HEPATOCARCINOGENESIS IN VITRO USING ACTIVATED FOS GENES

如果我们要了解肿瘤的分子病理学，一个 详细分析了激活带来的分子变化 癌基因必须在具有良好特征的 模型系统。活化癌基因的表达通常会导致 复杂的表型变化，包括非锚定生长和 致瘤性，其中一些变化可能涉及一种 癌基因产物对细胞基因表达的重新编程。近期 研究表明，几条癌基因通路可能汇聚在 核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，一个特征明确的核癌基因的影响 (即FOS)对活性变化的细胞基因表达的影响 在肝癌发生后始终如一。新生大鼠肝上皮细胞 (RLE)细胞为研究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，自 用化学致癌物或激活的ras基因转化 结果激活γ-谷氨酰转肽酶(γ-GT)和 肝癌变的两个标志物--谷胱甘肽-S转移酶-P 在活体内。我将使用一种金属可调节的金属硫蛋白-c-fos(MTcfos) 将融合基因导入培养的RLE细胞，鉴定 通过在软体中的生长来“转化”(即独立于锚定的)克隆 琼脂糖凝胶，并研究FOS转化之间的相关性 以及GT和GSTP的激活；初步结果表明 伽马GT而不是GSTP的表达在以下时间被激活 低水平MTcfos对RLE细胞的转化作用利用 金属诱导性的构造，我将建立最低水平 MTcfos mRNA(Northern Blotts)和蛋白质(Western Blots)的表达 诱导非锚定生长、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c-fos基因产物激活的机制 伽马GT基因的表达。我的努力将强调 C-fos的转录效应，因为最近的报告表明 C-fos产物可能与DNA上的转录调控位点结合。这个 将使用以下方法：i)在分离的核中进行研究 验证c-fos的转录效应；ii)5‘- 与猫(氯霉素)融合的γ-GT缺失突变体 乙酰基转移酶)基因用于确定GT的FOS依赖区域 基因；以及iii)基因组γGT基因的无细胞转录 确定FOS所需的任何细胞因子的活性- 依赖激活。

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.