Effect of Microtubule Disorganizing Or Overstabilizing Drugs on the Proliferation of Rat 3T3 Cells and Their Virally Induced Transformed Derivatives

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(CANCER RESEARCH 48. 4892-4896, September I, 1988] Effect of Microtubule Disorganizing or Overstabilizing Drugs on the Proliferation of Rat 3T3 Cells and Their Virally Induced Transformed Derivatives

Nodisi Kamech and Roland Seif Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France

ABSTRACT some transformed cells (25). The correlation, however, between transformation and alteration of the microtubule network has Drugs that disorganize or overstabilize cytoplasmic microtubules (col- not been endorsed by other investigators (26-29). It was argued chicine, vinblastine, griseofulvin, or taxol) can at certain concentrations that the differences in microtubule organization between nor totally block proliferation of SV40 and polyoma virus transformants with mal and transformed cells are only apparent and stem from only a minimal effect on the proliferation of the parental rat 3T3 cells. This difference in sensitivity is not due to a more active drug uptake by changes in the shape and degree of spreading of transformed transformed cells. Examination of cytoplasmic microtubules in actively cells. This fact renders difficult the documentation of the mi proliferating normal or transformed cells reveals two categories in each crotubule network. case: cells with microtubules and cells without distinct microtubules. The We have examined the effect of drugs that disorganize or proportion of cells without distinct microtubules did not differ much stabilize microtubules on the proliferation of rat 3T3 cells and between normal and transformed cells. However, transformed cells with on their transformed derivatives induced by polyoma virus or a clear microtubule network appear to have fewer microtubules than SV40. The SV40 transformants used in this study display all normal cells. This may contribute to the higher sensitivity of transformed the growth properties typical of maximal transformants but cells. These results render even more rational the use of antimicrotubule cannot be distinguished easily from the parental 3T3 cells on drugs in cancer chemotherapy. morphological grounds or on the basis of growth rate. They maintain the typical fibroblasta- elongated and bipolar shape INTRODUCTION and are neither smaller nor more retracted than the parental cells. The polyoma virus transformants used in this study are Microtubules, intermediate filaments, and actin microfila- more "progressed" in the pathway of transformation than the ments are the primary components of the cytoskeleton in mam SV40 transformants. They proliferate faster under conditions malian fibroblasts grown in culture. They are networks of restrictive for normal cell proliferation and have more profound cytoplasmic fibers that can be visualized by indirect immuno- membrane alterations (30, 31). fluorescence using antibodies directed against the protein mon omer that constitutes the fiber. MATERIALS AND METHODS Microfilaments are clearly implicated in the processes that ensure spreading and adhesion of the cell to the substratum and Cells, Medium, Chemicals, and Antibodies. The rat 3T3 cells and their derivatives transformed by polyoma virus or SV40 were all de in certain cell surface movements, namely the development of scribed previously (30, 31). They were grown at 33Â°Cin Dulbecco's lame lias and ruffling (1, 2). They also have a role in cell translocation and in the mobility of surface receptors (3-9). modified eagle medium supplemented with 10% newborn calf serum. Vinblastine (M, 909) and colchicine (M, 399) were purchased from Microtubules also play crucial roles in fundamental cell func Sigma. Griseofulvin (M, 352) was from Aldrich and taxol (M, 820) was tions, namely determination and maintenance of cell shape and a gift from Dr. Daniel Guenard, Institut de Chimie des Substances intracellular architecture, transport of vesicles, cell transloca Naturelles, Centre National de la Recherche Scientifique, Gif-sur- tion, and surface movements like extension and retraction of Yvette, France. Stock solutions were all in dimethyl sulfoxide. Pure processes, blebbing, and mobility of surface receptors. They are anti-tubulin sheep antibodies were prepared as described previously also essential elements of the mitotic spindle (5-8, 10-14). (32) and Texas red conjugated goat IgGs directed against sheep IgGs Microtubules may also have an additional major role; they were from Amersham. Tritium labeled vinblastine (10 Ci/mmol) was appear to be implicated in the control of cell proliferation and also from Amersham. Cell Proliferation. Actively proliferating normal or transformed cells in the mechanisms of oncogenic cell transformation. Indeed, a (10* cells/25 cm2) were treated with the drug at the indicated concen transient depolymerization of microtubules by drugs that pro trations; control cells were treated with dimethyl sulfoxide (1/1000 mote microtubule disassembly is sufficient to induce DNA dilution). The extent of cell proliferation was determined by calculating synthesis and division in resting cells cultivated without serum the logarithm of the ratio A3/M), the number of cells after 3 days of (15). Numerous reports had previously indicated that in the incubation in the presence of the drug over the number of cells on the presence of serum, microtubule depolymerization induces DNA day the drug was added. synthesis in resting cells or enhances the effect of various Uptake of Labeled Vinblastine. Actively proliferating normal or mitogens (3, 16-20). Furthermore, it was shown that in vivo transformed cells (IO5 cells/25 cm2) were incubated at 33Â°Cfrom 0 to 180 min with 25 x 10~8M tritium labeled vinblastine. At the end of the stabilization of microtubules by taxol inhibits the mitogenic effect of epidermal growth factor and thrombin on resting cells incubation period, the cells in the flask were washed four times with 10 ml PBS (phosphate buffered saline) and lysed with 2 ml l N sodium (21). Known tumor promoters like griseofulvin (22) disrupt hydroxide and the lysate was incubated for 30 min at 60Â°C.A 200-iil microtubules both in vivo and in vitro (23), and drugs like aliquot was used to determine the total protein content and the rest vinblastine, which prevent tubulin polymerization into micro was used to determine the total radioactivity retained by the cells. All tubules, increase, like tumor promoters, the frequency of cell points were in triplicate. transformation induced by polyoma virus (24). Indirect Immunofluorescence. The cells were fixed in 3.7% formal The network of microtubules was reported to be altered in dehyde for 30 min, permeabilized with 0.1% Triton for 5 min, and incubated with sheep anti-tubulin antibodies for 30 min and then with Received 4/7/86; revised 7/15/86. 3/21/88; accepted 6/1/88. goat anti-sheep antibodies for 30 min. After extensive washing the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in cytoplasmic microtubules were viewed by epifluorescence using a Zeiss accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Universal microscope. 4892

