CARCINOGEN METABOLIZING P450S--REGULATION BY CYTOKINES

描述：细胞因子可能是P450的生理调节剂。 水平，影响许多P450亚家族的成员。其中的许多， 包括多环芳香烃诱导的P450、P450 1A1和P450 1A2，代谢致癌物，促进其 解毒和/或激活。据报道，IL-1和过氧化氢已被 抑制P450 1A1和P450 1A2的转录激活 多环化合物。这种抑制可以通过提高 细胞内谷胱甘肽水平。细胞因子水平可能会有很大的差异 在不同的组织和器官内，也系统地 个体，由于特定的生理状态或遗传因素 (急性或慢性炎症或感染、吸烟、饮食、 免疫反应性等)。工作假说是， 细胞因子水平或P450基因对 细胞因子可能不同地影响致癌物的表达- 代谢P450，是导致 基因和/或环境决定的差异 个人对致癌物的易感性。两种DNA元素 据报道参与了IL-1和IL-1的作用 H_2O_2：(I)XRE，介导对芳基反应的元素 以及(Ii)一种名为IL-1反应元件的新元件， 伊尔雷。这个元素结合了一个因子ILRP，它迅速下降- 在用IL-1或H_2O_2处理的分离的肝细胞中进行调节。 这项研究检验的具体假设是，IL-1 依赖过氧化氢对P450 1A1转录的抑制作用 通过抑制ILREs与XREs的协作性和 由于ILRP结合活性下调而导致的其他增强剂，以及 通过抑制XRE之间的协作性。具体目标包括： 1.确定ILRE和XRE向下调节的机制- IL-1和H_2O_2对P450_1A1转录的调控DNA介导的 含不同种类基因的表达载体的基因转移 XRE、ILRE和其他增强剂的组合将用于 确定这些要素如何协作，以协调对 IL-1和H_2O_2。在分离的大鼠肝细胞中。2.序列特征 特异性和DNA-蛋白质相互作用对IL-1和H_2O_2很重要。 体外足迹和体内足迹联合应用的响应性 通过对野生型和野生型的凝胶迁移率分析 突变的ILRE。ILRP的组织分布及其调控 氧化剂、抗氧化剂、生长剂对ILRP结合活性的影响 因子、胰岛素、钒酸盐和其他因子将使用 电泳迁移率改变分析。3.克隆ILRP基因 表达克隆、斑块杂交或基于抗体的 程序。

DESCRIPTION: Cytokines may act as physiological modulators of P450 levels, influencing members of many P450 subfamilies. Many of these, including the polycyclic aromatic hydrocarbon-inducible P450s, P450 1A1 and P450 1A2, metabolize carcinogens, contributing to their detoxification and/or activation. IL-1 and H2O2 have been reported to inhibit the transcriptional activation of P450 1A1 and P450 1A2 by polycyclic compounds. This inhibition can be blocked by raising cellular glutathione levels. Cytokine levels can vary dramatically within different tissues and organs and also systemically between individuals, due to particular physiological states or genetic factors (acute or chronic inflammation or infection, cigarette smoking, diet, immunoreactivity, etc.). The working hypothesis is that differences in cytokine levels or differences in the responsiveness of P450 genes to cytokines may differentially affect the expression of carcinogen- metabolizing P450s and be an important factor contributing to genetically and/or environmentally determined differences among individuals in their susceptibility to carcinogens. Two DNA elements have been reported to be involved in mediating the effect of IL-1-and H2O2: (i) XREs, the elements that mediate responsiveness to aryl hydrocarbons, and (ii) a novel element termed IL-1 responsive element, ILRE. This element binds a factor, ILRP, that is rapidly down- regulated in isolated hepatocytes treated with either IL-1 or H2O2.. The specific hypothesis being tested by this research is that the IL-1 and H2O2-dependent suppression of P450 1A1 transcription is mediated by suppression of cooperativity of ILREs with XREs and other enhancers due to down regulation of ILRP binding activity, and by suppression of cooperativity between XREs. Specific objectives are: 1. Determine the mechanism by which ILREs and XREs mediate down- regulation of P450 1A1 transcription by IL-1 and H2O2.. DNA-mediated gene transfer of expression vectors containing various combinations of XREs, ILREs and other enhancers will be done to determine how these elements cooperate to mediate responsiveness to IL-1 and H2O2. in isolated rat hepatocytes. 2. Characterize sequence specificity and DNA-protein interactions important for IL-1 and H2O2. responsiveness using in vitro and in vivo footprinting in association with electrophoretic mobility shift analyses with wild type and mutant ILREs. The tissue distribution of the ILRP and the regulation of ILRP binding activity by oxidants, antioxidants, growth factors, insulin, vanadate, and other factors will be determined using electrophoretic mobility shift assays. 3. Clone ILRP cDNA using either expression cloning, plaque hybridization or antibody based procedures.