Chemicals and reagents
Dulbecco's minimum essential medium (DMEM), glutamine and penicillin/streptomycin/glutamine stock mix were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and charcoal-stripped FBS were from Invitrogen (Carlsbad, CA, USA). Fugene 6 and Dharmafect 1 were from Roche Diagnostics (Indianapolis, IN, USA) and Dharmacon (Thermo Scientific Dharmacon, Inc., Lafayette, CO, USA), respectively. ERα (J-003401-12), RARα (J-003437-07) and control (D-001810-02) small interfering RNA (siRNA) were purchased from Dharmacon (Thermo Scientific Dharmacon, Inc.). Affinity purified rabbit and mouse antibodies to human ERα (sc-543), RARα (sc-551), RARβ (sc-552), RARγ (sc-550) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-47724) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Peroxidase-conjugated secondary antibody was from Vector Laboratories (Burlingame, CA, USA). For standard PCR, HotStart Taq Plus DNA Polymerase was used (Qiagen, Germantown, MD, USA). Reagents for real time PCR, primers and TaqMan probes were purchased from Applied Biosystems (Branchburg, NJ, USA). PI/RNase staining buffer was from BD Pharmigen (San Diego, CA, USA). The Guava Nexin Reagent was purchased from Guava Technologies (Guava Technologies, Inc., Hayward, CA, USA). The protease inhibitor cocktail kit was obtained from Pierce Biotechnology (Thermo Scientific, Rockford, IL, USA). 17β-estradiol (E2), 4-hydroxytamoxifen (OH-Tam), all-trans-Retinoic acid (ATRA) and fulvestrant were purchased from Sigma Aldrich (Saint Louis, MO USA). ATRA stock solution (5 mmol/L) was made in a mixture of 50% ethanol and 50% DMSO (Fisher Chemical, Fair Lawn, NJ, USA). First strand cDNA from human peripheral blood leukocytes (PBL), thymus and spleen were obtained from Biochain Institute (Biochain Institute Inc., Hayward, CA, USA). Total RNA from normal human breast and human breast tumors were obtained from Biochain Institute Institute and Clonetech (Clonetech Laboratories Inc., Mountain View, CA, USA).
Cell culture and treatment with fulvestrant or ATRA
MCF-7 and T47 D (American Type Culture Collection) cells were cultured in DMEM supplemented with FBS (10%), penicillin (100 unit/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM). ZR-75-1 (American Type Culture Collection) cells were cultured in RPMI 1640 supplemented with FBS (10%), penicillin (100 unit/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM). Hormone depleted cells were grown in low glucose phenol-red free media supplemented with 5% charcoal-stripped FBS (v/v) and L-glutamine (2 mM) for 48 hours before the experiments. Hormone-depleted MCF-7 cells were grown to 60 to 70% confluency in six-well plates and treated with vehicle or ATRA (1 μM) for 24 hours. After 24 hours, the cells were harvested for total RNA isolation and mRNA profiling. Hormone-depleted MCF-7 cells were grown to 30 to 40% confluency in six-well plates and treated with vehicle or fulvestrant (100 nM) for up to 4 days. After 72 hours to 96 hours, the cells were harvested for isolation of total RNA and protein.
Transfection and gene silencing
Cells were plated at 20% confluence in low glucose phenol red free medium supplemented with 5% charcoal stripped FBS and glutamine 24 hours to 48 hours prior to transfection. Treatment with vehicle (ethanol), E2 (1 nM) or OH-Tam (100 nM) was begun an additional 24 hours later. Cells were transfected with control siRNA, ERα siRNA or RARα siRNA (100 pmol/mL) in 24-well microplates or 25 cm2 flasks using 2 μl and 12.5 μl of Dharmafect 1 (Thermo Scientific Dharmacon Inc.), respectively, according to the vendor's protocol. The cell culture medium was not replenished for the duration of the experiment. In the RARα1 rescue experiments, 2 × 106 cells were co-transfected with 2 μg of either the vector plasmid or RARα1 expression plasmid and with control siRNA or ERα siRNA by nucleofection using the Kit V Amaxa Nucleofection System (Amaxa Biosystems, Allendale, NJ, USA) according to the vendor's instructions. In the RARα1 rescue of fulvestrant- treated cells and RARα1 overexpression experiments, 2 μg RARα1 expression plasmid was introduced at a cell density of 2.5 × 105 cells per well in six-well plates using Fugene 6 according to the manufacturer's protocol.
Cell growth assay
Cells were seeded in 24-well microplates at 20% confluence in phenol-red free media supplemented with 5% charcoal-stripped FBS (v/v) and incubated at 37°C with 5% CO2 for 24 h. Cells were transfected with either control siRNA or siRNA targeting ERα using Dharmafect 1. Twenty-four hours after transfection the media was replaced with fresh phenol-red free media supplemented with 5% charcoal-stripped FBS (v/v) and the cells were treated with vehicle (ethanol), E2 (1 nM) or OH-Tam (100 nM) for the following five days; the culture media was not changed during this period but E2 and OH-Tam were replenished every 48 h. Viable cell counts were monitored using the trypan blue dye exclusion assay at intervals of 24 h.
