P493-6 cells were the generous gift of D. Eick at the Institute for Clinical Molecular Biology und Tumor Genetics, GSF-Research Centre, Munich, Germany. Cells were cultured in RPMI 1640, 10% fetal calf serum, and penicillin-streptomycin (Pen-Strep). To repress c-MYC expression, cells were treated with RPMI 1640-10% fetal calf serum-Pen-Strep supplemented with 0.1 μg/ml tetracycline (Sigma) for 72 h, washed two to three times in 1× phosphate-buffered saline (PBS), and then restimulated with regular RPMI medium for various time points. For desferrioxamine treatment, P493-6 cells were placed in RPMI medium containing 50 or 100 μM desferrioxamine (Sigma) at the same time as removal of tetracycline and collected at various time points. TGR-1 (wild-type rat fibroblasts) and HO15.19 (c-MYC knockout rat fibroblasts) cells (gift of J. Sedivy, Brown University) and c-MYC null cells reconstituted with W135E (gift of L. Z. Penn, University of Toronto) were cultured in Dulbecco's modified Eagle's medium (Gibco/Life Tech)-10% fetal bovine serum-Pen-Strep. HO15-MYC cells were made by stable transfection of a MYC expression construct driven by a murine leukemia virus promoter. For growth rate experiments under limiting conditions, 2.5 × 104 cells were plated and subsequently starved in 0.1% serum for 48 h. Cell were then washed, grown in 1% serum, and counted in triplicate wells every 24 h for 6 to 7 days.
Wright staining was performed according to the manufacturer's protocol using the HEMA 3 stain set (Fisher Scientific).
Western blot analysis.
For immunoblot analysis, cells were collected and lysed in incomplete Laemmli buffer at 95°C and protein was quantitated using a bicinchoninic acid kit (Pierce). Fifteen to twenty micrograms of protein was loaded on either a 8% or 10% polyacrylamide gel (for TFRC1 or c-MYC, respectively) and transferred to a nitrocellulose membrane. 9E10 c-Myc and TFRC1 mouse monoclonal antibodies were obtained from Zymed, Inc. (clone H68.4). α-Tubulin mouse monoclonal antibody was obtained from Oncogene Research Products.
In vivo analysis of TFRC1 in murine B-cell lymphomas.
Tumor-derived neoplastic lymphocytes were washed twice with PBS and resuspended in fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin in PBS) at a concentration of 1 × 106 cells/ml. One-hundred-microliter aliquots were incubated on ice for 45 min with 2 μl phycoerythrin-labeled anti-mouse CD71 (clone RI7 217.1.4; CALTAG, Burlingame CA). Samples were washed with PBS and subjected to flow cytometry. Unstained cells were used as a reference.
P493-6 cells untreated or treated with 0.1 μg/ml tetracycline for 72 h were used for chromatin immunoprecipitation (ChIP) assays. Cells were cross-linked with formaldehyde, and chromatin was immunoprecipitated as described previously (7
). The rabbit polyclonal c-Myc (sc-764; Santa Cruz Biotechnology) and human hepatocyte growth factor (sc-7949; Santa Cruz) antibodies were used to immunoprecipitate chromatin fragments. Total input controls were collected from the “no antibody” control supernatant. Mock control samples lacked chromatin but were treated the same as other samples.
Retroviral production and infection.
The TRS1 vector was a gift from Jim Basilion (Massachusetts General Hospital, Harvard University). The insert was first cloned into the pSG5 vector using the EcoRV and SalI restriction sites and then cloned into the pMSCV retroviral vector using the BamHI and BglII restriction sites. For retroviral transduction, the pMSCV vector containing the TRS1 insert was transfected into Phoenix amphotropic cells (at 80 to 90% confluence) using the CaPO4 method. Viral supernatant was collected every 24 h for 3 days. For infection of c-MYC-null cells, approximately 2 × 105 cells were plated per 10-cm dish and infected with 8 ml viral supernatant and 8 μl/ml polybrene 24 h later. Two subsequent rounds of infection were performed, and cells were then selected with puromycin for 14 days. Cell lysates were then collected 7 days after removal from puromycin.
