g(m2)-ganglioside and Neuroblastoma

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch14.18 at dose levels from 10 mg/m2 to 200 mg/m2. The plasma clearance of ch14.18 was biphasic. Following the first course of treatment t1/2,alpha was 3.4 +/- 3.1 h and t1/2,beta 66.6 +/- 27.4 h in 9/10 children. The t1/2,beta values were significantly less than those of 181 +/- 73 h previously reported in adult melanoma patients (P < or = 0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch14.18 in children and adults. After a second course of treatment, administered to 5/10 children, t1/2,beta decreased significantly from 72.9 +/- 19.8 h to 31.7 +/- 18.4 h (P = 0.015). We therefore conclude that the elimination kinetics of mAbs ch14.18 in children and adults are different, and furthermore that repeated administration of mAb ch14.18 to children with neuroblastoma leads to accelerated antibody clearance.

Topics: Adult; Animals; Antibodies, Monoclonal; Bone Neoplasms; Child; Child, Preschool; Dose-Response Relationship, Immunologic; Female; G(M2) Ganglioside; Half-Life; Humans; Immunoglobulin G; Immunoglobulin kappa-Chains; Immunotherapy; Infant; Male; Mice; Neuroblastoma; Osteosarcoma; Recombinant Fusion Proteins

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; G(M2) Ganglioside; Heterografts; Humans; Male; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; N-Acetylgalactosaminyltransferases; Neovascularization, Pathologic; Neuroblastoma; RNA-Seq; Skin Neoplasms; Transfection; Tumor Burden

Gal-PUGNAc (see picture), a highly selective inhibitor for beta-hexosaminidases HEXA and HEXB is cell-permeable and modulates the activity of HEXA and HEXB in tissue culture, increasing ganglioside GM2 levels. Gal-PUGNAc should allow the role of these enzymes to be studied at the cellular level without generating a complex chemical phenotype from concomitant inhibition of O-GlcNAcase.

Topics: beta-N-Acetylhexosaminidases; Cell Line, Tumor; Enzyme Inhibitors; G(M2) Ganglioside; Hexosaminidase A; Hexosaminidase B; Humans; Lysosomes; Neuroblastoma; Substrate Specificity

GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-beta-cyclodextrin (MbetaCD); the GM2-rich rafts and sensitivity to MbetaCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MbetaCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells. Until this study, GM2-rich rafts are poorly characterized and MbetaCD hypersensitivity, which may have clinical implications, has not been reported.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Apoptosis; beta-Cyclodextrins; Child; G(M2) Ganglioside; Humans; Membrane Microdomains; Neuroblastoma; Schwann Cells

Dendritic cell (DC) development and function is critical in the initiation phase of any antigen-specific immune response against tumours. Impaired function of DC is one explanation as to how tumours escape immunosurveillance. In the presence of various soluble tumour-related factors DC precursors lose their ability to differentiate into mature DC and to activate T cells. Gangliosides are glycosphingolipids shed by tumours of neuroectodermal origin such as melanoma and neuroblastoma. In this investigation we address the question of whether gangliosides suppress the development and function of monocyte-derived DC in vitro. In the presence of gangliosides, the monocytic DC precursors showed increased adherence, cell spreading and a reduced number of dendrites. The expression of MHC class II molecules, co-stimulatory molecules and the GM-CSF receptor (CD116) on the ganglioside-treated DC was significantly reduced. Furthermore, the function of ganglioside-treated DC was impaired as observed in endocytosis, chemotactic and T cell proliferation assays. In contrast to monocytic DC precursors, mature DC were unaffected even when higher doses of gangliosides were added to the culture. With regard to their carbohydrate structure, five different gangliosides (GM2, GM3, GD2, GD3, GT1b), which are typically shed by melanoma and neuroblastoma, were tested for their ability to suppress DC development and function. Suppression was induced by GM2, but not by the other gangliosides. These data suggest that certain gangliosides impair DC precursors, implying a possible mechanism for tumour escape.

