DNA Repair of Endogenous Lesions in Carcinogenesis

描述（由申请人提供）：该项目的广泛长期目标是阐明内源性修饰的DNA修复在预防癌症中的作用。我们的总体假设是内源性DNA损伤的修复，包括氧化碱基（活性氧（ROS）诱导）和环状DNA加合物（烯醛诱导），在致癌作用中起着关键作用。ROS和烯醛本身已被证明会引起肿瘤促进，但详细的分子机制需要阐明。本研究以Long Evans Cinnamon（LEG）大鼠为模型，探讨其作用机制。LEG大鼠在遗传上易患自发性肝细胞癌（HCC）。像威尔逊病（WD）患者一样，LEG大鼠在肝脏中积累过量的铜，因此器官经历严重的炎症和氧化应激。结果，LEG大鼠患上肝炎，存活的动物患上肝癌。一个重要的问题是慢性氧化应激是否损害LEG大鼠的修复能力，因此，增加的氧化损伤和环外加合物负荷在LEG大鼠肝脏的HCC中起着关键作用。我们的初步研究结果表明，DNA糖基化酶（如OGG 1和NTH 1）介导的切除活性的氧化碱基（8-氧代鸟嘌呤和二氢尿嘧啶）在不同阶段的HGG发展的LEG大鼠相比，在控制长伊文思Agglomerate（莱亚）大鼠肝脏显着改变，并因此促使我们提出了以下具体的目标。这些目标是：（1）研究LEG和莱亚大鼠肝脏中修复氧化碱基和乙烯加合物的碱基切除修复（BER）途径中修复DNA糖基化酶和/或其他酶（如果发现是限速酶）的活性和表达，并以肺作为对照;（2）研究LEG大鼠肝脏中NTH 1表达的调节机制;（3）通过评估金属的影响，检查DNA糖基化酶和/或其他限速BER酶的表达和/或活性（如果有的话）的变化机制（铜和铁），过氧化氢和烯醛在莱亚和LEG大鼠肝细胞中的作用，并与从Aim 1获得的组织中观察到的作用进行比较;（4）在LEG和作为对照的莱亚大鼠肝和肺中研究修复环丙烷加合物（Acr-、Cro-和HNE-dG）的能力。我们的初步研究结果表明，HNE-dG，一个主要的环丙烷加合物是通过核苷酸切除修复（NER）途径修复的哺乳动物细胞，包括莱亚和LEG肝脏。因此，我们还将通过评估烯醛在莱亚和LEG大鼠肝细胞中的作用并将其与组织中观察到的作用进行比较，来检查用于修复丙加合物的限速NER酶的表达和/或活性的变化机制（如果发现）。综上所述，本项目获得的数据将有助于阐明内源性DNA损伤（氧化碱基和环状DNA加合物）及其修复在突变和肝癌发生中的作用。

DESCRIPTION (provided by applicant): The broad long-term goal of this project is to elucidate the role of repair of endogenously modified DNA in the prevention of cancer. Our overall hypothesis is that the repair of endogenous DNA damage, including oxidized bases (Reactive Oxygen Species (ROS)-induced) and cyclic DNA adducts (enal-induced), plays a critical role in carcinogenesis. ROS and enals as such have been shown to elicit tumor promotion but detailed molecular mechanisms need to be elucidated. In this project we will use the Long Evans Cinnamon (LEG) rat as a model to investigate the mechanism. The LEG rat is genetically predisposed to spontaneous hepatocellular carcinoma (HCC). Like Wilson's disease (WD) patients, LEG rats accumulate excess copper in the liver and consequently the organ experiences severe inflammation and oxidative stress. As a result, the LEG rats develop hepatitis and the surviving animals get liver cancer. An important question is whether the chronic oxidative stress impairs repair capacity in LEG rats and as a consequence the increased oxidative damage and exocyclic adduct burden plays a critical role in HCC in LEG rat livers. Our preliminary results showed that DNA glycosylases (such as OGG1 and NTH1)-mediated excision activity of oxidized bases (8-oxoguanine and dihydrouracil) was significantly altered at different stages of HGG development in LEG rat compared to that in the control Long Evans Agouti (LEA) rat livers, and, thus prompted us to propose the following specific aims. These are to: (1) study activity and expression of repair DNA glycosylases and/or other enzymes, if found to be rate-limiting, in base excision repair (BER) pathway for repairing oxidized bases and etheno adducts in LEG and LEA rat livers, with lungs as a control; (2) study the mechanisms of regulation of NTH1 expression in LEG rat liver; (3) examine the mechanism of variation of expression and/or activity, if any, of DNA glycosylase(s) and/or other rate-limiting BER enzymes by assessing the effects of metals (copper and iron), hydrogen peroxide, and enals in hepatocytes of LEA and LEG rats and comparing the effects with that observed in tissues obtained from Aim 1; (4) study capacity to repair cyclic propano adducts (Acr-, Cro-, and HNE-dG) in LEG and LEA rat livers and lungs as control. Our preliminary results showed that HNE-dG, a major cyclic propano adduct is repaired by nucleotide excision repair (NER) pathway in mammalian cells including LEA and LEG livers. So, we will also examine the mechanism of variation, if found, of expression and/or activity of rate-limiting NER enzymes for repairing propano adducts by assessing the effects of enals in hepatocytes of LEA and LEG rats and comparing the effects with that observed in tissues. In summary, the data obtained by this project will be useful in elucidating the role of the endogenous DNA damage (oxidized bases and cyclic DNA adducts), and their repair in mutation and liver carcinogenesis.