Estrogen receptor positive human adenocarcinoma (MCF-7) breast cancer cells (obtained from American Type Culture Collection, Bethesda, MD) were maintained as monolayer cultures by weekly serial passage in 100 mm tissue culture plates in Dulbecco’s Modified Eagle medium (DMEM, Life Tech., Grand Island, NY) supplemented with 44 mM sodium bi-carbonate, 2 mM L-glutamine, 10.2 I.U/mL penicillin, 10.2 μg/mL streptomycin, 10 mM HEPES and 10% heat-inactivated fetal bovine serum (JRH Tech., Lenexa, KS) in a 95% air/5% CO2 humidified incubator. Cells were serum-starved by incubating in 2% serum containing complete growth medium for at least 16 h. Cell viability was monitored by the trypan blue dye exclusion method. When we co-cultured MCF-7 cells with BAEC cells, they shared same medium, MCDB-131 medium, so we adapted the MCF-7 cells into complete MCDB-131 medium for 3–4 passages. MCF-7 cells grew well in MCDB medium, and the doubling time (about 20 hours) became shorter than in DMEM medium (about 29 hours).
Primary Bovine aortic endothelial cells (BAEC; Clonetics/BioWhittaker, San Diego, CA) were cultured in MCDB-131 medium (Sigma, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, California) enriched with 250 ng/mL basic fibroblast growth factor (Pepro Tech, Rocky Hill, NJ), 1 mg/mL epidermal growth factor (PeproTech, Rocky Hill, NJ), 1 mg/mL hydrocortisone (Sigma, St. Louis, MO), 100 U/mL penicillin, and 100 mg/mL streptomycin (Mediatech, Herndon, VA). For all the experiments the cells were incubated in MCDB-131 medium with reduced serum concentration (2% FBS) for at least 16 h unless otherwise specified. Sterile 200 μL Gelatin solution (0.2%, 1 g gelatin to 500 mL endotoxin-free water, autoclave for 30 minutes to keep gelatin solubilize) was used for coating 100 mm cell culture plates (80 μL/well for 6-well plates) for the growth of endothelial cells, and the gelatin solution was left in the plates/wells at room-temperature for 2 hours. Cells at passages 3 were used to start the culture, cell passages 4–8 were used for all the experiments.
Exposure to ionizing radiation
For the selected doses of low Linear energy transfer (low-LET) radiation exposures, the cells were removed from 37 °C incubator and exposed to a clinically relevant dose (2 Gy) of 137Cs γ-rays at a dose rate of 1.03 Gy/min (Atomic Energy of Canada Ltd. Gamma Cell-40 Irradiator) at room temperature (22 °C). Immediately after exposures, the cultures were returned to the 37 °C incubator and harvested at selected time points specified for each experiment. Mock-irradiated control cells (0 Gy) were treated identically, except the cells were kept outside the exposure chamber.
Electrophoretic Mobility Shift Analysis (EMSA)
Nuclear proteins were extracted according to the method described in our earlier publication.27
The cell pellet was resuspended in 500 μL of cold buffer A (10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [HEPES] –KOH pH 7.9 at 4 °C, 10 mM KCl, 1.5 mM MgCl2
, 0.5 mM dithiothreitol (DTT), 1 mM of phenylmethylsulfonylfluoride (PMSF) and 1 mM of protease inhibitors (PI, Sigma, St. Louis, MO) followed by centrifugation for 10 seconds at 15,000 rpm in a microfuge centrifuge (Eppendorf, Houston, TX) at 4 °C. The cells were then lysed by incubating for 10 minutes at 4 °C with 50 μL of cold buffer A containing 0.1% NP-40, followed by centrifugation for 10 seconds at 15,000 rpm in the microfuge centrifuge at 4 °C. The nuclear pellet obtained after centrifugation was then resuspened in 20 μL of buffer B (20 mM HEPES– KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM of PMSF and 1 mM of PI) and incubated on ice for 10 minutes for high salt extraction. The samples were centrifuged again for 10 minutes at 15,000 rpm in a microfuge centrifuge at 4 °C. The clear supernatant was transferred to pre-chilled tubes and diluted with 30 μL of buffer C (20 mM HEPES–KOH, pH 7.9, 50 mM KCl, 0.2 mM EDTA and 20% glycerol). The total nuclear protein concentration in each sample was determined using the bicinchonic acid (BCA) method following the manufacturer’s protocol (Pierce, Rockford, IL). The color intensity was measured with an ELISA plate reader (Dynatech MR 5000, Chantilly, VA).
