. 57, 837-844 (1973) Sanger, ]. W., and Holtzer, H .: Cytochalasin B; effects on cell morphology, cell adhesion, and mucopolysaccharide synthesis. Proc. nat. Acad. Sci. (Wash.) 69, 253-257 (197 2) Sato, H., and Suzuki, M.: Studies on viability and deformability of tumor cells by transcapillary passage with special reference to metastasis in cancer. l!th Int. Cancer Congress, Abstracts, pp. 635-636 (1974) Vesely, P., and Boyde, A.: The significance of SEM evaluation of the cell surface for tumor cell biology. In: Johari and Corvin (Eds.), Scanning electron microscopy, IITRI pp. 69 0- 69 6 (1973)
In vitro Effects of Cytochalasin B on TA3 Tumor Cells . 14I Weiss, L.: The cell periphery, metastasis and other contact phenomena. North Holland Publishing Company, Amsterdam (I967) Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. 0., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M.: Microfilaments in cellular and developmental processes. Science 171, I35-I43 (I97I) Westermark, B.: Induction of a reversible GI block in human glia-like cells by Cytochalasin B. Exp. Cell Res. 82, 34I-350 (I973)
Received February 28, I977 . Accepted in revised form July 6, 1977
Key words: Ascites tumor - Cytochalasin B - DMSO - Microcinematography - Scanning electron microscopy Dr. W. Ryd, Institute of Pathology I, University of Goteborg, Goteborg, Sweden
10 Beitr. Path. Vol. 161
