Sensitivity of human melanoma cells to adherent leukocytes depends on the ratio between them, the activation status of adherent leukocytes and the metastatic potential of tumor cells

 This study examined the interaction of the poorly metastatic human melanoma cell line M4Be and the highly metastatic clone 4 derived from M4Be, with respect to fresh adherent leukocytes (AL) isolated from 17 different healthy blood donors. These AL contained 80% (73%–93%) monocytes, 15% (6%–20%) B lymphocytes and 5% (1%–8%) T lymphocytes. The survival of these tumor cells against the stress exerted by these AL was estimated with a clonogenic assay where isolated tumor cells were co-cultured for 14 days in contact with AL and lipopolysaccharide (LPS). For a given blood donor, AL either stimulates or inhibits the colony formation of the tumor cells (T) depending on the AL/T ratio, the AL activation status and the metastatic potential of tumor cells. At low AL/T ratios (<10/1) in the presence of low (8 ng/ml) and trace (8 pg/ml) levels of LPS, hydrogen peroxide (H₂O₂) release is significantly reduced, and tumor cells significantly increase their colony formation; the feeder effect of AL is suggested to be due to low concentrations of soluble tumor necrosis factor-alpha (TNF-α). At high AL/T ratios (>10/1), whatever the characteristics of the blood donor, clone 4 is significantly more sensitive than M4Be to AL activated with medium containing low (8 ng/ml) or high (1,000 ng/ml) levels of LPS; this killing effect is suggested to be due to TNF-α, both soluble and membrane-bound, but not to be due to release of H₂O₂. These data suggest that the regulatory role of AL, which remove the majority of human melanoma cells and stimulate the colony formation of a small fraction of them, is partly due to TNF-α. Received: 2 November 2000 / Accepted: 15 February 2001     

Gangliosides protect human melanoma cells from ionizing radiation-induced clonogenic cell death

With an experimental model of spontaneous lung metastases of melanoma developed in this laboratory, a range of sublines (variants and clones) with different metastatic potential and ganglioside expression was established from a single human melanoma cell line M4Be. Using anin vitro clonogenic assay and provided that cells were cultured for no more than five passages, variations in cellular radioresistance of M4Be and seven sublines derived from M4Be were detected. This study shows a positive correlation between the cell intrinsic radioresistance of M4Be and its seven sublines and their total ganglioside content. More precisely, the proportion of radioresistant cells in M4Be and the seven sublines correlated with the number of cells determined by flow cytometry that were positively labelled with a monoclonal antibody directed to GD3 disialoganglioside. Blocking the cellular biosynthesis of gangliosides with the inhibitor Fumonisin B1 or cleaving withVibrio cholerae neuraminidase the cell surface ganglioside-bound sialic acid in a radioresistant poorly metastatic subline increased its radiosensitivityin vitro. In contrast, enrichment of a radiosensitive metastatic subline with exogenous bovine brain GM1 increased its radioresistancein vitro. These results suggest that, in the radiation dose range important for radioprotection (0–1 Gy), membrane gangliosides radioprotect human melanoma cellsin vitro.Presented at the 43rd Annual Meeting of the Radiation Research Society, San Jose, USA, 1–6 April 1995 (Abstract).     

Selective shedding of gangliosides by cells of mouse ascitic sarcoma-37

The profile and content of gangliosides associated with tumour cells of mouse sarcoma-37 and exfoliated from the cell surface were determined. It was shown that 20% of tumour gangliosides shed from the cell surface and only about 7% of all the shed gangliosides were contained in plasma membrane fragments. When incubated in vitro at 37 degrees C for 1 hour, the tumour cells were found to release less than 2% of cellular gangliosides. The main components of cellular gangliosides corresponded in their chromatographic behaviour to GM2 (31-32% of the total ganglioside content), GD3 (17-18%), GD1a (9-11%), GD2 (16-17%) and hematosides (19-21%). The profiles of in vivo and in vitro shed gangliosides were similar but strongly differed from those of cellular gangliosides: the relative content of GM2 was 70-75% and the other gangliosides were found in negligible amounts. The data show that GM2 accumulation in extracellular spaces is rather the result of its selective shedding than the shedding of products of "incomplete" cellular ganglioside synthesis.     

