In situ assay for DNA cleavage by topoisomerase II

拓扑异构酶 II 的 DNA 切割原位测定

• 批准号: 8200230
• 负责人: BenXiao Zhang
• 金额: $ 14.85万
• 依托单位: VIVID TECHNOLOGIES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-26 至 2014-03-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8200230
• 关键词: Alzheimer' s Disease; Antineoplastic Agents; Apoptosis; Area; Biochemical; Biological Assay; Cell Death; Cell Nucleus; Cell Survival; Cells; Characteristics; Chromosome Segregation; Clinical; Cytoplasm; DNA; DNA Damage; DNA Double Strand Break; DNA biosynthesis; Detection; Development; Diagnostic; Disease; Doxorubicin; Drug Evaluation; Enzymes; Etoposide; Evaluation; Fluorescent Antibody Technique; Formaldehyde; Freezing; Generations; Genetic Transcription; Genome; Glioblastoma; Goals; Human; Imagery; In Situ; Individual; Intervention; Label; Light; Malignant - descriptor; Malignant Neoplasms; Measures; Medical; Molecular; Noise; Outcome; Paraffin Embedding; Pathology; Pharmaceutical Preparations; Pharmacotherapy; Procedures; Production; Protein Isoforms; Research; Residual state; Resolution; Role; Sampling; Sensitivity and Specificity; Side; Signal Transduction; Specificity; Spottings; Staining method; Stains; Stroke; Structure; Surface; Suspension substance; Suspensions; Technology; Testing; Tissue Sample; Tissues; Topoisomerase II; Topoisomerase-II Inhibitor; Type I DNA Topoisomerases; Vaccinia; Vaccinium; Work; abstracting; anticancer research; base; cancer therapy; clinical epidemiology; design; drug development; in vivo; innovation; prognostic; research study; response; tool; tumor

DESCRIPTION (provided by applicant): In situ assay for DNA cleavage by topoisomerase II Abstract The goal of the proposed research is to introduce an enabling technology and the first in situ assay for selective detection of DNA cleavage by topoisomerase II (TOPO II). TOPO II is the critical enzyme for DNA replication, transcription, and chromosome segregation. Although TOPO II is necessary for cell survival, it has a negative side and can itself trigger malignant transformation. Its role in several types of human malignancies is well-established. TOPO II can destabilize the genome because it unavoidably generates double-strand DNA breaks as part of its catalytic cycle. This underlies its high potential to damage cellular DNA and induce cell death. It, therefore, became the major target of anticancer therapies. Drugs that modify this activity of TOPO II, such as etoposide and doxorubicin, form a class of TOPO II inhibitors that act by triggering the generation of DNA breaks by TOPO II. In light of the profound significance of TOPO II, it is important to have a technology for specific in situ detection of DNA cleavage induced by this ubiquitous enzyme, especially at the individual cell level. However currently there are no assays detecting such activity of TOPO II in the tissue section format. In this proposal we will develop the first assay of this kind. It will use fluorescent oligoprobes and vaccinia topoisomerase I (VACC TOPO I) to label characteristic DNA breaks produced by TOPO II. The Specific Aims of this project are: 1. To develop the first oligoprobe for specific fluorescent detection of characteristic double- strand DNA breaks produced by TOPO II. To evaluate and optimize its sensitivity and specificity of detection in conditions with controlled production of TOPO II DNA breaks. 2. To develop the first in situ assay for detection of DNA cleavage activity of TOPO II in fixed and fresh-frozen tissues. To test and optimize its sensitivity, specificity and applicability in several in vivo conditions, where TOPO II breaks are generated. The new in situ assay will become a powerful tool and a useful commercial product with wide applications in both clinical and theoretical cancer research and in anticancer drug development and evaluation. PUBLIC HEALTH RELEVANCE: The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will allow precise evaluation of the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as various cancers, including glioblastoma, as well as stroke and Alzheimer's disease.

描述(申请人提供)：拓扑异构酶II切割DNA的原位检测摘要本研究的目的是介绍一种能够选择性检测拓扑异构酶II(Topo II)切割DNA的技术和第一个原位检测技术。Topo II是DNA复制、转录和染色体分离的关键酶。虽然Topo II是细胞生存所必需的，但它也有消极的一面，本身就会引发恶性转化。它在几种类型的人类恶性肿瘤中的作用是公认的。TOPO II可以破坏基因组的稳定，因为它不可避免地产生双链DNA断裂，作为其催化循环的一部分。这是其破坏细胞DNA和诱导细胞死亡的高潜力的基础。因此，它成为抗癌治疗的主要靶点。修饰Topo II这种活性的药物，如依托泊苷和阿霉素，形成了一类Topo II抑制剂，通过触发Topo II产生DNA断裂来发挥作用。鉴于Topo II的深远意义，有一种技术可以特异性地原位检测这种普遍存在的酶诱导的DNA切割，特别是在单个细胞水平上。然而，目前还没有在组织切片格式中检测Topo II活性的检测方法。在这项提案中，我们将开发这种类型的第一种测试。它将使用荧光寡核苷酸探针和痘苗病毒拓扑异构酶I(VACC Topo I)来标记Topo II产生的特征DNA断裂。本项目的具体目标是：1.建立第一个用于特异性荧光检测Topo II产生的特征双链DNA断裂的寡核苷酸探针，评估和优化其在控制Topo II DNA断裂产生的条件下检测的敏感性和特异性。2.首次建立了原位检测固定组织和新鲜冷冻组织中Topo II的DNA切割活性的方法。测试和优化其在几种体内条件下的敏感性、特异性和适用性，在这些条件下会产生Topo II断裂。这种新的原位检测方法将成为一种强有力的工具和有用的商业产品，在癌症的临床和理论研究以及抗癌药物的开发和评价中都有广泛的应用。 公共卫生相关性：拟议的项目将导致开发一种新的分析方法，以满足医疗诊断和病理学的需要。这项技术将允许准确评估细胞死亡和DNA损伤具有预后价值的疾病的治疗效果，例如各种癌症，包括胶质母细胞瘤，以及中风和阿尔茨海默病。