The objective of this project is to study the mechanism of carcinogenesis using quantitative two-dimensional gel electrophoresis (2D-gel). This technique lets us examine both qualitative and quantitative changes in the synthesis of thousands of cellular polypeptides as the cell undergoes neoplastic transformation. Research has focused on three areas: study of the effects of v-ras oncogenes on the synthesis of polypeptides in a diploid human fibroblast line (LG1); a study of the changes induced in dog plasma by the known urinary bladder carcinogen 4-aminobiphenyl (4- ABP); and an examination of the effects of an inducible c-raf proto- oncogene on the 2D-gel patterns in rat liver epithelial (RLE) cells. The 2D-gel patterns of LG1 cells have been compared with a non-tumorigenic, immortalized growth variant, VO, and with cell strains transformed with either v-H-ras or v-N-ras. LG1, VO and the ras transformed cells resulted in distinct 2D-gel patterns which were easily distinguished by cluster analysis techniques. Subtle, but consistent differences were also detected between the ras transformed cells. The second study, involving the effects of 4-ABP on dog plasma, was done to determine if there were deletions in plasma proteins specific to 4-ABP, and to identify such proteins. Two sets of proteins, which are greatly reduced during 4-ABP treatment, were observed. One was identified as the beta- chain of haptoglobin. A second set of three spots (MW = 65 kD, pI = 6.5- 6.6), disappears much more rapidly than the haptoglobin and recovers more quickly after dosing stops. The third study has employed RLE cells transfected with plasmids containing the c-raf-1 proto-oncogene under the control of an inducible metallothione promoter. It is anticipated that the raf gene can be induced in the transfectants by appropriate concentrations of zinc. We intend to examine proteins that are phosphorylated in response to increased raf synthesis in hopes of finding potential effector proteins for raf. Twenty subclones have been isolated and work is underway to determine the appropriate conditions for induc- tion of raf in these transfectants.
