N-烷基嘧啶DNA糖基化酶腺病毒提高人骨肉瘤化疗敏感性的研究

Effect of adenoviral N-methylpurine DNA glycosylase overexpression on chemosensitivity of human osteosarcoma cells

目的构建复制缺陷型腺病毒N-烷基嘧啶DNA糖基化酶(MPG)表达载体,观察人骨肉瘤细胞HOS过表达MPG及其对DNA损伤药物甲基甲磺酸酯(MMS)、N-甲基-N1-硝基-N-亚硝基鸟嘌呤(MNNG)和替莫唑胺(TMZ)敏感性的影响。方法应用流式细胞术、蛋白印迹和HEX标记寡核苷酸方法确定MPG腺病毒表达载体Ad5HA-MPG在人体骨肉瘤细胞HOS中的感染效率、MPG蛋白表达和MPG酶活性;MTS、硫氰酸盐B法和[3H]胸腺嘧啶掺入法检测经MMS(1、2μmol/L)、MNNG(25、50μmol/L)和TMZ(1、2mmol/L)处理过的转染细胞存活情况;PE-Annexinv/7-AAD流式细胞术检测细胞凋亡。结果10个感染增殖指数(MOI)的Ad5HA-MPG可使90%以上HOS感染,感染细胞高表达MPG并具有MPG酶活性。MPG过表达HOS显著地提高DNA损伤性化疗药物MMS、MNNG和TMZ的敏感性,其半数抑制浓度(IC50)值分别下降了6·0、4·5和2·5倍。结论Ad5HA-MPG瞬时MPG过表达,可能是一种提高骨肉瘤细胞对DNA损伤性化疗药物敏感性的潜在治疗方法。

Objective The overexpression of N-methylpurine DNA glycosylase （MPG） may imbalance the DNA base excision repair （BER） to sensitize tumor cells to current DNA damage chemotherapy. In an effort to improve the efficacy of cancer chemotherapy, we have constructed adenoviral vector of MPG, to study its ability to sensitize human osteosarcoma cell HOS to DNA damage agents. Methods The adenoviral infection and MPG expression, as well as enzyme activity were determined by flow cytometry, Western blot, and HEX labeled oligonucleotide-based assay respectively. The cell survival/ proliferation was measured using MTS, SRB, and [ ^3H ] thymidine incorporation assay. Apoptosis cell death was assayed by flow cytometry after treatment using phycoerythin （PE）-conjugated Annexin V and 7-aminoactinomycin （7-AAD）. Results A 10 MOI of recombinant nonreplicating adenovirus was found to infect more than 90% of HOS cells within 24 hours by EGFP fluorescence, in which the MPG overexpression and MPG enzyme activity were also detected. The MPG overexpression HOS cells were significantly more sensitive to the DNA damage agents, including MMS, MNNG, and TMZ, with changes in the IC50 of 6. 0, 4. 5, and 2.5 fold respectively. Condusious These data establish transient MPG overexpression as a potential therapeutic approach for increasing HOS cellular sensitivity to DNA damage agent chemotherapy.