Cytotechnology

, Volume 16, Issue 2, pp 89–100 | Cite as

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

  • Heike Brandt
  • Johannes Müthing
  • Jasna Peter-Katalinić
  • Jürgen Lehmann
Article

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 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 3μM and 30μM, giving evidence for strong inhibitory effects at 30μM GM3 and less but significant suppression after application of GM3 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 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.

Key words

Hybridoma growth inhibition gangliosides GM3 

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

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Andersson J, Möller G and Sjoberg O (1972) Selective induction of DNA synthesis in T and B lymphocytes. Cell. Immunol. 4: 381–393.Google Scholar
  2. Bethke U, Müthing J, Schauder B, Conradt P and Mühlradt PF (1986) An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms. J. Immunol. Methods 89: 111–116.Google Scholar
  3. Brandt H, Müthing J and Lehmann J (1993a) Exogenous ganglioside GM3 inhibits growth of hybridoma cells. Eur. J. Cell Biol. 37: 118.Google Scholar
  4. Brandt H, Müthing J and Lehmann J (1993b) Growth of hybridoma cells is inhibited by gangliosides. In: Spier RE, Griffiths JB, Berthold W (eds.), Animal Cell Technology: Products of Today, Prospects of Tomorrow, pp 170–172, Butterworth-Heinemann, London.Google Scholar
  5. Bremer EG, Hakomori SI, Bowen-Pope DF, Raines E and Ross R (1984) Ganglioside-mediated modulation of cell growth, growth factor binding and receptor phosphorylation. J. Biol. Chem. 259: 6818–6825.Google Scholar
  6. Bremer EG, Schlessinger J and Hakomori SI (1986) Ganglioside-mediated modulation of cell growth. J. Biol. Chem. 261: 2434–2440.Google Scholar
  7. Büntemeyer H, Lütkemeyer D and Lehmann J (1991) Optimization of serum-free processes for antibody production. Cytotechnology 5: 57–67.Google Scholar
  8. Büntemeyer H, Wallerius C and Lehmann J (1992) Optimal medium use for continuous high density perfusion processes. Cytotechnology 9: 59–67.Google Scholar
  9. Ciucanu I and Kerek F (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131: 209–218.Google Scholar
  10. Dyatlovitskaya EV, Koroleva AB, Suskova VS, Rozynov BV and Bergelson LD (1991) Influence of ganglioside GM3 and its breakdown products on lymphoblastic transformation and T-suppressor activity. Eur. J. Biochem. 199: 643–646.Google Scholar
  11. Hakomori SI (1981) Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu. Rev. Biochem. 50: 733–764.Google Scholar
  12. Hakomori SI (1984) Glycosphingolipids as markers for development and differentiation and as regulators of cell proliferation. In: Haber E (ed.) The Cell Membrane, pp 181–210, Plenum Press, New York.Google Scholar
  13. Hakomori SI (1990) Bifunctional role of glycosphingolipids. J. Biol. Chem. 265: 18713–18716.Google Scholar
  14. Hansen MB, Nielsen SE and Berg K (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119: 203–210.Google Scholar
  15. Hara S, Takemori Y, Yamaguchi M, Nakamura M and Ohkura Y (1987) Fluorometric high-performance liquid chromatography of N-acetyl-and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal. Biochem. 164: 138–145.Google Scholar
  16. Hashimoto Y, Otsuka H, Sudo K, Suzuki K, Suzuki A and Yamakawa T (1983) Genetic regulation of GM2 expression in liver of mouse. J. Biochem. 93: 895–901.Google Scholar
