人肿瘤细胞系Ligase Ⅳ基因突变与放射敏感性的关系研究

Relation between mutation of DNA-Ligase Ⅳ gene and radiosensitivity in human nasopharyngeal carcinoma cell lines

目的 研究人肿瘤细胞DNA LigaseⅣ基因突变与放射敏感性的关系。方法 常规集落形成方法测定人鼻咽鳞癌 (CNE)、肺腺癌 (SPC A1)和乳腺腺癌 (MCF 7)细胞系不同剂量照射后的细胞存活分数 ,采用聚合酶链式反应和克隆技术测定 3种细胞参与DNA损伤修复的DNAligaseⅣ基因序列 ,分析它们的同源性变化和突变对基因产物亲疏水性的影响。结果 3种细胞存活参数比较存在较大差异 ,CNE细胞较SPC A1和MCF 7细胞显示出较高的放射敏感性。CNE、SPC A1和MCF 7细胞LigaseⅣ基因同源性分别为 99.95 %、99.99%和 99.98% ,包括碱基转换和颠换突变。部分突变碱基导致氨基酸替代并影响基因产物的亲疏水结构。CNE细胞LigaseⅣp的 313aaHis→Arg、5 38aaGly→Arg、5 79aaLys→Arg和 5 85aaAsn→Ser替代与CNE细胞放射敏感性高于SPC A1和MCF 7细胞有关。结论 LigaseⅣp在非同源性末端连接 (NHEJ)途径中起关键和最终连接作用 ,该基因突变影响肿瘤细胞的双链断裂损伤修复能力和放射敏感性。

Objective To define the correlation between mutation of DNA-LigaseⅣ gene and radiosensitivity.Methods Nasopharyngeal squamous carcinoma cell line (CNE), lung adenocarcinoma cell line (SPC-A1) and breast adenocarcinoma cell line(MCF-7) after irradiation were assessed with specific biological parameters. Polymerase chain reaction, cloning and sequencing techniques were used to determine the sequence of DNA-LigaseⅣ gene in these three cell lines. Then the impact of homologous change and mutation on hydrophicity-hydrophobicity of genic products was analyzed.Results The surviving fractions derived from irradiation were different with more radiosensitivity in the CNE cell line than in the others. In three cell lines, the homology of LigaseⅣ gene were: 99.95%,99.99%,99.98%, respectively. Some mutations including transversion and transition were detected and led to alterations in the hydrophicity-hydrophobicity function of products. Higher radiosensitivity of CNE was associated with amino-acid substitutions: 313aa His→Arg,538aa Gly→Arg,579aa Lys→Arg and 585aa Asn→Ser.Conclusion These results suggest that LigaseⅣp play an important role in the ligation of DNA double strand breaks and certain mutations bring about changes in radiosensitivity.