Use of cholera toxin to isolate mutants of Balb/c 3T3 mouse fibroblasts with defective ganglioside biosynthesis.

Recent evidence has suggested that oncogenic transformation of mammalian tissue culture cells results in altered expression of several surface components. One of these changes is the reduced ability of transformed cells to synthesise complex gangliosides. This modification has been correlated with the other phenotypic alterations of transformed cells and with their tumorigenic potential. We have adopted a novel approach to elucidate the relationship between the impaired synthesis of complex gangliosides in transformed cells and other aspects of the transformed phenotype. Mutants of Balb/c 3T3 mouse fibroblasts, which normally contain substantial levels of the gangliosides GM3. GM2. GM1 and GD1a. have been selected for failure to express ganglioside GM1 at the cell surface. Other phenotypic properties of these mutants have been investigated, to determine whether the expression of any of the characteristics normally associated with transformed cells are dependent on the loss of ability to synthesise GM1 or the more complex gangliosides, GD1a and GTla. Cholera toxin, which is known to bind specifically to ganglioside GM1 with high affinity, has been used in the selection of these mutant cell lines. A method involving antibody-dependent complement-mediated lysis of cells able to bind cholera toxin was used as the one of choice. Previously, there has been some doubt as to whether any other cell surface receptors for cholera toxin exist in Balb/c 3T3 fibroblasts. Evidence is presented in this thesis, and in an accompanying paper, that the major receptor for cholera toxin in these cells is a ganglioside with similar characteristics to GM1. We have further demonstrated that a substantial proportion of this lipid is insoluble in neutral detergent and remains with Triton X-100 extracted cytoskeletons of Balb/c 3T3 cells. The possibility that GMlis indirectly associated with the cytoskeleton is discussed.