An Jing, Xu Qing-Zhi, Sui Jian-Li, Bai Bei, Zhou Ping-Kun
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Peoples Republic of China.
Int J Cancer. 2005 Nov 20;117(4):531-7. doi: 10.1002/ijc.21093.
DNA-dependent protein kinase (DNA-PK) has been intensively investigated for its roles in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair and maintenance of genomic stability. Its catalytic subunit, DNA-PKcs, a serine/threonine protein kinase, has recently been reported to be overexpressed in various human cancers, but its significance is unclear. In our study, we synthesized 3 small interfering RNA (siRNA) oligonucleotides, which separately target the translation initiation region, catalytic motif and a sequence between the scid-mutation region and the FATC motif of DNA-PKcs; 3 stable cell lines were generated from HeLa cells transfected with these siRNA constructs, respectively. All 3 siRNAs resulted in remarkable depression on DNA-PKcs expression in HeLa cells, and led to an increased sensitivity to 2 or 4 Gy of gamma-ray as well as 5 or 10 J/m(2) of ultraviolet (UV) irradiation. The siRNA targeting the catalytic motif of DNA-PKcs exhibited the greatest efficiency of radiosensitization. We demonstrated that c-myc protein level was suppressed more than 80% by siRNA-mediated silencing of DNA-PKcs. Using an E-box enhancer (c-myc binding element) driving a secreted alkaline phosphatase (SEAP) reporter strategy, we further found that the transcriptional activity of c-myc was extremely suppressed by silencing DNA-PKcs. The highest suppression effect on c-myc expression was observed in the cells transfected with the siRNA targeting the catalytic motif of DNA-PKcs. Moreover, a similar suppression on c-myc expression and activity was also detected in HeLa cells treated with wortmannin, a phosphatidylinositol (PI)-3 kinase inhibitor. However, silencing DNA-PKcs did not change the level of c-myc mRNA. We have further identified the interaction between DNA-PKcs and c-myc protein. Together, our results imply that DNA-PKcs activity is necessary or contributory to the expression of c-myc protein. Targeting DNA-PKcs is an attractive anticancer strategy, which can achieve through at least two mechanistic pathways: (i) sensitizing cancer cells to radiotherapy or chemotherapy of DNA-damaging agents and (ii) downregulation of c-myc protein.
DNA依赖性蛋白激酶（DNA-PK）因其在DNA双链断裂修复的非同源末端连接（NHEJ）途径及维持基因组稳定性中的作用而受到深入研究。其催化亚基DNA-PKcs是一种丝氨酸/苏氨酸蛋白激酶，最近有报道称其在多种人类癌症中过表达，但其意义尚不清楚。在我们的研究中，我们合成了3种小干扰RNA（siRNA）寡核苷酸，它们分别靶向DNA-PKcs的翻译起始区域、催化基序以及scid突变区域和FATC基序之间的序列；分别用这些siRNA构建体转染HeLa细胞，生成了3种稳定细胞系。所有3种siRNA均导致HeLa细胞中DNA-PKcs表达显著降低，并导致对2或4 Gy的γ射线以及5或10 J/m²的紫外线（UV）照射的敏感性增加。靶向DNA-PKcs催化基序的siRNA表现出最大的放射增敏效率。我们证明，通过siRNA介导的DNA-PKcs沉默，c-myc蛋白水平被抑制了80%以上。使用驱动分泌碱性磷酸酶（SEAP）报告基因的E-box增强子（c-myc结合元件）策略，我们进一步发现，沉默DNA-PKcs可极大地抑制c-myc的转录活性。在转染靶向DNA-PKcs催化基序的siRNA的细胞中，观察到对c-myc表达的抑制作用最强。此外，在用磷脂酰肌醇（PI）-3激酶抑制剂渥曼青霉素处理的HeLa细胞中，也检测到对c-myc表达和活性的类似抑制。然而，沉默DNA-PKcs并未改变c-myc mRNA的水平。我们进一步鉴定了DNA-PKcs与c-myc蛋白之间的相互作用。总之，我们的结果表明，DNA-PKcs活性对于c-myc蛋白的表达是必要的或有促进作用的。靶向DNA-PKcs是一种有吸引力的抗癌策略，可通过至少两种机制途径实现：（i）使癌细胞对DNA损伤剂的放疗或化疗敏感，以及（ii）下调c-myc蛋白。
