DNA-PK的活性与鼻咽癌细胞株CNE1/CNE2放射敏感性的关系

本文主要研究DNA依赖的蛋白激酶（DNA-dependent protein kinase,DNA-PK）与鼻咽癌细胞放射敏感性之间的关系。克隆形成实验分析鼻咽癌细胞CNEI／CNE2的剂量存活曲线,Signa TECT DNA-PK试剂盒检测DNA-PK活性,免疫荧光及激光显微共聚焦分析放疗前及放疗后15min、1h、6h、12h和24hCNE1／CNE2细胞中Kus及DNA-PKcs的亚细胞定位,Western blot分析两株细胞中Kus蛋白的表达。结果显示：CNE1细胞在每个剂量点的存活分数均高于CNE2细胞;同时发现放疗前后CNE1细胞中的DNA-PK活性也均高于CNE2细胞,但两株细胞中Ku70／Ku80蛋白表达无明显差异;放疗可使DNA-PK活性增加,且各个检测时间点CNE1细胞增加的幅度大于CNE2细胞;DNA-PK亚基可同时定位于胞浆和胞核,但主要位于胞核,细胞照射后Ku70、Ku80和DNA-PKcs从胞浆转运到胞核。结果表明：DNA-PK活性更高可能是CNE1细胞较CNE2细胞更能抵抗放射的原因之一;放疗所致DNA-PK活性增高可能与DNA-PK亚基从胞浆转运到胞核有关,而与Ku蛋白表达的总量无关。

DNA-dependent protein kinase activity and radiosensitivity of nasopharyngeal carcinoma cell lines CNE1/CNE2

The present study investigated the relationship between DNA-dependent protein kinase (DNA-PK) and radiosensitivity of nasopharyngeal carcinoma (NPC) cell lines. The dose-survival relationship for NPC cell lines, CNE1 and CNE2, was analyzed using clonogenic formation assay, the activity of DNA-PK of the two cell lines was measured using the Signa TECT DNA-PK assay kit, and the localization and expression of Kus (a heterodimer) and DNA-PKcs protein in CNE1 and CNE2 before irradiation and 15 min, 1 h, 6 h, 12 h, 24 h after 4 Gy irradiation were analyzed by immunofluorescence, laser scanning confocal microscope (LSCM) and Western blot. The results showed that the surviving fraction of CNE1 was higher than that of CNE2 at each dose. The DNA-PK activity of CNE1 was also significantly higher than that of CNE2 before and after irradiation (P<0.05), while the expression of total Ku70/Ku80 in CNE1 and CNE2 had no significant difference. Increasing translocation of Ku70 and Ku80 from the cytoplasm to the nuclei in the two cell lines was observed with increase of irradiation time as detected by Western blot, and the immunofluorescence of the DNA-PK complex subunits showed greater nuclear translocation in CNE1 than CNE2 after irradiation. The results suggest that the relatively higher radio-resistance of CNE1 correlates with the higher activity of DNA-PK as compared to that of more radiosensitive CNE2 (or lower radio-resistance) before and after irradiation. Thus, DNA-PK activity may be a useful predictor of radiosensitivity of NPC.