UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.     

Gangliosides from human melanoma immunomodulate response of T cells to interleukin-2

The gangliosides expressed by normal melanocytes are predominantly GM3 (greater than 90%) and GD3 (less than 5%). Malignant melanoma can express several other types of gangliosides in significant quantities, including GM2 and GD2. Melanoma patients can develop an immune response against some of these ganglioside antigens on autologous melanoma cells. The four major gangliosides expressed by human melanoma cells (GM3, GD3, GM2, and GD2) were examined for their immunomodulatory effect on lymph node lymphocytes from melanoma patients. Gangliosides were added exogenously to lymphocytes grown in the presence of IL-2. Preferential interactions of specific melanoma gangliosides on IL-2 stimulation were found. While GM2 and GD2 enhanced the lymphocyte response to IL-2, GM3 and GD3 significantly inhibited this response. GM2 and GD2 differ from GM3 and GD3 by the presence of a terminal N-acetylgalactosamine. Since different gangliosides can up-regulate and down-regulate lymphocyte responses to IL-2, the ganglioside phenotype of melanoma cells may play a major role in determining whether an individual tumor causes immune stimulation or suppression.     

Gangliosides of normal and neoplastic human melanocytes

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.     

Glycosyltransferase Activities in Cultured Endothelial Cells of Bovine Brain Microvascular Origin

Bovine brain microvascular endothelial cells (BMECs) express GM3 (NeuAc) and GM3 (NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b as well as sialosylparagloboside and sialosyllactosaminylparagloboside as the minor species. To investigate the metabolic basis of this ganglioside pattern, the activities of eight glycosyltransferases (GM3-, GD1a-, GD3-, LM1-, GM2 (NeuAc)-, GM2 (NeuGc)-, LacCer-, and GM1-synthases) in cultured BMECs were studied. It was found that BMECs possessed high activities of GM3- and GD1a-synthases, and low activities of GM2-, GM1-, and GD3-synthases. Thus, the present study provides evidence that endothelial cells are capable of synthesizing gangliosides in situ and that the high content of GM3 in BMEC is closely associated with high activities of GM3-synthase and low activities of GM2-, GM1-, and GD3-synthases.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

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.     

Ganglioside composition of astrocytes

Short-term and long-term (greater than 7 months) cultured astrocytes from 14-day-old rat brain were analyzed for ganglioside content. Analysis of the extracted gangliosides by HPTLC revealed that ganglioside GM1 was absent in 35 days and 235 days cultured astrocytes, and that the predominant ganglioside was GM3, showing a double band in both cases. A small amount of the disialogangliosides (GD3, GD1a) was also detected. More than 70% of radioactivities into ganglioside fractions by cultured astrocytes, in the presence of N-[3H]-acetylmannosamine, appeared in ganglioside GM3. The upper band component of GM3 increased 60% in long-term astrocyte cultures compared to 35-day-old cultures. Also, an increased GD3 content in long-term astrocyte cultures was detected. These results suggest that the increase of GD3 and upper band GM3 in long-term cultured astrocytes might be related to the appearance of small processes showing strong reactivity against GFAP and vimentin during astrocyte-subculture.     

Oncogene transgenic mice: an useful model to study in vivo the relationships between gangliosides and oncogenes

Several studies have demonstrated that transfer of oncogenes in cultured cells reproducibly induces transmissible alterations in their ganglioside profile; the transfection of the same oncogene into different cell lines and the different localization of the oncogene product result in a different ganglioside expression. In the present study the modifications of the ganglioside pattern in mammary carcinomas induced in transgenic mice by the activated form of the rat neu oncogene have been investigated. Whereas control mammary tissues contain quite exclusively GM3, all neoplastic samples show a substantial decrease of this ganglioside, an accumulation in variable amount of GM3-derived species (GM1, GD3, GD1a, GD1b, GT and GQ) and the appearance of new, not yet identified, sialic acid containing molecules. Interestingly, three out of 10 tumors analyzed, even if histologically comparable to the others but with a larger dimension, show a significative difference as regard to the GM1, GD3 and GD1a content. Our data suggest that an activated oncogene may induce also in vivo a specific and transmissible alteration in the ganglioside pattern, but this distribution could be susceptible to further modifications during the tumor progression.     

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.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Mechanism for the negative regulation of cell proliferation by ganglioside GM3 in high glucose-treated glomerular mesangial cells

We recently identified ganglioside GM3 as a modulator of glomerular hypertrophy in streptozotocin-induced diabetic rats (Life Sci., 72: 1997-2006, 2003). This study examined whether alteration of ganglioside GM3 expression could modulate the high glucose-induced proliferation of glomerular mesangial cells (GMCs). GMCs isolated from rat kidneys were cultured under normal (5.6 mM) or high (25 mM) glucose condition for 24-72 hrs. Cell proliferation was predominantly stimulated when GMCs were cultured with high glucose as well as 20 microM of d-threo-PDMP, an inhibitor of ganglioside biosynthesis, for 24 hrs, whereas raising ambient glucose significantly reduced the mesangial sialic acid contents. Based upon mobility on high-performance thin-layer chromatography (HPTLC), GMCs showed a complex pattern of ganglioside expression that consisted of three major components of gangliosides, mainly GM3. High glucose induced a significant reduction of ganglioside expression with apparent changes in the composition of major ganglioside GM3, and semi-quantitative analysis by HPTLC showed that ganglioside GM3 was reduced to 62% of GMCs cultured under normal glucose condition. A prominent immunofluorescence microscopy using anti-GM3 monoclonal antibody also showed a dramatic disappearance of immunoreactivity in high glucose-treated GMCs. Moreover, high glucose significantly lowered the Km values of GM3 synthase (16 microM vs. 49 microM), but did not change the Vmax. These results provide the pathophysiological relationship between the high glucose-induced proliferation of GMCs and the decreased expression of ganglioside GM3, indicating a mechanism for the negative regulation of mesangial proliferation by ganglioside GM3. This mechanism may play an important role in the development of diabetic glomerulopathy.     

