Carcinogenesis of Environmentally Relevant Ni Compounds

环境相关镍化合物的致癌作用

• 批准号: 6648316
• 负责人: Max Costa
• 金额: $ 53.7万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6648316
• 关键词: DNA methylation; acetylation; chemical carcinogen; chemical carcinogenesis; enzyme inhibitors; gene expression; gene induction /repression; histones; nickel; nucleic acid sequence; polymerase chain reaction; tissue /cell culture

In the past several years we have demonstrated that carcinogenic nickel compounds inactivate g ne expression by enhancing DNA methylation and inhibiting histone acetylation. The long-term goals of this grant proposal are to study the epigenetic mechanisms of nickel carcinogenesis. The short-term goals of the proposal are to study the mechanism by which nickel silences genes through DNA methylation and a newly discovered mechanism involving inhibition of histone H4 acetylation. We believe that these mechanisms involve nickel-binding to histidine at position 18 on the N-terminal tail of histone H4. In the absence of histone acetylation, an acidic region of H2A and H2B interact avidly with the basic 1ysines of the histone H4 tail. The specific aims of the grant proposal are to study the mechanism by which nickel inhibits the acetylation of lysines of H3 and H4 tails with the hypothesis that nickel-binding to histidine 18 on the N-terminal of H4 is responsible for a selective inhibition of H4 acetylation since H3 lacks a metal binding amino acid on its tail. We will study nickel-binding to H4 using the whole protein and model peptides with various analytical tools, such as XAS, in collaboration with inorganic chemists, Drs. Maria Zoroddu and Michael Maroney. We will also invest ate the relationship between histone acetylation and gene silencing in mammalian cells using histone deacetylase inhibitors, such as trichostatin A. We will examine whether trichostatin A can antagonize nickel-induced silencing and can revert nickel-silenced genes both in the G12/G10 mammalian system, as well as the URA3 gene which is silenced by nickel in yeast. We will utilize the chromosome immunoprecipitaton assay to investigate whether nickel effects gene-specific histone H3 or H4 acetylation. This assay involves crosslinking of histories to DNA with formaldehyde after which the DNA is fragmented to 0.5-2 Kb fragments, immunoprecipitated with antibodies against acetylated H3 and H4 and the crosslinked proteins are released and specific DNA sequences associated with acetylated histones are assessed by quantitative PCR. This assay will be applied tooth the G12 nickel-silenced system, as well as the URA3 gene in the nickel- silenced yeast system. Using the Affymetrix GeneChip, we will identify human genes turned-off following exposure to nickel compounds and reactivated by the histone deacetylase inhibitor trichostatin A. These human genes will then be further examined by the chromosome immunoprecipitaton assay and also will be studied in terms of the effects of DNA methylation on their expression. These studies should greatly enhance our body of knowledge concerning the epigenetic mechanisms of nickel carcinogenesis.

在过去的几年中，我们已经证明了致癌镍化合物通过增强DNA甲基化并抑制组蛋白乙酰化来使G NE表达失活。该赠款提案的长期目标是研究镍癌发生的表观遗传机制。该提案的短期目标是研究通过DNA甲基化沉默基因和一种涉及抑制组蛋白H4乙酰化的新发现的机制。我们认为，这些机制涉及组蛋白H4的N末端尾部的镍结合到组氨酸。在没有组蛋白乙酰化的情况下，H2A和H2B的酸性区域与组蛋白H4尾的碱性1肌胺狂热相互作用。赠款提案的具体目的是研究镍抑制H3和H4尾巴赖氨酸的乙酰化的机制，假设H4的N末端对组氨酸18的镍结合构成了H4乙酰化的选择性抑制，因为H3缺乏H3缺乏金属结合在其尾部上的金属结合氨基酸。我们将使用整个蛋白质和模型肽使用各种分析工具（例如XAS）与无机化学家DRS一起研究H4的镍结合。 Maria Zoroddu和Michael Maroney。 We will also invest ate the relationship between histone acetylation and gene silencing in mammalian cells using histone deacetylase inhibitors, such as trichostatin A. We will examine whether trichostatin A can antagonize nickel-induced silencing and can revert nickel-silenced genes both in the G12/G10 mammalian system, as well as the URA3 gene which is silenced by nickel in yeast.我们将利用染色体免疫蛋白酶测定法研究镍效应基因特异性组蛋白H3还是H4乙酰化。该测定法涉及与甲醛与DNA进行交联，然后将DNA碎片碎片至0.5-2 kb片段，用抗乙酰化H3和H4的抗体对免疫沉淀，并通过定量PCR释放与乙酰化组织酮相关的交联蛋白，并释放与乙酰化组织酮相关的特定DNA序列。该测定法将被牙齿添加到G12镍脱脂系统以及镍静音酵母系统中的URA3基因。使用Affymetrix Genechip，我们将在暴露于镍化合物后识别人类基因，并由组蛋白脱乙酰基酶抑制剂Trichostatin A重新激活。然后，这些人类基因将通过染色体免疫原子化测定进一步研究，并将以DNA甲基化对其表达的影响进行研究。这些研究应大大增强我们关于镍癌发生的表观遗传机制的知识。