Branda R F, Lafayette A R, O'Neill J P, Nicklas J A
Department of Medicine and the Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA.
Mutat Res. 1999 Jun 30;427(2):79-87. doi: 10.1016/s0027-5107(99)00095-0.
Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.
叶酸缺乏与烷化剂协同作用，增加中国仓鼠卵巢（CHO）细胞中次黄嘌呤-鸟嘌呤磷酸核糖转移酶（hprt）基因座的DNA链断裂和突变频率。为阐明这种协同作用的机制，对hprt突变体进行了分子分析。最近，我们实验室发现，与从叶酸充足培养基中获得的克隆相比，叶酸缺乏会增加暴露于甲磺酸乙酯（EMS）而非N-亚硝基-N-乙基脲（ENU）后具有基因内缺失的克隆百分比。本报告描述了对获得的37个不含缺失的hprt突变体克隆的分子分析。用EMS处理的叶酸缺乏细胞在非转录链的非CpG位点处有高频的G>A转换，特别是当这些碱基两侧均为G:C碱基对时。这些突变中有33%位于外显子3的六个G的序列中。用EMS处理的叶酸充足细胞的G>A转换百分比略低（但无显著差异），且具有相同的序列特异性。用ENU处理叶酸缺乏的CHO细胞导致相对于非转录链主要发生T>A颠换和C>T转换。这些发现提示了一个模型来解释叶酸缺乏与烷化剂之间的协同作用：（1）叶酸缺乏导致大量尿嘧啶掺入DNA；（2）碱基切除修复用于去除尿嘧啶和纠正烷化剂损伤的利用率大幅增加导致易出错的DNA修复。就EMS而言，这导致更多的基因内缺失和G:C到A:T突变，这是由于碱基切除修复过程中产生的单链断裂连接受损以及去除O6-乙基鸟嘌呤的能力降低。就ENU而言，还可见额外的T>A颠换和C>T转换，这可能是由于O2-乙基嘧啶的错配。纠正叶酸缺乏可能会降低烷化剂治疗期间这类遗传损伤的频率。
