Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.

Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.     

Altered sphingolipid metabolism in N-(4-hydroxyphenyl)-retinamide-resistant A2780 human ovarian carcinoma cells

In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

Role of Myelin-Associated Neuraminidase in the Ganglioside Metabolism of Rat Brain Myelin

The role of myelin-associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di- and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di- and polysialo-species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin-associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin.     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.     

Effect of gangliosides on binding, internalization and cytotoxic activity of ricin

The only gangliosides in Burkitt's lymphoma EB-3 cells is GM3. Treatment of Burkitt's lymphoma EB-3 cells with gangliosides GM1 or GM3 results in their binding to and partial incorporation into the cell membrane. About 25% of cell-associated ganglioside GM1 can interact with the ricin. However, such an increase in the number of binding sites does not enhance but rather decreases the cytotoxic effect of ricin. A similar protective effect was observed when the cells were pretreated with ganglioside GM3. In contrast, the increase in ricin biding sites caused by pretreatment of the cells with neuraminidase was accompanied by increase in ricin cytotoxicity. These differences may be related to observed differences in the rate of ricin-endocytosis by native and ganglioside-treated cells.     

Density-Dependent Changes in Gangliosides and Sialidase Activity of Murine Neuroblastoma Cells

Abstract: Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane- associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. G^M3 was the major ganglioside component of preconfluent S20Y cells, whereas G_DIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.     

Fluorescent derivatives of ganglioside GM1 function as receptors for cholera toxin

A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin-stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components.     

Coordinate Regulation of Ganglioside Glycosyltransferases in Differentiating NG108-15 Neuroblastoma X Glioma Cells

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.     

Isolation of an autocrine growth factor from hepatoma HTC-SR cells

A growth factor has been isolated from HTC-SR rat hepatoma tissue culture cells which specifically stimulates DNA synthesis and cell proliferation of the HTC cells that produce it. The factor can be isolated from HTC cell conditioned medium or from an HTC cell extract. This autocrine factor has been purified 640-fold from a postmicrosomal supernatant by successive steps, involving ethanol precipitation, heating at 80 degrees C for 10 min, chromatography on a DEAE Bio-Gel A column, and chromatography on a heparin-sepharose affinity column. The major peak of activity eluted from the heparin column migrates as a single band on SDS-PAGE with an apparent Mr of 60,000. The factor is resistant to acid, heat, and neuraminidase but sensitive to trypsin, papain, and protease. The autocrine nature of the factor is indicated by the finding that several other types of cells do not respond with increased DNA synthesis. Mouse L-cells, BHK cells, Novikoff hepatoma cells, hepatocytes in primary culture, and an epithelial-like rat liver-derived cell line (Clone 9) were tested, and none of the cells could be stimulated. Small amounts of the factor could be extracted from the Clone 9 cells, however. This material had the same physical and purification properties as the factor extracted from HTC cells, but it did not stimulate DNA synthesis in Clone 9 cells, only in HTC cells. Addition of the factor resulted in an almost immediate stimulation of DNA synthesis in a proliferating HTC cell population. When the factor was added together with [3H]thymidine for 2 h, a significant stimulation of DNA synthesis was observed, provided the addition was made between 18 and 48 h after the cells had been plated. Autoradiographic studies indicated that the factor both accelerates DNA synthesis in cells already making DNA and increases the number of cells entering the S period. The stimulation of DNA synthesis was completely inhibited by 10 mM hydroxyurea, whether the factor was present for 2, 24, or 48 h in the culture. A significant increase in cell number due to addition of the factor was also observed. This accelerated proliferation was detectable only after the cells had been in culture for at least 48 h with the factor present.     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

SV-40 transformation: effect on GM2 ganglioside in cultured cell lines.

In previous work, we observed the presence of substantially elevated levels of GM2 after Simian Virus 40 (SV-40) transformation of human fetal brain cells. This elevated level of GM2 contrasted with the reports of many other investigators who had often observed decreased levels of GM2 and a simplification of ganglioside pattern in various non-neural rodent cell lines. In order to determine if the increase in GM2 in the transformed human brain cells would also be found in transformed rodent brain cells, we analyzed ganglioside changes after transformation in mouse brain cell lines and observed the increase in GM3 and low levels or lack of GM2 usually noted in rodent SV-40 transformed cell lines. In addition, we analyzed changes after SV-40 transformation in three human fibroblast lines and found that all three lines contained substantially elevated levels of GM2 after SV-40 transformation. As a result of this study, our earlier work on SV-40 transformed human brain cells, and occasional other reports of high levels of GM2 in human SV-40 transformed cell lines, elevated levels of GM2 may be considered a marker for SV-40 transformed human cells of both fibroblastic and neural origin.     

Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ([1-14C]N-acetylmannosamine) and a radioactive precursor of ceramide ([3,3-3H2]serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.     

