Calléja F, Jansen J G, Vrieling H, Laval F, van Zeeland A A
MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, Netherlands.
Mutat Res. 1999 Apr 6;425(2):185-94. doi: 10.1016/s0027-5107(99)00034-2.
Exposure of mammalian cells to alkylating agents causes transfer of alkyl groups to N- as well as O-atoms of DNA bases. Especially the O-alkylated G and T bases have strong mutagenic properties, since they are capable of mispairing during replication. The mutagenic potential of N-alkylbases is less clear although specific base excision repair (BER) pathways exist which remove those lesions from the DNA. We investigated the relative contribution of N-alkylations to mutation induction at the Hprt gene in cultured Chinese hamster ovary cells (CHO). To this end BER activity in CHO cells was modulated by introduction of an expression vector carrying the rat N-alkylpurine-DNA glycosylase (APDG) gene, which codes for a glycosylase that is able to remove 3-methyladenine and 7-methylguanine from DNA thereby generating apurinic sites. Upon selection of a CHO clone which 10 times overproduced APDG compared to control CHO cells, mutation induction, the mutational spectrum, and cell survival were determined in both cell lines following treatment with methyl methanesulfonate (MMS). The results show that over-expression of APDG renders CHO cells more sensitive for mutation induction as well as cytotoxicity induced by MMS. The involvement of apurinic sites in induction of base pair changes at positions where 3-methyladenine was induced is inferred from the observation that the mutational spectrum of MMS-induced mutations in APDG-CHO cells showed twice as much base pair changes at AT base pairs (33.3%) compared to the spectrum of MMS-induced mutations in CHO-control cells (15.8%).
哺乳动物细胞暴露于烷化剂会导致烷基转移至DNA碱基的N原子和O原子上。尤其是O-烷基化的鸟嘌呤（G）和胸腺嘧啶（T）碱基具有很强的诱变特性，因为它们在复制过程中能够错配。尽管存在特定的碱基切除修复（BER）途径可从DNA中去除这些损伤，但N-烷基碱基的诱变潜力尚不清楚。我们研究了N-烷基化对培养的中国仓鼠卵巢细胞（CHO）中次黄嘌呤-鸟嘌呤磷酸核糖转移酶（Hprt）基因突变诱导的相对贡献。为此，通过引入携带大鼠N-烷基嘌呤-DNA糖基化酶（APDG）基因的表达载体来调节CHO细胞中的BER活性，该基因编码一种能够从DNA中去除3-甲基腺嘌呤和7-甲基鸟嘌呤从而产生脱嘌呤位点的糖基化酶。在选择了一个与对照CHO细胞相比APDG产量高出10倍的CHO克隆后，在用甲磺酸甲酯（MMS）处理后，测定了两种细胞系中的突变诱导、突变谱和细胞存活率。结果表明，APDG的过表达使CHO细胞对MMS诱导的突变诱导以及细胞毒性更敏感。从观察到APDG-CHO细胞中MMS诱导的突变谱在AT碱基对处的碱基对变化（33.3%）是CHO对照细胞中MMS诱导的突变谱（15.8%）的两倍，推断出脱嘌呤位点参与了3-甲基腺嘌呤诱导位置处碱基对变化的诱导。
