Antibodies, reagents, and cell lines
Mouse monoclonal β-catenin and N-cadherin antibodies were purchased from Transduction Laboratories (Mississauga, ON, Canada), a monoclonal E-cadherin antibody (anti-uvomorulin clone Decma-1) was purchased from Sigma (Oakville, ON, Canada), and monoclonal anti-ZO-1 and anti-vimentin antibodies were obtained from Chemicon International (Temecula, CA, USA) and ICN Biochemicals Inc. (Irvine, CA, USA), respectively. Polyclonal ErbB-1, ErbB-2, ErbB-3, and ErbB-4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), a monoclonal ErbB-2/Neu antibody was obtained from NeoMarkers (Fremont, CA, USA), and a monoclonal anti-phospho-tyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology (Charlottesville, VA, USA). Horseradish peroxidase conjugated secondary antibodies were obtained from Jackson Immuno Research Laboratories Inc (West Grove, PA, USA). Texas-red conjugated anti-rat and anti-mouse antibodies, Texas-red conjugated fungal toxin phalloidin, and Alexa-488 goat-anti-rat conjugates were purchased from Molecular Probes (Eugene, OR, USA). The FITC-conjugated anti-goat antibody was obtained from Santa Cruz Biotechnology and the rat anti-HA antibody was purchased from Boehringer-Ingelheim (Burlington, ON, Canada). Recombinant human epidermal growth factor (EGF) was obtained from Austral Biologicals (San Ramon, CA, USA), heregulin (HRG)-β1 from NeoMarkers, and recombinant human TGF-β1 and TGF-β2 were purchased from R&D Systems (Minneapolis, MN, USA).
Mink lung cells (Mv1Lu) were obtained from the American Tissue Culture Collection (ATCC; Mannasas, VA, USA) and routinely cultured in Dulbecco's modified Eagles medium (DMEM; Wisent Inc., St.-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories Inc., Logan, UT, USA). Murine myeloma NS0 cells (a kind gift from C Guilbault) were maintained in DMEM/NCTC 109 medium (4:1; vol/vol) containing 10% FBS. All cell lines were cultured at 37°C in a humidified 5% carbon dioxide atmosphere.
Isolation and in vitroculture of mammary tumor epithelial cell lines
FVB control and FVB MMTV/activated Neu transgenic mice were purchased from Charles River Laboratories (Wilmington, MA, USA). C3H×C57Bl/6 mice expressing MMTV/TβRII-AS  were backcrossed eight times with FVB mice and then interbred to generate mice expressing the MMTV/TβRII-AS on a congenic FVB background. These mice were mated with FVB MMTV/activated Neu transgenic animals to generate FVB MMTV/activated Neu + TβRII-AS bigenic animals. All offspring were screened for transgene expression by PCR using genomic mouse tail DNA. Neu (5'-GAAGGCACTGCCTCTCCGC-3') and TβRII-AS (5'-TTGTGGTTGATGTTGTTGG-3') specific primers were both used in combination with a polyadenylation signal specific primer (5'-CTCTGTAGGTAGTTTGTCC-3'), applying the following conditions: 94°C for 1 min; 34 cycles of 94°C for 30 s, 48°C (TβRII-AS) or 55°C (Neu) for 30 s, and 72°C for 1 min; followed by 72°C for 2 min.
Immediately after weaning, virgin MMTV/activated Neu and MMTV/activated Neu + TβRII-AS expressing female offspring were palpated twice a week for tumor formation. Mammary tumors were harvested, washed twice in ice-cold phosphate-buffered saline (PBS; Wisent Inc), cut into small pieces and incubated in trypsin/EDTA (Wisent Inc.) at 4°C for 30 min, while rocking. Cell suspensions were washed twice with ice-cold PBS, and resuspended in a mixture of DMEM and Ham's F12 (1:1) supplemented with 5% FBS and antibiotics/antimycotics (Wisent Inc.). The cells were further propagated in this medium, which is subsequently referred to as DF/5% unless otherwise stated.
The BRI-JM01 and BRI-JM04 cell lines were isolated by harvesting nonadherent cells 3 hours after plating the tumor suspension. These nonadherent cells were then reseeded and cultured for 3 days. Clusters of epithelial cells were then isolated and transferred to a 24-well plate for further propagation. The BRI-JM05 cell line was established by seeding the tumor cell suspension in growth factor reduced Matrigel without phenol red (Beckton Dickinson, San Jose, CA, USA). Cells were maintained in Matrigel until 'tree-like' structures appeared, which were then recovered using MatriSperse (Beckton Dickinson), rinsed twice with ice-cold PBS, resuspended in DF/5%, and further propagated on tissue-culture plastic.
