Polischouk A G, Cedervall B, Ljungquist S, Flygare J, Hellgren D, Grénman R, Lewensohn R
Biomedicine Unit, Swedish Radiation Protection Institute, Stockholm.
Int J Radiat Oncol Biol Phys. 1999 Jan 1;43(1):191-8. doi: 10.1016/s0360-3016(98)00362-9.
Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation.
Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting.
Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines.
The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.
肿瘤对放疗的敏感性差异与细胞修复辐射诱导的DNA双链断裂（DSB）的能力有关。DNA依赖性蛋白激酶（DNA-PK）和DNA连接酶可能会影响DNA双链断裂的重新连接。本研究旨在比较两种对电离辐射敏感性不同的人鳞状癌细胞系中辐射诱导的DSB重新连接率、DNA-PK和DNA连接酶活性。
通过体外克隆形成试验测定两种人鳞状癌细胞系UM-SCC-1和UM-SCC-14A的细胞存活率。使用脉冲场凝胶电泳（PFGE）研究DSB重新连接。使用BIOTRAK DNA-PK酶分析系统（Amersham）测定DNA-PK活性。使用[5'-33P]聚（dA）x（寡聚（dT）作为底物测量粗细胞提取物中的DNA连接酶活性。通过蛋白质印迹分析蛋白质的蛋白水解降解。
应用常用的线性二次方程来描述细胞存活率，S = e-alphaD-betaD2，两种细胞系的alpha值大致相同（约0.40 Gy（-1）），而UM-SCC-14A的beta值（0.067 Gy（-2）+/-0.007 Gy（-2）[SEM]）比UM-SCC-1（0.013 Gy（-2）+/-0.004 Gy（-2）[SEM]）高得多。此外，通过PFGE定量分析，与UM-SCC-14A相比，UM-SCC-1在重新连接X射线诱导的DSB方面更熟练。UM-SCC-1中DNA-PK活性的组成水平比UM-SCC-14A高1.6倍（<0.05）。两种细胞系中DNA连接酶活性的组成水平相似。
结果表明，DSB重新连接的熟练程度与DNA-PK活性有关，而与总DNA连接酶活性无关。