For EMSA analysis, a double-stranded oligonucleotide (5′ -AGT TGA GGG GAC TTT CCC AGG C- 3′) (Promega, Madison, WI) containing a tandem repeat of the consensus sequence 5 -GGG GAC TTT CC-3 was end-labeled with [γ-32P] ATP using T4 polynucleotide kinase. A push column device (Stratagene, La Jolla, CA) was used to separate the free unbound radioisotope from the bound probe. We first equilibrated a push column by adding 75 μL of STE (10 mM Tris-7.5, 1 mM EDTA, 100 mM NaCl) and forced the buffer to go through with a 10 cc disposable syringe (BD, Franklin Lakes, NJ) until one drop came out from the tip of the column. Then a beta shield device was used to mount the column. The reaction mix mentioned above, was added into the column, and gently pushed through the column. Seventy-five microliter of fresh STE was added to the top of the column and we repeated the steps mentioned above. The enriched probe separated from free nucleotide was collected in a 1.5 mL Eppendorf tube. Two microliters of the elution was added to four microliters of scintillation cocktail (PerkinElmer, Waltham, Massachusetts) and the specific activity determined. The binding reaction was performed by mixing nuclear extract (2 μg of total protein), 0.1 ng of poly (dI-dC), and 0.5 ng of [γ-32P] ATP labeled (111 TBq/mmol) NF-κB-specific oligonucleotide probe (Amersham, Arlington Heights, IL) in binding buffer containing 10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, and 20% (v/v) glycerol. All samples were incubated at room temperature (22 °C) for 20 min and subsequently electrophoresed at 100 V through non-denaturating 6% polyacrylamide gel in Tris-glycine buffer (25 mM Tris, 0.19 M glycine, and 1 mM EDTA, pH 8.3). The gels were then dried and autoradiographed at 70 °C with intensifying screens on hyperfilm (GE Health-care, Piscataway, NJ). After scanning the films using Epson Perfection V750, the total amount of NF-κB activation was determined by quantitative analysis using Adobe Photoshop image analysis software (Adobe, San Jose, CA).
For indirect co-culture, MCF-7 cells were cultured in the apical compartments of cell culture inserts (Pore size: 0.45 μm, growth area: 4.2 cm2, Falcon-BD Bioscience, Franklin Lakes, NJ) with endothelial cells in the basal compartment of 6-well plates (growth area: 9.6 cm2). Because MCF-7 cells have shorter doubling time than endothelial cells, different numbers of MCF-7 (2 × 105 cells) and endothelial cells (5 × 104 cells) were seeded to achieve equal numbers of each cell type at the treatment time. The semipermeable membrane of the insert allows the diffusion of secreted factors but prevents the cells from moving from one chamber to the other. There was no cell contact between the two sides of the two chambers. Complete MCDB medium with 10% FBS were shared by both the MCF-7 and endothelial cells. Sixteen hours after both kinds of cells were seeded, the inserts containing MCF-7 cells were placed on a separate 6-well plate (exposure plate) containing MCDB medium with reduced serum (2% FBS), after two hours, the MCF-7 cells were exposed to selected doses of radiation or sham-exposed to radiation. Immediately after exposure, the inserts were taken out of the exposure plate and placed on the 6-well plate containing BAEC cells (bottom chamber). After 16 hours, the expression of angiogenic growth factor receptors was measured in the co-cultured endothelial cells.
Concentration of the proteins in the medium
To enrich the proteins in the conditioned medium, the disposable Centrifugal Filter Devices containing anisotropic membranes (Millipore, Billerica, MA) were used. To concentrate VEGF and FGF-2 in the medium, filter devices containing the molecular weight cut off of 30,000 kDa (YM-10) and 10,000 kDa (YM-3), respectively were used. The conditioned medium after treatment or exposures were added to the pre-chilled sample reservoirs of the concentrator without damaging the membrane and centrifuged at 3,000 × g for 6 hours at 4 °C. The solvents and the low molecular weight solutes (<30 kDa for YM-30 and <10 kDa for YM-10) passing through the membrane were collected into the filtrate vial. Since the molecular weight of VEGF and FGF-2 are 43 kDa and 18 kDa, respectively, the macro-solute retained in the reservoir above the filter membrane were collected (as the concentrated medium) for further assays. The concentrated samples were recovered from the sample reservoir by placing the retentive vial over the sample reservoir, inverted the device, and centrifuged for 3 minutes at 2,000 × g at 4 °C. The samples concentrated using these centrifugal filter devices provided up to 100-fold sample enrichment with minimal loss of solute (by adsorption) into the device components.
