Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging
Journal article, 2020

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.

Author

Vandana Singh

Sahlgrenska University Hospital

Chalmers, Biology and Biological Engineering, Chemical Biology

Pegah Johansson

University of Gothenburg

Sahlgrenska University Hospital

Dmitry Torchinsky

Tel Aviv University

Yii Lih Lin

Chalmers, Biology and Biological Engineering, Chemical Biology

Robin Öz

Chalmers, Biology and Biological Engineering, Chemical Biology

Y. Ebenstein

Tel Aviv University

Ola Hammarsten

Sahlgrenska University Hospital

University of Gothenburg

Fredrik Westerlund

Chalmers, Biology and Biological Engineering, Chemical Biology

Translational Oncology

1936-5233 (ISSN)

Vol. 13 10 100822

Subject Categories

Biochemistry and Molecular Biology

Clinical Laboratory Medicine

Microbiology in the medical area

DOI

10.1016/j.tranon.2020.100822

More information

Latest update

8/20/2020