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

• 批准号: 8072619
• 负责人: Judy L Bolton
• 金额: $ 28.26万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-31 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072619
• 关键词: 4-Hydroxy-Tamoxifen; 4-OH-E(2); 4-hydroxy-equilenin; Affinity Chromatography; Agar; Alkylation; Alzheimer' 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; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stroke; Subcellular Fractions; Tetrachlorodibenzodioxin; Uncertainty; Vascular Dementia; Wild Type Mouse; Woman; Women' 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; success; tumor; tumorigenic

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.

说明(申请人提供)：雌激素过量暴露与荷尔蒙敏感组织(乳腺、子宫内膜)癌症的发展有明显的关联。该项目的中心假设是，亲电活性/氧化还原活性藜麦的形成是雌激素致癌的重要机制。邻喹酮类化合物是雌激素的已知代谢产物。我们的数据有力地表明，雌激素受体(ER)在雌激素邻苯二酚诱导的DNA损伤中起着重要作用。邻苯二酚选择性烷基化内质网会产生一种高度氧化还原活性的“特洛伊木马”，它选择性地针对雌激素敏感基因。其具体目的是：1.内质网在儿茶酚类雌激素诱导的DNA损伤中起什么作用？我们将首先用MALDI-TOF和LC-MS-MS实验研究雌激素邻苯二酚对纯化的内质网的修饰。通过凝胶移位分析研究ER的邻苯二酚/邻苯二酚结合/烷基化对ERE结合或对ERE寡核苷酸序列氧化DNA链断裂的影响。ER阳性的MCF-7细胞中，ER和ERE之间可能的雌激素效应或功能紊乱将通过ERE-荧光素酶分析来检验。最后，我们将用雌激素邻苯二酚处理的乳腺癌细胞中的ER抗体进行染色质免疫沉淀分析，以对比整个基因组DNA来分析雌激素敏感基因的DNA损伤。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，或者通过PCR扩增和随后的DNA测序来研究点突变。2.儿茶酚雌激素的蛋白质靶标是什么？对内质网、内质网共调节因子、组蛋白3和/或氧化还原敏感解毒酶的选择性？我们计划使用新的COATAG(隐蔽氧化激活标签)方法(即雌激素或生物素连接的儿茶酚代谢物)和“点击化学”或改进的Staudinger连接方法来研究大鼠乳腺亚细胞部分和MCF-7细胞中潜在的蛋白质共价修饰。目的蛋白用亲和层析法分离，2D电泳法分离，酶切后用MALDI-TOF和LC-MS-MS进行分析。3.内质网在体内细胞转化和DNA损伤中的作用是什么？转化研究将在ER阴性、ERA或ER阳性的MCF-10A细胞中进行。转化后的克隆将被移植到裸鼠体内，以研究它们诱导肿瘤形成的能力。将在小鼠乳腺器官培养(MMOC)模型中以及在体内使用野生型和ER基因敲除小鼠来评估ERs对儿茶酚雌激素诱导的DNA损伤的作用。这些数据将与AIM 1中描述的DNA损伤实验和AIM 2中确定的蛋白质靶标相关联，以便提供ER参与雌激素致癌的总体情况。

项目成果

Redox cycling of catechol estrogens generating apurinic/apyrimidinic sites and 8-oxo-deoxyguanosine via reactive oxygen species differentiates equine and human estrogens.

儿茶酚雌激素的氧化还原循环通过活性氧产生无嘌呤/无嘧啶位点和 8-氧代-脱氧鸟苷，从而区分马和人类雌激素。

• DOI: 10.1021/tx1001282
• 发表时间: 2010
• 期刊: Chemical research in toxicology
• 影响因子: 4.1
• 作者: Wang,Zhican;Chandrasena,EsalaR;Yuan,Yang;Peng,Kuan-wei;vanBreemen,RichardB;Thatcher,GregoryRJ;Bolton,JudyL
• 通讯作者: Bolton,JudyL