Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type


Degree Name



Molecular and Cellular Toxicology

First Advisor


Second Advisor



Estrogen sulfotransferase (SULT1E1) catalyzes the sulfonation of estrogens, which limits estrogen mitogenicity. TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERα and ERβ) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β-hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERα-positive luminal MCF7 breast cancer cells. Low levels of ERα and ERβ mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17βHSD1 mRNA was most abundant in neoplastic MCF10CA1a and cells, while 17βHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17βHSD1, and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

We recently reported that SULT1E1 expression is low in pre-confluent MCF10A breast epithelial cells but increases when the cells become confluent. Pulse-chase labeling experiments with bromouridine demonstrated that the confluence-mediated increase in SULT1E1 expression was due to increased mRNA synthesis. Because aryl hydrocarbon receptor (AhR) activation has been shown to suppress SULT1E1 expression and loss of cell-cell contact has been shown to activate AhR in other cell types, we tested whether the confluency-associated changes in SULT1E1 expression were mediated by the AhR. Relative to confluent MCF10A cells, pre-confluent cells had higher levels of CYP1A1 mRNA and greater activation of an AhR-responsive luciferase reporter, demonstrating that AhR was active in the pre-confluent cells. AhR and aryl hydrocarbon receptor nuclear translocator mRNA and protein levels were also higher in pre-confluent than in confluent cultures. Treatment of pre-confluent cells with the AhR antagonist, 3'-methoxy-4'-nitroflavone (MNF), or AhR knockdown significantly increased SULT1E1 expression. MCF10A cells stably transfected with a luciferase reporter containing ~7 kb of the SULT1E1 5'-flanking region showed both MNF- and confluence-inducible luciferase expression. Pre-confluent cells transiently transfected with the reporter showed both MNF treatment- and AhR knockdown-mediated luciferase induction, but mutation of a computationally predicted dioxin response element (DRE) at nt -3476 did not attenuate these effects. These results demonstrate that SULT1E1 expression in MCF10A cells is transcriptionally regulated by confluency through a suppressive action of the AhR, which is not mediated through a DRE at nt -3476.

Tobacco smoke has been implicated as a risk factor for breast cancer. We evaluated the effect of tobacco smoke condensate (TSC) on expression of the estrogen-inactivating enzyme SULT1E1 in the MCF10A human breast epithelial cell line. Because TSC contains components that are known aryl hydrocarbon receptor (AhR) agonists, effects of TSC treatment were compared to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and effects on SULT1E1 expression were compared to those on CYP1A1. Treatment for 24-72 h with 0.05-10 µg/ml TSC produced concentration-dependent increases in CYP1A1 mRNA content, decreases in SULT1E1 mRNA content, and increases in expression from a transfected AhR-responsive reporter plasmid. Treatment with 10 µg/ml TSC and 30 nM TCDD produced comparable increases in CYP1A1 mRNA levels (~300-fold) and decreases in SULT1E1 mRNA levels (~90%). Treatment with the AhR antagonist 3'-methoxy-4'-nitroflavone (PD168641, at 1 µM) completely inhibited TCDD-inducible CYP1A1 expression and partially reversed TCDD-mediated SULT1E1 suppression. PD168641 also inhibited TSC-mediated CYP1A1 induction and SULT1E1 suppression as indicated by rightward shifts of the TSC concentration-response curves. These findings support a role for the AhR in TSC-mediated regulation of CYP1A1 and SULT1E1 expression in human breast epithelial cells.