Silvestrol was kindly provided by Dr. A. Douglas Kinghorn. PKC412 was purchased from LC Laboratories (Woburn, MA, USA).
Cell lines and primary blasts
MV4-11 and THP-1 cells (ATCC, Manassas, VA) were cultured in RPMI 1640 medium supplemented with 10% calf serum. Blasts from AML patients were maintained in RPMI 1640 medium supplemented with 30% fetal bovine serum, 1% HEPES buffer, and 1× StemSpan CC100 (StemCell Technologies, Vancouver, BC, Canada) containing IL-3, IL-6, FLT3 ligand and SCF. All cells were incubated at 37°C with 5% CO2. Patient AML blasts were obtained from apheresis blood samples collected from patients treated at the Ohio State University (OSU) and stored in the OSU Leukemia Tissue Bank. Informed consent to use cells for investigational studies was obtained from each patient under an OSU Institutional Review Board-approved protocol, according to the Declaration of Helsinki. Authenticating tests of these cell lines was done using monoclonal antibodies and FLT3 mutational analysis.
Cells were suspended 30 min in 1 × lysis buffer (20 mM Hepes, 150 mM NaCl, 0.1% NP40) containing protease inhibitor cocktail III (Calbiochem, Darmstadt, Germany) and lysate was recovered by centrifugation. Lysates were separated using 4-20% SDS-PAGE and transferred to PVDF membrane (GE Healthcare, Piscataway, NJ). Membranes were blocked using 5% milk or BSA in 1 × TBS with 0.1% Tween 20 (1 × TBS-T) for 1 hour at room temperature with shaking, then incubated overnight at 4°C in the following primary antibodies diluted in 1 × TBS-T with 5% milk or BSA: actin (Santa Cruz Biotechnology, Santa Cruz CA), FLT3 (Cell Signaling, Danvers, MA), phosphorylated and total STAT5 (Cell Signaling), P65 antibody (Billerica, MA). Membranes were washed using 1 × TBS-T, incubated with HRP-conjugated secondary antibodies diluted in 1 × TBS-T with 5% milk or BSA, washed, and developed using ECL Western Blotting Detection reagents (GE Heathcare).
RNA immunoprecipitation (RIP), RNA extraction, Real-Time RT-PCR
MV4-11 cells were treated with 50 nM silvestrol for 3 hour, lysed (5 min) in 100 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH 7.0], 0.5% NP-40, 1 mM dithiothreitol (DTT), 100 units/ml RNase OUT (Invitrogen), 400 mM vanadyl-ribonucleoside complex and protease inhibitors (Roche, Mannheim. Germany). Extracts were clarified and stored at −80°C. Anti-eIF4E antibody (cell signaling) and goat IgG (Sigma, St. Louis, MO) were incubated with protein sepharose A/agarose G-coupled beads overnight. Beads were subsequently washed four times with 50 mM TRIS/HCl, pH 7.0, 150 mM NaCl, 1 mM MgCl2, and 0.05% NP-40, and twice after addition of 1 M urea. Precipitates were digested with proteinase K (55°C), and eIF4E-associated mRNAs were isolated using Trizol reagent (Invitrogen, Grand Island, NY). cDNA was synthesized using SuperScript III reagents (Invitrogen) and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Quantitative Real-Time RT-PCR for FLT3 and PU.1 genes and miR-155 and miR-34a expression was performed using commercially available TaqMan Gene Expression Assay primers and probes and the 7900HT Fast Real-Time PCR System (Applied Biosystems). The comparative cycle threshold (CT) method was used to determine the expression levels normalized by the internal control 18S for gene expression.
Clonogenic and viability analysis
Methylcellulose clonogenic assays were carried out by plating 2 × 104 primary blasts in 0.9% MethoCult (Stem Cell Technologies). Colonies (>100 mm) from cell lines and primary cells were scored 14 days later. Growth inhibition assays were performed. Briefly, 5.0 × 104 cells were incubated in triplicate in a 96-well plate in the presence or absence of the different concentrations of silvestrol in a final volume of 100 μl for 24, 48 and 72 hours at 37°C. Thereafter, 20 μl of the CellTiter 96® AQueous. One Solution Reagent which contains tetrazolium compound [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) (Promega, Madison WI) was added to each well. After 4 hours incubation at 37°C, the optical density at 490 nm was measured. Cell viability was calculated with respect to the control samples. At least three independent experiments were performed.