Cytoplasmic proteins from treated or exposed MCF-7 cells were extracted following the method described in our earlier publication.27
Culture medium was carefully removed from the cells and washed twice with cold 1 × PBS. The cells were lysed by adding 100 μL of cold RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a cocktail of protease and phosphatase inhibitors [1 mM of protease inhibitor, (PI; Sigma) and 1 mM of Phenylmethanesulfonyl fluoride (PMSF, Sigma)] to each 100 mm plates and scraped using cell scraper. The lysates were transferred to pre-chilled microcentrifuge tubes and centrifuged at 15,000 × g
for 15 minutes at 4 °C, and then transferred the clear supernatant to a new pre-chilled 1.5 mL Eppendorf tube for further analysis. Protein concentration was measured using the Bicinchonic acid (BCA) method as described in the manufacturer’s protocol (Pierce, Rockford, IL). Equal amount of protein samples from the cytoplasmic extract or from the concentrated medium (50 or 100 μg) were added with 1 × sample treatment buffer (125 mM Tris-Cl pH 6.8, 4% SDS, 20% glycerol and 10% 2-mercaptoethonol) and incubated at 96 °C for 10 min. The samples were then subjected to 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Separated proteins fractions were transferred to Hybond PVDF membrane (Amersham, Piscataway, NJ) by electro-transfer. The membrane was incubated in 1 × PBST (TBE; 10 mM Tris-Cl, pH 8.0, 150 mM NaCl; 0.05% Tween-20) containing 5% non-fat dry milk for 1 h to block non-specific binding sites. The blots were then incubated with anti-VEGF or anti-FGF-2 (1:1,000 dilution; Abcam, Cambridge, MA) for overnight at 4 °C. After the membrane was washed with 1 × PBST, the blots were then incubated with 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, CA) for 1 h at room temperature. The specific proteins were visualized via enzyme-linked chemiluminescence using the enhanced chemiluminescence (ECL) reagent (Amersham, Piscataway, NJ). The membranes were then exposed to BioMax ML film (Eastman Kodak, Rochester, NY). To monitor equal loading, the samples were probed with monoclonal antibodies for β-actin (Santa Cruz Biotechnology). The autoradiograms were scanned (Epson Perfection V750) and the protein expression was quantified using Adobe Photoshop Image-J software (Adobe, San Jose, CA).
To determine the expression of reciprocal receptor expression in the endothelial cells that were co-cultured with MCF-7 cells, Vascular endothelial growth factor receptor (VEGFR2) and fibroblast growth factors receptor (FGFR1) were examined. Western blotting was performed as described above using anti-VEGFR2 and anti-FGFR1 antibodies (1:1,000 dilution; Abcam, Cambridge, MA) as primary antibodies and a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA).
Tube formation assay in 3D matrigel matrix
To determine indirect effect of radiation on tubule formation, co-culture system were used. First, MCF-7 cells (4 × 104 cells/well) in complete MCDB medium (10% FBS) were seeded in the compartment of the cell culture inserts, which were in 24-well exposure plates overnight. Four hours before radiation exposure, the complete MCDB medium were replaced by plain MCDB medium in the MCF-7 cells, then were either mock irradiated or exposed to radiation. After radiation exposure, another 24-well plate was coated with 300 μL of Matrigel per well to the growth surface and incubate coated surface for 1 h at 37 °C to allow the gel to solidify. Endothelial cells (1 × 104 cells/well) in plain MCDB medium were then seeded in coated 24-well plate in the presence or absence of angiogenesis inducers (VEGF, 50 ng/mL) or inhibitors (NF-κB inhibitor, 10 μM). After 3 hours, cell culture inserts with MCF-7 cells were transferred to the endothelial cells plates. The tube formations were scored 8–10 hours later. Three random fields were viewed in triplicate wells for each test condition under high power Nikon ECLIPSE TE 2000-U microscope linked to a computer with Adobe Photoshop 7 software. Quantification of tubule formation was carried out by counting the number of tubule junction in the total area covered by tubules in each field using image analysis software Photoshop version 7.
Statistical design and methodologies
The proposed experiments were performed a minimum of three times in two sets. Data was expressed as mean ± SD. Statistical analyses were performed using paired and unpaired Student’s t-test to determine significant changes between test samples and controls. Paired data was used with repeated measures of analysis of variance. Statistical significance was defined as P < 0.05, P < 0.01.