This proposal is for research on the significance and biochemical mechanisms of progressive losses of sialoglycosphingolipids (gangliosides) from the mammalian cell surface during tumorigenesis. In the rat, with hepatomas induced by 2-acetylaminofluorene, the glycolipid simplifications occur as late events in the tumorigenic progression, the end result of an enzymatic cascade. Ganglioside losses correlate with metastatic potential, loss of normal adhesion characteristics and inability to bind certain adhesion (attachment) proteins (e.g. fibronectins). The higher gangliosides lost during tumorigenesis bind fibronectin. The binding constants are similar to those for binding to plasma membranes or for attachment of cells to an underlying stroma. Despite certain complexities, a relationship of ganglioside presence to fibronectin binding might help explain how cell surface changes facilitate metastasis through reduced cell adhesion. Relationships among ganglioside simplification, loss of fibronectin receptors, failure to bind fibronectins and other adhesion proteins and metastatic potential will be analyzed and correlated in a variety of transplanted tumors of varying metastatic ability. Comparisons will include metastatic/non-metastatic clones of known hepatic origin. Specifically, we propose to isolate and characterize a putative fibronectin binding protein that may be essential to cell attachment. Distributions of gangliosides and fibronectin binding proteins between the cell surface and internal membranes will be determined using a procedure for the facile preparation of highly purified fractions of plasma membrane and endoplasmic reticulum from transplanted tumors and cultured cells by preparative free-flow electrophoresis. New information concerning a proposed role for gangliosides as receptors on internal membranes to facilitate secretion and transport or as trafficking signals for adhesion proteins will be sought along with mechanisms whereby gangliosides are transported from sites of synthesis at the Golgi apparatus to sites of insertion at the plasma membrane and certain internal membranes. Basic mechanisms leading to ganglioside simplification will be sought along with the basis for an apparent build-up of higher ganglioside precursors and products of the monosialoganglioside biosynthetic pathway. These studies will be facilitated by liver cell lines temperature sensitive for transformation in which specific ganglioside alterations similar to those observed during tumorigenesis are induced reproducibly by the temperature shift.
