Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms
Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms
复制标题
DOI：
10.1021/bi034631s
复制
发表时间：
2003-08-26
期刊：
影响因子：
2.9
通讯作者：
Tang, MS
中科院分区：
文献类型：
--
作者：
Hu, WW;Feng, ZH;Tang, MS
在ras基因超家族中，K-ras基因的密码子12（-TGGTG-）是人类癌症中最常突变的密码子。最近，我们发现，在正常人支气管上皮（NHBE）细胞中，大体积化学致癌物优先在K-ras基因的密码子12和14（-CGTAG-）处形成DNA加合物。此外，在K-ras基因的密码子12处形成的DNA加合物与包括密码子14在内的其他密码子处形成的DNA加合物相比修复较差。这些结果表明，靶向致癌物-DNA加合物的形成是在人类癌症中观察到的K-ras基因的密码子12处的高突变频率的主要原因。在密码子12和14处的这种优先致癌物-DNA加合物的形成可能是由于（1）这些密码子及其周围密码子在K-ras基因中的一级序列，（2）染色质结构，和/或（3）表观遗传因素如C5胞嘧啶甲基化或这些密码子及其周围密码子处的其他DNA修饰的影响。为了区分这些可能性，我们在（1）从NHBE细胞分离的裸露完整基因组DNA，（2）通过限制性内切酶消化的片段化基因组DNA，和（3）体外合成的含有K-ras基因外显子1序列，其具有或不具有CpG位点处的胞嘧啶甲基化以及与密码子12和14的鸟嘌呤配对的胞嘧啶。致癌物-DNA加合物在K-ras基因中的分布在核苷酸序列水平上使用UvrABC核酸酶切割方法与或不与连接介导的聚合酶链反应技术进行映射。我们已经发现，致癌物优先形成加合物在密码子12和14的K-ras基因外显子1在完整的以及在片段化的基因组DNA。相反，在含有K-ras基因外显子1序列的PCR扩增DNA片段中没有观察到密码子12和14处的这种优先DNA加合物形成。密码子14的CpG位点的胞嘧啶甲基化，或密码子14的胞嘧啶与鸟嘌呤配对，大大增强了密码子14的致癌物-DNA加合物的形成，但不影响密码子12的致癌物-DNA加合物的形成。与密码子12的鸟嘌呤配对的胞嘧啶的甲基化也没有增强在密码子12的致癌物-DNA加合物的形成。此外，我们发现在NHBE细胞中密码子14的CpG位点的胞嘧啶高度甲基化。这些结果表明，胞嘧啶甲基化是K-ras基因密码子14处优先DNA损伤的主要原因，而胞嘧啶甲基化以外的表观遗传修饰可能导致K-ras基因密码子12处优先DNA损伤。
In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B 1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene.