阿霉素诱导鸟嘌呤-四链体形成对Tca8113细胞端粒酶介导的端粒延伸反应的影响

Effects of guanine-quadruplexes formation induced by adriamycin on telomeric extension reaction mediated by telomerase of Tca8113 cells

目的探讨阿霉素诱导端粒重复序列核苷酸形成G4及其抑制Tca8113细胞端粒酶介导的端粒延伸反应的功能作用。方法通过非变性聚丙烯酰胺凝胶电泳迁移实验来分析不同质量浓度的阿霉素对d(TTAGGG)3、d(TTAGGG)4和d(TTAGGG)5迁移速度的影响以及对d(TTAGGG)4、d(TTAGAG)4、d(TTAGGG)5和d(TTAGGGT)电泳的影响;用二甲基硫酸盐(DMS)甲基化保护实验分析d(TTAGGG)4和d(TTAGAG)4中G的甲基化作用;利用经典端粒重复序列扩增法(TRAP)和改良TRAP-G4检测法分析阿霉素抑制Tca8113细胞端粒酶介导的端粒延伸反应的特点。结果质量浓度为5.00μg/mL阿霉素可使部分线性d(TTAGGG)4和d(TTAGGG)5转变成有二级结构的复合物,呈现为新的高速迁移条带。质量浓度为1.25μg/mL阿霉素可保护端粒重复序列中G免遭甲基化作用。质量浓度为2.50、1.25μg/mL阿霉素在TRAP检测中可部分抑制端粒延伸反应,而在TRAP-G4检测中可完全抑制端粒延伸反应。结论阿霉素可通过诱导端粒重复序列形成分子内G4结构抑制端粒酶介导的端粒延伸反应。

Objective To study the effects of adramycin to disturb telomeric extention reaction mediated by telomerase of Tca8113 cells by inducing oligonucleotides that contain telomeric repeats to form guanine-quadruplex （G4） structures. Methods In the presence of adriamycin, d（TTAGGG）4, d（TTAGAG）4, d（TTAGGG）5 and d（TTAGGGT） were analyzed by electrophoretic mobility shift assay. The mobility of d （TTAGGG）3, d （TTAGGG）4 and d（TTAGGG）5 in native polyacrylamide electrophoresis were observed. Methylation protection experiments were performed to investigate the effects of adriamycin on methylation of guanine in d（TrAGGG）4 and d（TrAGAG）, The traditional telomeric repeats amplification protocol （TRAP） and modified TRAP-G4 assays were, respectively, used to analyze the different characteristcs of adriamycin＇s inhibiting telomeric extension mediated by telomerase of Tca8113 cells. Results At 5.00 μg/mL of adriamycin, conversion of some of linear d （TrAGGG）4 and d （TrAGGG）5 to the new, high-mobility bands formed by complex with special second structures were found in the mobility shift assay. Adriamycin at 1.25 μg/mL protected the G in d （TrAGGG）4 from methylating. Adriamycin at 2.50 μg/mL or 1.25 μg/mL partially inhibited the telomeric extension lengthened by telomerase of Tca8113 cells in TRAP assay, but completely did so in TRAP-G4 assay. Conclusion Adriamycin is able to disturb telomeric extention mediated by telomerase of Tca8113 ceils by inducing oligonucleotides that contain telomeric repeats to form intra-molecular G4 structures.