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Primitive neuroectodermal tumors (PNETs) are pediatric brain tumors that result from defects in signaling molecules governing the growth and differentiation of neural progenitor cells. We used the RCAS-TVA system to study the growth effects of three genetic alterations implicated in human PNETs on a subset of neural progenitor cells that express the intermediate filament protein, nestin. The genetic alterations tested were: 1) overexpression of the cellular oncoprotein, MYC; 2) activation of transcription factor, β-catenin; and 3) haploinsufficiency of Ptc, the hedgehog receptor gene. The RCAS-TVA system uses an avian retroviral vector, RCAS, to target gene expression to specific cell types in transgenic mice. To express exogenous genes in neural progenitor cells, we used Ntv-a mice. In these mice, the Nestin gene promoter drives expression of TVA, the cell surface receptor for the virus. Ectopic expression of MYC, but not activated β-catenin, promoted the proliferation of neural progenitor cells in culture and in the cerebral leptomeninges in vivo. These effects were equally penetrant in mice with Ptc+/- and Ptc+/+ genetic backgrounds. Although overexpression of MYC is not sufficient to cause intraparenchymal tumors, it may facilitate PNET formation by sustaining the growth of undifferentiated progenitor cells.
Primitive neuroectodermal tumors (PNETs) comprise a class of malignant central nervous system (CNS) neoplasms that afflict young children. Most often, these tumors arise in the cerebellum where they are called medulloblastomas. The cells comprising medulloblastomas and other PNETs closely resemble the neural progenitor cells found normally in the CNS. That observation suggests that signaling molecules governing the growth and differentiation of CNS progenitor cells mediate PNET formation.
The Myc family of proto-oncogenes plays a central role in cell proliferation and differentiation (reviewed in Ref. ). Overexpression of Myc may be a causative factor in PNET formation. In support of this, amplification and overexpression of the human MYC gene occur in medulloblastoma cell lines and primary tumors [2,3]. Furthermore, accumulation of MYC mRNA in medulloblastomas is an unfavorable prognostic indicator for patients . In cultured cells, Myc is expressed throughout the cell cycle, but the level of its expression rises and falls in response to agents that stimulate or repress mitosis. In situ hybridization studies in mouse embryos show that Myc mRNA levels do not correlate directly with proliferation in all tissues (reviewed in Ref. ). This suggests that during embryonic development, Myc may modulate cell activities in addition to proliferation. Down-regulation of Myc generally correlates with terminal differentiation. Myc immortalizes O-2A progenitor cells, but does not block their differentiation into oligodendrocytes and astrocytes . The effect of Myc overexpression on CNS progenitor cells in vivo is not known.
Another gene that may play a role in the pathogenesis of PNETs is CTNNB1, which encodes the transcription factor, β-catenin. β-catenin is a key downstream effector of the WNT signal transduction pathway, which is crucial for CNS development (reviewed in Ref. ). In the cytoplasm, β-catenin is held in a multiprotein complex, where it becomes phosphorylated by glycogen synthase kinase (GSK) and subsequently degraded via the ubiquitin-proteasome pathway. WNT signaling blocks β-catenin degradation and permits translocation of β-catenin to the nucleus, where it activates transcription of target genes. Activating mutations in the CTNNB1 gene have been reported in 4% of human medulloblastomas . These mutations encode N-terminal amino acid substitutions that stabilize the protein by removing phosphorylation sites critical for β-catenin degradation. Although the frequency of β-catenin mutations in medulloblastomas is low, pathways leading to its activation may be physiologically relevant to PNET biology.
Another signaling pathway that contributes to PNET formation is the Patched (Ptc) signaling pathway. PTC is the cell surface receptor for hedgehog, a class of secreted proteins that play key roles in embryonic pattern formation . Inherited mutations in the human PTC gene segregate in families with Gorlin's Syndrome, a condition wherein affected individuals develop neural tube defects, craniofacial abnormalities, and predisposition to various neoplasms, including medulloblastomas . PTC gene mutations have been reported in 3% to 14% of sporadic medulloblastomas [11–13]. Transgenic mice homozygous for inactive Ptc alleles die during embryonic development with open and overgrown neural tubes. Heterozygous (Ptc+/-) mice develop features of Gorlin's Syndrome, including medulloblastomas [14,15]. Northern analysis showed that full-length Ptc transcripts were present in these spontaneous tumors, indicating that haploinsufficiency, not a two-hit mechanism of Ptc gene inactivation, is promoting medulloblastoma growth [16,17]. We do not know if mutations in MYC, CTNNB1, and PTC overlap in human PNETs, or whether these mutations are found in separate subgroups of tumors.
