gq1b-ganglioside and Neuroblastoma

Low (< or = 35%) or absent expression of the complex 'b' pathway gangliosides GD1b, GT1b and GQ1b (CbG) correlates with an aggressive biological phenotype in human neuroblastoma tumors. To develop an in vitro model to probe mechanisms by which CbG may contribute to neuroblastoma behavior, we have comprehensively evaluated ganglioside expression in nine well-established human neuroblastoma cell lines, all derived from poor prognosis tumors. Total cellular ganglioside content ranged from 8 to 69 nmol/10(8) cells. High performance thin layer chromatography revealed that the simple disialoganglioside GD2 was prominent in eight of the cell lines (up to 60% of total gangliosides), whereas CbG were low (1-21%) in all nine cell lines. The structurally most complex 'b' pathway species, GQ1b, was not detected in any of the cell lines. The prominence of GD2 in neuroblastoma cell lines mirrors the high expression of GD2 that characterizes human neuroblastoma tumors, and the low CbG expression in the cell lines is analogous to that found in clinically and biologically unfavorable neuroblastoma tumors, thus establishing these neuroblastoma cell lines as valuable model systems for study of the role of CbG in the pathobiology of human neuroblastoma.

Topics: Cell Survival; Chromatography, Thin Layer; Gangliosides; Gene Expression Profiling; Humans; Neuroblastoma; Phenotype; Prognosis; Tumor Cells, Cultured

Ganglioside metabolism has been linked to the clinical and biological behavior of human neuroblastoma. This study investigated the importance of differences in complex "b" ganglioside (GD1b, GT1b, and GQ1b; designated CbG) expression in this tumor. Gangliosides of 74 neuroblastomas were analyzed by high-performance TLC. Associations of CbG expression with known prognostic markers and with event-free survival (EFS) were evaluated. Higher CbG expression characterized nonprogressive versus progressive tumors (median 41% versus 18% of total gangliosides; P = 0.001) and completely accounted for the observed higher overall "b" pathway ganglioside expression (median 81% versus 68%; P = 0.003). In contrast, expression of the structurally simpler "b" pathway gangliosides (GD2 and GD3) did not differ (median 31% versus 35%; P = 0.4). Absolute CbG content differed even more (median 93 versus 29 nmol/g among nonprogressive versus progressive tumors; P = 0.02) and was most striking in the case of GQ1b content (8-fold higher in nonprogressive tumors). High CbG (> or =35% of total gangliosides) expression was strongly predictive of a favorable outcome in: (a) the entire study population (90% versus 60% EFS at 25 months; P = 0.001); and (b) among patients assigned a low-risk status by a either single genetic or biochemical tumor marker (MYCN, DNA, NSE, or ferritin), or by both unamplified MYCN and aneuploid DNA (22-28% difference in EFS at 25 months). These data suggest that high tumor CbG content may substratify "good prognosis" neuroblastoma patients, identifying patients at very low risk of relapse or death, and that the biological roles of CbG in neuroblastoma will be of importance to define.

Topics: Carbohydrate Sequence; Cohort Studies; Gangliosides; Humans; Infant; Molecular Sequence Data; Neoplasm Staging; Neuroblastoma; Prognosis; Risk Factors

IgG antibodies to GQ1b ganglioside are found in > 90% of patients with the Miller Fisher Syndrome (MFS). MFS sera or IgG preparations have marked effects on neurotransmitter release at the neuromuscular junction, but their mode(s) of action remain unclear. To establish a cell-based system for investigating the mechanism of action of MFS serum preparations, we looked at neurotransmitter release from three cell lines. We failed to demonstrate substantial 14C-acetylcholine release from two motor-neuronal cell lines, VSC4.1 and NSC19, and therefore studied 3H-noradrenaline release from NGF-differentiated PC12 cells, a neural-crest derived catecholaminergic cell line. K(+)-induced release was inhibited by botulinum toxin and basal release was enhanced by alpha-latrotoxin, resembling that at the neuromuscular junction, although K(+)-induced release was dependent on L-type rather than P/Q-type calcium channels. The cells expressed polysialylated gangliosides on the cell surface. Incubation in heat-inactivated or untreated MFS preparations did not, however, affect basal or K(+)-induced release. Thus the PC12 cells do not appear to be sensitive to the effects of serum antibodies from MFS patients.

