EXPERIMENTAL

CELL RRSRARCH

189,13-21(1990)

The Bimodal Growth Response of Swiss 3T3 Cells to the B Subunit of Cholera Toxin Is Independent of the Density of Its Receptor, Ganglioside GM1 ’ NANCY E. BUCKLEY, GARY R. MATYAS**~ AND SARAH SPIEGEL~ Department of Biochemistry and Molecuhr Biology, Georgetown University Medical Center, Washington, D.C. 20007; and *Membrane Biochemistry Section, Laboratory of Molecular and Cellulnr Neurobiology, National Institute of Neurological and Communicative Disorders and Stroke, The National Institutes of Health, Bethesda, Maryland 20892

The B subunit of cholera toxin, a protein which binds specifically to cell surface ganglioside GMl, has been shown to have a bimodal effect on DNA synthesis in Swiss 3T3 fibroblasts. The B subunit induced cellular proliferation of confluent and quiescent cells while it inhibited the growth of the same cells when they were sparse and rapidly dividing. The amount of cell surface GM1 increased when the cells reached confluency. To examine the hypothesis that the variation in levels of GM1 was responsible for the bimodal effect, we increased GM1 levels in rapidly dividing cells by insertion of exogenous GM1 or by treatment of the cells with neuraminidase to convert polysialogangliosides to GMl. Even after the level of GM1 was increased to levels similar to those found in confluent cells, the B subunit still inhibited, rather than stimulated, their growth. Therefore, this result indicates that the bimodal response to the B subunit is not solely a function of the concentration of cell surface GMl; rather it is the growth stage that determines the fate of the signal transduced by the interaction of the B subunit and ganglioside GM 1. 8 1990 Academic Press, Inc.

INTRODUCI’ION Gangliosides, sialic acid containing glycosphingolipids, are ubiquitous components of the plasma membrane of mammalian cells [l] whose functions have still not been completely elucidated. There is evidence suggesting that gangliosides may play an important role in the regulation of transmembrane signaling and cellular proliferation. Changes in ganglioside composition and me1 This work was supported by Research Grant 1 R 29 GM 39718-02 from the National Institutes of Health. s Present address: Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, DC 20307. 3 To whom reprint requests should be addressed at Department of Biochemistry, Georgetown University Medical Center, 357 Basic Science Building, 3900 Reservoir Road NW, Washington, DC 20007.

tabolism have been observed during differentiation, oncogenic transformation and tumor progression, and density-dependent growth inhibition (reviewed in Refs. [24]). Recently, it has been shown that ganglioside GM3 undergoes growth-regulated turnover [5] and it has been suggested that an extracellular or membrane-bound sialidase may regulate the levels of GM3 [6]. Additionally, insertion of exogenous gangliosides or inhibition of their biosynthesis leads to alterations of cell growth and differentiation [3,5-121. Attempts have been made to determine whether endogenous gangliosides have physiological functions by the use of antibodies against gangliosides [ 13-161 or the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 [17, IS]. Initially, we found that the B subunit induces mitogenesis in rat thymocytes that is dependent on the direct interaction between the B subunit and ganglioside GM1 on the cell surface [ 171. The B subunit also has immunomodulatory properties as it potentiates the thymus-dependent antibody response [ 191. The ability of the B subunit to stimulate resting cells to divide is not limited only to lymphocytes as the B subunit also stimulates DNA synthesis and cell division in quiescent cultures of murine 3T3 fibroblasts (NIH 3T3, Balb/C 3T3, and Swiss 3T3) [l&20,21]. Extension of these studies to transformed cells revealed that in contrast to its effect on resting cells, the B subunit inhibits the growth of ras-transformed 3T3 fibroblasts (Ha-, Ki-, and N-ras) [ l&20,22 ] and C6 cells with elevated levels of GM1 [ 23 J. Recently, these studies were confirmed and further extended by showing that neuraminidase treatment of C6 cells which increases the amount of cell surface ganglioside GM1 also renders the cells susceptible to the inhibitory effect of the B subunit [24]. Furthermore, in astroglial cells, the B subunit not only inhibits their growth but induces marked differentiation [25]. Experiments with growing, normal, or untransformed 3T3 cells demonstrated that a bifunctional response pattern to the B subunit can be observed in the same cell line depending on their growth state [ 18,20 3. The B sub13

0014-4827/90 $3.00 Copyright 8 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.

14

BUCKLEY,

MATYAS,

unit stimulates DNA synthesis and cell division in confluent and quiescent 3T3 fibroblasts while inhibiting the growth of the cells when they are sparse and rapidly dividing. The biphasic response to the B subunit by the same 3T3 fibroblasts raised the possibility that endogenous gangliosides GM1 could function as bimodal regulators of cell growth signals. On the basis of the observation that the levels of cell surface GM1 and GDla are reduced in the growing ras-transfected cells and are increased as the untransfected NIH 3T3 cells become contact-inhibited [26], it has been suggested that the ability of ganglioside GM1 to modulate positive and negative signals may depend on its density on the cell surface. Our goals in this study were twofold: first, to quantitatively determine cell surface gangliosides, and in particular GMl, in Swiss 3T3 cells as a function of time in culture from initial plating to confluency; and second, to determine if there is a correlation between the amount of GM1 on the cell surface and the bimodal response to the B subunit. EXPERIMENTAL

