Antibodies and reagents
The function-blocking integrin antibodies against α1 (Ha31/8), α2 (HMα2), α6 (GoH3) and β1 (Ha2/5) subunits were purchased as sodium azide- and endotoxin-free reagents from PharMingen (San Diego, CA). The anti-ERα polyclonal antibody (MC-20) was purchased from Santa Cruz (Santa Cruz, CA) and the anti-ERα monoclonal antibody (NCL-ER-6F11) was purchased from NovoCastra (Newcastle, UK). The monoclonal anti-rat β-casein antibody was a gift from C. Kaetzel (Case Western Reserve University, Cleveland, OH). The anti-E-cadherin antibody (C20820) was purchased from Transduction Laboratories (Lexington, KY). Reconstituted basement membrane (rBM; Matrigel), purified laminin-1, purified collagen-IV and MatriSperse Cell Release Solution were purchased from Collaborative Biomedical Products (Bedford, MA). Collagen-I was purchased from Vitrogen (Palo Alto, CA). Poly(2-hydroxyethyl methacrylate) (polyHEMA) and fibronectin were purchased from Sigma Chemical. Collagenase type A and 5-bromo-2′-deoxy-uridine (BRdU) were purchased from Boehringer-Mannheim (Indianapolis, IN). Alamar blue was purchased from Accumed International (Westlake, OH).
Primary mammary epithelial cells were obtained by a procedure slightly modified from Kittrell et al. (Kittrell et al., 1992
). Briefly, after removal of the 4th inguinal mammary glands from nulliparous 12-week-old virgin BALB/c mice, the glands were minced by chopping with two razor blades in parallel. Mammary cells were dissociated by collagenase type A (2 mg/ml) in the presence of 5 μg/ml insulin (Sigma Chemical) and with antibiotics [600 U/ml nystatin (Sigma Chemical), 100 U/ml penicillin-streptomycin and 50 μg/ml gentamycin (Gibco, Rockville, MA)] in DMEM/F12 medium for 3 hours at 37°C with constant shaking (100 rpm). The resulting suspension was centrifuged at 1000 g
for 10 minutes, and the pellet resuspended in 4 ml DMEM/F12 containing 2 U/ml DNase (Sigma Chemical). After gently shaking for 2 minutes the DNase was diluted by adding 4% fetal bovine serum (FBS) in 4 ml of DMEM/F12 medium, and the final suspension (containing 2% FBS in DMEM/F12) was centrifuged again at 1000 g
for 10 minutes. The resulting pellet was resuspended in phosphate-buffered saline (PBS) containing 5% adult bovine serum (Atlanta Biologicals, Norcross, GA), and this procedure was repeated six times at 1500 g
for 15 seconds each time to remove stromal cells. This protocol yielded 90% or greater purity of epithelial cells (mostly as organoids of approximately 100 cells) as determined by immunofluorescence for keratin (data not shown). Each fraction, pellet (epithelial cells) and supernatant (mostly fibroblast cells, according to vimentin staining) was resuspended in growth medium (indicated below). The day of the isolation from the gland was considered time 0 in the culture period.
Scp2 is a functionally normal mouse mammary epithelial cell line established in our laboratory (Desprez et al., 1993
). The Scp2-ERE-TK-CAT cell line is a derivative of Scp2 that has been stably transfected by cotransfecting 30 μg of the pA2(−331/−87)tk-CAT8+ plasmid and 3 μg of pSV2neo plasmid. pA2(−331/−87)tk-CAT8+ contains the chloramphenicol acetyltransferase (CAT) enzyme as a reporter gene, under the control of a minimal thymidine kinase (TK) promoter containing an upstream consensus estrogen-response element (ERE). The ERE corresponds to the region −331 to −87 of the Xenopus vitellogenin A2
gene (Klein-Hitpass et al., 1986
). The resulting SCp2-ERE-TK-CAT cells were obtained by pooling neomycin-resistant colonies. They were selected under 400 μg/ml G418 (Gibco, Rockville, MA) and maintained under 40 μg/ml G418. These cells were used at passage 6-8 after transfection/selection.
