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Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro

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Abstract

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against ϰ-light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively G M3, substituted with C24∶1 and C16∶0 fatty acid and C18∶1 sphingosine, was found in this B cell derived cell line. A G M3 (NeuGc) to G M3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute G M3 amounts of about 0.3 mg 10−9 viable cells were determined. Exogenous application of G M3, 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 G M3. G M3 was applied in concentrations between 3μM and 30μM, giving evidence for strong inhibitory effects at 30μM G M3 and less but significant suppression after application of G M3 concentrations lower than 20μM. No cellular response was observed at the lowest concentration (3μM) 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 G M3 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 G M3 in high density hybridoma cell cultures.

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Abbreviations

DMB:

1,2-diamino-4,5-methylenedioxybenzene

FAB-MS:

fast atom bombardment-mass spectrometry

GSL(s):

glycosphingolipid(s)

HPLC:

high performance liquid chromatography

HPTLC:

high performance thin layer chromatography

MTT:

3,(4,5 dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide

NeuAc:

N-acetylneuraminic acid

NeuGc:

N-glycolylneuraminic acid

PBS:

phosphate buffered saline

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Authors and Affiliations

  1. Institute for Cell Culture Technology, University of Bielefeld, P.O. Box 100131, 33501, Bielefeld, Germany

    Heike Brandt, Johannes Müthing & Jürgen Lehmann

  2. Institute for Physiological Chemsitry, University of Bonn, Germany

    Jasna Peter-Katalinić

Authors
  1. Heike Brandt
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  2. Johannes Müthing
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  3. Jasna Peter-Katalinić
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  4. Jürgen Lehmann
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Additional information

The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Galß1–4Glcß1-1Cer; gangliotriaosylceramide or GgOse3Cer; GalNAcß1–4Galß1–4Glcß1-1 Cer; gangliotetraosylceramide or GgOse4Cer, Galß1–3GalNAcß1–4Galß1–4Glcß1-1Cer; G M3(NeuAc), II3NeuAc-LacCer; G M3(NeuGc), II3NeuGc-LacCer; G M2(NeuGc), II3NeuGc-GgOse3Cer; G M1 or G M1a, II3NeuAc-GgOse4Cer; G M1b, IV3NeuAc-GgOse4Cer.

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Brandt, H., Müthing, J., Peter-Katalinić, J. et al. Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro . Cytotechnology 16, 89–100 (1994). https://doi.org/10.1007/BF00754611

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