Proc. Nat. Acad. Sci. USAVol. 72, No. 12, pp. 4981-4985, December 1975Cell Biology

Cytoplasmic microtubules in normal and transformed cells inculture: Analysis by tubulin antibody immunofluorescence

(microtubule assembly/adenosine 3':5'-cyclic monophosphate)

B. R. BRINKLEY, G. M. FULLER, AND D. P. HIGHFIELDDivisions of Cell Biology and Human Genetics, Department of Human Biological Chemistry and Genetics, Graduate School of Biomedical Sciences, TheUniversity of Texas Medical Branch, Galveston, Texas 77550

Communicated by Keith R. Porter, September 2,1975

ABSTRACT Monospecific antibody directed against bo-vine brain tubulin was used as an immunofluorescent probeto evaluate the distribution of microtubules in normal andtransformed cells grown in tissue culture. The fluorescentstaining pattern of transformed and nontransformed cells issignificantly different and may be used in conjunction withother morphological features to identify transformants inmixed cell populations. Normal cells are flattened, elongated,and fibroblastic; they display numerous Colcemid-sensitivefluorescent cytoplasmic filaments, presumably microtubules.Transformed cells, however, are smaller, more polygonal inshape, and contain very few cytoplasmic tubules. During mi-tosis, the cytoplasmic microtubule complex of normal cellscompletely disappears, but reappears after cell division.Treatment of transformed cells with dibutyryl-adenosine 3'15'-cyclic monophosphate plus testosterone or theophylline re-stores the normal fibroblastic appearance of the cells andstimulates the assembly of numerous cytoplasmic microtub-ules. This study provides further evidence for two separatemicrotubule entities in cycling nontransformed cells: a cyto-plasmic microtubule complex and the microtubules of themitotic spindle. Although an interchange of tubulin dimersseems to exist between microtubules in the two systems, con-trol of tubule assembly may be under separate constraints.Stimulation of cytoplasmic microtubule assembly in trans-formed cells by derivatives of adenosine 3':5'-cyclic mono-phosphate suggests that impairment of the cytoplasmic mi-crotubule complex in these cells may be due to suboptimallevels of adenosine 3':5'-cyclic monophosphate.

Malignant transformation of normal cells in vitro is usuallycharacterized by alterations in cell form, surface topogra-phy, lectin binding features, and loss of density-dependentgrowth control. Although changes in cell morphology, espe-cially cell shape, are frequently cited as initial evidence fortransformation, the structural and biochemical bases formorphological 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 neuroblasto-ma cells with inhibitors such as colchicine, Vinca alkaloids,or cold temperatures leads to the disappearance of microtu-bules 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 reversedfrom a compact, randomly oriented state to more elongatedfibroblastic forms with derivatives of adenosine 3':5'-cyclicmonophosphate (cAMP). Moreover, such treatments havebeen reported to restore other normal features, includingcontact-inhibited growth (9, 10). Cytoplasmic microtubulesappear to be required in the reversion process, as indicatedby electron microscopic observation (4) and inhibition ofmorphological reversion by colchicine (5, 6, 8).Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate;Bt2cAMP, dibutyryl-adenosine 3':5'-cyclic monophosphate.

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Recently, Fonte and Porter (11) have provided directelectron microscopic evidence for diminished microtubulesin the cytoplasm of viral transformed cells; they relate thisphenomenon to altered cell surface topography and dimin-ished cAMP levels in transformed cells.

