X-Ray Induced Mutation Frequency at the iHypoxanthine Phosphoribosyltransferase/iLocus in Clinically Relevant Radioresistant Cells

摘要

To elucidate the molecular mechanisms underlying cellular radioresistance, clinically relevant radioresistant cell lines were established via long-term exposure to X-rays with stepwise dose escalation. Established cells continue to proliferate despite exposure to 2 Gy X-rays/day for more than 30 days, a standard protocol in cancer radiotherapy. DNA repair fidelity in radioresistant and the parental cells by evaluating the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus after exposure to X-rays was determined. Mutation spectrum at the HPRT locus was examined by multiplex polymerase chain reaction. Rejoining kinetics of X-ray-induced DNA double strand breaks (dsbs) was evaluated by the detection of phosphorylated histone H2AX (γH2AX) after X-irradiation. The fold increase in the HPRT mutation frequency due to acute radiation was similar between radioresistant and the parental cell lines. However, fractionated radiation (FR) consisting of 2 Gy X-rays/day increased the mutation frequency at the HPRT locus in parental but not in radioresistant cells. Analysis of the FR-induced mutations at the HPRT locus revealed a high frequency of deletion mutations (>70%) in parental but not in radioresistant cells. As assessed by γH2AX immunostaining, DNA dsbs induced by acute exposure to 10 Gy of X-rays were repaired to the control level within 7 days in radioresistant but not in the parental cells. Moreover, 2 Gy × 5 FR increased the number of γH2AX-positive cells in parental cultures but not in radioresistant cultures. DNA dsbs induced by 2 Gy/day FR are repaired with fidelity in radioresistant but not in parental cells.