The overall goal of this program is to develop an understanding of the role(s) played by sialoglycoproteins in mammary tumors. Ascites forms of the 13762 rat mammary adenocarcinoma have cell surfaces covered by a mucin-type sialoglycoprotein (ASGP-1). ASGP-1 is associated at the cell surface with a Concanavalin A-binding sialoglycoprotein (ASGP-2). Both of these glycoproteins have been purified to homogeneity. The presence of sialo-mucins at tumor cell surfaces has been implicated in escape from immune destruction. We will evaluate metastasis in tumors with and without ASGP-1. Antibodies against ASGP-1 and ASGP-2 will be prepared and used with other techniques to determine the nature of shed products from the tumor cell surfaces. The mechanism of clearance of shed products from the circulation will be investigated. The ability of soluble or particulate cell surface fractions and their immune complexes to inhibit tumor cell killing by lymphoid cells will be investigated. The expression of ASGP-1 and ASGP-2 in solid tumours, normal mammary gland and other tissues will be investigated using immunological techniques. We propose to investigate the biosynthesis of ASGP-1 and of complex using pulse-chase analyses, cell surface proteolysis and chromatographic fractionations. We have already determined the structures of the major oligosaccharides of ASGP-1. We will investigate their biosynthesis by determining the order of addition of peripheral oligosaccharide constituents, the kinetics of maturation of ASGP-1, kinetics of incorporation of various precursors, kinetics of expression at the cell surface and the evaluation of the cellular localization of various biosynthetic intermediates by cell fractionation and authoradiography. We will investigate kinetics of the formation of complex using lectin binding and pulse-chase analyses. The effects of biosynthesis inhibitors such as tunicamycin, FCCP and monensin will be determined. The protease involved in shedding of ASGP-1 in soluble form will be isolated for characterization. These investigations should give a clearer picture of the behavior and role of the sialo-mucins in the 13762 mammary adenocarcinoma and may provide insights into methods for inhibiting the expression of sialomucins.
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