TCDD and related compounds inhibit mammary tumor growth in rodent models and 17beta-estradiol (E2)-induced responses in the rodent uterus and human breast cancer cells. Mechanistic studies show that the mechanism of AhR-mediated antiestrogenicity involves interaction of the nuclear AhR complex with inhibitory dioxin responsive elements (iDREs) located in the 5'-promoter regions of target genes. Based on results of preliminary studies, Dr. Safe hypothesizes that functional iDREs in E2-responsive genes are critical genomic targets for ligand-dependent AhR-mediated antiestrogenicity. Moreover, he also hypothesizes that iDREs are enhancer sequences for ligand-independent regulation of some genes by the AhR complex. The following Specific Aims will test the validity of the hypotheses.
Aim 1 : Induction of progesterone receptor (PR), c-fos protooncogene and creatine kinase B (CKB) gene expression by E2 is inhibited by TCDD. Critical 5'-promoter regions associated with these responses (iDREs) have been identified and the role of iDREs in mediating AhR-induced antiestrogenicity will be determined using PR, CKB and fos gene promoter constructs.
Aim 2 : Multiple enhancer sequences associated with E2-induced transactivation and inhibition by TCDD have been identified in the cathepsin D gene promoter. Therefore, Aim 2A will focus on the crosstalk between transcription factors binding multiple iDREs and multiple E2-enhancer sequences.
Aim 2 B will investigate the role of the nuclear AhR complex (in the absence of ligand) in modulating these responses and will define a possible endogenous function for the AhR complex.
Aim 3 : Using a responsive region of the Bcl-2 gene promoter, Aim 3 will investigate the molecular mechanism of ER-mediated responses and crosstalk between the ER- and AhR-mediated pathways and thus complement studies proposed in Aims 1 and 2.
Aim 4 : Using a suppression substractive hybridization approach, several new E2-inducible genes which are inhibited by TCDD have been identified.
Aim 4 will continue to investigate mechanisms associated with crosstalk between the ER and AhR utilizing genes identified in these cloning studies. iDREs are important new 5'-promoter sequences which affect hormone-induced transactivation, and functional iDREs in critical E2-regulated genes could serve as important genomic target sequences for antiestrogen chemotherapy.
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