Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging
Autor: | Dmitry Torchinsky, Pegah Johansson, Yii-Lih Lin, Fredrik Westerlund, Ola Hammarsten, Vandana Singh, Robin Öz, Yuval Ebenstein |
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Jazyk: | angličtina |
Rok vydání: | 2020 |
Předmět: |
0301 basic medicine
Cancer Research Fluorescence-lifetime imaging microscopy Original article DNA damage SSB single-strand break lcsh:RC254-282 Ionizing radiation 03 medical and health sciences chemistry.chemical_compound Endonuclease 0302 clinical medicine AP site DSB double-strand break BER base excision repair Polymerase chemistry.chemical_classification PBMCs peripheral blood mononuclear cells DNA ligase biology APE1 apurinic/apyrimidinic endonuclease 1 lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens 3. Good health 030104 developmental biology Oncology chemistry 030220 oncology & carcinogenesis biology.protein Biophysics IR ionizing radiation DNA |
Zdroj: | Translational Oncology, Vol 13, Iss 10, Pp 100822-(2020) Translational Oncology |
ISSN: | 1936-5233 |
Popis: | Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent "spot" at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents. |
Databáze: | OpenAIRE |
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