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首页> 外文期刊>Biochemistry >Formation of Benzo[a] pyrene Diol Epoxide-DNA Adducts at Specific Guanines within K-ras and p53 Gene Sequences: Stable Isotope-Labeling Mass Spectrometry Approach
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Formation of Benzo[a] pyrene Diol Epoxide-DNA Adducts at Specific Guanines within K-ras and p53 Gene Sequences: Stable Isotope-Labeling Mass Spectrometry Approach

机译：苯并[a] Gene二醇环氧-DNA加合物在K-ras和p53基因序列内特定鸟嘌呤上的形成：稳定的同位素标记质谱法

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摘要

The mutagenicity of a prominent tobacco carcinogen, benzo [a] otrebe (B[a]O), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r, 8t-dihydroxy-c9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N~2-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a] pyrene (N~2-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR(LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within condons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M.F., Pao, A., Tang, M., and Pfeifer, G.P.Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, the performed a direct quantitative analysis of N~2-BPDE-dG originating from specific guanine nucleobases within p53-and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. ~(15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, thw two genes most frequently mutated in smoking-induced lung cancer. ~(15)N-labeled DNA was annaealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N~2-BPDE-dG lesions. The extent of adduct formation at ~(15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of ~(15)N-N~2-BPDE-dG and N~2-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and P53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N~2-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, and endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N~2-BPDE-dG adducts originated from the first position of the codon 12(GGT), consistent with the large number of G -> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N~2-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N~2-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.
机译：据信，一种突出的烟草致癌物，苯并[a] otrebe（B [a] O）的诱变性是由其二醇环氧代谢产物，（+）-抗-7r，8t-二羟基-c9、10-环氧7,8,9,10-四氢苯并[a]（（BPDE）和DNA，产生致突变的损伤，例如（+）-trans-anti-7R，8S，9S-trihydroxy-10S-（N〜2 -脱氧鸟苷基）-7,8,9,10-四氢苯并[a] pyr（N〜2-BPDE-dG）。先前的研究将DNA修复酶UvrABC核酸内切酶与连接介导的PCR（LMPCR）结合使用，证明BPDE对p53肿瘤抑制基因157、248和273位密码子中的鸟嘌呤核苷的反应性增强（Denissenko，MF，Pao， A.，Tang，M.和Pfeifer，GPScience 274，430-432）。这些位点也是在吸烟者的肺部肿瘤中观察到的突变的“热点”，表明B [a] P参与了肺癌的发生。但是，LMPCR方法依赖于修复酶切除BPDE诱导的病变的能力，因此，缓慢修复的病变可能无法检测。此外，无法确定BPDE-DNA加合物的结构和立体化学。在本工作中，通过使用我们实验室最近开发的稳定同位素标记质谱法，对源自p53和K-ras衍生的DNA序列中特定鸟嘌呤核碱基的N〜2-BPDE-dG进行了直接定量分析。 〜（15）N标记的dG被置于源自K-ras原癌基因和p53抑癌基因的DNA序列中的特定位置，这两个基因在吸烟诱发的肺癌中最常发生突变。将〜（15）N标记的DNA退火到互补链上，然后对N〜2-BPDE-dG病变进行BPDE处理和液相色谱-电喷雾串联质谱分析（HPLC-ESI-MS / MS）。直接由〜（15）N-N〜2-BPDE-dG和N〜2-BPDE-dG的HPLC-ESI-MS / MS峰面积比直接确定〜（15）N标记鸟嘌呤处加合物的形成程度。 BPDE诱导的鸟嘌呤加合物沿着K-ras和P53基因衍生的DNA序列非随机产生，取决于序列背景，加合物形成的差异超过5倍。 N〜2-BPDE-dG的产量由于胞嘧啶碱基与目标鸟嘌呤核苷碱基配对的5-Me取代基的存在以及p53基因内CpG二核苷酸的内源性DNA修饰而提高。在K-ras衍生的DNA序列中，大多数N〜2-BPDE-dG加合物起源于密码子12（GGT）的第一个位置，这与在该核苷酸中观察到的大量G-> T转换一致。吸烟引起的肺癌。相反，p53外显子5序列中N〜2-BPDE-dG形成的模式与肺癌的突变谱不相关，这表明除了N〜2-BPDE-dG形成以外的其他因素也参与了这些突变。 。这项工作中描述的稳定同位素标记HPLC-ESI-MS / MS方法普遍适用于研究其他致癌物和烷基化药物对分离的DNA的修饰。

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