For FLT3 detection, cells (5 × 105) were washed with phosphate-buffered saline (PBS) and resuspended in 50 μl binding buffer containing 5 μL FLT3 antibody (BD Biosciences, Billerica, MA). After 15 min incubation, cells were washed with PBS, resuspended in 400 μL flow buffer and analyzed on a FACSCalibur cytometer (BD Biosciences). To assess apoptosis, AML cells were incubated with 10, 30 and 50 nM silvestrol for 24 hours. Cells (5 × 105) were then washed with PBS and resuspended in 50 μl binding buffer containing 2 μL of annexin V-FITC stock (BioWhittaker, Inc, Walkersville, MD) and 5 μL propidium iodide (PI) (BD Biosciences). After 20 min incubation, fluorescence was quantified by flow cytometry on a FACSCalibur instrument.
MV4-11 xenograft murine model
This model was developed recently in our laboratory and described previously . Briefly, 4 ~ 6 week-old non-obese diabetic severe combined immunodeficient gamma (NSG) mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, The Jackson Laboratory, Bar Harbor, ME) were intravenously (i.v) injected via tail vein with 2 × 107 MV4-11 cells. Two months later, the spleen mononuclear cells (MNCs) were isolated from MV4-11-injected mice (1st-adapted MV4-11 cells in NSG mice). The adapted spleen MNCs were injected into a new cohort of NSG mice via tail vein. The loading dose of cells was reduced to 50% in this second transplantation. About a month later, the spleen MNC were isolated and a 3rd transplantation was performed using 0.5 × 107 cells/mouse spleen MNC from 2nd adapted NSG mice. The sequential transplants were performed using 0.5 × 106 cells/mouse spleen MNC from 3rd adapted NSG mice. This provides a more aggressive and fast onset of AML-like disease, thereby allows for rapid read-out of the experiment. Indeed, we observed development of leukemia at only two weeks from injection and a median survival of approximately 4 weeks after engraftment of the adapted MV4-11 cells from the 3rd transplant. The complete blood count and FACS analysis of CD45 and FLT3 expression and cytospin were examined weekly to monitor the progression of disease. All the experiments were conducted in accordance with the institutional guidelines for animal care and use.
Spleen cells (0.5 × 106) from MV4-11 transplanted NSG mice were intravenously (iv) injected into NSG mice via tail vein, and divided into groups for vehicle (hydroxypropyl beta-cyclodextrin in 30% sterile water; N = 6) or silvestrol (1.5 mg/kg in vehicle; N = 10) treatment. One control mouse (no leukemia/no treatment) was also included. Two to three weeks after engraftment, white blood count (WBC) and FLT3 expression by flow cytometry were assessed to confirm transplantation. Treatments with silvestrol or vehicle were initiated 21 days after engraftment (based on disease signs documented by WBC count and FLT3 expression). Administration was by intraperitoneal injection every 48 hours for up to three weeks or until euthanasia criteria were met. Expected median survival of untreated animals in this model is 28 days. Mice were weighed daily and checked for signs of dehydration, discomfort or toxicity. On day of administration, doses were recalculated for each animal after weighing to maintain 1.5 mg/kg. For the pharmacodynamic study, 6 mice were used (3 per group, vehicle and silvestrol). These mice were given 3 doses of either vehicle or silvestrol; and 48 hours following the third dose, spleens were isolated and mononuclear cells obtained for immunoblotting assay. For pathological examination, tissue sections from the liver, spleen and bone marrow sternum were fixed on formalin, embedded in paraffin blocks, and H&E stained.
Morphological signs of apoptosis were detected by Wright-Giemsa staining. Smears of control and treated cells were stained with Wright-Giemsa solution for 25 min, rinsed with distilled water and air dried. Cell morphology was studied by light microscopy.