Hollie I. Swanson - US grants

DESCRIPTION (from Applicant's Abstract): The Ah receptor (AHR) is a ligand activated transcription factor that binds ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and is thought to mediate many of the highly toxic and carcinogenic actions of TCDD. The AHR and its DNA binding partner ARNT (Ah receptor nuclear translocator) recognize specific DNA sequences, referred to as dioxin responsive element (DREs) resulting in the activation of gene transcription. Both the AHR and ARNT are basic/helix-loop-helix proteins. Basic/helix-loop-helix proteins are characterized by their ability to form homo- or hetero-dimers, contact DNA via their basic domains and form a variety of DNA binding species that recognize distinct DNA sequences and regulate specific sets of genes. Contrary to the generally accepted theory that TCDD exerts its adverse affects through the regulation of DRE responsive genes, we propose that TCDD actions include nuclear localization of the AHR, dimerization of ARNT and the subsequent removal of ARNT from the regulatory regions of other genes. This hypothesis suggests that ARNT, in the absence of the AHR regulates genes and that this regulation is adversely affected by the presence of TCDD and the AHR. Our preliminary data has shown that ARNT can form multiple partnerships. The ARNT homodimer recognizes the CACGTG sequence, the recognization site of the Myc/Max heterodimer involved in regulating cellular proliferation and differentiation. We will use Myc/Max gene regulation as a model of AHR-independent ARNT gene regulation. First, we will determine whether the ARNT homodimer will recognize the CACGTG site with high affinity required for biological significance and compare this binding affinity to that of Myc and Max. Then we will use transient transfection assays to determine whether theARNT homodimer formation may repress biological processes such as the induction of cytochrome P4501A1 and Myc induced cellular transformation. To determine why the AHR ARNT recognize different DNA half-sites, we will perform site-directed mutagenesis of their DNA binding regions followed by footprinting analysis. Finally, we will use cell-free transcription to identify the interacting transcription factors that facilitate AHR and ARNT gene regulation and determine whether different AHR and ARNT ratios found in vivo may affect the ability of the AHR and ARNT to heterodimerize and resulting differential gene regulation. The broad, long-term objectives of this proposal are to understand how the AHR and ARNT regulate and the mechanisms that dictate the tissue-specific effects of TCDD toxicity and carcinogenicity.

DESCRIPTION (provided by applicant): 2,3,7,8,Tetrachlorodibenzo-p-dioxin \(TCDD\) is a ubiquitous environmental contaminant that promotes tumor formation and modulates cellular differentiation via actions thought to involve the aryl hydrocarbon receptor (AHR) signaling pathway. Given that study of keratinocyte differentiation has been shown to lend important insights into the mechanism(s) by which tumor promoters exert their effects and that this model is amenable to a number of molecular techniques, we have initiated studies of differentiation to fulfill our long-term goal of understanding the role of TCDD in carcinogenesis. Using normal human keratinocytes (NHK), we have shown that administration of TCDD to NHK cells ultimately results in suppression of differentiation and that this effect is accompanied by decreased levels of the tumor suppressor protein, p27Kipl Additional studies using the ARR antagonist, 3'-methoxy-4'nitroflavone implied a role of the AHR in mediating TCDD's suppression of differentiation. Finally, it is apparent that TCDD suppresses differentiation by decreasing the number of differentiating cells while increasing the number of non-differentiating cells. These results and others, form the basis of our overall hypothesis that the ability of TCDD to suppress differentiation is mediated by a decrease in the expression levels of the tumor suppressor protein, p27Kipl thereby diminishing the p27Kipl differentiation signal. To test this hypothesis, we will use flow cytometry, western blot analysis, real time PCR and adenovirus-mediated over expression systems to determine whether: 1) the ability of TCDD to suppress differentiation requires suppression of the expression of p27Kipl, 2) TCDD regulates the p27Kipl protein at the transcriptional or post-transcriptional levels, 3) the ability of TCDD to suppress the expression of p27Kipl and differentiation requires the AHR and ARNT proteins, 4) suppression of differentiation by TCDD results in increased) proliferation, and 5) and the actions of TCDD on differentiation occur at the) genomic level.