To study the effect of Myc dysregulation, β-catenin activation, and Ptc haploinsufficiency on the growth of CNS progenitor cells, we modeled these genetic alternations in mice using the RCAS-TVA system [18,19]. This system uses an avian leukosis virus vector (RCAS) to target gene expression to specific cell types in transgenic mice. In these mice, cell type-specific gene promoters drive expression of TVA, the cell surface receptor for the virus. After infection, RCAS sequences integrate randomly into the host cell genome. There, the exogenous gene is expressed from a spliced message under control of the constitutive retroviral promoter, LTR. In mammalian cells, the retroviral genes are spliced out so that virus replication cannot occur.
In this study, we used a line of transgenic mice (Ntv-a) wherein the Nestin gene promoter drives expression of TVA . Nestin is an intermediate filament protein expressed by CNS progenitor cells in vivo prior to their commitment to either a neuronal or glial differentiation pathway . Ntv-a mice make it possible to express exogenous proteins in nestin-expressing, CNS progenitors both in primary brain cultures in vitro and inside the brain of live animals. Nestin-expressing progenitors give rise to glioblastomas in vivo with specific activation of the RAS and AKT signaling pathways  and to oligodendrogliomas with overexpression of platelet-derived growth factor . It is possible that these same progenitor cells can give rise to PNETs after stimulation of the appropriate signaling pathways.
We show here that ectopic expression of MYC, but not activated β-catenin, stimulates the proliferation of neural progenitor cells in culture and in the cerebral leptomeninges in vivo. These mitogenic effects were equally penetrant in mice with Ptc+/- and Ptc+/+ genetic backgrounds. Although overexpression of MYC is not sufficient to cause intraparenchymal tumors, it may facilitate PNET formation by sustaining the growth of neural progenitor cells.
Production of the Ntv-a transgenic mouse line has been described previously . Ntv-a/Ptc+/- mice were created by breeding Ntv-a mice with mice containing a targeted deletion of the Ptc gene . Therefore, the mice used in these experiments are mixtures of the following strains: C57B6, BALB/C, FVB/N, and CD1.
Primary brain cell cultures from newborn transgenic mice were obtained by mechanical dissociation of the whole brain, followed by digestion with trypsin (0.25% w/v) for 15 minutes at 37°C. Large debris was allowed to settle and single cells were plated in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum. To produce live virus, we used DF-1 cells, an immortalized line of chicken fibroblasts. DF-1 cells were cultivated in DMEM containing 5% fetal bovine serum, 5% calf serum, 1% chicken serum, and 0.2% tryptose phosphate broth.
The supernatant from DF-1 cells, transfected with RCAS vector and producing virus, was filtered (0.45 µm) and applied directly to subconfluent primary brain cultures from Ntv-a mice. After exposure to three changes of viral supernatant, brain cultures were harvested by trypsin digestion and plated into tissue culture plates in growth medium containing DMEM and 10% fetal bovine serum (heat-inactivated at 56°C for 30 minutes).
To initiate a growth curve, 105 cells were plated in triplicate and cultivated at 37°C in an atmosphere of 5% CO2 and 95% air. At successive time points, the cells in each plate were harvested and counted using a hemacytometer to determine the total number of cells (average of three plates). After each time point, a fresh plate was seeded with an aliquot of 105 cells. The percent change in cell number between successive time points was used to determine a running total cell number. For controls, we used parallel cultures of cells infected with an RCAS vector carrying the LACZ gene and cells that were not infected.
Anchorage-independent growth was determined using the method of Hamburger and Salmon . Cells (5x103) were suspended in culture medium (2 ml) containing 0.3% agar (Difco Bactoagar, Detroit, MI) and plated over a layer of 0.5% agar in the same culture medium. Triplicate cultures were incubated at 37°C under 5% CO2 and 95% air. Colonies were counted 30 days after plating by viewing under a microscope through a x20 objective and a graduated eyepiece reticle. Any colonies larger than 38 µm in diameter (>10 cells) were scored as positive. The efficiency of plating was defined as the number of visible colonies as a percentage of colonies plus single cells.
DF-1 cells, transfected with RCAS vector and producing virus, were harvested by trypsin digestion, collected by centrifugation, washed, and resuspended in phosphate-buffered saline. A single injection of 1 to 2 µl containing 105 cells was made with a gas-tight Hamilton syringe in the right frontal region of newborn mice, just anterior to the coronal suture of the skull. The mice were sacrificed 6 to 8 weeks after injection and their brains were removed and fixed with formaldehyde (4% w/v). Each brain was divided into quarters by parallel incisions in the coronal plane and embedded in paraffin. Tissue sections (4 µm), each containing all four brain quadrants, were mounted on glass slides for histochemical and immunocytochemical analysis.
