Repair kinetics of DNA double strand breaks induced in human mammary epithelial cells

The introduction of mammography screening programs in some countries initiated a continuous discussion about the benefit or possible accompanied risk of the used radiation qualities. Contributing to this discussion we used different methods, such as clonogenic survival and the analysis of chromosomal aberrations by FISH, to study biological effects with the human mammary epithelial cell line 184A1, which is of interest especially for the induction of breast cancer. Radiation qualities applied were 10 and 25 kV soft X-rays and a 200 kV X-ray tube as reference radiation source. Furthermore, we develop these methods and investigate the repair kinetics of DNA double strand breaks (DSB) with cell line 184A1 to learn more about the underlying processes.
The DNA damage response, following irradiation, includes an efficient DSB recognition system as well as different DSB repair mechanisms. Important contributions to these processes are the phosphorylation of histone H2AX (gH2AX) and the localization and phosphorylation of the p53 binding protein 1 (53BP1) at the sides of DNA DSBs. Both molecules are essential for the recognition of DNA DSBs and hence for the induction of repair processes. Using appropriate antibodies, the phosphorylated molecules can be visualized as nuclear foci and their time-dependent formation can be followed after irradiation.
The time-dependent formation of gH2AX- and 53BP1-foci was used in the present work to investigate the induction and rejoining of DNA DSBs up to 48 hours after irradiation. Two different antibodies were used to visualize and quantify the foci induced by two different doses of 0.25 and 2 Gy of 25 and 200 kV X-rays, respectively.
The kinetics obtained after irradiations with 2 Gy show a similar shape and therefore energy independence for both energies, which can be explained by the same underlying repair processes. Shortly after irradiation the number of foci per cell shows a steep increase, resulting in a maximum after 2 hours and a subsequent 2-phase decrease. Although the position of the maximum is the same, the number of foci per cell differs for 25 kV and 200 kV X-rays, being 7.99 ± 0.12 and 6.19 ± 0.10, respectively. The rejoining of DSBs, deduced by the decrease of number of foci per cell can be divided in a first phase, which seems to be faster, and a slower second one. During the first phase, the number of foci per cell is reduced to about 50 % of the maximum and in the second phase they are reduced to the control level, whereas for 25 kV a significant number of foci per cell remain. These results implicate that, on the one hand, the number of the induced DNA DSBs is significantly higher for 25 kV X-rays and, that on the other hand, some DSBs remain unrepaired. These DSBs can influence the genomic integrity and can result in chromosomal aberrations.
First experiments for the irradiation with 0.25 Gy of 25 kV show the same shape as that with higher dose, but the maximum is derived at 30 minutes after irradiation. The maximum number of foci per cell was 4.03, which is about the half compared to a dose of 2 Gy with the same energy.