Topics: Cell Adhesion; Cell Differentiation; Cells, Cultured; Chemotaxis, Leukocyte; Dendritic Cells; Endocytosis; Flow Cytometry; G(M2) Ganglioside; Gangliosides; HLA-D Antigens; Humans; Immunophenotyping; Lymphocyte Culture Test, Mixed; Melanoma; Monocytes; Neuroblastoma; Neuroectodermal Tumors; Tumor Escape

Anti-GM2 IgM antibodies have been reported in some patients with dysimmune neuropathy or lower motor neuron syndrome. To determine whether these antibodies can induce complement-dependent cytolysis we performed a cytotoxicity assay on neuroblastoma cells with sera from seven patients with demyelinating dysimmune neuropathies and high titers of anti-GM2 IgM. As controls we used sera from seven patients with other anti-neural reactivities, six with the same neuropathies but no anti-GM2 or other anti-neural reactivity and from eight normal subjects. Of the seven positive sera tested, six induced complement-mediated cytotoxicity, while none of the controls had any relevant effect on neuroblastoma cells. Preincubation of positive sera with purified GM2 removed cytotoxic activity. Affinity purified anti-GM2 IgM had the same cytotoxic anti-GM2 effect of whole serum while serum or complement alone did not have any effect. In four anti-GM2-positive patients the percentage of cell lysis correlated with anti-GM2 titers and with IgM staining of neuroblastoma cells while in two the cytotoxic effect was higher than expected from antibody titers. Complement-mediated cell lysis induced by anti-GM2 IgM antibodies may be a possible mechanism of neural damage in patients with dysimmune neuropathy and high titers of anti-GM2 IgM antibodies.

Topics: Chromatography, Affinity; Complement Activation; G(M2) Ganglioside; Guillain-Barre Syndrome; Humans; Immunoglobulin M; Motor Neuron Disease; Neuroblastoma; Tumor Cells, Cultured

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intranucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40-80 microM of lysosphingolipids for 24-48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 degrees C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses.

Topics: Amidohydrolases; Animals; Apoptosis; Caspase 3; Caspases; Cattle; Chromatin; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; G(M2) Ganglioside; Glycosphingolipids; Humans; Mice; Neuroblastoma; Oxidoreductases; Phosphatidylserines; Pseudomonas; Psychosine; Signal Transduction; Sphingolipids; Sphingosine; Tumor Cells, Cultured

Anti-GM2 IgM antibodies have been reported in some patients with dysimmune neuropathy or lower motor neuron syndrome, in whom they were often associated with a concomitant reactivity with GM1. To investigate the possible clinical and pathogenetic relevance of these antibodies we measured serum anti-GM2 IgM titers by ELISA in 224 patients with different neuropathies and motor neuron disease and examined their binding to SK-N-SH neuroblastoma cells by indirect immunofluorescence (IIF). High titers of anti-GM2 IgM antibodies were found in eight patients with dysimmune neuropathies including two with multifocal motor neuropathy (MMN), two with purely motor demyelinating neuropathy without conduction block (MN) and four with Guillain-Barré syndrome (GBS). In two MMN patients reactivity with GM2 was associated with anti-GM1 reactivity and in one MN patient with anti-GM1, -GD1a and -GD1b reactivity. All but one patient had a concomitant reactivity with GalNAc-GD1a. Serum IgM from all positive patients intensely stained by IIF the surface of SK-N-SH neuroblastoma cells. This reactivity was blocked by serum pre-incubation with GM2, was not observed with sera from patients without anti-GM2 antibodies including those with high anti-GM1 or other anti-glycolipid antibodies, and correlated with the presence of GM2 in the SK-N-SH neuroblastoma cells. These findings indicate that anti-GM2 antibodies, though infrequent, are strictly associated with dysimmune neuropathies and suggest that SK-N-SH neuroblastoma cells can be a suitable in vitro model to study the functional and biological effects of these antibodies.