神经节苷脂对脂多糖损伤大鼠嗜铬细胞瘤细胞的保护作用

目的：研究内源性神经节苷脂对大鼠嗜铬细胞瘤细胞株（PC12）脂多糖（LPS）损伤后的作用及机制。方法：培养PC12细胞,建立LPS损伤模型,采用MTT法检测不同浓度LPS对PC12细胞存活率的改变、葡萄糖神经酰胺合成酶抑制剂D（D-PDMP）耗竭内源性神经节苷脂时LPS对PC12细胞存活率的改变以及添加外源性神经节苷脂GM1后对PC12细胞存活率的保护作用;倒置显微镜和荧光显微镜观察细胞状态;RT-PCR检测NF-κB的相对表达含量。结果：LPS能导致PC12细胞形态学的改变及存活率下降,且随着LPS浓度的增加细胞存活率逐渐降低;神经节苷脂GM1能明显改善LPS所致的细胞形态学以及存活率的改变;工具药D-PDMP耗竭内源性神经节苷脂后,LPS对PC12细胞的损伤更严重,添加外源性神经节苷脂GM1,细胞形态学及存活率得到明显改善,细胞存活率上升;LPS损伤时细胞内NF-κB含量增加;D-PDMP预先耗竭内源性神经节苷脂时NF-κB含量增加更多;添加外源性神经节苷脂GM1后NF-κB含量降低。结论：内源性神经节苷脂对PC12细胞LPS损伤具有保护作用,可能是通过NF-κB信号通路来发挥作用的。     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.     

The role of gangliosides in the interaction of a growth inhibitor with mouse LM cells

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast

NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found. Our results further establish that the ability of a cell to organize fibronectin into an extracellular matrix is dependent on certain gangliosides, but they also indicate that cell adhesion to fibronectin is independent of these gangliosides. We suggest that matrix organization and cell attachment and spreading are based on separate mechanisms and that these functions are associated with different cell surface "receptors."     

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.     

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature- dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine- labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.     

Enhancement of metastatic potential of mouse B16-melanoma cells to lung after treatment with gangliosides of B-16-melanoma cells of higher metastatic potential to lung

Mouse B16LuF1 melanoma cells of lower metastatic potential to lung were treated in vitro with same concentration (50 microM) of gangliosides isolated from B16LuF5, B16LuF9 or B16LuF10 cells with higher metastatic potential to lung (LuF1< LuF5< LuF9< LuF10) and injected to groups of normal mice through tail vein. The number of metastatic tumor nodules formed in lung increased in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment. Metastatic potential of B16LuF1 cells gradually increased after treatment with gangliosides of B 16-melanoma cells of increasing metastatic potential to lung. The six major gangliosides isolated from B16LuF10 cells corresponded with standard gangliosides GT1b, GD1b, GD1a, GM1, GM2 and GM3 respectively on TLC-analysis. When B16LuF1 cells were treated in vitro with each of these six individual gangliosides and injected to groups of normal mice through tail vein the number of tumor nodules formed in lung varied. The four groups of mice receiving B16LuF1 cells treated with each of four gangliosides corresponding to GT1b, GD1b, GD1a or GM1 produced lung metastasis comparable to that of untreated control group. Only remaining two gangliosides which corresponded with standard gangliosides GM2 and GM3 increased metastatic potential of B16LuF1 cells. Thus, these results indicated that gangliosides GM2 and GM3 of B16-melanoma cells are definitely associated with metastatic potential of these tumor cells.     

