DNA Repair of Endogenous Lesions in Carcinogenesis

描述(申请人提供)：该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(ROS诱导)和环状DNA加合物(Enal诱导)，在癌症发生中起着关键作用。ROS和Enals本身已被证明可以促进肿瘤，但详细的分子机制还需要阐明。在本项目中，我们将使用Long Evans Cinnamon(LEG)大鼠作为模型来研究其机制。腿部大鼠在遗传上易患自发性肝细胞癌。与威尔逊病(WD)患者一样，腿部大鼠在肝脏中积累了过量的铜，因此该器官经历了严重的炎症和氧化应激。结果，腿部的大鼠患上了肝炎，幸存的动物患上了肝癌。一个重要的问题是，慢性氧化应激是否损害了腿部大鼠的修复能力，因此氧化损伤和外环加合物负荷的增加在大鼠腿部肝细胞癌的发生中起着关键作用。我们的初步结果表明，在腿大鼠HGG发育的不同阶段，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)的切割活性与对照Long Evans Agti(LEA)大鼠肝脏相比显著改变，从而促使我们提出以下具体目标。本研究的目的是：(1)研究修复DNA糖基酶和/或其他酶(如果被发现是限速酶)在修复大腿和LEA大鼠肝脏氧化碱基和乙烯加合物的碱基切除修复(BER)途径中的活性和表达，并以肺为对照：(2)研究大鼠腿部肝脏NTH1表达的调控机制；(3)通过检测金属(铜和铁)、过氧化氢和肠道对LEA和LEG大鼠肝细胞表达和/或活性的影响，并与目标1获得的组织中的影响进行比较，来研究DNA糖基酶(S)和/或其他限速BER酶的表达和/或活性(如果有)的变化机制；(4)研究LEA和LEA大鼠肝脏和肺组织中环丙氨酸加合物(ACR-、Cro-和HNE-DG)的修复能力作为对照。我们的初步结果表明，在包括LEA和腿部肝脏在内的哺乳动物细胞中，主要的环丙氨酸加合物HNE-DG可以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ENAL对LEA和LEG大鼠肝细胞的影响，并将其与组织中观察到的影响进行比较，来研究用于修复丙氨酸加合物的限速NER酶的表达和/或活性的变化机制。总之，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.