Chemicals, constructs, cells, and animals.
TCDD was prepared in this laboratory and shown to be >99% pure by gas chromatographic analysis. 17β-Estradiol (E), chloroquine, cycloheximide, cell culture chemicals, and media were purchased from Sigma. Calpain inhibitor II (Cal II), EST, MG132, PSI, PSII, bis-indolylmaleimide I, manumycin A, U0126, PP2, wortmannin, and KT5720 were purchased from Calbiochem. Primary antibodies for human ERα, AhR, Sp1, CYP1A1, c-Fos, and ubiquitin proteins were purchased from Santa Cruz. Alexa Fluor 594 goat anti-rabbit was purchased from Molecular Probes. Fluorescein isothiocyanate-conjugated goat anti-rabbit was purchased from Zymed Laboratories. The rat c-Fos pSP65 expression vector was kindly provided by Tom Curran (Roche Research Center), and the human phuAhR expression vector was a kind gift from Christopher Bradfield (University of Wisconsin School of Medicine). The pERE3
reporter construct containing three tandem consensus ERE sites linked to a luciferase gene was created by cloning an oligonucleotide containing this sequence into the Bam
dIII-cut pXP-2 plasmid (American Type Culture Collection, Manassas, Va.) 30 bp upstream from a TATA box. The sequence of the ERE was GGTCACAGTGACC. The ERα-GAL4-AD and AhR-GAL4-DBD fusion proteins were constructed using the mammalian Matchmaker two-hybrid kit (Clontech) pVP16 and pM fusion vectors. The expression plasmids for ERα with deletions of amino acids 1 to 178 (ERαΔAF1), 185 to 252 (ERαΔDBD), and 282 to 595 (ERαΔAF2) were kindly provided by Pierre Chambon (Institute de Genetique et Biologie Moleculaire et Cellulaire). T47D, MCF-7, and ZR-75 human breast cancer cells were obtained from the American Type Culture Collection and cultured as previously described (54
). B6C3F1 mice were obtained from Charles River Laboratories. The mice were kept in a temperature-controlled conditioned room with a 14-h light and 10-h dark photocycle. Rodent chow and water was supplied ad libitum.
Protein isolation and Western analysis.
Cells were seeded into 35-mm six-well tissue culture plates in phenol red-free medium (Dulbecco's modified Eagle's medium [DMEM] Ham F-12) containing 2.5% charcoal-stripped fetal bovine serum (FBS). After 24 h, cells were treated and harvested at designated time points and lysed in ice-cold lysis buffer (50 mM HEPES [pH 7.5], 500 mM NaCl, 10% [vol/vol] glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA) supplemented with protease inhibitor cocktail (Sigma). Equal amounts of protein from each treatment group were boiled in 1× Laemmli buffer (50 mM Tris-HCl, 2% sodium dodecyl sulfate [SDS], 0.1% bromphenol blue, 175 mM β-mercaptoethenol), separated by SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoresed to polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in Blotto (5% milk, Tris-buffered saline [10 mM Tris-HCl, pH 8.0, 150 mM NaCl], and 0.05% Tween 20) and probed with primary antibodies ERα G-20 (1:1,000), AhR N-19 (1:200), Sp1 PEP2 (1:5,000), CYP1A1 G-18 (1:1,000), and c-Fos H-125 (1:1,000). Following incubation with peroxidase-conjugated secondary antibody, immunoglobulins were visualized using the ECL detection system (NEN). Quantitation was performed using a Sharp JX-330 scanner and Zero-D Scanalytics software (Scanalytics Corp.). The human ERα, human phuAhR, and rat c-Fos pSP65 expression vectors were used to in vitro translate standards for Western blotting using the TNT T7 quick-coupled transcription/translation system (Promega). Human recombinant Sp1 protein (Promega) was added to the in vitro-translated mixtures as the Sp1 standard.