PROCEDURES

Materials. Silica gel 60-coated glass plates and aluminum sheets were from E. Merck. Vibrio cholerae sialidase (EC 3.2.1.18) was from Calbiochem-Behring Corp., (La Jolla, CA). ‘2sI-labeled cholera toxin was prepared using the chloramine-T procedure [27]. GM3 and GM2 were from dog erythrocytes and Tay Sachs brain, respectively; GM1 and GDla were from bovine brain, all isolated as described previously [28]. The B subunit of CT was purchased from Schwarz/Mann Biotech (Cleveland, OH). CT was from List Biological Labs (Campbell, CA). [nethyl-3H]Thymidine (55 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). Insulin, bombesin, and transferrin were from Collaborative Research (Lexington, MA). DMEM, PBS,’ and Waymouth medium were obtained from local sources [29]. Cell culture. Swiss 3T3 fibroblasts were from the American Type Culture Collection. Stock cultures of cells were maintained as described previously [29] in DMEM supplemented with 2 n&f glutamate, 2 mMpyruvate, penicillin (100 units/ml), streptomycin (100 pg/ ml), and 10% FCS, in a humidified atmosphere of 5% COz/95% air at 37°C [21]. Measurement of DNA synthesis in quiescent 3T3 cells. Swiss 3T3 cells were subcultured at a density of 1.5 X 10’ cells/cm’, refed with the same medium described above after 2 days and were used 5 days later when the cells were confluent and quiescent [21,30]. Cells were washed with DMEM to remove residual serum and 2 ml of DMEM/ Waymouth medium (l/l) supplemented with 20 pg/ml BSA and 5 r(pl ml transferrin were added [21]. Where indicated, the cells were treated with various growth factors or the B subunit of CT. After 18 h, the cells were pulsed with 0.5 &i of [3H]thymidine for 6 hand the incorporation of radioactivity into trichloroacetic acid-insoluble material was measured as described [ 201. Measurement of DNA synthesis in exponentiully growing 3T3 cells. Swiss 3T3 fibroblasts were subcultured at a density of 2 X lo3

4 Abbreviations used: EGF, epidermal growth factor; BSA, bovine serum albumin; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; CT, cholera toxin; PBS, phosphate-buffered saline; TPA, 120-tetradecanoylphorbol-13-acetate; Hepes, I-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The ganglioside nomenclature is used according to Svennerholm’s system [43].

AND SPIEGEL

cells/cm’ in DMEM containing 10% FCS. After 2 days, unless indicated differently, the cells were washed with DMEM, incubated in DMEM/Waymouth medium (l/l) and treated with various growth factors or the B subunit exactly as described above. After the last 18 h of treatment, DNA synthesis was measured as described above. Treatment of Swiss 3T3 cells with GMl. Cells were seeded in 35mm dishes at a density of 2 X lo3 cells/cm2 in DMEM containing 10% FCS as described above. After 2 days, the cells were incubated in the absence or in the presence of 10 FM GM1 at 37°C for 6 h. The cells were extensively washed with DMEM to remove residual GMl, incubated in DMEM/Waymouth medium (l/l), and treated with various growth factors or the B subunit exactly as described above. A different method to increase GM1 was also utilized. Cells were grown to confluency in 75-cm2 flasks, washed with DMEM, and then incubated without or with 5 &f GM1 at 37°C [31]. After 6 h the cells were washed with DMEM, trypsinized, and subcultured into 35-mm dishes at a density of 1.6 X 10’ cells/cm*. The cells were allowed to attach to the dish overnight in the presence of DMEM containing 10% FCS. The cells were then washed with DMEM, incubated in DMEM/Waymouth medium (l/l) and treated with various growth factors or the B subunit also as described above. Isolation and analysis of gangliosides. Fibroblasts were washed with ice-cold PBS (without Ca*+ and Mg2+), detached by incubating in PBS containing 1 n&f EDTA and 1 mM EGTA, pH 7.4, for 10 min at 4”C, and collected by centrifugation [26]. The cell pellets were sonicated in 2 ml of distilled water to make a homogeneous suspension and a portion was removed for protein determination. The lipids were extracted with 8 ml of chloroform/methanol (l/2, v/v) at 37°C for 1 h. After centrifugation, the residue was washed with 4 ml of the same solvent. The combined extracts were taken to dryness, desalted on Sephadex G-25 columns, and separated into neutral and acidic fractions on DEAE-Sephadex as described previously [32]. Gangliosides were isolated from the acidic fraction (solvent B) following alkaline hydrolysis, desalting on Sephadex G-25, and column chromatography on Unisil [32]. The Unisil column was eluted first with chloroform/ methanol (9/l, v/v) to increase the recovery of GM3 in the chloroform/methanol (2/3, v/v) fraction [32]. The gangliosides were resolved on high-performance silica gel 60 plates, visualized by resorcinol reagent, and quantified by scanning densitometry [33]. Analysis of gangliosides by overluy techniques. In order to identify gangliosides of the gangliotetrose series, the sensitive overlay technique was used [34]. Briefly, gangliosides purified from 0.5 mg of cell protein were separated on aluminum-backed silica gel sheets using chloroform/methanol/0.25% CaC12in water (60/35/8, v/v). The plates were fixed with 0.4% poly(isobuty1 methacrylate) in hexane and air dried. Where indicated, the chromatogram was overlaid with 100 mM sodium acetate buffer (pH 5.5) containing 150 mAf NaCl, 9 r&f CaClz and sialidase (0.1 units/ml) for 2 h at 25°C. After washing, the chromatogram was overlaid with 50 mM Tris-HCl (pH 7.4)-150 mA4 NaCl containing 1% bovine serum albumin and “?-cholera toxin (0.45 &i/ ml) for 30 min at 4’C. The chromatogram was extensively washed, then air dried, and the bound toxin detected by autoradiography using Kodak X-Omat AR-2 film. Measurement of binding of iodinated cholera toxin to 3T3 fibroblasts. Binding of cholera toxin to 3T3 fibroblasts was measured by two different methods. Briefly, each 16-mm well of cells was incubated with iodinated CT (20 nM; 125,000cpm/well) in 0.5 ml of serumfree medium buffered with 25 mA4 Hepes (pH 7.4) and supplemented with 0.1% bovine serum albumin. After 1 h at 37”C, the medium was removed and iodinated CT bound to the cells determined [31]. Nonspecific binding was determined by the addition of 0.2 pM unlabeled CT to the assay. Binding of CT to cells in suspension was measured as described [26]. Briefly, cells were grown in 25- or 75-cm* flasks and, where indicated, treated with sialidase (0.05 units/ml) for 2 h. The cells were detached from the flasks by incubation with 0.02% collagenase followed by addition of 0.01% trypsin/l mM EDTA/l mM EGTA. The cells were suspended in 25 mA4 Tris-HCl (pH 7.4), 127 n&f NaCl,

