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：
8037167
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/8037167
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 Vascular Dementia Wild Type Mouse Woman Women&apos s Health arm 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（通过应用提供）：在逃脱过度暴露与同性敏感组织（乳腺，子宫内膜）中的癌症发展之间存在明显的关联。该项目的中心假设是，亲电/氧化还原活性奎因的形成是逃脱的癌变的重要机制。 O- Quinones是已知的逃生代谢产物。我们的数据强烈表明，雌激素受体（ERS）在雌激素O-夸酮诱导的DNA损伤中起主要作用。 O-Quinones对ERS的选择性烷基化产生了高度氧化还原活性的“ Trojan马”，该马有选择地靶向雌激素敏感基因。具体目的是：1。ER在Catechol雌激素诱导的DNA损伤中的作用是什么？我们将首先通过使用MALDI-TOF和LC-MS-MS实验来研究通过雌激素O- Quinones对纯化ER的修饰。 ER的O-喹酮/儿茶素结合/烷基化对ERE寡核苷酸序列的结合或对ERE的DNA清洁的影响，将通过凝胶移位分析研究。 ER和ERE之间可能的雌激素作用或功能扰动将通过ER荧光酶测定在ER阳性MCF-7细胞中检查。最后，我们将在用雌激素O-奎因酮处理的乳腺癌细胞中进行染色质免疫沉淀测定法，以分析与整个基因组DNA相比，分析雌激素敏感基因的DNA损伤。将研究分离的雌激素敏感基因通过LC-MS-MS进行氧化/烷基化，或通过使用PCR扩增和随后的DNA测序来进行点突变。 2。儿茶酚雌激素的蛋白质靶标是什么？ ERS，ER组合剂，组蛋白3和/或氧化还原敏感解毒酶的选择性？我们计划采用新颖的外套（秘密氧化活化的TAG）方法（即链接到生物素的雌激素或儿茶酚代谢产物）和“点击化学”或改性的Staudinger连接方法，以检查大鼠乳腺乳细胞亚细胞骨骼和MCF-7细胞中潜在的蛋白质共价修饰。靶向蛋白将使用Avidin亲和色谱分离，并由2D电泳分离，并被MALDI-TOF和LC-MS-MS消化和分析。 3。ER在体内DNA损伤的细胞转化和诱导中的作用是什么？转化研究将在ER负，ERA或ER阳性的MCF-10A细胞中进行。转化后的克隆将植入无胸腺裸鼠，以研究其诱导肿瘤形成的能力。 ERS在小鼠乳腺器官培养（MMOC）模型和使用野生型和ER基因敲除小鼠中评估ERS在雌激素诱导的DNA损伤中的作用将在小鼠乳腺器官培养（MMOC）模型中评估。这些数据将与AIM 1中描述的DNA损伤实验以及AIM 2中鉴定的蛋白质靶标有关，以便整体描述ERS参与雌激素致癌作用。