Proc. Nat. Acad. Sci. USA
Vol. 72, No. 12, pp. 4981-4985, December 1975 Cell Biology
Cytoplasmic microtubules in normal and transformed cells in culture: Analysis by tubulin antibody immunofluorescence (microtubule assembly/adenosine 3':5'-cyclic monophosphate)
B. R. BRINKLEY, G. M. FULLER, AND D. P. HIGHFIELD Divisions of Cell Biology and Human Genetics, Department of Human Biological Chemistry and Genetics, Graduate School of Biomedical Sciences, The University of Texas Medical Branch, Galveston, Texas 77550
Communicated by Keith R. Porter, September 2,1975
ABSTRACT Monospecific antibody directed against bovine brain tubulin was used as an immunofluorescent probe to evaluate the distribution of microtubules in normal and transformed cells grown in tissue culture. The fluorescent staining pattern of transformed and nontransformed cells is significantly different and may be used in conjunction with other morphological features to identify transformants in mixed cell populations. Normal cells are flattened, elongated, and fibroblastic; they display numerous Colcemid-sensitive fluorescent cytoplasmic filaments, presumably microtubules. Transformed cells, however, are smaller, more polygonal in shape, and contain very few cytoplasmic tubules. During mitosis, the cytoplasmic microtubule complex of normal cells completely disappears, but reappears after cell division. Treatment of transformed cells with dibutyryl-adenosine 3'1 5'-cyclic monophosphate plus testosterone or theophylline restores the normal fibroblastic appearance of the cells and stimulates the assembly of numerous cytoplasmic microtubules. This study provides further evidence for two separate microtubule entities in cycling nontransformed cells: a cytoplasmic microtubule complex and the microtubules of the mitotic spindle. Although an interchange of tubulin dimers seems to exist between microtubules in the two systems, control of tubule assembly may be under separate constraints. Stimulation of cytoplasmic microtubule assembly in transformed cells by derivatives of adenosine 3':5'-cyclic monophosphate suggests that impairment of the cytoplasmic microtubule complex in these cells may be due to suboptimal levels of adenosine 3':5'-cyclic monophosphate.
Malignant transformation of normal cells in vitro is usually characterized by alterations in cell form, surface topography, lectin binding features, and loss of density-dependent growth control. Although changes in cell morphology, especially cell shape, are frequently cited as initial evidence for transformation, the structural and biochemical bases for morphological changes are poorly understood. The maintenance of cell form in most cells is thought to be due in part to the presence of cytoplasmic microtubules (1, 2). For example, treatment of fibroblasts or neuroblastoma cells with inhibitors such as colchicine, Vinca alkaloids, or cold temperatures leads to the disappearance of microtubules and a rapid alteration in cell shape (for review see ref. 3). The involvement of microtubules in virus-induced cell transformation has been implied in several earlier studies (4-6). The morphology of transformed cells can be reversed from a compact, randomly oriented state to more elongated fibroblastic forms with derivatives of adenosine 3':5'-cyclic monophosphate (cAMP). Moreover, such treatments have been reported to restore other normal features, including contact-inhibited growth (9, 10). Cytoplasmic microtubules appear to be required in the reversion process, as indicated by electron microscopic observation (4) and inhibition of morphological reversion by colchicine (5, 6, 8). Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; Bt2cAMP, dibutyryl-adenosine 3':5'-cyclic monophosphate. 4981
Recently, Fonte and Porter (11) have provided direct electron microscopic evidence for diminished microtubules in the cytoplasm of viral transformed cells; they relate this phenomenon to altered cell surface topography and diminished cAMP levels in transformed cells. In the present study, we have used tubulin antibody prepared from bovine brain (12) as an immunofluorescent probe to evaluate the patterns of cytoplasmic microtubules in a variety of normal and transformed cell lines. This method permits immediate resolution of microtubules by light microscopy and allows for rapid analysis of microtubule distribution in large populations of cells. Our findings are in agreement with those of Porter and coworkers (ref. 11 and K. R. Porter, personal communication) that an elaborate system of microtubules exists within the cytoplasm of most nonmalignant cells in culture. All transformed cell lines observed by means of the immunofluorescent procedure displayed a considerable reduction in the number of microtubules; this suggested an impairment in the ability of malignant cells to assemble cytoplasmic microtubules and maintain a fibroblastic appearance. Partial restoration of the cytoplasmic microtubule complex in viral transformed cells was initiated by exposure of cells to dibutyryl-adenosine 3': 5'-cyclic monophosphate (Bt2cAMP) plus testosterone or theophylline. Thus, the capacity to maintain cytoplasmic microtubule assembly in cells appears to be directly related to cAMP concentrations. MATERIALS AND METHODS
The cell lines HTC+, 6TG-11, MMT, C6, A9, LM(TK-), 3T3, SV 3T3, and RAG used in this study were grown as monolayer cultures in Dulbecco's modified Eagle's medium (Gibco, no. H-16) supplemented with 10% fetal calf serum. The CHO and HeLa lines were grown as monolayers in Hsu's modified McCoy's 5a medium supplemented with 10% fetal calf serum. The PtK1, HSF-CF, HSF-Normal, and SV CF cells were grown as monolayers in Ham's F-10 medium (catalogue no. 7712-00, Schwartz/Mann Biochemicals) supplemented with 10% fetal calf serum. We trypsinized cells from exponentially growing stock cultures, seeded aliquots into 60 mm petri dishes and incubated the aliquots for 24-48 hr to ensure an exponential growth phase. When chemicals were to be employed, the growth medium was aspirated from the petri dish and the appropriate prewarmed media-chemical solution was added. The chemicals used in this study were N6,02-dibutyryl adenosine 3':5'-cyclic monophosphoric acid monosodium salt, testosterone propionate (A4-androstene-17fl-propionate3-one, Sigma Chemical Co.) and Colcemid (CIBA Pharmaceutical Products, Inc.). Colcemid was used at a final concentration of 0.06 jig/ml in complete medium. A working
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FIGS. 1-6. (1) 3T3 cells showing numerous cytoplasmic microtubules that radiate out from the nucleus and extend into the major cell processes. X600. (2) Higher magnification of 3T3 cell. Note microtubules that appear bent (thin arrow) and those that extend in parallel bundles near the cell surface (thick arrows X930). (3) SV 3T3 cell that appears smaller, rounded, and polygonal in shape. Microtubules are essentially absent in these cells, but nuclear staining persists. X600. (4) 3T3 cells treated with Colcemid (0.06 gg/ml for 2A hr). Cytoplasm is essentially free of fluorescent filaments and cells have become rounded and.knobby in appearance. X600. (5) SV 3T3 control cell treated with ethanol prior to staining. X600. (6) SV 3T3 cell treated with Bt2cAMP and testosterone in ethanol for 6 hr. Note fibroblastic appearance and numerous cytoplasmic microtubules. X600.