RESPONSE

OF SWISS

3T3 CELLS

1 mM EDTA, 3 n&f NaN3, and a portion was removed for cell counting. ‘?-toxin (0.11 pCi, 10 r&f) was incubated with 2.5 X 10’ cells of 0.2 ml of the Tris buffer containing 0.1% BSA for 45 min at 25°C. Samples were then filtered under vacuum on 0.2~am Millipore EGWP filters. Nonspecific binding was determined in the presence of 0.2 &f unlabeled toxin.

TO CHOLERA

TOXIN

TABLE Ganglioside

Composition Growing

Confluent RESULTS Ganglioside

Different Effects of the B Subunit of Cholera Toxin on DNA Synthesis in Confluent versus Rapidly Growing 3T3 Cells The B subunit of CT has been shown previously to have a bimodal effect on DNA synthesis in nontransformed 3T3 fibroblasts derived from Swiss, Balb/c, and NIH mouse embryos [ 1620,221. The B subunit induces cellular proliferation of confluent and quiescent 3T3 cells while it inhibits the growth of the same cells when they are sparse and rapidly dividing. However, these studies were conducted in cultures which still contained residual amounts of serum [ l&20,22]. To avoid complications due to the complex nature of serum and its multiplicity of actions, the effects of the B subunit on DNA synthesis were examined in Swiss 3T3 fibroblasts which can be cultured in serum-free, chemically defined medium [21,30,35]. In agreement with our previous results, the B subunit alone stimulated DNA synthesis and also potentiated the response to insulin and FCS in contact-inhibited cells (Table 1). In contrast, the B subunit inhibited DNA synthesis in exponentially growing cells and also reduced the mitogenic response to insulin and to a lesser extent to FCS (Table 1). The present results indicate that a bifunctional response pattern to the B subunit can be observed even in chemically defined medium. TABLE Effects

of the B Subunit Exponentially

on DNA Growing

1 Synthesis in Quiescent 3T3 Fibroblasts

and

[3H]Thymidine incorporation (percent of control) Stimulator None B I I+B FCS FCS+B

Confluent lOOk 4 15Ok 8 242+ 7 762 3~ 4 2592 f 13 3787 + 18

Exponentially

growing

lOOf 6O-c5 26529 166+3 2039 + 7 1884fl

Note. Confluent or exponentially growing Swiss 3T3 cells cultured as described under Experimental Procedures were exposed to the indicated mitogens and [3H]thymidine incorporation was measured. Each value is the mean & SD of triplicate determinations from a representative experiment. Similar results were obtained in at least five additional experiments. The concentrations of the agents were as follows: B subunit of CT (B), 1 pg/ml; insulin (I), 2 *g/ml; FCS 10% (v/v).

B SUBUNIT

GDla GM1 GM2 GM3 Total

+ 0.6 + 0.1 + 0.2 ?I 0.6

2

of Quiescent and Exponentially 3T3 Fibroblasts

Exponentially growing

(nmol sialic acid/mg protein) 5.2 0.9 1.3 4.9 12.3

15

1.2 f 0.04 ND ND 2.8 i 0.1 4.0

Confluent

Exponentially growing

(% of total ganglioside sialic acid) 42.2 7.3 10.5 39.8

30 ND ND 70

Note. Cells were harvested at a density of 3.9 X 10’ and 1 X 10’ cells/ cm2 for confluent and exponentially growing cultures, respectively. Gangliosides were isolated, separated on high-performance silica gel plates in chloroform/methanol/0.25% CaCI, in water (60/35/8), and analyzed as described under Experimental Procedures. Values are the means + SD of four separate preparations and are given as nmol of sialic acid per mg of protein. ND, below detectable limits.

