REGULATION OF CYTOCHROME P-450 BIOSYNTHESIS

本提案的总体目标是了解分子 经典诱导剂苯巴比妥增加基因表达的机制 细胞色素P450的表达。 细胞色素P450形成一个超家族， 负责氧化激活或失活的酶 各种内源性和外源性化合物的代谢。 的 许多药剂的最终治疗或毒性活性由以下因素决定： 不同的催化剂催化的活化和失活之间的平衡 细胞色素P450 这种平衡可以通过归纳法来显著改变 这些酶的子集。 本建议的具体目标是确定顺式和 兔体内介导苯巴比妥诱导的反式作用因子 CYP 2C亚家族。 该亚家族包含至少8个基因， 不同的组织特异性表达， 对苯巴比妥的反应 缺乏合适的连续细胞 培养模型系统阻止了对苯巴比妥的分析， 基因中的响应元件及其相关转录 因素 为了避免这个问题，转录活性的 通过引入CYP 2C启动子评估CYP 2C基因- 荧光素酶杂合基因导入转基因小鼠和大鼠肝细胞， 原代培养 荧光素酶活性的检测是一种简单、快速， 启动子活性的灵敏测定。 转基因小鼠提供了一种 肝脏苯巴比妥调节机制完整的体内模型 并且其中调节元件的活动通常更多 比在细胞培养测定中更真实。 一次转基因株系表达 已经建立了CYP 2C-荧光素酶杂合基因， CYP 2C基因表达和苯巴比妥的个体发育 将研究诱导。 大鼠肝细胞接种在基质胶上， 显示维持苯巴比妥诱导表达的条件 将用CYP 2C杂合基因转染内源性CYP 2C基因。 调控元件将通过以下方式中的缺失和点突变来定义： CYP 2C基因序列。 蛋白质与苯巴比妥的结合- 将通过凝胶阻滞和DNA酶I研究响应元件 足迹分析和这些蛋白质将被分离或其cDNA 将被克隆。 在使用类似技术的研究中，基底和组织- 将通过转染来研究特定的调节元件和蛋白质 或者将杂交基因导入HepG 2和肾细胞。 这些研究应该 导致对基础的，组织特异性的， 和苯巴比妥诱导的CYP 2C基因表达， 分子水平。

The overall goal of this proposal is to understand the molecular mechanisms by which the classical inducer, phenobarbital, increases gene expression of cytochromes P450. Cytochromes P450 form a superfamily of enzymes responsible for activation or inactivation by oxidative metabolism of a wide variety of endogenous and foreign compounds. The ultimate therapeutic or toxic activity of many agents is determined by the balance between activation and inactivation catalyzed by different cytochromes P450. This balance can be dramatically altered by induction of subsets of these enzymes. The specific objectives of this proposal are to identify the cis-and trans-acting factors that mediate phenobarbital induction in the rabbit CYP2C subfamily. This subfamily contains at least 8 genes which exhibit different tissue-specific expression and have strikingly different responses to phenobarbital. The lack of a suitable continuous cell culture model system has prevented analysis of the phenobarbital- responsive elements in the genes and their associated transcription factors. To circumvent this problem, the transcriptional activity of CYP2C genes will be assessed by the introduction of CYP2C promoter - luciferase hybrid genes into transgenic mice and rat hepatocytes in primary culture. Detection of luciferase activity is a simple, rapid, sensitive assay for promoter activity. Transgenic mice provide an in vivo model in which liver phenobarbital regulatory mechanisms are intact and in which the activities of regulatory elements are often more authentic than in cell culture assays. Once transgenic lines expressing the hybrid CYP2C-luciferase genes have been established, tissue-specific expression and the ontogeny of CYP2C gene expression and phenobarbital induction will be studied. Rat hepatocytes plated on matrigel, under conditions shown to maintain phenobarbital-inducible expression of endogenous CYP genes, will be transfected with the CYP2C hybrid genes. Regulatory elements will be defined by deletion and point mutations in the CYP2C gene sequences. Binding of proteins to phenobarbital- responsive elements will be studied by gel retardation and DNase I footprint analyses and these proteins will be isolated or their cDNAs will be cloned. In studies using similar techniques, basal and tissue- specific regulatory elements and proteins will be studied by transfection or the hybrid genes into HepG2 and kidney cells. These studied should result in a comprehensive understanding of the basal, tissue-specific, and phenobarbital-inducible expression of the CYP2C genes at the molecular level.