solution of Bt2cAMP in warm complete medium at a final concentration of 0.3 mM was prepared on the day of the experiment. From a stock solution of testosterone at a concentration of 1.5 mM in ethanol, an appropriate volume was added to the Bt2cAMP media to yield a final testosterone concentration of 0.015 mM. Theophylline (Sigma Chemical Co.) was used at a final concentration of 1 mM. We used as controls cultures treated with ethanol alone to ensure that none of the effects observed were due to the solvent. Bovine tubulin antibody was purified and used in indirect
immunofluorescence as previously described (12). Briefly, coverslips with attached cells were rinsed in phosphate-buffered saline and fixed with formaldehyde at concentrations of 1, 2, or 3%, followed by acetone at -10°. The coverslips were then incubated with the antibody (rabbit immunoglobulin, 0.2 mg/ml in phosphate-buffered saline) for 35 min at 370. The preparations were rinsed again in the same buffer and then incubated in a 1:1.5 dilution of fluorescein-tagged goat antiserum against rabbit immunoglobulin G (Meloy Laboratories, Springfield, Va.) and phosphate-buffered sa-
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perinents with microtubule inhibitors. When cells were treated with Colcemid for 2 hr, the filaments essentially disappeared or became greatly diminished (Fig. 4). Concomitantly, the cells lost their fibroblastic shape and became rounded and knobby in appearance. Exposure of cells to cold media (00 for 30 min) resulted in a similar disruption of most fluorescent filaments. Because both treatments are known to disrupt microtubules (22), we can assume that the fluorescent filaments seen in the 3T3 cells are indeed cytoplasmic microtubules. In this regard, the pattern of fluorescence in 3T3 cells is essentially identical to the pattern of microtubules seen in the cytoplasm of these cells by high voltage electron microscopy (ref. 7 and K. R. Porter, personal communication). The nucleus of most cells displayed fluorescent staining either in the form of discrete patches or more generalized staining throughout the nucleus. Although the fluorescence
FIGS. 7 AND 8. (7) Metaphase, rat kangaroo fibroblast (strain PtK1). Cytoplasmic microtubules have all disappeared; fluorescence has become concentrated in the spindle. X704. (8) Late telophase of 3T3 cell with brightly fluorescent stem korper (arrow) extending between two daughter cells. Cytoplasmic microtubules have begun to reappear at opposite poles of cells near the centrosphere. X704.
line. Finally, they were rinsed in the same buffer and mounted on glass slides in a drop of phosphate-buffered saline:glycerol (1:9 adjusted to pH 8.5-10) for viewing in a Leitz microscope adapted for darkfield ultraviolet microscopy. Photographs were recorded on Kodak Tri-X Pan film.
RESULTS The immunofluorescent staining pattern of 3T3 cells is shown in Figs. 1 and 2. The cytoplasm is laced with numerous fluorescent filaments, presumably microtubules, which radiate out from the nucleus and extend in parallel bundles along the major axes of the cell. As shown in the inset in Fig. 1, the fluorescent filaments frequently appear to focus at a region near the nucleus, presumably the centrosphere. Often, the filaments are bent to conform to the shape of the cell surface (thin arrow, Fig. 2) and in some instances bundles of filaments extend in parallel array along the cell margin (thick arrows, Fig. 2). Evidence that the fluorescent filaments seen in these preparations represented intact microtubules came from ex-
was more intense in cells fixed in 3% formaldehyde, staining was still apparent in cells fixed in 1% formaldehyde. Thus, we do not believe that the staining is due to fixation artifact. In all cells examined, the nucleolus was constantly free of stain. Although the nature of nuclear fluorescence is not known, it was resistant to both Colcemid and cold treatments and was present in transformed cells as well as their normal counterparts. Fate of Cytoplasmic Microtubules during Mitosis. In all cells observed, the extensive cytoplasmic microtubule complex disappeared during mitosis. The tubulin antibody immunofluorescent staining properties of the mitotic spindles of mammalian cells have been described elsewhere (12), and the staining of spindles of normal and transformed cells in the present study was essentially the same as described in our earlier study. However, as the cell entered early prophase, as evidenced by the condensation of chromatin, the cytoplasmic tubules began to disappear. By metaphase, microtubules in the cytoplasm had completely disappeared, and most of the fluorescence had become localized within the mitotic spindle (Fig. 7). During the late telophase-early G1 phase, the cytoplasmic tubules reappeared in each daughter cell (Fig. 8). Initially, the microtubules formed near the centrosphere and then became distributed throughout the cell. Immunofluorescence of Transformed Cells. As shown in Figs. 3 and 5, SV 3T3 cells have very few microtubules within their cytoplasm. Also, the cells are smaller than their normal counterparts and assume a more polygonal shape, especially when they become crowded. Essentially, the same staining patterns and morphological features were apparent in all transformed lines included in this study (Table 1). In each population, the cytoplasmic microtubules were shorter, fewer in number, and randomly distributed throughout the cell. In all transformed cells studied, the sparsely distributed microtubules appeared to have no apparent relationship to the shape or geometry of the cells. The paucity of cytoplasmic microtubules in transformed cells, when used in conjunction with other morphological features, enabled us to identify correctly transformed cells in cultures which contained known mixtures of transformed and nontransformed cells. In our experiments, 3T3 cells were mixed with SV 3T3 cells in seven specific ratios and plated onto coverslips. After the cells became attached, the coverslips were processed for antitubulin immunofluorescence and scored microscopically for the number of cells with or without a cytoplasmic microtubule complex. In double blind experiments, 500 cells were scored in each mixture, and in five out of seven mixtures the correct ratio was identified (Table 2).