Scp2, Scp2-ERE-TK-CAT and primary mammary cells were cultured at a density of ~50,000 cells/cm2 or ~100,000 cells/cm2 (for cultures on top rBM and on polyHEMA, see below) in DMEM/F12 medium containing 50 μg/ml gentamycin, 5 μg/ml of insulin, 1 μg/ml of hydrocortisone and 3 μg/ml of prolactin (Sigma Chemical). For primary cultures, the growth medium was supplemented with epidermal growth factor (EGF, 5 ng/ml; Sigma), linoleic acid (5 μg/ml; Sigma) and bovine serum albumin (BSA, 5 mg/ml; Sigma). Attachment and spreading of the cells to the covered-glass chamber slide (for immunofluorescence) or the plastic dish were performed for 24 hours of culture in the presence of 2% FBS. The cells were then grown for the period indicated in each case with fresh serum-free medium containing insulin, hydrocortisone and prolactin, with or without addition of ECM components (see below). In experiments where the ER activity was measured (CAT reporter assays) we used charcoal-treated FBS (HyClone, Logan, Utah) and phenol red-free DMEM/F12 medium to avoid interference from exogenous estrogens. When indicated, 10−8 M of 17β-estradiol (Sigma Chemical), 10−7 M of the antagonist ICI 182,780 (Tocris Cookson, Ellisville, MO) or the same volume of ethanol (vehicle) were added to the medium for the last 48 hours.
The culture conditions for cell lines or primary cells consisted of untreated tissue culture plastic or plastic covered by a thick layer (50 μl/cm2) of growth factor-reduced rBM derived from Englebreth-Holm-Swarm tumor (Matrigel). For this last condition, the cells were seeded on top of the gel (on top rBM) and covered with the corresponding serum-free medium (see above). Matrigel was previously allowed to solidify at 37°C for 40 minutes. For assays in pre-rounded cells, primary or Scp2 cells were cultured in suspension by placing ~100,000 cells/cm2 in a culture dish coated with the nonadhesive substratum polyHEMA in serum-free medium. PolyHEMA-coated dishes were prepared using a solution of 6 mg/ml polyHEMA in 95% ethanol added to culture plates at 0.05 ml/cm2 and allowed to evaporate to dryness.
For the ‘dripping’ conditions, soluble rBM or purified ECM components laminin-1, collagen-I, collagen-IV or fibronectin were diluted in the culture medium, and were added as an overlay to previously attached and spread cells in the case of Scp2 cells or immediately after isolation from the gland in the case of primary cultures. In the case of polyHEMA cultures, when indicated, rBM was mixed in the medium with the cells. For rBM we tested 1%, 2% and 5% dilution from a 10 mg/ml protein concentration of Matrigel. Because the most effective dilution was 2%, we estimated the final concentration for the ECM components corresponding to their relative proportion in 2% Matrigel. The final concentrations used were: 150 μg/ml of laminin-1, 20 μg/ml of collagen-I, 20 μg/ml of collagen-IV and 10 μg/ml fibronectin. Under these conditions, the components form a precipitate covering the cultured cells.
Cells were treated for the required number of days (as indicated in results and legends to figures), with one change of medium every 2 days, and at the end of the culture period, cells were lysed and extracted for protein or RNA analysis. For lysis and extraction, cells were rinsed once with PBS; for protein extraction, we used the protein extraction reagent for mammalian cells (M-PER; Pierce, Rockford, IL) and for total RNA extraction, we used the RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer’s directions. For cells growing on top of rBM, cells were removed from the gel by using enzymatic digestion with MatriSperse for 1 hour on ice, followed by a centrifugation for 5 minutes at 1000 g. The resulting pellet was then subjected to protein or RNA extraction. For cells growing on polyHEMA-coated dishes, they were transferred to Eppendorf tubes, centrifuged and lysed.
Scp2 cells or primary mammary epithelial cells were grown on plastic or in the presence of rBM, collagen-IV or laminin-1 for 3 days in the presence of 10 μg/ml of mouse IgG (control, c) or in the presence of 10 μg/ml of α1, α2 or α6 or 5 μg/ml of β1 integrin blocking antibodies. The antibodies were diluted in the corresponding medium at the time of plating the cells on top of rBM, or after 24 hours of plating under other conditions to let them attach and spread. At the end of the experiment, cell lysis followed by protein extraction was performed as described above. Cell viability using Alamar blue vital dye assay was carried out in parallel cultures according to manufacturer’s instructions.
