The long term objective of the studies outline in this proposal is to increase our understanding of the molecular mechanisms involved in the transition of hepatocytes from the reversible stage of promotion to the irreversible, karyotypically unstable stage of progression. We will utilize a model of multistage hepatocarcinogenesis in the rat, chemically- induced in wild type and transgenic rats, the latter bearing the albumin SV-40 T antigen gene construct as transgene. The experiments to be undertaken involve 1) a careful study of the region of common duplication (chromosome 1q3.7-q4.3) seen in chemically-and transgenically-induced hepatic neoplasms using molecular technologies to define the smallest region of duplication common to all hepatic neoplasms and determination of the expression of genes found in this region as it relates to multistage hepatocarcinogenesis. 2) The effect of treatment with chemical carcinogenesis on the multistage development of neoplastic lesions in these transgenic rats will be quantitated and related to mutational changes in the p53 gene during early and late stages. The effect of hormonal ablations as well as the structure of the transgene on the differential growth of neoplastic lesions in male and female rats bearing the alb-SV-40 Tag transgene will be investigated. The determination of the reversible or irreversible nature of hepatic lesions in the transgenic animals will be determined using a fasting regimen of periodic fasting. 3) The specificity of the expression of TGFalpha and c-myc to the stage of progression as determined by a relationship to genomic instability will be analyzed using studies of allelic imbalance and gene amplification during multistage, chemically-induced hepatocarcinogenesis in nontransgenic animals. 4) Studies of the mechanism of altered gene expression during chemically-induced multistage hepatocarcinogenesis and in the expression of the alb-SV-40 Tag transgene will be investigated by determination of cytosine methylation using bisulfite reaction and sequencing of potentially involved regions. The methylation and expression of imprinted gene in or near duplicated region of chromosome 1 will be investigated by this technique and in animals exhibiting allelic heterozygosity as judged by RFLP at these loci. From these studies we hope to develop a morphologic, karyotypic and molecular understanding of the transition of hepatocytes from the stage of promotion to that of progression in rat hepatocarcinogenesis.
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