Saleh Ekram M Y, El-Awady Raafat A E
Biochemistry Unit, Tumor Biology Department, National Cancer Institute, Cairo University.
J Egypt Natl Canc Inst. 2005 Jun;17(2):93-102.
The aim of the present study is to investigate whether differences between tumor cells in radiosensitivity are related to misrejoined- or residual DNA-double strand breaks.
An assay that allows measurement of absolute induction frequencies for DNA double strand breaks (DSBs) in defined regions in the genome, and that quantitates rejoining of correct DNA ends has been used to study repair of DSBs in three human tumor cell lines. DNA double-strand breaks (DSBs) were measured within a 3.5-Mbp Not 1 fragment on chromosome X of human tumor cell lines with different radiosensitivities. Correct rejoining of DSBs was measured by hybridization of single-copy DNA probe to Not 1 restriction fragments separated according to size by pulsed field gel electrophoresis (PFGE). Induction of DSBs is quantified from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of DSBs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis technique (FDR assay) that detects all rejoining events, it was possible to quantitate the misrejoining frequency after 50Gy of X irradiation. Residual breaks were measured 24h after irradiation.
In terms of clonogenic assay, squamous cell carcinoma cell line (4451) was the most radiosensitive, followed by the breast carcinoma cell line (BB) while the bladder carcinoma cell line (RT112) was the most radioresistant. Twenty-four hours after irradiation, 4451 cell line accumulated the highest level of residual (non-repairable) DSB followed by BB and then RT112 cell line, which showed the lowest level of residual DSB. This was the same rank as in the radiosensitivity assay. Regarding DSB misrejoining, RT112 cell line showed the highest percent of incorrectly repaired DSB, which does not agree with the results of the radiosensitivity assay.
From our data, it could be concluded that residual (non repairable) DSB is more important in terms of radiosensitivity than incorrectly repaired DSB.
本研究旨在探究肿瘤细胞放射敏感性的差异是否与DNA双链断裂错接或残留有关。
一种能够测量基因组特定区域DNA双链断裂（DSB）绝对诱导频率，并对正确DNA末端重接进行定量的检测方法，已被用于研究三种人类肿瘤细胞系中DSB的修复情况。在具有不同放射敏感性的人类肿瘤细胞系的X染色体上一个3.5兆碱基对的Not 1片段内测量DNA双链断裂（DSB）。通过将单拷贝DNA探针与经脉冲场凝胶电泳（PFGE）按大小分离的Not 1限制性片段杂交，来测量DSB的正确重接。DSB的诱导通过杂交限制性片段强度的降低以及条带下方 smear 的积累来定量。只有当正确的DNA末端连接时，DSB的重接才会导致完整限制性片段的重建。通过将该技术的结果与检测所有重接事件的传统电泳技术（FDR检测）的结果进行比较，能够对50Gy X射线照射后的错接频率进行定量。照射后24小时测量残留断裂。
就克隆形成试验而言，鳞状细胞癌细胞系（4451）放射敏感性最高，其次是乳腺癌细胞系（BB），而膀胱癌细胞系（RT112）放射抗性最强。照射后24小时，4451细胞系积累的残留（不可修复）DSB水平最高，其次是BB，然后是RT112细胞系，其残留DSB水平最低。这与放射敏感性试验中的排序相同。关于DSB错接，RT112细胞系显示出错误修复的DSB百分比最高，这与放射敏感性试验的结果不一致。
从我们的数据可以得出结论，就放射敏感性而言，残留（不可修复）DSB比错误修复的DSB更重要。