Patterns of endogenous gangliosides and metabolic processing of exogenous gangliosides in cerebellar granule cells during differentiation in culture

The qualitative and quantitative pattern of endogenous gangliosides and the routes of metabolic processing of exogenous GM1,³H labeled in the sphingosine moiety (Sph-³H GM1) were studied in cerebellar granule cells during differentiation in vitro. During the first 7–8 days in culture the ganglioside content markedly increased, and the qualitative pattern showed, in percentage terms, a drastic decrease of GD3 and a marked increase of GD2, O-Ac-GT1b, O-Ac-GQ1b and GQ1b. After pulse with (Sph-³H) GM1, at all the investigated days in culture, different radiolabelled lipids were formed indicating that taken up exogenous GM1 was degraded and that its catabolic fragments, and partly GM1 itself, were used for biosynthetic purposes; moreover radioactive water was measured in the culture medium during chase indicating that labelled sphingosine underwent also degradation. The uptake of exogenous GM1 and the extent of its metabolic processing per cell unit increased during differentiation: a) GM2 was the major metabolic product and was relatively more abundant at 2 than 7 days in culture; b) the percentage of metabolites of biosynthetic origin over total metabolites increased during differentiation, especially at the short pulse times; c) among the metabolites of anabolic origin sphingomyelin equalled gangliosides at 2 days, whereas it was largely overcome by gangliosides at 7 days in culture; d) at 4 and 7 days in culture a radioactive substance, not yet identified, was present, whereas no trace of it was found at 2 days. In conclusion, cerebellar granule cells in culture feature a different pattern of endogenous gangliosides and display different ability to metabolically process exogenous GM1 ganglioside in the undifferentiated and fully differentiated stage.Abbreviations used: this article follows the ganglioside nomenclature of Svennerholm [J. Lipid Res., 5, 145–155, (1964)] and the IUPAC-IUP recommendations for lipid nomenclature [Lipids, 12, 455–468, (1977)] NeuAc N-Acetylneuraminic acid; sph, sphingosine - O-Ac O-acetylated - TLC thin layer chromatography     

Immunosuppression by human gangliosides. II. Carbohydrate structure and inhibition of human NK activity.

Gangliosides shed by tumors enhance tumor formation, possibly by suppressing host antitumor immune function, and gangliosides purified from animal tissues and cultured cells inhibit human cellular immune function in vitro. Determination of immunosuppressive activity of highly purified gangliosides, to uncover structure-activity relationships, is therefore important. Here we have studied a series of gangliosides obtained from human tissue and determined their effects on human natural killer (NK) activity. Total gangliosides from human brain tissue were moderately inhibitory; 100 nmol/ml reduced NK activity of human nonadherent PBMC by 43%. The influence of carbohydrate structure upon inhibitory activity was determined by study of eight highly (HPLC) purified individual gangliosides. Of these, we unexpectedly found that the two minor brain gangliosides with the simplest carbohydrate structures, GM2 and GM3, were very active inhibitors (75 and 47%, respectively, at 50 nmol/ml). In contrast, the structurally more complex major species, GM1, GD1a, GD1b, GT1b, and two other minor gangliosides, GD2 and GD3, were inactive. Reduced effector-target binding in a single-cell binding assay by GM2 but not GM3 suggests different mechanisms of inhibition by these two active gangliosides. Since GM2 and GM3 are present in high concentrations in, and are shed by, several common human tumors (e.g., neuroblastoma, melanoma, and glioma), their ability to inhibit NK cytotoxicity supports the hypothesis of a role of shed tumor gangliosides in the enhancement of tumor formation.     

Fibronectin binding to gangliosides and rat liver plasma membranes

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.     

Lack of specificity of brain gangliosides in the modulation of lymphocyte activation

A potential role for glycolipid gangliosides to act as immunomodulating agents has been suggested. Most studies have employed brain gangliosides. We have systematically investigated highly purified murine brain gangliosides for their ability to modulate lymphocyte activation. All sialic acid classes of ganglioside inhibited lipopolysaccharide (LPS)-induced antibody secretion and all polysialated gangliosides inhibited LPS-induced DNA synthesis. Monosialated gangliosides had no effect on DNA synthesis induced by LPS. 8-BrcGMP-induced DNA synthesis was also inhibited, suggesting that a negative signal was delivered to B lymphocytes by co-cultivation with exogenous gangliosides. The lack of specificity with respect to sialic acid class observed in these studies suggests that further investigation of an immunomodulatory role for gangliosides focus on endogenous lymphocyte gangliosides.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