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Table 1. Immunofluorescent staining properties of various cell lines
Cell line 3T3 PtK1
HSF-CF HSF-Normal SV CF
HTC4 6TG-1 1 MMT
C6 A9 LM(TK -) SV 3T3 RAG
CHO
HeLa
Cell type
Swiss mouse embryo fibroblast Rat kangaroo fibroblast Human skin fibroblast from cystic fibrosis patient Normal human skin fibroblast Simian virus transformed human skin fibroblast from cystic fibrosis patient Rat hepatoma Rat hepatoma Mouse mammary gland carcinoma Rat glial cell Mouse L cell (azaguanine resistant) Mouse L cell (BrdUrd resistant) Simian virus transformed 3T3 Mouse renal adenocarcinoma Chinese hamster ovary Human cervical carcinoma
Table 2. Microscopic identification of transformed cells in mixed cell populations
Cytoplasmic microTrans- tubule formed complex -
+
-
+
-
+
-
+
+
-
+ +
-
+ +
-
+
-
+
-
+
-
+
-
+
-
+
-
-
-
+, presence of cytoplasmic microtubule complex (see Figs. 1 and 2). -, diminished cytoplasmic microtubule complex (see Figs. 3 and 5). Cell lines HTC+ and 6TG-11 were derived from Morris rat hepatoma 7288c; SV 3T3 and SV CF were transformed by SV-40 virus; and the remaining transformed lines were spontaneous transformants.
Cyclic Nucleotide Stimulation of Microtubules. To evaluate the effect of cyclic nucleotide derivatives with our system, we stained both CHO and SV 3T3 cells for antitubulin immunofluorescence after exposure to Bt2cAMP plus testosterone or theophylline according to the procedures of Hsie and Puck (8) and Johnson et al. (9). Although we observed dramatic changes in morphology of both cell types after treatment, the most pronounced effect was apparent in the SV 3T3 cells. As shown in Fig. 6, the treated cells assumed a more fibroblastic appearance and displayed numerous fluorescent tubules within the cytoplasm. Thus, our results confirm previous reports (4-6, 8) and establish that morphological changes stimulated by cAMP derivatives are due largely to microtubule assembly. DISCUSSION The simplicity of the antitubulin immunofluorescence procedure permits immediate resolution of cytoplasmic mi-
Slide
Cells with CMtC*
Cells lacking CMtC
Expected ratio (T/NT)t
Observed ratio (T/NT)
A B C D E F G
499 249 71 82 2 400 469
1 252 437 418 498 102 31
1:0 1:1 1:5 1:10 0:1 5:1 10:1
1:0 1:1 1:6 1:5 0:1 4:1 15:1
Exponentially growing cultures of 3T3 and SV 3T3 cells were trypsinized from the culture flask and maintained in suspension. We gently dispersed each suspension several times through a 25 gauge needle to ensure singlet cells. The suspensions were counted in a Coulter counter, and the cultures were mixed appropriately to yield the desired ratio of 3T3 to SV 3T3 cells. We then plated the cells onto coverslips and incubated the coverslips with frequent agitation for 60 min to ensure attachment of single cells to the substrate. The coverslip preparations were stained and mounted onto a glass slide. Double blind scoring of each slide was carried out with a Leitz ultraviolet microscope with a darkfield condenser and a Zeiss 40x apochromatic objective. *CMtC, cytoplasmic microtubule complex (as shown in Figs. 1 and 2). tT/NT, ratio of transformed (SV 3T3) to nontransformed (3T3) cells.
crotubules in cultured mammalian cells. The images produced by this technique correlate closely with transmission electron microscope images of cytoplasmic microtubules (7, 11). Because the preparation can be viewed with conventional ultraviolet optics, large populations of cells can be examined with relative ease and efficiency. Through use of this approach, we have identified an elaborate microtubular complex within the cytoplasm of normal cultured fibroblasts which confirms in another way the observations of Porter (personal communication). The distribution and orientation of cytoplasmic microtubules conform to the major cell axes, and the tubules probably play an important role in the maintenance of cell shape, as suggested by previous investigators (1-3). The dissolution of the cytoplasmic microtubule network during mitosis coincides with the loss of fibroblastic shape of cells and the appearance of the mitotic apparatus. From these observations, it seems likely that most of the tubulin dimers of cytoplasmic microtubules are reutilized in the assembly of spindle microtubules. Reciprocally, the reappearance of cytoplasmic microtubules in late telophase-early Gi phase suggests that spindle tubule subunits may then, in turn, be recycled into cytoplasmic microtubules. The apparent cyclic transition of cytoplasmic microtubules to spindle tubules appears to be an important feature of normal proliferating mammalian cells, but is somehow lacking in transformed cells. The results of our experiments with mixed populations of 3T3 and SV 3T3 cells suggest that tubulin antibody immunofluorescence may be useful, in conjunction with other morphological features, in identifying transformants in cultured cell populations. Transformed cells displayed few cytoplasmic microtubules; the paucity of microtubules appears to be related to the major morphological changes which accompany transformation. Further relationship of the reduced number of cytoplasmic microtubules to other familiar tumor cell properties, such as cell surface changes and loss of contact inhibition, are also implied in the experiments pre-
