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损伤的原位检测 摘要 拓扑异构酶2（Top2）是DNA复制和转录所必需的，但这种酶 在正常的催化循环中产生双链DNA断裂。Top2的这个活动是一个关键 在几种肿瘤类型中是癌症发生和发展的贡献者。巧合的是， Top2 DNA损伤成为最成功的癌症治疗策略的基础。它 生产了一些最好的治疗人类恶性肿瘤的药物。几乎每一种形式 可以用靶向Top2的方案治疗。新型无毒、 选择性Top2抑制剂成为精确抗癌药物的优先靶点之一。 因此，检测和定量Top2产生的DNA断裂的技术将被广泛应用。 在分子和临床研究领域都很重要。最有利的方法应该是检测 它的目标直接在正常和癌组织的部分。这样的原位测定将使得能够有效地 破译癌症起始机制，并将促进发现新的Top2抗癌药物 毒品然而，目前还没有特异性针对Top2介导的DNA断裂的原位测定。 该项目的目标是引入第一个Top2 DNA损伤的原位测定。新 这项技术将直接在组织切片中定量显示Top2产生的DNA断裂， 单个细胞。这种新的能力对于分子病理学研究和评估 抗癌疗法。该测定将具有高商业潜力，因为它将提供以下优点： 新的检测格式，速度，特异性和成本超过目前的生化方法。它将 使用一种新的分子标记方法，特异性的签名Top2断裂，携带同源二聚体 加合物连接到DNA的5-端上的突出的4-核苷酸突出端。 该项目的具体目标是： 1.在商业应用的背景下开发和验证第一种组织切片技术 用5 ′ Top2加合物和4-碱基突出端特异性标记Top2产生的DNA断裂。的 技术将使用新的和原始的原位5'酪氨酸激活标记方法。采用模型 具有Top2 DNA断裂的受控产生的系统，以验证新的 这是一种新的方法，并确保其足够的灵敏度和检测的可靠性。 2.将新的成像技术应用于固定的组织切片。在以下情况下验证测定法 用于癌症相关模型的商业用途。评估和验证特定的、稳健的和敏感的 通过使用具有Top2 DNA断裂的组织切片检测Top2 DNA切割。测试和优化 灵敏度，特异性和商业适用性的新检测。