The goal of this project is to evaluate the hypothesis that oncogene expression occurs as a step in radiation carcinogenesis. This possibility is being evaluated using a rat thyroid carcinogenesis model capitolizing 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 are being 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 are being 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 are being subjected to restriction endonuclease digestion and Southern blot analysis utilizing a complete battery of nick translated retroviral oncogene probes. Using DNA isolated from a radiation-induced thyroid carcinoma we have been able to identify mutations and DNA rearrangements in the rask, myc, and abl oncogenes. This DNA is also capable of transfecting NIH/3T3 cells in the DNA mediated gene transfer assays. At present molecular cloning and sequencing techniques are being used to localize the dominant transforming oncogene. Once the effected oncogenes are isolated their individual and collaborative effects will be tested in the transfection assays using recombinant DNA molecules harboring cDNA copies of these oncogenes in eukaryotic expression vectors. Northern blot analysis is being utilized to determine the level of expression of these 3 oncogenes in normal and tumor tissue. cDNA copies of the affected oncogenes are being cloned in bacterial plasmids and oncogene-specific proteins are being characterized using specific antisera against myc and rask protein products. These proteins are being characterized with respect to localization, molecular weight, protein-kinase activity, GTPase and GTP-binding activity. These parameters will be compared with those of known retroviral transforming proteins.
