MPNST cell lines
The six human MPNST cell lines used in this study included FU-SFT8611, FU-SFT9817, HS-Sch-2, NMS-2, NMS-2PC, and FMS-1. The FU-SFT8611 and FU-SFT9817 cell lines were established in our department, as described previously . The HS-Sch-2 cell line was established at Gifu University School of Medicine from a left thigh MPNST in a 54-year-old woman with no clinical evidence of NF1 . The NMS-2 cell line was established at Niigata University from an MPNST in the right thigh of a 30-year-old man with NF1, and NMS-2PC cells were derived from a retroperitoneal metastasis diagnosed 9 months later in the same patient . The patient received pre- and post-operative chemotherapy. The FMS-1 cell line was established at Fukushima Medical University from a right axillary MPNST of a 69-year-old woman with NF1 .
FU-SFT8611, FU-SFT9817, and HS-Sch-2 cells were maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Rockville, MD, USA) and Ham’s F12 (Nissui Seiyaku, Tokyo, Japan), while FMS-1, NMS-2, and NMS-2PC cells were cultured in Roswell Park Memorial Institute 1640 (RPMI1640) medium. Both types of media, at pH 7.35, were supplemented with 10% fetal calf serum (FCS), L-glutamine (746 μg/ml), sodium bicarbonate (0.2%), streptomycin (90 μg/ml) and penicillin G (90 μg/ml), used as the growth medium. All cell lines were maintained under a humidified 5% CO2 atmosphere at 37°C and the medium was replaced every 3 days.
Imatinib mesylate (STI571) was kindly provided by Novartis Pharma AG (Basel, Switzerland). The drug was diluted in sterile distilled water to a stock concentration of 10 mM. The stock solution was stored at −20°C and protected from light. Dilutions of this stock solution were prepared immediately before use in cell culture medium and added directly to the cells. Anti-PDGFR-β, PDGFR-α, phospho-PDGFR-β, phospho-PDGFR-α, and α-tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Recombinant human PDGF-BB was obtained from R&D Systems (Minneapolis, MN, USA).
Detection of PDGFR and phosphorylated PDGFR by western blotting
All six MPNST cell lines were cultured in growth medium in 25 cm2 culture bottles. After stabilization of the cells, the serum-containing medium was aspirated and replaced with serum-free medium for 24 h. After with or without pretreatment for 60 min with 10 μM imatinib mesylate, the cells were stimulated for 30 min, with 25 ng/ml of PDGF-BB in the presence or absence of imatinib mesylate. After removal of the medium, the cells were washed and scraped in phosphate-buffered saline (PBS) and collected by centrifugation at 1,000 rpm for 5 min. The cells were then lysed with a cell lysis buffer consisting of 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1 mM Na3VO4, and protease inhibitor cocktail tablets (Complete Mini, Roche Applied Sciences, Penzberg, Germany). Lysed cells were sonicated on ice for 15 min and centrifuged at 15,000 rpm for 20 min at 4°C. The resultant supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after measurement of their protein concentrations using the Bio-Rad protein assay (Hercules, CA, USA). After electrophoresis, the proteins were transferred electrophoretically to Immobilon membrane (Millipore, Bedford, MA, USA). Non-specific sites were blocked with 2% bovine serum albumin (BSA) in 0.05% Tween-20/Tris-buffered saline, pH 7.6 (TBS-T) at 37°C for 1 h and membranes were incubated overnight at 4°C with antibodies against PDGFR-α, PDGFR-β, phospho-PDGFR-α, phospho-PDGFR-β and α-tubulin dissolved in TBS-T containing 1% BSA. After washing with TBS-T, the membrane was incubated for 1 h with peroxidase-conjugated goat anti-rabbit (for anti-PDGFR-α, phospho-PDGFR-α, and phospho-PDGFR-β and α-tubulin antibodies) or anti-mouse IgG (for anti-PDGFR-β antibody). Color was developed with chemiluminescence reagents according to the instructions supplied by the manufacturer (DuPont NEN, Boston, MA, USA). The bands on the film were subjected to image analysis (Image J version 1.44 software, National Institute of Health, Bethesda, MD, USA). Statistical analysis was performed using the Student’s t-test.
RNA extraction and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from all six MPNST cell lines using the High Pure RNA Tissue Kit, 2033674 (Roche Applied Science, Indianapolis, IN, USA) according to the instructions supplied by the manufacturer. Next, One microgram of total RNA from each sample was reverse-transcribed using the PrimeScript II 1st standard cDNA Synthesis Kit (Takara Bio, Otsu, Japan). Real-time monitoring of PCR reactions was performed using the Light-Cycler system (Roche Applied Science) and SYBRVRPremix Ex Taq TMII (Takara Bio), according to the instructions supplied by the manufacturer. Gene-specific oligonucleotide primer pairs for PDGFR-β, PDGFR-α and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were purchased from Takara Bio; PDGFRB forward (F): GCCCTTATGTCGGAGCTGAAGA, reverse (R): GTTGCGGTGCAGGTAGTCCA; PDGFRA (F): CTGACATTGACCCTGTCCCTGA, (R): GATGAAGGTGGAACTGCTGGAAC; and GAPDH (F): GCACCGTCAAGGCT GAGAAC, (R): TGGTGAAGACGCCAGTGGA.