Topics: Adolescent; Adult; Autoantibodies; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; G(M2) Ganglioside; Humans; Immunoblotting; Immunoglobulin M; Male; Middle Aged; Motor Neuron Disease; Myelin Sheath; Neuroblastoma; Polyradiculoneuropathy; Sural Nerve; 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

We have examined ganglioside compositions and the presence of sulfated glucuronyl glycolipids of immortalized motor neuron-like cell lines, neuroblastoma-spinal cord (NSC) hybrid cell lines established by fusing mouse neuroblastoma N18TG2 with motor neuron-enriched embryonic spinal cord cells. Among NSC cell lines, only NSC-34 aggregates acetylcholine receptors on co-cultured myotube and expresses a receptor for S-laminin, a neuromuscular junction specific basal lamina protein. GM2, which is only a minor ganglioside component of CNS, was the major component in NSC-34 occupying almost 75% of total gangliosides, whereas GD1a and GM3 were major species in the parental N18TG2, which had only 8.5% GM2. These results indicated that NSC lines have unique ganglioside pattern that is distinctive from other nervous tissues, and this pattern, especially that of NSC-34 cells, might reflect the characteristics of mouse spinal motor neuron gangliosides. Sulfated glucuronyl paragloboside was demonstrated to be present in N18TG2, however, it could not be detected in either of NSC cell lines. Even though the pathogenesis of amyotrophic lateral sclerosis remains unknown, autoimmunological participation has been suggested. Because high-titered antibody against GM2 has been observed in a patient with amyotrophic lateral sclerosis-like disease, GM2 which is possibly expressed on the surface of motor neurons might serve as a potential target antigen in this disorder.

Topics: Animals; Cell Line; Cholera Toxin; Chromatography, Thin Layer; G(M1) Ganglioside; G(M2) Ganglioside; Immunohistochemistry; Mice; Motor Neurons; Neuroblastoma; Phenotype; Protein Binding; Spinal Cord

Gangliosides GM1 [3H-labeled at the sphingosine (Sph) moiety] and GM2 [3H-labeled at the Sph or N-acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1-4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed 3H-metabolites of [Sph-3H]GM1 were GM2, asialo-GM1 asialo-GM2, and lactose-ceramide, and those of [Sph-3H]GM2 were asialo-GM2 and lactose-ceramide. The asialo-GM1 and asialo-GM2 formed were isolated and chemically characterized. [3H]Asialo-GM2 was produced in identical amounts after treatment with equimolar [Sph-3H]GM2 and [GalNAc-3H]GM2. At low temperature or in the presence of chloroquine, the formation of all 3H-metabolites, including asialo-GM2 and asialo-GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis- and lysosome-dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1-->GM2-->asialo-GM2-->-->Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1-->asialo-GM-1-->asialo-GM2-->-->Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase-mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.

Topics: Animals; G(M1) Ganglioside; G(M2) Ganglioside; Mice; Neuraminidase; Neuroblastoma; Tumor Cells, Cultured

Disialoganglioside GD2 is widely expressed among neuroblastomas, melanomas, small-cell lung carcinoma, sarcomas and brain tumors. Immunity directed against this antigen may have anti-tumor utility. Since GD2 is poorly immunogenic, anti-idiotypic antibodies may serve as alternative tumor vaccines. Monoclonal antibody 3F8, a murine IgG3 specific for GD2, has shown excellent tumor-targeting ability in vitro and in vivo. LOU/CN rats were immunized with 3F8 and their spleens were used in somatic-cell hybridization, using SP2/0, P3 and Y3 as fusion partners. Six anti-idiotypic (anti-id) MAbs (C2D8, Idio-2, AIG4, C2H7, C4E4, A2A6) were selected based on their reactivity with 3F8 and non-reactivity with murine IgG3 myelomas. Specificity of each anti-id was demonstrated by using various ELISA: (i) lack of direct binding to solid phase myelomas and serum proteins; (ii) inability of other myelomas to inhibit anti-id binding to 3F8; (iii) absence of cross-reactivity of other myelomas to solid-phase anti-id; (iv) lack of inhibition by anti-id of binding of other ganglioside antibodies to their antigens. Antigen specificity was further examined by inhibition of binding of 3F8 to GD2 on immuno-thin-layer chromatography, and by inhibition of 3F8 immunostaining of neuroblastoma cell lines. These 6 antibodies were demonstrated to be distinct, in view of their cross-reactivity, fusion partners and relative strength of binding to 3F8. Anti-GD2 antibodies were induced after immunization with these anti-id antibodies in C57Bl/6 mice. These rat anti-3F8-idiotypic antibodies with exquisite specificity for anti-GD2 antibodies may be useful in vaccine construction.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Cross Reactions; Enzyme-Linked Immunosorbent Assay; G(M2) Ganglioside; Gangliosides; Humans; Immunization; Immunotherapy; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Myeloma Proteins; Neuroblastoma; Rats; Rats, Inbred Strains

