g(m3)-ganglioside and Neuroblastoma

CD40 expression by dendritic cells (DCs) critically regulates their maturation/antitumor activity. CD40-CD40 ligand (CD40L) signaling stimulates DC-mediated IL-12 production/cytotoxicity. Recent studies suggest that neuroblastoma (NB)-derived gangliosides impair DC maturation, IL-12 secretion, and NK/T-cell activity. Neuroblastoma ganglioside-mediated abrogation of CD40 expression by DC and tumor-induced tolerance has not been studied. The purpose of this study is to determine if NB inhibits DC IL-12 production via CD40. The contributory role of the NB-derived ganglioside GM3 in this process is also examined.. Dendritic cells were generated from bone marrow of mice injected with saline (control) or murine NB. Control DCs were matured with or without GM3. Dendritic cells were cocultured with NB cells treated with or without a ganglioside synthesis inhibitor. Dendritic cell groups were analyzed for maturation/costimulatory markers. Control and tumor-derived DC were stimulated with CD40L or Staphylococcus aureus and studied for IL-12 expression.. CD40 expression on DC generated from NB bearing mice decreased by 64% (P < .001). GM3 down-regulated DC maturation and CD40 expression. Only CD40-dependent IL-12 production was abrogated (60%, P < .01) in DC derived from NB-bearing mice. Dendritic cell capacity to synthesize IL-12 remained intact.. Neuroblastoma-induced inhibition of DC function may result from ganglioside-mediated CD40 signaling deficiency. Strategies to bypass/augment CD40-CD40L signaling may improve current NB immunotherapies.

Topics: Animals; CD40 Antigens; CD40 Ligand; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Down-Regulation; G(M3) Ganglioside; Interleukin-12; Mice; Mice, Inbred Strains; Nervous System Neoplasms; Neuroblastoma; Signal Transduction

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.

Topics: Animals; Carbohydrate Sequence; Cell Division; Dogs; Epidermal Growth Factor; ErbB Receptors; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Molecular Sequence Data; Neuroblastoma; Phosphorylation; Structure-Activity Relationship; Tumor Cells, Cultured; Tyrosine

The orientation of the catalytic site of a ganglioside-specific sialidase in the plasma membrane of SK-N-MC neuroblastoma cells was probed using water-soluble GD1a-neoganglioprotein substrate on intact cells and GM1-product detection by cholera toxin B. Desialylation of substrate was readily observed, whereas specific sialidase inhibitors prevented the reaction, and conditioned medium was inactive. Inhibitors of endocytosis and acidification had no effect on substrate degradation, and lowering temperature to 18 degrees C reduced activity but did not abolish it. We conclude that the ganglioside sialidase activity is cell surface-orientated and displays an in situ specificity that mirrors enzyme preparations in vitro.

Topics: Catalytic Domain; Cell Membrane; Cholera Toxin; Culture Media, Conditioned; Enzyme Activation; Enzyme Inhibitors; Extracellular Space; G(M3) Ganglioside; Gangliosides; Glycoproteins; Humans; Neuraminidase; Neuroblastoma; Substrate Specificity; Temperature; Tumor Cells, Cultured

Hemopoiesis is disturbed in bone marrow-involving cancers like leukemia and neuroblastoma. Shedding of gangliosides by tumor cells may contribute to this tumor-induced bone marrow suppression. We studied in vitro the inhibitory effects of murine neuroblastoma cells (Neuro-2a and C1300) and their gangliosides on hemopoiesis using normal murine hemopoietic progenitor colony-forming assays. Transwell cultured neuroblastoma cells showed a dose-dependent inhibition on hemopoiesis, indicating that a soluble factor was responsible for this effect. Furthermore, the supernatant of Neuro-2a cultured cells inhibited hemopoietic proliferation and differentiation. To determine whether the inhibitory effect was indeed due to shed gangliosides and not, for instance, caused by cytokines, the effect of DL-threo-1 -phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) on Neuro-2a cells was studied. DL-PDMP is a potent inhibitor of glucosylceramide synthase, resulting in inhibition of the synthesis and shedding of gangliosides. The initially observed inhibitory effect of supernatant of Neuro-2a cells was abrogated by culturing these cells for 3 days in the presence of 10 microM DL-PDMP. Moreover, gangliosides isolated from Neuro-2a cell membranes inhibited hemopoietic growth. To determine whether the described phenomena in vitro are a reflection of bone marrow suppression occurring in vivo, gangliosides isolated from plasma of neuroblastoma patients were tested for their effects on human hemopoietic progenitor colony-forming assays. These human neuroblastoma-derived gangliosides inhibited normal erythropoiesis (colony-forming unit-erythroid/burst-forming unit-erythroid) and myelopoiesis (colony-forming unit-granulocyte/macrophage) to a higher extent compared with gangliosides isolated from control plasma. Altogether these results suggest that gangliosides shed by neuroblastoma cells inhibit hemopoiesis and may contribute to the observed bone marrow depression in neuroblastoma patients.

