人O6-烷基鸟嘌呤-DNA烷基转移酶突变体对O6-苄基鸟嘌呤耐药性的研究

目的　探讨人O6 烷基鸟嘌呤 DNA烷基转移酶 (O6 alkylguanine DNAalkyltransferase ,O6 AGT)中单个氨基酸的改变对O6 苄基鸟嘌呤 (O6 benzylguanine ,O6 BG)抑制作用敏感性的影响。方法应用定点诱导突变的方法 ,将人AGT的第 15 6位点的甘氨酸突变为丙氨酸 ,第 140位点的脯氨酸突变为丙氨酸 ,并检测了重组蛋白的活性。结果　点突变G15 6A和P140A的重组蛋白的AGT活性分别为(2 44± 90 )fmol/mg和 (2 31± 48)fmol/mg蛋白 ,与野生型AGT活性 (2 2 3± 35 )fmol/mg蛋白相近 ,并且对O6 BG具有耐药性。O6 BG对突变后的G15 6A AGT和P140A AGT的IC50 分别为 7.2 0 μg/ml和 0 .92 μg/ml,而对野生型AGT的IC50 为 0 .0 6 8μg/ml,耐药性分别提高了 10 5 .8及 13.5倍。 结论　应用G15 6A和P140A基因转导 ,可克服BG与烷化剂联合化疗的骨髓抑制作用

Mutation of human O~6-alkylguanine DNA alkyltransferase confers resistance to O~6-benzylguanine

Objective O 6 alkylguanine DNA alkyltransferase (O 6 AGT),capable of repairing DNA damage, is responsible for tumor cell resistance to nitrosourea. While O 6 benzylguanine as a selective inhibitor of AGT helps reverse drug resistance, it would aggravate myelo suppression. This investigation is to generate AGT mutant and see if it would confer resistance to O 6 benzylguanine induced inhibition but leave its alkyltransferase activity intact.Methods Human O 6 methylguanine DNA methyltansferase (MGMT) cDNA was mutated by site directed mutagenesis. The mutant cDNA was transferred into E.coli and the protein expressed was purified. The activity of the mutant MGMT was determined in vitro with O 6 ( 3H) methylguanine DNA as substrate.Results Two mutant MGMT proteins were obtained: G156A and P140A, with glycine to alanine mutation at position 156 and proline to alanine mutation at position 140, respectively. The AGT activity of both mutants was similar to that of the wild type MGMT. However, their resistance to O 6 benzylguanine was significantly increased up to 105.8 and 13.5 fold, respectively as compared to that of the wild type MGMT. Conclusion The results suggested that transduction of the mutant MGMT herein reported into hematopoietic progenitor cells may lead to their selective resistance to the combined use of O 6 benzylguanine and alkylating agents designed to overcome tumor resistance to nitrosourea treatment.