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Date of Award

2006

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

William K. Chan

First Committee Member

James Uchizono

Second Committee Member

Craig A. Vierra

Third Committee Member

Timothy J. Smith

Fourth Committee Member

David Thomas

Abstract

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous protein serving as a required dimerization partner for the aryl hydrocarbon receptor (AhR) and hypoxia inducible factor-1α (HIF-1α) transcription factors. Additionally it serves as a potent co-activator for estrogen receptor (ER) signaling. We sought to take advantage of these cross-talk mechanisms by designing an AhR construct that can influence the regulation of these pathways by sequestering ARNT. CΔ553 is a truncated form of the AhR lacking the C-terminal 553 amino acids which harbors the complete transactivation (TAD) and significant portions of the ligand binding (LBD) domains. Altering the LBD allows CΔ553 to become constitutively active and has been shown to associate with ARNT and bind DNA. Without the TAD, CΔ553 cannot recruit co-activators to the promoter so that no activation of gene transcription may occur. Transient transfection studies using a corresponding luciferase reporter plasmid in MCF-7 cells showed that CΔ553 effectively suppressed the AhR, HIF-1α, and ER signaling pathways. RT/real-time QPCR data showed that CΔ553 blocked the up-regulation of the target genes controlled by AhR ( CYP1A1 ), HIF-1α ( VEGF, aldolase C , and LDH-A ), and ER ( GREB1 ) in breast cancer cells. Since both HIF-1α and ER are highly active in ER-positive breast cancers, CΔ553 has the potential to be developed as a protein drug to treat breast cancer by blocking these two signaling pathways. Seeking to determine if complete suppression of genes is possible with CΔ553, a tetracycline regulated retroviral expression system is investigated along with the possibilities for cellular administration via the HIV-1 Tat protein transduction domain. Since ARNT dimerizes with both AhR and HIF-1α, we present further studies looking into the role protein factors play in the activation of each system. Previously within our lab p23 and Cyp40, two components of the hsp90 chaperon complex, were found to facilitate the formation of the AhR•ARNT•DNA binding complex. Analysis of these proteins within the hypoxia signaling pathway found that only p23 was capable of generating the HIF-1α•ARNT•DNA binding complex.

Pages

153

ISBN

9780542897658

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