RESULTS

100h Effect of Microtubule Disrupting Agents on Cell Proliferation. Proliferating rat 3T3 cells, SV40 transformants (SV3T3) or TAXOL polyoma virus transformants (PY3T3) were treated with various â€¢3T3 doses of vinblastine (5 to 50 x 10~8 M), colchicine (1 to 6 x â€¢SV3T3 10~7 M), or griseofulvin (5 to 50 x 10~6 M) and their prolifera â€¢PY3T3 tion was assessed 3 days later (Fig. 1). In the case of SV40 or 50 polyoma virus transformants a 50% inhibition of proliferation cc was observed at 7.5 x 10~8 M vinblastine, 1.5 x 10~7M colchi LU cine, and 12.5 x IO"6 M griseofulvin. In the case of normal cells o a 50% inhibition of proliferation required higher doses: 27.5 x oc 10~8 M vinblastine; 2.5 x 10~7 M colchicine; and 30 x 10~6 M CL griseofulvin. Moreover, proliferation of SV40 or polyoma virus 13579 transformants was totally blocked at 15 x 10~8 M vinblastine, 3 X 10~7 M colchicine, and 15 x 10~6 M griseofulvin. At those CONCENTRATION (xlOw) doses the normal cells could still proliferate well during 7 Fig. 2. Proliferation of normal (3T3) and transformed cells (SV3T3, PY3T3) in the presence of various doses of taxol. additional days; the medium containing the drug was renewed every 3 days. Normal cell proliferation was blocked only with ation occurred at 5 x 10~7 M taxol. At this dose the normal 50 x 10~8M vinblastine; 7 x 10~7 M colchicine; and 40 x 10~6 cells could still proliferate well during 7 additional days. A total M griseofulvin. inhibitory effect on normal cell proliferation required a dose of Six additional transformed cell lines induced either by po 10 x 10~7M taxol. Six additional transformed cell lines induced lyoma virus or SV40 (30) were assayed for their ability to by either polyoma virus or SV40 (30) were similarly sensitive proliferate in the presence of microtubule disrupting agents. None could proliferate in the presence of 15 x 10~8 M vinblas to the inhibitory effect of taxol. Thus transformed cells were tine, 3 x 10~7 colchicine, or 15 x 10~6 M griseofulvin. Thus also more sensitive than normal cells to taxol, a microtubule stabilizing agent. transformants derived from rat 3T3 appear to be more sensitive Uptake of Vinblastine by Normal and Transformed Cells. The than their parental cells to the inhibitory effect on proliferation possibility that transformed cells are more sensitive to vinblas of vinblastine, colchicine, or griseofulvin. All three agents act tine because of a more active transport of this drug across the directly on tubulin and interefere with the autopolymerization membrane was considered. process that generates microtubules (11). Transformed cells are usually reported to take up metabolites Effect of Taxol on Cell Proliferation. Taxol is an agent that from the medium at a higher rate than normal cells. This overstabilizes microtubules (33, 34). It considerably diminishes difference, however, usually comes from the fact that crowded the critical concentration of free tubulin monomer required for but actively proliferating transformed cells are compared to polymerization (35). Proliferating normal or transformed cells were treated with taxol at doses ranging from 1 to 10 x 10~7M crowded but mostly resting normal cells. When actively prolif erating normal and transformed cells are compared, differences (Fig. 2). A 50% inhibition of proliferation required a dose of 3 x 10~7 M for the transformed cells and a dose of 5 X 10~7 M in metabolite uptake usually disappear. Moreover, proliferating normal cells compared to resting cells have a more active for normal cells. Total inhibition of transformed cells prolifer- metabolism and uptake of metabolites. Radiolabeled vinblastine was therefore added to a final con centration of 25 x 10~8 M to the medium of actively prolifer 100 ating normal or SV40 transformed cells. Fig. 3 shows that normal and transformed cells transport vinblastine at a similar rate and accumulate this drug to a similar level. Differences in sensitivity towards vinblastine do not therefore come from an increased poisoning of transformed cells due to a more active transport. Morphology and Microtubules in Proliferating Normal and Transformed Cells. The normal rat 3T3 cells used in this study are regular fibroblasts which when sparse are mostly elongated, 100 GRISEOFULVIN bipolar, and flat (Fig. 4A). The SV40 induced transformed derivatives do not differ much from their parental cells, they f remain mostly elongated and bipolar with only narrower cell o extensions and are well spread on the substrate (Fig. 4B). On K 50 < the other hand, the polyoma virus induced transformants are multipolar but are still flat and well spread on the substrate (Fig. 4C). Previous reports had indicated that the microtubule o E networks are less well organized in transformed cells. This view, o. 25 45 however, was seriously challenged by other investigators on the x10Â°M grounds that the differences were only apparent and resulted CONCENTRATION from the fact that transformed cells have less well spread Fig. 1. Proliferation of normal (3T3) and transformed cells (SV3T3, PY3T3) cytoplasm (28). in the presence of various doses of vinblastine (a), colchicine (o), or griseofulvin (c). Actively proliferating cells (10'/25 cm2) were treated with the drug at various The organization of cytoplasmic microtubules was examined concentrations. All the cells were counted after 3 days. by indirect immunofluorescence in actively proliferating normal 4893