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sented here. When SV 3T3 or CHO cells were exposed to Bt2cAMP plus testosterone or theophylline, they underwent a morphological transition from a rounded, clumped appearance to a more fibroblastic form containing numerous cytoplasmic microtubules. Although we did not evaluate the growth characteristic of our cells, similar treatment of spontaneous transformants of 3T3 (strain 3T6) and polyoma virus transformants (strain PyV-3T3) led to the restoration of contact inhibited growth (10). The loss of the cytoplasmic microtubule complex in viral transformed cells suggests that conditions that restrict microtubule assembly exist in these cells. Factors controlling microtubule assembly both in vitro and in vivo are being identified by numerous investigators (3, 13-17, 19). Cyclic nucleotides, including cAMP and guanosine 3':5'-cyclic monophosphate (cGMP), have been implicated in regulating microtubule assembly (23, 24). Weisenberg's discovery (15) of the inhibitory role of Ca++ in the bovine brain tubulin assembly system suggests that a similar regulatory function could exist within intact cells, although direct evidence is lacking. If Ca++ is a natural regulator of assembly, cyclic nucleotides could function in turn to regulate Ca++ flux within various cellular compartments (18). Recently, Olmstead and Borisy (19) suggested that a regulatory role by Ca++ in tubule assembly be viewed with caution since their data showed magnesium-GTP association involved in tubulin polymerization in vitro. Their findings suggest that Ca++ can be either stimulatory or inhibitory of in vitro assembly, depending upon the ionic conditions of the system. The reports that cyclic nucleotide levels are unusually low in malignant cells (20, 21) are also consistent with the findings of the present study. Thus, the diminished number of cytoplasmic microtubules in transformed cells is apparently due in part to low levels of cAMP in these cells. The precise role of cyclic nucleotides in regulating cytoplasmic microtubule assembly is yet to be determined. Finally, our observations permit a distinction to be made between the cytoplasmic microtubule complex of interphase cells and the microtubules of the mitotic apparatus. Electron microscope studies show that the morphology of the tubules in the two systems appears identical (22). Because the cytoplasmic microtubules disappear with the onset of spindle formation and vice yersa, it is likely that tubulin components of one system can be recycled into the other. If so, control of tubule assembly in the two systems appears to be under separate constraints. Thus, from studies with immunofluorescent staining it is apparent that there is no defect in the assembly of the mitotic spindle in transformed cells, yet the cytoplasmic microtubule complex is greatly diminished. Treatment of transformed cells with Bt2cAMP stimulates the cytoplasmic microtubule assembly and restores the normal fibroblastic appearance of the cells. Therefore, we believe that the cytoplasmic microtubule complex should be viewed as a separate entity from the mitotic spindle. The role of this complex in the maintenance of cell form, as well as the many other functions attributed to cytoplasmic microtubules, merit further study. The loss of a cytoplasmic microtubule complex seems to be a common feature of transformed cells that may account for many of the characteristic morphological and growth related properties in malignant cells.
4985
In an earlier study, Weber et al. (25) found that antibody made against tubulin from the outer doublets of sea urchin sperm flagella decorated a reticulum of fine filaments in the cytoplasm of cells derived from a variety of sources. The images of normal 3T3 cells reported in our study are essentially identical to those presented by Weber and co-workers. Transformed cells from many sources, however, display a greatly diminished cytoplasmic microtubule complex. We are grateful to Dr. Robert Klebe for providing many of the transformed cell lines used in this study. We thank Ms. Joan Ellison for excellent technical assistance in antibody preparation. Appreciation is extended to Mrs. Shirley Brinkley for editorial assistance. This work was supported in part by NCI Grant CA 14675 and a grant from the Dow Chemical Co. 1. Porter, K. R. (1966) in Principles of Biomolecular Organization, eds. Wolstenholme, G. E. & O'Conner, M. (Little, Brown, Boston), p. 308. 2. Tilney, L. G. & Porter, K. R. (1967) J. Cell Biol. 34, 327-343. 3. Olmsted, J. B. & Borisy, G. (1973) Annu. Rev. Biochem. 42, 507-540. 4. Porter, K. R., Puck, T. T., Hsie, A. W. & Kelley, D. (1974) Cell 2, 145-162. 5. Hsie, A. W., Jones, C. & Puck, T. T. (1971) Proc. Nat. Acad. Sci. USA 68,1648-1652. 6. Puck, T. T., Waldren, C. A. & Hsie, A. W. (1972) Proc. Nat. Acad. Sci. USA 69, 1943-1947. 7. Buckley, I. K. (1975) Tissue Cell 7, 51-72. 8. Hsie, A. & Puck, T. T. (1971) Proc. Nat. Acad. Sci. USA 68,
358-361. 9. Johnson, G. E., Friedman, R. M. & Pastan, I. (1971) Proc. Nat. Acad. Sci. USA 68, 425-429. 10. Sheppard, J. R. (1971) Proc. Nat. Acad. Sci. USA 68, 13161320. 11. Fonte, V. & Porter, K. R. (1974) in Eighth Int. Cong. Elect. Micro., eds. Sanders, J. V. & Goodchild, D. J. (Aust. Acad. Sci., Canberra), Vol. II, p. 334. 12. Fuller, G. M., Brinkley, B. R. & Boughter, J. M. (1975) Science
187,948-950. 13. Inoue, S. & Sato, H. (1967) J. Gen. Physiol. Suppl. 50, 259288. 14. Rosenbaum, J. L. & Child, F. M. (1967) J. Cell Biol. 34, 345364. 15. Weisenberg, R. C. (1972) Science 177, 1104-1105. 16. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA 70,765-768. 17. Borisy, G. G. & Olmsted, J. B. (1972) Science 177, 1196-1197. 18. Borle, A. B. (1974) J. Membr. Biol. 16,221-236. 19. Olmsted, J. B. & Borisy, G. G. (1975) Biochemistry 14, 29963004. 20. Monahan, T. M., Marchand, N. W., Fritz, R. R. & Abell, C. W. (1975) Cancer Res. 35, 2540-2547. 21. Monahan, T. M., Fritz, R. R. & Abell, C. W. (1973) Biochem. Biophys. Res. Commun. 55,642-646. 22. Brinkley, B. R. & Cartwright, J., Jr. (1975) Ann. N.Y. Acad. Sci. 253, 428-439. 23. Soifer, D., Laszio, A. & Scotto, J. (1972) Biochim. Biophys. Acta 271, 182-192. 24. Oliver, J. M., Zurier, R. B. & Berlin, R. D. (1975) Nature, 253, 471-473. 25. Weber, K., Pollack, R. & Bibring, T. (1975) Proc. Nat. Acad.
Sci. USA, 72,459-463.
