HEPATOCARCINOGENESIS IN VITRO USING ACTIVATED FOS GENES

如果我们要了解肿瘤的分子病理学， 详细分析了激活后所带来的分子变化 癌基因的研究必须在特征明确的 模型系统 激活的癌基因的表达通常导致 复杂的表型变化，包括锚定非依赖性生长， 致瘤性，并且这些变化中的一些可能涉及 癌基因产物对细胞基因表达的重编程。 最近 研究表明，几种致癌基因通路可能在肿瘤细胞中会聚， 通过特定细胞的转录激活， 基因. 因此，直接分析是合乎逻辑和令人信服的， 在分子水平上，一个特征明确的核癌基因的作用 (i.e., fos）对活性改变的细胞基因表达的影响 在肝癌发生后一直如此。 新生大鼠肝上皮 (RLE)细胞提供了一个有用的体外模型系统， 与肝癌发生相关的分子机制， 用化学致癌物或激活的ras基因转化 导致γ-谷氨酰转肽酶（γ GT）的活化， 谷胱甘肽-S-转移酶-P（GSTP），肝癌发生的两个标志物 in vivo. 我将使用金属调节的金属硫蛋白-c-fos（MTcfos） 融合基因以在培养物中转染RLE细胞，以鉴定任何 “变换的”（即，锚定独立）克隆生长在软 琼脂糖凝胶，并探讨fos转化之间的相关性， 以及γ GT和GSTP的激活;初步结果表明， γ GT的表达而不是GSTP的表达在 通过低水平的MTcfos转化RLE细胞。 利用 金属感应的结构，我将建立最低水平 MTcfos mRNA（北方印迹）和蛋白质（蛋白质印迹） 诱导锚定非依赖性生长、γ GT表达和GSTP 表情 在这些研究的第二阶段，我将分析 详细说明了c-fos基因产物激活的机制 γ GT基因的表达。 我的努力将强调 c-fos的转录作用，因为最近的报告表明， c-fos产物可与DNA上的转录控制位点结合。 的 将使用以下方法：i）在分离的细胞核中进行研究， 验证c-fos的转录作用; ii）用5 '- 与CAT（氯霉素）融合的γ GT缺失突变体 乙酰转移酶）基因来鉴定γ GT的fos依赖性区域 iii）基因组γ GT基因的无细胞转录， 确定FOS所需的活性的任何细胞因子- 依赖性激活

If we are ever to understand the molecular pathology of neoplasia, a detailed analysis of the molecular changes brought about by activated oncogenes must be undertaken within the context of well-characterized model systems. Expression of activated oncogenes usually results in complex phenotypic changes including anchorage-independent growth and tumorigenicity, and it is likely that some of these changes involve a reprogramming of cellular gene expression by oncogene products. Recent studies suggest that several oncogene pathways may converge in the nucleus through the transcriptional activation of specific cellular genes. It is therefore logical and compelling to analyze directly, at the molecular level, the effects of a well-characterized nuclear oncogene (i.e., fos) on the expression of cellular genes whose activity changes consistently after hepatocarcinogenesis. Newborn-rat liver epithelial (RLE) cells provide a useful in vitro model system in which to study molecular mechanisms associated with hepatocarcinogenesis, since transformation with either chemical carcinogens or an activated ras gene results in the activation of gamma-glutamyl transpeptidase (gamma GT) and glutathione-S-transferase-P (GSTP), two markers of liver carcinogenesis in vivo. I will use a metal-regulatable, metallothionein-c-fos (MTcfos) fusion gene to transfect RLE cells in culture, to identify any "transformed" (i.e., anchorage-independent) clones by growth in soft agarose, and to investigate the correlation between fos-transformation and the activation of gamma GT and GSTP; preliminary results indicate that expression of gamma GT but not GSTP is activated after transformation of RLE cells by low levels of MTcfos. Utilizing the metal-inducibility of the constructs, I will establish the minimum levels of MTcfos mRNA (northern blots) and protein (western blots) necessary for induction of anchorage independent growth, gamma GT expression, and GSTP expression. During the second phase of these studies, I will analyze in detail the mechanisms by which the c-fos gene product activates expression of the gamma GT gene. My efforts will emphasize transcriptional effects of c-fos, since recent reports suggest that the c-fos product may bind to transcriptional control sites on DNA. The following approaches will be used: i) studies in isolated nuclei to verify transcriptional effects of c-fos; ii) transfection with 5'- deletion mutants of gamma GT fused to the CAT (chloramphenicol acetyltransferase) gene to identify fos-dependent regions of the gamma GT gene; and iii) cell-free transcription of the genomic gamma GT gene to identify any cellular factors whose activity is required for fos- dependent activation.