Characterization of Gangliosides in ConfEuent and Rapidly Growing 3T3 Cells Since the levels of gangliosides have been shown to be markedly increased as normal 3T3 cells reach confluency [36,37], it is possible that the bimodal response to the B subunit could be related to differences in ganglioside composition during growth. In agreement with these previous studies, we also found substantial changes in ganglioside composition when the cells became contact inhibited and quiescent. Confluent cells had almost threefold higher content of total gangliosides than did the rapidly dividing cells (Table 2). The major gangliosides were GM3 and GDla (Fig. 1A). Gangliosides GM2 and GM1 were present in lower amounts in confluent cells and were not detectable by resorcinol spray in rapidly dividing cells.5 As GMl, the receptor for the B subunit, is present in only trace amounts, especially in exponentially growing cells, two other methods were used to identify and quantify GMl. Using the sensitive overlay technique with iodinated CT [34], GM1 was detected in both the confluent and the rapidly growing cells. However, there was clearly a much greater amount of GM1 in the confluent cells (Fig. 1B). Treatment of the chromatogram with sialidase revealed that GDla was the only ganghoside converted to GM1 by this enzyme (Fig. 1C) and that both confluent and rapidly dividing cells have more GDla than GMl. However, the absolute amounts of GM1 or GDla could not be determined quantitatively with this method because there was not a linear correlation be5 It should be noted that the gangliosides resolve into doublets on thin layer chromatograms due to heterogeneity in the ceramide moiety [441.

16

BUCKLEY,

MATYAS,

AND SPIEGEL

FIG. 1. Thin layer chromatogram of ganglioeides extracted from quiescent and exponentially growing cultures of Swiss 3T3 fibroblasts. Cells were harvested at a density of 3.9 and 1 X 10’ cells/cm2 for confluent and exponentially growing cultures, respectively. (A) Gangliosides were isolated from cells, separated on silica gel-coated glass plates, and visualized with resorcinol as described under Experimental Procedures. (Lane 1) Gangliosides extracted from exponentially growing cultures (2 mg of protein). (Lane 2) Gangliosides extracted from contact-inhibited and quiescent fibroblasts (2 mg of protein). (Lane 3) Standard gangliosides (from top to bottom 1 nmol each of GM3, GM2, GMl, and GDla). (B) Detection of ganglioside GM1 in quiescent and exponentially growing cells by the overlay technique. Gangliosides were separated by thin layer chromatography on an aluminum-backed silica gel sheet and overlain with ‘*I-CT, and the bound toxin was detected by autoradiography (18 h exposure). (Lane 1) Gangliosides isolated from exponentially growing cultures (0.5 mg of protein). (Lane 2) Gangliosides isolated from quiescent fibroblasts (0.5 mg of protein). Arrows indicate location from top to bottom of GM3, GM2, GMl, and GDla as detected by orcinol spray. (C) Detection of gangliosides GM1 and GDla. Gangliosides were separated by thin layer chromatography on an aluminum-backed silica gel sheet. The chromatograms were treated with neuraminidase and then overlain with 1261-CT,and the bound toxin was detected by autoradiography (18 h exposure). Lanes 1 and 2 as in B.

tween the concentration of GM1 on the plate and the density of the autoradiographic spot [32]. To quantify the amounts of GM1 and GDla on the cell surface, CT binding to intact attached cells and to cells in suspension was measured. As shown in Table 3, confluent cells bound two- to threefold more toxin than did exponentially growing cells and thus have more cell surface GMl. TABLE

3

Binding of Iodinated CT to Quiescentand Exponentially Growing 3T3 Fibroblasts ‘261-CT Bound (pmol/lO’ cells) Exponentially growing

Treatment

Confluent

Intact attached cells Control Sialidase

32.5 f 1.9 189.7 + 8.6

10.9 + 2.7 92.2 zk2.7

Cells in suspension Control Sialidase

34.8 z!T 1.2 202.2 f 11.7

16.2 + 3.3 119.8 r 6.6

Note. Confluent (3.9 X 10’ cells/cm2) or exponentially growing (1 10’ cells/cm2) Swiss 3T3 cells were washed with DMEM and incubated in the absence or presence of sialidase (0.004 units/ml). After 2 h, specific binding of iodinated CT to attached cells in situ or to cells in suspension was measured exactly as described under Experimental Procedures. X

Both binding assays gave similar results showing lower values for attached cells. This is probably due to latent CT binding sites where the cells are attached to the substratum. Nonspecific binding with attached cells is much larger (20%) than that for cells in suspension (40%) due to the binding of the toxin to the plastic dish. As the latter method allows a more accurate determination of specific iodinated toxin binding, especially for lower number of cells, this was the method of choice when low numbers of cells were used. Treatment of both confluent and rapidly growing cells with sialidase enhanced toxin binding over fivefold, thus indicating that, in both growth states, the cells contained more surface GDla than GMl. By taking the difference in binding between untreated and sialidase-treated cells, we were also able to quantify the level of cell surface GDla. In agreement with the thin-layer chromatographic analysis (Fig. 1 and Table 2) the expression of both GDla and GM1 on the cell surface significantly increased when the cells reached confluency. Effect of Cell Density on Levels of GM1 and GDla Since it had been shown previously that there are changes in gangliosides, in particular GDla, at the early stage of cell contact [37], it was of interest to compare in detail the changes in ganglioside GM1 and GDla as a function of cell growth when starting with sparse cul-

