Role of electrophilic/redox active quinoids in estrogen carcinogenesis

描述（由申请人提供）：过度暴露于雌激素与激素敏感组织（乳腺、子宫内膜）中癌症的发生之间存在明显的相关性。该项目的中心假设是，亲电/氧化还原活性醌类化合物的形成是雌激素致癌的重要机制。邻醌是已知的雌激素代谢物。我们的数据有力地表明，雌激素受体（ER）在雌激素邻醌诱导的DNA损伤中起着重要作用。雌激素受体通过邻醌的选择性烷基化产生高度氧化还原活性的“特洛伊木马”，其选择性靶向雌激素敏感基因。具体目标是：1.雌激素受体在儿茶酚雌激素诱导的DNA损伤中的作用是什么？我们将首先利用MALDI-TOF和LC-MS-MS实验研究雌激素邻醌对纯化的ER的修饰。将通过凝胶位移试验研究ER的邻醌/邻苯二酚结合/烷基化对与ERE结合或对ERE寡核苷酸序列的氧化DNA链切割的影响。在ER阳性MCF-7细胞中，通过ERE-荧光素酶试验检查ER和ERE之间可能的雌激素效应或功能干扰。最后，我们将在用雌激素邻醌处理的乳腺癌细胞中用ER抗体进行染色质免疫沉淀测定，以分析与全基因组DNA相比雌激素敏感基因的DNA损伤。将通过LC-MS-MS研究分离的雌激素敏感基因的氧化/烷基化，或通过PCR扩增和随后的DNA测序研究点突变。2.儿茶酚雌激素的蛋白质靶点是什么？对ER、ER辅调节剂、组蛋白3和/或氧化还原敏感解毒酶的选择性？我们计划采用新的COATag（隐蔽氧化激活标签）方法（即，雌激素或连接到生物素的儿茶酚代谢物）和“点击化学”或改良的Staudinger连接方法来检查大鼠乳腺亚细胞级分和MCF-7细胞中潜在的蛋白质共价修饰。采用亲和素亲和层析法分离目的蛋白，二维电泳分离，酶切，MALDI-TOF和LC-MS-MS分析。ER在体内细胞转化和诱导DNA损伤中的作用是什么？将在ER阴性、ER α或ER β阳性的MCF-10A细胞中进行转化研究。将转化的克隆植入无胸腺裸鼠中以研究其诱导肿瘤形成的能力。ER对儿茶酚雌激素诱导的DNA损伤的作用将在小鼠乳腺器官培养（MMOC）模型中和使用野生型和ER敲除小鼠的体内进行评估。这些数据将与目标1中描述的DNA损伤实验和目标2中确定的蛋白质靶点相关，以便全面了解ER参与雌激素致癌作用的情况。

DESCRIPTION (provided by applicant): There is a clear association between excessive exposure to estrogens and the development of cancer in hormone sensitive tissues (breast, endometrium). The central hypothesis of this project is that the formation of electrophilic/redox active quinoids is an important mechanism of carcinogenesis for estrogens. o-Quinones are known metabolites of estrogens. Our data strongly suggests that estrogen receptors (ERs) play a major role in estrogen o-quinone-induced DNA damage. Selective alkylation of ERs by the o- quinones generates a highly redox active "Trojan horse" which selectively targets estrogen sensitive genes. The specific aims are: 1. What is the role of ERs in catechol estrogen induced DNA damage? We will first investigate modification of purified ERs by estrogen o-quinones with MALDI-TOF and LC-MS-MS experiments. The influence of o-quinone/catechol binding/alkylation of ER on binding to the ERE or on oxidative DNA strand cleavage of ERE oligonucleotide sequences will be studied by gel shift assays. A possible estrogenic effect or functional perturbations between ER and ERE will be examined by ERE-luciferase assays in ER positive MCF-7 cells. Finally, we will conduct chromatin immuno-precipitation assays with ER antibodies in breast cancer cells treated with estrogen o-quinones to analyze DNA damage of estrogen sensitive genes compared to the whole genomic DNA. The isolated estrogen sensitive genes will be investigated for oxidative/alkylation by LC-MS-MS or for point mutation by amplification with PCR and subsequent DNA sequencing. 2. What are the protein targets of catechol estrogens? Selectivity for ERs, ER coregulators, histone 3 and/or redox sensitive detoxification enzymes? We plan to employ novel COATag (covert oxidized activated tag) methodology (i.e., estrogens or catechol metabolites linked to biotin) and the "click chemistry" or modified Staudinger ligation approaches to examine potential protein covalent modification in rat mammary subcellular fractions and MCF-7 cells. The targeted proteins will be isolated using avidin affinity chromatography, separated by 2D electrophoresis, digested, and analyzed by MALDI-TOF and LC-MS-MS. 3. What is the role of ERs in cellular transformation and induction of DNA damage in vivo? Transformation studies will be performed in MCF-10A cells that are either ER negative, ERa, or ER¿ positive. The transformed clones will be implanted into athymic nude mice to investigate their ability to induce tumor formation. The role of ERs on catechol estrogen-induced DNA damage will be assessed in the mouse mammary organ culture (MMOC) model and in vivo using wild type and ER knockout mice. These data will be correlated with the DNA damage experiments described in Aim 1 and the protein targets identified in Aim 2 in order to give an overall picture of the involvement of ERs in estrogen carcinogenesis.