17β-estradiol (E2), 4-hydroxytamoxifen (OHT) and Leptomycine B (LMB) were purchased from Sigma-Aldrich (St. Louis, MO). ICI 182,780 (ICI) was purchased from Zeneca Pharmaceuticals. RU39,411 (RU39) and RU58,668 (RU58) were kindly provided by Dr. J.M. Renoir (Paris, France). Stock solutions of E2, OHT, ICI, RU39 and RU58 were prepared in ethanol. Stock solution of LMB was prepared in methanol. The solution of proteasome inhibitor acetyl-leucyl-leucyl-norleucinal (ALLN) was purchased from Calbiochem.
Rabbit polyclonal anti-ERα (HC-20), rabbit polyclonal anti-lamin A (H-102), rabbit polyclonal anti-cytokeratine 18 (H-80) were purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal anti-GAPDH (MAB374) was purchased from Chemicon International, mouse monoclonal anti-GFP from Roche, mouse monoclonal anti-α-tubulin (clone DM1A) from Sigma-Aldrich. Mouse monoclonal anti-20S proteasome subunit α2 (clone MCP21) was gift from Dr. M.P. Bousquet (IPBS, Toulouse, France).
All cell culture products were obtained from Invitrogen.
Cell culture and generation of stable GFP-ERα cell line
Human breast cancer cell lines were maintained in Dulbecco's modified Eagle's medium F-12 (DMEM F-12) with Glutamax containing 50 μg/ml gentamicin, 1 mM sodium pyruvate and 10% heat-inactivated fetal calf serum. All cells were grown at 37°C in a humidified atmosphere containing 5% CO2. The stably transfected GFP-ERα reporter SK19 cell line was generated from ERα-positive breast cancer MCF-7 cells (ATCC). 2nd passage cells were transfected with a GFP-ERα expression vector (pEGFP-C2-hERα) using FuGENE® HB Transfection Reagent (Roche Applied Science) and G418 resistant clones were selected at the concentration 1 mg/ml. GFP-ERα expressing clones were isolated, ERα protein expression in response to estradiol and to anti-estrogens was quantified using fluorescence microscopy and western blot. Expression of ERα-regulated genes was tested by qRT-PCR and compared to gene-expression regulation in MCF-7 cells. The clone SK19 in which GFP-ERα behavior was comparable to endogenous ERα was selected for further investigation.
To study the effects of estrogens and antiestrogens, cells were grown for 3 days in medium containing phenol red-free DMEM F-12 supplemented with 5% charcoal-stripped fetal calf serum, without gentamicin and sodium pyruvate. Cells were subsequently treated or not with 10 nM E2, 1 μM ICI, 1 μM OHT, 1 μM RU39, 1 μM RU58 for the indicated times. To study ERα degradation by the proteasome, cells were pre-treated 30 min with 100 μM ALLN, a proteasome inhibitor, or 10 nM LMB, a nuclear export inhibitor.
Cell extracts and Western blots
MCF-7 cells grown in 6-well plates were treated as indicated, washed with ice-cold PBS and collected by centrifugation. Total cell lysates were prepared by resuspension of cells in 100 μl lysis buffer (50 mM Tris pH = 6.8, 2% SDS, 5% glycerol, 2 mM EDTA, 1.25% β-mercaptoethanol, 0.004% Bromophenol blue). The samples were boiled for 20 min at 95°C and cleared by centrifugation at 12 000 × g
for 10 min. Protein concentration was determined by an Amido Schwartz assay when the samples contained SDS. Samples were subjected to SDS-PAGE and proteins transferred onto nitrocellulose membranes. Western blot analysis was performed as previously described [48
] using ERα and GAPDH antibodies and quantified using the TINA PC-Base Software from FUJI.
Total RNAs were extracted using TRIzol reagent (Invitrogen) following the manufacturer's protocol. 1-5 μg of total RNA was reverse transcribed in a final volume of 20 μl using SuperScript™III Reverse Transcriptase (Invitrogen). cDNA was stored at -80°C. All target transcripts were detected using quantitative RT-PCR (SYBRGreen SuperMix, Invitrogen) assays on a Mastercycler Realplex device (Eppendorf) using TBP or RPLP0 genes as endogenous control for normalization of the data. The following primer pairs were used for amplification:
RPLP0: (Fwd) 5'-TGGCAGCATCTACAACCCTGAA-3'
TFF1: (Fwd) 5'-CCCCTGGTGCTTCTATCCTAAT-3'
PGR: (Fwd) 5'-CTTAATCAACTAGGCGAGAG-3'
The results were analyzed using Mastercycler Realplex and qBASE software.
