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 我们计划在解决这个问题时使用的方法 （1）检测铬（VI）对细胞增殖的影响 基因表达在14天的鸡胚，一个简单的明确的体内 （2）比较铬（VI）对模型基因的影响， 具有不同类型的一般和特异性调节元件（GC- 盒，糖皮质激素反应元件（GRES），cAMP反应性 元件和金属响应元件（MRE），以及各自的特定 转录因子（AP 1、SP1、糖皮质激素受体、cAMP结合 蛋白质和金属活化因子）：和（3）确定 铬（VI）对转录因子与其 识别序列 铬（VI）化合物已被证明 优先攻击特定的DNA序列， 影响可诱导基因与不可诱导基因的转录。 因此，我们计划测试铬（VI）诱导的DNA 损伤通过改变基因表达， 转录因子与基因的启动子区域。 具体 我们建议测试铬（VI）干扰 调控区域内调节蛋白的正常相互作用 而不是其他组成型基因。 的 本研究的具体目标是：（1）铬对植物生长的影响 （VI）诱导的DNA损伤对组成性和诱导性稳态的影响 金属硫蛋白的mRNA水平和转录率， 磷酸烯醇丙酮酸羧激酶和白蛋白基因将被确定 在体内用铬（VI）处理的鸡胚肝脏中。 的 铬（VI）处理对基础和诱导稳定的影响， 这些基因表达的状态水平将通过溶液 杂交和北方印迹分析。 铬（VI）的影响 这些基因的基础和诱导转录率将是 通过核“流出”转录测定确定。 (2)的影响 铬（VI）诱导的DNA损伤对转录的相互作用 与金属硫蛋白的启动子区域的因子， 磷酸烯醇丙酮酸羧激酶和白蛋白基因将被确定 在体内用铬（VI）处理的14日龄鸡胚的肝脏中。 体内DNA-蛋白质相互作用将使用连接测定。 介导的基于聚合酶链反应（PCR）的基因组足迹法 分析这些基因的启动子区域。 远程 本研究项目的目的是通过以下方式了解其机制： 其中铬（VI）化合物作为致癌物。 拟议的研究 应提供证据证明铬（VI）对 基因的重要调控区域，并阐明对 DNA结构和功能，从而提供了深入了解的机制， 铬（VI）引发癌症。 这反过来又可以提供 基本见解的整体机制启动的 遗传毒性致癌物引起的肿瘤过程。

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.