The human GIST cell lines, GIST62 with KIT-negative and GIST48 with a homozygous c-KIT exon 11 V560D mutation and a heterozygous c-KIT exon 17 D820A mutation, were gifts from Dr. Li-Tzong Chen (National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan). The human GIST cell line with exon 11 deletion, GIST-T1 was purchased from Cosmo Bio Co., Ltd. Both GIST62 and GIST48 were maintained in RPMI 1640 medium (Hyclone) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific), 15 mM HEPES (Biological Industries), 2 mM L-glutamine (Caisson Laboratories), 1x antibiotic-antimycotic solution (1000 units/L penicillin, 2.5 µg/L amphotericin B, and 1000 µg/L streptomycin; Caisson Laboratories). GIST-T1 cells were maintained in Dulbecco's Modified Eagles Medium (Hyclone) supplemented with 10% FBS (Thermo Fisher Scientific), 15 mM HEPES (Biological Industries), 2 mM L-glutamine (Caisson Laboratories), 1 × antibiotic-antimycotic solution (1000 units/L penicillin, 2.5 µg/L amphotericin B, and 1000 µg/L streptomycin; Caisson Laboratories). All cell lines cultured in a humidified atmosphere with 5% of CO2 at 37 °C.
GIST62 cells in 6-well plates were transfected with 2 µg of plasmids, including pcDNA3.1b-KIT exon 11 △557–558, pcDNA3.1b-KIT wild type, pcDNA3.1b-KIT exon11 V560D, and empty pcDNA3.1b vector as control using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After transfection for 48 hours, we harvested cells for further study. We also used geneticin (800 µg/mL) for selection to establish stable cell lines.
Cells in culture dishes were washed with ice-cold PBS and were collected to microcentrifuge tube using cell scraper. The cells were lysed with ice-cold RIPA cell lysis buffer (Millipore Corporation) containing protease inhibitor cocktail (Millipore Corporation) and phosphatase inhibitor cocktail (Sigma-Aldrich). The cell lysate was centrifuged at 13,000 rpm at 4 °C for 30 minutes, and then the supernatant was collected to a new 1.5 mL microcentrifuge tube. The protein concentration of the samples was measured by BCA protein assay (Thermo Fisher Scientific), and all samples were stored at -80 °C.
Equal amounts of protein in 1 × SDS loading buffer were heated at 95 °C for 10 minutes. Samples were separated by 8 or 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corporation) at 100V for 100 minutes. The membrane was then blocked with 5% milk diluted in 0.05% TBST for 1 hour at room temperature. After blocking, membrane was probed by primary antibodies diluted in 0.05% TBST overnight at 4 °C. Next day, the membrane was washed 10 minutes for three times with 0.05% TBST followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. The membrane was washed 10 minutes for three times with 0.05% TBST. The blots signal was developed by using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation) according to the manufacturer’s instruction and captured by Biospectrum Imaging System (UVP).
Immunohistochemical (ihc) Analysis
The formalin-fixed paraffin embedded tissues were cut into 5 µm-thick sections. Antigen retrieval was performed by autoclaving the sections at 121 °C for 10 minutes in sodium citrate buffer (10 mM, pH 6.0). After blocking with the blocking buffer (Thermo Fisher Scientific) at room temperature for 30 minutes, the sections were incubated with specific primary antibodies at 4 °C overnight and then with biotinylated secondary antibodies at room temperature for 30 minutes. The immunoreaction was visualized using a DAB chromogen system (DAKO). The cell nuclei were stained with hematoxylin.
Immunofluorescence (if) Staining
Cells (3 × 104 cells/well) were seeded in an 8-well chamber slide (Millipore Corporation). After 24 hours of growth, cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.05% Triton X-100 for 15 minutes. Cells were then blocked in Super Blocking Buffer (Pierce) for 1 hour and incubated with primary antibodies overnight. Next day, cells were incubated with fluorochrome-conjugated secondary antibodies for 1 hour at room temperature. Nuclei were counter-stained with DAPI. All the images were visualized using a fluorescence microscope (BX51, OLYMPUS).
Seven weeks old male NOD/SCID mice were subcutaneously injected with GIST62 V cells or GIST62 Δ cells (4 × 106 cells in 50 µL serum free medium with 50µL matrix gel). The tumor volume was calculated using the formula of (length × width2)/2. When tumor volume was > 100 mm3, the drug treatments started (every 3 days for 15 days). After the treatments, we sacrificed these mice and collected the tumors. During this study, we raised and cared the animals according to the guidelines set up by the National Science Council, Taiwan. The Institutional Animal Care and Use Committee in NCKU Laboratory Animal Center approved animal experiments.
Cell Viability Analysis
GIST62V and GIST62Δ cells (8 × 103 cells/well) were seeded on a 96-well plate. After drug treatment, medium was removed and the cells were fixed with methanol for 10 minutes and stained with 0.05% of methylene blue for 1 hour. The cells were then washed with water and air-dried overnight. Hydrochloric acid (0.5N) was added to each well to dissolve the methylene blue stain and the absorbance was measured with the SpectraMax M5 microplate reader (Molecular Device) at 595 nm wavelength and normalized to the DMSO control group.
Patients And Specimens
Two hundred and fifty-one GISTs from National Cheng Kung University Hospital (NCKUH) were included, and their clinical records were retrospectively analyzed in this study. The study protocol was reviewed and approved by the Institutional Review Board of NCKUH (ER-104-157). Anonymous archived samples of GISTs were obtained from Human Biobank of NCKUH.
Statistical evaluation was conducted using GraphPad Prism software version 5.01 (GraphPad, Inc.). Values are expressed as means ± SEM. Differences between groups were determined by Student’s t test and ANOVA. The survival probability was calculated with the Kaplan-Meier method. P value less than 0.05 was considered to be statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).