CELL, MOLECULAR AND GENETIC CHARACTERIZATION OF PROGRESSION OF A HEPATOCARCINOGEN

这项建议的研究大纲的长远目标是： 增加我们对生物多样性的分子机制的了解 肝细胞从可逆的促进期过渡到 不可逆的，核型不稳定的进展阶段。我们会 利用大鼠多阶段肝癌形成模型，化学- 在野生型和转基因大鼠中诱导，后者携带白蛋白 构建以SV-40T抗原基因为转基因载体。将要进行的实验是 所采取的措施包括：1)仔细研究共同重复的区域 (染色体1q3.7-q4.3)在化学和转基因诱导下可见 使用分子技术定义最小的肝肿瘤 所有肝肿瘤常见的重复区域和测定 在该区域发现的与多阶段有关的基因的表达 肝癌的发生。2)药剂处理的效果 肿瘤病变多阶段发展过程中的致癌作用 这些转基因大鼠将被量化并与突变相关 早期和晚期P53基因的变化。的影响 荷尔蒙消融以及转基因的结构 雄性和雌性孕鼠肿瘤病变的差异生长 将对白蛋白-SV-40标签转基因进行研究。这一决定决定了 转基因动物肝脏病变的可逆性或不可逆性 动物将使用定期禁食的禁食方案来确定。3) 肿瘤生长因子α和c-myc的表达对肿瘤分期的特异性 由基因组不稳定关系决定的进展将是 利用等位基因不平衡和基因扩增的研究分析 多阶段、化学诱导的非转基因肝癌的发生 动物。4)基因表达变化机制的研究 化学诱导的多阶段肝癌发生及其表达的研究 将通过检测白蛋白-SV-40标签转基因的 利用亚硫酸氢盐反应实现胞嘧啶甲基化及其测序 可能涉及的地区。印迹蛋白的甲基化及其表达 1号染色体重复区或接近重复区的基因将通过 这项技术和在被判断为等位基因杂合性的动物中 在这些座位上进行RFLP分析。从这些研究中，我们希望开发出一种 细胞转化的形态、核型和分子理解 大鼠肝细胞从促进期到进展期 肝癌的发生。

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.