ZR-75 cells were seeded onto 12-well plates at a concentration of 2.75 × 105 cells per well in phenol red-free DMEM Ham F-12 supplemented with 2.5% charcoal-stripped FBS. After 18 h, cells were transfected by the calcium phosphate method with 500 ng of pERE3 luciferase reporter plasmid and 250 ng of pCDNA3.1-β-gal (Invitrogen) as the control vector. Cells were treated for 36 h and assayed for luciferase (Promega) and β-galactosidase (Tropix) activity in a Packard luminometer according to the manufacturer's instructions. Protein-protein interactions between ERα and AhR were examined in ZR-75 cells by using the ERα-GAL4-AD (vpER) and AhR-GAL4-DBD (pmAhR) fusion constructs. ZR-75 cells were transfected by the calcium phosphate method with 500 ng of 5XGAL-luciferase, 250 ng of pmAhR, 100 ng of vpER, 250 ng of pCDNA3.1-β-gal as the control vector, or empty pM and pVP vectors as DNA mass balance controls. Cells were treated for 36 h, harvested, and assayed as described above. The effects of TCDD on proteasome-dependent degradation of ERα mutants were determined in T47D cells. Cells were seeded onto 35-mm six-well culture plates at a concentration of 6 × 105 cells per well in DMEM Ham F-12 without phenol red, supplemented with 2.5% charcoal-stripped FBS. After 18 h, cells were transfected by the calcium phosphate method with 500 ng of ERαΔAF1, ERαΔDBD, or ERαΔAF2 expression plasmids. After a 12-h recovery period, cells were treated with dimethyl sulfoxide (DMSO; control) or 10 nM TCDD for 6 h. Cells were collected for transfected and endogenous ERα protein analysis using Western blotting buffers as described in “Protein isolation and Western analysis.” Fifty-microgram protein aliquots were electrophoresed for each treatment and transfection group.
Nuclear extract preparation and EMSA.
MCF-7 and ZR-75 cells were seeded at a density of 5 × 106 cells/plate in 60-mm tissue culture plates using DMEM Ham F-12 without phenol red, supplemented with 2.2 g of sodium bicarbonate/liter and antibiotic-antimycotic solution, and 2.5% charcoal-stripped FBS, pH 7.4. After 24 h, cells were treated for 3 h with 10 nM E or 10 nM E plus 10 nM TCDD following a 30-min pretreatment with DMSO, 10 μM MG132, or 10 μM Cal II. Nuclear extracts were obtained using the NE-PER nuclear and cytoplasmic extraction kit (Pierce) according to the manufacturer's instructions. Three micrograms of nuclear protein from each treatment group was incubated for 10 min at 25°C with 500 ng of poly(dI-dC) in 30 μl of HEGDK+ (25 mM HEPES, 1.5 mM EDTA, 10% glycerol, 1 mM DTT, 100 mM KCl) and 32P-end-labeled ERE probe (5′-GTC CAA AGT CAG GTC ACA GTG ACC TGA AAG TT-3′). ERα D-12 antibody and normal mouse immunoglobulin G (IgG) were used for supershift controls. Samples were electrophoresed on a 5% polyacrylamide gel at 120 V in 90 mM Tris, 90 mM borate, 2 mM EDTA (pH 8.0), dried, and visualized by autoradiography.
Northern blot analysis.
T47D cells were seeded into 100-mm tissue culture plates and, after 24 h, cells were treated for 6 h with DMSO or 10 nM TCDD following a 30-min pretreatment with DMSO, 10 μM MG132, or 10 μM PSI. Thirty micrograms of total RNA from each treatment was loaded onto a 1.2% agarose, 10% formaldehyde gel and electrophoresed in 1× morpholinepropanesulfonic acid (MOPS) buffer. Separated RNA was transferred to Zeta-Probe GT (Bio-Rad) membrane by capillary action in 1× MOPS for 48 h, UV cross-linked for 15 min, dried at 80°C for 1 h, and probed at 42 to 65°C according to the Zeta-Probe GT protocol. Membranes were scanned on a STORM 860 PhosphorImager (Molecular Dynamics). Band intensities of AhR, Arnt, and ERα were normalized to values obtained for the β-tubulin loading control. Expression vectors used for generating RNA probes for Northern blot analysis were generously supplied by the following: AhR (Kristy Dolwick, Northwestern University Medical School), Arnt (Rosally Agbunag, University of California), hERα (Ming-Jer Tsai, Baylor College of Medicine), and β-tubulin (Masahito Negishi, National Institute of Environmental Health Sciences, National Institutes of Health).