Preannealled small interfering RNA (siRNA) duplexes directed against TFRC1 (and a scrambled control) were purchased from Dharmacon, Inc., and used according to the manufacturer's instructions (Option C). TFRC1 oligonucleotides used were GGAUGGUAACCUCAGAAAGdTdT (sense) and dTdTCCUACCAUUGGAGUCUUUC (antisense). Scrambled JTV1 control oligonucleotides were CACGCUCGGUCAAAAGGUUdTdT (sense) and dTdTGUGCGAGCCAGUUUUCCAA (antisense). RNA interference (RNAi) oligonucleotides were transfected by electroporation. siRNA duplexes were first added to a 4-mm-gap cuvette (BTX/Cambrige Pharmaceuticals) and then mixed with 3 × 106 P493-6 cells (in a total volume of 500 μl). Electroporation settings used for P493-6 cells were as follows: 240 V and 1500 μF. Cell viability was assessed immediately following electroporation by trypan blue exclusion (and determined to be approximately 30%). Mock-transfected cells and cells transfected with a scrambled siRNA duplex were used as controls. Cells were collected and assayed for gene function approximately 72 h after electroporation.
HO15 and TGR cells were first trypsinized and counted. Approximately 1 × 106 cells were washed two times in 1× PBS and then resuspended in 2 ml serum-free Dulbecco's modified Eagle's medium. Samples were then serum starved for 2 h with rotation at 37°C to clear transferrin (present in culture medium) from the transferrin receptors. Alexa633-labeled transferrin (Molecular Probes) was then added to each sample in a final concentration of 5 μg/ml, followed by incubation for 30 min, rotating at 37°C. Cells were then washed three times with 1× PBS, resuspended in chilled 1% paraformaldehyde, and analyzed using a Beckman Dickinson FACScalibur machine.
Nude mouse experiments.
Cells (5 × 106) in 100 μl of sterile Hanks balanced salt solution (Gibco) were injected subcutaneously into the right flank of male homozygous nude mice at 4 to 6 weeks of age. Tumor volume was measured using calipers every 3 to 5 days until the tumor mass reached 1,500 mm3. Tumor volume was calculated using the following formula: [length (mm) × width (mm)]2/2. Two independent experiments were performed (n = 4 to 5 mice per cell line for experiment 1 and n = 15 mice per cell line for experiment 2). All experiments were approved by the Johns Hopkins School of Medicine Animal Care and Use Committee.
Correlation of TFRC1 and c-MYC mRNA levels using the Atlas Gene Expression database. TFRC1
mRNA levels were examined in 91 human tissues and cell lines using the Gene Expression Atlas database (http://expression.gnf.org/cgi-bin/index.cgi
). We first log transformed the expression values and then performed linear regression analysis (using SPSS version 11.0 for Windows) on log-transformed mRNA expression values with log TFRC1
expression levels as the dependent variable and log c-MYC
expression values as the independent variable. An r2
value was determined which measured the extent to which expression of c-MYC
For cell size analysis, P493 cells were collected, washed two to three times, and then resuspended in 1× PBS. For propidium iodide staining, approximately 1 × 106
cells were washed in 1× PBS, trypsinized for 10 min at room temperature (RT) (solution A), neutralized with trypsin inhibitor-RNase A for 10 min at RT (solution B), and then stained with propidium iodide/spermine tetrahydrochloride (solution C) for 10 min at RT (54
). Cells were filtered and then analyzed using a Becton Dickinson FACScan or FACSCalibur flow cytometer.
For anti-human CD71 labeling of P493 lymphocytes, cells were washed in 1× PBS and then resuspended in staining buffer (1× PBS, 2% fetal bovine serum) to a concentration of 2 × 107 cells per ml. Twenty microliters fluorescein isothiocyanate (FITC)-labeled CD71 antibody (BD Biosciences) was added to 1 × 106 cells (in 50-μl aliquots) and incubated on ice in the dark for 30 min. After washing with cold 1× PBS, cells were resuspended in 500 μl 1× PBS and analyzed by flow cytometry.
mRNA quantitation (real-time PCR).
Total RNA was extracted from P493-6 cells using Trizol (Invitrogen) or the RNeasy kit (QIAGEN). Quantitative real-time PCR expression was performed using the ABI 7700 sequence detection system. TFRC1, p53, and p21 mRNA expression levels were determined using predeveloped mixtures of specific probe and primers (PE Applied Biosystems) and the TaqMan One-step RT-PCR Master Mix kit (PE Applied Biosystems). A predeveloped probe and primers specific to 18S rRNA levels were used for normalization. All PCRs were performed in triplicate.
