Single Molecule Imaging of Ionizing Radiation-Induced DNA Damage

Abstract

Ionizing radiation and hyperthermia are part of cancer treatment to control or kill malignant cells. Here, we adapted a new method, the Direct DNA Damage assay (D3 assay), to determine the amount of single stranded DNA damage caused by ionizing radiation. We show that the D3 assay directly determines the amount of damage and that the amount of damage increases with prior hyperthermia treatment before administering gamma irradiation.

Introduction

Cancer leads to the formation of abnormal cells that grow beyond their usual boundaries. Hyperthermia and radiotherapy are designed to kill cancer cells by inducing DNA damage. Detection of single strand breaks (SSBs) is cumbersome, gel-based, and error-prone. Previous methods used to assess DNA damages are for example the comet assay and nick-end labeling assay. Notably, in 2014 Zirkin et al. studied the repair dynamics in response to UV irradiation utilizing single molecule labeling and microscopy, the D3 assay. Here we used the D3 assay to visualize the effect on SSBs induced by ionizing radiation and hyperthermia.

Materials and Method

Lymphocytes were isolated from blood samples and treated with radiation and/or hyperthermia. The D3 assay was performed with 500 ng of DNA, 100 μM of dATP, dGTP, dCTP, 10 μM dTTP and 10 μM Aminoallyl-dUTP-ATTO-647N in 1X nick translation buffer with a cocktail of DNA repair enzymes.

Results and Discussion

SSBs increased around 2.5 times with radiation dose ranging from 0.5 Gy to 2.5 Gy. Approximately, 57.8% of the damages caused by gamma irradiation persist for 45 min, indicating the lethality of the damage. Cells treated prior with 42°C hyperthermia for 30 min and 2 Gy radiation showed around 5.8 times increase in the number of SSBs as compared to the control sample. Inhibition of DNA repair, as well as the formation of SSBs, explains the additive effect of radiation and hyperthermia treatment. APE1 is the key player in repair of ROS induced damage. APE1 increased our ability to detect the SSBs. SSBs increased around 1.2 times when the enzymatic cocktail is supplemented with APE1 enzyme in case of 2 Gy dose, and much more at higher doses.

Conclusion

D3 assay shows an increase in the number of SSBs with increase in temperature,γ-rays, and the hyperthermia-γ-ray treatment. This demonstrates the usefulness of the D3 assay for determining DNA damage and ability to give mechanistic details of biochemical processes concerning cancer therapy.