Proc. Natl. Acad. Sci. USA Vol. 74, No. 8, pp. 3433-3437; August 1977

Cell Biology

Changes in surface properties of normal and transformed cells caused by tunicamycin, an inhibitor of protein glycosylation (cell-surface glycoproteins/cell adhesion/lectins/agglutinability)

DAN DUKSIN AND PAUL BORNSTEIN Departments of Biochemistry and Medicine, University of Washington, Seattle, Washington 98195

Communicated by Edmond H. Fischer, May 23,1977

ABSTRACT Normal and virally transformed mouse (3T3) and human (WI-38) cells were treated with tunicamycin, an inhibitor of lipid-carrier-dependent glycosylation of proteins. Incubation of cells with tunicamycin (1 jug/ml) caused detachment and death of simian virus 40- and polyoma-transformed cells within 24 hr; these effects were not seen with nontransformed cell lines. However, the proliferation of 3T3 cells was inhibited by tunicamycin and, after a few days, a distinct change from an epithelioid to an abnormally elongated shape was observed. Both inhibition of growth and the morphological changes were reversible. A marked decrease in concanavalin A agglutinability was observed in virally transformed cells treated with tunicamycin (0.5 gg/ml), but agglutination by wheat germ agglutinin or soybean agglutinin was unaffected. Analysis of biosynthetically labeled proteins showed that a high-molecular-weight protein, presumed to be related to fibronectin, is markedly reduced in the medium of cells cultured in the presence of tunicamycin. These results suggest that tunicamycin interferes with the insertion or function of one or more cell-surface glycoproteins. Su~,h cell-surface changes could affect a number of cellular properties, including attachment, cell shape, and agglutinabilityby some lectins.

Many aspects of the social behavior of cells are influenced by the composition, arrangement, and interaction of cell-surface macromolecules (1). Thus, alterations in plasma membrane composition and structure in malignant and transformed cells, compared with normal cells, appear to contribute to differences in such characteristics as cell adhesion, contact inhibition of movement, and tumorogenicity (2). Cell-surface glycoproteins, in particular, participate in a number of membrane-modulated phenomena, including responsiveness to hormones, agglutination by lectins, and recognition by antibodies (3, 4); these properties are frequently altered following transformation (5, 6). Extensive studies have been made of cell-surface glycoproteins and of the alterations that occur after transformation (5, 6), but little is known about the function of the carbohydrate moieties in these proteins or of the significance of transformation-associated changes in their structure (7-9). A prominent finding in transformed cells has been the absence or marked reduction of a high-molecular-weight cell-surface glycoprotein variously known as LETS protein, fibroblast surface antigen, or fibronectin (6). We have examined the influence of tunicamycin, an antibiotic that inhibits protein glycosylation (10-12), on the behavior of normal and virally transformed cells in culture. Tunicamycin blocks the transfer of GlcNAc from UDP-GIcNAc to a polyisoprenyl lipid carrier (13-15). Consequently, synthesis and transfer of the core oligosaccharide to asparaginyl side The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

