Utzat Christopher D, Clement Cristina C, Ramos Leilani A, Das Arunangshu, Tomasz Maria, Basu Ashis K
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.
Chem Res Toxicol. 2005 Feb;18(2):213-23. doi: 10.1021/tx049813h.
Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.
丝裂霉素C（MC）是一种具有细胞毒性和致突变性的抗肿瘤药物，它能使DNA烷基化并形成交联。这些作用依赖于MC的还原性生物活化。2,7 - 二氨基丝裂霉素（2,7 - DAM）是MC在肿瘤细胞中的主要代谢产物，由MC还原产生。2,7 - DAM在细胞内原位使DNA烷基化，在2'-脱氧鸟苷（2,7 - DAM - dG - N7）的N7位形成加合物。为了确定这种加合物的生物学效应，我们合成了一种含有单个2,7 - DAM - dG - N7加合物的寡核苷酸，并将其插入M13噬菌体基因组中。该构建体在具有修复能力的大肠杆菌中复制表明，该加合物毒性较弱，与对照相比产生的子代约为50%。在对来自SOS诱导或未诱导宿主细胞的4000多个子代噬菌体进行分析后，未分离到突变体；因此，我们估计在大肠杆菌中2,7 - DAM - dG - N7的突变频率小于2×10⁻⁴。随后，为了确定这种加合物在哺乳动物细胞中是否可能具有致突变性，将其掺入单链穿梭噬菌粒载体pMS2中，并在猴肾（COS - 7）细胞中复制。对后代的分析表明，位点特异性2,7 - DAM - dG - N7的突变频率不高于猴肾细胞中的自发突变频率。在无细胞系统的平行实验中，含有单个2,7 - DAM - dG - N7加合物的模板寡核苷酸在Klenow（外切酶缺失）DNA聚合酶的催化下，引导5'-³²P标记的引物链在与加合鸟嘌呤相对的位置选择性掺入胞嘧啶。加合模板也支持Klenow（外切酶缺失）和T7（外切酶缺失）DNA聚合酶对引物链的完全延伸以及DNA聚合酶η的部分延伸。2,7 - DAM - dG - N7单加合物在体内和体外的无害行为与早期报道的有毒MC - dG - N2单加合物形成鲜明对比。
