The ganglioside composition of mouse ascites hepatoma (MAH) cells, the ascites fluid and cell-conditioned media were determined and found to be qualitatively identical, but quantitatively different.The ganglioside content of the ascites fluid and the medium conditioned by MAH-cells at the native cell concentration (1 O8 cells/ml) comprised respectively 74.9 :d and 23 % of the cell-associated gangliosides. When incubated at lower cell-density (lo6 cells/ml) the cells were found to be release about three-times higher amounts of ganglioside per cell than during incubation at the native concentration. Centrifugation of the dense-cell-conditioned medium revealed the major part of the released gangliosides to be associated with a 150000 x g pellet that probably contains shed plasma membrane fragments. In the 150000 x g pellet of the extracellular fluids the relative content of the most polar cell ganglioside corresponding chromatographically to GT1, was about ten-times higher than in the cells. The possibility is raised that the more intense shedding of gangliosides from less crowded MAH cells may play a role in the self protection of the tumor from host immune rejection during initial stages of growth.Gangliosides repeatedly have been suggested to exert immunosuppressing activity [I -61. Although the major part of the cell gangliosides are believed to reside in the outer leaflet of the plasma membrane certain amounts are found in non-cell associated form in the blood serum, where they may be present as free molecules, micelles, protein-bound complexes or membrane fragments. It has been speculated that serum gangliosides released by tumor cells may participate in the selfprotection of those cells from host immune rejection [I].However up to now only few data on the shedding of gangliosides from tumor cells have been published [7 -91.In the present study we examine the gangliosides secreted by mouse ascites hepatoma (MAH) cells and demonstrate that the amount of gangliosides shed depends specifically on the cell density. A preliminary communication has been published [lo]. MATERIALS AND METHODSwere redistillated prior to use. All chemicals were of analytical reagent grade and solvents CellsMale CBAxC5,BL/6F1 mice of 18-22 g weight were used throughout. The MAH-cells were grown in the peritoneal cavity and maintained for 9 days. The ascites fluid was separated from the cells by centrifugation at 800 x g (3000 rpm) for 3 min. The cell pellet was washed three times with physiological salt solution. For subsequent incubation the cell pellet was suspended in Eagle's complete medium and diluted with physiological salt solution to obtain a final concentration of 17 mg protein/ml or 0.17 mg protein/ml corresponding to lo8 or 106 celIs/mI respectively.Abbreviations. MAH, mouse ascite hepatoma 22a; the designation of gangliosides follows the nomenclature system of Svennerholm as listed in [IS].The intensity of protein synthesis was determined by studying incorporation of [14C]leucine (Amersham; specific activity 330 Ci/mol; acti...
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