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Epigenetic toxicity of polycyclic aromatic hydrocarbons

多环芳烃的表观遗传毒性

基本信息

批准号：
7417349
负责人：
Brad L. Upham
金额：
$ 0.54万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-16 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7417349
关键词：
Affect Apoptosis Aromatic Hydrocarbons Aromatic Polycyclic Hydrocarbons Arts Atherosclerosis Biological Markers Carcinogens Cell Line Cell Proliferation Cell membrane Cells Chromatography Cigarette Class Classification Connexin 43 Data Development Diet Disease Dominant-Negative Mutation End Point Environment Environmental Tobacco Smoke Enzyme Inhibitor Drugs Enzyme Inhibitor Gene Enzyme Inhibitors Epigenetic Process Epithelial Epithelial Cells Event Excision Gene Expression Gene Silencing Glycyrrhetinic Acid Growth Growth and Development function High Pressure Liquid Chromatography Intercellular Communication Induction Isomerism Link Lipids Maintenance Malignant Neoplasms Marijuana Smoking Membrane Membrane Lipids Mitogen Activated Protein Kinase 1 Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinases Molecular Molecular Weight Pathway interactions Phospholipase Play Polycyclic Hydrocarbons Property Proteins Proteomics Proto-Oncogene Proteins c-akt RNA Interference Regulation Research Research Personnel Role Second Messenger Systems Series Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Molecule Signaling Protein Small Interfering RNA Stem cells Stimulation of Cell Proliferation Structure-Activity Relationship Techniques Technology Telomerase Testing Therapeutic Tobacco Toxic effect Toxicology Tumor Promotion U-0126 base cell growth cigarette smoking design human disease inhibitor/antagonist intercellular communication light scattering phospholipase inhibitor programs research study response second messenger

项目摘要

DESCRIPTION (provided by applicant): Cigarette smoking is an important cause of cancers of the lung, larynx, pharynx, esophagus, bladder, kidney, pancreas and more recently determined, the liver. Considerable toxicological research on polycyclic aromatic hydrocarbons (PAHs), which are prevalent compounds in cigarette smoke that contribute to cancer, has focused on their genotoxic attributes. However, many human diseases, such as cancer, are not solely the consequence of non-reversible mutagenic events but also include reversible, epigenetic events (altered expression of genes at transcriptional, translational and post-translational levels). Thus, there is a need to reassess the toxicity of PAHs at the epigenetic level. Gap junctional intercellular communication (GJIC) plays a central role in modulating signal transduction pathways that epigenetically alter gene expression. There is considerable evidence linking abnormal regulation of GJIC with the nongenotoxic steps of tumor promotion. Mitogen activated protein kinases (MAPKs) are also known to play a central role in cell signaling. We will use a series of tobacco smoke-relevant PAHs and determine structure activity relationships with inter- and intracellular signaling mechanisms in pluripotent mammalian, including human, epithelial cell lines. We will specifically test the hypothesis SA#1 that phospholipases are the upstream regulators of GJIC and MAPK in response to cigarette smoke-relevant PAHs by using specific phospholipase inhibitors and the emerging and powerful technique of silencing genes using small interfering RNA (siRNA). By using chromatographic techniques, we will test the hypothesis SA#2 that the phospholipases will release lipid-derived second messengers from the plasma membrane, and also the hypothesis that these messengers will activate signal transduction proteins that regulate GJIC and MAPK activity by using state of the art proteomic techniques to identify these unknown signaling proteins. We will also test the hypothesis SA#3 that alteration of GJIC will contribute to the mitogenic process and inhibit apoptosis and cell differentiation by monitoring genes and proteins specific to these biological endpoints using flow cytometry, and Western & Northern blot analyses in normal cell lines as well as those transfected with dominant negative gap junction genes or gap junction-siRNA. Our final hypothesis SA#4 to be tested will be that the effects of these PAHs on GJIC and MAPK will be the same on epithelial stem cell types from different tissues and species, which all express the same major gap junction protein. We will use our well-established F344-WB rat liver epithelial stem cell line, our newly isolated human liver stem cell line, a mouse lung epithelial cell line and a human bronchial epithelial cell line. We would like to note that the use of the biologically active 1-methyl isomer vs the inactive 2-methylisomer of anthracene, a tobacco smoke PAH, for all of the aims allows us to systematically identify molecular events that are specific to the regulation of GJIC and MAPK and subtract out non-specific events. Overall, determining the effect of tobacco-relevant PAHs on key signaling and gene expression events would provide invaluable mechanistically based information on the epigenetic toxicity of these compounds, thereby aiding in the development of preventative and therapeutic strategies of controlling human diseases such as cancer.

描述（由申请人提供）：吸烟是肺癌、喉癌、咽癌、食道癌、膀胱癌、肾癌、胰腺癌以及最近确定的肝癌的重要原因。多环芳烃 (PAH) 是香烟烟雾中常见的致癌化合物，对其进行了大量毒理学研究，重点关注其基因毒性属性。然而，许多人类疾病，例如癌症，不仅是不可逆诱变事件的结果，还包括可逆的表观遗传事件（转录、翻译和翻译后水平上基因表达的改变）。因此，有必要在表观遗传水平上重新评估PAHs的毒性。间隙连接细胞间通讯（GJIC）在调节表观遗传改变基因表达的信号转导途径中发挥着核心作用。有大量证据表明 GJIC 的异常调节与肿瘤促进的非基因毒性步骤有关。丝裂原激活蛋白激酶 (MAPK) 也被认为在细胞信号传导中发挥着核心作用。我们将使用一系列与烟草烟雾相关的多环芳烃，并确定多能哺乳动物（包括人类上皮细胞系）细胞间和细胞内信号传导机制的结构活性关系。我们将通过使用特定的磷脂酶抑制剂和使用小干扰 RNA (siRNA) 的新兴强大基因沉默技术，专门测试假设 SA#1，即磷脂酶是 GJIC 和 MAPK 的上游调节剂，以响应与香烟烟雾相关的 PAH。通过使用色谱技术，我们将测试 SA#2 的假设，即磷脂酶将从质膜释放脂质衍生的第二信使，以及这些信使将通过使用最先进的蛋白质组学技术来识别这些未知的信号蛋白，从而激活调节 GJIC 和 MAPK 活性的信号转导蛋白的假设。我们还将测试 SA#3 假设，即 GJIC 的改变将有助于有丝分裂过程并抑制细胞凋亡和细胞分化，方法是使用流式细胞术监测这些生物学终点特异的基因和蛋白质，并在正常细胞系以及转染显性阴性间隙连接基因或间隙连接-siRNA 的细胞系中进行 Western & Northern 印迹分析。我们要测试的最终假设 SA#4 是，这些 PAH 对 GJIC 和 MAPK 的影响对于来自不同组织和物种的上皮干细胞类型是相同的，它们都表达相同的主要间隙连接蛋白。我们将使用我们成熟的 F344-WB 大鼠肝上皮干细胞系、我们新分离的人肝干细胞系、小鼠肺上皮细胞系和人支气管上皮细胞系。我们要指出的是，为了实现所有目标，使用蒽（一种烟草烟雾 PAH）的生物活性 1-甲基异构体与无活性 2-甲基异构体，使我们能够系统地识别 GJIC 和 MAPK 调节特异的分子事件，并剔除非特异事件。总体而言，确定与烟草相关的多环芳烃对关键信号传导和基因表达事件的影响将为这些化合物的表观遗传毒性提供宝贵的基于机制的信息，从而有助于制定控制癌症等人类疾病的预防和治疗策略。