To analyze protein expression in tissue sections, we used an immunoperoxidase staining method described previously . Brain tissue sections, mounted on glass slides, were deparaffinized with toluene, and then hydrated through a descending series of ethanol. Sections were immersed in an antigen-unmasking solution (Vector Laboratories, Burlingame, CA) and exposed to two power bursts in a microwave oven at full power (750 W) for 5 minutes each. Treatment with H2O2 (1% v/v) was carried out for 10 minutes to quench endogenous peroxidase activity. After immersion in normal goat serum (2%), sections were incubated with primary antibody in a humid chamber at 4°C overnight. The avidin-biotin complex technique was used to localize immunoreactivity (IR). Sections were developed with diaminobenzidine (0.075% w/v)-H2O2 (0.002% v/v), then counterstained with toluidine blue (0.1% w/v). When using mouse monoclonal antibodies, we followed the MOM kit protocol (Vector Laboratories) to lower the background staining due to endogenous mouse immunoglobulins.
To detect human MYC and CTNNB1 mRNA in mouse cells infected with RCAS vectors, we used total RNA (100 ng) as template in one-step reverse transcription polymerase chain reaction (RT-PCR) (Gibco BRL Superscript) with oligonucleotide primers specific for human MYC and CTNNB1 coding sequences within the integrated RCAS provirus. After 35 cycles of amplification, the PCR products were resolved on 1.0% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light.
For analysis of protein expression, whole cell lysates of culture cells were prepared by dounce homogenization in lysis buffer containing 100 mM NaCl, 30 mM Tric-Cl (pH 7.6), 1% (v/v) NP40, 30 mM NaF, 1 mM EDTA, 1 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Lysates were incubated on ice for 45 minutes and cellular debris was removed by centrifugation. Protein samples (40 µg) were resolved by SDS-PAGE using 10% gels and transferred to nitrocellulose filters. Filters were incubated in a blocking solution containing nonfat dry milk (5% w/v), 120 mM NaCl, 50 mM Tris-Cl (pH 7.4), and Tween-20 (0.1% v/v). Primary antibody was applied to filters in the blocking solution described above for 2 hours at room temperature. Detection was carried out using peroxidase-conjugated secondary antibody (goat antimouse IgG) and enhanced chemiluminescence.
To detect protein expression by Western blotting and/or immunocytochemistry, we used the following mouse monoclonal antibodies obtained from the indicated commercial sources: 9E10 — MYC (Santa Cruz Biotechnology, Santa Cruz, CA); 401 — nestin (Becton Dickinson PharMingen, San Diego, CA); 35-Z6 — CD45 (Santa Cruz Biotechnology); C19220 — β-catenin (Becton Dickinson Transduction Laboratories, San Diego, CA); 2F11 — 70 kDa neurofilament protein (Dako, Carpinteria, CA); TuJ1 — βIII tubulin (Sigma, St. Louis, MO); A6 — notch1 (Lab Vision, Fremont, CA). To detect GFAP expression, we used a rabbit, anticow polyclonal antibody (Dako).
We prepared primary cultures of the whole brain from two different strains of Ntv-a mice: one heterozygous for an inactive Ptc gene (Ptc+/-) and a second with wild-type genetic background (Ptc+/+). Brain cultures were infected in vitro with avian leukosis viruses (derived from RCAS vectors) carrying the coding sequences of the genes, MYC and CTNNB1. The RCAS-MYC vector contained full-length, wild-type, human MYC cDNA (Figure 1A). The RCAS-β-catenin vector contained a mutant β-catenin allele encoding an S37A amino acid substitution. This mutation renders β-catenin refractory to phosphorylation by GSK and hence resistant to degradation . Mutations in the human CTNNB1 gene encoding serine 37 substitutions occur in sporadic medulloblastomas . We demonstrated MYC and β-catenin expression in infected cultures by Western blotting and by RT-PCR analysis using oligonucleotide primers specific for the human MYC and CTNNB1 sequences in the respective RCAS vectors (Figure 1B and andCC).