Topics: Animals; Cell Differentiation; Gangliosides; Humans; Hybrid Cells; Immune Sera; Immunoglobulin G; Mice; Miller Fisher Syndrome; Nerve Growth Factors; Neuroblastoma; Neurotransmitter Agents; PC12 Cells; Rats; Spinal Cord; Tumor Cells, Cultured

It was reported recently by our group that the transfection of GD3 synthase cDNA into Neuro2a cells, a neuroblastoma cell line, caused cell differentiation with neurite sprouting (Kojima et al., 1994; J. Biol. Chem., 269, 30451-30456). To further explore this phenomenon in detail, we applied tetracycline-regulated system to control the expression of GD3 synthase cDNA in Neuro2a cells. Under this system, the process of Neuro2a cell differentiation was rather slow, about 3 weeks of cell culturing in the absence of tetracycline was required for most cells to extend the neurite-like structures. The RNase protection assay indicated that the mRNA of GD3 synthase gene was first detected between 4 h and 8 h after the gene was activated and kept at approximately the same level through the process. Furthermore, time-course analysis of total ganglioside expressions has shown that GD3 and GT1b gangliosides appeared on the cell surface early in the process and reached the maximum level around day 6. We also found that the amounts of GD3 and GT1b on the cell surface started to decrease after day 6 and returned gradually to the basal values after 3 weeks. On the other hand, GQ1b and GD1b were started to be synthesized at early stage and the amounts were continuously to increase through the whole Neuro2a morphological change process. In addition, time-course analysis by flow cytometry method for GD3 and GQ1b suggested that the conversions of simple gangliosides to more complex gangliosides may be required to induce the Neuro2a differentiation. Our results indicated that the combination of cDNA transfection and regulated gene expression is a powerful tool to study the function of glycolipids and should have a general application to the glycobiology field.

Topics: Animals; Cell Differentiation; DNA, Complementary; Flow Cytometry; Gangliosides; Gene Expression Regulation; Kinetics; Mice; Neuroblastoma; RNA, Messenger; Sialyltransferases; Tetracycline; Transfection; Tumor Cells, Cultured

Previously, we reported that ganglioside GQ1b specifically promoted neuritogenesis of human neuroblastoma cells (GOTO), and also that is specifically stimulated the phosphorylation of several cell surface proteins on the same cells. To disclose the relationship between the two events, we examined them using a novel protein kinase inhibitor, K-252b, which is a derivative of K-252a and cannot pass through cell membrane. K-252b inhibited the GQ1b-dependent neuritogenesis as well as the GQ1b-stimulated phosphorylation. This suggests the direct coupling between the two cell events and the occurrence of a new biosignal transduction system.

Topics: Carbazoles; Cell Membrane Permeability; Gangliosides; Humans; Indole Alkaloids; Neurites; Neuroblastoma; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Tumor Cells, Cultured

Seven monoclonal antibodies (MAbs) directed to tetrasialoganglioside (GQ1b) were established, purified GQ1b being used for immunization and hybridoma screening. All of the MAbs reacted strongly with GQ1b, although they also reacted with other gangliosides, with different specificities and reactivities. Some MAbs (1H10, 2C7, and 3F4) reacted with GD3, GT1a, GQ1b, and GP1c. MAb 1H4 showed broad specificity. It reacted with GD3, GD1b, GD2, GT1a, GT1b, GO1b, GQ1c, and GP1c. MAbs 7F5, 4E7, and 4F10 recognized GT1a, GQ1b, and GP1c. MAb 4F10 was more specific for GQ1b than the other MAbs. Using MAb 4F10, we determined, by means of an immunoassay, the quantities of endogenous GQ1b in some neuronal and adrenal cell lines, GOTO (human neuroblastoma), Neuro2a (mouse neuroblastoma), and PC12 (rat pheochromocytoma). PC12 and Neuro2a cells contained at least 5.1 X 10(6) and 3.9 X 10(5) molecules/cell of GQ1b, respectively. In contrast, no GQ1b was detected in GOTO cells, which are known for their specific neuritogenic response to this particular ganglioside when exogenously added.

