Genotoxicity and Repair of Tobacco-Specific Nitrosamine DNA Adducts

描述（由申请人提供）：吸烟是美国癌症死亡的单一主要原因，也是许多癌症的危险因素，包括肺癌、上气消化道、膀胱癌和胰腺癌。烟草烟雾中最强大的致癌物之一是4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）。NNK生物活性强的亲电试剂反应形成甲基和4-（3-吡啶基）-4-氧丁基（POB） DNA加合物。甲基DNA加合物在致癌中的作用已经被很好地描述了，目前的范式是甲基DNA加合物在烟草致癌的病因学中比POB-DNA加合物更重要。然而，最近发现O2-POB-dT加合物是nnk处理啮齿动物中最持久的POB加合物。我们的初步结果表明，O2-POB-dT在人类细胞中修复效率低下，在sos诱导的大肠杆菌和哺乳动物细胞中具有诱变性。本应用程序的目的是确定O2-POB-dT在哺乳动物细胞中形成突变和修复的机制。这些目标将在两个具体目标中进行研究：(1)确定参与O2-POB-dT精确和诱变旁路的聚合酶；(2)确定pob加合物修复的机制。参与旁路的聚合酶将在特定聚合酶被siRNA下调的细胞中确定，并在体外用纯化的聚合酶确定。在翻译合成过程中，链转换的作用将用无细胞提取物进行检验。修复机制将通过离体和体外实验相结合进行评估。研究人员将利用体外修复蛋白缺失的细胞，利用HPLC- MS/MS测定DNA加合物的水平，评估NER和BER的作用。通过NER和BER对O2-POB-dT的修复将在体外使用合成的寡脱氧核苷酸进行研究。转录偶联NER对O6-POB-dG和O2- POB-dT的作用将通过一种新的修饰宿主细胞再激活来探索。

DESCRIPTION (provided by applicant): Tobacco-smoking is the single major cause of cancer mortality in the US, and is a risk factor for a number of cancers including lung, upper aero-digestive tract, bladder and pancreas. One of the most powerful carcinogens in tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is bioactivated to potent electrophiles that react to form methyl and 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts. Role of methyl-DNA adducts in carcinogenesis have been well characterized and the current paradigm is that methyl- DNA adducts are more important than POB-DNA adducts in the etiology of tobacco-induced cancers. However, recently it was found that O2-POB-dT adduct is the most persistent POB adduct in NNK-treated rodents. Our preliminary results show that O2-POB-dT is inefficiently repaired in human cells and is mutagenic in SOS-induced E. coli and mammalian cells. The objective of this application is to determine the mechanisms by which O2-POB-dT forms mutations and is repaired in mammalian cells. These goals will be examined in two specific aims: (1) to determine the polymerases involved in accurate and mutagenic bypass of O2-POB-dT and (2) to determine the mechanisms by which the POB-adducts are repaired. The polymerases involved in the bypass will be determined in cells in which specific polymerases are down regulated by siRNA, and in vitro with purified polymerases. The role of strand switching during translesion synthesis will be examined with cell- free extracts. The repair mechanisms will be evaluated by a combination of ex vivo and in vitro experiments. The roles of NER and BER will be evaluated ex vivo using cells with deficient repair proteins using a HPLC- MS/MS assay to measure levels of DNA adducts. The repair of O2-POB-dT via NER and BER will examined in vitro using synthetic oligodeoxynucleotides. The role of transcription-coupled NER for O6-POB-dG and O2- POB-dT will be probed with a novel modified host cell reactivation.