To quantify the population of nestin-expressing cells susceptible to infection by RCAS vectors, we prepared separate, primary brain cultures from six mice at P0. Three days later, we carried out immunoperoxidase staining with antibodies for nestin and GFAP. The percentage of nestin+ cells averaged 44% (range=28% to 54%) and the percentage of GFAP+ cells averaged 57% (range=41% to 59%). To determine the efficiency of RCAS infection, we infected three brain cultures with RCAS-LACZ and performed a colorimetric assay for the expressed gene product 3 days later. The percentage of LACZ+ cells averaged 47% 2(range 32% to 60%) These results indicated that primary brain cultures from newborn Ntv-a mice contain an abundant pool of nestin-expressing cells that can be infected with RCAS vectors with high efficiency.
We studied the growth properties of infected cultures in two ways: 1) plotting cell growth curves and 2) measuring colony-forming efficiency in soft agar media. Cultures infected with RCAS-MYC showed a growth advantage compared to those infected with RCAS-β-catenin or uninfected controls. This effect was observed in six independent brain cultures (three from Ntv-a/Ptc+/- mice and three from Ntv-a/Ptc+/+ mice). Figure 2A and andBB show the results with two cells lines, R4 and WT3.
After 23 days in culture, cells infected with RCAS-β-catenin and uninfected controls had a similar growth rate of one population doubling per 24 hours. By contrast, infection with RCAS-MYC stimulated this rate three-fold and six-fold in Ptc+/- and Ptc+/+ cells, respectively. Although we observed variation in basal growth rates of uninfected cells from different mice, RCAS-MYC infection significantly enhanced the growth rate in every case. Comparison of the growth curves from Ptc+/- and Ptc+/+ brain cultures (mock-infected with RCAS-LACZ) showed that Ptc+/- cells had a slightly higher growth rate initially (Figure 2C). After 15 days in culture, however, the growth rates of Ptc+/- and Ptc+/+ cultures had become identical. A positive control virus carrying the polyoma middle T oncogene stimulated cell growth in every case (data not shown).
Consequent to infection with RCAS-MYC a distinct, morphologic change occurred in the brain cell cultures. Flat, polygonal cells in early passage cultures acquired a refractile, spindle-shaped appearance and a marked tendency to form multicellular aggregates (Figure 2D). Compared to β-catenin-expressing cells or uninfected controls (Figure 2E), MYC-expressing cells lost contact inhibition and grew to very high cell density in culture.
In soft agar, we observed a low level of anchorage-independent growth following infection with RCAS-MYC (1.7% and 8.0% colony-forming efficiencies in Ptc+/+ and Ptc+/- cells, respectively) (Table 1). This effect was far weaker than that of RCAS-MTA, carrying the potent transforming oncogene, polyoma middle T. The observed increase in colony-forming efficiency of cells infected with RCAS-MYC compared to uninfected control cells was not statistically different (P=.245 and .407 by t-test in Ptc+/+ and Ptc+/- cells, respectively). A trend toward anchorage-independent growth after infection with RCAS-MYC was observed in Ptc+/- cells compared to Ptc+/+ cells (P=.120 by t-test).
Immunocytochemical analysis of late-passage brain cultures (at the end of the growth curve experiments) showed that nearly 100% of cells was nestin+ and only occasional clusters of cells were GFAP+ (Figure 3E). This staining pattern was observed in cultures infected with RCAS-MYC, RCAS-β-catenin, and in uninfected cultures. These results indicate that the culture conditions (including serum-containing media) do not induce terminal differentiation, but rather sustain an undifferentiated phenotype of neural progenitor cells. Furthermore, genotyping by PCR of late-passage brain cultures infected with RCAS-MYC, RCAS-β-catenin, and in uninfected controls showed that both wild-type and recombinant Ptc alleles were present.
We concluded from these in vitro studies that ectopic expression of MYC selects for cells with marked proliferative capacity and morphologic changes in culture. Furthermore, heterozygous loss of Ptc cooperates with MYC overexpression to promote anchorage-independent growth of CNS progenitor cells in culture.
To determine the effect of transferring RCAS-MYC into nestin-expressing progenitor cells in vivo, we injected ALV producer cells directly into the brains of 53 newborn Ntv-a mice. These test animals comprised six litters arising from matings between Ntv-a/Ptc+/- parents. Six to 8 weeks later, the mice were sacrificed and their brains were removed for histologic and immunocytochemical analysis. Five mice were sacrificed 3 to 4 weeks after injection because they showed signs of increased intracranial pressure. The brains from 51 animals were available for analysis. After genotyping for Ptc, we divided the mice into two groups, Ptc+/- (n=36) and Ptc+/+ (n=15). These proportions reflect the expected distribution of alleles arising from matings between heterozygous parents (Ptc nullizygotes die early in embryonic development).
