Reagents, Cell Culture and Mice
Short hairpin (sh)RNAs targeting eIF4E were designed and tested as previously described (Paddison et al., 2004
). Two of these, sh4E.389 (5'
) and sh4E.610 (5'
), as well as shFLuc.1309 (a neutral shRNA targeting Firefly luciferase (Premsrirut et al., 2011
)) were targeted to the murine Col1A1 locus using a Flp/FRT recombinase-mediated cassette exchange (RMCE) strategy in pre-engineered ES cells (Hochedlinger et al., 2005
). All ES cells were selected and maintained on irradiated (40 Gy) MEFs derived from the DR4 mouse strain. ES cells were cultured in knockout Dulbecco's modified Eagle's medium (Cellgro Mediatech) supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin, non-essential amino acid, LIF (leukemia inhibitory factor), and 55μM β-mercaptoethanol. Electroporations with KH2 ES cells were performed with 50 μg pColTGM (aka CTGM) and 25 μg pCAGGS-FLPe at 400V and 125 μF as previously described (Premsrirut et al., 2011
). ColAI-targeted clones were selected in hygromycin, tested for GFP inducibility, and transgenic mice derived using tetraploid embryo complementation.
Eμ-Myc mice were crossed to 4E.389/rtTA, 4E.610/rtTA, or FLuc.1309/rtTA mice to generate triple transgenic mice. Genotypes were obtained at the expected Mendelian inheritance ratios. The Eμ-Myc transgene was detected by genomic PCR amplification of a 600-bp product using the primers 5'-GGACAGTGCTTAGATCCAAGGTGA-3' and 5'-CCTCTGTCTCTCGCTGGAATTACT-3'. Genotyping for R26-rtTA was performed using the primers 5'-AAAGTCGCTCTGAGTTGTTAT-3', 5'-GCGAAGAGTTTGTCCTCAACC-3', and 5'-GGAGCGGGAGAAATGGATATG-3'. Genotyping for R26-rtTA yields two PCR products of ~500 bp (wild-type ROSA26 allele) and ~300 bp (R26-rtTA allele). Genotyping for 4E.389 by PCR used the primers 5'-AATTACTAGACAACTGGATTGCCT-3' and 5'-GAAGAACAATCAAGGGTCC-3' (~200 bp product), whereas genotyping for 4E.610 by PCR used the primers 5'-GCCACAGATGTATTTAGCTCTAAC-3' and 5'-GAAGAACAATCAAGGGTCC-3' (~200 bp product). Genotyping for FLuc.1309 used the primers 5'-CACCCTGAAAACTTTGCCCC-3' and 5'-AAGCCACAGATGTATTAATCAGAGA-3' (~300 bp product).
All mice strains were maintained on a C57BL/6 background. Activation of shRNAmir production in mice was performed in 4 wk old transgenic mice by supplying DOX (1mg/ml) in the drinking water (+5% sucrose) for the indicated periods of time. DOX-supplemented water was changed every 4 days. To assess the impact of silvestrol on lymphoma onset, 4 wk old Eμ-Myc mice were treated with vehicle (5.2% PEG 400/5.2% Tween-80) or 0.2 mg/kg silvestrol (daily intraperitoneal injections) for 23 consecutive days. All mice were monitored twice a week for signs of morbidity and lymphoma development, the latter scored by peripheral lymph node palpation. Tumor-free survival is defined as the time from birth to the time of appearance of a palpable lymphoma. Data were analysed in the Kaplan-Meier format using the log-rank (Mantel-Cox) test for statistical significance. All animal studies were approved by the McGill University Faculty of Medicine Animal Care Committee.
Fresh cell suspensions were isolated in PBS+ 2% FBS. Erythrocytes were removed by lysis in ACK buffer (150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA). Remaining cells were collected by centrifugation and resuspended in 1 ml PBS + 2% FBS. Blocking was performed by incubating samples with purified anti-CD16/CD32 antibody (clone: 2.4G2; BD Biosciences) for 5 min on ice before labelling cells with fluorochrome conjugated substrate specific antibodies (see Supplemental Experimental Procedures). The forward and side light-scatter gate excluded small apoptotic cells and granular cells, whereas large cells were included.
Antibodies used to identify monocytes and granulocytes were: Ly-6G (Gr-1) PECy7 (clone 1A8; BD Biosciences) and CD11b PE (clone M1/70; BD Biosciences). Antibodies used to identify T and B lymphocytes were: CD4 PE (clone RM4–5; BD Biosciences) and CD45R/B220 PE (clone RA3–6B2; BD Biosciences). Incubations were performed in the dark on ice for 20 min before data acquisition and analysis were conducted on a FACSAria II (BD Biosciences). Erythrocytes, dead cells and debris were excluded with gating based on forward/side scatter characteristics. The percent B and T lymphocytes, monocytes and granulocytes for each sample was expressed as a percentage of total gated cells analyzed.
