Hickson I, Fairbairn L J, Chinnasamy N, Dexter T M, Margison G P, Rafferty J A
CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK.
Gene Ther. 1996 Oct;3(10):868-77.
Low levels of expression in haemopoietic cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (A Tase), is associated with the dose-limiting sensitivity of these cells to the chemotherapeutic chloroethylating and related methylating agents. Thus, the use of agents which deplete ATase such as O6-benzylguanine (O6-beG), as a tumour sensitisation strategy is likely further to potentiate collateral toxicity in bone marrow. In order to address this problem, we have engineered two mutants of human ATase (hAT) for resistance to O6-beG and characterised the in vitro properties of the proteins. In one mutant protein (hATPA), the proline at position 140 was changed to an alanine, whilst in the other (hATPA/GA) an additional mutation (glycine 156 to alanine) was also introduced. The I50 values for O6-beG of hAT, hATPA and hATPA/GA are 0.16, 2.5 and > 500 microM respectively, indicating that hATPA is resistant and hATPA/GA effectively refractory to O6-beG inactivation. Both mutant proteins retain comparable methyl transfer kinetics to those of nonmutant hAT and although they are thermally less stable in vitro than the wild-type protein, both can be substantially stabilised by DNA. Expression of either hAT or hATPA/GA following gene transfer into RJKO cells, raised the D37 value for mitozolomide from 0.35 microgram/ml for control cells to 10 micrograms/ml in the absence of O6-beG. However, whilst hAT-mediated protection was ablated by 20 microM O6-beG, the hATPA/GA protein provided protection against mitozolomide under the same conditions. Similar observations were made with chlorozotocin. The data suggest that transfer and expression of O6-beG resistant ATase in normal progenitor cells, should be a useful therapeutic strategy to protect the cells from the cytotoxic effects of the O6-alkylating agents even when used in combination with tumour sensitising agents such as O6-beG.
DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶（A Tase）在造血细胞中的低水平表达，与这些细胞对化疗性氯乙基化及相关甲基化剂的剂量限制性敏感性相关。因此，使用诸如O6-苄基鸟嘌呤（O6-beG）等能消耗A Tase的药物作为肿瘤致敏策略，可能会进一步增强骨髓中的附带毒性。为了解决这个问题，我们构建了两个人类A Tase（hAT）的突变体以使其对O6-beG产生抗性，并对这些蛋白质的体外特性进行了表征。在一个突变蛋白（hATPA）中，第140位的脯氨酸被替换为丙氨酸，而在另一个（hATPA/GA）中还引入了额外的突变（第156位的甘氨酸替换为丙氨酸）。hAT、hATPA和hATPA/GA对O6-beG的I50值分别为0.16、2.5和>500 microM，表明hATPA具有抗性，而hATPA/GA对O6-beG失活有效耐受。两种突变蛋白都保留了与非突变hAT相当的甲基转移动力学，并且尽管它们在体外的热稳定性低于野生型蛋白，但两者都能通过DNA得到显著稳定。将hAT或hATPA/GA基因转移到RJKO细胞后表达，在不存在O6-beG的情况下，使米托蒽醌的D37值从对照细胞的0.35微克/毫升提高到了10微克/毫升。然而，虽然hAT介导的保护作用在20 microM O6-beG存在时被消除，但hATPA/GA蛋白在相同条件下提供了对米托蒽醌的保护。用氯脲霉素也得到了类似的观察结果。数据表明，在正常祖细胞中转移和表达对O6-beG有抗性的A Tase，应该是一种有用的治疗策略，可保护细胞免受O6-烷基化剂的细胞毒性作用，即使与诸如O6-beG等肿瘤致敏剂联合使用时也是如此。
