Enzymatic basis for increased expression of GD3 on human melanoma cells derived from metastatic lesions

Abstract

AbstractThe expression of disialoganglioside GD3 is examined in human melanoma cell lines derived from primary (WM 115 and WM 75) or metastatic (WM 266 and WM 373) lesions resected from the same two patients. GD3 is demonstrated to be preferentially expressed by the metastatic cell lines relative to their primary cell line counterparts as shown by thin layer chromatography and flow cytometric analysis. The enhanced expression of GD3 in the metastatic cell lines is due to the increased activity of its biosynthetic enzyme, cytidine‐5′‐monophospho‐N‐acetylneuraminic acid:GMS sialyltransferase (GD3 synthetase), as evident from assays of GD3 synthetase activity of these cell lines. The activity of this enzyme in membrane extracts of all 4 cell lines is dependent on protein concentration and time, with peak activity occurring after 30 min. GD3 synthetase in both WM 115 and WM 266 exhibits a relatively broad pH optima between 6.2 and 6.6, is stimulated by Mn2+ and Mg2+, and inhibited by EDTA and Hg2+. Subcellular fractionation indicates that GD3 synthetase activity is localized primarily within the Golgi‐enriched fraction. The enhanced expression of GD3 on the metastatic melanoma cell lines WM 266 and WM 373 relative to that of their primary cell line counterparts cannot be attributed to blocked synthesis of other gangliosides utilizing GD3 as a precursor, but is primarily due to the increased activity of GD3 synthetase.