Cell cycle analysis
Cells were trypsinized and harvested in phenol red free medium supplemented with charcoal stripped FBS. Cells (1 × 106) were washed and resuspended in 500 μl PBS. The cells were fixed by adding 500 μl 100% ice cold ethanol, drop-wise with agitation and incubated on ice for 20 minutes. The cells were sedimented by brief centrifugation at 200 xg for five minutes and the excess ethanol decanted. After the remaining ethanol was dried off, the cells were resuspended in 500 μl of PI/RNase solution. The cells were incubated in the dark at room temperature for 20 minutes and the cell cycle distribution determined by flow cytometric analysis using a FACSCalibur cell analyzer (BD Biosciences, San Jose, CA, USA). The data were acquired with BD CellQuest Pro software and analyzed using ModFit LT software.
Early stage apoptosis of cells was measured by Guava Nexin analysis using the Guava Nexin Reagent staining kit according to the manufacturer's instructions. Briefly, 8 × 104 cells were incubated for 20 minutes at room temperature with the Guava Nexin Reagent and 2,000 cells per sample were analyzed using the Guava System.
Cells were harvested by trypsinization, lysed in a high salt-detergent buffer (400 nM NaCl; 10 nM Tris, pH 8.0; 1 mM EDTA; 1 mM EGTA; β-mercaptoethanol; and 0.1% Triton x-100) containing a protease inhibitor cocktail kit and incubated on ice for 30 minutes. Cell lysates were heated to 95°C for five minutes. Protein samples (10 to 20 μg) were resolved by electrophoresis on 8% sodium dodecylsulfate-polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore Corporation, Bedford, MA, USA). The blots were probed with the appropriate primary antibody and the appropriate horseradish peroxidase conjugated secondary antibody and the protein bands visualized using enhanced chemiluminescence as described [31
]. The chemiluminescent signals were quantified using the FluorChem HD2 imaging system (Alpha Innotech/Cell Biosciences, Inc., Santa Clara, CA, USA) and normalized to GAPDH.
RNA isolation, reverse transcription PCR and Real time PCR
Total RNA from cells was isolated using the RNeasy mini kit (Qiagen, Georgetown, MD, USA). Reverse transcription PCR reactions were performed using 500 ng of total RNA and the high capacity complementary DNA Archive kit (Applied Biosystems) according to the vendor's protocol. cDNAs of RARα1 and RARα2 were amplified by competitive PCR. The upstream and downstream primers used for amplification of RARα1 and RARα2 were as follows: RARα1, 5'-GCCAGGCGCTCTGACCACTC-3' and 5'-AGCCCTTGCAGCCCTCACAG-3'; RARα2, 5'-ACTCCGCTTTGGAATGGCTCAAAC-3' and 5'-AGCCCTTGCAGCCCTCACAG-3'. The cDNA for the house keeping gene glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was amplified and the primer sequences used were as follows: 5'-TGGTCACCAGGGCTGCTTTT-3' and 5'-GGTGAAGACGCCAGTGGACT-3'. The cycling parameters were: 95°C for 15 minutes; 94°C for 30 sec; 60°C for 30 sec; 72°C for 30 sec and 72°C for 10 minutes. RARα1 and RARα2 cDNAs were amplified in the same reaction, yielding products of 222 bp and 182 bp, respectively. PCR products were separated in ethidium bromide-stained 2% agarose gels by electrophoresis. cDNA was also measured by quantitative real time PCR in the 7500 StepOne Plus Real time PCR System (Applied Biosystems). Primers and TaqMan probes for the human ERα, CCNA, CDKN1, ERBB2, ERBB3, MUC20, LYPD1, RARα and GAPDH genes were obtained from the Applied Biosystems inventory. All samples were measured in triplicate and normalized to GAPDH values.
The Affymetrix chips were purchased from Affymetrix (Santa Clara, CA, USA) DNA microarray analysis using Affymetrix was performed as a full service global gene expression study at the transcriptional profiling core facility of the Cancer Institute of New Jersey. Total RNA samples were used to generate labeled cRNAs, which were hybridized to human U133 Plus2.0 Affymetrix microarrays. The expression data were analysed initially using Affymetrix GeneChip Operating Software to create CEL files. The CEL files were imported into the Bioconductor program affylmGUI [32
]. The probe set level intensities were quantified and normalized using robust multiarray averaging and quantile normalization. Differential expression between treatments was determined using the limma linear modeling method, and the significance of differences was ranked by the moderated t
-statistic. The values for signal intensities were corrected for siRNA transfection efficiencies determined using a Green Fluorescent Protein (GFP) reporter expression plasmid. To identify genes differentially expressed under the different treatments, the fold-changes were calculated by dividing the average signal of the treatment by the control, and genes with a fold-change greater or lesser than a given threshold were chosen. The advantage of this approach is that rejection of many false negatives is avoided, compared to requiring a statistically significant difference in expression, but has the potential drawback of including false positives. When we limited the genes in Tables and to those showing significant differential expression at the P
= 0.05 level by the linear modeling method, 25/54 genes in Table and 24/68 genes in Table were retained. In the reduced sets of genes, similar percentages of genes showed RARα peaks as in the larger gene set, confirming the generality of our result. The Affymetrix data are deposited in GEO (Accession number: [GEO:GSE26298]).
Tamoxifen Insensitive genes supported by the Apo-ER -> Apo-RARα Axis
Tamoxifen Insensitive genes Repressed by the Apo-ER -> Apo-RARα Axis
Experimental values are presented as mean ± standard deviation (s.d.). The statistical significance of differences (P-value) between values being compared was determined using analysis of variance. In all cases, the differences noted in the text are reflected by a P-value of <0.001.