Hu Q, Hill R P
Experimental Therapeutics Division, Ontario Cancer Institute, University of Toronto, Canada.
Radiat Res. 1996 Dec;146(6):636-45.
The relationship of cell survival to induction and repair of DNA double-strand breaks (DSBs), as assessed by the neutral comet assay, was studied in two pairs of CHO cell lines, repair-deficient mutants xrs-5-11 and V3, and their respective parent lines K-1 and AA8, treated at two dose rates, 10.5 and 0.09 Gy/min. A marked difference in cell survival after irradiation was found between each pair of lines. For initial DNA damage, there was a significant difference between each pair of lines after the low-dose-rate treatment but not with high-dose-rate irradiation. Initial DSBs were dependent on dose at both dose rates. When residual damage at 2 h after irradiation was assessed, the 5-11 cells showed significantly more damage than K-1 cells after both high- and low-dose-rate irradiation. The V3 cells also showed more residual damage than the AA8 cells, but this difference was significant only after high-dose-rate irradiation. The results indicate that low-dose-rate irradiation can differentiate better between the DSB rejoining capacity of the sensitive and resistant cells and are consistent with the idea that it is the slow component of repair of DSBs which is different between them. Further studies with 5-11 and K-1 cells showed that radiation-induced apoptosis was dependent on dose, with a higher fraction of apoptotic cells in 5-11 than in K-1 cells after exposure to a given high-dose-rate radiation dose. However, the levels and time course of induction of apoptosis were similar for doses which gave equal levels of clonogenic survival. Radiation treatment was found to delay the progression of 5-11 and K-1 cells through the cell cycle to the same extent, with accumulation of cells in S phase and G2 phase, at 4 and 12 h after irradiation, respectively. There was no evidence for a G1-phase arrest. Western blotting revealed that there were higher levels of p53 and Waf1 protein in nonirradiated 5-11 than in K-1 cells, and that a dose of 5 Gy of high-dose-rate radiation up-regulated the expression of p53 and Waf1 protein to similar levels in both cell lines. There was no change in levels of the Gadd45 and Bcl2 proteins in either cell line after irradiation. These results suggest that the accumulation of p53 and Waf1 protein does not cause early G1-phase arrest in these cells.
通过中性彗星试验评估细胞存活与DNA双链断裂（DSB）诱导和修复之间的关系，在两对CHO细胞系中进行了研究，即修复缺陷型突变体xrs - 5 - 11和V3及其各自的亲本系K - 1和AA8，分别以10.5和0.09 Gy/min的两种剂量率进行处理。发现每对细胞系在照射后的细胞存活存在显著差异。对于初始DNA损伤，低剂量率处理后每对细胞系之间存在显著差异，但高剂量率照射时则没有。初始DSB在两种剂量率下均依赖于剂量。当评估照射后2小时的残留损伤时，无论是高剂量率还是低剂量率照射后，5 - 11细胞显示出的损伤均显著多于K - 1细胞。V3细胞也比AA8细胞显示出更多的残留损伤，但这种差异仅在高剂量率照射后显著。结果表明，低剂量率照射能够更好地区分敏感细胞和抗性细胞的DSB重新连接能力，并且与它们之间DSB修复的慢成分不同这一观点一致。对5 - 11和K - 1细胞的进一步研究表明，辐射诱导的凋亡依赖于剂量，在暴露于给定的高剂量率辐射剂量后，5 - 11细胞中的凋亡细胞比例高于K - 1细胞。然而，对于产生相同水平克隆存活的剂量，凋亡诱导的水平和时间进程相似。发现辐射处理在相同程度上延迟了5 - 11和K - 1细胞通过细胞周期的进程，分别在照射后4小时和12小时，细胞在S期和G2期积累。没有证据表明存在G1期停滞。蛋白质免疫印迹分析显示，未照射的5 - 11细胞中p53和Waf1蛋白水平高于K - 1细胞，并且5 Gy的高剂量率辐射剂量上调了两种细胞系中p53和Waf1蛋白的表达至相似水平。照射后两种细胞系中Gadd45和Bcl2蛋白水平均无变化。这些结果表明，p53和Waf1蛋白的积累不会导致这些细胞早期G1期停滞。
