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Role of electrophilic/redox active quinoids in estrogen carcinogenesis

亲电/氧化还原活性醌类化合物在雌激素致癌作用中的作用

基本信息

批准号：
7786288
负责人：
Judy L Bolton
金额：
$ 29.14万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-31 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7786288
关键词：
4-Hydroxy-Tamoxifen 4-OH-E(2)4-hydroxy-equilenin Affinity Chromatography Agar Alkylation Alzheimer&apos s Disease Antibodies Avidin Base Pairing Binding Biological Assay Biology Biotin Breast Breast Cancer Cell Carcinogenesis Mechanism Catechol Estrogens Catechols Cell Line Cells Chemistry Chromatin Complex Coronary heart disease DNA DNA Damage DNA Sequence Data Development Dose Drug Formulations Drug Metabolic Detoxication Electrophoresis Electrophoretic Mobility Shift Assay Endometrium Enzymes Epidemiologic Studies Epithelial Cells Equus caballus Estrogen Receptors Estrogen Replacement Therapy Estrogen Replacements Estrogen receptor negative Estrogen receptor positive Estrogens Exposure to Female Genes Genomics Goals Histones Hormones Hydroxylation Implant In Vitro Incidence Isoenzymes Knockout Mice Laboratories Letters Ligation Link Luciferases MCF7 cell Malignant Neoplasms Mammary Gland Parenchyma Mammary gland Menarche Menopausal Symptom Menopause Methodology Modification Mus National Institute of Environmental Health Sciences Nuclear Extract Nude Mice Oligonucleotides Organ Culture Techniques Osteoporosis Oxidation-Reduction Parents Pharmaceutical Preparations Phase Phenols Phenotype Play Point Mutation Positioning Attribute Postmenopause Precipitation Process Progestins Property Prospective Studies Proteins Pulmonary Embolism Quinones Raloxifene Rattus Reactive Oxygen Species Reporting Reproductive History Research Personnel Response Elements Risk Role Selective Estrogen Receptor Modulators Site Specificity Stroke Subcellular Fractions Tetrachlorodibenzodioxin Uncertainty Upper arm Vascular Dementia Wild Type Mouse Woman Women&apos s Health cancer risk carcinogenesis cell transformation cellular targeting in vivo in vivo Model insight malignant breast neoplasm metaplastic cell transformation novel oxidation oxidative damage programs research study response semiquinone success tumor tumorigenic

项目摘要

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.

描述（由申请人提供）：过量接触雌激素与激素敏感组织（乳腺、子宫内膜）发生癌症之间存在明显的关联。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。邻醌是雌激素的已知代谢物。我们的数据强烈表明雌激素受体 (ER) 在雌激素邻醌诱导的 DNA 损伤中发挥着重要作用。邻醌对 ER 进行选择性烷基化，产生高度氧化还原活性的“特洛伊木马”，选择性地针对雌激素敏感基因。具体目标是： 1. ER 在儿茶酚雌激素诱导的 DNA 损伤中起什么作用？我们将首先通过 MALDI-TOF 和 LC-MS-MS 实验研究雌激素邻醌对纯化 ER 的修饰。 ER 的邻醌/儿茶酚结合/烷基化对与 ERE 结合或对 ERE 寡核苷酸序列的氧化 DNA 链切割的影响将通过凝胶位移测定来研究。将通过 ER 阳性 MCF-7 细胞中的 ERE 荧光素酶测定来检查 ER 和 ERE 之间可能的雌激素效应或功能扰动。最后，我们将在用雌激素邻醌处理的乳腺癌细胞中使用 ER 抗体进行染色质免疫沉淀测定，以分析雌激素敏感基因的 DNA 损伤与整个基因组 DNA 的比较。分离的雌激素敏感基因将通过 LC-MS-MS 进行氧化/烷基化研究，或通过 PCR 扩增和随后的 DNA 测序进行点突变研究。 2. 儿茶酚雌激素的蛋白质靶点是什么？对 ER、ER 共调节因子、组蛋白 3 和/或氧化还原敏感解毒酶的选择性？我们计划采用新型 COATag（隐蔽氧化激活标签）方法（即与生物素连接的雌激素或儿茶酚代谢物）和“点击化学”或改良的施陶丁格连接方法来检查大鼠乳腺亚细胞组分和 MCF-7 细胞中潜在的蛋白质共价修饰。将使用亲和素亲和层析分离靶蛋白，通过 2D 电泳分离、消化并通过 MALDI-TOF 和 LC-MS-MS 进行分析。 3. ER在体内细胞转化和诱导DNA损伤中起什么作用？转化研究将在 ER 阴性、ERa 或 ER¿ 阳性的 MCF-10A 细胞中进行。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) 模型中以及使用野生型和 ER 敲除小鼠进行体内评估。这些数据将与目标 1 中描述的 DNA 损伤实验和目标 2 中确定的蛋白质靶标相关联，以便全面了解 ER 参与雌激素致癌作用的情况。