PDGF-B overexpression promotes proliferation of both GFAP-expressing astrocytes and nestin-expressing CNS progenitors in cellculture

To test the mitogenic functions of PDGF-B in specific cell types, we infected primary brain cell cultures from both Gtv-a (astrocytes) and Ntv-a (glial progenitors) transgenic mice with RCAS–PB, RCAS–PBIG, and RCAS–lacZ vectors respectively. After infection, these cells were passaged in culture for 40 d and their proliferative capacity was measured by growth curves (Holland et al. 1998b). Infection of either Gtv-a (Fig. ​(Fig.1A)1A) or Ntv-a cell cultures (data not shown) with PDGF-encoding viruses resulted in a substantial increase in the growth rate of these cells relative to infection with the control LacZ-encoding vector. A similar growth rate was seen for both RCAS–PBIG and RCAS–PB indicating that the expression of GFP did not affect the growth potential of these cells. Cells infected with RCAS–PBIG were visualized by green fluorescence secondary to the expression of eGFP (Fig. ​(Fig.1B).1B).

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Figure 1

Dedifferentiation of cultured astrocytes to glial progenitors caused by overexpression of PDGF-B chain. (A) The proliferation rate of primary astrocytes (from Gtv-a mouse) was significantly increased by overexpression of PDGF-B. The details of generating growth curve are described in Material and Methods. (B) Giemsa staining shows the morphology of cultured cells infected with RCAS–LacZ and RCAS–PBIG from Gtv-a, Ink4a–Arf^+/+ mouse. Fluorescence microscopy shows that the cells infected with RCAS–PBIG are expressing green fluorescent protein. Both photomicrographs are 100× magnification; bars, 100 μm. (C) Western blot analyses show the increased expression of molecular markers for glial precursors in the astrocytes after RCAS–PBIG infection. Two lines of RCAS–lacZ-infected astrocytes were used as control. (PLP) Myelin proteolipid protein; (PDGFR) platelet-derived growth factor receptor; (GFP) green fluorescent protein; (Id4) inhibitor of binding 4, a dominant negative HLH protein. α-Tubulin was used as loading control.

PDGF/PDGFR autocrine loop stimulation dedifferentiates GFAP-expressing astrocytes to glial precursor-like cells inculture

In addition to the enhanced proliferative capacity induced by PDGF signaling, PDGF also induced morphological and gene expression characteristics of undifferentiated cells. These changes were most striking in the astrocytes from Gtv-a mice. After 40 d in culture, the control RCAS–lacZ-infected Gtv-a cells showed a large, flat morphology expected for cultured astrocytes. In contrast, both RCAS–PB and RCAS–PBIG infected Gtv-a cells showed a small, elongated, bipolar or simple multipolar morphology, characteristics of glial progenitor cells (Fig. ​(Fig.11B).

Western blot and immunocytochemical analyses showed gene expression patterns similar to that of glial precursors (Fig. ​(Fig.1C).1C). Expression of myelin proteolipid protein (PLP) (Asakura et al. 1998; Spassky et al. 1998), PDGFRα (Hart et al. 1989; Hall et al. 1996) and β, A2B5 (Williams et al. 1985; Dubois-Dalcq 1987; data not shown), all known as markers for glial progenitors, were up-regulated in cells infected with PDGF-encoding vectors. Moreover, these cells also showed elevated expression of Id4, an HLH gene whose expression is shown to correlate inversely with differentiation status of oligodendrocyte precursors (Andres-Barquin et al. 1999; Kondo and Raff 2000; Nogueira et al. 2000). By contrast, these cells did not express O4 antigen, which is expressed in premature and mature oligodendrocytes (Sommer and Schachner 1981), implying that they are not differentiating to oligodendrocytes. Of note, NG2, which is expressed in bipotential O2A progenitors (Stallcup and Beasley 1987), was not expressed in these cells, nor was the expression of neuronal markers such as neurofilament (NF) or synaptophysin (data not shown). Therefore, the expression pattern of cellular markers, along with the morphology and the proliferation capacity, suggest an early glial precursor stage for these cells, similar but not identical to the well-known O-2A progenitors (Raff et al. 1983). The potential for autocrine loop stimulation of PDGF and PDGFR is demonstrated by the elevated expression of both PDGFRα and β compared with the RCAS–lacZ-infected cells.

