Promotion of cell-substratum adhesion of clonal rat pheochromocytoma cells (PC12) by factors contained in glioma-conditioned medium (GCM): separation of two active factors contained in GCM

Culture medium conditioned over C6 glioma cells (GCM) contains factors which induce neurite outgrowth from clonal rat pheochromocytoma (PC12) cells. The effects of GCM on the cell-substratum adhesion of PC12 cells, which is an early event required for the neurite outgrowth, were investigated. The results obtained are as follows. Addition of GCM promoted the adhesion of PC12 cells specifically to collagen-coated tissue culture dish. The GCM-promoted adhesion of PC12 cells was prevented by the treatment of the cells with cytochalasin B, concanavalin A and glycosidase mixture suggesting the contribution of microfilaments and cell surface carbohydrates in the cell adhesion. GCM did not increase significantly the intracellular content of cAMP and the extent of cell adhesion promoted by cAMP or dibutyryl-cAMP was much less than that by GCM. Two active factors contained in GCM were separated by either gel filtration or chromatofocusing using the cell adhesion assay as an index. The first factor with an apparent mol. wt. around 40,000 had the abilities to induce the neurite outgrowth and to enhance the choline acetyltransferase activity in addition to the ability to promote the adhesion of PC12 cells. The second factor with an apparent mol. wt. around 10,000 was devoid of the ability to induce the neurite outgrowth, but had the abilities to enhance the choline acetyltransferase activity and to promote the adhesion of PC12 cells. Both factors were sensitive to trypsin digestion and relatively heat stable. The significance of these factors in the neuronal differentiation was discussed.