Glycosphingolipid Glycosyltransferases in Human Fetal Brain

The developmental pattern of gangliosides in human fetal brain should reflect the activities of the respective glycosyltransferases. LA2-synthase activity, along with that of GM3-, GD3-, GM2-, and GM1-synthases, was determined in human fetal brain at 10-22 weeks of gestation. LA2-synthase is the pivotal enzyme in lacto series ganglioside formation. LA2-synthase activity decreased during the study period, mirroring a similar temporal decline in levels of the lacto series gangliosides, particularly 3'-isoLM1. The developmental profiles of the ganglio series glycosyltransferase activities demonstrate distinct changes that correspond to the ganglioside pattern between fetal weeks 10 and 22. In particular, the marked increase in GM2-synthase activity at 20 and 22 weeks of gestation and the decline in GD3-synthase activity after 15 weeks could explain the prominent expression of the a series gangliosides in this period of rapid neuronal outgrowth. However, a similar decline (two- to 2.5-fold) in GM3-synthase activity suggests a more likely conclusion, namely, that the two sialyltransferase activities are derived mainly from astroglial cells, which show a marked proliferation during the 10-15th fetal weeks. The data do not negate the hypothesis that GM3- and GD3-synthase are the critical enzymes in the regulation of ganglioside biosynthesis but do indicate a need to reevaluate the significance of GM2-synthase in expression of the a series gangliosides.     

Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis

Using blood monocytes and lymphocytes from atherosclerotic patients and healthy subjects we have investigated a role of ganglioside GM3 in monocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC). The results showed that activity of GM3 synthase and cellular levels of ganglioside GM3 in blood mononuclear cells from atherosclerotic patients were several-fold higher than those from healthy subjects. In monocytes the activity of GM3 synthase was one order of magnitude higher than in lymphocytes from both groups studied; this suggests the major contribution of monocytes to enhanced biosynthesis and levels of GM3 in mononuclear cells in atherosclerosis. Enrichment of monocytes from healthy subjects with ganglioside GM3 by their incubation in the medium containing this ganglioside increased adhesion of these monocytes to HUVEC up to the level typical for monocytes from atherosclerotic patients. In addition, an increase in CD11b integrin expression comparable to that seen in lipopolysaccharide-activated monocytes was observed. It is suggested that in atherosclerosis the enhanced cellular levels of GM3 in monocytes and lymphocytes may be an important element of cell activation that facilitates their adhesion to endothelial cells and penetration into intima.     

Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.     

Ganglioside GM3 inhibits hepatoma cell motility via down‐regulating activity of EGFR and PI3K/AKT signaling pathway

Two related sublines derived from murine ascites hepatoma cell lines Hca‐F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3‐suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. J. Cell. Biochem. 114: 1616–1624, 2013. © 2013 Wiley Periodicals, Inc.     

Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100 degrees C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.     

神经节苷脂GM_3对人单核样白血病J6-2细胞中磷脂代谢的影响

用50μmol/L GM_3处理J6-2细胞6天,经组化与细胞学检查,证实细胞沿单核巨噬细胞途径分化。以[~(32)P]Pi或[CH_3-~(14)C]胆碱冲激(Pulse)后,将洗净的细胞进行Folch分配。取下相进行薄层层析,再做放射自显影。结果表明:GM_3的处理能促进[~(32)P]Pi或[CH_3-~(14)C]胆碱向磷脂酰胆碱的参入,而抑制向其他磷脂组分的参入。用[CH_3-~(14)C]胆碱冲激的Folch分配上相含水溶性胆碱代谢物,经TLC,结果表明CM_3的处理使[CH_3-~(14)C]胆碱向CDP-胆碱的参入减少。本研究证实,GM_3能调节J6-2细胞的磷脂代谢,其调节机制值得研究。     

Inhibition of DNA Synthesis in C6 Glioma Cells Following Cellular Incorporation of GM1 Ganglioside and Choleragenoid Exposure

The B subunit of cholera toxin, which is multivalent and binds specifically to GM1 ganglioside on the cell surface, has previously been used as a ganglioside-specific probe to regulate DNA synthesis in thymocytes and fibroblasts. To explore in more detail this growth-regulatory action of gangliosides, C6 glioma cells (which are GM1 ganglioside deficient) were used as a model system. When cultures of C6 cells were first treated with GM1, followed by exposure to the B subunit, proliferation was inhibited, as measured by 3H-labeled thymidine incorporation into DNA. Pretreatment of the cells with 50 microM GM1 for 15 min (followed by washing with fetal calf serum) and incubation with 1 microgram/ml of B subunit for 21 h was sufficient to reduce DNA synthesis to 15% of control values (and confirmed by autoradiographic analysis), although maximal inhibition could be achieved with as little as 30 min exposure to B, followed by washing. Furthermore, the B subunit inhibited the response of the C6 cells to basic fibroblast growth factor only following GM1 pretreatment. The B subunit-induced inhibition of DNA synthesis was specific for the ganglioside GM1, and was unrelated to increases of cyclic AMP. These results demonstrate that cell-incorporated GM1 ganglioside may act as a receptor capable of undergoing a specific ligand interaction, subsequently affecting molecular processes at the nuclear level.