chains in glycoproteins is inhibited (16). In the presence of tunicamycin, a striking cytotoxic effect and a reduction in agglutinability with concanavalin A (Con A) were observed with transformed cells. Normal cells, on the other hand, were inhibited in their growth and developed an altered morphology (17). We suggest that some of these effects result from impaired synthesis or secretion of cell-surface glycoproteins or from insertion of carbohydrate-poor glycoproteins in the cell membrane. The differential effect of tunicamycin on normal and transformed cells may be a consequence of the altered metabolism of membrane glycoproteins in the latter cells, a state which is reflected in the altered morphology, adhesiveness, and other properties characteristic of viral transformation. MATERIALS AND METHODS Tunicamycin (batch T-12-06) was a gift from Gakuzo Tamura, Department of Agricultural Chemistry, University of Tokyo. Trypsin (1:250) was from ICN Pharmaceuticals, Inc.; L-[U14C]proline (200 Ci/mol), L-[3,5-3H]tyrosine (50 Ci/mmol), and [G-3H]-concanavalin A ([3H]Con A) (40.9 Ci/mmol) were from New England Nuclear; D-[2-3H]mannose (2 Ci/mol) was from Amersham/Searle Co. Con A and wheat germ agglutinin (WGA) were from Miles-Yeda, and soybean agglutinin (SBA) was a gift from Nathan Sharon, Department of Biophysics, The Weizmann Institute of Science. Cell Lines and Cell Culture. 3T3 cells, WI-38 cells, and WI-38 cells transformed by simian virus 40 (SV40-WI38 or WI-38VA13, subline 2RA) were obtained from the American Type Culture Collection. SV40-transformed (SV4O-3T3) and polyoma virus-transformed (Py-3T3) 3T3 cells were obtained from John M. Keller, Department of Biochemistry, Chicago Medical School. Cells were grown as described previously (12). Cultures were transferred every 3-4 days by trypsinization with 0.25% trypsin and seeded at a concentration of 5 X 105 cells per 60-mm plate. Growth curves were determined by counting viable cells (those that excluded 0.02% trypan blue) in a hemocytometer. The values reported are the means obtained by counting triplicate plates twice. Agglutination and [3H]Con A Binding to Cells. Agglutination tests were performed on tunicamycin-treated (0.5 ,ug/ml for 24 hr) and control cells. The attached cells tested for agglutinability were viable by dye exclusion test. Subsequently, the cells were detached from the surface with either trypsin or EDTA solutions (18) and washed three times with Ca2+- and Mg2+-free phosphate-buffered saline. The cells were suspended in this buffer at 2 X 106 cells per ml and incubated with gentle shaking at room temperature for 30 min with 5-500,ug/ml of the lectins Con A, SBA, or WGA. At the end of the incubation period, the appearance of the cells was observed with a binocAbbreviations: Con A, concanavalin A; SBA, soybean agglutinin; WGA, wheat germ agglutinin; SV40, simian virus 40; Py, polyoma virus.

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Proc. Nati. Acad. Sci. USA 74 (1977)

Cell Biology: Duksin and Bornstein

3434

Table 1. Incorporation of proline and mannose into trichloroacetic acid-insoluble fractions in the presence of tunicamycin

L-['4C] Proline Culture

Cell line 3T3

Py-3T3 WI38

SV40-WI38

Total incorporation, dpm/mg cell protein X 10-3

D-[2-3H]Mannose Inhibition,

Total incorporation, dpm/mg cell protein X 10-3

Inhibition,

fraction

Control

Tunicamycin*

%

Control

Tunicanycin*

%

Medium Cell layer Medium Cell layer Medium Cell layer Medium Cell layer

9.2 87 35 75 6.8 47.4 5 130

6 47 14 42 4.5 41.6 2 62

35 46 60 44 34 22 60 52

25 160 725 360 17 510 264 1533

0.25 1 7.3 17 0.5 28 13 134

99 99 99 95 97 95 95 91

* Cultures were preincubated for 4 hr in the presence of tunicamycin at 0.5 jg/ml, washed, and labeled for 2 hr with radioactive proline and mannose.