Vol. 72, No. 12, pp. 4981-4985, December 1975 Cell Biology
Cytoplasmic microtubules in normal and transformed cells in culture: Analysis by tubulin antibody immunofluorescence (microtubule assembly/adenosine 3':5'-cyclic monophosphate)
B. R. BRINKLEY, G. M. FULLER, AND D. P. HIGHFIELD Divisions of Cell Biology and Human Genetics, Department of Human Biological Chemistry and Genetics, Graduate School of Biomedical Sciences, The University of Texas Medical Branch, Galveston, Texas 77550
Communicated by Keith R. Porter, September 2,1975
ABSTRACT Monospecific antibody directed against bovine brain tubulin was used as an immunofluorescent probe to evaluate the distribution of microtubules in normal and transformed cells grown in tissue culture. The fluorescent staining pattern of transformed and nontransformed cells is significantly different and may be used in conjunction with other morphological features to identify transformants in mixed cell populations. Normal cells are flattened, elongated, and fibroblastic; they display numerous Colcemid-sensitive fluorescent cytoplasmic filaments, presumably microtubules. Transformed cells, however, are smaller, more polygonal in shape, and contain very few cytoplasmic tubules. During mitosis, the cytoplasmic microtubule complex of normal cells completely disappears, but reappears after cell division. Treatment of transformed cells with dibutyryl-adenosine 3'1 5'-cyclic monophosphate plus testosterone or theophylline restores the normal fibroblastic appearance of the cells and stimulates the assembly of numerous cytoplasmic microtubules. This study provides further evidence for two separate microtubule entities in cycling nontransformed cells: a cytoplasmic microtubule complex and the microtubules of the mitotic spindle. Although an interchange of tubulin dimers seems to exist between microtubules in the two systems, control of tubule assembly may be under separate constraints. Stimulation of cytoplasmic microtubule assembly in transformed cells by derivatives of adenosine 3':5'-cyclic monophosphate suggests that impairment of the cytoplasmic microtubule complex in these cells may be due to suboptimal levels of adenosine 3':5'-cyclic monophosphate.
Malignant transformation of normal cells in vitro is usually characterized by alterations in cell form, surface topography, lectin binding features, and loss of density-dependent growth control. Although changes in cell morphology, especially cell shape, are frequently cited as initial evidence for transformation, the structural and biochemical bases for morphological changes are poorly understood. The maintenance of cell form in most cells is thought to be due in part to the presence of cytoplasmic microtubules (1, 2). For example, treatment of fibroblasts or neuroblastoma cells with inhibitors such as colchicine, Vinca alkaloids, or cold temperatures leads to the disappearance of microtubules and a rapid alteration in cell shape (for review see ref. 3). The involvement of microtubules in virus-induced cell transformation has been implied in several earlier studies (4-6). The morphology of transformed cells can be reversed from a compact, randomly oriented state to more elongated fibroblastic forms with derivatives of adenosine 3':5'-cyclic monophosphate (cAMP). Moreover, such treatments have been reported to restore other normal features, including contact-inhibited growth (9, 10). Cytoplasmic microtubules appear to be required in the reversion process, as indicated by electron microscopic observation (4) and inhibition of morphological reversion by colchicine (5, 6, 8). Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; Bt2cAMP, dibutyryl-adenosine 3':5'-cyclic monophosphate. 4981
Recently, Fonte and Porter (11) have provided direct electron microscopic evidence for diminished microtubules in the cytoplasm of viral transformed cells; they relate this phenomenon to altered cell surface topography and diminished cAMP levels in transformed cells. In the present study, we have used tubulin antibody prepared from bovine brain (12) as an immunofluorescent probe to evaluate the patterns of cytoplasmic microtubules in a variety of normal and transformed cell lines. This method permits immediate resolution of microtubules by light microscopy and allows for rapid analysis of microtubule distribution in large populations of cells. Our findings are in agreement with those of Porter and coworkers (ref. 11 and K. R. Porter, personal communication) that an elaborate system of microtubules exists within the cytoplasm of most nonmalignant cells in culture. All transformed cell lines observed by means of the immunofluorescent procedure displayed a considerable reduction in the number of microtubules; this suggested an impairment in the ability of malignant cells to assemble cytoplasmic microtubules and maintain a fibroblastic appearance. Partial restoration of the cytoplasmic microtubule complex in viral transformed cells was initiated by exposure of cells to dibutyryl-adenosine 3': 5'-cyclic monophosphate (Bt2cAMP) plus testosterone or theophylline. Thus, the capacity to maintain cytoplasmic microtubule assembly in cells appears to be directly related to cAMP concentrations. MATERIALS AND METHODS
The cell lines HTC+, 6TG-11, MMT, C6, A9, LM(TK-), 3T3, SV 3T3, and RAG used in this study were grown as monolayer cultures in Dulbecco's modified Eagle's medium (Gibco, no. H-16) supplemented with 10% fetal calf serum. The CHO and HeLa lines were grown as monolayers in Hsu's modified McCoy's 5a medium supplemented with 10% fetal calf serum. The PtK1, HSF-CF, HSF-Normal, and SV CF cells were grown as monolayers in Ham's F-10 medium (catalogue no. 7712-00, Schwartz/Mann Biochemicals) supplemented with 10% fetal calf serum. We trypsinized cells from exponentially growing stock cultures, seeded aliquots into 60 mm petri dishes and incubated the aliquots for 24-48 hr to ensure an exponential growth phase. When chemicals were to be employed, the growth medium was aspirated from the petri dish and the appropriate prewarmed media-chemical solution was added. The chemicals used in this study were N6,02-dibutyryl adenosine 3':5'-cyclic monophosphoric acid monosodium salt, testosterone propionate (A4-androstene-17fl-propionate3-one, Sigma Chemical Co.) and Colcemid (CIBA Pharmaceutical Products, Inc.). Colcemid was used at a final concentration of 0.06 jig/ml in complete medium. A working
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FIGS. 1-6. (1) 3T3 cells showing numerous cytoplasmic microtubules that radiate out from the nucleus and extend into the major cell processes. X600. (2) Higher magnification of 3T3 cell. Note microtubules that appear bent (thin arrow) and those that extend in parallel bundles near the cell surface (thick arrows X930). (3) SV 3T3 cell that appears smaller, rounded, and polygonal in shape. Microtubules are essentially absent in these cells, but nuclear staining persists. X600. (4) 3T3 cells treated with Colcemid (0.06 gg/ml for 2A hr). Cytoplasm is essentially free of fluorescent filaments and cells have become rounded and.knobby in appearance. X600. (5) SV 3T3 control cell treated with ethanol prior to staining. X600. (6) SV 3T3 cell treated with Bt2cAMP and testosterone in ethanol for 6 hr. Note fibroblastic appearance and numerous cytoplasmic microtubules. X600.
