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Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging.
Vandana Singh1, Pegah Johansson2, Dmitry Torchinsky3
1Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden; Laboratory of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Translational Oncology
|July 12, 2020
Summary
This study introduces a sensitive assay to quantify DNA damage from ionizing radiation (IR) using fluorescence imaging. Including human apurinic/apyrimidinic endonuclease 1 (APE1) significantly enhanced detection of IR-induced DNA damage.
Area of Science:
- Molecular Biology
- Biophysics
- Cancer Research
Background:
- Ionizing radiation (IR) is a cornerstone of cancer therapy, primarily inducing cell death through DNA damage.
- Quantifying DNA damage is crucial for understanding IR efficacy and potential side effects.
Purpose of the Study:
- To develop and optimize a fluorescence-based assay for quantifying DNA damage induced by IR in single DNA molecules.
- To evaluate the impact of human apurinic/apyrimidinic endonuclease 1 (APE1) on assay sensitivity.
- To investigate the combined effect of IR and hyperthermia on DNA damage levels.
Main Methods:
- Isolation of DNA from irradiated lymphocytes.
- Enzymatic processing of DNA lesions using a cocktail including APE1.
- Incorporation of fluorescent nucleotides at damage sites via polymerase and ligase.
- Fluorescence imaging and counting of individual DNA damage spots along stretched DNA molecules.
Main Results:
- The developed assay successfully quantifies IR-induced DNA damage at the single-molecule level.
- Inclusion of APE1 significantly increased the assay's sensitivity for detecting DNA damage.
- The optimized assay detected a synergistic increase in DNA damage when IR was combined with mild hyperthermia.
Conclusions:
- The optimized single-DNA-molecule fluorescence imaging assay provides a sensitive method for quantifying IR-induced DNA damage.
- APE1 inclusion enhances the detection of DNA damage, improving assay performance.
- This method holds potential for identifying patients susceptible to IR and other DNA-damaging agents, aiding personalized cancer therapy.
