The specific aims of our research are: (1) to purify the uncombined and combined subunits of human chorionic gonadotropin (hCG) subunits produced by malignant trophoblastic cells and normal placenta; (2) to characterize biochemically the hCG subunits produced by these tissues; and (3) to determine the rate-limiting steps involved in the processing and secretion of hCG subunits by these tissues. Methods include pulse-chase labeling of cultured trophoblastic cell lines and organ cultures of normal placenta with radioactive amino acids and carbohydrates (in the presence and absence of various glycosidase inhibitors and drugs that affect intracellular cation and pH gradients); determination of carbohydrate structure and amino acid composition; and sequence analysis of the glycoprotein subunits of hCG produced by these tissues. Data obtained to date indicate that: (1) free hCG-beta subunit is present in cells and media from cultured choriocarcinoma cells and placental explants and in the sera of pregnant women and of patients with choriocarcinoma; (2) free alpha, but not hCG-alpha, contains an O-linked oligosaccharide chain; (3) there is a pH gradient, apparently within the Golgi apparatus, that regulates the oligosaccharide processing and secretion of hCG and its free subunits; and (4) the kinetics of secretion of free alpha, free beta, and hCG dimer are similar even though the kinetics of processing of their N-linked oligosaccharides are somewhat different. Long-term objectives are to learn what controls the secretion of hCG from normal and malignant trophoblastic cells and to determine what differences, if any, exist between the glycoprotein hormone subunits produced by normal and malignant cells. The results of these studies will have potential significance for the use of hCG subunits as tumor markers and in fertility control. (A)
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