Anabolic sialosylation of gangliosides in situ in rat brain cortical slices

Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of gangliosides in glucosamine-induced proliferation decrease of retinal pericytes

The hexosamine pathway (HP) is a biochemical hypothesis recently proposed explaining cellular alterations occurring during diabetic microvascular complications. Diabetic retinopathy is a common microvascular complication of diabetes, and it is known that cell proliferation is severely affected during the development of the disease. Particularly, early stages are characterized by death of the retinal microvascular cells, pericytes. Gangliosides have often been described to regulate cell growth; however, very few studies focused on the potential role of gangliosides in diabetic microvascular alterations. The aim of this article was to investigate the effect of the HP activation on pericyte proliferation and determine the potential implication of gangliosides in this process. Results indicate first that HP activation, mimicked by glucosamine treatment, decreased pericyte proliferation. Second, glucosamine treatment induced a modification of gangliosides pattern, particularly GM1 and GD3 were significantly increased. Next, results showed that exogenous addition of a-series gangliosides (GM3, GM2, GM1, GD1a) and b-series ganglioside (GD3) caused a decrease of pericyte proliferation, whereas nonsialylated precursors glucosylceramide and lactosylceramide were without effect. Furthermore, when ganglioside biosynthesis was blocked using PPMP, a glucosylceramide synthase inhibitor, the effects of glucosamine on pericyte proliferation were partially reversed. Our results suggest that in retinal pericytes, gangliosides and particularly GM1 and GD3 that are increased in response to glucosamine, are involved in the antiproliferative effect of glucosamine. These observations also underlie the potential involvement of gangliosides in a pathological context, such as diabetic microvascular complications.     

Inhibition of TLR activation and up-regulation of IL-1R-associated kinase-M expression by exogenous gangliosides

Gangliosides, sialic acid-containing glycosphingolipids present in the outer leaflet of plasma membranes, are produced at high levels by some tumors, are actively shed into the tumor microenvironment, and can be detected in high concentrations in the serum of cancer patients. These tumor-shed molecules are known to be immunosuppressive, although mechanisms remain to be fully elucidated. In this study, we show that membrane enrichment of human monocytes with purified exogenous gangliosides potently inhibits ligand-induced activation and proinflammatory cytokine production induced by a broad range of TLRs, including TLR2, TLR3, TLR6, and TLR7/8, in addition to a previously identified inhibitory effect on TLR4 and TLR5. Inhibition of TLR activation is reversible, with complete restoration of TLR signaling within 6-24 h of washout of exogenous gangliosides, and is selective for certain gangliosides (GM1, GD1a, and GD1b), whereas others (GM3) are inactive. To characterize the inhibition, we assessed the expression of the TLR signaling pathway inhibitor, IL-1 receptor associated kinase-M (IRAK-M). In response to ganglioside enrichment alone, we observed striking up-regulation of IRAK-M in monocytes, but without concomitant proinflammatory cytokine production. This contrasts with endotoxin tolerance, in which IRAK-M up-regulation follows proinflammatory cytokine expression caused by LPS exposure. We hypothesize that ganglioside treatment induces a state of tolerance to TLR signaling, leading to blunted activation of innate immune responses. In the tumor microenvironment, shed tumor ganglioside enrichment of APC membranes may likewise cause these cells to bypass the normal TLR signaling response and progress directly to the inhibitory state.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Certain phosphorylated sugars can prevent the mRNA-induced inhibition of Met-tRNA f Met binding to initiation factor eIF-2

When mouse myeloid leukemia M1 cells were induced to differentiate into macrophages by bacterial lipopolysaccharide (LPS), phospholipids and gangliosides of the cells changed markedly. The amounts of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol per mg protein increased 30%, 20% and 30%, respectively, during differentiation, but the others, phosphatidylserine and sphingomyelin, did not increase significantly. Three species of gangliosides constituted of major portions of gangliosides in M1 cells. Several-fold increase in monosialoganglioside GM1 was observed in the LPS-treated cells with a concomitant decrease in disialogangliosides. Based upon the treatment with sialidase, this GM1 was identified to be GM1b, which was recently found in rat ascites hepatoma cells and human erythrocyte membranes.