DNA Repair of Endogenous Lesions in Carcinogenesis

描述（由申请人提供）：本项目的长期目标是阐明内源性修饰DNA的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DNA加合物（醛诱导），在致癌过程中起着关键作用。ROS和enals已被证明可以促进肿瘤，但详细的分子机制需要阐明。在本项目中，我们将以朗埃文斯肉桂（LEG）大鼠为模型来研究其机制。LEG大鼠在遗传上易患自发性肝细胞癌（HCC）。像威尔逊氏病（WD）患者一样，LEG大鼠在肝脏中积累了过量的铜，因此器官经历了严重的炎症和氧化应激。结果，LEG大鼠患上肝炎，幸存的动物患上肝癌。一个重要的问题是慢性氧化应激是否会损害LEG大鼠的修复能力，从而导致氧化损伤和外环加合物负担的增加在LEG大鼠肝脏HCC中起关键作用。我们的初步结果显示，与对照组Long Evans Agouti （LEA）大鼠肝脏相比，DNA糖基酶（如OGG1和NTH1）介导的氧化碱基（8-氧鸟嘌呤和二氢尿嘧啶）的切除活性在LEG大鼠HGG发育的不同阶段发生了显著变化，因此我们提出了以下具体目标。这些是：(1)研究修复DNA糖基酶和/或其他酶的活性和表达，如果发现是限速的，在LEG和LEA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，以肺为对照；(2)研究NTH1在LEG大鼠肝脏中的表达调控机制；(3)通过评估金属（铜和铁）、过氧化氢和enals对LEA和LEG大鼠肝细胞的影响，并将其与Aim 1中获得的组织中观察到的影响进行比较，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）变化的机制；(4)作为对照，研究LEG和LEA大鼠肝脏和肺部修复环丙基加合物（Acr-、Cro-和HNE-dG）的能力。我们的初步研究结果表明，在包括LEA和LEG在内的哺乳动物细胞中，主要的环丙基加合物HNE-dG通过核苷酸切除修复（NER）途径进行修复。因此，我们还将通过评估enals在LEA和LEG大鼠肝细胞中的作用，并将其与组织中观察到的效果进行比较，来研究用于修复丙烯加合物的限速NER酶的表达和/或活性的变异机制。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.