Epigenetic toxicity of polycyclic aromatic hydrocarbons

描述(申请人提供)：吸烟是肺癌、喉癌、咽癌、食道癌、膀胱癌、肾癌、胰腺癌和最近确定的肝脏癌症的重要原因。多环芳烃(PAHs)是香烟烟雾中常见的致癌化合物，对其进行了大量的毒理学研究，重点放在它们的遗传毒性属性上。然而，许多人类疾病，如癌症，不仅仅是不可逆转的诱变事件的后果，还包括可逆的表观遗传事件(基因在转录、翻译和翻译后水平上的表达改变)。因此，有必要在表观遗传水平上重新评估多环芳烃的毒性。缝隙连接细胞间通讯(GJIC)在调节表观遗传学改变基因表达的信号转导通路中起着核心作用。有相当多的证据表明，GJIC的异常调节与肿瘤促进的非遗传毒性步骤有关。丝裂原活化蛋白激酶(MAPK)也被认为在细胞信号转导中发挥核心作用。我们将使用一系列与烟草烟雾相关的多环芳烃，并在多能哺乳动物中确定结构活性与细胞间和细胞内信号机制的关系，包括人类和上皮细胞系。我们将通过使用特定的磷脂酶抑制剂和使用小干扰RNA(SiRNA)沉默基因的新兴而强大的技术，具体测试假设SA#1，即磷脂酶是GJIC和MAPK的上游调节因子，以响应香烟烟雾相关的多环芳烃。通过使用层析技术，我们将检验假设SA#2，即磷脂酶将从质膜中释放脂质衍生的第二信使，以及假设这些信使将激活调节GJIC和MAPK活性的信号转导蛋白，通过最新的蛋白质组学技术来鉴定这些未知的信号蛋白。我们还将测试假设SA#3，即GJIC的改变将有助于有丝分裂过程和抑制细胞分化，方法是使用流式细胞仪监测这些生物终点的特异性基因和蛋白质，并对正常细胞系以及那些转导显性负间隙连接基因或间隙连接-siRNA的细胞进行Western&Northern印迹分析。我们要验证的最后一个假设SA#4将是，这些多环芳烃对GJIC和MAPK的影响将在来自不同组织和物种的上皮干细胞类型上相同，它们都表达相同的主要缝隙连接蛋白。我们将使用我们建立的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。我们想指出的是，为了所有目的，使用具有生物活性的1-甲基异构体和不活跃的2-甲基菲(一种烟草烟雾多环芳烃)，使我们能够系统地识别调控GJIC和MAPK的分子事件，并减去非特异性事件。总体而言，确定与烟草相关的多环芳烃对关键信号和基因表达事件的影响将提供关于这些化合物表观遗传毒性的宝贵机械信息，从而有助于制定控制癌症等人类疾病的预防和治疗策略。

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.