  17. Igarashi Y, Nojiri H, Hanai N and Hakomori SI (1989) Gangliosides that modulate membrane protein function. Methods Enzymol. 179: 521–541.Google Scholar
  18. IUPAC-IUB recommendations (1977) The nomenclature of lipids. Eur. J. Biochem. 79: 11–21.Google Scholar
  19. Jäger V, Lehmann J and Friedl P (1988) Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors. Cytotechnology 1: 319–329.Google Scholar
  20. Keenan TW, Schmid E, Franke WW and Wiegandt H (1975) Exogenous glycosphingolipids suppress growth rate of transformed and untransformed 3T3 mouse cells. Exp. Cell Res. 92: 259–270.Google Scholar
  21. Laine RA and Hakomori SI (1973) Incorporation of exogenous glycosphinglipids in plasma membranes of cultured hamster cells and concurrent change of growth behavior. Biochem. Biophys. Res. Commun. 54: 1039–1045.Google Scholar
  22. Ladisch S, Gillard B, Wong C and Ulsh L (1983) Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides. Cancer Res. 43: 3808–3813.Google Scholar
  23. Ledeen RW & Yu RK (1982) Gangliosides: structure, isolation and analysis. Methods Enzymol. 83: 139–191.Google Scholar
  24. Ledeen RW (1989) Biosynthesis, metabolism, and biological effects of gangliosides. In: Margolis RU and Margolis RK (eds.) Neurobiology of Glycoconjugates. Plenum Press, New York and London.Google Scholar
  25. Magnani JL, Nilsson B, Brockhaus M, Zopf D, Steplewski AZ, Koprowski H and Ginsburg V (1982) A monoclonal antibodydefined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentanose II. J. Biol. Chem. 257: 14365–14369.Google Scholar
  26. Müthing J, Egge H, Kniep B and Mühlradt PF (1987) Structural characterization of gangliosides from murine T lymphocytes. Eur. J. Biochem. 163: 407–416.Google Scholar
  27. Müthing J and Mühlradt PF (1988) Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment. Anal. Biochem. 173: 10–17.Google Scholar
  28. Müthing J, Schwinzer B, Peter-Katalinić J, Egge H and Mühlradt PF (1989) Gangliosides of murine T lymphocyte subpopulations. Biochemistry 28: 2923–2929.Google Scholar
  29. Müthing J, Peter-Katalinić J, Hanisch FG and Neumann U (1991) Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry. Glycoconjugate J. 8: 414–423.Google Scholar
  30. Müthing J and Neumann U (1993) Selective detection of terminally α2–3 and α2–6 sialylated neolacto-series gangliosides by immunostaining on thin layer chromatograms. Biomed. Chromatogr. 7: 158–161.Google Scholar
  31. Müthing J, Steuer H, Peter-Katalinić J, Marx U, Bethke U, Neumann U and Lehmann J (1994) Expression of gangliosides GM3 (NeuAc) and GM3 (NeuGc) in myelomas and hybridomas of mouse, rat and human origin. J. Biochemistry 116: 64–73.Google Scholar
  32. Nagai Y and Iwamori M (1989) A new approach to the analysis of ganglioside molecular species. In: Svennerholm L, Mandel P, Dreyfus H and Urban PF (eds.) Structure and Function of Gangliosides, pp 13–21, Plenum Press, New York and London.Google Scholar
  33. Peter-Katalinić J and Egge H (1990) Desorption mass spectrometry of glycosphingolipids. Methods Enzymol. 193: 713–733.Google Scholar
  34. Pörtner A, Peter-Katalinić J, Brade H, Unland F, Büntemeyer H and Müthing J (1993) Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes. Biochemistry 32: 12685–12693.Google Scholar
  35. Prokazova NV, Dyatlovitskaya EV and Bergelson LD (1988) Sialylated lactosylceramides. Possible inducers of non-specific immunosuppression and atherosclerotic lesions. Eur. J. Biochem. 172: 1–6.Google Scholar
  36. Radsak K, Schwarzmann G and Wiegandt H (1982) Studies on the cell association of exogenously added sialo-glycolipids. Biol. Chem. Hoppe Seyler 363: 263–272.Google Scholar