solution of Bt2cAMP in warm complete medium at a final concentration of 0.3 mM was prepared on the day of the experiment. From a stock solution of testosterone at a concentration of 1.5 mM in ethanol, an appropriate volume was added to the Bt2cAMP media to yield a final testosterone concentration of 0.015 mM. Theophylline (Sigma Chemical Co.) was used at a final concentration of 1 mM. We used as controls cultures treated with ethanol alone to ensure that none of the effects observed were due to the solvent. Bovine tubulin antibody was purified and used in indirect
immunofluorescence as previously described (12). Briefly, coverslips with attached cells were rinsed in phosphate-buffered saline and fixed with formaldehyde at concentrations of 1, 2, or 3%, followed by acetone at -10°. The coverslips were then incubated with the antibody (rabbit immunoglobulin, 0.2 mg/ml in phosphate-buffered saline) for 35 min at 370. The preparations were rinsed again in the same buffer and then incubated in a 1:1.5 dilution of fluorescein-tagged goat antiserum against rabbit immunoglobulin G (Meloy Laboratories, Springfield, Va.) and phosphate-buffered sa-
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perinents with microtubule inhibitors. When cells were treated with Colcemid for 2 hr, the filaments essentially disappeared or became greatly diminished (Fig. 4). Concomitantly, the cells lost their fibroblastic shape and became rounded and knobby in appearance. Exposure of cells to cold media (00 for 30 min) resulted in a similar disruption of most fluorescent filaments. Because both treatments are known to disrupt microtubules (22), we can assume that the fluorescent filaments seen in the 3T3 cells are indeed cytoplasmic microtubules. In this regard, the pattern of fluorescence in 3T3 cells is essentially identical to the pattern of microtubules seen in the cytoplasm of these cells by high voltage electron microscopy (ref. 7 and K. R. Porter, personal communication). The nucleus of most cells displayed fluorescent staining either in the form of discrete patches or more generalized staining throughout the nucleus. Although the fluorescence
FIGS. 7 AND 8. (7) Metaphase, rat kangaroo fibroblast (strain PtK1). Cytoplasmic microtubules have all disappeared; fluorescence has become concentrated in the spindle. X704. (8) Late telophase of 3T3 cell with brightly fluorescent stem korper (arrow) extending between two daughter cells. Cytoplasmic microtubules have begun to reappear at opposite poles of cells near the centrosphere. X704.
line. Finally, they were rinsed in the same buffer and mounted on glass slides in a drop of phosphate-buffered saline:glycerol (1:9 adjusted to pH 8.5-10) for viewing in a Leitz microscope adapted for darkfield ultraviolet microscopy. Photographs were recorded on Kodak Tri-X Pan film.
RESULTS The immunofluorescent staining pattern of 3T3 cells is shown in Figs. 1 and 2. The cytoplasm is laced with numerous fluorescent filaments, presumably microtubules, which radiate out from the nucleus and extend in parallel bundles along the major axes of the cell. As shown in the inset in Fig. 1, the fluorescent filaments frequently appear to focus at a region near the nucleus, presumably the centrosphere. Often, the filaments are bent to conform to the shape of the cell surface (thin arrow, Fig. 2) and in some instances bundles of filaments extend in parallel array along the cell margin (thick arrows, Fig. 2). Evidence that the fluorescent filaments seen in these preparations represented intact microtubules came from ex-
was more intense in cells fixed in 3% formaldehyde, staining was still apparent in cells fixed in 1% formaldehyde. Thus, we do not believe that the staining is due to fixation artifact. In all cells examined, the nucleolus was constantly free of stain. Although the nature of nuclear fluorescence is not known, it was resistant to both Colcemid and cold treatments and was present in transformed cells as well as their normal counterparts. Fate of Cytoplasmic Microtubules during Mitosis. In all cells observed, the extensive cytoplasmic microtubule complex disappeared during mitosis. The tubulin antibody immunofluorescent staining properties of the mitotic spindles of mammalian cells have been described elsewhere (12), and the staining of spindles of normal and transformed cells in the present study was essentially the same as described in our earlier study. However, as the cell entered early prophase, as evidenced by the condensation of chromatin, the cytoplasmic tubules began to disappear. By metaphase, microtubules in the cytoplasm had completely disappeared, and most of the fluorescence had become localized within the mitotic spindle (Fig. 7). During the late telophase-early G1 phase, the cytoplasmic tubules reappeared in each daughter cell (Fig. 8). Initially, the microtubules formed near the centrosphere and then became distributed throughout the cell. Immunofluorescence of Transformed Cells. As shown in Figs. 3 and 5, SV 3T3 cells have very few microtubules within their cytoplasm. Also, the cells are smaller than their normal counterparts and assume a more polygonal shape, especially when they become crowded. Essentially, the same staining patterns and morphological features were apparent in all transformed lines included in this study (Table 1). In each population, the cytoplasmic microtubules were shorter, fewer in number, and randomly distributed throughout the cell. In all transformed cells studied, the sparsely distributed microtubules appeared to have no apparent relationship to the shape or geometry of the cells. The paucity of cytoplasmic microtubules in transformed cells, when used in conjunction with other morphological features, enabled us to identify correctly transformed cells in cultures which contained known mixtures of transformed and nontransformed cells. In our experiments, 3T3 cells were mixed with SV 3T3 cells in seven specific ratios and plated onto coverslips. After the cells became attached, the coverslips were processed for antitubulin immunofluorescence and scored microscopically for the number of cells with or without a cytoplasmic microtubule complex. In double blind experiments, 500 cells were scored in each mixture, and in five out of seven mixtures the correct ratio was identified (Table 2).