In the present study, we have used tubulin antibody pre-pared from bovine brain (12) as an immunofluorescentprobe to evaluate the patterns of cytoplasmic microtubulesin a variety of normal and transformed cell lines. This meth-od permits immediate resolution of microtubules by lightmicroscopy and allows for rapid analysis of microtubule dis-tribution in large populations of cells. Our findings are inagreement with those of Porter and coworkers (ref. 11 andK. R. Porter, personal communication) that an elaborate sys-tem of microtubules exists within the cytoplasm of mostnonmalignant cells in culture. All transformed cell lines ob-served by means of the immunofluorescent procedure dis-played a considerable reduction in the number of microtub-ules; this suggested an impairment in the ability of malig-nant cells to assemble cytoplasmic microtubules and main-tain a fibroblastic appearance. Partial restoration of the cy-toplasmic microtubule complex in viral transformed cellswas initiated by exposure of cells to dibutyryl-adenosine 3':5'-cyclic monophosphate (Bt2cAMP) plus testosterone ortheophylline. Thus, the capacity to maintain cytoplasmicmicrotubule assembly in cells appears to be directly relatedto 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 mono-layer 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 inHsu's modified McCoy's 5a medium supplemented with10% fetal calf serum. The PtK1, HSF-CF, HSF-Normal, andSV CF cells were grown as monolayers in Ham's F-10 medi-um (catalogue no. 7712-00, Schwartz/Mann Biochemicals)supplemented with 10% fetal calf serum. We trypsinizedcells from exponentially growing stock cultures, seeded ali-quots into 60 mm petri dishes and incubated the aliquots for24-48 hr to ensure an exponential growth phase. Whenchemicals were to be employed, the growth medium was as-pirated from the petri dish and the appropriate prewarmedmedia-chemical solution was added.The chemicals used in this study were N6,02-dibutyryl

adenosine 3':5'-cyclic monophosphoric acid monosodiumsalt, testosterone propionate (A4-androstene-17fl-propionate-3-one, Sigma Chemical Co.) and Colcemid (CIBA Pharma-ceutical Products, Inc.). Colcemid was used at a final con-centration of 0.06 jig/ml in complete medium. A working

4982 Cell Biology: Brinkley et al.

FIGS. 1-6. (1) 3T3 cells showing numerous cytoplasmic microtubules that radiate out from the nucleus and extend into the major cellprocesses. X600. (2) Higher magnification of 3T3 cell. Note microtubules that appear bent (thin arrow) and those that extend in parallelbundles near the cell surface (thick arrows X930). (3) SV 3T3 cell that appears smaller, rounded, and polygonal in shape. Microtubules areessentially absent in these cells, but nuclear staining persists. X600. (4) 3T3 cells treated with Colcemid (0.06 gg/ml for 2A hr). Cytoplasmis essentially free of fluorescent filaments and cells have become rounded and.knobby in appearance. X600. (5) SV 3T3 control cell treatedwith ethanol prior to staining. X600. (6) SV 3T3 cell treated with Bt2cAMP and testosterone in ethanol for 6 hr. Note fibroblastic appear-ance and numerous cytoplasmic microtubules. X600.

solution of Bt2cAMP in warm complete medium at a finalconcentration of 0.3 mM was prepared on the day of the ex-periment. From a stock solution of testosterone at a concen-tration of 1.5 mM in ethanol, an appropriate volume wasadded to the Bt2cAMP media to yield a final testosteroneconcentration of 0.015 mM. Theophylline (Sigma ChemicalCo.) was used at a final concentration of 1 mM. We used ascontrols cultures treated with ethanol alone to ensure thatnone 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-buff-ered saline and fixed with formaldehyde at concentrationsof 1, 2, or 3%, followed by acetone at -10°. The coverslipswere then incubated with the antibody (rabbit immunoglob-ulin, 0.2 mg/ml in phosphate-buffered saline) for 35 min at370. The preparations were rinsed again in the same bufferand then incubated in a 1:1.5 dilution of fluorescein-taggedgoat antiserum against rabbit immunoglobulin G (MeloyLaboratories, Springfield, Va.) and phosphate-buffered sa-

Proc. Nat. Acad. Sci. USA 72 (1975)

Proc. Nat. Acad. Sci. USA 72 (1975) 498.3

FIGS. 7 AND 8. (7) Metaphase, rat kangaroo fibroblast (strainPtK1). Cytoplasmic microtubules have all disappeared; fluores-cence has become concentrated in the spindle. X704. (8) Late tel-ophase of 3T3 cell with brightly fluorescent stem korper (arrow)extending between two daughter cells. Cytoplasmic microtubuleshave begun to reappear at opposite poles of cells near the centro-sphere. X704.

line. Finally, they were rinsed in the same buffer andmounted on glass slides in a drop of phosphate-buffered sa-

line:glycerol (1:9 adjusted to pH 8.5-10) for viewing in a

Leitz microscope adapted for darkfield ultraviolet microsco-py. Photographs were recorded on Kodak Tri-X Pan film.