RESPONSE

OF SWISS

3T3 CELLS

TO CHOLERA

TOXIN

B SUBUNIT

17

0123456769’ DAYS IN CULTURE

DAYS IN CULTURE

FIG. 2. Changes in cell surface ganglioside GM1 and GDla as a function of cell growth. (A) Changes in cell density with days in culture starting with a cell density of 0.2 X 10’ cells/cm*. (B) Changes in the level of cholera toxin binding to GM1 and GDla on the cell surface. Iodinated CT binding before (-) and after sialidase treatment (- - -) was measured as described under Experimental Procedures.

tures. Figure 2A shows that starting with a cell density of 0.2 X lo4 cells/cm2, cells reached confluency and stopped dividing after 4-5 days in culture. The level of GM1 fell during the first 2 days and then increased as the cells became contact inhibited (Fig. 2B). However, although the amount of GM1 doubled between Day 2 and Day 5, there was a much larger increase during succeeding days after the cells were confluent. Similarly, the level of GDla also increased as the cells reached confluency, became maximum at Day 7, but, in contrast to GMl, then declined. Effect of Cell Density on the Mitogenic Response Since there were marked changes in ganglioside GM1 and GDla levels with time in culture, we decided to examine whether there was also a correlation with the ability of the cells to respond to various mitogens. Figure 3 depicts the effects of the B subunit, bombesin, and EGF on DNA synthesis as a function of the days in culture starting with a cell density of 0.2 X lo4 cells/cm2. As ex-

pected (Table l), during the first days in culture when the cells were sparse, the B subunit had an inhibitory effect on DNA synthesis (Fig. 3A). As the cell density increases and the cells reach confluency (Day 5), the B subunit began to have an opposite, stimulatory effect on DNA synthesis. Even after 8 days in culture, the stimulatory effect of the B subunit had not reached a plateau and appeared to follow the increase in GMl. In contrast, neither bombesin (Fig. 3B) nor EGF (Fig. 3C) had an initial inhibitory effect and both had a maximum mitogenie effect on DNA synthesis when the cells became contact inhibited. These experiments were all carried out in the presence of insulin, which increased the viability of the cells from 87 to 95%. Similar results were obtained in the absence of insulin (data not shown). The B Subunit Still Inhibits DNA Synthesis in Rapidly Growing 3T3 Cells after Increasing Levels of GM1 Since there appears to be a correlation between the level of ganglioside GM1 on the cell surface and the bi-

300 250-

B subunit

23456769 DAYS IN CULTURE

300

Bombesin

h

23456789 DAYS IN CULTURE

x

600 1I 700

El3

23456769 DAYS IN CULTURE

FIG. 3. DNA synthesis in response to the B subunit (A), bombesin (B), and EGF (C) after various days in culture. Cells were seeded at a density of 0.2 X 10’ cells/cm2. On the indicated days, cells were washed with DMEM and incubated in DMEM/Waymouth (l/l), supplemented with BSA and transferrin, in the presence of insulin and the indicated mitogens: B subunit (1 pg/ml); bombesin (100 nM); EGF (5 rig/ml). [aH]Thymidine incorporation was measured as described under Experimental Procedures. The results are the means + SD of three determinations and are expressed as percentage of control compared to cells treated with insulin alone.

18

BUCKLEY,

MATYAS,

AND

None

SPIEGEL

Insulin

FCS

FIG. 4. Effect of insertion of ganglioside GM1 on iodinated CT binding and on DNA synthesis in exponentially growing cells. Insertion of GM1 into Swiss 3T3 cells was carried out by the second method as described under Experimental Procedures. Exponentially growing control cells (open bars) or after insertion of GM1 (hatched bars) or confluent cultures (solid bars) were washed with DMEM, and specific iodinated CT binding (A) was measured as described under Experimental Procedures. For [3H]thymidine incorporation (B) the washed cells were incubated in DMEM/Waymouth, in the presence or absence of the B subunit (1 pg/ml) and the indicated mitogens. [eH]Thymidine incorporation results are expressed as percentage of control + SD compared to percentage of cells not treated with the B subunit. The concentrations of the agents were as follows: B subunit of CT (B), 1 @g/ml; insulin (I), 2 pg/ml; FCS 5% (v/v).

modal effect observed with the B subunit, it was of intereat to examine whether increasing the level of GM1 to a level similar to that found in confluent cells caused subsequent changes in the response to the B subunit. It has been shown that ganglioside GM1 can be readily taken up by a variety of cells and inserted into the plasma membrane in a physiological manner as shown by a corresponding increase in CT binding and responsiveness [38, 391. Thus, ?he level of membrane-associated GM1 in exponentially growing 3T3 cells was increased by the insertion of exogenous GMl. In sparse 3T3 cells treated with GMl, the B subunit reduced DNA synthesis by 45%. When added in conjunction with insulin or FCS, the B subunit reduced DNA synthesis by 58 and 53%, respectively, compared to that of cells treated with insulin or FCS alone. Thus insertion of ganglioside GM1 into exponentially growing 3T3 fibroblasts did not change their response to the B subunit. This approach, however, had some limitations because some of the GM1 adhered to the dish, thus increasing the binding of the B subunit to the dish instead of binding to GM1 on the cell surface. To avoid complications due to this potential problem, another set of experiments was carried out in which confluent cells were first treated with the exogenous GM1 and then replated. Similar results were obtained by this method. That is, increasing the levels of GM1 on the surface of exponentially growing cells to the levels found in confluent cells, as assayed by a corresponding increase in iodinated CT binding (Fig. 4A), did not change the responsiveness of exponentially growing cells to the B subunit (Fig. 4B). The B subunit still decreased DNA synthesis and inhibited the mitogenic effect of other mitogens, such as insulin or FCS. In contrast, the B subunit stimulated DNA synthesis and enhanced the mito-