  37. Rösner H, Greis CH and Rodemann HP (1990) Density-dependent expression of ganglioside GM3 by human skin fibroblasts in an all-or-none fashion, as a possible modulator of cell growth in vitro. Exp. Cell Res. 190: 161–169.Google Scholar
  38. Saito M (1989) Bioactive sialoglycosphingolipids (gangliosides): potent differentiation-inducers for human myelogenous leukemia cells. Develop. Growth & Differ. 31: 509–522.Google Scholar
  39. Schauer R (1988) Sialic acids as antigenic determinants of complex carbohydrates. Adv. Exp. Med. Biol. 228: 47–72.Google Scholar
  40. Schneider-Jakob HR and Cantz M (1990) Lysosomal and plasma membrane ganglioside GM3 sialidase of cultured human fibroblasts. Biol. Chem. Hoppe Seyler 272: 443–450.Google Scholar
  41. Schwarzmann G, Hoffmann-Bleihauer P, Schubert J, Sandhoff K and Marsh D (1993) Incorporation of ganglioside analogues into fibroblast cell membranes. A spin label study. Biochemistry 22: 5041–5048.Google Scholar
  42. Schwarzmann G, Marsh D, Herzog V and Sandhoff K (1987) In vitro incorporation and metabolism of gangliosides. In: (ed.) Rahmann H, Gangliosides and Modulation of Neuronal Functions, Springer Verlag.Google Scholar
  43. Schwarzmann G and Sandhoff K (1990) Metabolism and intracellular transport of glycosphingolipids. Biochemistry 29: 10866–10871.Google Scholar
  44. Stults CLM, Sweeley CC and Macher BA (1989) Glycosphingolipids: structure, biological source, and properties. Methods Enzymol. 179: 167–214.Google Scholar
  45. Suzuki M, Nakamura K, hashimoto Y, Suzuki A and Yamakawa T (1986) Mouse liver gangliosides. Carbohydr. Res. 151: 213–223.Google Scholar
  46. Svennerholm L (1957) Quantitative estimation of sialic acids: a colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta 24: 604–611.Google Scholar
  47. Svennerholm L (1963) Chromatographic separation of human brain gangliosides. J. Neurochem. 10: 613–623.Google Scholar
  48. Thompson TE and Tillack TW (1985) Organization of glycosphingolipids in bilayer and plasma membranes of mammalian cells. Ann. Rev. Biophys. Chem. 14: 361–386.Google Scholar
  49. Ueno K, Ando S and Yu RK (1978) Gangliosides of human, cat, and rabbit spinal cords and cord myelin. J. Lipid Res. 19: 863–871.Google Scholar
  50. Usuki S, Lyu SC and Sweeley CC (1988a) Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside. J. Biol. Chem. 263: 6847–6853.Google Scholar
  51. Usuki S, Hoops P and Sweeley CC (1988b) Growth control of human foreskin fibroblasts and inhibition of extracellular sialidase activity by 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid. J. Biol. Chem. 263: 10595–10599.Google Scholar
  52. Yelton DE, Desaymard C and Scharff MD (1981) Use of monoclonal anti-mouse immunoglobulin to detect mouse antibodies. Hybridoma 1: 5–11.Google Scholar
  53. Yohe JHC, Cuny CL, Macala LJ, Saito M, McMurray WJ and Ryan WJ (1991) The presence of sialidase-sensitive sialylgangliotetraosyl ceramide GM1b in stimulated murine macrophages. J. Immunol. 146: 1900–1908.Google Scholar
  54. Zeller CB and Marchase RB (1992) Gangliosides as modulators of cell function. Am. J. Physiol. 262: C1341-C1355.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Heike Brandt
    • 1
  • Johannes Müthing
    • 1
  • Jasna Peter-Katalinić
    • 2
  • Jürgen Lehmann
    • 1
  1. 1.Institute for Cell Culture TechnologyUniversity of BielefeldBielefeldGermany
  2. 2.Institute for Physiological ChemsitryUniversity of BonnGermany

Personalised recommendations