Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Vandana Singh; Pegah Johansson; Dmitry Torchinsky; Yii-Lih Lin; Robin Öz; Yuval Ebenstein; Ola Hammarsten; Fredrik Westerlund

doi:10.1016/j.tranon.2020.100822

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Transl Oncol. 2020 Oct;13(10):100822. doi: 10.1016/j.tranon.2020.100822. Epub 2020 Jul 8.

Authors

Vandana Singh¹, Pegah Johansson², Dmitry Torchinsky³, Yii-Lih Lin⁴, Robin Öz⁴, Yuval Ebenstein³, Ola Hammarsten², Fredrik Westerlund⁵

Affiliations

¹ Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden; Laboratory of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
² Laboratory of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden; Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
³ Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Israel.
⁴ Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
⁵ Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. Electronic address: fredrikw@chalmers.se.

Abstract

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.