Topics: Animals; Female; G(M3) Ganglioside; Gangliosides; Hematopoiesis; Humans; Mice; Mice, Inbred C57BL; Neuroblastoma

Exogenously added gangliosides are known to promote neurite outgrowth in a variety of cell types, including some neuroblastoma cell lines. To study neuritogenesis in SH-SY5Y human neuroblastoma we serum starved the cells for 24 hr and exposed them to gangliosides (GM1, GM3, or GT1b), platelet-derived growth factor (PDGF), insulin, nerve growth factor (NGF), insulin-like growth factor I (IGF-I), or combinations of these for 3 days. We measured four parameters of neurite outgrowth using image analysis. PDGF induced neurite outgrowth in SH-SY5Y and GM1 inhibited this. Both phenomena were dose-dependent with neurites/cell and neurite length being below controls with 100 microM GM1, and percent of neurite-bearing cells being below controls with 25, 50, and 100 microM GM1. Similar but more inhibitory results were obtained with GM3 and GT1b. Insulin and IGF-I induced a neuritogenic response that was less potent than that of PDGF and was also inhibited by gangliosides. NGF had no effect on neurite outgrowth but gangliosides were still inhibitory even in cells not treated with growth factors. From this we conclude that gangliosides inhibit spontaneous and trophic factor-induced neurite outgrowth in SH-SY5Y cells. For GM1 and GT1b, but not GM3, this probably involves inhibition of trophic factor receptor function.

Topics: Culture Media, Serum-Free; Dose-Response Relationship, Drug; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Growth Inhibitors; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Kinetics; Nerve Growth Factors; Neurites; Neuroblastoma; Platelet-Derived Growth Factor; Tumor Cells, Cultured

Gangliosides are important components of the cell membrane that are usually shed in the surrounding microenvironment by neoplastic cells. Gangliosides can also modulate the angiogenic response of microvessels stimulated by angiogenic factors. The experiments reported here make a contribution to the assessment of the nature of this angiogenic modulation, by demonstrating that a) GM3 gangliosides can block the proliferation of endothelium induced by neoplastic cells from human tumors of five different origins; b) this block also occurs when the endothelial cells are preincubated with GM3 and disappears when the cells are returned to a medium poor in GM3; c) in the presence of GM3 the capacity of the endothelial cells to bind to fibronectin and to collagen types I and IV was sharply reduced; d) concentrations of GM3 able to block endothelial cell growth are counteracted by addition to the medium of GT1b ganglioside. The data suggest that the prevalence of a microenvironment rich in GM3 prevents proliferation of vascular endothelium, but the appropriate presence of another ganglioside, such as GT1b, nullifies the effect. Modulation of the angiogenic response of vascular endothelium to angiogenic factors released by tumors is probably dependent on the distribution and activity of growth factor receptors on the endothelial cell surface. The nature and concentration of the gangliosides in the endothelial microenvironment have a decisive influence on this event and possibly on the progression of tumor-induced angiogenesis.

Topics: Angiogenesis Inducing Agents; Cell Adhesion; Cell Division; Cell Membrane; Collagen; Culture Media; Disease Progression; Endothelium, Vascular; Fibronectins; G(M3) Ganglioside; Gangliosides; Humans; Microcirculation; Neovascularization, Pathologic; Neuroblastoma; Protein Binding; Radiopharmaceuticals; Receptors, Growth Factor; Thymidine; Tritium; Tumor Cells, Cultured

Prosaposin has been recently identified as a neurotrophic factor eliciting differentiation in neuronal cultured cells (NS20Y). In this paper we investigate whether prosaposin and its active peptide (prosaptide) may modify the ganglioside pattern in neuroblastoma cells. The analysis by high performance thin layer chromatography did not reveal qualitative changes in the ganglioside pattern of NS20Y cells incubated in the presence of prosaposin, compared to control cells, but it did reveal an increase of the content of all three major resorcinol positive bands (GM3, GM2, GD1a). Cytofluorimetric and immunofluorescence microscopic analysis revealed that the increase of the ganglioside content was at the plasma membrane level. These findings suggest that the neurotrophic activity of prosaposin on NS20Y neuroblastoma cells might be mediated in part by the increase of cell surface gangliosides.

