In situ assay for topoisomerase 2-mediated DNA damage

拓扑异构酶 2 介导的 DNA 损伤的原位测定

• 批准号: 10323156
• 负责人: KM Wahidur Rahman
• 金额: $ 23.67万
• 依托单位: VIVID TECHNOLOGIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2023-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323156
• 关键词: Antibodies; Antineoplastic Agents; Apoptosis; Area; Biochemical; Biological Assay; Biological Models; Brain; Cell Nucleus; Cells; Characteristics; Clinical Research; Complex; DNA; DNA Adducts; DNA Damage; DNA Double Strand Break; DNA biosynthesis; Detection; Development; Drug Targeting; Ensure; Enzymes; Genetic Transcription; Goals; Histologic; Human; Imaging technology; Immune; Immunohistochemistry; In Situ; Individual; Label; Legal patent; Link; Malignant Neoplasms; Mediating; Medicine; Methods; Modeling; Molecular; Molecular Biology Techniques; Molecular Profiling; Monoclonal Antibodies; Noise; Nucleotides; Organ; Paraffin Embedding; Pathology; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Phosphotyrosine; Production; Regimen; Reproducibility; Research; Sampling; Sensitivity and Specificity; Signal Transduction; Specificity; Speed; Spottings; Structure; Suspensions; Techniques; Technology; Testing; Therapeutic; Therapeutic Agents; Tissues; Topoisomerase; Toxin; Tyrosine; Visualization; Work; adduct; anti-cancer; base; cancer cell; cancer initiation; cancer therapy; carcinogenesis; cell type; commercial application; cost; design; ds-DNA; in vivo; inhibitor/antagonist; innovation; molecular pathology; new technology; novel; novel anticancer drug; novel strategies; preservation; prevent; side effect; tumor; tumor progression; tumorigenesis; virtual

In situ assay for topoisomerase 2-mediated DNA damage Abstract Topoisomerase 2 (Top2) is essential for DNA replication and transcription, but this enzyme generates double-strand DNA breaks during its normal catalytic cycle. This activity of Top2 is a key contributor to cancer initiation and progression in several tumor types. Paradoxically, amplification of Top2 DNA damage became the basis of the most successful strategy for cancer treatment. It produced some of the best therapeutic agents used to treat human malignancies. Virtually every form of cancer can be treated with regimens targeting Top2. The development of novel non-toxic and selective Top2 inhibitors became one of the prioritized targets in precision anticancer medicine. Therefore, a technology which detects and quantifies Top2-produced DNA breaks will be broadly important in both molecular and clinical research fields. The most advantageous method should detect its target directly in sections of normal and cancer tissues. Such an in situ assay would enable efficient deciphering of cancer initiation mechanisms and would enhance discovery of new Top2 anticancer drugs. However, currently there are no in situ assays specific for Top2-mediated DNA breakage. The goal of this project is to introduce the first in situ assay for Top2 DNA damage. The new technology will quantitatively visualize Top2-generated DNA breaks directly in tissue sections and in individual cells. This new ability is essential for molecular pathology studies and for the assessment of anticancer therapies. The assay will have high commercial potential because it will offer advantages of the new detection format, speed, specificity and cost over the current biochemical approaches. It will use a novel molecular labeling method specific for signature Top2 breaks, which carry homodimeric adducts linked to extruding 4-nucleotide overhangs on the 5-end of DNA. The Specific Aims of this project are: 1. To develop and validate in the context of commercial use the first tissue section technology specifically labeling Top2-produced DNA breaks with 5’Top2 adducts and 4-base overhangs. The technology will use the new and original in situ 5’ tyrosine enabled labeling method. To employ model systems with controlled production of Top2 DNA breaks to validate labeling specificity of the new approach, and to ensure its adequate sensitivity and reliability of detection. 2. To apply the new imaging technology to fixed tissue sections. To validate the assay in the context of commercial use in models related to cancer. To assess and verify the specific, robust, and sensitive detection of Top2 DNA cleavage by using tissue sections with Top2 DNA breaks. To test and optimize sensitivity, specificity and commercial applicability of the new assay.

拓扑异构酶 2 介导的 DNA 损伤的原位测定 抽象的 拓扑异构酶 2 (Top2) 对于 DNA 复制和转录至关重要，但这种酶 在其正常催化循环期间产生双链 DNA 断裂。 Top2的这个活动很关键 导致多种肿瘤类型的癌症发生和进展。矛盾的是，放大 Top2 DNA 损伤成为最成功的癌症治疗策略的基础。它 生产了一些用于治疗人类恶性肿瘤的最佳治疗药物。几乎每种形式 的癌症可以通过针对 Top2 的方案进行治疗。新型无毒、 选择性Top2抑制剂成为精准抗癌医学的优先靶点之一。 因此，检测和量化 Top2 产生的 DNA 断裂的技术将得到广泛应用。 在分子和临床研究领域都很重要。最有利的方法应该检测 它直接靶向正常组织和癌组织的切片。这种原位测定将能够有效 破译癌症起始机制并将促进新的 Top2 抗癌药物的发现 药物。然而，目前还没有专门针对 Top2 介导的 DNA 断裂的原位测定。 该项目的目标是推出首个针对 Top2 DNA 损伤的原位检测方法。新的 技术将直接在组织切片中定量可视化 Top2 生成的 DNA 断裂 单个细胞。这种新能力对于分子病理学研究和评估至关重要 抗癌疗法。该测定法将具有很高的商业潜力，因为它具有以下优点： 与当前生化方法相比，新的检测形式、速度、特异性和成本。它将 使用一种新颖的分子标记方法，专门针对带有同源二聚体的特征 Top2 断裂 与 DNA 5 端突出的 4 核苷酸突出端相连的加合物。 该项目的具体目标是： 1. 在商业用途的背景下开发和验证第一个组织切片技术 用 5'Top2 加合物和 4 碱基突出端专门标记 Top2 产生的 DNA 断裂。这 技术将使用新的、原创的原位5’酪氨酸标记方法。雇用模型 控制生产 Top2 DNA 断裂的系统，以验证新标签的特异性 方法，并保证其足够的检测灵敏度和可靠性。 2.将新的成像技术应用于固定组织切片。为了在以下背景下验证测定 在与癌症相关的模型中的商业用途。评估和验证特定的、稳健的和敏感的 使用具有 Top2 DNA 断裂的组织切片检测 Top2 DNA 切割。测试和优化 新检测方法的敏感性、特异性和商业适用性。