genie effect of insulin and FCS in confluent cells which contain similar levels of GM1 (Fig. 4B). Another approach to increase levels of GM1 in exponentially growing cells is to convert endogenous ganglioside GDla to GM1 by using sialidase treatment. As shown by the lz51-CT overlay technique on thin layer chromatography, GDla was the only ganglioside converted to GM1 by sialidase (Fig. 1C). There was a ninefold increase in GM1 on the cell surface as measured by iodinated CT binding (Table 4). However, DNA synthesis in the sialidase treated cells was still reduced by the B subunit. The inhibitory effects of the B subunit were observed regardless of whether insulin, EGF, or FCS were added to the medium, even though these factors by themselves increased [3H]thymidine incorporation (Table 4). Thus, the inhibition of growth of rapidly dividing 3T3 fibroblasts by the B subunit is apparently not directly related to the levels of membrane-bound GMl. DISCUSSION In previous studies, we demonstrated that the B subunit of cholera toxin, which binds solely to the plasma membrane ganglioside GMl, stimulates DNA synthesis and cell division in quiescent, nontransformed murine 3T3 fibroblasts [18,20,21]. However, the B subunit had opposite effects on the growth of ras-transformed 3T3 cells and on rapidly dividing normal 3T3 cells [ 18, 201. Our present studies confirmed and extended this finding to 3T3 fibroblasts cultured in serum-free medium, thus avoiding any complications due to uncharacterixed growth factors present in serum. In distinct contrast to other mitogens, such as insulin, epidermal growth factor,

RESPONSE

OF SWISS

3T3 CELLS

TO CHOLERA

TABLE

TOXIN

19

B SUBUNIT

4

Effects of Sialidase Treatment on DNA Synthesis Induced by the B Subunit in Exponentially Growing 3T3 Fibroblasts [3H]Thymidineincorporation (percentof control) Stimulator None B BSA I I+B EGF EGF+B FCS FCS+B

Siahdase treatment

100 2 64+96k 254 iz 189+ 162 f 114+ 1334 + 1206 k

?-CT bound (pmol/lO’ cells)

+ 11 5 2 11 8 8 4 24 13

120 + 54* 95+ 289 + 152 + 182+ 114 + 1327 + 1077+

13 4 4 25 13 7 18 23 7

-

+

9.2 f 1.6 0.9 * 0.1 9.7 + 1.9 9.8 f 1.6 1.1 + 0.2 ND ND ND ND

102.3 + 16.9 21.3 + 5.9 103.8 zk 0.9 110.4 f 18.7 20.7 + 5.3 ND ND ND ND

Note. Exponentially growing Swiss 3T3 cells were washed with DMEM and incubated in DMEM/Waymouth in the absence (-) or presence (+) of siahdase (0.005 U/ml) and the indicated mitogens, and [sH]thymidine incorporation was measured as described under Materials and Methods. Each value is the mean + SD of triplicate determinations from a representative experiment. Similar results were obtained in three additional experiments. The data are expressed as percentages of the values obtained in the absence of any mitogen. The concentrations of the mitogenic agents were as follows: B, 1 pg/ml; BSA, 1 pg/ml; insulin, 2 pg/ml; EGF, 5 rig/ml; FCS, 5%. Iodinated CT binding was measured in duplicate cultures as described under Materials and Methods. Nonspecific ‘“I-CT bound to the untreated cells or cells treated with sialidase was 2.2 + 0.1 and 21.0 + 2.2 pmol/lO’ cells, respectively.

or bombesin, the B subunit had dual effects on cellular proliferation of Swiss 3T3 fibroblasts, cultured in chemically defined medium, depending on their state of growth. It has been reported that the levels of gangliosides in Swiss 3T3 fibroblasts increase as the cells reach confluency [37]. Thus, it was possible that the bimodal response to the B subunit might be related to the amount of gangliosides on the cell surface. In agreement with the previous report [37], we found that confluent cells had more than two-fold higher content of cellular gangliosides than rapidly dividing cells. The major gangliosides were GM3 and GDla, while gangliosides GM2 and GM1 were present in barely detectable amounts in rapidly dividing cells. We were also able to quantify the cell surface ganglioside GM1 and GDla using a sensitive binding assay. Cell surface GM1 and GDla also increased significantly as the cells reached confluency. Surprisingly, there was a much larger increase in the level of both GM1 and GDla on the cell surface during succeeding days in culture, even after the cells were already confluent. This is in contrast to the previous finding [37], where the enhanced GDla concentration was observed at the early stage of cell contact (touching) when cell growth was still not inhibited. Although the explanation for this discrepancy is not known, it might be due to differences in culture conditions or to differences in the methods of ganglioside analyses. However, it should be emphasized that the levels of cell surface gangliosides GM1 and GDla were measured in this study by methods that were not available earlier. Although at first glance, from these results, it seems that the ability of ganglioside GM1 to modulate negative

