Significant inhibition of hybridoma cells by exogenous application of ganglioside GM3, a possible modulator of cell growth in vitro.

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against kappa-light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively GM3, substituted with C24:1 and C16:0 fatty acid and C18:1 sphingosine, was found in this B cell derived cell line. A GM3(NeuGc) to GM3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute GM3 amounts of about 0.3 mg 10(-9) viable cells were determined. Exogenous application of GM3, which has been isolated from large cell preparations, to 187.1 cells showed growth inhibition in a concentration dependent manner. Using the MTT-assay and the [3H]thymidine incorporation assay, the cells exhibited a strong reduction in metabolic and proliferative activity, respectively, after exposure of cells to GM3. GM3 was applied in concentrations between 3 microM and 30 microM, giving evidence for strong inhibitory effects at 30 microM GM3 and less but significant suppression after application of GM3 concentrations lower than 20 microM. No cellular response was observed at the lowest concentration (3 microM) used in this study. Hybridoma cells as well as other cell types like fibroblasts, muscle cells and endothelial cells, are in general characterized by high expression of the GM3 ganglioside, which is known to act as a modulator of cellular growth in monolayer cultures of adherent cells. Since gangliosides are released to the culture medium by cell lysis, i.e. cell death, and/or by active membrane shedding, the results obtained in this study suggest a growth regulatory role of GM3 in high density hybridoma cell cultures.