REGULATION OF AFP GENE EXPRESSION IN HEPATOMA CELLS IN VITRO AND IN VIVO

这项研究的长期目标是了解 发育和肿瘤过程中的差异基因表达； 甲胎蛋白基因在肝细胞中的表达/抑制机制我们 将继续致力于这样一种假设，即这种监管是由 体液因子与转录因子的相互作用。我们也是 会问正常的发育建立的控制机制 在肿瘤转化过程中会发生改变。 我们建议对AFP的诱导机制进行定义 在正常成人肝细胞和微小异常肝细胞中的表达 肝癌细胞，或-HEPA，在本实验室建立。成年C，H小鼠将成为 用250万个细胞接种IP以产生腹水形式的 肿瘤。两周后，给宿主小鼠注射L-乙硫氨酸 (0.5杯/体重)在成人肝脏中被发现能诱导甲胎蛋白。这项实验 我们在早期研究中开发的模型允许对 同一实验条件下对正常细胞和转化细胞的影响 条件。甲胎蛋白在宿主肝和腹水中的表达 24-96小时后的细胞将与其他肝脏的表达相关 放射免疫化学和斑点/Northern杂交检测基因(Trf和Alb)。 核连续分析将被用来评估转录水平 包括癌基因(c-fos、c-Ha-ras、c-jun)和生长的一组基因 因子(IGF-II、转化生长因子-β、转化生长因子-α)。此外，核蛋白来自 这两个组织将通过与AFP微基因结合，然后进行凝胶检查 移位机动性和DNaseI保护分析检测特定的反式作用 各种因素。用于比较与胎儿核蛋白类似的实验 都会完成的。 使用动物模型更好地了解基本的细胞机制 与人类健康问题相关的是一种被广泛接受的实验方法。 通过这次调查获得的信息将有助于澄清 癌细胞和其他细胞/遗传学的病因和特征 人类疾病，因此，具有潜在的治疗意义 目的。 该项目的另一个主要目标是继续为学生提供 在移动和通信领域的重要领域的最新培训和指导 生物医学科学的分子生物学。

The long term goal of this research is to understand the regulation of differential gene expression during development and neoplasia; specifically the expression/repression mechanism of the AFP gene in liver cells. We will continue working on the hypothesis that this regulation is mediated by the interaction of humoral factors with transcription factors. Also we will ask whether the normal developmentally established control mechanism is altered during neoplastic transformation. We propose to define the mechanism involved in the induction of AFP expression in normal adult liver cells and in the minimally deviant hepatoma cells, OR-HEPA established in this lab. Adult C,H mice will be inoculated IP with 2.5 million cells to produce the ascites form of this tumor. After two weeks, host mice will be injected with L-ethionine (0.5mug/body weight) found to induce AFP in adult liver. This experimental model developed in our earlier studies allows comparative analysis of the effects on normal and transformed cells under the same experimental condition. The expression of AFP in both the host liver and the ascites cells, after 24-96 hr, will be correlated to expression of other liver genes (Trf and Alb) by radio-immunochemistry and dot/northern blots. Nuclear run-on assays will be used to assess the transcriptional levels of a battery of genes including oncogenes (c-fos, c-Ha-ras, c-jun) and growth factors (IGF-II, TGF-beta, TGF-alpha). Additionally, nuclear proteins from both tissues will be examined by binding to AFP mini-genes followed by gel shift mobility and DNaseI protection assays to detect specific trans-acting factors. For comparison similar experiments with fetal nuclear proteins will be done. The use of animal models to better understand basic cellular mechanisms related to human health problems is a well accepted experimental approach. The knowledge obtained through this investigation will help to clarify the etiology and characteristics of the cancer cell and other cellular/genetic human diseases and thus, has potential implications for therapeutic purposes. Another main goal of this project is to continue offering the students an up-to-date training and guidance in the important areas of cellular and molecular biology of biomedical sciences.