Primary cell culture and cell lines
Human tumor specimens were obtained and stored in accordance with the human subject research protocols approved by the institutional review board. The processing of tumor specimens has been described previously [32, 33]. Briefly, after resection, a portion of each tumor was sent for routine histopathological analysis. The remainder of all samples was immediately used to establish primary cultures. Tumor fragments were dispersed into individual cells by treatment with Dispase 1 for 15 to 30 min at 37°C. From each tumor, 1 × 106 cells were then plated in a 100 mm tissue culture dish in low-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin mixture. The cells were grown to confluence and then harvested, aliquoted, and stored in liquid nitrogen for future use. The U251, U87, U373, and SW1088 glioma cell lines were obtained from American Type Culture Collection (Manassas, VA). The NG-1 glioma cell line was a gift from Dr. TF Liu (University of Texas MD Anderson Cancer Center). U251, U87, U373, SW1088 and NG-1 glioma cell lines were grown in DMEM high glucose, L-glutamine medium with 10% FBS and 1% penicillin/streptomycin mixture. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.
Recombinant adenovirus vectors
Ad/gTRAIL were constructed as described previously[34, 35]. Ad/CMV-GFP was provided by Dr. T.J. Liu in our institution. Virus titers were determined by optical absorbance at A260 and by plaque assay. Particle/plaque ratios normally fell between 30:1 and 100:1. Based on a report by others on evaluation of the concentration , and our own experience, vector titers determined by A260 were used in this study while titers determined by plaque assays were used as additive information. Thus, the multiplicity of infection (MOI) of 1000 VPs was equivalent to an MOI of 10-30 infectious units. Unless otherwise specified, Ad/CMV-GFP was used as the vector control, and PBS was used as a mock control. All viral preparations were free of contamination by E1+ adenovirus and endotoxin.
For the infectivity analyses, human benign and malignant tumor cells (5 × 105 ) were infected with 1000 MOI Ad/g-TRAIL, 1000 MOI Ad/CMV-GFP, or PBS, in which Ad/g-TRAIL, Ad/CMV-GFP-infected cells express GFP. 72 hours after infection, the cells were treated with 0.05% trypsin for 5 minutes and washed twice with phosphate-buffered saline (PBS). The cells were then counted for GFP-positive cells by flow cytometry as described below, or visualized and photographed by using a Nikon Eclipse TE300 inverted fluorescence microscope (Nikon, Melville, NY) and were analyzed with MetaMorph imaging software (Universal Imaging Corp Downington, PA).
Cell viability assay
Human benign and malignant tumor cells were seeded in at a density of 3 × 103 cells/well in 96 well plates and allowed to grow for 20 hours at 37°C. Cells were then infected with 1000 MOI Ad/g-TRAIL, 1000 MOI Ad/CMV-GFP, or PBS at 7 days respectively. Cell viability was determined by 3-bis-[2-methoxy-4-nitro-5 sulfenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Cell Proliferation Kit II; Roche Molecular Biochemicals, Indianapolis, IN) following manafacturer's instructions. Each experiment was performed in quadruplicate and repeated at least three times.
Real-time quantitative RT-PCR
Total RNA was extracted using the Mini-prep RNeasy kit (Qiagen). cDNA synthesis was constructed from high-grade RNA from all samples using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time Q PCR was performed in the ABI Prism 7700 Sequence Detection System according to the protocol of the manufacture. Typical amplification mixes (25μl) contained the sample DNA (or cDNA), 10× TaqMan Buffer (2.5μl), 200μm dATP, dcP, dGTP, and 400μM dUTP, 5 mM MgCl2, 0.65 units of Ampli Taq Gold, 0.25 units of AmpErase uracil N-glycosyladse (UNG), 200 nM each primer and 100 nM probe. The thermal cycling conditions consist of 1 cycle at 2 min for 50°C and 10 min for 95°C, and 50 cycles of 95°C 15 s and 60°C for 1 min. All reactions were performed in duplicates. After the reaction, we used the built-in software in the 7700 system to perform analyses of the data and generate the standard curve, the Ct value of each testing sample and their corresponding starting quantity based on the relative standard curve.