In order to investigate GM2 expression in gliomas, the GM2-positive human glioma cell line (HGL) D-54 MG, which contains 0.6 nmol GM2/mg protein, representing 77% of the total monosialoganglioside fraction, was used as an immunogen for the production of anti-GM2 monoclonal antibodies. For ganglioside designations, see IUPAC-IUB (Eur. J. Biochem., 79: 11-21, 1977) and Svennerholm (J. Neurochem., 10: 613-623, 1963). Five IgM monoclonal antibodies (DMAb-1 through DMAb-5) specifically recognizing the GalNAc beta1-4(NeuAc alpha 2-3)Gal-terminal epitope common to GM2 and GalNAC-GD1a are reported. The antibodies did not react with GM1, GM3, GD2, GD3, GD1a, GD1b, and GQ1b. Purified anti-GM2 MAbs were used to define the expression of the "GM2" terminal epitope by cultured human malignant and normal cells by radioimmunoassay and membrane immunofluorescence. Among neuroectodermal tissue-derived cell lines, DMAb-3, at an optimal concentration of 5 micrograms/ml, showed high reactivity (radioimmunoassay binding ratios greater than 20) with 9 of 19 HGLs, 3 of 5 medulloblastoma, 4 of 5 neuroblastoma, and 1 of 3 melanoma lines. Moderate reactivity (binding ratio, 10-20) was exhibited by 3 HGL, 2 medulloblastoma, and 1 neuroblastoma lines and low reactivity (binding ratio, 3-10) by 5 HGL lines; no reactivity was detected with 2 HGL and 2 melanoma lines. Densitometric evaluation of monosialoganglioside extracts from human glioma and medulloblastoma cell lines in conjunction with immunostaining on thin-layer chromatograms showed that GM2 represents the major monosialoganglioside in 8 of 10 HGL and in 3 of 4 Med lines. In these lines the amount of GM2 ranged from less than 0.1 to 0.6 nmol/mg protein. These results indicate that GM2 represents a proportionally increased ganglioside of most glioma, medulloblastoma, and neuroblastoma cells in vitro.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Dose-Response Relationship, Immunologic; Epitopes; G(M2) Ganglioside; Gangliosides; Glioma; Humans; Medulloblastoma; Melanoma; Neuroblastoma

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

Concentration of gangliotriaose-series glycosphingolipids, including GA2, GM2, GD2 and GT2, was measured in human sera by a thin-layer chromatography/enzyme-immunostaining method. By this method, as little as 5-10 ng/ml of these glycolipids in serum could be determined simultaneously. Although GD2 ganglioside could be consistently detected in normal cord blood (1-2 ng/ml of serum), the ganglioside was never detected in normal adult serum. However, the same ganglioside was found to be present in large quantity in preoperative sera of 6/9 patients with neuroblastomas (25-658 ng/ml of serum). In addition to GD2, gangliosides GM2 and GA2 increased concomitantly than usual. It is concluded that this highly sensitive quantification of the tumor-associated glycolipids circulating in serum of neuroblastoma patients could be useful in their diagnosis.

Topics: Adult; Chromatography, Thin Layer; Fetal Blood; G(M2) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Immunoenzyme Techniques; 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

Topics: Animals; Calcium; Cells, Cultured; Chemical Phenomena; Chemistry; Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase; G(M2) Ganglioside; Gangliosides; Glycosphingolipids; Hexosyltransferases; Kinetics; Magnesium; Manganese; Mice; Neuroblastoma; Sialyltransferases; Transferases

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