The construction of RCAS-PB and RCAS-PBIG vectors has been described before [15
]. RCAS-lacZ plasmid was kindly provided by Yi Li (Baylor College of Medicine, Houston, TX). RCAS-Akt/HA was a gift from Peter Vogt (The Scripps Research Institute, La Jolla, CA). RCAS-Kras (G12D) plasmid was kindly provided by Galen Fisher (Medical University of South Carolina, Charleston, SC).
Establishment and Infection of Primary Brain Cell Cultures
Newborn tv-a transgenic mice were sacrificed and the whole brains were mechanically dissociated into small pieces in sterile PBS (Ca2+- and Mg2+-free, pH 7.4) followed by digestion with 1 ml of 0.25% trypsin-1 mM EDTA in HBSS (Gibco BRL, Carlsbad, CA) in sterile tubes and incubation in 37°C water bath for 15 minutes with gentle shaking. After incubation, fresh medium was added to terminate trypsin digestion and large debris was settled. The single cells were pelleted, resuspended in DMEM with 10% fetal calf serum (Gibco BRL), and plated. The supernatants containing various RCAS virons from DF-1 cell cultures producing various RCAS viruses were collected in sterile syringes and filtered through 0.22-µm filters, followed by transfer into 70% to 80% confluent primary brain cell cultures, which had been plated and grown in DMEM with 10% fetal calf serum. Infections were repeated three times with 12-hour intervals.
The cells were fixed in -20°C methanol and washed with PBS (pH 7.4) thrice. The dishes were blocked by using 5% normal horse serum diluted in PBS (pH 7.4) for 1 hour at room temperature with shaking. Monoclonal anti-GFAP antibody (1:1000; Boehringer Mannheim, Indianapolis, IN) was diluted in PBS-0.05% Tween 20 with 5% normal horse serum and incubated with cells at room temperature for 2 hours or 4°C overnight. Secondary goat antimouse IgG-fluorescin conjugate (1:200; Vector Laboratories, Burlingame, CA) was diluted in PBS-0.05% Tween 20 with 5% normal horse serum and incubated with cells at room temperature for 1 hour. The nuclei were counterstained with DAPI (Sigma, St. Louis, MO). The fluorescence was visualized using a fluorescence microscope (Leica, Wetzlar, Germany) or a confocal laser microscope (Memorial Sloan-Kettering Cancer Center Cytology Core Facility, New York, NY).
Blocking of PDGF Signaling
The cells infected with RCAS-PBIG were cultured in DMEM supplemented with 10% FCS, penicillin and streptomycin, and l-glutamine. A total of 1 x 105 cells was plated and the experiment groups were treated with PTK787 (stock solution dissolved in DMSO) with the final concentration of 1 µM. The control groups were treated with the same volume of DMSO.
Western Blot Analysis
Whole cell protein extracts were prepared by using cold lysis buffer consisting of: M-Per mammalian protein extraction reagent (Pierce Biochemical, Rockford, IL), 30 mM sodium fluoride, 1 mM sodium vanadate, and protease inhibitor cocktail tablets (Boehringer Mannheim). Samples were incubated on ice for 10 minutes and supernatants were recovered by centrifuging at 14,000 rpm at 4°C for 20 minutes. Protein concentrations were determined by the BCA method (Pierce Biochemical). Proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane (Osmonics, Minnetonka, MN). Blocking reagent was 5% nonfat dry milk in PBS (pH 7.4). Washing buffer was PBS (pH 7.4) with 0.1% Tween 20. Polyclonal rabbit anti-phospho-AKT (Ser437), anti-phospho-ERK1/2 (Ser217/221), anti-phospho-p38MAPK (Thr180/Tyr182), anti-phospho-STAT3 (Tyr705), anti-phospho-PKCα/βII (Thr638), anti-AKT, anti-ERK1/2 (Cell Signaling Technology, Beverly, MA), polyclonal rabbit anti-PDGFRα and anti-PDGFRαβ (Upstate Biotechnology, Lake Placid, NY), polyclonal goat antiactin, monoclonal anti-p21/CIP1, polyclonal rabbit anti-FLK1/VEGFR2 (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti-PCNA (Chemicon, Temecula, CA), and monoclonal anti-GAPDH were used as primary antibodies. The respective HRP-conjugated secondary antibodies were purchased from Boehringer Mannheim. Signals were visualized by using ECL chemiluminescence (Amersham, Indianapolis, IN) or Supersignal chemiluminescence (Pierce Biochemical) and Kodak X-OMAT films.