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O BO 120 180 minutes Fig. 3. Uptake of tritium labeled vinblastine by normal (A) and SV40 trans formed cells (SV3T3) (B). Actively proliferating cells (10'/25 cm2) were incubated with 'H labeled vinblastine. Error bars vary from 4 to 16%. or SV40 transformed cells (Fig. 5). Two categories could be clearly distinguished among normal cells (Fig. 5; Table 1): cells with numerous microtubules; and cells without distinct microtubules with an intense diffuse background of free tubulin. The same results were obtained using the procedure described by Osborn and Weber (28) where cell fixation and extraction are made in a microtubule stabilizing buffer. In the case of SV40 transformants, two categories were also clearly distinguished (Fig. 5; Table 1): cells with microtubules; and cells without distinct microtubules. The proportion of cells with microtubules was similar in normal and transformed cells. There is a slight shift in transformed cell population toward cells without distinct microtubules. The immunofluorescent Fig. 4. Morphology of normal and transformed cells. /. normal 3T3 cells; B, SV40 transformants; C, polyoma virus transformants. Bar, 30 pm. visualization of these cells also suggested that each transformed cell that had distinct microtubules appeared to have fewer microtubules than normal cells. eration of transformed cells with only a minimal effect on Microtubules in Resting Cells. When the cells were allowed normal cell proliferation. This difference is not due to a more to reach confluence in 10% serum or were incubated in 1% active uptake of these drugs by transformed cells. Examination serum for 2 days while still isolated, the great majority (>95%) of cytoplasmic microtubules in actively proliferating normal were found to have a well developed microtubule network (Fig. and transformed cells shows that the transformed cells do have 6; Table 1). This strongly argues (a) against the presence of two microtubules arranged in networks, but the number of micro distinct deniable populations and (h) against the possibility of tubules per cell is reduced compared to normal cells. The a fixation artifact. The data, however, appear to favor the reduced number of microtubules per cell may contribute to the possibility that microtubules are massively disorganized during a certain period of the cell cycle which lasts longer than the higher sensitivity of transformed cells to microtubule disrupting immediate premitosis phase. or overstabilizing drugs. Previous reports had indicated that the number, the organi zation, and the integrity of microtubules are different in trans DISCUSSION formed cells (25). The validity of these findings was later This paper reports that drugs which disorganize or stabilize doubted. The following arguments were raised (28). When large microtubules can at certain concentrations totally block prolif- and well spread normal cells are compared to small and re- 4894