RESULTS

The immunofluorescent staining pattern of 3T3 cells isshown in Figs. 1 and 2. The cytoplasm is laced with numer-

ous fluorescent filaments, presumably microtubules, whichradiate out from the nucleus and extend in parallel bundlesalong 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 thecell surface (thin arrow, Fig. 2) and in some instances bun-dles of filaments extend in parallel array along the cell mar-

gin (thick arrows, Fig. 2).Evidence that the fluorescent filaments seen in these

preparations represented intact microtubules came from ex-

perinents with microtubule inhibitors. When cells weretreated with Colcemid for 2 hr, the filaments essentially dis-appeared or became greatly diminished (Fig. 4). Concomi-tantly, the cells lost their fibroblastic shape and becamerounded and knobby in appearance. Exposure of cells tocold media (00 for 30 min) resulted in a similar disruption ofmost fluorescent filaments. Because both treatments areknown to disrupt microtubules (22), we can assume that thefluorescent filaments seen in the 3T3 cells are indeed cyto-plasmic microtubules. In this regard, the pattern of fluores-cence in 3T3 cells is essentially identical to the pattern ofmicrotubules seen in the cytoplasm of these cells by highvoltage electron microscopy (ref. 7 and K. R. Porter, person-al communication).The nucleus of most cells displayed fluorescent staining

either in the form of discrete patches or more generalizedstaining throughout the nucleus. Although the fluorescencewas more intense in cells fixed in 3% formaldehyde, stainingwas 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 ofstain. Although the nature of nuclear fluorescence is notknown, it was resistant to both Colcemid and cold treat-ments and was present in transformed cells as well as theirnormal counterparts.

Fate of Cytoplasmic Microtubules during Mitosis. In allcells observed, the extensive cytoplasmic microtubule com-plex disappeared during mitosis. The tubulin antibody im-munofluorescent staining properties of the mitotic spindlesof mammalian cells have been described elsewhere (12), andthe staining of spindles of normal and transformed cells inthe present study was essentially the same as described inour earlier study. However, as the cell entered early pro-phase, as evidenced by the condensation of chromatin, thecytoplasmic tubules began to disappear. By metaphase, mi-crotubules in the cytoplasm had completely disappeared,and most of the fluorescence had become localized withinthe mitotic spindle (Fig. 7). During the late telophase-earlyG1 phase, the cytoplasmic tubules reappeared in eachdaughter cell (Fig. 8). Initially, the microtubules formednear the centrosphere and then became distributed through-out the cell.Immunofluorescence of Transformed Cells. As shown in

Figs. 3 and 5, SV 3T3 cells have very few microtubules with-in their cytoplasm. Also, the cells are smaller than their nor-mal counterparts and assume a more polygonal shape, espe-cially when they become crowded. Essentially, the samestaining patterns and morphological features were apparentin all transformed lines included in this study (Table 1). Ineach population, the cytoplasmic microtubules were shorter,fewer in number, and randomly distributed throughout thecell. In all transformed cells studied, the sparsely distributedmicrotubules appeared to have no apparent relationship tothe shape or geometry of the cells. The paucity of cyto-plasmic microtubules in transformed cells, when used inconjunction with other morphological features, enabled us toidentify correctly transformed cells in cultures which con-tained known mixtures of transformed and nontransformedcells. In our experiments, 3T3 cells were mixed with SV 3T3cells in seven specific ratios and plated onto coverslips. Afterthe cells became attached, the coverslips were processed forantitubulin immunofluorescence and scored microscopicallyfor the number of cells with or without a cytoplasmic mi-crotubule complex. In double blind experiments, 500 cellswere scored in each mixture, and in five out of seven mix-tures the correct ratio was identified (Table 2).

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4984 Cell Biology: Brinkley et al.

