In vivo detection and genome-wide location analysis of DNA-adducts

DNA 加合物的体内检测和全基因组定位分析

• 批准号: 8547505
• 负责人: STEPHEN B HOWELL
• 金额: $ 23.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-03 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8547505
• 关键词: Address; Alkylating Agents; Antineoplastic Agents; Apoptosis; Base Pairing; Biological; Biological Assay; Cancer Biology; Carcinogen exposure; Cell Death; Cell Line; Cell physiology; Cells; Chlorambucil; Chromatin Structure; Cisplatin; Clinical; Computer Analysis; DNA; DNA Adduction; DNA Adducts; DNA Damage; DNA Methylation; DNA Repair; DNA Sequence; DNA lesion; DNA-Directed DNA Polymerase; Data; Data Set; Detection; Development; Doctor of Philosophy; Drug Metabolic Detoxication; Environmental Carcinogens; Environmental Risk Factor; Epigenetic Process; Event; Exposure to; Genes; Genome; Genomics; Goals; Homeostasis; Implant; In Vitro; Individual; Kinetics; Knowledge; Lead; Libraries; Ligation; Location; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Methods; Molecular; Mus; Mutation; Normal tissue morphology; Nucleotides; Pathway interactions; Pattern; Performance; Pharmaceutical Preparations; Pharmacology; Physiological; Platinum; Predisposition; Process; Proteins; Protocols documentation; Reading; Reproducibility; Research; Research Personnel; Resolution; Sampling; Scientist; Single-Stranded DNA; Specificity; Specimen; Tail; Testicular Germ Cell Tumor; The Cancer Genome Atlas; Therapeutic; Time; Tissue Sample; Tissues; Tumor Tissue; UV induced DNA damage; Ultraviolet Rays; Validation; adduct; anticancer research; base; cancer cell; carcinogenesis; cell type; chemotherapeutic agent; chemotherapy; drug carcinogenesis; drug efficacy; drug sensitivity; epigenome; experience; genetically modified cells; genome-wide; improved; in vivo; innovation; novel; preference; public health relevance; repaired; research study; response; tumor; tumor xenograft; ultraviolet damage

DESCRIPTION (provided by applicant): DNA adducts are the hallmark and most common form of DNA damage in the cell. They result from environmental carcinogen exposure (such as UV) or during chemotherapy using DNA modifying agents like cisplatin (cDDP) or alkylators such as chlorambucil (CLB). While mechanisms underlying sensitivity, agent homeostasis, detoxification, DNA repair and apoptosis, have been well investigated, the central molecular event, the formation of adducts, is not well understood in vivo. Evidence suggests that the epigenetic landscape and the structure of the chromatin influences the formation of adducts and mediates drug sensitivity. Therefore, there is a need to better identify DNA adducts and understand the association between the epigenetic marks in the cell. Currently there is no method to determine the exact location of DNA adducts in vivo nor at a high-resolution across the genome. In order to address this, we propose to develop a method, TdT-Seq, that will identify these adducts genome-wide at the single base pair resolution. The expertise of the investigators include knowledge in cancer biology and platinum drug pharmacology (Drs. Howell and Abada) as well as experience in high-throughput genomic assays and computational analysis (Dr. Harismendy); expertise that will be needed to successfully develop the assay. The TdT-Seq assay relies on adduct-mediated inhibition of the DNA polymerase in vitro. The resulting single strand DNA will be captured by a specific TdT mediated ligation, enriched, then sequenced in high throughput. We propose to establish the technical validity of the assay by determining 1) sensitivity at various cDDP concentrations and read depth, 2) specificity by the development of a locus specific method (Strand Specific Adduct Detection) and independent analysis of 50 adduct loci, and 3) quantativity using increasing cDDP concentrations and known spike-in controls. We will also perform specific experiments to establish TdT-Seq's use for clinical cancer research. In particular, we will optimize the protocol for the identification of UVor chlorambucil (CLB) induced adducts to broaden its applicability. We will also develop the protocol for low amounts of DNA originating from mouse tissues or heterogeneous tissue specimens. Finally, we will analyze the ability of TdT-Seq to measure the kinetics of DNA repair using genetically modified cell lines. TdT-Seq's development will therefore lead to a robust and innovative assay, with demonstrated performance and utility for cancer research. TdT-Seq will generate an entirely new type of data, which can be used in combination of other whole genome datasets from the ENCODE or TCGA consortium to provide a more precise and comprehensive description of the mechanism of DNA damage and repair in vivo in various cell types and cancers. The long-term benefits of such research include the prediction of drug sensitivity or the study of epigenetic modifying compounds to rationalize combinations for optimal drug efficacy.

描述(由申请人提供)：DNA加合物是细胞中DNA损伤的标志和最常见的形式。它们是由环境致癌物暴露(如紫外线)或在使用DNA修饰剂如顺铂(CDDP)或烷化剂如氯氨丁苯(CLB)进行化疗时产生的。虽然药物敏感性、药物动态平衡、解毒、DNA修复和细胞凋亡的机制已经被很好地研究了，但在体内对加合物的形成这一中心分子事件还没有很好的了解。证据表明，表观遗传景观和染色质的结构影响加合物的形成，并调节药物敏感性。因此，有必要更好地鉴定DNA加合物，并了解细胞中表观遗传标记之间的关联。目前还没有方法确定DNA加合物在体内的确切位置，也没有在整个基因组中以高分辨率确定的方法。为了解决这个问题，我们建议开发一种方法，TDT-SEQ，它将在单碱基对分辨率下在全基因组范围内识别这些加合物。研究人员的专业知识包括癌症生物学和白金药物药理学方面的知识(Howell和Abada博士)以及高通量基因组分析和计算分析方面的经验(Harismendy博士)；成功开发这种分析所需的专业知识。TDT-Seq分析依赖于加合物介导的DNA聚合酶体外抑制。产生的单链DNA将被特定的TDT介导的连接捕获，浓缩，然后高通量测序。我们建议通过确定1)在不同cDDP浓度和阅读深度下的敏感性，2)通过发展位点特异性方法(链特异性加合物检测)和对50个加合物基因座的独立分析来确定该方法的技术有效性，以及3)通过增加cDDP浓度和已知的掺入对照来确定定量。我们还将进行具体的实验，以确定TDT-Seq用于临床癌症研究。特别是，我们将优化紫外线或百菌清(CLB)诱导加合物的鉴定方法，以扩大其适用性。我们还将开发从小鼠组织或异种组织样本中提取低量DNA的方案。最后，我们将分析TDT-Seq使用转基因细胞系测量DNA修复动力学的能力。因此，TDT-Seq的开发将带来一种稳健和创新的分析方法，具有被证明的性能和用于癌症研究的实用性。TDT-Seq将生成一种全新类型的数据，可与ENCODE或TCGA联盟的其他全基因组数据集结合使用，以便更准确和全面地描述各种细胞类型和癌症中体内DNA损伤和修复的机制。这类研究的长期益处包括预测药物敏感性或研究表观遗传修饰化合物，以使组合合理化，以获得最佳的药物疗效。