Topics: Animals; Cell Line; Cell Membrane; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Flow Cytometry; Fluorescent Antibody Technique; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Glycoproteins; Membrane Lipids; Mice; Nerve Growth Factors; Neuroblastoma; Protein Precursors; Saposins; Tumor Cells, Cultured

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Cholera Toxin; Dihydropyridines; Egtazic Acid; Fura-2; G(M1) Ganglioside; G(M3) Ganglioside; Manganese; Mice; Microscopy, Fluorescence; Neuroblastoma; Nicardipine; Nickel; Potassium; Tumor Cells, Cultured

Neuro-2a neuroblastoma cells, when differentiated via a cAMP-dependent pathway by treatment with anti-GM3 monoclonal antibody, accumulated a high level of pp60c-src protein and pp60c-src kinase activity just before the onset of neurite formation. The specific kinase activity of the accumulated c-src protein was found to be comparable to that of normal cerebellar neurons, but was about 6- to 8-fold higher than that of normal astrocytes. These results, and migrations of peptide fragments in the SDS-polyacrylamide gels after V8 proteolysis, strongly indicate the accumulation of the neuron-specific isoform of the c-src protein (pp60c-srcN) in the GM3 antibody-treated Neuro-2a cells. Similar high levels of pp60c-src protein and pp60c-src kinase activity were observed in the Neuro-2a cells differentiated via a cAMP-dependent pathway by treatment with dibutyryl cAMP, but not in the same cell line when differentiated via a cAMP-independent pathway with 5-bromo-2'-deoxyuridine. These results demonstrate that the accumulation of high levels of the neuron-specific isoform of the pp60c-src protein (pp60c-srcN) in the Neuro-2a neuroblastoma cells depends on the specific signal transduction pathway involved during the differentiation of these cells.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Cell Differentiation; Cyclic AMP; G(M3) Ganglioside; Mice; Nerve Tissue Proteins; Neuroblastoma; Neurons; Proto-Oncogene Proteins pp60(c-src); Time Factors; Tumor Cells, Cultured

beta-D-Xylosides are often used to competitively inhibit proteoglycan synthesis by serving as primers for free glycosaminoglycan (GAG) chain assembly. Quite unexpectedly, we found that when human melanoma cells and Chinese hamster ovary cells are labeled with [3H] galactose in the presence of 4-methyl umbelliferyl beta-D-xyloside (Xyl beta 4MU), a large portion of the labeled acceptor does not consist of the expected GAG chains, but of the novel GM3 ganglioside-like structure: Sia-alpha 2,3-[3H]Gal beta 1, 4Xyl beta 4MU. Moreover, formation of this derivative is associated with an inhibition of glycosphingolipid synthesis by up to 78% without affecting synthesis of other [3H]Gal-labeled glycoconjugates. Inhibition occurs rapidly and equally for all glycolipid species and is partially abrogated by brefeldin A. Inhibition requires the addition of a single galactose residue to the xyloside within the lumen of the Golgi apparatus. This addition appears to be carried out by galactosyl transferase I that normally synthesizes the core region of GAG chains. Although alpha-xyloside does not inhibit proteoglycan synthesis, it is galactosylated, but not sialylated, and is nearly as effective as a beta-xyloside at inhibiting glycolipid biosynthesis. Similar results were obtained for human macrophage U937, and differentiated or undifferentiated PC12 cells. However, in neuroblastoma cell line MR23, no low molecular weight xyloside products were made and glycolipid synthesis was not inhibited. These results suggest that some of the previously documented effects of beta-xylosides might result, in part, from their inhibition of glycolipid synthesis. The mechanism of inhibition is not a direct competition for glycolipid synthesizing enzymes; rather, it is an unexplained result of formation of Gal beta 1,4Xyl-1 (alpha or beta)4MU.

