In situ assay for DNA cleavage by topoisomerase II

描述（由申请人提供）：拓扑异构酶II的DNA切割的原位分析摘要所提出的研究的目标是引入一种使能技术和第一个原位分析，用于选择性检测拓扑异构酶II（TOPO II）的DNA切割。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，但它也有负面作用，本身也会引发恶性转化。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性，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目标。改变TOPO II活性的药物，如依托泊苷和阿霉素，形成一类TOPO II抑制剂，其通过触发TOPO II产生DNA断裂而起作用。鉴于TOPO II的深远意义，重要的是要有一种技术，用于特异性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。然而，目前没有检测组织切片形式中TOPO II的这种活性的测定法。在本提案中，我们将开发第一种此类检测方法。它将使用荧光寡聚探针和牛痘拓扑异构酶I（VACC TOPO I）标记由TOPO II产生的特征性DNA断裂。该项目的具体目标是：1。目的研制第一种特异性荧光检测TOPO Ⅱ引起的特征性双链DNA断裂的寡核苷酸探针。评价并优化其在TOPO II DNA断裂受控生产条件下检测的灵敏度和特异性。2.建立首次在固定和新鲜冷冻组织中原位检测TOPO Ⅱ的DNA切割活性的方法。测试并优化其在几种体内条件下的灵敏度、特异性和适用性，其中产生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一种有用的商业产品，在临床和理论癌症研究以及抗癌药物的开发和评价中具有广泛的应用。 公共卫生相关性：拟议项目将导致开发一种新的测定方法，以满足医学诊断和病理学的需求。该技术将允许精确评估细胞死亡和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。

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.