Quantitation of ChIP fragments was performed using the SYBR Green core reagent kit (PE Applied Biosystems) according to the manufacturer's instructions. TFRC1-specific primers were designed using the OMIGA program or Primer Express software (listed in Table S1 in the supplemental material). PCR parameters were optimized using the Failsafe Real-time PCR PreMix selection kit (EpiCentre, Madison, WI). Known quantities of 10-fold dilutions of total input DNA were used to generate standard curves for each primer pair. Relative amounts of each ChIP sample (expressed as a percentage of total input) were determined in the linear range according to their CT value. For each primer set, melting curves were used to verify the correct PCR product.
For microarray validation, mRNA samples were first reverse transcribed and then subjected to SYBR Green quantitative real-time PCR. Primers were designed to cross exon-exon junctions and span less than 400 bp of the target mRNA. Primer sequences are shown in Table S6 in the supplemental material.
Expression profiling using DNA microarrays.
Seventy-two hours after TFRC1 siRNA transfection, P493 cells were stained with the CD71-FITC antibody as described above and then sorted into TFRC1-negative and -positive populations using a Becton Dickinson cell sorter. Approximately 40 independent TFRC1 siRNA transfections were pooled for sorting. Samples from two independent biological replicates were then processed at the JHMI microarray facility and used to probe an Affymetrix U133 Plus 2.0 array. Total RNA was isolated from cells using the RNeasy minikit (QIAGEN). Five micrograms of starting total RNA from control and experimental cell preparations (two independent replicates for each) was processed using single-round RNA amplification protocols, following Affymetrix specifications (Affymetrix GeneChip Expression Analysis Technical Manual). Briefly, 5 μg of total RNA was used to synthesize first-strand cDNA using oligonucleotide probes with 24 oligo(dT) plus T7 promoter as a primer (Proligo LLC) and the SuperScript Choice system (Invitrogen). Following double-stranded cDNA synthesis, the product was purified by phenol-chloroform extraction, and biotinylated antisense cRNA was generated through in vitro transcription using the BioArray RNA High Yield transcript labeling kit (ENZO Life Sciences Inc). Fifteen micrograms of the biotinylated labeled cRNA was fragmented at 94°C for 35 min (100 mM Tris-acetate, pH 8.2, 500 mM potassium acetate, 150 mM magnesium acetate), and 10 μg of total fragmented cRNA was hybridized to the Affymetrix Human Genome GeneChip array U133Plus 2.0 for 16 h at 45°C with constant rotation (60 rpm). An Affymetrix Fluidics Station 450 instrument was then used to wash and stain the chips, removing the nonhybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinylated cRNA. The staining was then amplified using goat immunoglobulin G as a blocking reagent and biotinylated antistreptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate. Fluorescence was detected using the Affymetrix GeneChip scanner (GS 3000), and image analysis of each GeneChip was done through the GeneChip operating system software from Affymetrix (GCOS1.1.1), using the standard default settings. For comparison between different chips, global scaling was used, scaling all probe sets to a user-defined target intensity of 150.
To ascertain the quality control of the total RNA from the samples, we used the Agilent Bioanalyzer, Lab on a Chip technology and confirmed that all the samples had optimal rRNA ratios and clean run patterns. Likewise, this technology is used to confirm the quality of the RNA in the form of cRNA and fragmented cRNA. To assess the quality control of the hybridization, GeneChip image, and comparison between chips, we confirmed the following parameters: resulting scaling factor values within comparable range, background within average, significant percentage of present calls, 3′/5′ ratios of glyceraldehyde-3-phosphate dehydrogenase as a representation of housekeeping genes, and presence of internal spike controls.
The initial analysis of the expression results was based on pairwise comparisons among the different experimental conditions represented by the samples. Any transcript that showed an arbitrary value of at least a twofold change in expression level between the experimental sample and the control sample was considered significantly differentially expressed. All analysis was perfomed using the Li and Wong method. A lower bound of 2.0 was used as the cutoff for a significant change in gene expression.