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：
7303189
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/7303189
关键词：
4-Hydroxy-Tamoxifen 4-OH-E(2)4-hydroxy-equilenin Affinity Chromatography Agar Alkylation Alzheimer&apos s Disease Antibodies Appendix 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 Nuclear Extract Nude Mice Oligonucleotides Organ Culture Techniques Osteoporosis Oxidation-Reduction Parents Pharmaceutical Preparations Phase Phenols Phenotype Play Point Mutation Polymerase Chain Reaction Positioning Attribute Postmenopause Precipitation Process Progestins Property Prospective Studies Proteins Pulmonary Embolism Quinones Raloxifene Range Rattus Reactive Oxygen Species Reporting Reproductive History Research Personnel Response Elements Risk Role Selective Estrogen Receptor Modulators Site Specificity Stroke Subcellular Fractions Tetrachlorodibenzodioxin Thinking Uncertainty Upper arm Vascular Dementia Wild Type Mouse Woman Women&apos s Health benzoquinone cancer risk carcinogenesis cell transformation cellular targeting in vivo in vivo Model insight malignant breast neoplasm metaplastic cell transformation novel oxidation 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（通过施用）：屁股在雌激素中暴露于雌激素（乳房，子宫内膜）。 ERS）在雌激素O-喹酮 - 碘酮诱导的DNA损伤中起主要作用。损坏我们将首先研究用MALDI-TOF和LC-MS实验对雌激素O-Quinones纯化的纯化。移动测定。与整个基因组DNA相比，分离的雌激素敏感基因将通过LC-MS-MS进行ION或使用PCR的点突变和随后的DNA测序。 /或氧化还原敏感的解毒酶？ 7个细胞将使用2D电泳分离的Avidin亲和色谱分离，LC -MS -MS。 -10a细胞帽子是负面的，eera或er¿阳性的克隆将植入小裸体，以研究其雌激素诱导的DNA损伤的能力与以目标N顺序描述的损伤实验相关，以整体描述雌激素致癌作用的反应。