Blocking PDGF autocrine signaling reverses the PDGF-induced phenotype invitro

The PDGF-induced alterations described above could either be direct effects of elevated PDGF signaling or a result of selection for growth advantage in culture. To address this issue, we treated long-term cultured PDGF-transformed astrocytes (from Gtv-a mice) with a tyrosine kinase inhibitor (PTK787/ZK222584) (Wood et al. 2000) that blocks the activation of PDGF receptors. At 1μM drug concentration, the proliferation rate of the PDGF-expressing cells was dramatically inhibited (Fig. ​(Fig.2A,B)2A,B) and the progenitor-like morphology reverted back to a large, flat, astrocyte-like morphology (Fig. ​(Fig.2C).2C). Western blot analyses of RCAS–PBIG-infected astrocytes showed that the expression levels of PLP and Id4 were down-regulated by treatment with PTK787 implying a direct effect of PDGF signaling in control of these genes. In contrast, the elevated PDGFRα and PDGFRβ levels seen in the PDGF overexpressing astrocytes were not reduced by PTK787, suggesting that the elevated expression of PDGFRs may be due to selection during extended culture for cells with high level of PDGFR expression that can maximally respond to overexpression of PDGF. The expression level of eGFP pre- and post-drug treatment was similar, indicating that the effects of PTK787 were not due to decreased expression of virally encoded PDGF-B (Fig. ​(Fig.2D).2D). We demonstrated that this effect of PTK787 was not a general property of this drug by its effect on astrocytes infected with an RCAS vector carrying the coding sequence for polyoma middle T antigen (RCAS–MTA). MTA expression can activate similar signal transduction pathways as PDGF and infection with RCAS–MTA causes increased proliferation rate of astrocytes in cell culture and induces oligoastrocytomas from astrocytes in vivo (Holland et al. 2000b). At the same concentration of PTK787 that reversed the phenotype of RCAS–PBIG-infected cells, there was no effect on the proliferation capacity or morphology of RCAS–MTA-infected cells, implying specificity of the PTK787 effect for signaling through PDGFR in these glial cells.

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Figure 2

Reversion of PDGF-induced phenotypic effects by blocking PDGF receptor kinase. Astrocytes (1×10⁵) were plated and grown in culture either with or without 1 μM PTK787 that inhibits the PDGF receptor kinase. The cells were counted after 14 d. (A) The proliferation of RCAS–PBIG-infected cultured astrocytes was inhibited by 1 μM PTK787 treatment. In contrast, the proliferation of RCAS–MTA-infected astrocytes was not inhibited by PTK787 (B). (MTA) Polyoma virus middle T antigen. (C) Giemsa staining shows the morphology of RCAS–PBIG-infected astrocytes with and without 1 μM PTK787 for 14 d. 100× magnification; bars, 100 μm. (D) Western blot analyses show the changes in expression pattern of molecular markers after blocking autocrine PDGF stimulation. (PLP) Myelin proteolipid protein; (PDGFR) platelet-derived growth factor receptor; (GFP) green fluorescent protein; (Id4) inhibitor of binding 4, a dominant negative HLH protein. α-Tubulin was used as loading control.

Gene transfer of PDGF-B into nestin-expressing neural progenitors induces oligodendroglioma formation inmice

To investigate the effect of PDGF autocrine loop stimulation on these same cell types in vivo, we infected Ntv-a and Gtv-a transgenic mice with RCAS–PBIG by a single intracranial injection of about 10⁴ DF-1 cells producing this vector. All the mice were sacrificed at the end of week 12, or earlier if they showed signs or symptoms of intracranial disease such as macrocephaly or lethargy. The brains were fixed and sections were analyzed by standard hemotoxylin and eosin (H&E) staining. Previous experiments have demonstrated that gene transfer of the marker gene alkaline phosphatase, activated Ras, or activated Akt alone to either of these mouse lines by this procedure does not induce tumors or histologic abnormalities (Holland and Varmus 1998; Holland et al. 2000a).

