Isomeric HRT estrogen-DNA adducts: Structure and Repair

异构 HRT 雌激素-DNA 加合物：结构和修复

• 批准号: 6857312
• 负责人: Nicholas E Geacintov
• 金额: $ 30.03万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857312
• 关键词: DNA directed DNA polymerase; DNA repair; adduct; biotechnology; breast neoplasms; cancer risk; cell free system; chemical conjugate; drug resistance; enzyme activity; estradiol; estrogens; hormone related neoplasm /cancer; molecular dynamics; nucleic acid structure; oligonucleotides; p53 gene /protein; polymerase chain reaction; postmenopause; protein purification; stereoisomer; women' s health

DESCRIPTION (provided by applicant): A widely used hormone replacement therapy (HRT) formulation (~ 57 million prescriptions in 2003 alone) contains the conjugated equine estrogens equilenin (EN), equilin, and 8,9-dehydro-estrone (total ~ 54% composition), and the endogenous estrone and 17beta-estradiol. Although the long term risks of cancers of the breast and other hormone-sensitive tissues have been well publicized, women continue to use this formulation because of the significant health benefits associated with the relief of post-menopausal systems. The metabolic activation of equine and endogenous estrogens to genotoxic metabolites has been implicated in the etiology of cancers associated with HRT. It is known that the catechol 4-hydroxyequilenin (4-OHEN) is the most significant metabolite among all three equine estrogens, and that it forms unusual cyclic, stable DNA adducts in vitro, in rat mammary tissue model systems, and in human breast tissue. Adducts of 4-OHEN with cytosine, adenine, and guanine in DNA have been recognized, and each type of adduct is characterized by four stereoisomeric forms. If not removed by cellular DNA repair mechanisms, such adducts may be incorrectly replicated by DNA polymerases, thus causing mutations that may ultimately lead to the development of tumors. Nucleotide excision repair (NER) is the major repair mechanisms that removes bulky adducts from DNA in human cells with efficiencies that depend on the structural properties of the adducts and the distortions they cause in the DNA structure. However, there is no information on how the 12 different 4-OHEN-DNA adducts are processed by NER enzymes. The goal of this project is to determine how the conformations of the different stereoisomeric forms of these adducts influence the efficiencies of their removal by human NER enzymes. In Aim 1, site-specific oligonucleotides with single 4-OHEN-DNA lesions will be constructed. In Aim 2, their structural features will be analyzed by high resolution NMR and computational techniques, while in Aim 3, the resistance to DNA repair will be evaluated using cell free extracts in cells, and in several different types of cells in culture treated with 4-OHEN.The identification of the 4-OHEN-DNA adducts that are resistant to DNA repair in human cells is significant because this information could help to (1) develop biomarkers for identifying women at risk to the adverse effects of HR7, and (2) stimulate the design of new, modified estrogen replacement drugs that are less resistant to nucleotide excision repair enzymes at the DNA adduct level, and thus less active as potential cancer initiating agents.

描述（由申请人提供）：一种广泛使用的激素替代疗法 (HRT) 配方（仅 2003 年就有约 5700 万张处方）含有结合的马雌激素马烯雌酮 (EN)、马烯雌酮和 8,9-脱氢雌酮（总计约 54 %成分），以及内源性雌酮和17β-雌二醇。尽管乳腺癌和其他激素敏感组织的癌症的长期风险已广为人知，但由于与绝经后系统缓解相关的显着健康益处，女性继续使用这种配方。马和内源性雌激素代谢激活为基因毒性代谢物，与激素替代疗法相关癌症的病因学有关。众所周知，儿茶酚 4-羟基马萘雌酮 (4-OHEN) 是所有三种马雌激素中最重要的代谢物，并且它在体外、大鼠乳腺组织模型系统和人类乳腺组织中形成不寻常的环状、稳定的 DNA 加合物。 4-OHEN 与 DNA 中的胞嘧啶、腺嘌呤和鸟嘌呤的加合物已被识别，每种类型的加合物都具有四种立体异构形式。如果不通过细胞 DNA 修复机制去除，此类加合物可能会被 DNA 聚合酶错误地复制，从而引起突变，最终可能导致肿瘤的发展。核苷酸切除修复 (NER) 是从人类细胞 DNA 中去除大量加合物的主要修复机制，其效率取决于加合物的结构特性以及它们在 DNA 结构中引起的扭曲。然而，没有关于 NER 酶如何处理 12 种不同的 4-OHEN-DNA 加合物的信息。该项目的目标是确定这些加合物的不同立体异构形式的构象如何影响人类 NER 酶去除它们的效率。在目标 1 中，将构建具有单个 4-OHEN-DNA 损伤的位点特异性寡核苷酸。在目标 2 中，将通过高分辨率 NMR 和计算技术分析它们的结构特征，而在目标 3 中，将使用细胞中的无细胞提取物以及用 4 处理的培养物中的几种不同类型的细胞来评估对 DNA 修复的抵抗力。 -OHEN。对人类细胞中对 DNA 修复具有抵抗力的 4-OHEN-DNA 加合物的鉴定具有重要意义，因为该信息可以帮助 (1) 开发生物标志物来识别面临 HR7 不利影响风险的女性，以及 (2 ） 刺激设计新的改良雌激素替代药物，这些药物在 DNA 加合物水平上对核苷酸切除修复酶的抵抗力较低，因此作为潜在癌症引发剂的活性较低。