In situ assay for DNA cleavage by topoisomerase II

描述（由申请人提供）：拓扑异构酶II DNA切割的原位分析摘要本研究的目标是引入一种启用技术和第一个用于选择性检测拓扑异构酶II (TOPO II) DNA切割的原位分析。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，它本身可以引发恶性转化。它在几种人类恶性肿瘤中的作用已得到证实。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。这是它极有可能破坏细胞DNA和诱导细胞死亡的原因。因此，它成为抗癌治疗的主要目标。修饰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，通过触发TOPO II产生DNA断裂起作用。鉴于TOPO II的深远意义，有必要建立一种特异性的原位检测这种普遍存在的酶诱导的DNA切割的技术，特别是在单个细胞水平上。然而，目前还没有检测TOPO II在组织切片格式中的这种活性的方法。在这个建议中，我们将开发第一个这种试验。它将使用荧光寡聚探针和牛痘拓扑异构酶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。开发首个用于特异性荧光检测TOPO II产生的特征双链DNA断裂的寡聚探针。评价并优化其在TOPO II DNA断裂控制条件下检测的灵敏度和特异性。2. 建立首个原位检测TOPO II在固定和新鲜冷冻组织中的DNA裂解活性的方法。在几种产生TOPO II断裂的体内条件下，测试并优化其敏感性、特异性和适用性。这种新的原位分析方法将成为一种强有力的工具和有用的商业产品，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。

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.