Topics: Animals; Anions; Brefeldin A; CHO Cells; Cricetinae; Cyclopentanes; G(M3) Ganglioside; Galactose; Glucuronidase; Glycolipids; Glycosides; Humans; Hymecromone; Macrophages; Melanoma; Neuroblastoma; PC12 Cells; Tritium; Tumor Cells, Cultured

A monoclonal antibody against GM3 ganglioside (GM3Ab) was found to trigger differentiation of Neuro-2a cells in culture. The differentiation of Neuro-2a cells by GM3Ab was accompanied by increased levels of intracellular serotonin and amino acid neurotransmitters viz. aspartate, glutamate, glutamine, glycine and taurine. Further study indicated that the increase in the serotonin level was not due to a higher rate of serotonin synthesis but rather to a higher rate of active transport of serotonin from the medium. Studies on the cell surface gangliosides revealed that unlike the proliferating cells, the GM3Ab-mediated differentiated cells contained higher gangliosides in addition to GM3 and GM2 gangliosides. Analysis of total cellular proteins indicated the appearance of a 25 kDa protein, pI 5.4, in the GM3Ab-treated cells--a small amount of this protein was observed in dibutyryl cAMP (Bt2cAMP)-treated cells, however, the protein was totally absent in the 5-bromo-2'-deoxyuridine (BrdU)-treated cells. Investigation of the mode of action of GM3Ab indicated that the cellular differentiation was due to increased cAMP accumulation resulting from an increase in the adenylate cyclase activity. Further studies with different agents affecting protein kinase C (PKC) activity and direct assay of PKC ruled out the possibility that GM3Ab mediated its effect via PKC. This GM3Ab-induced differentiation could be inhibited by protein kinase A (PKA) inhibitor, H8, but could not be inhibited by sphingosine, an inhibitor of PKC. Pertussis toxin could mimic the effect of GM3Ab, suggesting that GM3Ab caused the elevation in the adenylate cyclase activity by reducing the Gi-protein inhibition of the adenylate cyclase. The data suggests that GM3Ab, after interaction with cell surface GM3, elevated intracellular cAMP level by withdrawing the inhibitory effect of some undefined factor(s) present in culture medium which normally keeps adenylate cyclase activity low through activation of Gi-protein.

Topics: Amino Acids; Animals; Antibodies, Monoclonal; Bromodeoxyuridine; Bucladesine; Catecholamines; Cell Differentiation; Cell Division; Cell Line; DNA Replication; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; G(M3) Ganglioside; Gangliosides; Kinetics; Methionine; Mice; Models, Biological; Molecular Weight; Neoplasm Proteins; Neuroblastoma; Serotonin; Thymidine

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.

Topics: Alprostadil; Animals; Bucladesine; Cell Differentiation; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Galactosyltransferases; Ganglioside Galactosyltransferase; Gangliosides; Glioma; Hybrid Cells; Kinetics; Mass Spectrometry; Mice; N-Acetylgalactosaminyltransferases; Neuroblastoma; Polypeptide N-acetylgalactosaminyltransferase; Rats; Theophylline; Tumor Cells, Cultured

Gangliosides are shed in substantial amounts by some tumors, including human neuroblastoma, and these molecules modulate experimental tumor formation in vivo. We now demonstrate that neuroblastoma tumor gangliosides have potent immunoregulatory activity. Gangliosides of every one of 17 tumors studied were highly inhibitory for the normal in vitro human lymphoproliferative responses to the soluble antigen, tetanus toxoid; 30 nmol ganglioside/ml caused 43% to greater than 99% inhibition and the mean concentration causing 50% inhibition was only 17.3 nmol/ml. Furthermore, gangliosides isolated from clinically more aggressive tumors (Stage III or IV) were up to twice as immunosuppressive as those of the generally less aggressive tumors (Stage I or II) (p less than 0.05). Taken together with the lack of immunosuppressive activity of normal plasma gangliosides, the potent activity of neuroblastoma gangliosides supports the hypothesis that one mechanism by which these shed molecules may act to enhance tumor formation in vivo is through abrogation of the host cellular immune response at the site of tumor formation.

