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: 6998966
• 负责人: Nicholas E Geacintov
• 金额: $ 27.3万
• 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/6998966
• 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（仅在2003年就有5700万个处方）包含共轭的马雌激素平衡（EN），Equilin和8,9-脱氢雌激素的雌激素（总含量）（总计〜54％的组成），以及Endenogenos ostrone and EntenososEtrad Estradiol Ettradiol andiol Ettradiol。尽管乳腺癌和其他对激素敏感组织的癌症的长期风险已得到充分宣传，但由于与绝经后系统的缓解有关的重大健康益处，妇女继续使用这种配方。马和内源性雌激素对遗传毒性代谢产物的代谢激活与与HRT相关的癌症的病因有关。众所周知，Catechol 4-羟基均衡素（4-OHEN）是所有三种马雌激素中最重要的代谢物，并且在大鼠乳腺组织模型系统和人乳腺组织中形成了体外，体外形成异常的环状，稳定的DNA加合物。已经识别出具有胞嘧啶，腺嘌呤和鸟嘌呤的4-OHEN的加合物，并且每种类型的加合物都以四种立体异构体形式为特征。如果不通过细胞DNA修复机制去除，则这种加合物可能会被DNA聚合酶错误地复制，从而导致突变最终可能导致肿瘤的发展。核苷酸切除修复（NER）是主要的修复机制，它具有依赖加合物的结构特性及其在DNA结构中引起的扭曲的效率的人类细胞中DNA的庞大加合物。但是，没有关于如何通过NER酶处理12种不同的4-OHEN-DNA加合物的信息。该项目的目的是确定这些加合物的不同立体异构体形式的构象如何影响人类NER酶去除的效率。在AIM 1中，将构建具有单个4-OHEN-DNA病变的位点特异性寡核苷酸。在目标2中，将通过高分辨率NMR和计算技术来分析它们的结构特征，而在AIM 3中，将使用细胞中的无细胞提取物以及4-OHEN治疗的几种不同类型的细胞来评估对DNA修复的抵抗力。对4-OHEN-DNA的识别，对人类细胞的识别是有效的（1），因为人类均可识别（1），因为人类的抗性可能有助于（1），因为（1）可能有助于（1），因为（1）可能有助于（1），因为它可能有助于（1），因为它可能有助于（1）有助于（1），因为它可能有助于（1）有助于（1），因为它可能有助于（1）有助于（1）。 HR7的不良影响和（2）刺激新的，改良的雌激素替代药物的设计，这些药物对DNA加合物水平上对核苷酸切除修复酶的耐药性较低，因此作为潜在癌症引发剂的活性较低。