Initially, we directly infected a total of 34 newborn Ntv-a transgenic mice. Over 60% of this population of mice harbored gliomas. Histologically, these tumors were diffusely infiltrating neoplasms composed of small tumor cells with monotonous, regular round nuclei surrounded by cleared cytoplasm (“perinuclear halos”) (Fig. ​(Fig.3B).3B). These morphologic features are identical to those seen in human oligodendrogliomas. The tumors did not express neuronal differentiation markers, such as NeuN and synaptophysin, as demonstrated by immunohistochemistry (data not shown). Further, also similar to human oligodendrogliomas, except for scattered entrapped reactive astrocytes the majority of tumor cells were immunonegative for GFAP, suggesting the absence of astrocytic differentiation (Fig. ​(Fig.3C).3C). In addition to the morphologic and immunohistochemical similarity of the individual tumor cells to human oligodendrogliomas, the infiltrative behavior and interactions with normal host cellular constituents also closely resembled that seen in human gliomas. The murine gliomas displayed all of the classical morphologic formations seen in infiltrating human oligodendrogliomas referred to as secondary structures of Scherer (1938); including intrafascicular queuing in white matter tracts of the corpus callosum and subpial infiltration, perivascular satellitosis, and perineuronal satellitosis in areas of cortical invasion (Fig. ​(Fig.4).4). The expression of exogenous PDGF in these tumors was verified by positive immunostaining for eGFP expression (data not shown).

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Figure 3

Histologic analysis of oligodendrogliomas induced from neural progenitors. PDGF-induced gliomas in Ntv-a mice with either Ink4a–Arf^+/+ (A–C) or Ink4a–Arf^−/− (D–F) genetic backgrounds. (A) Low magnification of H&E-stained section illustrates the diffuse infiltrating low-grade tumor (indicated by the arrow). (B) H&E-stained section shows small, homogenous tumor cells with perinuclear “halo” appearance. (C) GFAP immunostaining shows negative oligodendroglioma cells and a few positive, trapped reactive astrocytes. (D) Low magnification of H&E-stained section illustrates a high-grade oligodendroglioma. The arrow indicates the necroses. (E) H&E-stained section shows microvascular proliferation (indicated by the arrow), a criterion for diagnosing high-grade gliomas. (F) H&E-stained section shows the palisading necrosis (indicated by the arrow), a criterion for diagnosing high-grade gliomas.

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Figure 4

Invasion of normal brain structures by mouse glioma cells. Similar to what is seen in human gliomas, these mouse tumors migrate along the white matter tracts (A), and surround neurons (B), and collect adjacent to the pial surface of the brain and blood vessels (C,D). Arrows indicate the listed structures.

In the experiment reported here, both low- and high-grade tumors arose from PDGF gene transfer in vivo. The high-grade oligodendrogliomas, called anaplastic oligodendrogliomas in the current World Health Organization Classification of Tumours (WHO 2000), were characterized by dense cellularity without intervening neuropil, easily identifiable mitotic figures, cellular and nuclear pleomorphism and microvascular proliferation (Fig. ​(Fig.3D–F).3D–F). These anaplastic oligodendrogliomas frequently were additionally characterized by the presence of foci of tumor necrosis with surrounding tumor cell pseudopalisading (Fig. ​(Fig.3F).3F). The majority of the PDGF-induced gliomas, arising in either Gtv-a or Ntv-a mice having a wild-type genetic background, were low-grade tumors that lacked the features just described for high-grade oligodendrogliomas.

