Allelic Discrimination Assay
We tested for the presence of RNase L variants R462Q and E541D as potential indicators of a genetic risk factor for IBC, using the TaqMan allelic discrimination assay (Life Technologies, Carlsbad, CA). Primer probes were identical to those designed by Shook et al.
. The primers and probes for R462Q were: forward primer 5'-GGAAGATGTGGAAAATGAGGAAGA-3', reverse primer 5'-TGCA- GATCCTGGTGGGTGTA-3', and probes 5'-VIC-CAGGACATTTCGGG- CAA-MGB and 5'-FAM-CAGGACATTTTGGGCAA-MGB. The primers and probes for E541D were: forward primer 5'-TCTATGTGGTAAAGAAGGGAAGCA-3', reverse primer 5'-TTGAACCACCTCTTCATTACTTTGAG-3', and probes 5'-VIC-TTTCAGATCCT-CAAAT-MGB and 5'-FAMTTTCAGCTCCTCAAAT-MGB 31
We extracted genomic DNA from 12 cell lines using the QIAamp DNA purification kit (Qiagen, Valencia, CA). We used 20 ng of DNA per reaction in 96-well plate format. All reactions were conducted in triplicate using an ABI 7500 Fast RT PCR System and analyzed using SDS 2.0 software (Life Technologies). P values for the cell line SNP comparisons were calculated using the online contingency table from VassarStats (http://vassarstats.net
A comparative search was performed on the NCBI GEO site (http://www.ncbi.nlm.nih.gov/geo/
) in order to assess differences in expression of RNase L between IBC and non-IBC tumor tissues. The GSE5847 entry originally published by Boersma et al.
provided a suitable data set with 15 IBC and 35 non-IBC tumor samples, with 2 normal breast tissue samples as a control 35
. The decision to use this data set was based on lack of available entries for IBC samples, as well as the consistent nature of the expression profile of stromal tissue relative to tumor samples. Moreover, because we were looking for a DNA-based marker of genetic susceptibility, and tumor cells are highly heterogeneous, we selected the stromal data set for this analysis.
To assess SNP prevalence between IBC and non-IBC breast stromal tissue samples, a computational script was written in the language R based on the sample size analysis recommendations made by Pfeiffer et al
. The script was then independently verified using the Bioinformatics Institute's (BII) Online Sample Size Estimator (http://osse.bii.a-star.edu.sg/
). Using a case-control design, based on the lowest minor allele frequency (MAF) for SNP rs486907; we based these estimates on the MAF for rs486907, because the lower allele frequency will require a larger sample size. We calculated the required sample sizes to test a significant risk ratio between normal samples and IBC or non-IBC samples, as well as the risk ratio between IBC and non-IBC samples.
Cell Culture SUM149 cells, BT474 cells, and MDA-MB-231 cells were used in this study. All cell lines were acquired from the American Type Culture Collection (ATCC, Manassas, VA) except the SUM149 cell line sourced from Asterand (Detroit, MI). All cell lines were subjected to genotyping with the ABI Identifiler Assay (Life Technologies) for validation of cell line identity. SUM149 cells were grown in Ham's F-12 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics/antimycotics (Invitrogen, Carlsbad, CA). BT474 cells were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS and 1% antibiotics/antimycotics (Invitrogen). MDA-MB-231 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% antibiotics/antimycotics (Invitrogen). All cell lines were maintained in a humidified incubator with 5% CO2 atmosphere at 37o C. Cells were plated in triplicate at a density of 5,000 cells per well in 96-well tissue culture plates and grown to greater than or equal to 30% confluence at the time of treatment. Purified DNA from the SUM190 cell line was graciously provided by Dr. Cunliffe from the Translational Genomics Research Institute (TGen) in Phoenix, AZ.
