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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA018801-07
Application #
3165044
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1978-03-01
Project End
1989-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
7
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Hartel-Schenk, S; Minnifield, N; Reutter, W et al. (1991) Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat. Biochim Biophys Acta 1115:108-22
Morre, D J; Wilkinson, F E; Keenan, T W (1990) Gangliosides depleted in plasma membrane are directed to internal membranes of rat hepatomas: evidence for a glycolipid sorting defect in hepatocarcinogenesis. Biochem Biophys Res Commun 169:192-7
Navas, P; Nowack, D D; Morre, D J (1989) Isolation of purified plasma membranes from cultured cells and hepatomas by two-phase partition and preparative free-flow electrophoresis. Cancer Res 49:2147-56
Morre, D J (1988) Free-flow electrophoresis: preparative applications to cell-free analyses of exocytotic membrane traffic. Prog Clin Biol Res 270:7-19
Morre, D J; Morre, D M; Mollenhauer, H H et al. (1987) Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices. Eur J Cell Biol 43:235-42
Matyas, G R; Morre, D J (1987) Subcellular distribution and biosynthesis of rat liver gangliosides. Biochim Biophys Acta 921:599-614
Minnifield, N; Creek, K E; Navas, P et al. (1986) Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes. Eur J Cell Biol 42:92-100
Matyas, G R; Evers, D C; Radinsky, R et al. (1986) Fibronectin binding to gangliosides and rat liver plasma membranes. Exp Cell Res 162:296-318
Sun, I; MacKellar, W C; Crane, F L et al. (1985) Decreased NADH-oxidoreductase activities as an early response in rat liver to the carcinogen 2-acetylaminofluorene. Cancer Res 45:157-63
Morre, D J; Minnifield, N; Mollenhauer, H H (1985) Kinetics of monensin-induced swelling of Golgi apparatus cisternae of H-2 hepatoma cells. Eur J Cell Biol 37:107-10