ular microscope. Suspensions of cells (2 X 106 cells per ml), treated with tunicamycin or control cultures, were incubated with [3HJCon A at room temperature with gentle shaking for 30 min. The cells were washed four times with phosphatebuffered saline and assayed for radioactivity in a liquid scintillation spectrometer. Methyl a-D-mannoside (0.01 M; Sigma) or nonradioactive Con A at 50,ug/ml was added together with [3H]Con A to inhibit binding. Subsaturating concentrations of [3HICon A were used to distinguish small changes in 3H binding. Incorporation of Amino Acids and Sugars. The effect of tunicamycin on the incorporation of ['4C]proline and D-[23Hlmannose into macromolecules by cells in culture was tested as described previously (12). Briefly, cell cultures were preincubated in the absence or presence of tunicamycin (0.5 jug/ml) for 4 hr. The cultures were then labeled, in the absence or presence of tunicamycin, for 2 hr with D-[2-3H]mannose at 2 jCi/ml and ['4C]proline at 0.2 ,Ci/ml in serum-free Dulbecco-modified Eagle's medium that was supplemented with sodium ascorbate, glutamine, (f-aminopropionitrile, and sodium pyruvate (10 mM), but contained a reduced glucose concentration (50 mg/liter) (19, 20). Total incorporation into trichloroacetic acid-insoluble material in medium and cell layer was measured as described earlier (12). Counting efficiencies were 32% for 3H and 66% for 14C. Total protein was measured by a modification (21) of the Lowry method, using bovine serum albumin as a standard. Slab Gel Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis. 3T3, SV40-3T3, and Py-3T3 cells were preincubated for 4 hr in 5 ml of serum-free, tyrosine-free, Dulbecco-modified Eagle's medium or in the same medium containing tunicamycin at 0.2 ,ug/ml. The cultures were washed, medium containing Trasylol (Calbiochem) at 20 units/ml and L-[3,5-3Hjtyrosine at 12.5 ,Ci/ml and having a reduced tyrosine content (0.04 mM) was added, and the cultures were further incubated for 24 hr in the absence or presence of tunicamycin. At the end of the incubation period, the culture media were collected and protein was precipitated with trichoroacetic acid (10% final concentration). Medium proteins were examined by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis in gels containing 0.5 M urea (22, 23). After electrophoresis, slabs were permeated with dimethyl sulfoxide and 2,5-diphenyloxazole for fluorescent autoradiography (24). Gels were then dried on paper and exposed at -700 to RP Royal X-Omat film (Kodak) previously sensitized for quantitation of radioactivity (25).

RESULTS Effect of Tunicamycin on Protein Synthesis. When normal and transformed cells were incubated in the presence of tunicamycin, there was a marked inhibition of incorporation of D-[3H~mannose into macromolecules, both in the culture medium and in the cell layer (Table 1). The small residual incorporation may reflect metabolic conversion of mannose to fucose (which retains the 3H label) and assembly of mannose-containing oligosaccharide side chains whose synthesis is insensitive to tunicamycin (see Discussion). The incorporation of proline into macromolecules was decreased to a lesser extent, by-about 3040%o in the normal cells (3T3 and WI-38) and by 50-60% in the transformed lines (Table 1). This reduction cannot be attributed to changes in amino acid transport, because uptake of the radioactive label by cells was not affected by tunicamycin (data not shown). Effect of Tunicamycin on Cell Viability and Morphology. Higher levels of tunicamycin (1 ,ug/ml) produced a marked decrease in the rate of growth of 3T3 cells (Fig. 1) but caused little or no cell death in either sparse or confluent cultures after 107

3T3

SV40-3T3

Py-3T3

7,

I0

.

E z

Days in culture

FIG. 1. Growth curves of cells treated with tunicamycin. Cells were grown in Dulbecco-modified Eagle's medium supplemented with 10% fetal calf serum and treated with tunicamycin (1 /Ag/ml; arrows pointing down). In the case of 3T3 cells, tunicamycin-containing medium was replaced by fresh medium (arrow pointing up). Cells were counted in the presence of trypan blue in a hemocytometer. *-*, Growth in the absence of tunicamycin; O--- - -0, growth in the presence of tunicamycin.

Cell Biology: Duksin and Bomstein

Proc. Natl. Acad. Sci. USA 74 (1977)

3435

.I

in. I

C

*.4

.wb .A~

a it

Jx

s~ ~ ~ ~ ~ ~ ~ ~ M

,I__g

A=b 0O

FIG. 3. Morphological changes caused by tunicamycin. 3T3 cells incubated with tunicamycin (1 ug/ml) for 2 days (a), 3 days (b), 4 days (c), and 4 days with tunicamycin followed by 2 days in fresh medium (d). (X70.) were

,1~f. -W_Alt e W.%O f I4 FIG. 2. Differential cytotoxic activity of tunicamycin. (a and b) 3T3 cells; (c and d) SV40-transformed 3T3 cells; (e and f) polyoma virus-transformed 3T3 cells. (a, c, and e) Control cultures; (b, d, and f) cultures treated with tunicamycin (1 Ag/ml) for 24 hr in Dulbecco-inodified Eagle's medium supplemented with 10% fetal calf serum. (X70.) 24 hr. When tunicamycin was removed, 3T3 cells resumed their normal rate of growth (Fig. 1). In contrast, distinct cytotoxicity was observed in SV40- and polyoma-transformed 3T3 cells (Figs. 1 and 2). Similar cytotoxicity was observed in SV40transformed WI-38 cells. Transformed cells, but not normal 3T3 cells, when treated with tunicamycin became rounded and then detached from the surface (Fig. 2). The detached cells stained with trypan blue and did not attach after seeding in fresh medium. While only about 90% of transformed cells were killed by tunicamycin, the surviving cells were not genetically resistant to the drug, because retreatment, after plating and growth of survivors, again produced an equivalent degree of cytotoxicity.

