The overall objective of this research proposal is, using hepatocarcinogenesis as a model system, to test the hypothesis that DNA sequence changes, including loss and rearrangement, occur during carcinogenesis. Specifically, we propose: (1) to use two animal protocols to produce liver nodules and tumors representing clonal populations of cells at different stages in the process of hepatocarcinogenesis; (2) to prepare a rat liver DNA probe representing minor reiterated sequence components; (3) to determine the inter-animal and inter-strain variability in the hybridization pattern of this probe with electropheretically separated EcoRI digested total rat genomic DNA; (4) to compare the hybridization patterns of the probe with electrophoretically separated EcoRI digested genomic DNA obtained from the individual nodules and tumors obtained in 1; (5) to determine whether hepatocarcinogenesis and/or aging results in the loss of highly repetitive DNA sequences; (6) to determine whether DNA isolated from hepatoma cell lines and individual liver nodules and tumors can transform NIH 3T3 cells; and (7) to determine, using restriction endonuclease sensitivity and mapping, whether the same or different transforming DNA sequences are obtained from different hepatoma cell lines and individual rat liver nodules and tumors. The results of these studies using well characterized and controlled protocols for hepatocarcinogenesis in inbred rats should provide new information regarding whether DNA sequence changes occur during the course of carcinogenesis and if so whether they occur early or late in tumor development. Later in the project we will attempt to isolate and characterize transforming genes from DNA of individual liver tumors.
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