Powell S N, McMillan T J
Radiation Research Unit, Institute of Cancer Research, Sutton, Surrey, UK.
Int J Radiat Oncol Biol Phys. 1994 Jul 30;29(5):1035-40. doi: 10.1016/0360-3016(94)90399-9.
The accuracy of DNA repair may play a role in determining the cytotoxic effect of ionizing radiation. Repair, as measured by DNA strand breakage, often shows little difference between tumor cell lines of widely different radiosensitivity. The mechanism by which DNA fragments are rejoined is poorly understood. This study used plasmid transfection as a probe to assess the balance between correct repair and misrepair.
Using techniques described, a double-strand break was introduced into a coding sequence of circular plasmid DNA using a restriction endonuclease as a model for a radiation-induced double-strand break; it was then transfected as a linear molecule into human tumor cells, and the subsequent cell-mediated restoration of the coding sequence, evidenced by intact gene function, was documented. The plasmid used in these experiments, pPMH16, is known to integrate into genomic DNA. Gene function was tested by the ability to grow colonies in selection media. The plasmid also contains a second selectable marker gene that was used to identify transfected cells, before the function of the damaged gene was tested. The proportion of transfected cells that had correctly restored the damaged gene gave a measure of repair fidelity.
A general trend for sensitive cells to show lower repair fidelity relative to resistant cells was observed. The type of double-strand cleavage of the plasmid (staggered or blunt) made little difference to the measured repair fidelity, in contrast to published studies in which restriction-enzyme breaks had been introduced into DNA within chromatin. Specific comparison of parent lines and their radiosensitive clones showed significant differences in repair fidelity for a relatively small change in radiation response, which was in line with the overall correlation. These same pairs have previously been shown to have no difference in the loss of DNA fragmentation with time after irradiation, and Southern analysis had confirmed the integrated plasmid copy number was similar in the cell lines compared. The number of intact copies of the damaged gene relative to the undamaged gene mirrored the observed repair fidelity. However, in one cell line out of the 10 studied, an exception to the observed trend was found. In a comparison of two equally radioresistant bladder cancer cell lines, large differences in repair fidelity were observed. Again, no difference in the integrated copy number was found, and the damaged gene was highly rearranged or deleted in the cell line with low repair fidelity.
These studies have shown repair fidelity to correlate closely with radiosensitivity, including the comparison of genetically related lines. It is suggested that repair fidelity can be, but is not invariably, a measure of correct repair relative to misrepair, resulting from the processing of double-strand breaks and, hence, the response to ionizing radiation.
DNA修复的准确性可能在决定电离辐射的细胞毒性作用中发挥作用。通过DNA链断裂来衡量的修复，在放射敏感性差异很大的肿瘤细胞系之间通常差异不大。DNA片段重新连接的机制尚不清楚。本研究使用质粒转染作为探针来评估正确修复和错误修复之间的平衡。
利用所述技术，使用限制性内切酶在环状质粒DNA的编码序列中引入双链断裂，作为辐射诱导双链断裂的模型；然后将其作为线性分子转染到人类肿瘤细胞中，并记录随后由完整基因功能证明的细胞介导的编码序列恢复情况。这些实验中使用的质粒pPMH16已知可整合到基因组DNA中。通过在选择培养基中生长菌落的能力来测试基因功能。该质粒还包含第二个选择标记基因，在测试受损基因的功能之前，用于鉴定转染细胞。正确恢复受损基因的转染细胞比例给出了修复保真度的度量。
观察到一个总体趋势，即敏感细胞相对于抗性细胞显示出较低的修复保真度。与已发表的将限制性酶切位点引入染色质内DNA的研究相反，质粒的双链切割类型（交错或平端）对测量的修复保真度影响不大。亲代细胞系及其放射敏感克隆的具体比较显示，对于辐射反应相对较小的变化，修复保真度存在显著差异，这与总体相关性一致。之前已经证明，这些相同的细胞系在照射后随时间的DNA片段丢失方面没有差异，Southern分析证实，所比较的细胞系中整合质粒的拷贝数相似。受损基因的完整拷贝数相对于未受损基因反映了观察到的修复保真度。然而，在所研究的10个细胞系中，有一个细胞系是观察到的趋势的例外。在比较两个同等放射抗性的膀胱癌细胞系时，观察到修复保真度存在很大差异。同样，在整合拷贝数方面没有发现差异，并且在修复保真度低的细胞系中受损基因高度重排或缺失。
这些研究表明修复保真度与放射敏感性密切相关，包括对遗传相关细胞系的比较。有人认为，修复保真度可以，但并非总是，作为相对于错误修复的正确修复的一种度量，这是由双链断裂的处理以及因此对电离辐射的反应导致的。
