| Research Abstract |
Introduction : In general, the dose rate of sparsely ionizing radiation is reduced over a period of time the biological effect of a given total dose is reduced. This low dose-rate effects (LDRE) has been generally accepted to result from the sub-lethal damage (SLD) recovery. To study the molecular mechanism of LDRE, we analyzed the knock-out mutants KU70^<-/->, RAD54^<-/->, and KU70^<-/->/RAD541^<-/->of the chicken B-cell line, DT40 (Cell (1997) 89:185-193,EMBO J (1998) 17:5497-5508), since our recent studies on SLD recovery, using these DT40 cells showed that the recovery of SLD is due to DSB repair mediated by homologous recombination (HR) (Radiat.Res (2001) 155,680-686).
Materials and Methods : Our results were obtained with these knockout mutant clones of DT40. The length of cell cycle is almost the same,〜8 h, between the four types of cells. LDR radiation was delivered at under 1Gy/day using a ^<137>Cs g-ray source (Sangyo Kagaku Co., Ltd., Tokyo) at the RBC, Kyoto University. Cell
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s were irradiated in suspension continuously at 39.5℃ in an incubator set in front of the ^<137>Cs source. Subsequently, serially diluted cells were inoculated into several individual culture dishes (Iwaki, Japan) containing 6 ml of 1.5% (w/v) methylcellulose (Aldrich, Milwaukee, WI) containing D-MEM/F-12 (Gibco-BRL), 15% FCS, 1.5% chicken serum and 10mM b-mercaptoethanol for each radiation dose. The cells were allowed to grow for 7 days.
Results and Discussions : Survival enhancement by LDR irradiation was observed in parent DT40 and RAD54^<-/-> cells but not in non-homologous end-joining (NHEJ) deficient KU70^<-/-> and KU70^<-/->/RAD54^<-/-> cells. Under continuous LDR irradiation, NHEJ-deficient cells will be irradiated in G1 phase and killed since there is no repair system of DSBs in G1 phase. In LDRE, NHEJ pathway shall be more important than HR pathway because NHEJ pathway is working in whole cell cycle. We studied further LDRE using the deficient KU70^<-/-> and KU70^<-/->/53BP1^<-/-> cells since 53BP1 is reported to play a role in new NHEJ repair (Genes to Cells (2006) 11:935-948). KU70^<-/->/53BP1^<-/-> cells were sensitive than KU70^<-/-> cells under 1 Gy/day dose rate irradiation. These results suggest that 53BP1 play a role in a pathway distinct form Ku-dependent NHEJ pathways, and that both NHEJ pathway is important for LDRE. Less
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