Induction of DNA double-strand breaks at various stages of the cell cycle using the comet assay

ABSTRACT

The present study reports improvements in the current protocols of the neutral comet assay to measure radiation induced DNA damage. Different biochemical steps, including mierogel preparation, lysis and enzymatic/chemical treatments have been modified and optimized to estimate the fraction of cells and DNA damage, at different stages of the cell cycle, in a higher dose range of X-rays. Among all tail parameters that were used to measure the DNA damage induced by ionizing radiation, the tail intensity was found to be the most sensitive. In this study DNA damage induced by X-ray doses as low as 5 Gy was detected. A developed analytical method has been used for the simultaneous estimation of the fraction of cells and DNA damage, at different stages of the cell cycle. The results of bivariate analysis of tail intensity versus DNA content showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increase slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters may not be relevant to measure DNA damage in cells at different stages of the cell cycle. The results revealed a highly significant decrease in comet area, at all stages of the cell cycle, after irradiation with all doses, indicating the high sensitivity of this parameter to X-rays. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose, at all stages of the cell cycle. This reduction in comet fluorescence was attributed to partial unwinding of DNA at the strand break sites during the alkaline treatment after electrophoresis, with the consequent reduction in ethiduim bromide stainibitliy and fluorescence of DNA. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this method showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates which vary in the following order G0> G2/M> S > G 1. These results suggest that G0 and G2IM cells are the most sensitive to X-rays among all phases of the cell cycle and synchronization of tumor cells in these phases during radiotherapy may be more efficient in treatment of cancers