Quantification of DNA double-strand break induced by radiation in cervix cancer cells: in vitro study

Abstract

AbstractAimDNA double-strand break (DSB) results in the phosphorylation of the protein, H.2AX histone. In this study, the effect of radiotherapy and chemotherapy on DNA DSB in cervical cancer cells is analysed by the phosphorylation of the protein.MethodsThe cervical cancer cells (HeLa cells) were cultured and exposed to ionising radiation. Radiation sensitivity was measured by clonogenic survival fraction after exposing to ionising radiation. Since the phosphorylation of H.2AX declines with time, the DNA damage was quantified at different time points: 1 hour, 3 hours and 1 week after exposed to the radiation. The analysis of γ-H.2AX was done by Western-blot technique. The protein expression was observed at different dose of radiation and combination of both radiation and paclitaxel.ResultsLow-dose hypersensitivity was observed. By 1 week after radiation at 0·5, 0·8 and 2 Gy, there was no expression of phosphorylated H.2AX. Previous experiments on the expression of phosphorylated H.2AX (γ-H.2AX) in terms of foci analysis was found to peak at 1 hour and subsequently decline with time. In cells treated with the DNA damaging agents, the expression of phosphorylated H.2AX decreases in a dose-dependent manner when treated with radiation alone. However, when combined with paclitaxel, at 0·5 Gy, the expression peaked and reduces at 0·8 Gy and slightly elevated at 2 Gy.FindingsIn this study, the peak phosphorylation was observed at 3 hour post irradiation indicating that DSBs are still left unrepaired.