A High-Throughput Assay for DNA Repair Activity in the Presence of AberrantBRCA1

存在异常 BRCA1 时 DNA 修复活性的高通量检测

• 批准号: 7993434
• 负责人: JAMES M. FORD
• 金额: $ 16万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993434
• 关键词: Address; Adenoviruses; Base Excision Repairs; Basic Science; Biological Assay; Breast; Cancer-Predisposing Gene; Cells; Chemoprevention; Code; DNA Damage; DNA Repair; DNA Repair Pathway; Degenerative Disorder; Disease; Eligibility Determination; Epigenetic Process; Estrogen receptor negative; FDA approved; Gene Mutation; Genetic; Genomic Instability; Hereditary Breast and Ovarian Cancer Syndrome; Hydrogen Peroxide; Lead; Libraries; Malignant Neoplasms; Malignant neoplasm of ovary; Measures; Molecular Bank; Molecular Profiling; Monitor; Mutation; Nonhomologous DNA End Joining; Nucleotide Excision Repair; Pathway interactions; Pharmaceutical Preparations; Phenotype; Prevention; Prevention strategy; Process; Production; Raloxifene; Reporter; Reporter Genes; Reproducibility; Research; Screening procedure; Tamoxifen; Testing; Tumor Suppressor Genes; Tumor Suppressor Proteins; base; cell growth; high throughput screening; homologous recombination; malignant breast neoplasm; novel; oxidative DNA damage; oxidative damage; prevent; public health relevance; repaired; response; small molecule; tool; tumorigenesis

DESCRIPTION (provided by applicant): Tumorigenesis arises when DNA damage exceeds DNA repair. The most common form of DNA damage is endogenous oxidative damage, which is repaired by base excision repair (BER), and in some cases nucleotide-excision repair (NER), homologous recombination (HR) and/or non- homologous end-joining (NHEJ). BRCA1, a 220kD tumor suppressor protein, has been implicated in preventing breast cancer by maintaining genetic stability through its ability to prevent oxidative DNA damage and promote DNA repair, including BER, NER, HR, and NHEJ. Upon mutation or inactivation of BRCA1, excessive DNA damage leads to the accumulation of mutations and genetic instability, and ultimately formation of BRCA1-associated malignancies. BRCA1-associated malignancies include hereditary breast and ovarian cancers due to mutations in BRCA1, sporadic breast cancers due to epigenetic silencing of BRCA1, and a subset of breast cancers with the basal-like phenotype (i.e. triple-negative for ER/PR/HER2), which have a similar expression profile to cancers with mutations in BRCA1. They comprise 30-50% of breast cancer cases. These malignancies often do not respond to current chemoprevention agents, suggesting a need for effective prevention strategies. This study will initiate a novel, targeted approach to preventing BRCA1-associated malignancies. The hypothesis states: high-throughput (HT) screening of a compound library may identify small molecules that activate DNA repair of oxidative DNA damage in the presence of aberrant BRCA1. Lead compounds can then be used to identify potential chemoprevention agents for BRCA1-associated malignancies or as tools in basic science research. The following specific aims will be addressed: (1) validate a DNA repair assay for high- throughput format., (2) carry out a quantitative high-throughput pilot screen using a DNA repair assay, and (3) carry out a quantitative high-throughput counter screen using an oxidative DNA damage response assay. First, a cell-based assay that uses adenovirus containing oxidative DNA damage within the coding region of a GFP reporter gene to monitor for activation of DNA repair by host-cell reactivation will be developed for HT-screening. Therefore, optimal cell growth and treatment conditions will be determined, and plate acceptance for the DNA repair assay will be carried out in 384-well format. Second, a small, diverse compound library and the quantitative high-throughput screening format will be used to carry out a pilot screen and to determine assay reproducibility. Finally, we will adapt and validate an H2O2 sensitivity assay for high-throughput format, test the assay in a quantitative high-throughput pilot screen, and compare the hits obtained from the pilot screens using the DNA repair and H2O2 sensitivity assays. Overall, a DNA repair assay will be developed, adapted, and validated for high-throughput screening of a compound library to identify potential chemoprevention agents for BRCA1-associated malignancies. PUBLIC HEALTH RELEVANCE: We will initiate the discovery of potential drugs for preventing a subset of breast cancer known as BRCA1-associated malignancies. Furthermore, this study may lead to advances in treating other diseases that result from oxidative DNA damage (e.g. repair-deficient cancers, degenerative diseases, and various sclerotic diseases), and may generate tools for basic science research.

描述（由申请人提供）：当DNA损伤超过DNA修复时发生肿瘤。DNA损伤的最常见形式是内源性氧化损伤，其通过碱基切除修复（BER）修复，并且在某些情况下通过核苷酸切除修复（NER）、同源重组（HR）和/或非同源末端连接（NHEJ）修复。BRCA 1是一种220 kD的肿瘤抑制蛋白，通过其防止氧化DNA损伤和促进DNA修复（包括BER、NER、HR和NHEJ）的能力维持遗传稳定性，从而参与预防乳腺癌。当BRCA 1突变或失活时，过度的DNA损伤导致突变的积累和遗传不稳定性，并最终形成BRCA 1相关的恶性肿瘤。BRCA 1相关恶性肿瘤包括由于BRCA 1突变引起的遗传性乳腺癌和卵巢癌、由于BRCA 1表观遗传沉默引起的散发性乳腺癌以及具有基底样表型（即ER/PR/HER 2三阴性）的乳腺癌亚组，其具有与BRCA 1突变癌症相似的表达谱。它们占乳腺癌病例的30-50%。这些恶性肿瘤通常对目前的化学预防剂没有反应，这表明需要有效的预防策略。这项研究将启动一种新的，有针对性的方法来预防BRCA 1相关的恶性肿瘤。该假设指出：化合物文库的高通量（HT）筛选可以鉴定在异常BRCA 1存在下激活氧化DNA损伤的DNA修复的小分子。然后，先导化合物可用于识别BRCA 1相关恶性肿瘤的潜在化学预防剂，或作为基础科学研究的工具。本研究的主要目的是：（1）验证高通量DNA修复检测方法，(2)使用DNA修复测定进行定量高通量中试筛选，和（3）使用氧化DNA损伤反应测定进行定量高通量计数筛选。首先，将开发一种基于细胞的测定法用于HT筛选，该测定法使用在GFP报告基因的编码区内含有氧化DNA损伤的腺病毒来监测通过宿主细胞再活化而激活的DNA修复。因此，将确定最佳细胞生长和处理条件，并在384孔板中进行DNA修复试验的平板验收。其次，将使用小型多样化合物库和定量高通量筛选格式进行中试筛选并确定试验重现性。最后，我们将调整和验证H2 O2灵敏度测定的高通量格式，测试在定量高通量中试筛选的测定，并比较使用DNA修复和H2 O2灵敏度测定从中试筛选获得的命中。总体而言，将开发、调整和验证DNA修复试验，用于化合物文库的高通量筛选，以鉴定BRCA 1相关恶性肿瘤的潜在化学预防剂。 公共卫生关系：我们将开始发现潜在的药物，用于预防乳腺癌的一个子集，称为BRCA 1相关的恶性肿瘤。此外，这项研究可能导致治疗其他由氧化DNA损伤引起的疾病（例如修复缺陷型癌症，退行性疾病和各种皮肤病）的进展，并可能为基础科学研究提供工具。