The thrust of this study is to elucidate the genetic mechanisms of neoplastic transformation of normal tissues and the cellular immune defense mechanisms against neoplastic cells. Two experimental systems were used: 1) A human oncogene derived from a hepatocellular carcinoma established in culture (MAH); 2) IL-2, an autocrine of T-cells, and CCDF (cytotoxic cell differentiation factor), produced by macrophages for the generation of lymphokine-induced cytotoxic cells. A systematic screening with DNA transfection assay, preferential AFB-1 binding and repeated molecular clonings, have been carried out and led to the successful isolation of a human hepatocellular carcinoma oncogene, hHC, which transforms NIH 3T3 cells at very low efficiency. Its transformation capability was, however, enhanced by more than 300 fold upon AFB-1 activation. One hHC clone, carried in Puc 8, is 3.1 kb and shares homology with human c-fes, c-raf and k-ras. We have established a restriction map of hHC and resolved the nucleotide sequence of the entire gene. By using a modified Maxam/Gilbert sequencing technique, the deoxyguanine targets in the hHC DNA sequence under controlled binding conditions with AFB-1 have been identified. This yields some relevant information regarding the nucleotide sequence specificity influencing AFB-1 activation of hHC. A cellular homolog of hHC, nhl, has been isolated from normal liver DNA. Restriction mapping of nhl suggests that, whereas homology between hHC and nhl is evident in the termini, recombination and rearrangement in the central portion of nhl has probably occurred to account for the structure of hHC. After extensive purification, the function and relationship of two oncogene products, IL-2 and CCDF, have been analyzed. In the absence of antigenic or mitogenic stimulation, IL-2 provides the first signal to activate the cytotoxic precursors and prepares these cells to differentiate into cytotoxic effectors in the absolute presence of CCDF. The synergistic action of IL-2 and CCDF is found critical for the generation of lymphokine-induced cytotoxicity, a cellular process considered pivotal in cellular immune defense mechanisms against tumor cells.
