These studies were undertaken to approach an understanding of gene derepression in tumor cells which is manifest in the ectopic expression of polypeptide hormones. The glycoprotein hormone alpha subunit secreted by HeLa cells has been purified about 60,000-fold and is 90% pure as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A partially purified preparation elutes ahead of urinary human chorionic gonadotropin alpha-subunit (hCG-alpha) on Sephadex chromatography but migrates coincident with it on SDS-PAGE. Unlike urinary alpha, the HeLa protein does not recombine with CG-beta, indicating a functional as well as structural difference in the tumor protein. The carbohydrate composition and tryptic peptide fingerprint of the HeLa subunit are currently under investigation. When further material is purified, the amino acid composition also will be determined and compared to that of urinary CG-alpha. These studies will demonstrate for the first time whether the immunoreactive alpha-like protein secreted by HeLa cells (a nontrophoblastic tumor) is indeed a glycoprotein hormone subunit, and if so, how closely the ectopic product resembles its placental counterpart. Preliminary studies have suggested that protein glycosylation may be correlated with subunit synthesis in HeLa cells. For example, the proportion of label from [3H] mannose associated with dolicholpyrophosphoryl oligosaccharide andintracellular glycopeptides is lower in cells treated with sodium butyrate (which increases alpha accumulation 10-fold), while label in secreted glycoproteins was increased in the same cultures. The induction of alpha by butyrate is inhibited by 2-deoxyglucose and does not occur when cells are cultured in the absence of glucose, two conditions known to inhibit protein glycosylation. We\have also demonstrated that the HeLa protein contains fucose, whereas the CG subunit does not. Taken together, these results suggest that the production of this ectopic hormone may in part be modulated at the level of post-translational modification in addition to (or instead of) gene activation as has been commonly proposed. (C)