Loss of Ink4a–Arf enhances tumor initiation and tumor progression in Ntv-a transgenicmice

The above data suggest that autocrine stimulation of PDGF signaling, in the absence of other experimentally induced genetic alterations, could be sufficient to induce low-grade gliomas from neural progenitors. Human high-grade gliomas have additional mutations that result in disruption of cell cycle arrest pathways such as loss of Ink4a–Arf (Cairncross et al. 1998) To elucidate the roles of disruption of G1 cell cycle arrest pathway in PDGF-induced gliomagenesis, we compared the incidence and characteristics of gliomas occurring in both Ink4a–Arf^+/+ and Ink4a–Arf^−/− mice. At 12 wk of age, 61% of Ink4a–Arf^+/+ and 57% of Ink4a–Arf^−/− mice developed gliomas respectively (Fig. ​(Fig.5A).5A). All of these tumors were oligodendrogliomas according to the above histopathological criteria and as described in the WHO 2000 classification. Although the overall glioma incidence was similar between these two groups, over half of the gliomas forming in Ink4a–Arf^+/+ mice were low-grade and asymptomatic at the 12-wk time point whereas only about 10% of tumors arising from Ink4a–arf^−/− mice were low grade. By contrast, using the same criteria these Ink4a–Arf^−/− tumors not only were more likely to be malignant but were also more likely to be symptomatic, resulting in earlier sacrifice and analysis (Fig. ​(Fig.5A,B).5A,B). Therefore, experimental disruption of the Ink4a–Arf locus appears not essential for the induction of these gliomas from nestin-expressing neural progenitors; however, it does either facilitate the progression of gliomas toward a more malignant phenotype, or it selects for a more aggressive tumor cell that outgrows the population in vivo.

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Figure 5

Role of Ink4a–Arf loss in PDGF-induced gliomagenesis from neural progenitors. (A) Tumor-free survival curve indicating percent of mice developing symptomatic tumors over time. All mice were sacrificed at 12 wk; clinically occult tumors are indicated by the vertical line at 12 wk. The total number of mice without detectable tumors at 12 wk was ~40% in both animal groups as indicated by the arrows. (B) Ink4a–Arf loss significantly enhanced tumor malignancy. The grading criteria are described in the text. (O) Oligodendroglioma (WHO classification); (AO) anaplastic oligodendroglioma (WHO classification). AO tumors having the histologic feature of pseudopalisading necrosis are listed as “anaplastic with necrosis.”

Gene transfer of PDGF-B into GFAP-expressing astrocytes induces either oligodendrogliomas or mixed oligoastrocytomas inmice

The experiments described thus far were performed in Ntv-a mice, that is, gene transfer to nestin-expressing neural progenitors. To test the influence of cell of origin on PDGF-induced gliomagenesis, we infected Gtv-a transgenic mice (astrocytes) with RCAS–PBIG. The total incidence of glioma formation was somewhat lower (~40%), and the tumors were either oligodendrogliomas or gliomas of mixed histology containing both oligodendroglioma and astrocytoma components (oligoastrocytoma). Figure ​Figure6A6A shows a representative example of an oligoastrocytoma in which distinct oligodendroglioma and astrocytoma components are present. The oligodendroglioma component exhibits morphologic features identical to those seen in human oligodendrogliomas, including monotonous, uniform round nuclei and scant-to-nondetectable cytoplasm. By contrast, in the astrocytoma component the constituent tumor cells are more pleomorphic and atypical, with irregular nuclei and prominent eosinophilic cytoplasm that is strongly immunoreactive for GFAP (Fig. ​(Fig.6B,C).6B,C). In addition, multinucleated giant cells are intimately associated with the astrocytoma component (data not shown), as is sometimes seen in human astrocytomas. These gliomas arising in Gtv-a mice also occurred in low-grade and malignant forms. However, over 70% of these tumors were low grade in mice with a wild-type Ink4a–Arf genetic background.

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Figure 6

Histology of mixed gliomas induced from GFAP-expressing astrocytes. (A) Low magnification of GFAP-immunostained section illustrates the coexistence of both positive astrocytoma cells and negative oligodendroglioma cells. (B) H&E-stained section shows astrocytic tumor cells with irregular nuclei and eosinophilic cytoplasm. (C) GFAP immunostaining illustrates the strongly positive astrocytoma cells; the staining pattern distinguishes them from reactive astrocytes.

