Lipid peroxidation product induced DNA damage in the p53 gene and liver cancer

描述(申请人提供)：肝细胞癌是世界范围内的一种主要恶性肿瘤。流行病学研究表明，乙肝病毒感染和膳食黄曲霉毒素B1(AFB1)是人类肝细胞癌的两个主要病因。然而，这两种药物如何诱导肝癌发生的潜在机制尚不清楚。胆碱缺乏饮食(CDD)可诱发大鼠肝细胞癌。CDD诱导的大鼠肝细胞癌的发病机制类似于人类肝细胞癌的发病机制：早期为肝炎，随后是脂肪变性、肝硬变，最后是肝细胞癌。因此，CDD大鼠为研究肝细胞癌提供了一个很好的模型。研究发现，CDD大鼠肝细胞脂质过氧化(LPO)水平及LPO产物诱导的DNA损伤明显增加，LPO在肝细胞癌发生中可能起重要作用。LPO是一种细胞过程，通常发生在正常的生理条件下，当细胞处于氧化应激状态时，这一过程变得重要。LPO产生多种醛，如丙烯醛(ACR)、巴豆醛、丙二醛(MDA)和反式-4-羟基-2-壬烯醛(4-HNE)，这些加合物能与DNA相互作用，主要诱导G到T的颠倒。醛也能与含硫醇基的蛋白质相互作用。我们最近发现，4-HNE和丙二醛能够显著抑制细胞核苷酸切除修复(NER)。根据这些结果，我们推测这些醛可能通过两种作用在癌症发生中发挥重要作用：通过它们与DNA的相互作用诱导突变和通过它们与修复蛋白的相互作用抑制DNA修复。通过在序列水平上定位4-HNE-DNA加合物在人P53基因片段和基因组DNA中的分布，我们发现4-HNE优先与-GAGGC/A-序列结合，包括P53基因的249密码子，这是肝癌中唯一的突变热点。我们的结果提出了4-HNE可能在大鼠和人类的肝癌发生中发挥特别重要的作用的强烈可能性。我们建议使用大鼠和培养的人肝细胞模型来检验这些假设。我们将确定P53基因中的4-HNE-DNA加合物是如何处理的，以及P53基因在醛介导的人类细胞DNA修复抑制中的作用。我们还将确定CDD对大鼠肝细胞修复能力、4-HNE和其他醛诱导的DNA加合物在P53基因中形成的影响以及在CDD诱导的大鼠肝细胞癌中P53突变谱的影响。此外，我们还将确定4-HNE-DG在GAGGC位点的立体结构对修复和突变的影响。这些研究的结果将大大增强我们对LPO在肝癌发生中的作用的理解。

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) is a major malignancy worldwide. Epidemiological studies have suggested that hepatitis B virus infection and dietary aflatoxin B1 (AFB1) are the two major etiological agents for human HCC. However, the underlying mechanisms of how these two agents induce hepatocarcinogenesis remain unclear. In rats HCC can be induced by a choline-deficient diet (CDD). The pathogenesis of CDD-induced HCC in rats mimics the pathogenesis of human HCC: hepatitis at an early stage, followed by steatosis, cirrhosis and finally HCC. Rats on CDD therefore provide an excellent model for studying HCC. It has been found that the lipid peroxidation (LPO) level and LPO product-induced DNA damage are significantly increased in liver cells of rats on CDD; LPO has thus been suspected of playing an important role in hepatocarcinogenesis. LPO is a cellular process that commonly takes place under normal physiological conditions, and this process becomes significant when cells are under oxidative stress. LPO generates a variety of aldehydes, such as acrolein (Acr), crotonaldehyde, malondialdehyde (MDA), and trans-4-hydroxy- 2-nonenal (4-HNE), that are able to interact with DNA; these adducts induce mainly G to T transversion. Aldehydes are also able to interact with proteins with a thiol group. We have recently found that 4-HNE and MDA are able to significantly inhibit cellular nucleotide excision repair (NER). Based on these results we hypothesize that these aldehydes may play important roles in carcinogenesis through two effects: induction of mutations by their interactions with DNA and inhibition of DNA repair by their interactions with repair proteins. By mapping the 4-HNE-DNA adduct distribution at the sequence level in human p53 gene fragments and genomic DNA we have found that 4-HNE preferentially binds to -GAGGC/A- sequences, including codon 249 of the p53 gene, the sole mutational hotspot in liver cancer. Our results raise the strong possibility that 4-HNE may play a particularly important role in hepatocarcinogenesis in both rats and humans. We propose to test these hypotheses using both the rat and cultured human hepatocyte model. We will determine how 4-HNE-DNA adducts in the p53 gene are processed, and the role of the p53 gene in aldehyde-mediated inhibition of DNA repair in human cells. We will also determine the effect of CDD on the repair capacity in rat liver cells, the formation of 4-HNE- and other aldehyde-induced DNA adducts formed in the p53 gene and the p53 mutational spectrum in CDD-induced HCC in rats. In addition, we will determine the effect of stereostructure of 4-HNE-dG at GAGGC sites on repair and mutagenicity. Results from these studies should greatly enhance our understanding of the role of LPO in hepatocarcinogenesis.