IFN-α Treatment IFN-α (Imgenex, San Diego, CA) was dissolved in phosphate-buffered saline (PBS) with 5% fetal bovine serum to a final stock concentration of 100 µg/ml. Before treatment, the complete medium was removed and the cell monolayers were rinsed once with PBS. Cells were then treated with 0, 500, 1000, 2500, and 5000 U/ml of IFN-α for 24 hours and 48 hours. At each time point, we removed the IFN-α, rinsed the cells with PBS, and evaluated cell proliferation. For IFN-α block experiments, we pre-incubated the cells for 15 minutes with 1 µg/ml IFN-α specific antibody (Sigma, St. Louis, MO).
Proliferation Assays Cell proliferation was evaluated in triplicate for all treatments with the CyQUANT cell proliferation assay kit (Invitrogen), per the manufacturer's instructions. Briefly, IFN-α treatment was removed and the cells washed once with PBS. 100 µl CyQUANT cell proliferation assay working reagent was then added to the cells and incubated for 1 hour at 37o C. Emission (directly proportional to proliferation) was recorded on the BioTek FLx800 plate reader (Winooski, VT). Proliferation data was analyzed by one-way analysis of variance using the Tukey multiple comparisons test and GraphPad Prism software (GraphPad Prism Software, Inc., La Jolla, CA). All assays were performed in at least triplicate and were analyzed together at the same time point.
Primer Design and PCR
Putative HMTV sequence were obtained from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/sites/entrez
). Specific primers were designed to the env
/LTR and late LRT regions. The primers used for PCR of the env
/LTR region were as follows: 5'TCT GCG TTA CAC CAC TAC CG 3' and 5'TGA ACT CGA CCT TCC TCC TG 3'. The primers used for PCR of the late LTR region were as follows: 5'
ACC TTC CTC CTG AGC CTA GC 3' and 5'TTT ATT AGC CCA ACC TTG CG 3'. For reverse-transcription polymerase chain reaction (RT-PCR), total RNA was isolated from SUM149, BT474 and MDA-MB-231 cells using the RNeasy purification kit (Qiagen) and cDNA produced using the first strand cDNA synthesis kit from MBI Fermentas (Glen Burnie, MD). To conduct PCR, we used MBI Fermentas reagents and evaluated products on a 1% Tris-borate-EDTA (TBE)/agarose gel. Gel images were acquired using a Gel Logic 200 Imaging System with Kodak 1D 3.6 software (Carestream Molecular Imaging, Rochester, NY).
Cloning and SequencingPCR products of interest were excised from agarose gels and the DNA purified using the QIAquick gel extraction kit (Qiagen). Purified PCR products were then ligated into the pGEM-T Easy Vector (Promega, Madison, WI) overnight at 4o C and transformed into TOP10 Chemically Competent E. coli (Invitrogen). Blue-white colony selection was used to screen for recombinant plasmids containing ligated PCR fragments (recombinants resulting in disrupted β-galactosidase function, preventing metabolism of X-gal substrate). Recombinant plasmids were evaluated by Sanger sequencing, performed by the Genomic Analysis and Technology Core Facility at the BIO5 Institute at the University of Arizona (Tucson, AZ).
Western Blot Analysis Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% gradient minigel (Bio-Rad, Hercules, CA), using the Mini-PROTEAN 3 Cell, run at 100 V for 1.5 hours at 25° C. 15 μg of total protein from cytoplasmic/membrane extracts were resolved and transferred to nitrocellulose membranes using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Efficient transfer of proteins was confirmed by SYPRO Ruby protein blot stain (Bio-Rad) and Kaleidoscope molecular weight markers (Bio-Rad). Nitrocellulose membranes were blocked for 2 hours in blocking buffer (4.0% bovine serum albumin [BSA], 10 mM PBS, 0.05% Triton X-100, pH 7.4) at 25° C. Membranes were next incubated with antihuman-specific rabbit monoclonal IFN receptor alpha IFNAR1 antibody (ab45172, Abcam, Cambridge, MA), diluted 1:20,000 v/v in blocking buffers overnight at 4° C with gentle agitation. Following incubation with primary antibody, membranes were washed 3 times in 10 mM PBS, 0.05% Triton X-100, pH 7.4 then incubated in donkey anti-rabbit secondary antibody with alkaline phosphatase (AP) conjugate (1:1000 v/v; Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 hour. Nitrocellulose membranes were washed 3 times in 10 mM PBS, 0.05% Triton X-100 pH 7.4; and protein products visualized after 1 to 5 minutes following addition of AP substrate (MBI Fermentas). Quantification of a specific protein band was established with GelQuant.NET software provided by biochemlabsolutions.com. In the densitometry analysis, relative pixel density of the IFN receptor wells was normalized to that of β-actin.
