依托泊苷影响HEK293细胞同源重组修复体系

抗肿瘤药物疗效的研究多集中在肿瘤细胞，目前针对正常细胞的研究颇少，有必要建立能进行定量分析的同源重组定量修复体系。我们已建立的模型可以探讨肿瘤药物化疗后对HEK293细胞DSBs修复的继发性后果。通过构建含有带I-SceⅠ酶切位点的同源介导的重组修复底物（homologous direct recombination, HDR），或单链退火修复（single strand annealing, SSA）底物的细胞株，定量检测依托泊苷 (etoposide,VP-l6)对同源性重组修复(homologous recombination, HR)通路的影响。成功构建了可用于定量检测DNA双链断裂(double-strand break, DSBs)诱导的SSA和HDR修复的正常人HEK293细胞应用模型。细胞毒结果证实，与SSA/293对照组对比，VP-16给药组 16 μmol/L（0.475±0.029 vs 1.000±0.000, P<0.001)细胞活力明显降低；与HDR/293对照组相比，VP-16 给药组16 μmol/L（0.458±0.188 vs 1.000±0.000, P<0.05)细胞活力降低。此外，本研究证实，VP-16抑制SSA修复，VP-16给药组 2 μmol/L与SSA/293对照组相比（0.575%±0.177% vs 1.352%±0.195%, P<0.05），修复效率降低；VP-16抑制HDR修复，VP-16给药组1 μmol/L与HDR/293对照组修复效率相比（0.305%±0.078% vs. 0.635%± 0.049%，P<0.05)，修复效率降低。VP-16诱导DNA损伤的同时，抑制HDR修复和SSA修复，修复效率呈现剂量依赖性。本研究结果可为抗肿瘤药物的临床应用提供某些指导。

Effects of Etoposide on the Homologous Recombination Repair System of HEK293 Cells

Researches on the therapeutic effects of anticancer drugs mostly concentrated on tumor cells, but few studies were on normal cells. It is necessary to establish homologous recombination repair systems that can perform quantitative analysis. The models established can explore the secondary consequences of double-strand break (DSB) repair after chemotherapy on HEK293 cells. The effects of etoposide (VP-l6) on the homologous recombination (HR) pathway were quantitatively detected by constructing cell lines that contain homologous direct recombination (HDR) or single strand annealing (SSA) substrates with the I-SceⅠ restriction site. We successfully constructed normal human HEK293 cell models, which could be applied in the quantitative detection of DSB-induced SSA and HDR repairs. Cytotoxicity results confirmed that the cell viability of the VP-16-treated groups were significantly decreased at 16 μmol/L compared with the SSA/293 control groups (0.475±0.029 vs1.000±0.000, P<0.001), and decreased compared with the HDR/293 control groups (0.458±0.18 vs 81.000±0.000, P<0.05). In addition, this study confirmed that VP-16 inhibited SSA and HDR in HEK293 cells. The SSA repair efficiency was decreased in VP-16-treated groups at 2 μmol/L than the control SSA/293 cells (0.575%±0.177% vs 1.352%±0.195%, P<0.05), and the HDR repair efficiency was decreased in VP-16-treated groups at 1 μmol/L than the control HDR/293 groups (0.305% ± 0.078% vs 0.635% ± 0.049%，P<0.05). VP-16 induced DNA damages and inhibited HDR and SSA repairs, while their repair efficiencies were dose dependent. The results of this study may provide some guidance for the clinical application of anti-tumor drugs.