Topics: G(M3) Ganglioside; Gangliosides; Humans; Immunosuppression Therapy; Lymphocyte Activation; Neoplasm Staging; Neuroblastoma

A variety of naturally occurring ganglioside structures were previously shown to be effective agents for inducing neurite outgrowth of primary neurons and neuroblastoma lines. We report here the results of similar experiments with a synthetic epimer of GM3 (epi-GM3) possessing a neuraminidase-resistant beta-ketosidic linkage. This substance was found to enhance neuritogenesis toward two transformed cell lines (neuro-2A, PC-12) and one primary neuronal tissue (dorsal root ganglia). The results indicate that the stereochemistry of the ketoside linkage is not critical and that metabolism of exogenous ganglioside by the treated cells is not involved directly in the neuritogenic phenomenon.

Topics: Animals; Cell Count; Cell Line; Chick Embryo; Dendrites; G(M1) Ganglioside; G(M3) Ganglioside; Ganglia, Spinal; Gangliosides; Mitogens; Molecular Conformation; Neuroblastoma; Stereoisomerism; Tumor Cells, Cultured

An abnormal circulating ganglioside was found in patients with neuroblastoma. This ganglioside appeared as a single band by resorcinol-HCl staining of thin-layer chromatograms of purified total gangliosides isolated from as little as 1 ml of patient plasma. It is a major ganglioside of neuroblastoma tumour tissue and was present (250-1500 pmol lipid-bound sialic acid/ml) in the plasma of five patients with widespread neuroblastoma. In contrast, the ganglioside was not detected (less than 50 pmol/ml) in plasma samples of six patients in complete remission, nor in plasma samples of seventeen healthy children and adults. Measurement of this circulating tumour-associated ganglioside should be clinically useful in neuroblastoma, offering a new approach to the detection of tumour and the evaluation of therapy.

Topics: Adolescent; Adult; Child; Child, Preschool; Chromatography, Thin Layer; Female; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Male; Middle Aged; Neuroblastoma

Rat glioma X mouse neuroblastoma hybrid neurotumor cells (NG108-15), synchronized by amino acid deprivation, showed a cell-cycle-dependent peak of activity of a ganglioside N-acetylgalactosaminyl transferase 14-24 h following release from the cell cycle block (S/G2 phase). Maximal expression of two typical lysosomal hydrolases, N-acetyl-beta-hexosaminidase and beta-galactosidase, occurred between 18 and 21 h following release (S phase), declining to G1 phase levels during the peak of N-acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G2 phase cells showed an increase in apparent Vmax (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) (32 versus 14 microM) when compared to transferase activity in the G1 phase. However, the opioid peptide enkephalin [D-Ala2, D-Leu5], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108-15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G1 phase) than in cells 20 h after release (G2 phase), with 50% inhibition occurring at 2 X 10(-9) M and 2 X 10(-7) M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both Vmax and Km changes are observed, and that the cyclic AMP-dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent.

Topics: Animals; Cell Cycle; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; G(M2) Ganglioside; G(M3) Ganglioside; Galactosyltransferases; Gangliosides; Glioma; Hybrid Cells; Kinetics; Mice; N-Acetylgalactosaminyltransferases; Neuroblastoma; Rats

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones). The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.

Topics: Bromodeoxyuridine; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Culture Media; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Neoplasms, Experimental; Neuroblastoma

Topics: Animals; Astrocytes; Cells, Cultured; Cricetinae; Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Galactose; Gangliosides; Glucosyltransferases; Humans; Mice; Neuroblastoma; Neuroglia; Sialic Acids

The reaction sequence for the biosynthesis of gangliosides by mouse neuroblastoma cells has been investigated by studying the pattern of incorporation of labeled precursors into sialoglycosphingolipids. Cultured NB41A cells incorporated N-[3H]acetylmannosamine into the sialic acid moiety of GM3 in less than 10 min. Labeled GM2 was not detected in cells incubated for less than 30 min, while measurable radioactivity did not appear in GM1 until after 60 to 90 min. Analogous experiments were carried out using [14C]galactose. No significant amount of labeled hexose was incorporated into asialo-GM2 during 60 min of culture. These studies are in accord with results of previous studies on glycosyltransferases of NB41A cells (Kemp, S. F., and Stoolmiller, A. C. (1976), J. Neurochem. 26, 723-732), and further support the concept that the pathway of synthesis of gangliosides proceeds via GM3 leads to GM2 leads to GM1.

Topics: Animals; Cell Line; Culture Media; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Galactose; Glucose; Glycosphingolipids; Hexosamines; Kinetics; Mice; Neuroblastoma; Sialic Acids