Radiosensitization by inhibiting DNA-protein kinase and microenvironmental factor

比较了低pH微环境和中性pH微环境中Wortmannin对人肿瘤细胞的放射增敏作用。A-172人胶质母细胞瘤细胞、A-549人腺癌细胞和HMV-I人黑色素瘤细胞在照射前2 h用20 um Wortmannin处理，检测细胞存活率。在Wortmannin处理细胞之前，将细胞置于低pH培养液中24 h，以模拟低pH微环境。用脉冲场凝胶电泳法测定了低pH和中性条件下50次照射后，Wortmannin对DNA双链断裂(DSB)修复的影响。用Western印迹分析比较了DNA依赖蛋白激酶催化亚单位(DNA-PKcs)在低pH和中性pH条件下的表达。在低pH培养条件下，Wortmannin对所有细胞系的放射增敏作用均强于中性pH培养条件。在低pH条件下，DNA DSB修复的快速重合部分受到的抑制比在中性pH条件下更强，尽管DNA-PKcs在两组之间的表达差异不大。低pH培养条件是为了模拟活跃增殖的实体瘤中心肿块的微环境，通过抑制DNA DSB修复的快速连接部分和延长未修复的DNA DSB的保留时间来增强Wortmannin的放射增敏作用。

The radiosensitizing effect of wortmannin on human tumor cells in a low-pH microenvironment was compared with that in a neutral-pH environment. A-172 human glioblastoma cells, A-549 human adenocarcinoma cells, and HMV-I human melanoma cells were treated with 20 uM wortmannin 2 h before irradiation, and cell survival was examined. A low-pH microenvironment was simulated by exposing cells to low-pH culture medium for 24 h before wortmannin treatment. The effect of wortmannin on the repair of DNA double-strand breaks (dsbs) after 50 Gyirradiation in both low-and neutral-pH conditions were measured by pulse-field gel electrophoresis. Expression of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs)in low-pH conditions was also compared with that in neutral-pH conditions was also compared with that in neutral-pH conditions by Western blot analysis. The radiosensitizing effect of wortmannin was greater in low-pH cultures than in neutral-pH cultures for all cell lines. The faster-rejoining component of DNA dsb repair was inhibited more strongly in low-pH than in neutral pH conditions, although there was little difference in DNA-PKcs expression between groups. The low-pH culture condition, which was designed to mimic the microenvironment of the central tumor mass in actively proliferating solid tumors, enhanced the radiosensitizing effect of wortmannin by inhibiting the fast-rejoining component of DNA dsb repair and by prolonging the retention of nonrepaired DNA dsbs.