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. ?
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