Regulation of basal and inducible CYP1A1 gene expression in mammalian cancer cell lines
Previous in vivo and in vitro studies have demonstrated that a range of estrogen-induced responses are antagonized by the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Relatively non-toxic analogues of TCDD, such as 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) an...
Saved in:
| Main Author: | |
|---|---|
| Format: | Dissertation |
| Language: | English |
| Published: |
ProQuest Dissertations & Theses
01-01-1997
|
| Subjects: | |
| Online Access: | Get full text |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | Previous in vivo and in vitro studies have demonstrated that a range of estrogen-induced responses are antagonized by the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Relatively non-toxic analogues of TCDD, such as 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) and indole-3-carbinol (I3C) are weak agonists of the toxic and biochemical responses induced by AhR ligands, and moderately potent antiestrogens. Studies have suggested that the estrogen receptor (ER) is necessary for AhR function and cotransfection of wildtype ER can restore TCDD-inducibility to the prototypical marker of AhR-mediated toxicity, the CYP1A1 gene. In our studies, 10$\sp{-5}$ M cycloheximide caused a 31- and 32-fold increase in basal, and 60- and 53-fold increase in TCDD-inducible CYP1A1 mRNA levels in ER-MDA-MB-231 and ER+ MCF7 breast cancer cells, respectively. In MDA-MB-231 cells, cycloheximide-enhanced induction of CYP1A1 mRNA was dose dependent, with maximal mRNA levels achieved at 10$\sp{-8}$ M TCDD. Time course studies demonstrated a greater than 3-fold induction of CYP1A1 mRNA occurred by 6 hrs of cycloheximide addition. In the ovarian carcinomas, PEO1, PEO4, and PEO6, cycloheximide alone did not cause a significant increase in basal CYP1A1 mRNA levels. However, pre-treatment with 10$\sp{-5}$ M cycloheximide, and treatment with 10$\sp{-8}$ M TCDD, resulted in $-$3, $-$5- and 14-fold induction of CYP1A1 mRNA levels, for PEO1, PEO4 and PEO6 cells, respectively. In transient transfections using CAT constructs containing sequences from $-$1140 to +2340 of the human CYP1A1 gene, deletion of the negative regulatory element did not affect basal promoter activity, but did result in a 6-fold induction of CAT activity in PEO4 cells treated with (10$\sp{-8}$ M). Gel retardation assays demonstrated a general lack of correlation between CYP1A1 gene expression and in vitro DRE binding The technique of ligation-mediated polymerase chain reaction (LMPCR) was utilized to investigate the interactive differences between TCDD and MCDF on the 5$\sp\prime$-regulatory region of the CYP1A1 gene of Hepa1c1c7 and MCF7 genomic DNA. Results demonstrate that, in both Hepa1c1c7 and MCF7 cells, a key guanine of the DRE (CGCACT), which is protected in TCDD-treated cells, is not protected in cells treated with MCDF or I3C. |
|---|---|
| ISBN: | 9780591500110 0591500116 |