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells*

Gangliosides of the plasma membrane are important modulatorsof cellular functions. Previous work from our laboratory hadsuggested that a plasma membrane sialidase was involved in growthcontrol and differentiation in cultured human neuroblastomacells (SK-N-MC), but its substrates had remained obscure. Wenow performed sialidase specificity studies in subcellular fractionsand found ganglioside GM3 desialylating activity in presenceof Triton X-100 to be associated with the plasma membrane, butabsent in lysosomes. This Triton-activated plasma membrane enzymedesialylated also gangliosides GDla, GD1b, and GT1b, therebyforming GM1; cleavage of GM1 and GM2, however, was not observed.Sialidase activity towards the glycoprotein fetuin with modifiedC-7 sialic acids and towards 4-methylumbelliferyl neuraminatewas solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in ganglioside desialylationof living cells was examined by following the fate of [³H]galactose-labelledindividual gangliosides in pulse-chase experiments in absenceand presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminicacid. When the plasma membrane sialidase was inhibited, radioactivityof all gangliosides chased at the same rate. In the absenceof inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degradedat a considerably faster rate in confluent cultures, whereasthe GM1-pool seemed to be filled by the desialylation of highergangliosides. The results thus suggest that the plasma membranesialidase causes selective ganglioside desialylation, and thatsuch surface glycolipid modification triggers growth controland differentiation in human neuroblastoma cells. ganglioside neuroblastoma cells plasma membrane sialidase     

Assembly of hereditary amyloid beta-protein variants in the presence of favorable gangliosides

The mechanisms underlying regional amyloid beta-protein (Abeta) deposition in brain remain unclear. Here we show that assembly of hereditary variant Dutch- and Italian-type Abetas, and Flemish-type Abeta was accelerated by GM3 ganglioside, and GD3 ganglioside, respectively. Notably, cerebrovascular smooth muscle cells, which compose the cerebral vessel wall at which the Dutch- and Italian-type Abetas deposit, exclusively express GM3 whereas GD3 is upregulated in the co-culture of endothelial cells and astrocytes, which forms the cerebrovascular basement membrane, the site of Flemish-type Abeta deposition. Our results suggest that regional Abeta deposition is induced by the local gangliosides in the brain.     

Glycolipids of mouse erythroleukemia cells (Friend cells) and their alteration during differentiation

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.     

Alteration of ganglioside composition by stable transfection with antisense vectors against GD3-synthase gene expression.

Gangliosides are ubiquitous components of mammalian cells. Their expression is frequently altered in many tumor types. We previously showed that alteration of the ganglioside composition often resulted in changes in cellular morphology and differentiation of cultured cells. In this study, we targeted sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetylated GD3 (OAc-GD3) in the neuroblastoma F-11 cells greatly reduced the tumor growth in nude mice. The sense and antisense vectors containing either a 5' end fragment or the entire sequence of the cDNA coding for GD3-synthase were prepared and used in separate experiments to transfect the F-11 cells which express high levels of gangliosides GD3 and OAc-GD3. Single clones were isolated and expanded. Both the activity of the GD3-synthase and the concentrations of GD3 and OAc-GD3 in the antisense-transfected cells were dramatically decreased as a result of transfection with the antisense expression vectors. Further characterization of the antisense-transfected cells showed reduced rates of cell growth and neurite formation and changes in cellular morphology. When the cells were inoculated in athymic nude mice, the tumor growth rate was remarkably suppressed although the tumor incidence was not affected by the altered ganglioside composition. These results indicate that the tumor-associated ganglioside(s) is(are) involved in regulation of tumor growth, probably through the stimulation of angiogenesis of the tumor.     

Adaptation of Zajdela ascitic hepatoma cells to monolayer growth: change in the cell ganglioside pattern

The Zajdela hepatoma is a transplantable ascitic tumor of the rat, characterized by a very simple ganglioside pattern, GM3 being the main compound. When these cells are adapted to monolayer culture, they undergo a maturation process and the total cellular ganglioside concentration increases progressively; GM2, GM1 and GD3 amounts rose and GD1a accumulated. These modifications in the ganglioside pattern complexity are not affected by the addition of ascitic fluid to the cultures, nor by growth in serum free, hormone-supplemented medium. They are totally reversible when the cultured hepatoma cells are reinjected into a rat and developed an ascitic tumour. Cell growth control and adhesion processes could be related to the maturation process of these hepatoma cells growing in monolayer, which may constitute a convenient model for further investigations on the regulation of membrane glycolipid composition by the external environment.     

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I)

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform–methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.     

Lipid composition of human malignant melanoma tumors at various levels of malignant growth.

The lipid pattern of thirteen human melanoma tumors from various tissues were investigated. In seven of the tumors, an estimate was given about the proportion of malignant melanocytes to the total cell population, and a reverse correlation was determined between the proportion of malignant cells in these tumors and their neutral lipid content. The phospholipids did not show any modification, nor did the cholesterol in the cancerous tissues. The ganglioside pattern was found to be similar in all analyzed samples, with GM3, GM2 and GD3 as major components, although no correlation was found between the malignant level and the ganglioside content of the tumors.