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.
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