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:
    7491755
  • 负责人:
  • 金额:
    $ 29.14万
  • 依托单位:
  • 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

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):在激素敏感组织(乳房,子宫内膜)中,过度暴露于雌激素与癌症的发展之间存在明确的关联。该项目的中心假设是亲电/氧化还原活性类醌的形成是雌激素致癌的重要机制。邻醌是雌激素的代谢物。我们的数据强烈表明,雌激素受体(er)在雌激素-醌诱导的DNA损伤中起主要作用。o-醌选择性烷基化内质网产生高度氧化还原活性的“特洛伊木马”,选择性靶向雌激素敏感基因。具体目标是:1。内质网在儿茶酚雌激素诱导的DNA损伤中的作用是什么?我们将首先通过MALDI-TOF和LC-MS-MS实验研究雌激素-醌修饰纯化的er。邻醌/儿茶酚结合/ ER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移位试验进行研究。在ER阳性MCF-7细胞中,将通过ER -荧光素酶检测ER和ERE之间可能的雌激素效应或功能扰动。最后,我们将在雌激素-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试验,分析雌激素敏感基因与全基因组DNA的DNA损伤情况。分离的雌激素敏感基因将通过LC-MS-MS进行氧化/烷基化或通过PCR扩增和随后的DNA测序进行点突变研究。2. 儿茶酚类雌激素的蛋白靶点是什么?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?我们计划采用新的COATag(隐蔽氧化活化标签)方法(即雌激素或儿茶酚代谢物与生物素相关联)和“点击化学”或改良的Staudinger连接方法来检测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。目的蛋白将通过亲和层析分离,通过2D电泳分离,消化,并通过MALDI-TOF和LC-MS-MS分析。3. 内质网在体内细胞转化和诱导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。转化后的克隆将被植入胸腺裸鼠体内,以研究其诱导肿瘤形成的能力。我们将在小鼠乳腺器官培养(MMOC)模型以及野生型和ER敲除小鼠的体内实验中评估ER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中确定的蛋白质靶点相关联,以便全面了解雌激素致癌作用中er的参与情况。

项目成果

期刊论文数量(0)
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科研奖励数量(0)
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Judy L Bolton其他文献

Judy L Bolton的其他文献

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{{ truncateString('Judy L Bolton', 18)}}的其他基金

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:
    7786288
  • 财政年份:
    2007
  • 资助金额:
    $ 29.14万
  • 项目类别:
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:
    8037167
  • 财政年份:
    2007
  • 资助金额:
    $ 29.14万
  • 项目类别:
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
  • 财政年份:
    2007
  • 资助金额:
    $ 29.14万
  • 项目类别:
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:
    7303189
  • 财政年份:
    2007
  • 资助金额:
    $ 29.14万
  • 项目类别:
MECHANISMS OF ACTION IN MENOPAUSE
更年期的作用机制
  • 批准号:
    6954972
  • 财政年份:
    2005
  • 资助金额:
    $ 29.14万
  • 项目类别:
Symposium on Mechanisms of estrogen carcinogenesis
雌激素致癌机制研讨会
  • 批准号:
    6415051
  • 财政年份:
    2001
  • 资助金额:
    $ 29.14万
  • 项目类别:
Estrogenic Agents--In Vitro and In Vivo Evaluation
雌激素药物——体外和体内评价
  • 批准号:
    6357004
  • 财政年份:
    2000
  • 资助金额:
    $ 29.14万
  • 项目类别:
Carcinogenic Metabolites formed from Antiestrogens
抗雌激素形成的致癌代谢物
  • 批准号:
    6582106
  • 财政年份:
    1999
  • 资助金额:
    $ 29.14万
  • 项目类别:
Carcinogenic Metabolites formed from Antiestrogens
抗雌激素形成的致癌代谢物
  • 批准号:
    7175312
  • 财政年份:
    1999
  • 资助金额:
    $ 29.14万
  • 项目类别:
Carcinogenic Metabolites Formed from Antiestrogen
抗雌激素形成的致癌代谢物
  • 批准号:
    7588719
  • 财政年份:
    1999
  • 资助金额:
    $ 29.14万
  • 项目类别:
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