In situ assay for topoisomerase 2-mediated DNA damage Topoisomerase 2 (Top2) is essential for DNA replication and transcription, but this enzymegenerates double-strand DNA breaks during its normal catalytic cycle. This activity of Top2 is a keycontributor to cancer initiation and progression in several tumor types. Paradoxically, amplification ofTop2 DNA damage became the basis of the most successful strategy for cancer treatment. Itproduced some of the best therapeutic agents used to treat human malignancies. Virtually every formof cancer can be treated with regimens targeting Top2. The development of novel non-toxic andselective 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 broadlyimportant in both molecular and clinical research fields. The most advantageous method should detectits target directly in sections of normal and cancer tissues. Such an in situ assay would enable efficientdeciphering of cancer initiation mechanisms and would enhance discovery of new Top2 anticancerdrugs. 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 newtechnology will quantitatively visualize Top2-generated DNA breaks directly in tissue sections and inindividual cells. This new ability is essential for molecular pathology studies and for the assessment ofanticancer therapies. The assay will have high commercial potential because it will offer advantages ofthe new detection format, speed, specificity and cost over the current biochemical approaches. It willuse a novel molecular labeling method specific for signature Top2 breaks, which carry homodimericadducts 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 technologyspecifically labeling Top2-produced DNA breaks with 5'Top2 adducts and 4-base overhangs. Thetechnology will use the new and original in situ 5' tyrosine enabled labeling method. To employ modelsystems with controlled production of Top2 DNA breaks to validate labeling specificity of the newapproach, 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 ofcommercial use in models related to cancer. To assess and verify the specific, robust, and sensitivedetection of Top2 DNA cleavage by using tissue sections with Top2 DNA breaks. To test and optimizesensitivity, specificity and commercial applicability of the new assay.
Public Health Relevance Statement: Narrative Statement
The purpose of this project is to introduce an enabling method to analyze many varieties
of cancer. This will produce a new and useful histological assay with broad commercial
applications in the development of more efficient therapies and for molecular research
in cancer and other pathologies where tissues sections are routinely used.
Project Terms: inhibitor/antagonist ; inhibitor ; Antibodies ; Monoclonal Antibodies ; Clinical Treatment Moab ; mAbs ; Antineoplastic Agents ; Anti-Cancer Agents ; Antineoplastic Drugs ; Antineoplastics ; Cancer Drug ; Neoplastic Disease Chemotherapeutic Agents ; Tumor-Specific Treatment Agents ; anti-cancer drug ; anticancer agent ; anticancer drug ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Brain ; Brain Nervous System ; Encephalon ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cell Nucleus ; Nucleus ; Cells ; Cell Body ; Clinical Research ; Clinical Study ; DNA ; Deoxyribonucleic Acid ; DNA Damage ; DNA Injury ; Pharmaceutical Preparations ; Drugs ; Medication ; Pharmaceutic Preparations ; drug/agent ; Enzymes ; Enzyme Gene ; Goals ; Human ; Modern Man ; Immunohistochemistry ; Immunohistochemistry Cell/Tissue ; Immunohistochemistry Staining Method ; Medicine ; Methods ; Biological Models ; Biologic Models ; Model System ; Noise ; Nucleotides ; Legal patent ; Patents ; Pathology ; Patients ; Pharmacology ; Production ; Research ; Sensitivity and Specificity ; Signal Transduction ; Cell Communication and Signaling ; Cell Signaling ; Intracellular Communication and Signaling ; Signal Transduction Systems ; Signaling ; biological signal transduction ; Specificity ; Suspensions ; Suspension substance ; Technology ; Testing ; Tissues ; Body Tissues ; Toxin ; Genetic Transcription ; Gene Transcription ; RNA Expression ; Transcription ; Tyrosine ; Work ; Phosphotyrosine ; Tyrosine-O-phosphate ; Paraffin Embedding ; Mediating ; Apoptosis ; Apoptosis Pathway ; Programmed Cell Death ; base ; Organ ; tumor progression ; cancer progression ; neoplasm progression ; neoplastic progression ; Label ; Area ; Histologic ; Histologically ; Biochemical ; Link ; Ensure ; Individual ; DNA Adducts ; Carcinogen-DNA Adducts ; Therapeutic ; Therapeutic Agents ; Double-Stranded DNA ; dsDNA ; ds-DNA ; Spottings ; Malignant Cell ; cancer cell ; Immunes ; Immune ; Complex ; In Situ ; cell type ; Pattern ; Techniques ; adduct ; Cancer Induction ; carcinogenesis ; molecular pathology ; DNA Replication ; DNA Synthesis ; DNA biosynthesis ; Speed ; Structure ; novel ; novel technologies ; new technology ; Modeling ; Sampling ; Cancer Treatment ; Malignant Neoplasm Therapy ; Malignant Neoplasm Treatment ; anti-cancer therapy ; anticancer therapy ; cancer-directed therapy ; cancer therapy ; preventing ; prevent ; Oncogenesis ; tumorigenesis ; Topoisomerase ; DNA Double Strand Break ; Detection ; Molecular Biology Techniques ; Molecular Fingerprinting ; molecular profile ; molecular signature ; Molecular Profiling ; Reproducibility ; in vivo ; Characteristics ; Molecular ; Development ; developmental ; cost ; virtual ; design ; designing ; novel strategies ; new approaches ; novel approaches ; novel strategy ; Imaging technology ; innovation ; innovate ; innovative ; commercial application ; tumor ; cancer initiation ; Regimen ; Drug Targeting ; novel anticancer drug ; new anti-cancer agent ; new anticancer agent ; new anticancer drug ; new antineoplastic ; new cancer drug ; novel anti-cancer agent ; novel anti-cancer drug ; novel anticancer agent ; novel antineoplastic ; novel cancer drug ; preservation ; anti-cancer ; anticancer ; side effect ; Visualization ;