Fig. 5. Cyloplasmic microtubules in proliferating normal or SV40 transformed cells. A, B, normal cells; C, D, SV40 transformed cells. Bar, 15 Â¡im.

Table 1 Cytoplasmic microtubules in actively proliferating normal, SV40 be interpreted as diffuse fluorescence caused by free tubulin. To transformed, and quiescent rat 3T3 cells avoid this problem, we have used transformants that do not with distinct without distinct differ from the normal cells by their shape or degree of spread CellsProliferating microtubules(%)5042Â°>95Cellsmicrotubules(%)46 ing and found that they apparently contain fewer microtubules. 3T3 Proliferating SV3T3 61 We also report that in an actively proliferating normal cell Quiescent 3T3Cells <5 population, not all cells have a well developed cytoplasmic Â°Each transformed cell had fewer microtubules than a normal cell (Fig. 5, A microtubule network. This is most probably a cell cycle related and C). phenomenon, not a fixation artifact, since the great majority of cells resting at confluence or in low serum medium have a well tracted transformed cells it is difficult to rule out that the developed microtubule network. alteration of the microtubules network is only apparent and The difference in sensitivity towards vinblastine or taxol, results from the difference in shape and degree of cell spreading. between normal and transformed cells is not related to their Indeed, in large and well spread normal cells most microtubules rate of proliferation. Indeed SV40 transformants do not differ are present and can be photographed in the same focal plane, from normal cells on the basis of growth rate (31 ). Furthermore, whereas in retracted cells, microtubules are located in many polyoma virus transformants proliferate faster than SV40 trans different focal planes. When such cells are photographed in a formants (30) yet they have an equal sensitivity. single focal plane, only some microtubules appear, and the out When the normal rat 3T3 cells are treated with limited of focus fluorescence due to the remaining microtubules may amounts of vinblastine that affect only slightly their prolifera- 4895

5. Edelman, G. Surface modulation in cell recognition and cell growth. Science (Wash. DC). 192: 218-226, 1976. 6. Goldman, R. D. The role of three cytoplasmic fibers in BHK 21 cell motility. I. Microtubules and the effect of colchicine. J. Cell Biol., 51: 752-762, 1971. 7. Goldman, R. D., and Knife, D. M. Functions of cytoplasmic fibers in non muscle cell motility. Cold Spring Harbor Symp. Quant. Biol., 37: 523-534, 1972. 8. Goldman, R. D., Pollard, T., and Rosenbaum, J. (eds.). Cell Motility. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1976. 9. Lazarides, E., and Weber, K. Actin antibody: the specific visualization of actin filaments in non muscle cells. Proc. Nati. Acad. Sci. USA, 71: 2268- 2272, 1974. 10. Freed, J. J.. and Lebowitz, M. M. The association of a class of saltatory movements with microtubules in cultured cells. J. Cell Biol., 45: 335-354, 1970. 11. Olmsted, J.. and Borisy, G. Microtubules. Annu. Rev. Biochem., 42: 634- 647. 1973. 12. Osborn, M., and Weber, K. Tubulin specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum. Exp. Cell Res., 103: 331-340, 1976. 13. Vasiliev, J., Gelfand, L, Domina, L., Ivanova, O., Komm, S., and Olshev- skaja, L. Effect of Colcemid on the locomotory behavior of fibroblasts. J. Embryol. Exp. Morphol., 24: 625-640, 1970. 14. Weber, K., Pollack, R., and Bibring, T. Antibody against tubulin: the specific visualization of cytoplasmic microtubules in tissue culture cells. Proc. Nati. Acad. Sci. USA, 72: 459-463, 1975. 15. Crossin, K. L., and Carney, D. H. Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis. Cell, 23: 61-71, 1981. 16. McClain, D. A., and Edelman, G. M. Density-dependent stimulation and Fig. 6. Cytoplasmic microtubules in resting normal cells. Bar, 15 >

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Nédia Kamech and Roland Seif

Cancer Res 1988;48:4892-4896.

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