O-GLYCOSYLATION OF HCG AND CANCER

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There are three aims to the proposed research, each with its own methodology but all with a common clinical objective - to identify and develop carbohydrate-specific tumor markers, with emphasis on hCG and the differential diagnosis of trophoblastic neoplasms. 1. Distribution of O-linked sugar units on pregnancy- and tumor-derived hCG. On hCG in pools of urine from normal pregnancy, 12-14% of the O-linked sugar units (4 total) are a hexasaccharide structure, NeuAc-Gal-GlcNAc- (NeuAc-Gal)-GalNAc-. In contrast, pilot studies show that the structure of those on hCG in the urines of choriocarcinoma subjects (2 examined) and in the media from choriocarcinoma cells were around 50% this hexasaccharide structure. The only published findings of this structure have been on cancer proteins. To confirm that hCG abundant in hexasaccharides is a choriocarcinoma marker, the structures of O-linked sugars on hCG molecules in the urines of 12-15 additional subjects will be examined (methods: immunoprecipitation of hCG, beta-elimination/labeling of O-linked sugar units, chromatographic comparison with oligosaccharide standards). To determine if the abundance of hexasaccharide is correlated with the malignance of trophoblastic disease, that on urinary hCG molecules from subjects with the more benign invasive/partial/complete mole will also be examined. O-linked sugar units on hCG from nontrophoblastic neoplasms, and on other urinary and tissue glycoproteins will be examined. 2. Development on trophoblast disease-specific assays. Although trophoblast disease is rare in the U.S.A. (1 in 1500 pregnancies), a 10 X higher occurrence is found in several African and Asian nations. To generate a disease-specific assay, the beta subunit C-terminal peptide containing the hexasaccharides on choriocarcinoma hCG will be isolated and utilized as immunogen for generating sheep polyclonal antisera, for monoclonal antibody approaches, and as domain for a lectin-antibody system. 3. Glycosyltransferase activities in normal, neoplastic, and induced or transformed cells. To investigate the pathways or mechanisms by which changes in protein glycosylation accompany cancer, glycosyltransferase activities will be examined in trophoblast explant cultures. Effects of virus and chemical transformation, growth factors, and other inducers/suppressants of cellular differentiation on enzyme activities will be examined; where appropriate kinetic aspects of activity changes and effects of DNA, RNA and protein synthesis inhibitors will be investigated.