Proc. Nati. Acad. Sci. USA Vol. 84, pp. 141-145, January 1987 Cell Biology Gangliosides as bimodal regulators of cell growth (mitogen/cholera toxin/transformed cells) SARAH SPIEGEL AND PETER H. FISHMAN Membrane Biochemistry Section, Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 Communicated by Roscoe 0. Brady, September 18, 1986 (receivedfor review August 4, 1986) ABSTRACT The B subunit of cholera toxin, which binds directly examine potential functions for endogenous gan- specifically to several molecules of ganglioside galactosyl- gliosides, we have developed a different approach (14). The (,B1--3)-N-acetylgalactosyminyl(fi1---4)-[N-acetylneuramin- B subunit of cholera toxin, which is pentavalent and binds yl(a2-+3)]-galactosyl(fi1--+4)glucosyl(fl-d)ceramide (GM1) only to ganglioside galactosyl(31--3)-N-acetylgalacto- on the cell surface, stimulated DNA synthesis and cell division syminyl(p1--4)-[N-acetylneuraminyl(a2--+3)]-galactosyl- in quiescent, nontransformed mouse 3T3 cells in a dose- (/31--4)glucosyl(,81-l)ceramide (GM1) on the cell surface dependent manner. In addition, the B subunit potentiated the (2), was used as a ganglioside-specific probe to induce response of the 3T3 cells to other mitogens, such as epidermal proliferation of thymocytes (14). We have now extended this growth factor, platelet-derived growth factor, and insulin. This approach to other types of cells, such as normal and trans- synergistic effect indicates that the B subunit does not act formed murine 3T3 cells, to evaluate the role of membrane identically to any of these growth factors but probably modu- gangliosides in the regulation of cell growth in general. lates a common effector system crucial for cell proliferation. In distinct contrast, the B subunit inhibited the growth of ras- transformed 3T3 cells as well as rapidly dividing normal 3T3 MATERIALS AND METHODS cells. Thus, the same cells, depending on their state of growth, Materials. The B subunit of cholera toxin was purchased exhibited a bimodal response to the B subunit. We conclude from List Biologicals (Campbell, CA). [3H]Thymidine (55 that endogenous gangliosides may be bimodal regulators of Ci/mmol; 1 Ci = 37 GBq) was purchased from ICN. Epider- positive and negative signals for cell growth. mal growth factor (EGF), insulin, and platelet-derived growth factor (PDGF) were obtained from Collaborative Gangliosides, sialic acid-containing glycosphingolipids, have Research (Waltham, MA). Other materials and chemicals long been recognized as characteristic constituents of the were obtained from commercial sources. plasma membrane of mammalian cells (1, 2). Although Cell Lines and Cell Cultures. Swiss 3T3, BALB/c 3T3, NIH gangliosides have been identified as receptors for bacterial 3T3, and all of the ras-transformed NIH 3T3 cell lines were toxins (2) and viruses (3), relatively little is known about their generous gifts ofStuart Aaronson (National Cancer Institute, normal function in membranes. In recent years, interest in Bethesda, MD). All ofthe latter were established from clones these membrane constituents has increased due to the dis- ofNIH 3T3 cells transfected with human cellular Ha-ras (15), covery that many monoclonal antibodies raised against tumor Ki-ras (16) and N-ras (17) oncogenes. Stock cultures of cells recognize carbohydrate sequences present on ganglio- murine 3T3 cells were maintained in Dulbecco's modified sides and these determinants appear to be tumor-specific Eagle's medium (DMEM), supplemented with 10% (vol/vol) antigens (reviewed in refs. 4-6). Furthermore, the alterations fetal bovine serum in a humidified atmosphere of 5% in ganglioside metabolism and organization related to C02/95% air at 37°C. oncogenic transformation, cell cycle, and density-dependent Measurement of DNA Synthesis in Quiescent 3T3 Cells. growth inhibition (1, 6) raised the possibility that gangliosides Swiss, NIH, and BALB/c 3T3 cells were subcultured into play an important role in the regulation of cell growth. Such 24-well tissue culture plates (Costar, Cambridge, MA) at a apossibility was reinforced by observations that exogenously density of2.5 x 104 cells per well in 1 ml ofDMEM containing added gangliosides altered the growth of a variety of cell 10% fetal bovine serum and refed with the same medium after types (7-13). In particular, exogenous gangliosides inhibited 2 days. Such cultures were used at least 5 days after the last the action of several growth factors as well as the tyrosine change of medium when the cells were confluent and quies- kinase activity associated with the growth factor receptors (8, cent (18). To the quiescent cultures, 1 ml of DMEM was 9), suppressed the proliferation of lymphocytes induced by added without or with the B subunit and the various growth lectins, antigens, and interleukin 2 (4, 10), and sensitized factors as indicated in the figure and table legends. After 20 tumor cells to growth inhibitors (11). In contrast, exogenous hr, the cells were pulsed with 0.5 ,Ci of [3H]thymidine for 4 gangliosides stimulated the proliferation ofastroglial (12) and hr and then washed two times with 1 ml of ice-cold phos- neuroblastoma cells (13). phate-buffered saline and two times with ice-cold 5% tri- Thus, exogenous gangliosides have been reported to cause chloroacetic acid. The insoluble material was dissolved in 0.5 ml of 0.25 M NaOH, which was transferred to glass scintil- opposite effects on cell growth. This apparently conflicting lation vials containing 10 ml of Ready-Solv HP (Beckman) aspect of ganglioside action has made it difficult to determine and analyzed for radioactivity. whether gangliosides play a role as membrane transducers of Measurement of DNA Synthesis in Exponentially Growing positive or negative growth signals. In addition, little prog- 3T3 Cells. Cells were seeded at a density of 3 x 103 cells per ress has been made in clarifying the significance of these well as described above and grown in DMEM containing 10% observations with respect to the function of endogenous fetal bovine serum. After 2 days (NIH 3T3 and Swiss 3T3) or gangliosides in the process of cellular proliferation. To Abbreviations: EGF, epidermal growth factor; PDGF, platelet- The publication costs of this article were defrayed in part by page charge derived growth factor; GM1, galactosyl(831- 3)-N-acetylgalacto- payment. This article must therefore be hereby marked "advertisement" syminyl(pf1-4)-[N-acetylneuraminyl(a2--*3)]-galactosyl(,B1--4)glu- in accordance with 18 U.S.C. §1734 solely to indicate this fact. cosyl(,B1-l)ceramide. Downloaded by guest on October 2, 2021 141 142 Cell Biology: Spiegel and Fishman Proc. Natl. Acad. Sci. USA 84 (1987) 3 days (ras-transformed NIH 3T3), the cells were treated with Table 1. Stimulation of DNA synthesis in quiescent normal the B subunit or with growth factors and were pulsed 40 hr mouse 3T3 cells by the B subunit of cholera toxin and later with [3H]thymidine for 1 hr. The incorporation of3I into growth factors trichloroacetic acid-insoluble material was determined as Other additions described above. B subunit EGF PDGF FBS FBS RESULTS Cell line (1 Ag/ml) None (4 ng/ml) (2 units/ml) (2.5%) (5%) NIH - 1.9 25.0 3.8 13.5 17.8 The B Subunit of Cholera Toxin Stimulates DNA Synthesis + 13.5 30.1 17.5 36.3 33.1 and Cell Division in Quiescent Nontransformed Mouse 3T3 Swiss - 4.8 19.5 13.6 35.8 51.5 Cells. Quiescent cells can be stimulated to synthesize DNA + 13.0 75.4 23.9 89.7 96.8 and proliferate by the addition of fresh serum or combina- BALB/c - 3.9 19.8 8.5 ND 19.3 tions of growth factors (19). To investigate the potential + 19.3 40.4 22.9 ND 48.1 functions of endogenous gangliosides in cell growth, we Quiescent cultures were exposed to the indicated mitogens in the exposed quiescent mouse cells (NIH, BALB/c, and Swiss absence (-) and presence (+) of the B subunit and assayed for 3T3 cells) to the B subunit of cholera toxin. The B subunit incorporation of [3H]thymidine; data are expressed as cpm of stimulated incorporation of [3H]thymidine by all three cell [3H]thymidine incorporated per well x 10-3. Results are the means lines in a concentration-dependent fashion (Fig. lA). A of triplicate determinations that varied <10%o and are from a significant effect was observed at 50 ng/ml (=1 nM) and representative experiment. Similar results were obtained with each maximum mitogenic stimulation was achieved at 500 ng/ml. cell line in at least five additional experiments. FBS, fetal bovine Maximal [3H]thymidine incorporation occurred at 20-24 hr serum; ND, not determined. (data not shown). The B subunit not only stimulated DNA synthesis but also caused cell division (Fig. 1B). A maximal B subunit was comparable to that caused by other known increase in cell numbers was observed after a 48-hr exposure mitogens, such as unfractionated serum, EGF, and PDGF. to the B subunit and was comparable to the increase mediated Addition of the B subunit in the presence of EGF, PDGF, or by 2% fetal bovine serum. Thus, the kinetics of the response fetal bovine serum further enhanced the stimulation of DNA ofquiescent 3T3 cells to the B subunit ofcholera toxin appear synthesis due to the growth factors alone (Table 1). There to be similar to other mitogens. were some differences among the three mouse 3T3 cell lines. Synergistic Effects Between the B Subunit of Cholera Toxin The response to PDGF was potentiated the most by the B and Other Growth-Promoting Factors. As shown in Table 1, subunit in NIH 3T3 cells, whereas EGF stimulation was most stimulation of [3H]thymidine incorporation in response to the enhanced by the B subunit in Swiss 3T3 cells.