CELL, MOLECULAR AND GENETIC CHARACTERIZATION OF PROGRESSION OF A HEPATOCARCINOGEN

本建议中概述的研究的长期目标是 增加我们对参与的分子机制的理解， 肝细胞从可逆的促进阶段过渡到 不可逆的、核型不稳定的进展阶段。我们将 利用大鼠多阶段肝癌发生模型，化学- 在野生型和转基因大鼠中诱导，后者携带白蛋白 SV-40 T抗原基因构建体作为转基因。这些实验 1）仔细研究常见重复的区域 （染色体1q3.7-q4.3）在化学和转基因诱导的 利用分子技术确定最小的肝肿瘤 所有肝肿瘤共有的重复区域和确定 在这个区域发现的基因的表达，因为它涉及多阶段 肝癌发生2)化学处理的效果 致癌作用对肿瘤病变多阶段发展的影响 这些转基因大鼠将被定量并与突变相关。 p53基因在早期和晚期的变化。的影响 激素消融以及转基因的结构， 雄性和雌性大鼠肿瘤病变的生长差异 将研究alb-SV-40标签转基因。的确定 转基因小鼠肝损伤的可逆性或不可逆性 使用定期禁食的禁食方案确定动物。第三章 TGF α和c-myc的表达与肿瘤分期的特异性 由与基因组不稳定性的关系确定的进展将是 使用等位基因不平衡和基因扩增的研究进行分析， 非转基因小鼠多阶段化学诱导肝癌发生 动物4)基因表达改变机制的研究 化学诱导的多阶段肝癌发生， 将通过测定alb-SV-40标签转基因的 使用亚硫酸氢盐反应和测序的胞嘧啶甲基化 可能涉及的地区。印迹基因的甲基化和表达 将通过以下方法研究1号染色体重复区域中或附近的基因： 这种技术和在显示出等位基因杂合性的动物中， 在这些基因座上进行RFLP分析。通过这些研究，我们希望开发出一种 形态学、核型和分子生物学的理解， 大鼠肝细胞从促进阶段到进展阶段 肝癌发生

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.