Table 1. Immunofluorescentstaining properties of various cell lines

Cyto-plasmicmicro-

Trans- tubuleCell line Cell type formed complex

3T3 Swiss mouse em-bryo fibroblast - +

PtK1 Rat kangaroo fibro-blast - +

HSF-CF Human skin fibro-blast from cysticfibrosis patient - +

HSF-Normal Normal human skinfibroblast - +

SV CF Simian virus trans-formed humanskin fibroblastfrom cystic fibro-sis patient + -

HTC4 Rat hepatoma + -

6TG-1 1 Rat hepatoma + -

MMT Mouse mammarygland carcinoma + -

C6 Rat glial cell + -

A9 Mouse L cell(azaguanineresistant) + -

LM(TK -) Mouse L cell(BrdUrd resistant) + -

SV 3T3 Simian virus trans-formed 3T3 + -

RAG Mouse renal adeno-carcinoma + -

CHO Chinese hamsterovary + -

HeLa Human cervicalcarcinoma + -

+, presence of cytoplasmic microtubule complex (see Figs. 1 and2).-, 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-40virus; and the remaining transformed lines were spontaneoustransformants.

Cyclic Nucleotide Stimulation of Microtubules. To eval-uate the effect of cyclic nucleotide derivatives with our sys-tem, we stained both CHO and SV 3T3 cells for antitubulinimmunofluorescence after exposure to Bt2cAMP plus testos-terone or theophylline according to the procedures of Hsieand Puck (8) and Johnson et al. (9). Although we observeddramatic changes in morphology of both cell types aftertreatment, the most pronounced effect was apparent in theSV 3T3 cells. As shown in Fig. 6, the treated cells assumed amore fibroblastic appearance and displayed numerous fluo-rescent tubules within the cytoplasm. Thus, our results con-firm previous reports (4-6, 8) and establish that morphologi-cal changes stimulated by cAMP derivatives are due largelyto microtubule assembly.

DISCUSSIONThe simplicity of the antitubulin immunofluorescence pro-cedure permits immediate resolution of cytoplasmic mi-

Table 2. Microscopic identification oftransformed cells in mixed cell populations

Cells Cells Expected Observedwith lacking ratio ratio

Slide CMtC* CMtC (T/NT)t (T/NT)

A 499 1 1:0 1:0B 249 252 1:1 1:1C 71 437 1:5 1:6D 82 418 1:10 1:5E 2 498 0:1 0:1F 400 102 5:1 4:1G 469 31 10:1 15:1

Exponentially growing cultures of 3T3 and SV 3T3 cells weretrypsinized from the culture flask and maintained in suspension.We gently dispersed each suspension several times through a 25gauge needle to ensure singlet cells. The suspensions were countedin a Coulter counter, and the cultures were mixed appropriately toyield the desired ratio of 3T3 to SV 3T3 cells. We then plated thecells onto coverslips and incubated the coverslips with frequentagitation for 60 min to ensure attachment of single cells to the sub-strate. The coverslip preparations were stained and mounted ontoa glass slide. Double blind scoring of each slide was carried out witha Leitz ultraviolet microscope with a darkfield condenser and aZeiss 40x apochromatic objective.*CMtC, cytoplasmic microtubule complex (as shown in Figs. 1and 2).

tT/NT, ratio of transformed (SV 3T3) to nontransformed (3T3)cells.

crotubules in cultured mammalian cells. The images pro-duced by this technique correlate closely with transmissionelectron microscope images of cytoplasmic microtubules (7,11). Because the preparation can be viewed with conven-tional ultraviolet optics, large populations of cells can be ex-amined with relative ease and efficiency. Through use ofthis approach, we have identified an elaborate microtubularcomplex within the cytoplasm of normal cultured fibroblastswhich confirms in another way the observations of Porter(personal communication). The distribution and orientationof cytoplasmic microtubules conform to the major cell axes,and the tubules probably play an important role in themaintenance of cell shape, as suggested by previous investi-gators (1-3).The dissolution of the cytoplasmic microtubule network

during mitosis coincides with the loss of fibroblastic shape ofcells and the appearance of the mitotic apparatus. Fromthese observations, it seems likely that most of the tubulin di-mers of cytoplasmic microtubules are reutilized in the as-sembly of spindle microtubules. Reciprocally, the reappear-ance of cytoplasmic microtubules in late telophase-early Giphase suggests that spindle tubule subunits may then, inturn, be recycled into cytoplasmic microtubules. The appar-ent cyclic transition of cytoplasmic microtubules to spindletubules appears to be an important feature of normal prolif-erating mammalian cells, but is somehow lacking in trans-formed cells.The results of our experiments with mixed populations of