Screening for gene expression using RT-PCR
PCR and RT-PCR analysis were used, respectively, to screen the harvested mammary tumors and generated cell lines for the presence and expression of the activated Neu and TβRII-AS transgenes. Total RNA was isolated from frozen tumors or 2.5 × 106 BRI-JM01, BRI-JM04, and BRI-JM05 cells using Trizol (Invitrogen-Life Technologies, Burlington, ON, Canada). PolyA+ RNA was obtained using Oligotex resin (Qiagen, Mississauga, ON, USA), further purified using DNAse I (NEN-Life Technologies, Boston, MA, USA), and quantified using Ribogreen (Molecular Probes). First strand cDNA was synthesized in a final volume of 20 μl using 1 μg polyA+ RNA, an 18-base poly(dT) primer, and 200 units Superscript II (Life Technologies). Of this first strand reaction, 2 μl was used in a PCR reaction using Neu or TβRII-AS specific primers under the conditions described for screening of the transgenic mice. Screening of the BRI-JM01 cell line for the presence of TβRIII mRNA transcripts was carried out using two sets of primers (set 1: 5'-CCT-CCT-CCA-CAG-ATT-TT-CCA-3' and 5'-TGA-GTG-CTC-CCT-ATG-CTG-TG-3'; set 2: 5'-ACG-ATC-CAT-GAC-AGT-GAC-CA-3' and 5'-GAA-GCT-CAG-GAG-AAT-GGT-G-3'). For the RT-PCR reactions we used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control (5'-ACC-ACA-GTC-CAT-GCC-ATC-AC-3' and 5'-TCC-ACC-ACC-CTG-TTG-CTG-TA-3'). The conditions used for both GAPDH and TβRIII PCR reactions are similar to those described for the Neu amplification.
Plating efficiency, population doubling time, soft agar, and Matrigel assays
Plating efficiency of the three cell lines was determined by seeding 40, 75, 150, 300, 600, and 1250 cells/well using six-well plates. After 7 days the wells were rinsed with PBS, the adherent colonies were stained with crystal violet (0.2% weight/vol in 100% ethanol) for 5 min and rinsed with water, and colonies visible were counted. The plating efficiency was calculated as follows: (number of colonies/number of cells seeded) × 100.
Population doubling time of the three cell lines was determined by seeding 1 × 105 cells/well using six-well plates. Cells were trypsinized after 24, 48, 72 and 96 hours of culturing and counted using a hemacytometer.
The anchorage-independent growth of the three cell lines was studied by plating 105 cells in 0.3% agarose on top of a layer of 0.6% agarose, both in DF/5%, using 100 mm dishes. Cells were fed every week and colony growth was evaluated by light microscopy after 7, 14, and 21 days of culturing.
The ability of the BRI-JM01, BRI-JM04, and BRI-JM05 cell lines to grow in three-dimensional culture was evaluated by seeding 5 × 104 cells in growth factor reduced Matrigel (Becton Dickinson) using 12-well plates. In addition to these cells, we also seeded BRI-JM01 cells that had been exposed to TGF-β1 for 48 hours and subsequently cultured for more than five passages in the absence of this growth factor. Cellular structures were evaluated 1, 2, and 3 weeks after seeding by light microscopy and documented using a Leitz Labovert (Leitz, Wetzlar, Germany) inverted microscope.
The cell cycle distribution of the three cell lines was determined by flow cytometry. Briefly, exponentially growing cells were trypsinized, washed and resuspended in ice-cold PBS, and fixed in 70% ethanol (4°C). Cells were washed the next day with ice-cold PBS, treated with 1 mg/ml RNAse in PBS (15 min at 37°C), and stained (30 min at room temperature) with propidium iodide (Molecular Probes) to a final concentration of 50 μg/ml in PBS before analysis. Cell populations were selected by forward and side scattering using a 488 nm dichroic long pass filter, and doublets were discriminated using the peak versus the integrated signal of propidium iodide red fluorescence (645 nm dichroic long pass and 620 nm band pass filter set). Data were analyzed using Multicycle Software (Phoenix Flow Systems, San Diego, CA, USA).