Topics: Adrenal Gland Neoplasms; Animals; Antibodies, Monoclonal; Gangliosides; Humans; Neuroblastoma; Pheochromocytoma; Tumor Cells, Cultured

Recent studies, including ours, on bioactive gangliosides revealed that certain gangliosides have an interesting ability to modulate a variety of cell functions. For instance, we demonstrated that a tetrasialoganglioside, GQ1b, promotes neurite outgrowth when added in nanomolar concentrations to cells from two human neuroblastoma cell lines. Also, phosphorylation of several cell surface proteins was observed on addition of ATP. Several lines of evidence indicated that this phosphorylation is probably catalysed by a novel cell surface membrane-bound protein kinase which is specifically activated by a particular ganglioside (Gg). Because of its location on the cell surface we proposed calling this type of kinase(s) ecto-Gg kinase. A procedure to inhibit the phosphorylation of the cell surface protein resulted in suppression of the GQ1b-dependent promotion of neuritogenesis, strongly suggesting that these two cellular events are intricately coupled. Other evidence also indicated that the GQ1b-dependent neuritogenesis is mediated through a receptor-coupled process of the cell surface membrane. Thus, it is likely that this represents a new type of biosignal transduction that is mediated through cell surface carbohydrate recognition (ecto biosignal transduction system).

Topics: Cell Line; Cell Membrane; Gangliosides; Humans; Membrane Glycoproteins; Nerve Growth Factors; Neuroblastoma; Oligosaccharides; Phosphorylation; Protein Kinases; Signal Transduction

A ganglioside-stimulated ecto-type protein phosphorylation system (ecto-Gg-kinase) was detected on the cell surface of a human neuroblastoma cell line (GOTO). When intact cells were incubated with [gamma-32P]ATP, at least 28 cell surface proteins were phosphorylated, as evident on SDS-PAGE (4-20%) analysis. Exogenously added gangliosides specifically stimulated the phosphorylation of at least three cell surface associated proteins of Mr = 64,000, 60,000, and 54,000. Phosphorylation was directed toward Thr and Ser residues, respectively, as revealed on acid hydrolysis followed by electrophoresis. GQ1b, at 5 nM, was the most potent among the several gangliosides tested and was more effective when added to cells before [gamma-32P]ATP administration. The simultaneous addition of an excess amount of the saccharide portion of GQ1b (oligo-GQ1b) inhibited the GQ1b-stimulated phosphorylation, indicating the necessity of the sialosaccharide moiety. These results strongly suggest that phosphorylation of the three proteins may be closely associated with the highly specific neuritogenic effect of GQ1b previously reported.

Topics: Cell Line; Gangliosides; Humans; Membrane Proteins; Neuroblastoma; Phosphorylation; Protein Kinases; Tumor Cells, Cultured

Individual ganglioside species (possessing the gangliotetrose oligosaccharide) were purified from bovine brain gray matter and applied in varying concentrations to the culture medium of mouse neuroblastoma cells (N2A) in vitro. After 48 hr of incubation, the cells were stained, and the neuritogenic response quantitated with a video analysis system, employing a program to measure three parameters of neuroblastoma differentiation: neurites per cell (sprouting), neurite length (extension), and degree of neurite branching (arborization). All the individual gangliosides tested promoted neurite extension in a dose-dependent fashion. Asialogangliosides ("neutral" glycosphingolipids) were without effect, which suggests that sialic acid (N-acetylneuraminic acid) is necessary to elicit this cellular response. With increasing concentrations of GM1 (5 to 500 micrograms/ml), the average cellular neurite length increased significantly, whereas the number of neurites per cell decreased. With the trisialoganglioside GT1b, neurite length did not increase to the extent seen with GM1, but an increase in the number of neurites per cell (sprouting) and branch points per neurite (arborization) was observed. These results suggest that the in vitro neuronal response to exogenous gangliosides may combine specific responses to individual species making up the total.