When 3T3 cells were exposed to tunicamycin for a longer period (2-4 days), they underwent striking changes from an epithelioid to an elongated and spindle-shaped morphology (Fig. 3). This alteration in morphology was accompanied by decreased adhesiveness to the surface of the plate, although the 3T3 cells did not detach. These changes were slowly reversed by removal of tunicamycin (Fig. 3d); the morphological reversioh was accompanied by a resumption in growth (Fig. 1). Both 3T3 and transformed 3T3 cells, exposed to low concentrations of tunicamycin for 24 hr, developed extensive surface ruffles and blebs visible by scanning electron microscopy. * In order to determine whether a reduction in protein synthesis, per se, was responsible for the cytotoxicity and morphological changes observed with tunicamycin, cells were treated with low concentrations (0.1 sg/ml) of cycloheximide. This level of cycloheximide produced the same degree of inhibition of protein synthesis (30-50%), but no changes in morphology, adhesiveness, or agglutinability. Agglutination. The agglutinability of normal and transformed cells by Con A, WGA, and SBA was tested after treatment with tunicamycin (0.5 ,qg/ml). Trypsinized or EDTA*

D. Duksin, K. Holbrook, and P. Bornstein, unpublished data.

detached, virally transformed cells showed a marked reduction in agglutination by Con A (Table 2). Tunicamycin-treated SV40-transformed 3T3 or WI-38 cells and Py-3T3 cells failed to agglutinate until Con A concentrations of 500-1000 ,ug/ml were used, whereas similar agglutination in control cells required Con A at only 5-10 yg/ml. This inhibition was not due to a direct effect of tunicamycin, because transformed cells agglutinated normally when tunicamycin, even at concentrations of 10 ,ig/ml, was added at the time of agglutination. The agglutination of 3T3 cells, detached from plates by trypsin or EDTA treatment, was unchanged by treatment with tunicamycin (Table 2). Similarly, tunicamycin did not affect the agglutination of normal or virally transformed cells by WGA or SBA (Table 2). Binding of [3HJCon A to Tunicamycin-Treated Cells. To determine whether the lack of agglutinability of transformed cells by Con A resulted from a lack of binding of the lectin, studies with [3H]Con A were performed. Both tunicamycintreated 3T3 and virally transformed 3T3 cells bound approximately 20-30% less [3H]Con A than did control cells (Fig. 4). The binding was specific, because it was inhibited by methyl a-D-mannopyranoside (0.01 M) and by Con A (50' g/ml). Thus, the differences in Con A agglutinability of tunicamyTable 2. Agglutinability of tunicamycin-treated cell lines by lectins*

Cells 3T3

SV4O-3T3 Py-3T3

W138

SV40-WI38

Tunicamycint

Con A

Lectin WGA

SBA

-

+++

++

++

+

+++

++

++

-

++++

+++

+++

+

-

+++

+++

-

++++

+++

+++

+

-

+++

+++

-

+++

++

+++

+

+++

++

+++

-

++++

+++

+++

+

-

+++

++

* Agglutination was estimated visually using a five-point scale: -, no agglutination, to ++++, maximal agglutination. Cells were at 2 X 106 per ml. Lectin concentrations were: Con A, 250 gg/ml; WGA, 5 jig/ml; SBA, 10 gg/ml. t Cells were treated for 24 hr with tunicamycin at 0.5 jig/ml prior to. the agglutination test.

3436

Proc. Nati. Acad. Sci. USA 74 (1977)

Cell Biology: Duksin and Bornstein

2

1 C

3

4

5

6

-0

oz114 l o

of0S

-

---~~~~~~~~~~~~6 .4

d'~~~~~~~~0 cells 102Aj OO uiayi 05z/l-rae 104

10

['HI Con FIG. 4.

cells;

A added,

el.Clswr

106, dpmn/l06

e 106

.

