The goal of this project is to evaluate the hypothesis that oncogene expression occurs as a step in radiation carcinogenesis. This possibility will be evaluated using a rat thyroid carcinogenesis model capitilizing on the exquisite sensitivity of thyroid epithelium to the carcinogenic effects of ionizing radiation. Dominant transforming oncogenes identified in the genome of cells from radiation-induced adenomas and carcinomas will be characterized and compared to those isolated from the DNA of normal thyroid cells and cells from spontaneous thyroid tumors. Likewise, the mRNA transcripts and protein products associated with the oncogenes mentioned above will be analyzed. The structure and function of these macromolecules will be compared with those associated with the known retroviral oncogenes. The cellular DNA from radiation-induced and spontaneous thyroid tumors as well as from normal thyroid cells will be subjected to restriction endonuclease digestion and Southern blot analysis utilizing a complete battery of nick translated retroviral oncogene probes. The presence of mutations and DNA rearrangements will thus be detected. The restriction endonuclease fragment harboring the oncogene will be identified by hybridization and will be tested for its ability to transfect NIH 313 cells. Once localized, the oncogene contained in this fragment will be characterized by molecular cloning and sequencing techniques. To test the hypothesis that transformation is the result of augmented oncogene expression, the level of mRNA transcription in normal and neoplastic thyroid tissues will be studied by Northern blot analysis. Quantitative estimations of oncogene specific mRNAs in the begin and malignant tumor tissues (radiation-induced and spontaneous) will be made and compared with that of normal thyroid tissue to assess the extent of mRNA transcription. The mRNA species will be characterized with regard to size and correlation with other known oncogene-related mRNAs. cDNA copies will be cloned in bacterial plasmids and characterized by nucleotide sequence analysis. Oncogene specific proteins will be detected by immunoprecipitation using antisera from tumor-bearing rats or specific antisera against synthetic peptides coded for by the oncogene specific mRNAs. These oncogene proteins will be quantitated and compared with the known retroviral transforming proteins as to their size, molecular weight, protein kinase activity and cross-reactivity with antibodies.