In Gtv-a mice, loss of Ink4a–Arf increases tumor incidence and tumormalignancy

To test the effects of Ink4a–Arf loss on the formation of the mixed gliomas arising from astrocytes in Gtv-a mice, we compared PDGF-induced glioma formation in Gtv-a, Ink4a–Arf^+/+ and Gtv-a, Ink4a–Arf^−/− mice. Similar to the Ntv-a mice, gliomas arising from Gtv-a mice with Ink4a–Arf deficiency were of higher grade and shorter latency than those in mice with an Ink4a–Arf wild-type background (Fig. ​(Fig.7A).7A). In contrast to Ntv-a mice, in which a higher percentage of animals developed gliomas and no increase in tumor incidence was seen with Ink4a–Arf loss, loss of Ink4a–Arf in Gtv-a mice resulted in a nearly twofold increase in the incidence of PDGF-induced gliomas at 12 wk of age (Fig. ​(Fig.7A).7A). The most malignant gliomas with the shortest latency were seen with the combination of both neural progenitor cell of origin and loss of Ink4a–Arf (Fig. ​(Fig.7B).7B).

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Figure 7

Role of Ink4a–Arf loss, p53 loss in PDGF-induced gliomagenesis from Gtv-a mice. (A) Ink4a–Arf loss, but not p53 loss, resulted in a shortened tumor latency and an increased total tumor incidence (70% vs. 39%); (B) Ink4a–Arf loss also significantly enhanced the malignancy of gliomas induced from Gtv-a mice. In summary, the most malignant gliomas arose from the combination of both neural progenitor cell of origin and loss of Ink4a–Arf. (O) Oligodendroglioma (WHO classification); (AO) anaplastic oligodendroglioma (WHO classification). Final tumor incidence for each genetic background is indicated by the arrows.

Deletions of 1p, 19q, and 10q are not present in PDGF-inducedgliomas

It is not clear whether PDGF signaling is sufficient for the induction of oligodendrogliomas or whether additional chromosomal abnormalities during tumor initiation or progression are required to cooperate with PDGF signaling in gliomagenesis. We investigated this question by analyzing these tumors for allelic loss of the mouse chromosomes syntenic to regions that are frequently lost in human gliomas. Approximately half of human oligodendrogliomas demonstrate deletions of chromosomes 1p and 19q, and malignant gliomas often show loss of chromosome 10q (Ransom et al. 1992; Reifenberger et al. 1994; von Deimling et al. 1992; Bello et al. 1995; Maier et al. 1997). Such regions would be likely candidates for additional mutations that might be required for PDGF-induced glioma formation. We addressed this issue by using fluorescence in situ hybridization (FISH) for murine loci that are syntenic to the candidate tumor suppressor regions on human chromosomal arms 1p, 10q, and 19q. Murine chromosomal arms (murine 3, 84.9 cM and murine 4, 46.6 and 81.5 cM, for human 1p; murine 7, 4–5.5 and 23.0 cM for human 19q; and murine 19, 26.0 cM for human 10q), and corresponding centromeres were studied in 21 PDGF-induced gliomas of various grades. For each case at each locus, the chromosomal counts ranged from 1.71 and 2.08, satisfying the criteria for two chromosomal copies (Fig. ​(Fig.8).8). Thus, in no case was there evidence for loss of any of these regions. Therefore, those common mutations implicated in human oligodendrogliomas appear not to be required for PDGF-induced glioma formation. These data may imply that PDGF signaling achieves the same biologic effect as 1p and 19q loss and that there is no selective pressure for generating these mutations, or that that this model may simulate those human oligodendrogliomas without such mutations.

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Figure 8

Fluorescence in situ hybridization analysis of PDGF-induced gliomas. (A) Two copies of murine chromosome 4 syntenic to human chromosome 1p. Green and red signals indicate the locus of murine chromosome 4, centromere and 81.5 cM, respectively. (B) Two copies of murine chromosome 7 syntenic to human chromosome 19q. Green signals are for the centromere of murine chromosome 7, and red for chromosome 7, 23 cM.