Western blot analysis
The cells treated with 1000 MOI Ad/CMV-GFP or 1000 MOI Ad/g TAIL at 3 days, the cell protein extraction was performed with Laemmli lysis buffer. Equal amounts of lysate were separated using 10% SDS-PAGE and transferred to Hybond enhanced chemiluminescence membranes (Amersham, Piscataway, NJ). The membranes were blocked with PBS-T containing 5% non-fat milk for 1 h or overnight at 4°C, and incubated with primary antibodies for 1 h at room temperature. After washing three times with PBS containing 0.05% Tween, the membranes were incubated with peroxidase-conjugated secondary antibodies and developed using a chemiluminescence detection kit (ECL kit; Amersham).
Fluorescence-activated cell sorting (FACS) and flow cytometric analysis
Cells were seeded at 1 × 105/well in 6-well plates, and after an overnight incubation, cells were either treated with 1000 MOI Ad/CMV-GFP or Ad/gTRAIL or complete media (control cells) for 24 hours. 72 hours later, both adherent and floating cells were harvested by trypsinization, washed with PBS, and fixed in 70% ethanol overnight at 4°C. Before analysis, cells were stained with propidium iodide for 30 min. The apoptosis induction was quantified by flow cytometric analysis. All experiments were performed in the Core Laboratory of the M. D. Anderson Cancer Center.
Intracranial xenografting of human meninglioma and glioma cells
Protocol for animal use was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine, and was in accordance with National Institutes of Health guidelines (NIH publication number 85-23).
A total of 24 female, 10-weeks-old, nu/nu athymic mice (Charles River Lab) were used. Human meningioma M6 and glioma U87 cell lines (at a concentration of 1 × 106 cells/5 μL) were resuspended in PBS and injected into the right frontal lobe of nude mice using a guide-screw system implanted within the skull as described previous [21, 37, 38]. On day 3, after the implantation of tumor cells, animals were divided into four groups, and the each group is treated with one single intratumoral injection (1.5 × 108 viral particles in 5 μL) with following: the first group animals (n = 6) were treated with Vectors Ad/CMV-GFP for meningioma M6; the second group animals (n = 6) were treated Vectors Ad/gTRAIL for meningioma M6; the third group animals (n = 6) were treated with Vectors Ad/CMV-GFP for glioma U87; the fourth group animals (n = 6) were treated with Ad/gTRAIL for glioma U87. Mice were anesthetized with xylazine/ketamine during the procedure. Mice showing general or local symptoms of toxicity were killed. When the animals became moribund due to tumor progression, they were euthanized and the brains were removed, fixed in 4% formaldehyde for 24 h at room temperature
Histology and tumor analysis
Brain tissue fixed for 24 h in 10% formalin solution, transferred to a 70% ethanol solution, and processed for paraffin embedding. Serial sections (6 μm) were prepared and stained with H&E according to standard histopathologic techniques. Stained sections were examined under light microscope (×100 magnification).
To detect apoptotic cells in tumor, we used an in situ cell death detection kit, POD (Roche Applied Science, Indianapolis, IN). The staining was performed according to the manufacturer's instructions, counterstained with haematoxylin, and viewed under a light microscope (×400 magnification). Brown staining indicates oligonucleosome cytoplasmic release resulting from apoptosis-induced DNA fragmentation. Counting was performed in randomly chosen fields, and the apoptosis was calculated as a percentage of at least 1,000 scored cells. Data analyzed with ImageJ software. Images segmented and the number of apoptotic cells quantified.
The standard fluorescein isothiocyanate-dependent apoptosis assay techniques (TUNEL or Annexin V) could not be used in this study because Ad/gTRAIL-infected cells express GFP, which interferes with fluorescein isothiocyanate detection by means of flow cytometry.
Differences among the experimental groups were analyzed by analysis of variance (AOV) using statistical software (StatSoft, Tulsa, OK). A difference was considered statistically significant when the P value was 0.05 or less. Differences in tumor growth in vivo among the treatment groups were assessed by AOV with a repeated measurement module. AOV was performed to determine statistical significance between each treatment group by using the SAS procedure with the SAS version 6.12 software. Survival was assessed by using the Kaplan-Meier method. Survival in different treatment groups was compared using the log-rank test.