or positive growth signals correlates with its density on the cell surface, further studies revealed that this is a premature conclusion. Using the unique ability of gangliosides to be functionally inserted into the plasma membrane of cells, it was found that even after the levels of GM1 on the surface of exponentially growing cells was increased to levels found on confluent cells, the responsiveness to the B subunit was not altered. Furthermore, similar results were obtained when the level of endogenous GM1 was increased by conversion of GDla to GM1 after sialidase treatment. Thus, this finding leads to the conclusion that the bimodal response to the B subunit is not a function of the amount of cell surface ganglioside GMl. Rather, it is the growth stage that determines the fate of the signals transduced by the interaction of the B subunit and ganglioside GMl. In this regard, it is interesting to note that the dual action of the B subunit was recently observed in quiescent Swiss 3T3 fibroblasts, depending on the context of other growth factors [40]. While the B subunit potentiates the effect of EGF, insulin, bombesin, platelet-derived growth factor, and unfractionated FCS [Zl], it inhibits DNA synthesis induced by TPA via protein kinase C [40]. The dual effects of the B subunit are also related in this case to different stages of the cell cycle. The ability of the B subunit to potentiate the mitogenic effect of insulin is greatest when it is added 2 h prior to insulin. Whereas, its ability to inhibit the mitogenic effect of TPA is greatest when it is added 2-4 h afteraddition of TPA [40]. Similar results were obtained in quiescent rat thyroid (FRTL-5) cells [41]. In these cells, the B subunit markedly enhances DNA synthesis induced by insulin and inhibits it when induced by thyrotropin, dibutyryl

20

BUCKLEY,

MATYAS,

CAMP, or phorbol-12-myristate-13-acetate [41]. In both cell lines, bimodal response to the B subunit is dependent on the presence of other growth factors. It is known that the response of the cells to growth-promoting agents is dependent not only on the growth factor itself but also on the total set of stimulatory and inhibitory factors that are operating on the cell at a certain time [42]. The total context of autocrine growth regulators is different for confluent compared to rapidly growing cells. Therefore, differing effects of the B subunit might reflect these different sets of growth regulators. The B subunit has proved to be useful for studying the molecular mechanisms underlying the action of ganglioside GM1 [18,21,22,29,40,45,46]. The binding of the B subunit to endogenous ganglioside GM1 does not elicit the classical intracellular second messenger systems, such as CAMP, diacyl glycerol (an endogenous activator of protein kinase C), or inositol trisphosphate (which mobilizes Ca2+from internal stores). This conclusion is based on the demonstrations that the B subunit does not activate adenylate cyclase, Na+/H+ exchange, phospholipase C, or protein kinase C [18, 21, 22, 451. However, the B subunit mediated a large increase of intracellular free calcium resulting from a net influx from extracellular sources [21, 451. The rise in [Ca’+]i alone is not sufficient to explain the effects of the B subunit on cellular proliferation, as Ca2+ionophores do not increase the synthesis of numatrin [46], a nuclear protein whose synthesis is closely correlated to cellular commitment for mitogenesis, and do not stimulate DNA synthesis [21]. In contrast, not only is the interaction of the B subunit with ganglioside GM1 accompanied by a large increase in the synthesis of numatrin [46] but also this is sufficient to induce the progression of the cells into the S phase [21]. Recently, it has been proposed that metabolites of gangliosides (sphingosine and lysogangliosides) may be endogenous inhibitors of protein kinase C, an enzyme which plays a key regulatory role in signal transduction in cellular proliferation [47]. Thus, it is important to note that in contrast to the inhibitory effects of ganglioside metabolites on protein kinase C activity and binding of phorbol ester [47], the B subunit inhibited DNA synthesis induced through activation of protein kinase C without affecting the binding of phorbol ester or the phosphorylation of its specific substrate [40]. Thus, endogenous ganglioside GM1 must modulate signal transduction mediated through protein kinase C at a step subsequent to phosphorylation of its endogenous substrate. This is consistent with our observation that significant inhibition of the TPA-induced increase in DNA synthesis was still observed even when the B subunit was added 6 h after TPA [40]. This finding demonstrates the importance of still unelucidated events which are distal to phosphorylation of the 80-kDa protein and implies that the cross-talk between signal transduction induced

AND

SPIEGEL

through endogenous gangliosides and protein kinase C occurs at a late step in mitogenesis [40]. Similarly, it has been shown that a pertussis toxin-sensitive GTP-binding protein is involved in a late event of DNA synthesis mediated through interaction of endogenous ganglioside and the B subunit [29]. DNA synthesis induced by bombesin also proceeds via this same pertussis toxinsensitive pathway even though different early cellular events precede the arrival to this point in the process [29]. The late pertussis toxin-sensitive events allude to the importance of late signals that have not previously received much attention. Our recent data imply that ganglioside GM1 modulates cellular proliferation through another uncharacterized signal transduction pathway which can cross talk with other second messenger systems. We thank Dr. Peter Fishman for his valuable advice during the course of this study and for reviewing the manuscript.

REFERENCES 1. 2. 3. 4. 5.

Wiegandt,

H. (1982) Adv. Neurochem. 4,149-223.

Hakomori,

S. (1981) Annu. Rev. Biochm. 50,733-764.

Hakomori,

S. (1985) Cancer Res. 45,2405-2414.

Fishman, Usuki,

P. H., and Brady, R. 0. (1976) Science 194,906-915.

S., Lyu, S.-C., and Sweeley, C. C. (1988) J. Biol. Chm.

263,6847-6853. 6. Sweeley, C. C., and Usuki, S. (1988) in New Trends in Ganglioside Research: Neurochemical and Neuroregenerative Aspects (Ledeen, R. W., Hogan, E. L., Tettamanti, G., Yates, A. J., Eds.), Fidia Research Series, Vol. 14, pp. 307-315, Liviana Press, Padova, Italy.