Transfection of DF-1cells

DF-1 cells were grown in DMEM medium complemented with 10% fetal calf serum. RCAS–PB, RCAS–PBIG, RCAS–MTA, and RCAS–lacZ plasmid DNAs were transfected into chicken DF-1 cells by using calcium phosphate precipitation methods and the vectors were allowed to replicate within the producer cell population.

Tv-a transgenic mice

The Gtv-a mouse line that expresses tv-a from the GFAP promoter and the Ntv-a mouse line that expresses tv-a from the nestin promoter have been published (Holland and Varmus 1998; Holland et al. 1998a). The mice are a mixed genetic background including contribution from C57Bl6, 129, Balb/C, and FVB/N.

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⁺, Mg²⁺ free (pH 7.4), followed by digestion with 1 mL of 0.25% Trypsin–1 mM EDTA in HBSS (GIBCO BRL) in sterile tubes and incubation in 37°C water bath for 15 min 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.

Infection of primary brain cell culture and growth rateanalysis

The supernatants containing various RCAS virons from DF-1 cell cultures transfected with various RCAS vectors were collected using sterile syringes and filtered through 0.22 μm filters, followed by transferring into 70%–80% confluent primary brain cell cultures that had been plated and grown in DMEM with 10% fetal calf serum. Infections were repeated 3 times with 12-h intervals. After infection, the proliferation rates were measured. Briefly, the cells were harvested by trypsin digestion and counted using a hemocytometer. Total 1×10⁵ cells were replated and grown in DMEM with 10% fetal calf serum until the cells reached confluence; the same procedure was repeated over time. Finally, the total accumulated cell numbers were calculated and plotted as growth curves.

Fluorescence microscopy

DF-1 cells transfected with RCAS–PBIG and primary brain cell cultures infected with the same vector were seeded and grown on sterilized glass coverslips. First, the cells were fixed by immersing the coverslips in 2 mL of 4% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. After washing with PBS three times, the coverslips were mounted on slides using Fluoromount G (EMS) and visualized under fluorescence microscope (Nikon Eclipse) using conventional FITC filter sets (Nikon B-1E, EX470–490, DM505, BA520–560) for green fluorescence. The red fluorescence of brain sections stained with anti-GFP antibody (Santa Cruz Biotech) was visualized by using Texas Red filter set (Nikon G-1B, EX540/10, DM580, BA590).

Giemsa staining of culturedcells

The medium was aspirated from cells at 80% confluence and the cells were washed with PBS (pH 7.4), followed by fixing in methanol at room temperature for 10 min. Then, 1 mL of Giemsa stain (LabChem Inc) was applied for 5 min. The stain was discarded and the cells were washed with PBS several times and visualized under a bright field microscope.

Immunocytochemistry

The cells infected with RCAS–PBIG or RCAS–lacZ were fixed in either 4% paraformaldehyde in PBS (pH 7.4) or cold methanol. The dishes were blocked by using 5% normal horse serum diluted in PBS (pH 7.4) for 1 h at room temperature with shaking. Monoclonal anti-A2B5 antibody (Chemicon), polyclonal anti-NG2 antibody (Chemicon), monoclonal anti-O4 antibody (Chemicon), monoclonal anti-GFAP antibody (Boehringer Mannheim), monoclonal anti-NeuN antibody (Chemicon), and monoclonal anti-neurofilament antibody (Boehringer Mannheim) were used as primary antibodies. Appropriate biotinylated secondary antibodies and avidin-conjugated peroxidase were purchased from Vector Lab. The signal was visualized by using DAB substrate kit (Vector Lab) and examined under microscopy.

Blocking of PDGFsignaling

The cells infected with RCAS–PBIG were cultured in DMEM supplemented with 10% FCS. Cells (1×10⁵) were plated and the experiment groups were treated with PTK787 (stock solution dissolved in DMSO) with the final concentration of 1 μM for 2 wk. The control groups were treated with the same volume of DMSO. After two wk, the cell numbers were counted, and the morphology was visualized by Giemsa staining.

