The thrust of this study is to elucidate the genetic mechanism of neoplastic transformation of normal tissues. The experimental system focuses on a 3.1 kb moderately cell-transforming human oncogene, hhcM, isolated from an African hepatocellular carcinoma cell line, Mahlavu. Several related genomic DNA clones have now been isolated from other human hepatomas. Recently we have also isolated four cDNA clones each from human hepatoma and fetal liver DNA libraries. The cell transformation capability of hhcM on both NIH3T3 cells and buffalo rat liver cells has been studied and the tumorigenicity of the transformants has also been described. Determination of the nucleotide sequence of hhcM has now been completed. The hhcM nucleotide sequence shares no significant homology with other known oncogenes. It predicts an uninterrupted open reading frame specifying a message for a polypeptide of 467 amino acids, ca 52 kD. This was supported by the observation of a 52-55 kD polypeptide obtained in hybrid-select cell-free translation studies. This ORF was used in a chimeric construct with the lacZ promoter sequence, and production of the 52 kD protein is now scaled up significantly. The chimeric protein is being used to generate antibodies to be employed in characterization of the protein in cultured cells as well as in clinical samples of human urine, serum, and liver. Since earlier deletion analysis suggested that interruption of the nucleotide sequence within this ORF abolished the cell transformation capability, the possible direct involvement of this 52 kD protein in cell transformation is being explored.