The epithelial character of the cell lines was evaluated by cell surface E-cadherin expression. Cells were harvested using nonenzymatic cell dissociation buffer (Sigma), then washed twice with 10% FBS containing ice-cold PBS (PBS/10%), and incubated (20 min at 4°C). Cells were pelleted, resuspended in PBS/10%, divided into three equal portions, and incubated with an E-cadherin antibody (1:1600), a control isotype equivalent antibody (anti-HA; 1:1600), or a secondary antibody Alexa-488 (1:400) in PBS/10% (1 hour at 4°C). The cells were rinsed twice in ice-cold PBS/10%, incubated with Alexa-488 secondary antibody in the dark (1 hour at 4°C), washed twice with ice-cold PBS/10%, and finally resuspended in 1 ml PBS. Viable cell populations were selected using forward and side scattering parameters with a 488 nm dichroic long pass filter. FITC or Alexa green fluorescent emission was detected using a 550 nm dichroic long pass and 525 nm band pass filter. Both cell cycle distribution and the cell surface E-cadherin expression of the three cell lines were analyzed using a Coulter EPICS™ XL-MCL flow cytometer (Beckman-Coulter, Fullerton, CA, USA) equipped with a 15 mW at 488 nm argon ion laser as the excitation source.
[3H]thymidine incorporation assays
Mv1Lu and BRI-JM01 cells (2 × 104 cells/well), and BRI-JM04 and BRI-JM05 cells (4 × 104 cells/well) were seeded in 24-well plates. The next day, cells were incubated for 4 hours in either serum-free (recombinant human EGF and HRG-β1) or medium containing 1% FBS (TGF-β1), after which serial dilutions of recombinant human EGF, HRG-β1 or TGF-β1 were added. After 24 hours, cells were pulse-labeled for 3 hours by adding 0.5 μCi [3H]thymidine/well (Amersham, Piscataway, NJ, USA). Cells were then rinsed with PBS, trypsinized, and harvested using a cell harvester (Cambridge Technology, Cambridge, MA, USA). [3H]thymidine incorporation was determined by scintillation counting (Universol; ICN Biochemicals Inc.) using a β-counter (LKB-Wallac, Turku, Finland).
[125I]TGF-β cross-link affinity labeling studies
Mv1Lu and BRI-JM01 cells were seeded at a density of 1 × 105cells/well, and BRI-JM04 and BRI-JM05 cells at a density of 2 × 105cells/well, using 12-well plates. The next day, cells were cross-link affinity labeled with 100 pmol/l [125I]TGF-β1 (NEN-Life Science Products) or [125I]TGF-β2 (iodination according to Philip and O'Connor-McCourt ) in the absence or presence of 100× excess unlabeled TGF-β1 in 300 μl binding buffer (D-PBS/Ca2+/Mg2+, 1 mg/ml BSA; pH7.4). Receptor bound ligand was cross-linked using Bis(sulfosuccinimidyl) suberate (Pierce Biotechnology, Rockford, IL, USA), as described previously . Cells were solubilized (1% Triton X100, 10% glycerol, 1 mmol/l EDTA, 20 mmol/l Tris-HCl; pH 7.4) and equal amounts of total protein were resolved on a linear 3–12% gradient SDS-PAGE. Immunoprecipitations of the labeled receptor complex were performed using antisera directed against activin receptor-like kinase (ALK)-1, ALK-5 (Tβ RI) or ALK-7 (antisera kindly provided by Dr P ten Dijke ), TβRII and TβRIII, before SDS-PAGE. For treatment of the cells with glycosaminoglycan-cleaving enzymes, subconfluent monolayers were treated with 100 mU heparinitase I, II and III, and 10 mU chondroitinase ABC (Ibex Technologies, Montreal, PQ, USA) in 200 μl binding buffer (3 hours at 37°C) before cross-link affinity-labeling with 100 pmol/l [125I]TGF-β1 or [125I]TGF-β2. Gels were fixed (1 hour, 10% HAc/30% MeOH) at room temperature and dried, and [125I]TGF-β cross-link affinity labeled proteins were imaged using a PhosphorImager 401E and analyzed using ImageQuant software (both purchased from Molecular Dynamics, Sunnyvale, CA, USA).
Cell motility was determined using two independent assays. For the wound closure assay, cells were seeded at a density of 2 × 105 (BRI-JM01) and 4 × 105 (BRI-JM04 and BRI-JM05) cells/well in 12-well plates in DF/5% and grown to confluency. Monolayers were then 'wounded' with a pipet tip and the ability of the cells to migrate into this 'wound area' was monitored in the absence or presence of 100 pmol/l TGF-β1.
For the 'black ink assay', 24-well plates were coated with ink according to Al-Moustafa and coworkers , with modifications. Briefly, 24-well plates were coated with 0.1% gelatin (30 min at 37°C). Excess gelatin was aspired from the wells, and a black ink solution (Demco, St.-Lambert, PQ, Canada; 1:3 dilution in sterile ddH2O) was added, covering the bottom of the wells. After 5 min, the excess ink was removed and the wells were rinsed vigorously with sterile ddH2O and finally PBS. Cells were seeded immediately after the ink-coated wells were prepared at a density of 2 × 104 cells/well in DF/5% in the absence or presence of 100 pmol/l TGF-β1.