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Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. Immunofluorescent staining properties of various cell lines
Cell line 3T3 PtK1
HSF-CF HSF-Normal SV CF
HTC4 6TG-1 1 MMT
C6 A9 LM(TK -) SV 3T3 RAG
CHO
HeLa
Cell type
Swiss mouse embryo fibroblast Rat kangaroo fibroblast Human skin fibroblast from cystic fibrosis patient Normal human skin fibroblast Simian virus transformed human skin fibroblast from cystic fibrosis patient Rat hepatoma Rat hepatoma Mouse mammary gland carcinoma Rat glial cell Mouse L cell (azaguanine resistant) Mouse L cell (BrdUrd resistant) Simian virus transformed 3T3 Mouse renal adenocarcinoma Chinese hamster ovary Human cervical carcinoma
Table 2. Microscopic identification of transformed cells in mixed cell populations
Cytoplasmic microTrans- tubule formed complex -
+
-
+
-
+
-
+
+
-
+ +
-
+ +
-
+
-
+
-
+
-
+
-
+
-
+
-
-
-
+, presence of cytoplasmic microtubule complex (see Figs. 1 and 2). -, diminished cytoplasmic microtubule complex (see Figs. 3 and 5). Cell lines HTC+ and 6TG-11 were derived from Morris rat hepatoma 7288c; SV 3T3 and SV CF were transformed by SV-40 virus; and the remaining transformed lines were spontaneous transformants.
Cyclic Nucleotide Stimulation of Microtubules. To evaluate the effect of cyclic nucleotide derivatives with our system, we stained both CHO and SV 3T3 cells for antitubulin immunofluorescence after exposure to Bt2cAMP plus testosterone or theophylline according to the procedures of Hsie and Puck (8) and Johnson et al. (9). Although we observed dramatic changes in morphology of both cell types after treatment, the most pronounced effect was apparent in the SV 3T3 cells. As shown in Fig. 6, the treated cells assumed a more fibroblastic appearance and displayed numerous fluorescent tubules within the cytoplasm. Thus, our results confirm previous reports (4-6, 8) and establish that morphological changes stimulated by cAMP derivatives are due largely to microtubule assembly. DISCUSSION The simplicity of the antitubulin immunofluorescence procedure permits immediate resolution of cytoplasmic mi-
Slide
Cells with CMtC*
Cells lacking CMtC
Expected ratio (T/NT)t
Observed ratio (T/NT)
A B C D E F G
499 249 71 82 2 400 469
1 252 437 418 498 102 31
1:0 1:1 1:5 1:10 0:1 5:1 10:1
1:0 1:1 1:6 1:5 0:1 4:1 15:1
Exponentially growing cultures of 3T3 and SV 3T3 cells were trypsinized from the culture flask and maintained in suspension. We gently dispersed each suspension several times through a 25 gauge needle to ensure singlet cells. The suspensions were counted in a Coulter counter, and the cultures were mixed appropriately to yield the desired ratio of 3T3 to SV 3T3 cells. We then plated the cells onto coverslips and incubated the coverslips with frequent agitation for 60 min to ensure attachment of single cells to the substrate. The coverslip preparations were stained and mounted onto a glass slide. Double blind scoring of each slide was carried out with a Leitz ultraviolet microscope with a darkfield condenser and a Zeiss 40x apochromatic objective. *CMtC, cytoplasmic microtubule complex (as shown in Figs. 1 and 2). tT/NT, ratio of transformed (SV 3T3) to nontransformed (3T3) cells.
crotubules in cultured mammalian cells. The images produced by this technique correlate closely with transmission electron microscope images of cytoplasmic microtubules (7, 11). Because the preparation can be viewed with conventional ultraviolet optics, large populations of cells can be examined with relative ease and efficiency. Through use of this approach, we have identified an elaborate microtubular complex within the cytoplasm of normal cultured fibroblasts which confirms in another way the observations of Porter (personal communication). The distribution and orientation of cytoplasmic microtubules conform to the major cell axes, and the tubules probably play an important role in the maintenance of cell shape, as suggested by previous investigators (1-3). The dissolution of the cytoplasmic microtubule network during mitosis coincides with the loss of fibroblastic shape of cells and the appearance of the mitotic apparatus. From these observations, it seems likely that most of the tubulin dimers of cytoplasmic microtubules are reutilized in the assembly of spindle microtubules. Reciprocally, the reappearance of cytoplasmic microtubules in late telophase-early Gi phase suggests that spindle tubule subunits may then, in turn, be recycled into cytoplasmic microtubules. The apparent cyclic transition of cytoplasmic microtubules to spindle tubules appears to be an important feature of normal proliferating mammalian cells, but is somehow lacking in transformed cells. The results of our experiments with mixed populations of 3T3 and SV 3T3 cells suggest that tubulin antibody immunofluorescence may be useful, in conjunction with other morphological features, in identifying transformants in cultured cell populations. Transformed cells displayed few cytoplasmic microtubules; the paucity of microtubules appears to be related to the major morphological changes which accompany transformation. Further relationship of the reduced number of cytoplasmic microtubules to other familiar tumor cell properties, such as cell surface changes and loss of contact inhibition, are also implied in the experiments pre-
Cell Biology:
Proc. Nat. Acad. Sci. USA 72 (1975)
Brinkley et al.
