EFFECT OF CHROMIUM ON GENE EXPRESSION

本研究项目的总体目标是了解 致癌铬(VI)化合物影响基因的机制 表情。我们计划用来解决这个问题的方法 主要有：(1)考查铬(VI)的差异性影响能力 基因在14日龄鸡胚胎中的表达，一种简单而明确的体内表达 (2)比较铬(VI)对模型基因的影响。 具有不同类型的一般和特定监管要素(GC- 糖皮质激素反应元件(GRE)盒，cAMP反应元件 元素和金属响应元素(MRE)，以及各自特定的 转录因子(AP1、SP1、糖皮质激素受体、cAMP结合 蛋白质和金属激活因子)：和(3)决定 铬(VI)对转录因子与转录因子正常结合的影响 识别序列。铬(VI)化合物已被证明是 优先攻击特定的DNA序列并区分 影响可诱导基因与非诱导基因的转录。 因此，我们计划测试铬(VI)诱导DNA的假设 损伤通过改变相互作用来影响基因的表达 带有基因启动子区域的转录因子。特指 我们建议检验铬(VI)干扰 调控蛋白在控制区内的正常相互作用 可诱导基因，而不是其他构成基因。这个 拟议研究的具体目标是：(1)铬的影响 (Vi)诱导性DNA损伤对构成态和诱导态的影响 金属硫蛋白的mRNA水平和转录速率， 将确定磷酸烯醇式丙酮酸羧酸激酶和白蛋白基因 在体内用铬(VI)处理的鸡胚的肝脏中。这个 铬(VI)处理对基础和诱导稳定性的影响 这些基因的状态表达水平将由解决方案确定 杂交和Northern杂交分析。铬(VI)的影响 这些基因的基础和诱导转录速率将是 由核“径流”转录分析确定。(2) 铬(VI)诱导DNA损伤对转录相互作用的影响 影响金属硫蛋白启动子区域的因素， 将确定磷酸烯醇式丙酮酸羧酸激酶和白蛋白基因 在体内用铬(VI)处理14日龄雏鸡胚胎的肝脏。 体内的DNA-蛋白质相互作用将通过连接- 基于介导的聚合酶链式反应的基因组足迹 分析这些基因的启动子区域。长距离的 本研究项目的目的是通过以下方式了解其作用机制 哪些铬(VI)化合物具有致癌作用。建议进行的研究 应为铬(VI)对环境的不同影响提供证据 基因的重要调控区，并阐明对 DNA结构和功能，从而提供了对机制的洞察 铬(VI)通过这种物质引发癌症。这反过来又可以提供 对启动的总体机制的基本见解 遗传毒性致癌物的致癌过程。

The overall objective of this research project is to understand the mechanism by which carcinogenic chromium(VI) compounds affect gene expression. The approaches we plan to use in attacking this problem are: (1) examine the ability of chromium (VI to differentially affect gene expression in 14 day chick embryos, a simple well-defined in vivo system; (2) compare the effects of chromium (VI) on model genes which have different types of general and specific regulatory elements (GC- boxes, glucocorticoid responsive elements (GREs), cAMP-responsive elements, and metal responsive elements (MREs), and respective specific transcription factors (AP1,SP1, glucocorticoid receptor, cAMP-binding protein, and metal-activated factors): and (3) determine the effect of chromium (VI) on the normal binding of transcription factors to their recognition sequences. Chromium (VI) compounds have been shown to preferentially attack specific DNA sequences and to differentially affect the transcription of inducible vs. non-inducible genes. Therefore, we plan to test the hypothesis that chromium (VI)-induced DNA damage affects gene expression by altering the interaction of transcription factors with promoter regions of the gene. Specifically we propose to test the hypothesis that chromium (VI) interferes with the normal interaction of regulatory protein within the controlling regions of inducible genes and not those of other constitutive genes. The specific aims of the proposed research are: (1) The effects of chromium (VI)-induced DNA damage on the constitutive and inducible steady-state mRNA levels and transcription rates of metallothionein, phosphoenolpyruvate carboxykinase, and albumin genes will be determined in the liver of chick embryos treated with chromium (VI) in vivo. The effects of chromium (VI) treatment on the basal and inducible steady- state levels of expression of these genes will be determined by solution hybridization and Northern blot analyses. The effects of chromium (VI) on the basal and inducible rates of transcription of these genes will be determined by nuclear "run-off" transcription assays. (2) The effect of chromium (VI)-induced DNA damage on the interaction of transcription factors with the promoter regions of the metallothionein, phosphoenolpyruvate carboxykinase, and albumin genes will be determined in the liver of 14 day chick embryos treated with chromium (VI) in vivo. The DNA-protein interactions in vivo will be determined using ligation- mediated polymerase chain reaction (PCR)-based genomic footprinting analysis of the promoter regions of these genes. The long-range objective of this research project is to understand the mechanism by which chromium (VI) compounds act as carcinogens. The proposed studies should provide evidence for differential effects of chromium (VI) on important regulatory regions of genes and elucidate critical effects on DNA structure and function, and thus, provide insight into the mechanism by which chromium (VI) initiates cancer. This, in turn, could provide fundamental insights into the overall mechanism of initiation of the neoplastic process by genotoxic carcinogens.