-

......

cells

Binding of [3HJCon A to cultured cells. @-@, Control -0, tunicamycin (0.5 jig/ml)-treated cells. Cells were re-

moved from the dishes by trypsinization, washed, and incubated with increasing amounts of [3H]Con A. The cells were then washed again and radioactivity was measured.

cin-treated normal and transformed cells are not due to gross differences in binding of the lectin. A subset of Con A-binding proteins may be deleted in tunicamycin-treated transformed cells or intracellular changes may be responsible for the differences in agglutination. Labeled Proteins from Tunicamycin-Treated 3T3 and SV40-Transformed 3T3 Cells. [3H]Tyrosine-labeled proteins in the culture medium of tunicamycin-treated and control 3T3, SV40-3T3, and Py-3T3 cells were examined by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis (Fig. 5). A selective reduction was observed in a number of secreted proteins, primarily in a high-molecular-weight (230,000) glycoprotein known to be a cell-surface component by external labeling (26). Similar results were observed when [3H]proline-labeled proteins were studied (data not shown). DISCUSSION Treatment of normal and virally transformed cells with tunicamycin has been shown to have the following effects: (i) inhibition of growth of normal cells (Fig. 1) and cytotoxicity of transformed cells (Figs. 1 and 2); (ii) reduced adhesion to a substratum, and, in normal 3T3 cells after extended exposure, profound morphological changes (Fig. 3); (iii) reduced agglutination of transformed cells by Con A (Table 1); and (iv) a reduction in the secretion of several proteins, including a high-molecular-weight glycoprotein, probably related to fibronectin (Fig. 5). Many of these effects can be attributed to the inhibition of synthesis and secretion, or the failure of insertion, of functionally normal fibronectin and other glycoproteins in the cell surface. Tunicamycin has been shown to inhibit the synthesis of Nacetylglucosaminylpyrophosphorylpolyisoprenol (15), and thus the further synthesis and transfer of oligosaccharides that are initiated by a chitobiose unit, to asparaginyl side chains in glycoproteins (27). It is currently thought that addition of sugars to the N-acetylglucosamine-lipid carrier is not affected by tunicamycin (13, 15). In yeast, synthesis of oligosaccharide chains linked to seryl or threonyl residues also appears to involve a lipid carrier (27), but an effect of tunicamycin on the process has not been established. The fate of glycoproteins synthesized in the presence of tunicamycin may vary. Some, such as procollagen, may be secreted in an underglycosylated form (12). In the case of others, such as fibronectin, secretion, and possibly also synthesis, may be hampered by lack of the oligosaccharide chains (12). Alternatively, fibronectin synthesized and secreted in the presence of tunicamycin may be unstable and highly susceptible to proteolytic degradation. We have recently found that lacto-

FIG. 5. Composite fluorescent autoradiogram of [3H]tyrosirelabeled proteins isolated from medium of cultured 3T3 and virally transformed 3T3 cells. Proteins were electrophoresed on dodecyl sulfate/acrylamide slab gels under reducing conditions. Lane 1, 3T3 control; 2, tunicamycin (0.2 gg/ml)-treated 3T3; 3, SV40-3T3 control; 4, tunicamycin-treated SV40-3T3; 5, Py-3T3 control; 6, tunicamycin-treated Py-3T3. The arrow indicates the position of migration of fibronectin.