Three hours after incubation with ERα ligands, SK19 cells were washed with ice-cold PBS, scraped and centrifuged at 1,500 rpm for 5 min at 4°C. The pellets were resuspended in 150 μl digitonin lysis buffer containing 1% digitonin and 1 mM EDTA in PBS, immediately centrifuged at 13,000 rpm for 20 min at 4°C, to obtain the cytosolic fraction (C). The pellets were resuspended in 150 μl HEPES lysis buffer containing 1% Triton X-100, 10% glycerol, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mM PMSF, 1 mM Na3VO4 and 50 mM NaF in HEPES buffer (25 mM HEPES, 0.3 M NaCl, 1.5 mM MgCl2, 20 mM β-glycerol-phosphate, 2 mM EDTA, 2 mM EGTA and 1 mM DTT), kept 15 min on ice and centrifuged at 13,000 rpm for 15 min at 4°C, to obtain the soluble nuclear fraction (SN). The pellets from the previous step were resuspended in 100 μl of a third buffer containing 95% Laemmli buffer and 5% β-mercaptoethanol and incubated 5 min on ice and boiled for 20 min at 95°C to obtain the insoluble nuclear fraction (IN). The different fractions were stored at -80°C until use. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad).
Immunofluorescence and Fluorescence microscopy
For indirect immunofluorescence experiments, SK19 cells were grown for 3 days on coverslips in DMEM without phenol red, containing 5% charcoal-stripped fetal serum. After 3 days, cells were treated for 1 h with the following ligands: 10 nM E2, 1 μM ICI, 1 μM OHT, 1 μM RU39, 1 μM RU58. Cells were then washed twice with PBS, fixed in 4% paraformaldehyde/PBS for 10 min at room temperature, subsequently permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature, counterstained with DAPI (4',6-diamidino-2-phenylindole) and mounted on microscopy slides.
To study co-localization of ERα and proteasome by immunofluorescence, SK19 cells were grown for 3 days on coverslips in DMEM without phenol red, containing 5% charcoal-stripped fetal serum and next treated for 3 h with drugs as indicated above. To block proteasome-mediated ERα degradation, the cells were incubated 30 min with 100 μM ALLN prior to treatment with ICI or RU58. Before immunostaining, cells were fixed in 4% paraformaldehyde/PBS for 30 minutes at room temperature, washed three times in PBS, quenched in 75 mM NH4Cl containing 20 mM glycine and permeabilized with 0.5% Triton X-100 in PBS for 30 minutes. Next, cells were washed with PBS, blocked for 1 h at room temperature in 5% dry milk in TBS-T (20 mM TRIS-HCl, 150 mM NaCl, 0.1% Tween 20, pH = 7.4) and incubated overnight at 4°C with anti-20S proteasome antibody at a final concentration 2 μg/ml in 5% dry milk in TBS-T followed, after washing, by incubation with the Alexa Fluor® 647 goat anti-mouse secondary antibody (1:1000, Invitrogen, Molecular Probes) for 90 min in the dark at room temperature. Finally, cells were washed with TBS-T, counterstained with DAPI and mounted on microscopy slides.
Cells were examined by fluorescence microscopy using an Olympus IX-81 microscope, equipped with a CoolSNAP HQ camera (Roper Scientific) and imaged through an Olympus oil-immersion objective 100x PLANAPO NA1.4. Images were recorded and deconvolved using Metamorph software (Universal Imaging). All images were processed for presentation using Adobe Photoshop 9.0.2.
MCF-7 cells were grown and treated as described above. For immune-electron microscopy cells were fixed with 4% paraformaldehyde in Na cacodylate buffer (pH 7.4), dehydrated in a graded series of ethanol and embedded in acrylic resin (LRWhite). 80 nm ultrathin sections were mounted on Nickel grids, incubated with 2% BSA/PBS and incubated overnight at 4°C with a mixture of primary antibodies (anti -20S proteasome antibody at final concentration 2 μg/ml and anti-ERα antibody (dilution 1/500)) in 2% BSA/PBS, washed 5 times for 5 mins in 1% BSA/PBS and then labeled for 1 h with 6 nm goat anti-mouse and 10 nm goat anti-rabbit gold conjugated particles in 1% BSA/PBS. Grids were finally washed 4 times for 5 mins in 1% BSA/PBS, incubated for 15 mins in 1% glutaraldehyde/PBS, washed 2 times for 5 mins in PBS, 3 times in distilled water and dried at room temperature. The samples were visualized using 120 kV Jeol electron microscope at 80 kV and images were captured using a digital camera AMT.