Two very different kinds of observations suggest that sialic acid may play a role in the mechanism of or be indicative of human cancer. First, sialic acid-rich mucins have been shown to mask cell-surface antigens and increased metastasis and resistance to natural killer (NK) cell-mediated lysis have been correlated with increased sialic acid content. Second, a specific sialic acid derivative, N-glycolylneuraminic acid (NeuGl), is not a constituent of normal human tissue, but has been associated with tissues or sera from patients with lymphoma, leukemia, neuroblastoma, and colon cancer. The proposed work will address two questions: 1) What is the relationship between susceptibility to NK-mediated cytolysis and expression of sialic-acid rich mucin? 2) How is the synthesis and incorporation of NeuGl regulated? Rat mammary tumor ascites cell (13762 MAT-Bl and MAT-Cl) will be used as the system of study, because greater than 80% of the sialic acid in both lines is associated with a major mucin-type glycoprotein. Expression of this glycoprotein can be reversibly inhibited upon culturing in vitro. Also, the MAT-Bl and MAT-Cl glycoproteins differ in the presence of NeuGl. The relationship of NK susceptibility and expression of mucin will be determined by monitoring, as a function of time in culture, a) glycoprotein production by glucosamine labeling and density gradient centrifugation, b) NK-susceptibility to normal rat spleen cells, c) expression of histocompatibility complex by determination of the adsorptive capacity for rat anti-MHC; d) furthermore, the mechanism for resistance of ascites cells to NK-mediated cytolysis will be determined. Regarding the mechanism of NeuGl formation, the proposed work will e) generate a new and improved method for assaying the monooxygenase, f) identify the preferred substrate, requirements and subcellular localization of the enzyme in the ascites cells, and g) determine whether exogenous N-glycolylmannosamine can be incorporated into NeuGl in cells where NeuGl is not normally synthesized. The results will establish whether cell surface mucins contribute to resistance to NK-mediated cytolysis in this system, and provide information on the mechanisms by which levels of sialic acid derivatives are regulated. Ultimately through alteration of sialic acid metabolism, increased recognition and destruction of tumor cells may be accomplished.