peroxidase-catalyzed iodination of cell-surface fibronectin on 3T3 cells is markedly reduced by tunicamycin. * A number of recent findings implicate membrane glycoproteins in general, and fibronectin more specifically, in maintenance of normal cell-surface functions. Reduced adhesiveness and contact inhibition of movement, as well as morphological changes, were observed in mutant 3T3 cells that lacked the ability to synthesize N-acetylglucosamine (28, 29). Some of these mutant 3T3 cells, selected for reduced adhesiveness, have been shown to have reduced levels of a number of cell-surface proteins, including fibronectin (29). Altered cyclic AMP levels, which are also known to affect adhesiveness of cells (30), do not appear to be responsible for the reduced adhesiveness of these cells (28). Cell extracts containing fibronectin, added to cultures of such mutant 3T3 cells or to virally transformed cells, which in general possess low levels of surface fibronectin (5, 6), partially restored adhesiveness, contact inhibition of movement, and normal morphology to these cells (31). Finally, baby hamster kidney (BHK) fibroblasts (32) and Chinese hamster ovary (CHO) cells (33), which are resistant to the cytotoxic effects of ricin and WGA, respectively, showed morphological changes and reduced adhesiveness in comparison with the parent cell lines. A number of different defects in oligosaccharide synthesis have been identified in different clones of these cells (32, 33) and several of the ricin-resistant fibroblast clones have been shown to be deficient in cell-surface fibronectin (32). We suggest that the reduced adhesion to a substratum and the morphological changes caused by tunicamycin result, in part, from reduced synthesis and secretion or secretion of a carbohydrate-poor, functionally defective fibronectin. The greater sensitivity of virally transformed cells to the drug may be due to the marked preexisting deficiency of cell-surface fibronectin in transformed cells (34, 35). However, the differential cytotoxicity observed with transformed cells in the presence of higher concentrations of tunicamycin (Fig. 2) is probably not the consequence solely of detachment, because the growth of many SV40-transformed 3T3 cells was shown to be anchorage independent (36). Similarly, this effect cannot be explained by the partial inhibition of protein synthesis caused

Cell Biol

:

Duksin an Bornstein

by tunicamycin (Table 1, see Results). The use of tunia i as an antitumor agent in the investigation of the neoplastic process therefore deserves further study, although its broad effects on normal cells probably preclude its clinical application. The marked reduction in Con A agglutinability of transformed cells caused by tunicamycin may also be due to inhibition of synthesis of carbohydrate side chains in specific Con A-binding proteins. However, the basis for the modulation of lectin agglutination of cells is clearly complex. The agglutination of 3T3 cells, which were equally susceptible to inhibition of glycosylation by tunicamycin (Table 1), was unaffected by the drug (Table 2). ATP levels, the degree of organization of cortical cytoplasmic microfilaments and microtubules, and other factors regulating membrane fluidity may all contribute to the agglutination of cells by some lectins (37), and these factors may differ in normal and transformed cells. It is interesting to note that the agglutinability of transformed cells by WGA and SBA was unaffected by tunicamycin (Table 2). There is some evidence to indicate that agglutination of cells by these lectins is less dependent on ATP levels or on the degree of membrane fluidity than is agglutination by Con A (38). In preliminary experiments, tunicamycin inhibited mitogenesis of lymphocytes by Con A without changing [3H]Con A binding (unpublished observation). The drug may therefore be of considerable use in probing the chemical nature of cell surface receptors and the role of membrane glycoproteins in a wide variety of cell-surface functions. Note Added in Proof. After this paper was submitted for publication, we were informed of the existence of an abstract in the Japanese literature [Takatsuki, A., Nishimura, M., Kohno, K., Onodera, K. & Tamura, G. (1976) Abstracts of the Annual Meetings of the Agricultural Chemical Society of Japan, p. 131] indicating a differential effect of tunicamycin on normal (C3H and WIRL) and SV4O-transformed (SV-C3H and SV-WIRL) cells.

The excellent technical assistance of Ms. Kathleen Williams-Geiger is gratefully acknowledged. We thank Dr. G. Tamura for his generous gift of tunicamycin. We also thank Dr. Peter Byers for his helpful discussions and critical reading of the manuscript. This work was supported by National Institutes of Health Grants DE-02600 and AM-11248. 1. Edelman, G. M. (1976) Science 192, 218-226. 2. Dulbecco, R. (1970) Nature 227, 802-806. 3. Lis, H. & Sharon, N. (1973) Annu. Rev. Biochem. 43, 541574. 4. Cuatrecasas, P. & Hollenberg, M. D. (1976) Adv. Protein Chem. 30, 251-451. 5. Nicolson, G. L. (1976) Biochim. Biophys. Acta 458, 1-72. 6. Hynes, R. 0. (1976) Biochim. Blophys. Acta 458,73-107.

Proc. Natl. Acad. Sci. USA 74 (1977)

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