Kinase inhibitors and cycloheximide studies.
ZR-75 cells were seeded as described in the Western immunoblotting protocol above and treated for 3 h with DMSO, E, TCDD, or ET following a 30-min pretreatment with DMSO, 10 μM PP2, 1 μM bis-indolylmaleimide I, 1 μM KT5720, 2 μM manumycin A, 5 μM U0126, 400 nM wortmannin, methanol (MeOH), or 25 μM cycloheximide. Cells were harvested and immunoblotted as described above in the “Protein isolation and Western analysis” protocol.
MCF-7 and ZR-75 cells were seeded into 150-mm tissue culture plates in maintenance medium and allowed to grow to approximately 90% confluence. Cells were treated with DMSO or 10 nM TCDD for 30 min, and nuclear extracts for each treatment group were obtained using the NE-PER nuclear and cytoplasmic extraction kit with the addition of protease inhibitor cocktail. Duplicate aliquots of 550 μg (MCF-7) and 525 μg (ZR-75) of equal volume were used for the experiments. The nuclear protein was diluted fivefold in ice-cold phosphate-buffered saline (PBS) containing protease inhibitor cocktail to a final volume of 1 ml, followed by the addition of 30 μl of protein A/G PLUS-agarose beads (Santa Cruz). The reactions were placed on a rocker at 4°C for 3 h, followed by centrifugation at 600 × g at 4°C for 5 min. A 900-μl aliquot of supernatant was removed from each sample and placed into a new Eppitube on ice. Mouse monoclonal anti-ERα D-12 (1 μg) or normal mouse IgG (1 μg) was added to either replicate treatment set, followed by the addition of 30 μl of protein A/G PLUS-agarose beads. The samples were then placed on a rocker at 4°C for 12 h, followed by centrifugation at 600 × g at 4°C for 5 min. The supernatant was removed by aspiration, and the pellets were washed once with 1 ml of ice-cold PBS followed by centrifugation at 600 × g at 4°C for 5 min. The agarose pellet was then resuspended in 50 μl of 1× Laemmli buffer, boiled, and centrifuged. The supernatant was separated by SDS-10% PAGE, electrophoresed to PVDF membrane, and visualized by ECL as described above.
Ubiquitinated ERα immunoprecipitation.
ZR-75 cells were seeded as described in the coimmunoprecipitation protocol above and treated with DMSO or 10 nM E, 10 nM TCDD or ET, or 10 nM ICI 182,780 for 6 h. Duplicate aliquots of 300 μg were immunoprecipitated with anti-Sp1 PEP2 (1 μg) or anti-ERα D-12 (1 μg) as described above. Immunoprecipitates were washed with two cycles of 1 ml of ice-cold radioimmunoprecipitation assay buffer followed by 1 ml of ice-cold PBS using centrifugation at 600 × g at 4°C for 5 min. The agarose pellet was resuspended in 50 μl of 1× Laemmli buffer, boiled, and centrifuged, The supernatant was separated by SDS-10% PAGE, electrophoresed to PVDF membrane, blotted with anti-ubiquitin P4D1 (Sigma), and visualized by ECL as described above. The membrane was then stripped using stripping buffer (62.5 mM Tris-HCl, 112 mM 2-mercaptoethanol, 20% SDS [wt/vol]; pH 6.8) at 60°C for 1 h and reprobed with anti-ER α D-12 and anti-Sp1 PEP2 consecutively.
Effects of siRNA for the AhR.
