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

点击翻译按钮获取中文摘要

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.