Radiosensitization by inhibiting DNA-protein kinase and microenvironmental factor

比较了wortmannin在低ph微环境和中性ph环境下对人肿瘤细胞的放射增敏作用。在照射前2小时用20 uM wortmannin处理A-172人胶质母细胞瘤细胞、A-549人腺癌细胞和HMV-I型人黑色素瘤细胞，观察细胞存活率。在wortmannin处理前，将细胞暴露在低ph培养基中24 h，模拟低ph微环境。采用脉冲场凝胶电泳法测定了wortmannin在低ph和中性ph条件下对DNA双链断裂（dsbs）修复的影响。Western blot分析了低ph条件下dna依赖性蛋白激酶催化亚基（DNA-PKcs）与中性ph条件下的表达情况，并比较了中性ph条件下的表达情况。在所有细胞系中，wortmannin在低ph培养中比在中性ph培养中具有更大的放射增敏作用。DNA dsb修复的快速重连接组分在低pH条件下比在中性pH条件下受到更强烈的抑制，尽管各组之间DNA- pkcs的表达差异不大。低ph培养条件旨在模拟活跃增殖实体瘤中心肿瘤块的微环境，通过抑制DNA dsb修复的快速重新连接成分和延长未修复的DNA dsb的保留，增强了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.