NON RAS ONCOGENES IN CHEMICALLY INDUCED RAT LIVER TUMORS

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Hepatic cell carcinoma is one of the major tumors of man. The rat offers one of the best characterized model systems for examining the development of chemically-induced hepatic cell carcinoma. Despite the rapid advances in isolating and characterizing activated proto-oncogenes in a number of other systems, there have been relatively few such reports of hepatic tumors, particularly in the rat. Methyl (acetoxymethyl) nitrosasmine (DMN-OAc) is a "conditional" liver carcinogen producing tumors only when injected into animals undergoing active liver regeneration. We have enxamined DNA from 8 primary rat tumors induced by DMN-OAc for transforming ability upon transfection into NIH3T3 cells. In 7 of the 8 primary tumors we have detected transforming activity. The morphologically transformed NIH3T3 cells grow in soft agar and those which have been tested are tumorigenic upon inoculation into nude mice. Since we have previously characterized the expression of several proto-oncogenes during regenerative growth in rat liver, we examined DNA from the NIH3T3 transformants for altered ras (N- ras, rasH or rasR), myc, erbA, erb, V-raf, A-raf or neu genes and have found no evidence for any involvement of these genes. Preliminary restriction endonuclease sensitivity profiles of the primary transformant DNA suggest that there are at least 3 dinstinct genes activated whithin the set of tumors we are examining. The major focus of this research project will be to isolate each of these genes by molecular cloning and to characterize the genes with respect to expression in both normal rat tissues and during the course of DMN-OAc induced carcinogenesis. Further studies characterizing these genes will include transfection of the genes into rat liver "oval" cell lines and into hepatocytes with enhanced proliferative capability. In the later stages of the project we will express the protein products of these genes using the baculovirus expression system and characterize the oncogene proteins with respect to cellular and tissue localization by immunofluorescence. Ultimately we hope to define the role of these proto-oncogenes in both normal rat development and the development of hepatic cell carcinoma.