TWO-DIMENSIONAL GEL ANALYSIS OF CARCINOGENESIS

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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 two areas: 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 of rat liver epithelial (RLE) cells. The first 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 were greatly reduced during 4-ABP treatment, were observed. One was identified as the beta-chain of haptoglobin. We hypothesize that 4-ABP binds to hemoglobin, causing hemolysis. This ABP-hemoglobin complex then binds to haptoglobin and is purged from the blood stream. 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. Despite intense efforts, we have not been able to identify this protein. The second study has employed RLE cells transfected with a plasmid containing the c-raf-1 proto-oncogene under the control of an inducible metallo-thionein promoter. We anticipated that the raf gene would be induced in the trans- fectants by appropriate concentrations of zinc and by examining proteins that were phosphorylated in response to increased raf synthesis we might identify potential effector proteins for raf. Twenty subclones were isolated and at least two were shown to be inducible by high concentrations of zinc. However, no consistent changes were observed in the 2D-gel pattern of induced versus uninduced cells. Western blot analysis with three different anti-raf antibodies failed to demonstrate the presence of induced raf protein. It appears, then, that the plasmid raf mRNA is induced, but not translated in vivo.