Topics: Animals; Cell Line; G(M1) Ganglioside; Gangliosides; Glycosphingolipids; Mice; Neuroblastoma; Neurons

A ganglioside-stimulated protein phosphorylation system was discovered in plasma membrane fractions of human neuroblastoma cells (GOTO). Gangliosides (GQ1b, GT1a, GT1b, GD1a, GD1b, GD3, and GM1) could stimulate this system. GQ1b showed the most effective stimulation among these gangliosides. The substrate specificity was rather broad. Not only some (de novo) proteins of the membranes but also purified histones and tubulin were phosphate-acceptable. This protein phosphorylation system specifically depended upon Ca2+ (optimum concentration: 50-100 microM). The optimum pH was 7.0-7.5. GQ1b/Ca2+ could not directly activate well known protein kinases (Ca2+/phospholipid-activated protein kinase, Ca2+/calmodulin-activated protein kinase, and cyclic nucleotide-dependent protein kinases). Furthermore, GQ1b could replace neither phospholipids nor calmodulin. Thus, an unknown, new type of protein kinase(s) may be involved in this system. Alternatively, GQ1b may activate some known protein kinase(s) in cooperation with another unknown factor which may be removed during the preparation of the partially purified known protein kinase used in this experiment.

Topics: Animals; Calcium; Cell Line; Cell Membrane; Enzyme Activation; Gangliosides; Guinea Pigs; Histones; Humans; Neuroblastoma; Phosphorylation; Protein Kinases; Substrate Specificity; Tubulin

The novel effects of gangliosides from human brain on the number of nuclei of nerve cells and neurite outgrowth were studied in cultures of human neuroblastoma cell lines GOTO and NB-1. Ganglioside GQ1b at a nanomolar level stimulated cell proliferation and neurite outgrowth during culture for 24 hours, as reported previously [J Biochem 94: 303-306, 1983]. Although the neurite promoting activity of GQ1b was similar to those of total human brain gangliosides (GS) and nerve growth factor (NGF), its activity on cell proliferation did not persist on longer culture; that is, the number of GQ1b-treated cells rapidly decreased to the control level during culture for 48 or 72 hours. In contrast, on treatment with GS or NGF, the number of cell nuclei increased continuously during prolonged culture. These results showed that either some other molecular species of ganglioside(s) than GQ1b or other substances such as proteins present in the GS fraction were responsible for the long-term activity. Studies on the GS fraction after its treatment with proteases and neuraminidases revealed that ganglioside GD1a (20 ng/ml) had the ability to prolong the activity of GQ1b. Namely, GQ1b and GD1a gangliosides cooperated in maintaining the number of nuclei in long-term cultures of neuroblastoma cell lines.

Topics: Cell Differentiation; Cell Division; Cell Line; Cell Nucleus; Gangliosides; Humans; Nerve Growth Factors; Neuroblastoma; Neurons

To clarify the role of gangliosides in the morphological and biochemical differentiation of neuronal cell cultures, the model cell culture system represented by two neuroblastoma cell lines, GOTO and NB-1, which were established from adrenal gland and metastatic neck lymph node, respectively, was examined. We found that the total ganglioside fraction from human brain had two remarkable effects on these cell lines, which are similar to those of nerve growth factor (NGF): (a) an increase in the cell number, and (b) an increase in the neurite number and the total length of neurites. In these cases, the genuine effector in total gangliosides could not be ascribed to a possibly contaminating NGF-like protein, but rather to a particular molecular species of the gangliosides, GQ1b, which could completely replace the effector function not only qualitatively but also quantitatively. Our results provide direct evidence for the participation of gangliosides in such functions.

Topics: Cell Line; Gangliosides; Humans; Neoplasms, Experimental; Nerve Growth Factors; Neuroblastoma