To measure apoptosis in vivo, TUNEL assays were performed on freshly isolated splenic cells from indicated transgenic mice treated with vehicle or DOX for 6 days following the manufacturer's instructions (In Situ Cell Death Detection Kit, TMR red, Roche).
For cell cycle analysis, freshly isolated splenic B220+ cells from vehicle or DOX-treated transgenic mice were incubated with 1 ml DNA staining buffer (0.3% Triton-X 100, 50 μg/mL propidium iodine, 20 μg/ml RNAase A and 4mM sodium citrate). Cell cycle distribution was analyzed by flow cytometry using a Guava EasyCyte (Millipore).
For silvestrol-treated C57BL/6 and Eμ-Myc mice, freshly isolated splenic B220+ cells (106 cells/ml) were washed, fixed in 75% ethanol solution for 1 hour at 4°C and stained with propidium iodide (Sigma) (50 μg/ml propidium iodide, 3.8 mM sodium citrate, and 500 μg/ml RNase A) for 3 hours at 4°C. Cells were then analyzed for DNA content using a FACScan (BD Biosciences).
For Western blotting, cells were lysed in RIPA lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM DTT, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml each of leupeptin, pepstatin, and aprotinin). Protein concentrations were determined using the Bio-Rad Protein assay. Total protein lysates (30 μg) were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), probed with the indicated antibodies, and visualized using enhanced chemiluminescence (ECL) detection (Amersham). Primary antibodies were: anti-cyclin D1 was from Cell Signaling Technology (#2926, Beverly, MA), and anti-Mcl-1 was purchased from AbD Serotec (#AHP1249, Oxford, UK). Anti-GFP (#sc-9996), anti-eIF4E(#sc-9976), anti-p27 (#sc-528) and anti-c-Myc (# sc-764) antibodies were obtained from Santa Cruz Biotechnology. Anti-β-actin (#A5316) and anti-tubulin (#T5268) antibodies were purchased from Sigma.
For metabolic studies, 2 × 105 B220+ cells were isolated from vehicle or DOX-treated triple transgenic mice and seeded in 24 well plates. Cells from DOX-treated mice were maintained in 1μg/ml DOX. Cells were cultured for 45 min in methionine-free medium, followed by 60 min in [35S]methionine-containing medium (150–220 μCi/ml) supplemented with 10% dialyzed FCS, washed and lysed in RIPA buffer. Proteins were TCA precipitated onto 3MM Whatman paper and the amount of radioactivity quantitated by scintillation counting. Values were normalized to total protein levels as determined by the Bradford assay.
For m7GTP Sepharose pull-down assays, freshly isolated cells were harvested in 300 μl of Lysis Buffer (20 mM Hepes7.5, 100 mM KCl, 1.0 mM EDTA, 1 mM DTT, 1 mM PMSF and 0.2% Tween 20, 10 mM NaF and 20 mM β-glycerophosphate), and then subjected to 3 cycles of freeze-thaw. The lysate was then incubated with 50 μl of 50% slurry of m7GTP-Sepharose 4B (GE Healthcare, United Kingdom) for 2 hrs at 4°C. The resin was washed three times with 1 ml of Lysis Buffer and one time with buffer A containing 200 μM GDP. Finally, proteins bound to the resin were eluted with 80 μl of m7GDP (1 mM) for 10 min on ice. Aliquots of the eluted fractions (25 μl) were resolved by SDS-PAGE (10% polyacrylamide) and analyzed by Western blotting.
Immunohistochemistry and TUNEL staining
Tissues were harvested, fixed in 10% formalin, and embedded in paraffin. Sections (5 μm) were then dewaxed and rehydrated through a graded series of alcohol washes to water. They were placed in 10 mM citric acid buffer (pH 6.0) and subjected to antigen retrieval by boiling for 15 minutes. Immunohistochemistry was performed using HRP/DAB Detection Kit (ab64261, Abcam) according to the manufacturer's instruction. Briefly, after incubation with blocking buffer for 1 hour and 3% hydrogen peroxide for 10 minutes, rabbit anti-eIF4E (Cat# 9742, Cell Signaling) or rabbit anti-GFP (Cat# 2555, Cell Signaling) was applied overnight at 4°C. Sections were washed with TBS-T (1 M Tris-HCl, pH 7.5, 1.5 M NaCl, and 1% Tween-20) and incubated with biotinylated goat anti-rabbit IgG for 30 minutes at room temperature. After washing with TBS-T, streptavidin peroxidase was added for 30 minutes at room temperature. The signals were developed using DAB chromogen as substrate at room temperature for 5 minutes. Sections were counterstained with hematoxylin, dehydrated and mounted with permount. Tissue sections were analyzed using an Aperio Scanscope XT (Aperio Technologies, Inc, Vista, CA, USA).