ZR-75 cells were cultured in DMEM Ham F-12 containing 5% FBS in six-well plates until 50 to 60% confluent. Based on results of ongoing studies, a maximal decrease in the AhR protein was observed using 7 μl of a 20 μM solution of the small inhibitory RNA (siRNA), and this amount was transfected into ZR-75 cells using oligofectamine reagent (Invitrogen, Carlsbad, Calif.). The final concentration of siRNAs in each well was 140 nM. Thirty-six hours after transfection, cells were treated with DMSO, 10 nM E2, or 10 nM TCDD for 5 h, and nuclear extracts were obtained and analyzed by Western blot analysis for AhR, ERα, and Sp1 proteins essentially as described elsewhere (1
). Replicate (three) experiments were carried out to quantitate the effects of siRNA for the AhR on TCDD-induced downregulation of ERα. The siRNA oligonucleotides for the AhR and scrambled siRNA were as follows: scramble siRNA, 5′-GCG CGC UUU GUA GGA UUC G TT and TT CGC GCG AAA CAU CCU AAG C-5′; siRNA for AhR, 5′-UAC UUC CAC CUC AGU UGG C TT and TT AUG AAG GUG GAG UCA ACC G-5′; siRNA for lamin A/C, 5′-CUG GAC UUC CAG AAG AAC A TT and TT GAC CUG AAG GUC UUC UUG U-5′.
For uterine immunohistochemistry, 25-day-old mice were injected intraperitoneally with 200 ng of E in 100 μl of corn oil, 1 μg of TCDD in 100 μl of corn oil, ET, or corn oil alone. Twelve hours after treatment, mice were euthanized by CO2 asphyxiation. Uteri were removed, fixed in 4% paraformaldehyde overnight, washed with 70% ethanol, paraffin embedded, and sectioned at a 5-μm thickness onto positively charged slides and, after subsequent processing, slides were immunostained with ERα H-184 antibodies and analyzed by immunofluorescence as indicated below.
For immunocytochemistry, ZR-75 cells were seeded onto four-well glass chamber slides at a density of 75,000 cells per well in RPMI maintenance medium. After 24 h, cells were treated with DMSO, 10 nM E, 10 nM TCDD, or ET for 24 h. Slides were then fixed for 10 min in −20°C MeOH, air dried, and washed for 5 min in PBS-0.3% Tween. Slides were blocked for 1 h with 5% goat serum in antibody dilution buffer (1% bovine serum albumin-PBS-0.3%Tween-31% glycerol [vol/vol] [pH to 8.0] with 0.5 M Na2CO3 [pH 9.5]). A 1:100 dilution of anti-ERα H-184-5% goat serum-antibody dilution buffer, or 5% goat serum-antibody dilution buffer alone (control) was added to the samples and placed in a humidified chamber overnight at 4°C. Slides were then washed three times for 30 min in PBS-Tween and blocked again for 1 h with 5% goat serum-antibody dilution buffer. Alexa Fluor 594 goat anti-rabbit secondary antibody was added at a 1:1,000 dilution in 5% goat serum-antibody dilution buffer to all samples for 1 h at room temperature. Slides were washed three times for 30 min in PBS-Tween and once for 15 min in deionized water and mounted as above. Immunofluorescence preparations were evaluated with a Zeiss Axioplan2 microscope (Carl Zeiss) fitted with a Hamamatsu-C5810 chilled 3CCD color camera (Hamamatsu Corporation). Images of at least three different fields from three different sections per treatment group containing uterine luminal epithelium and stromal cells were captured using identical settings. Fluorescence intensity measurements of ER in both epithelial and stromal cells were obtained following subtraction of background staining determined from the control prepared without primary antibody. Values of mean fluorescence intensity ± the standard error (SE) were analyzed statistically.
All quantitative data were analyzed by an analysis of variance followed by Fisher's protected least significant difference test for significance (P < 0.05). Data are expressed as means ± SE (n ≥ 3).