Immunofluorescence for ERα
For ERα detection, cells were fixed with −20°C methanol:acetone (1:1) solution for 5 minutes, air dried for 10 minutes, rehydrated in PBS, blocked with Super Block Blocking Buffer in PBS (Pierce) and incubated with ERα monoclonal antibody (NCL-ER-6F11) followed by FITC-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA), mounted and observed under fluorescence microscopy. Before mounting, 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was used to stain DNA. Control experiments were carried out omitting the primary antibody. Images were captured using Spot RT camera and software (Technical Instruments, Burlingame, CA). Cellular labeling indices for ERα were determined by counting at least 100 cells from randomly selected visual fields and calculating the intensity of labeling in the cells by using Simple PCI imaging software (Compix Imaging Systems, Cranberry Township, PA).
Cell proliferation assay
To study the influence of cell proliferation on ERα expression, Scp2 cells cultured for 2 days on plastic or in the presence of rBM were treated with increasing amounts of insulin (between 0 and 10 μg/ml). The cells were maintained for another 24 hours, including a 6 hours labeling period with 10 μM BrdU (BrdU labeling and detection kit to measure DNA synthesis) according to the manufacturer’s instructions. Nuclear labeling indices were determined by counting at least 100 cells from randomly selected visual fields and calculating the percentage of cells with labeled nuclei. Parallel experiments were performed to detect ERα levels by western blot.
We used the nonradioactive FLASH CAT Assay kit (Stratagene, La Jolla CA) to measure the CAT activity in cell lysates from Scp2-ERE-TK-CAT cells. Briefly, we mixed 5 μg protein/sample quantified by protein assay (Protein Assay DC, Bio Rad, Hercules, CA) with the fluorescent derivative of chloramphenicol BODIPY (borondipyrromethane difluoride fluorophore). This substrate is converted to a single monoacetylated product by CAT that is separated from the substrate by thin layer chromatography (TLC). The TLC plates were scanned using STORM fluoroimager (Amersham Biosciences, Sunnyvale, CA) and quantitation was performed using ImageQuant (Amersham).
Western blot for ERα and β-casein detection
Equal amounts of protein (20 μg of cellular extracts) were treated with reducing protein sample buffer and were size-fractionated in a 10% SDS-PAGE gel. The resulting gel slabs were electrotransferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH), and the membranes incubated for 2 hours at room temperature in blocking buffer containing 5% non-fat milk in 0.1% Tween-PBS, pH 7.5. The blots were incubated with specific primary antibodies for 1 hour at room temperature. The antibody used for loading control recognizes the 120 kDa transmembrane glycoprotein E-cadherin. To detect ERα, we used a polyclonal antibody raised against the C-terminus of the protein, MC-20 (Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes a band of ~67 kDa. The monoclonal antibody used to detect β-casein recognizes a band of ~30 kDa. The blots were washed appropriately with 0.1% Tween-PBS followed by the addition of the appropriate horseradish-peroxidase-conjugated secondary antibody. After 1 hour of incubation and appropriate washes, the signal was detected using the SuperSignal West Dura detection kit (Pierce, Rockford, IL). The intensity of each band was quantified using the ChemiImager (Alpha Innotech Corporation, San Leandro, CA) scanning densitometry equipment.
cDNA was prepared with 2 μg of total RNA using M-MLV reverse transcriptase and oligo-dT primer (Gibco Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Quantification was done using LightCycler and the DNA Master Syber Green I kit (Roche, Indianapolis, IN). The set of primers used in the PCR (forward primer 5′ AGACCGCCGAGGAGGGAGAATGTT 3′ and reverse primer 5′ GGAGCGCCAGACGAGACCAATC 3′) amplify the region between +783 and +1197 of ERα mRNA corresponding to the C-terminus of the protein. To normalize the values of ERα we performed a quantification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, forward primer 5′ CCCCTGGCCAAGGTCATCCATGAC 3′ and reverse primer 5′ CATACCAGGAAATGAGCTTGACAAAG 3′) in the same samples. The reactions for both amplifications were carried out for 40 cycles with an annealing temperature of 59°C.
All values presented in this study are means±the standard error of the mean (s.e.m.) of at least three independent experiments. Comparisons between groups were performed employing one-way analysis of variance, and differences between means were determined by a Student-Newman-Keuls multiple comparison test.