Zinc Chemoprevention of Arsenic Co-Carcinogenesis

描述(由申请人提供)：有强有力的证据表明，除了已知的高浓度砷的直接致癌作用外，低水平、非致癌水平的砷还与其他DNA损伤剂，如苯并[a]芘和紫外线辐射(UVR)协同作用，促进致癌。这一观察结果导致确定砷是一种共同致癌物质。解释砷共同致癌的一种作用模式是抑制含有锌指DNA结合基序的DNA修复蛋白。我们实验室的研究表明：i)带有E3半胱氨酸残基的锌指蛋白是亚砷酸盐结合的直接分子靶点，包括A组干皮病(XPA)和多聚ADP核糖聚合酶(PARP)-1，它们分别是核苷酸切除修复(NER)和碱基切除修复(BER)的关键蛋白质；ii)低水平的亚砷可以增强紫外线诱导的光产物和DNA氧化损伤；以及iii)补充锌可以阻止亚砷对PARP-1的抑制作用，增强UVR诱导的DNA损伤和突变。我们假设补锌可以通过恢复BER和NER通路的DNA修复能力来阻止亚砷酸盐对UVR诱导的皮肤肿瘤的增强。这项拟议的研究将探讨一种新的机制，即锌可能通过与DNA修复途径中不同的分子靶点相互作用来防止砷增强的癌症发生。在目标1中，我们将使用多种分析方法来明确地研究亚砷酸盐和锌与代表PARP-1和XPA锌指结构域的合成肽以及与纯化蛋白的竞争性相互作用。这些发现将与功能分析相结合，以确定亚砷酸盐对靶蛋白和锌相互作用亲和力的任何已识别差异的后果。目的2分析从处理细胞中分离出的PARP-1和XPA，以测定砷和锌与蛋白质的结合及其相应的功能后果，以揭示锌逆转砷效应的具体机制。在目标3中，我们将使用生物化学方法在体内验证PARP-1和XPA作为砷的靶点，并验证锌可以防止砷增强UVR诱导的DNA损伤和皮肤肿瘤的假设。拟议研究的结果有望证明锌恢复PARP-1和XPA活性的能力，从而改善基因组完整性，减少砷对共同致癌的贡献。虽然锌主要通过其一般的抗氧化活性被认为具有化学保护作用，但这一建议是创新的，它专注于一种新的机制，通过该机制，锌可以阻止亚砷酸盐与特定的锌指DNA修复蛋白靶标结合，从而逆转亚砷酸盐对DNA修复的抑制。本研究将为明确补锌如何降低砷共癌的发生机制提供坚实的基础，并为补锌预防砷共癌的潜力提供理论依据。如果得到验证，补充锌可以代表一种低成本和易于实施的策略，用于砷暴露人群的化学预防。

DESCRIPTION (provided by applicant): There is strong evidence that in addition to the known direct carcinogenic actions of high arsenic concentrations, low, non-carcinogenic levels of arsenic synergize with other DNA-damaging agents such as benzo[a]pyrene and ultraviolet radiation (UVR) to enhance carcinogenesis. This observation has led to the identification of arsenic as a co-carcinogen. One mode of action to account for arsenic co-carcinogenesis is inhibition of DNA repair proteins containing zinc finger DNA binding motifs. Research from our lab demonstrates that: i) zinc finger proteins with e3 cysteine residues are direct molecular targets for arsenite binding, including xeroderma pigmentosum group A (XPA) and poly(ADP-ribose)polymerase (PARP)-1, which are the critical proteins for nucleotide excision repair (NER) and base excision repair (BER) respectively; ii) low levels of arsenite enhance UVR-induced photoproducts and oxidative DNA damage; and iii) supplemental zinc prevents arsenite effects on PARP-1 inhibition, enhancement of UVR-induced DNA damage and mutations. We hypothesize that zinc supplement will prevent arsenite enhancement of UVR-induced skin tumors by restoring the DNA repair capabilities in both BER and NER pathways. The proposed study will address a novel mechanism by which zinc may act to prevent arsenic-augmented carcinogenesis through interaction with distinct molecular targets in DNA repair pathways. In Aim 1, we will use multiple analytical approaches to definitively examine the competitive interaction of arsenite and zinc with synthetic peptides representing the PARP-1 and XPA zinc finger domains and with purified protein. These findings will be coupled with functional assays to establish the consequences of any identified differences in arsenite affinity for target proteins and zinc interactions. Aim 2 will analyze PARP-1 and XPA isolated from treated cells to measure arsenic and zinc binding to protein and the corresponding functional consequences to reveal the specific mechanism by which zinc reverses the arsenic effects. In Aim 3, we will validate PARP-1 and XPA as arsenic targets in vivo using biochemical methods, and test the hypothesis that zinc will prevent arsenic enhancement of UVR-induced DNA damage and skin tumors. The outcomes from the proposed studies are expected to demonstrate the capability of zinc to restore PARP-1 and XPA activity leading to improved genomic integrity and a decrease in the arsenic contribution to co-carcinogenesis. Although zinc has been considered chemoprotective largely through its reported general antioxidant activities, this proposal is innovative by focusing on a novel mechanism by which zinc prevents arsenite binding with specific zinc finger DNA repair protein targets, thus reversing the arsenite inhibition of DNA repair. This study will provide the solid foundations for a clear mechanistic understanding of how supplemental zinc reduces arsenic co-carcinogenesis, and provide the proof of principle for the potential of zinc supplement to prevent arsenic co-carcinogenesis. If validated, supplemental zinc could represent a low cost and easily implemented strategy for chemoprevention in arsenic exposed populations.