7. Bremer, E. G., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. &em. 259,6818-6825. 8. Ledeen, R. W. (1984) J. Neurosci. Res. 12,147-159. 9. Okada, Y., Radin, N. S., and Hakomori, S. (1988) FEBS Lett. 235,25-29. 10. Usuki, S., Hoops, P., and Sweeley, C. C. (1988) J. Biol. Chem. 263,10595-10599. 11. Bremer, E. G., Schlessinger, J., and Hakomori, S. (1986) J. Biol. Chem. 261,2434-2440. 12. Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S. (1988) J. Biol. Chem. 263.6296-6301. 13. Lingwood, C. A., and Hakomori, S. (1977) Exp. Cell Res. 108, 385-391.

14. Lingwood, C. A., Ng, A., and Hakomori, S. (1978) Proc. Natl. Acad. Sci. USA 75,6049-6053. 15. Mandel, P., Dreyfus, H., Yusufi, A. N., Sarlieve, L., Robert, J., Neskovic, N., Harth, S., and Rebel, G. (1980) Adv. Exp. Med. Biol. 125,515-531. 16. Okada, Y., Matsuura, H., and Hakomori, S. (1985) Cancer Res. 45,2793-2801. 17. Spiegel, S., Fishman, P. H., and Weber, R. J. (1985) Science 230, 18.

1285-1287. Spiegel, S. (1988) in New Trends in Ganglioside Research: Neurochemical and Neuroregenerative Aspects (Ledeen, R. W., Hogan, E. L., Tettamanti, G., Yates, A. J., Eds.), Fidia Research Series, Vol. 14, pp. 405-421, Liviana Press, Padova, Italy.

RESPONSE OF SWISS 3T3 CELLS TO CHOLERA 19. Campbell, K. S., and Munson, A. E. (1987) J. Phormucol. Exp. Themp. 242,895-904. 20. Spiegel, S., and Fishman, P. H. (1987) Proc. Natl. Acad. Sci. USA 84,141-147. 21. Spiegel, S., and Panagiotopoulos, C. (1988) Exp. Cell Res. 177, 414-427. 22. Spiegel, S. (1989) in Gangliosides in Cancer (Oettgen, H. F., Ed.), pp. 17-29, VCH Publishers, New York. 23. Spiegel, S. (1988) Biochim. Biophys. Acta 969,249-256. 24. Skaper, S. D., Facci, L., Favaron, M., and Leon, A. (1988) J. Neurochem. 61,688-696. 25. Facci, L., Skaper, S. D., Favaron, M., and Leon, A. (1988) J. Cell. Biol. 106,821-828. 26. Matyas, G., Aaronson, S., Brady, R., and Fishman, P. (1987) Proc. Natl. Acad. Sci. USA 84,6065-6068. 27. Cuatrecasas, P. (1973) Biochemistry 12,3547-3558. 28. Pacuszka, T., DuBard, R., Nishimura, R. N., Brady, R. O., and Fishman, P. H. (1978) J. Bid. Chem. 253,5839-5846. 29. Spiegel, S. (1989) J. Biol. Chem. 264,6766-6772. 30. Dicker, P., and Rozengurt, E. (1980) Nature (London) 287,607612. 31. Spiegel, S. (1985) Biochem&ry 24,5947-5952. 32. Spiegel, S. Matyas, G., Cheng, L., and Sacktor, B. (1988) Bio&em. Biophys. Acta 938,270-278. Received November 6,1989 Revised version received February 20,199O

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33. Fishman, P. H., Quarles, R. H., and Max, S. R. (1979) in Densitometry in Thin Layer Chromatography: Practice and Applications (Touchston, J. C., and Sherman, J. A., Eds.), pp. 315-327, Wiley, New York. 34. Spiegel, S., Handler, J. S., and Fishman, P. H. (1988) J. Bid. Chem. 261,15755-15760. 35. Rozengurt, E. (1986) Science 234,161-166. 36. Hakomori, S. (1970) Proc. Natl. Acad. Sci. LfSA 67,1741-1747. 37. Yogeeswaran, G., and Hakomori, S. (1975) Biochemistry 14, 2151-2156. 38. Fishman, P. H. (1982) J. Membr. Biol. 69,85-97. 39. Spiegel, S., Kassis, S., Wilchek, M., and Fishman, P. H. (1984) J. Cell Biol. 99,1575-1581. 40. Spiegel, S. (1989) J. Biol. Chem. 264,16,512-16,517. 41. Tetsumoto, T., Takada, K., Amono, N., and Miyai, K. (1988) Biothem. Biophys. Res. Commun. 167,605-610. 42. Sporn, M. B., and Roberts, A. B. (1988) Nature (London) 332, 217-219. 43. Svennerholm, L. (1963) J. Neurochm. 10,613-623. 44. Yogeeswaran, G., Sheinin, R., Wherrett, J. R., and Murray, R. K. (1972) J. Bid. Chem. 247,5146-5158. 45. Dixon, S. J., Stewart, D., Grinstein, S., and Spiegel, S. (1987) J. CeU.Bid. 105,1153-1161. 46. Feuerstein, N., Spiegel, S., and Mond, J. J. (1988) J. Cell Biol. 107,1629-1642. 47. Hannum, Y. A., and Bell, R. M. (1987) Science 235,670-673.