sented here. When SV 3T3 or CHO cells were exposed to Bt2cAMP plus testosterone or theophylline, they underwent a morphological transition from a rounded, clumped appearance to a more fibroblastic form containing numerous cytoplasmic microtubules. Although we did not evaluate the growth characteristic of our cells, similar treatment of spontaneous transformants of 3T3 (strain 3T6) and polyoma virus transformants (strain PyV-3T3) led to the restoration of contact inhibited growth (10). The loss of the cytoplasmic microtubule complex in viral transformed cells suggests that conditions that restrict microtubule assembly exist in these cells. Factors controlling microtubule assembly both in vitro and in vivo are being identified by numerous investigators (3, 13-17, 19). Cyclic nucleotides, including cAMP and guanosine 3':5'-cyclic monophosphate (cGMP), have been implicated in regulating microtubule assembly (23, 24). Weisenberg's discovery (15) of the inhibitory role of Ca++ in the bovine brain tubulin assembly system suggests that a similar regulatory function could exist within intact cells, although direct evidence is lacking. If Ca++ is a natural regulator of assembly, cyclic nucleotides could function in turn to regulate Ca++ flux within various cellular compartments (18). Recently, Olmstead and Borisy (19) suggested that a regulatory role by Ca++ in tubule assembly be viewed with caution since their data showed magnesium-GTP association involved in tubulin polymerization in vitro. Their findings suggest that Ca++ can be either stimulatory or inhibitory of in vitro assembly, depending upon the ionic conditions of the system. The reports that cyclic nucleotide levels are unusually low in malignant cells (20, 21) are also consistent with the findings of the present study. Thus, the diminished number of cytoplasmic microtubules in transformed cells is apparently due in part to low levels of cAMP in these cells. The precise role of cyclic nucleotides in regulating cytoplasmic microtubule assembly is yet to be determined. Finally, our observations permit a distinction to be made between the cytoplasmic microtubule complex of interphase cells and the microtubules of the mitotic apparatus. Electron microscope studies show that the morphology of the tubules in the two systems appears identical (22). Because the cytoplasmic microtubules disappear with the onset of spindle formation and vice yersa, it is likely that tubulin components of one system can be recycled into the other. If so, control of tubule assembly in the two systems appears to be under separate constraints. Thus, from studies with immunofluorescent staining it is apparent that there is no defect in the assembly of the mitotic spindle in transformed cells, yet the cytoplasmic microtubule complex is greatly diminished. Treatment of transformed cells with Bt2cAMP stimulates the cytoplasmic microtubule assembly and restores the normal fibroblastic appearance of the cells. Therefore, we believe that the cytoplasmic microtubule complex should be viewed as a separate entity from the mitotic spindle. The role of this complex in the maintenance of cell form, as well as the many other functions attributed to cytoplasmic microtubules, merit further study. The loss of a cytoplasmic microtubule complex seems to be a common feature of transformed cells that may account for many of the characteristic morphological and growth related properties in malignant cells.
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In an earlier study, Weber et al. (25) found that antibody made against tubulin from the outer doublets of sea urchin sperm flagella decorated a reticulum of fine filaments in the cytoplasm of cells derived from a variety of sources. The images of normal 3T3 cells reported in our study are essentially identical to those presented by Weber and co-workers. Transformed cells from many sources, however, display a greatly diminished cytoplasmic microtubule complex. We are grateful to Dr. Robert Klebe for providing many of the transformed cell lines used in this study. We thank Ms. Joan Ellison for excellent technical assistance in antibody preparation. Appreciation is extended to Mrs. Shirley Brinkley for editorial assistance. This work was supported in part by NCI Grant CA 14675 and a grant from the Dow Chemical Co. 1. Porter, K. R. (1966) in Principles of Biomolecular Organization, eds. Wolstenholme, G. E. & O'Conner, M. (Little, Brown, Boston), p. 308. 2. Tilney, L. G. & Porter, K. R. (1967) J. Cell Biol. 34, 327-343. 3. Olmsted, J. B. & Borisy, G. (1973) Annu. Rev. Biochem. 42, 507-540. 4. Porter, K. R., Puck, T. T., Hsie, A. W. & Kelley, D. (1974) Cell 2, 145-162. 5. Hsie, A. W., Jones, C. & Puck, T. T. (1971) Proc. Nat. Acad. Sci. USA 68,1648-1652. 6. Puck, T. T., Waldren, C. A. & Hsie, A. W. (1972) Proc. Nat. Acad. Sci. USA 69, 1943-1947. 7. Buckley, I. K. (1975) Tissue Cell 7, 51-72. 8. Hsie, A. & Puck, T. T. (1971) Proc. Nat. Acad. Sci. USA 68,
358-361. 9. Johnson, G. E., Friedman, R. M. & Pastan, I. (1971) Proc. Nat. Acad. Sci. USA 68, 425-429. 10. Sheppard, J. R. (1971) Proc. Nat. Acad. Sci. USA 68, 13161320. 11. Fonte, V. & Porter, K. R. (1974) in Eighth Int. Cong. Elect. Micro., eds. Sanders, J. V. & Goodchild, D. J. (Aust. Acad. Sci., Canberra), Vol. II, p. 334. 12. Fuller, G. M., Brinkley, B. R. & Boughter, J. M. (1975) Science
187,948-950. 13. Inoue, S. & Sato, H. (1967) J. Gen. Physiol. Suppl. 50, 259288. 14. Rosenbaum, J. L. & Child, F. M. (1967) J. Cell Biol. 34, 345364. 15. Weisenberg, R. C. (1972) Science 177, 1104-1105. 16. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA 70,765-768. 17. Borisy, G. G. & Olmsted, J. B. (1972) Science 177, 1196-1197. 18. Borle, A. B. (1974) J. Membr. Biol. 16,221-236. 19. Olmsted, J. B. & Borisy, G. G. (1975) Biochemistry 14, 29963004. 20. Monahan, T. M., Marchand, N. W., Fritz, R. R. & Abell, C. W. (1975) Cancer Res. 35, 2540-2547. 21. Monahan, T. M., Fritz, R. R. & Abell, C. W. (1973) Biochem. Biophys. Res. Commun. 55,642-646. 22. Brinkley, B. R. & Cartwright, J., Jr. (1975) Ann. N.Y. Acad. Sci. 253, 428-439. 23. Soifer, D., Laszio, A. & Scotto, J. (1972) Biochim. Biophys. Acta 271, 182-192. 24. Oliver, J. M., Zurier, R. B. & Berlin, R. D. (1975) Nature, 253, 471-473. 25. Weber, K., Pollack, R. & Bibring, T. (1975) Proc. Nat. Acad.
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