Cell motility in both assays was determined 24 hours after TGF-β addition using a Leitz Labovert inverted microscope. The ink particle-free tracks, produced by the migrating cells in the black ink assay, of five microscopic fields were quantified using ImageJ freeware http://rsb.info.nih.gov/ij/. Differences in endogenous and TGF-β1 induced motility between the three cell lines was expressed as the relative fold/cell per 24 hours using the least motile BRI-JM05 cell line as a standard.
BRI-JM01 cells were seeded in glass chamber slides (Lab-Tek, NalgeNunc International, Rochester, NY, USA) at a density of 1 × 104 cells/0.8 cm2 in DF/5%. A total of 100 pmol/l TGF-β1 was added and the cells were incubated for an additional 24 hours. Cells were rinsed with PBS (repeated after each of the following steps), fixed in ice-cold 100% methanol (10 min), and permeabilized with 0.1% Triton X100 in PBS (5 min at room temperature). Nonspecific sites were blocked (30 min at room temperature) with 1.5% nonfat milk-powder in PBS (PBS/1.5% milk), followed by an incubation with antibodies raised against ZO-1 (1/500), E-cadherin (1/500), β-catenin (1/1000), N-cadherin (1/1000), and vimentin (1/50), all in PBS/1.5% milk. This was followed by incubation with a fluorescently conjugated secondary antibody (1/500 in PBS/1.5% milk) for 1 hour at room temperature. F-actin filaments were stained, after fixation of the cells in 4% paraformaldehyde, with Texas-red conjugated phalloidin (30 min at room temperature). Nuclei were counter stained on all slides (5 min at room temperature) using 0.4 μg/ml 4,6-diamidino-2-phenylindole (Sigma) in PBS. Slides were mounted using antifade (Molecular Probes) and fluorescent images were captured using a cooled Coolsnap CCD digital camera mounted on a Leitz upright microscope.
Immunoprecipitations and western blot analysis
Semiconfluent monolayers of BRI-JM01, BRI-JM04, and BRI-JM05 cells were rinsed with PBS before lysis in CSK buffer (150 mmol/l NaCl, 10 mmol/l Pipes, 30 μmol/l MgCl2, 300 mmol/l sucrose, 2% Triton X-100) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Laval, PQ, USA). The total ErbB-1, ErbB-2, ErbB-3, and ErbB-4 content of the cell lines was determined by resolving 20 μg total protein by 7.5% SDS-PAGE followed by western blotting. The phosphorylation status of the ErbB-2/Neu receptor was determined by immunopreciptation of ErbB-2/Neu, followed by western blotting with a phosphotyrosine specific antibody. Briefly, semiconfluent monolayers of BRI-JM01, BRI-JM03, and BRI-JM04 cell lines were lysed in lysis buffer (150 mmol/l NaCl; 50 mmol/l Tris-HCl, pH 8.0; 0.55% NP-40; 50 mmol/l NaF). Total protein (500 μg) was precleared with protein G sepharose (4 hours at 4°C) and the ErbB-2/Neu receptor was precipitated (16 hours at 4°C) using 2 μg monoclonal ErbB-2/Neu antibodies coupled to protein G sepharose. The next day precipitates were rinsed five times with lysis buffer and resolved by 7.5% SDS-PAGE.
The E-cadherin, N-cadherin, and β-catenin content of the BRI-JM01 cells was determined by lysing BRI-JM01 cells in CSK buffer after 24, 72, and 120 hours of TGF-β1 treatment (100 pmol/l final concentration) and resolving 20 μg total protein by 10% SDS-PAGE followed by western blotting.
All proteins were transferred to nitrocellulose, nonspecific sites were blocked using 5% non-fat milk powder in TBS (20 mmol/l Tris-HCl, pH 7.6; 137 mmol/l NaCl) containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, and filters were then incubated with primary antibody (E-cadherin, ErbB-1, ErbB-2, ErbB-3, and ErbB-4, 1:500; N-cadherin and β-catenin, 1:1000; phospho-tyrosine, 1 μg/ml) in TBS-T/5% nonfat milk (16 hours at 4°C). Filters were washed with TBS-T (3 × 10 min), incubated with the appropriate secondary HRP-conjugated antibody (1:10,000, 1 hour at room temperature) in TBS-T/5% nonfat milk, and washed with TBS-T (3 × 10 min). Immunoreactive bands were visualized by chemiluminescence (Perkin Elmer, Boston, MA, USA).