MPNST cell proliferation was evaluated using the One-Solution Cell Proliferation Assay with the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (CellTiter 96 Aqueous, Promega, Madison, WI, USA). The MTS compound is bioreduced to formazan by reduced NADPH or reduced NADH produced by metabolically active dehydrogenases of cells, which can be detected at 490 nm. After treatment of MPNST cells with 100 μl medium in each well of a flat-bottomed 96-well plate, 20 μl of MTS solution was added to each well and incubated at 37°C for 1 h. The 96-well plate was then placed in a kinetic microplate reader (Benchmark, Bio-Rad) and absorbance was measured at 490 nm.
The effects of imatinib mesylate on MPNST cell proliferation and survival were determined in 96-well plates by the MTS assay. The six MPNST cell lines were seeded onto the wells of flat-bottomed 96-well plate at each appropriate number for proliferation (FU-SFT8611 and FU-SFT9817 cells: 1 × 103 cells per well, HS-Sch-2, NMS-2, NMS-2PC and FMS-1 cells: 3 × 103 cells per well) in growth medium. Cells were allowed to adhere to the substratum overnight and then the medium was replaced by treatment medium containing 2% FCS in the presence of different concentrations of imatinib mesylate (1, 5, 10 and 20 μM) (day 0). The control groups were incubated with fresh imatinib mesylate-free 2% FCS containing medium. The test medium containing 2% FCS without or with different concentrations of imatinib mesylate was changed every 2 days. At days 0, 1, 3, and 5 of the experiment, the cell proliferation was determined by the MTS assay.
Small interfering RNA (siRNA)
MPNST cells were seeded in 6-well plates in antibiotic-free medium containing 10% FCS at a density of 1.5 × 105 cells per well. After stabilization of the cells overnight, the cells were transfected with pooled siRNA for PDGFR-βor negative control siRNA (B-Bridge International, Sunnyvale, CA, USA) using LipofectAMIN 2000 (Invitrogen, Carlsbad, CA) according to the instructions supplied by the manufacturer. After 72 h transfection, the cells were lysed and analyzed by western blotting with anti-PDGFR-β, PDGFR–α, and α-tubulin antibodies to confirm knock down at the protein level. To examine the effect of siRNA for PDGFR-β on proliferation of MPNST cells, cells were similarly transfected with siRNA in flat-bottomed 96-well plate (3 × 103 cells per well). Growth medium containing 2% FCS was replaced every 72 h, followed by assessment of cell proliferation at days 1, 3, 5, and 7 using MTS assays as described above.
Soft agar colony formation assay
The effect of imatinib mesylate on the anchorage-independent growth of 3 MPNST cell line was examined using a soft agar colony formation assay. After the addition of 50 μl per well of 0.6% agar solution containing 10% FCS containing medium to the bottom of flat-bottomed 96-well plate, as a basal agar layer, 75 μl per well of 0.4% agar solution with 7% FCS containing 6.5 × 103 MPNST cells were transferred onto the basal agar layer as a cell agar layer. Imatinib mesylate was added to the cell agar layer at final concentrations of 5 or 10 μM. After solidification of the cell agar layer, 2% FCS medium (100 μl) with or without imatinib mesylate (5 or 10 μM) was overlaid onto the layers. After 7-days incubation at 37°C, the numbers of anchorage-independent tumor cell colonies growing in the soft agar were counted using a phase contrast microscope.
Animal xenograft model and treatment with imatinib mesylate
The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Fukuoka University School of Medicine. In these experiments, 5 × 106 cells from each of three MPNST cell lines, HS-Sch-2, FMS-1 and NMS-2PC, were transplanted into the subcutis of the right flank of 6–8 week-old female NOD/SCID mice. When the tumor size reached 100 mm3 (about 28 days after cell inoculation), the mice were randomly divided into two groups: one group received water (control group), and the other was treated orally with imatinib mesylate (100 mg/kg/day) (treatment group). The latter group was closely watched for unusual symptoms or behaviors in order to evaluate systemic toxicity of imatinib mesylate, and the body weight was measured once a week.
Tumor size was measured once a week. The tumor volume was calculated according to the formula: L × W2 × 0.5, where L is the longest diameter and W is the width. At each time point, the mean tumor volume was compared between the two groups with a two-tailed Student’s t-test. The level of statistical significance was set at P < 0.05. After euthanasia, the tumors and internal organs were harvested and immediately fixed in 10% formalin. Non-necrotic portions of the tumor were excised and frozen. Various organs were prepared for histopathological analysis.
DNA extraction, PCR and DNA sequencing
Genomic DNA was extracted from the cell lines by using a GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) based on the procedure recommended by the manufacturer, and PDGFR-β exons 12 and 18 were analyzed by PCR as described previously . The primer sequences used were as follows; PDGFR-β exon 12 primer forward (F): TGTCCTAGACGGACGAACCT, reverse (R): CCAACTTGAGTCCCCACACT; exon 18 (F): GAAGGGTCTTTCCCCACAAT, (R): CACACTGGTCAGGAGGGAAT. The PCR products were purified using a QiAquick PCR purification kit (Qiagen Inc., Hilden, Germany). Direct sequencing of PCR products was performed using Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
Fluorescence in situ hybridization (FISH)
FISH of 6 MPNST cell lines was performed by labeling bacterial chromosomes (BACs) centromeric (RP11-1149B8 and RP11-348I17) and telomeric (RP11-101B10 and RP11-434E5) to the PDGFB with Spectrum Green and Spectrum Red (Abbott Molecular Inc., Des Plaines, IL, USA). Detection of the labeled probes with Spectrum Red and Spectrum Green was performed with streptavidin Alexa 594 (Molecular Probes, Eugene, OR, USA) and fluorescein isothiocyanate anti-diogoxigenin (Roche).