REGULATION OF CYTOCHROME P-450 BIOSYNTHESIS

这项提议的总体目标是理解分子 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 多种内源和外源化合物的代谢。这个 许多药物的最终治疗或毒性活性由以下因素决定 不同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来显著改变。 这些酶的亚群。 这项建议的具体目标是确定独联体和 介导兔苯巴比妥诱导的反式作用因子 细胞色素P450 2 C亚家族。这个亚家族包含至少8个基因，这些基因表现出 不同的组织特异性表达和有显著不同 对苯巴比妥的反应。缺乏合适的连续电池 培养模型系统阻止了对苯巴比妥的分析- 基因中的反应元件及其相关转录 各种因素。为了绕过这个问题，转录活性 将通过引入CYP2C启动子来评估CYP2C基因。 荧光素酶杂合基因导入转基因小鼠和大鼠肝细胞 原生文化。荧光素酶活性的检测是一种简单、快速、 启动子活性的灵敏分析。转基因小鼠提供了一种 肝脏苯巴比妥调节机制完整的活体模型 其中监管要素的活动往往更多 比细胞培养检测结果更可靠。曾经表达过的转基因株系 已建立具有组织特异性的杂合细胞色素C-荧光素酶基因 CYP2C基因表达与苯巴比妥的个体发育 将学习归纳法。大鼠肝细胞接种于基质材料上，下 维持苯巴比妥诱导表达的条件 内源性CYP基因，将其与CYP2C杂合基因一起导入。 将通过删除和点突变来定义调控元素 细胞色素P450 2 C基因序列。蛋白质与苯巴比妥的结合- 响应元件将通过凝胶延迟和DNase I进行研究 足迹分析和这些蛋白质将被分离或它们的cDNA 将被克隆。在使用类似技术的研究中，基底和组织- 特定的调节元件和蛋白质将通过转基因进行研究。 或者将杂交基因植入HepG2和肾脏细胞。这些研究应该 导致对基础的、特定于组织的、 和苯巴比妥诱导的细胞色素P450 2 C基因的表达 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.