Activated Ras Pull-Down Assay
The cells infected with RCAS-PBIG, RCAS-Kras (G12V), or RCAS-lacZ were cultured as above. To block PDGF signaling, RCAS-PBIG-infected cells were treated with 1 µM PTK787 for 7 days. For the activated Ras pull-down assay, cells were transferred to reduced serum media (1% FBS) and whole cell extracts were prepared with ice-cold lysis buffer containing: 50 mM Tris (pH 7.6), 500 mM NaCl, 0.1% SDS, 0.5% DOC, 1% Triton X-100, 0.5 mM MgCl2, 30 mM NaF, 1 mM Na3VO4, 0.5 mM PMSF, and protease inhibitor cocktail tablets (Boehringer Mannheim). Samples were incubated on ice for 30 minutes and supernatants were collected following centrifugation at 55,000 rpm for 15 minutes at 4°C. Protein concentration was determined using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). One milligram of total protein was then used for activated Ras pull-down with 10 µg of glutathione-conjugated Raf-1 GST-RBD beads (Upstate Biotechnology). Samples were incubated for 45 minutes at 4°C with agitation. Beads were then washed three times with wash buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5 mM MgCl2, 30 mM NaF, 1 mM Na3VO4, 0.5 mM PMSF, and protease inhibitor cocktail tablets) and analyzed by Western Blot on SDS-PAGE as described above. Monoclonal mouse anti-Kras antibody was from Santa Cruz Biotechnology.
Infection of tv-a Transgenic Mice
DF-1 cells producing various RCAS virons were trypsinized and pelleted by centrifugation; the pellets were resuspended in approximately 50 µl of DMEM medium and placed on ice before injection. Using a 10-µl gas-tight Hamilton syringe, a single intracranial injection of 1 µl of cell suspension (about 105 cells) was made in the right frontal region of newborn mice, with the tip of the needle just touching the skull base.
Brain Sectioning, Hematoxylin and Eosin (H&E) Staining, and Immunohistochemistry
Mice were sacrificed before (due to early symptoms) or at 12 weeks of age, and the whole brains were fixed in 4% formaldehyde in PBS for at least 36 hours with shaking. Five sections of each brain were cut and embedded in paraffin; 5-µm sections were cut with a Leica microtome. The sections were stained with H&E. Immunostaining of paraffin sections was performed using ABC kits (Vector Laboratories). Briefly, deparaffinized slides were first treated with antigen unmasking reagent (Vector Laboratories) with heating in a steamer for 30 minutes, followed by immersion in 10% hydrogen peroxide in methanol for 20 minutes to inactivate the endogenous peroxidases. Then the sections were blocked with 1.5% normal horse serum in PBS (pH 7.4) for 1 hour at room temperature. Monoclonal anti-GFAP (1:1000; Boehringer Mannheim) and polyclonal rabbit anti-HA (1:200; Santa Cruz Biotechnology) were diluted in PBS-0.05% Tween 20 with 5% normal horse serum (Vector Laboratories) and incubated with sections at 37°C for 1 hour; polyclonal rabbit anti-phospho-AKT (Ser473) and anti-pERK1/2(Ser217/221) (Cell Signaling Technology) were diluted at 1:100 in PBS-0.05% Tween 20 with 5% normal horse serum (Vector Laboratories) and incubated with sections at 4°C overnight; mouse monoclonal anti-Olig2 antibody (a gift of John Alberta, Dana-Farber Cancer Institute, Boston, MA) was diluted at 1:200 in PBS-0.05% Tween 20 with 5% normal horse serum (Vector Laboratories) and incubated with sections at 4°C overnight. After washing with PBS-0.05% Tween 20, appropriate biotinylated secondary antibodies (Vector Laboratories) diluted in the same antibody dilution buffer were incubated with sections at 37°C for 60 minutes. Then, after washing, avidin-conjugated peroxidase (Vector Laboratories) diluted in PBS containing 1.5% normal horse serum was incubated with sections for 60 minutes at room temperature. Finally, after exclusive PBS-T washing, DAB substrate (Vector Laboratories) was added to develop the color. After terminating the staining reaction, the sections were counterstained with hematoxylin and mounted. The negative controls were included with the same procedure, except replacing primary antibodies with antibody dilution buffer.