3T3 and SV 3T3 cells suggest that tubulin antibody immu-nofluorescence may be useful, in conjunction with othermorphological features, in identifying transformants in cul-tured cell populations. Transformed cells displayed few cy-toplasmic microtubules; the paucity of microtubules appearsto be related to the major morphological changes which ac-company transformation. Further relationship of the re-duced number of cytoplasmic microtubules to other familiartumor cell properties, such as cell surface changes and loss ofcontact inhibition, are also implied in the experiments pre-

Proc. Nat. Acad. Sci. USA 72 (1975)

Proc. Nat. Acad. Sci. USA 72 (1975) 4985

sented here. When SV 3T3 or CHO cells were exposed toBt2cAMP plus testosterone or theophylline, they underwenta morphological transition from a rounded, clumped ap-pearance to a more fibroblastic form containing numerouscytoplasmic microtubules. Although we did not evaluate thegrowth characteristic of our cells, similar treatment of spon-taneous transformants of 3T3 (strain 3T6) and polyoma virustransformants (strain PyV-3T3) led to the restoration of con-tact inhibited growth (10).The loss of the cytoplasmic microtubule complex in viral

transformed cells suggests that conditions that restrict mi-crotubule assembly exist in these cells. Factors controllingmicrotubule assembly both in vitro and in vivo are beingidentified by numerous investigators (3, 13-17, 19). Cyclicnucleotides, including cAMP and guanosine 3':5'-cyclic mo-nophosphate (cGMP), have been implicated in regulatingmicrotubule assembly (23, 24). Weisenberg's discovery (15)of the inhibitory role of Ca++ in the bovine brain tubulin as-sembly system suggests that a similar regulatory functioncould exist within intact cells, although direct evidence islacking. If Ca++ is a natural regulator of assembly, cyclicnucleotides could function in turn to regulate Ca++ fluxwithin various cellular compartments (18). Recently, Olm-stead and Borisy (19) suggested that a regulatory role byCa++ in tubule assembly be viewed with caution since theirdata showed magnesium-GTP association involved in tubu-lin 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 thefindings of the present study. Thus, the diminished numberof cytoplasmic microtubules in transformed cells is appar-ently due in part to low levels of cAMP in these cells. Theprecise role of cyclic nucleotides in regulating cytoplasmicmicrotubule assembly is yet to be determined.

Finally, our observations permit a distinction to be madebetween the cytoplasmic microtubule complex of interphasecells and the microtubules of the mitotic apparatus. Electronmicroscope studies show that the morphology of the tubulesin the two systems appears identical (22). Because the cyto-plasmic microtubules disappear with the onset of spindleformation and vice yersa, it is likely that tubulin componentsof one system can be recycled into the other. If so, control oftubule assembly in the two systems appears to be under sep-arate constraints. Thus, from studies with immunofluores-cent staining it is apparent that there is no defect in the as-sembly of the mitotic spindle in transformed cells, yet thecytoplasmic microtubule complex is greatly diminished.Treatment of transformed cells with Bt2cAMP stimulates thecytoplasmic microtubule assembly and restores the normalfibroblastic appearance of the cells. Therefore, we believethat the cytoplasmic microtubule complex should be viewedas a separate entity from the mitotic spindle. The role of thiscomplex in the maintenance of cell form, as well as themany other functions attributed to cytoplasmic microtub-ules, merit further study. The loss of a cytoplasmic microtu-bule complex seems to be a common feature of transformedcells that may account for many of the characteristic mor-phological and growth related properties in malignant cells.

In an earlier study, Weber et al. (25) found that antibodymade against tubulin from the outer doublets of sea urchinsperm flagella decorated a reticulum of fine filaments in thecytoplasm of cells derived from a variety of sources. The im-ages of normal 3T3 cells reported in our study are essentiallyidentical to those presented by Weber and co-workers.Transformed cells from many sources, however, display agreatly diminished cytoplasmic microtubule complex.

We are grateful to Dr. Robert Klebe for providing many of thetransformed cell lines used in this study. We thank Ms. Joan Ellisonfor excellent technical assistance in antibody preparation. Apprecia-tion is extended to Mrs. Shirley Brinkley for editorial assistance.This work was supported in part by NCI Grant CA 14675 and agrant from the Dow Chemical Co.

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