Southern Blot Analysis 10 µg purified SUM149 genomic DNA was digested with 5U FastDigest BamHI restriction enzyme (MBI Fermentas) at 37o C for 30 minutes, then heat inactivated at 80o C for 5 minutes. DNA was subsequently extracted with an equal volume of isopropyl alcohol and resuspended in 10 µl nuclease-free water at room temperature for 15 minutes. The entire volume was electrophoresed through a 1% agarose gel in 1X TBE buffer for 6 hours at 3 V/cm (until the bromphenol blue marker reached the bottom of the gel). Halfway through the gel electrophoresis, we loaded a positive control synthetic fragment (IDT Technologies, San Diego CA) encoding for a 172 base-pair region of the HMTV env region 5' TAT GAT TTT ATC TGC GTT ACA CCA CTA CCG TAT AAT GCT TCT GAG AGC TGG GAA AGA ACC AAG GCT CAT TTA CTG GGC ATT TAA AAT AAC AAT GAG ATT TCA TAT AAC ATA CAA AAA TTA ACC AAC CTA ATT AGT GAT ATG AGC AAA CAA CAT ATT GAC GCA GTG GAC CTT A 3'. Before blotting, the gel was rinsed in deionized water, incubated in denaturing solution for 30 minutes at room temperature with shaking, rinsed again in deionized water and incubated in neutralization for 15 minutes at room temperature with shaking. We repeated this procedure and then transferred the DNA by traditional upward capillary action for 18 hours at room temperature. After transfer, the membrane was washed in 2X SSC solution to remove any residual agarose, dried at room temperature, and fixed by UV crosslinking for 2 minutes.
Probe Synthesis HMTV env DNA (500 ng) was labeled using the Biotin DecaLabel DNA Labeling Kit (MBI Fermentas). The HMTV env DNA template was combined with 5x decanucleotide reaction buffer and nuclease-free water. The tube was vortexed, pulse-spun for 5 seconds, incubated in a boiling water bath for 10 minutes, and quickly cooled on ice. Biotin Labeling Mix and 5U of Klenow fragment were added and the reaction incubated for 1 hour at 37° C. The reaction was stopped by adding 1μl 0.5M EDTA, pH 8.0. The labeled DNA was directly used for hybridization.
Hybridization and Detection The membrane was incubated in a pre-hybridization solution containing 5X SSC/5X Denhardt's, 0.5% SDS, 100 µg/ml nonspecific DNA (Sigma) at 42° C for 4 hours with agitation in a ProBlot 12 hybridization oven (Labnet International, Woodbridge, NJ). During this time, the biotin-labeled probe was denatured at 100° C for 5 minutes and chilled on ice. The denatured probe was added to the pre-hybridization solution to obtain a final probe concentration of 100 ng/ml and incubated it overnight at 42° C with shaking. After hybridization, the membrane was washed twice with 2X SSC, 0.1% SDS for 10 minutes at room temperature, then twice with 0.1X SSC, 0.1% SDS for 20 minutes at 65º C. Excess liquid was removed from the membrane by briefly placing it on filter paper. The biotin-labeled DNA was detected using the Biotin Chromogenic Detection Kit (MBI Fermentas), according to the manufacturer's directions. Color development started to be visible after 1 hour. We acquired both gel and membrane images using the Gel Logic 200 Imaging System with Kodak 1D 3.6 software (Carestream Molecular Imaging, Rochester, NY).