trisialoganglioside-gt1 and Neuroblastoma

The focus of this review is the ganglio-series of glycosphingolipids found in neuroblastoma (NB) and the myriad of unanswered questions associated with their possible role(s) in this cancer. NB is one of the more common solid malignancies of children. Five-year survival for those diagnosed with low risk NB is 90-95%, while that for children with high-risk NB is around 40-50%. Much of the survival rate reflects age of diagnosis with children under a year having a much better prognosis than those over two. Identification of expression of GD2 on the surface of most NB cells led to studies of the effectiveness and subsequent approval of anti-GD2 antibodies as a treatment modality. Despite much success, a subset of patients, possibly those whose tumors fail to express concentrations of gangliosides such as GD1b and GT1b found in tumors from patients with a good prognosis, have tumors refractory to treatment. These observations support discussion of what is known about control of ganglioside synthesis, and their actual functions in NB, as well as their possible relationship to treatment response.

Topics: Child; Disease-Free Survival; Gangliosides; Humans; Neuroblastoma; Risk Factors; Survival Rate

Since gangliosides play many important roles in neural systems, we investigated whether gangliosides are involved in glutamate release from neural cells. Differentiated neruro2a cells were treated with gangliosides, including GM3, GM1, GD1a, GD3, GD1b, or GT1b, for 30 min, and glutamate concentration in the culture media was measured using o-phthalaldehyde derivatization. Among the tested gangliosides, GT1b significantly increased the glutamate concentration when compared with untreated cells. Moreover, GT1b increased the glutamate concentration in the culture media of neuroblastoma × dorsal root ganglion neuron hybrid F11 cells. These results suggested that gangliosides are important in regulating extracellular glutamate concentration in the nervous system.

Topics: Amino Acids; Cell Line, Tumor; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; Gangliosides; Glucosyltransferases; Glutamates; Humans; Hybrid Cells; Morpholines; 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

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

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

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

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

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