Western analysis

Whole-cell protein extracts were prepared by using cold lysis buffer consisting of: 100 mM NaCl, 30 mM Tris-HCl (pH 7.6), 1% NP-40, 30 mM sodium fluoride, 1 mM EDTA, 1 mM sodium vanadate, 0.5 mM PMSF, and protease inhibitor cocktail tablets (Boerhinger Mannheim). Samples were incubated on ice for 30 min and supernatants were recovered by centrifuging at 14,000 rpm at 4°C for 20 min. Protein concentrations were determined by BCA method (Pierce Biochemical). Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane (Osmonics). Blocking reagent was 5% non-fat dry milk in TBS (pH 7.4). Washing buffer was TBS (pH 7.4) with 0.1% Tween-20. Polyclonal rabbit anti-PLP (Oncogene Research), polyclonal rabbit anti-PDGFRα and β (Upstate Biotech), polyclonal goat anti-vimentin (Chemicon), polyclonal rabbit anti-Id4 (Santa Cruz Biotech), polyclonal rabbit anti-GFP (Santa Cruz Biotech), and monoclonal anti-α-tubulin (Sigma) were used as primary antibodies. Respective HRP-conjugated secondary antibodies were purchased from Boehringer Mannheim. Signals were visualized by using ECL chemiluminescence (Amersham) and Kodak X-OMAT films.

Infection of tv-a-transgenicmice

DF-1 cells producing various RCAS virons were trypsinized and pelleted by centrifugation; the pellets were resuspended in ~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 (~10⁴ cells) was made in the right frontal region of newborn mice, with the tip of the needle just touching the skull base.

Brain sectioning, H&E staining, andimmunohistochemistry

Mice were sacrificed before (because of early symptoms) or at 12 wk of age and the whole brains were fixed in 4% formaldehyde in PBS for at least 36 h with shaking. Five sections of each brain were cut and embedded in paraffin, 5-micron sections were cut with a Leica microtome. The sections were stained with H&E. Immunostaining was performed using ABC kits (Vector Lab). Briefly, deparaffinized slides were first treated with antigen unmasking reagent (Vector Lab) with heating in a steamer for 30 min, followed by immersing in 10% hydrogen peroxide in methanol for 20 min to inactivate the endogenous peroxidases. Then the sections were blocked with 1.5% normal horse serum in PBS (pH 7.4) for 1 h at room temperature. Monoclonal anti-GFAP (Boehringer Mannheim), monoclonal anti-NeuN (Chemicon), polyclonal rabbit anti-synaptophysin (Biomeda), and polyclonal rabbit anti-GFP (Santa Cruz Biotech) were diluted in ChemMate antibody dilution buffer (Ventana Medical Systems) and incubated with sections at 37°C for 1 h. After washing with PBS/0.05% Tween 20, appropriate biotinylated secondary antibodies (Vector Lab) diluted in the same antibody dilution buffer were incubated with sections at 37°C for 30 min. Then, after washing, avidin-conjugated peroxidase or alkaline phosphatase (Vector Lab) diluted in PBS containing 1.5% normal horse serum was incubated with sections for 30 min at room temperature. Finally, after exclusive PBS-T washing, DAB or alkaline phosphatase red substrate (Vector Lab) 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.

We thank Yi Li (Memorial Sloan Kettering Cancer Center) for the RCAS–LacZ vector and Dr. Timothy S. Schaefer (MD Anderson Cancer Center, Houston, TX) for the pSM-1 clone. Mice with targeted deletions of Ink4a–Arf were obtained from Ron DePinho (Dana Farber Cancer Center) and those with targeted deletions for p53 were obtained from Larry Donehower (Baylor University). The PDGF receptor kinase inhibitor PTK787/ZK222584 was a gift of Jeanette Wood (Novartis). This work was supported by NIH grant UO1CA894314-1 and the Searles Scholars Program.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.