DESCRIPTION (provided by applicant): The broad, long term goals of this proposal are to use a novel class of antagonists of the aryl hydrocarbon receptor (AHR), AHR-Protacs, developed herein as 1) a research tool to delineate the mechanisms by which activation of the AHR pathway by environmental agents leads to carcinogenesis in the human population and identify roles of the AHR in other disease processes and 2) a therapeutic agent to treat cancers, diabetes and cardiovascular diseases. Exposures to environmental factors, such as benzo[a]pyrene and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) are thought to play important roles in the development of human cancers. While these agents are known to exert many of their carcinogenic actions via activation of the AHR, the mechanisms by which the AHR elicits the key events are unknown. Further, it has not yet been established whether targeting the AHR would be an effective chemopreventive approach. Finally, emerging evidence implicates a role for the AHR in not only cancer, but also diabetes and cardiovascular diseases. The immediate goals of the current proposal are to optimize and characterize novel AHR antagonists, some of which have already been developed in our laboratories, as appropriate AHR antagonists. Specific Aims: (1) Synthesis of optimum AHR Protacs. (2) Determine that the optimum AHR Protac molecule(s) inhibits AHR signaling in cultured cells with high efficacy. (3) Determine whether the optimum AHR Protac(s) inhibits the ability of TCDD to alter cell cycle, apoptosis and senescence. (4) Determine whether the optimum AHR Protac(s) alters the AHR pathway with high specificity. The work proposed herein will develop a novel research tool that can be used to identify how inappropriate activation of the AHR by environmental carcinogens leads to human cancers and to understand the role of the AHR in normal biological processes. In addition, this work will set the stage for the development of drugs that target the AHR and may be clinically useful not only as agents that may prevent cancers, but may also be effective in the treatment of diabetes and cardiovascular diseases.

[unreadable] DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) may play in important role in the etiology of tobacco smoke induced lung cancer and may also be an important target for effective chemopreventive approaches. Our initial data performed in human lung epithelial cells indicates that constituents present in cigarette smoke condensate can act as AHR agonists and that several dietary flavonoids, in particular, apigenin, emodin and kaempferol appear to act as effective AHR antagonists. The hypothesis to be tested in this proposal is that the AHR is an important pharmacological target of dietary flavonoids that will be effective for chemoprevention of tobacco smoke induced lung cancer. Specific Aims: (1) Determine the extent to which cigarette smoke condensate activates and flavonoids inhibit the AHR and cell transformation. (2) Determine whether apigenin inhibits cigarette smoke condensate-induced malignant transformation of human lung epithelial cells in vivo. [unreadable] [unreadable] [unreadable]

Abstract The overall goal of this proposal is to develop and implement a 10-week summer program (Summer Undergraduate Research in Environmental Health Sciences (SURES) that will immerse upper-level undergraduates in environmental health science research. The program is designed to introduce students to the highly interdisciplinary and collaborative nature of environmental health science research that is focused on solving key global health problems. An additional focus is preparing underprivileged and under-represented students for successful careers in relevant fields. The SURES program will leverage the substantial and ongoing efforts at the University of Kentucky driven to enhance the undergraduate experience with the robust environmental health science research programs of involved faculty mentors. Students will be recruited from the Commonwealth of Kentucky with an emphasis on those from the Appalachian region. The SURES program will commence with a week-long session that introduces students to research, laboratory safety and scientific ethics. The students will then experience hands-on research by performing experiments in the laboratories of their chosen mentors. Professional development and a sense of belonging to the environmental health sciences community will be built via student participation in weekly breakfasts, lunches, net- working opportunities, tours of research facilities and social activities including ?movie nights? where films depicting environmental issues will be viewed and discussed. The SURES program will culminate in a poster session attended by the faculty, staff and students of the participating departments. Dissemination of the approaches used in this program and their outcomes will build upon the expansion of the traditional environmental health sciences research training from the graduate and post-doctoral level to the undergraduates at a national level.