We have recently identified by monoclonal antibodies a cell surface protein (p50) consistently found in hepatitis B virus - related primary hepatocellular carcinomas. p50 was undetectable in normal adult and fetal tissues except in the adrenal glands. A very similar protein has also been identified and partially purified from bovine adrenals. We wish to study the structure and cellular function(s) of this protein and evaluate its possible role(s) in malignant transformation. In this proposal we will focus on three major aspects of p50 as follows: 1) Primary structure: In this regard we plan to purify p50 from human hepatoma cells and bovine adrenals to homogeneity by affinity chromatography. We will determine its primary structure by N-terminal sequencing. We will also analyze immunodominant epitopes by generating new monoclonal antibodies to the purified protein. 2) Molecular biology: In this regard we have constructed cDNA libraries from hepatoma cell polyA(+) RNAs in procaryotic and eucaryotic expression vectors. We plan to use our specific monoclonal antibodies for immunoselection of p50 cDNA's. We will determine genomic organization of cDNA inserts of interest by dideoxynucleotide sequencing. 3) Functional analysis: We will investigate p50 expression at mRNA and protein level in normal and regenerative liver as well as human tumors compared to adrenal glands. We will transfect cDNA clone(s) into mammalian cells to obtain model systems for transient and constitutive expression of p50. To test whether p50 overproduction could alter biological properties of established cells, representative cell lines will be studied in terms of changes of cell shape, loss of contact inhibition, immortalization and tumorigenicity in immunodeficient nude mice. These investigations will permit a better understanding of the biological significance of the hepatoma-associated expression of p50 and the pathogenesis of hepatocellular carcinoma As p50 was also found in all human tumor cell lines thus far studied, such investigations may have a broader significance to the malignant transformation of mammalian cells in general.