We found aggregates of cells with hyperchromatic nuclei within the leptomeninges of the brain in 27/36 (75%) Ptc+/- specimens and in 11/15 (73%) Ptc+/+ specimens (Figure 3A). In no case did we see a solid tumor mass within the brain parenchyma. Microscopic examination of brains from 18 mice injected with RCAS-β-catenin showed neither tumors nor leptomeningeal cell aggregates in any mouse.
To determine whether these cell aggregates arising in mice infected with RCAS-MYC were the result of virally expressed human MYC, we carried out immunoperoxidase staining of brain sections using monoclonal antibody, 9E10. This antibody recognizes human MYC, but not endogenous mouse MYC, in formalin-fixed, paraffin-embedded tissue sections. Cells within the leptomeningeal aggregates showed 9E10 IR, indicating expression of MYC from the integrated RCAS provirus. In these cells, we observed both nuclear and cytoplasmic IR (Figure 3B and C). To exclude the possibility that these cells might be inflammatory leukocytes, we stained adjacent sections with monoclonal antibody, 35-Z6, specific for CD45, a transmembrane protein widely expressed in hematopoietic cells. Only a few scattered cells were CD45+ (Figure 3D).
To characterize these leptomeningeal aggregates further, we stained brain sections with antibodies for nestin, βIII tubulin, and notch1 — markers for neural progenitor cells. Approximately 10% to 20% of cells within the leptomeningeal aggregates was nestin+ (Figure 3F). The cell type specificity of the RCAS-TVA system is limited to the original gene transfer event. Afterwards, the transferred gene is expressed from the viral LTR. Therefore, there is no selective pressure for infected cells to maintain nestin expression in vivo.
In contrast to the variable expression of nestin, 100% of the cells expressed notch1 (Figure 3G). Notch1 is a cell surface receptor whose activation in CNS progenitors inhibits neuronal and oligodendroglial differentiation [27,28]. In addition, approximately 10% of the leptomeningeal aggregates contained βIII tubulin+ cells (Figure 3H). Expression of βIII tubulin by CNS progenitor cells is an early marker of neuronal differentiation . We did not detect expression of GFAP or neurofilament protein, markers for terminally differentiated astrocytes and neurons. Taken together, these immunocytochemistry results indicate that the MYC-expressing, leptomeningeal cells are undifferentiated CNS progenitors. Expression of βIII tubulin suggests a capacity to differentiate along a neuronal pathway. Furthermore, the Ptc genetic background does not appear to alter this effect.
We show here that ectopic expression of MYC stimulates the growth of CNS progenitor cells and leads to their accumulation in the cerebral leptomeninges. The leptomeningeal pattern of cell growth induced by overexpressed MYC in mice mimics the tendency of human medulloblastomas to invade the subarachnoid spaces of the brain. In fact, subarachnoid tumor spread correlates with shortened survival in patients (reviewed in Ref. ). Also, increased expression of MYC is an unfavorable prognostic factor for medulloblastoma patients . These clinical correlations suggest that MYC or signaling pathways that upregulate MYC may mediate this leptomeningeal pattern of tumor growth.
In vivo, RCAS-MYC promoted growth of progenitor cells only in the leptomeninges. One possible explanation for this phenomenon is that physical contact with leptomeningeal cells may be required for sustained growth of CNS progenitor cells. In support of this, cultivation of human medulloblastoma cells on a monolayer of leptomeningeal cells prolongs cell viability compared to growth on plastic surfaces .
Numerous cell signaling pathways converge upon β-catenin (reviewed in Ref. ). The occurrence of mutations that activate β-catenin in human medulloblastomas implicates one or more of these pathways in PNET formation. We did not find a proliferative effect of β-catenin in our mouse model system. Kolligs et al., using retroviral expression, showed that activated β-catenin, lacking GSK phosphorylation sites, failed to transform mouse NIH3T3 fibroblasts, rat IEC-18 intestinal epithelial cells, or human 1811 squamous epithelial cells. However, these β-catenin mutants were able to transform a rat kidney cell line (RK3E) that had been immortalized by adenovirus E1A . These experiments imply that β-catenin is capable of cooperating with molecules, like E1A, that disrupt cell cycle arrest. In addition, expression of activated β-catenin in Ntv-a mice may have a weak genetic effect detectable only after a longer period of observation. For these reasons, we cannot conclude from our experiments that β-catenin is not involved in the pathogenesis of PNETs. If β-catenin does contribute to PNET formation, it most likely does so through collaboration with other signaling molecules.
Further evidence that multiple signaling pathways cooperate in PNET formation is the fact that medulloblastomas arise spontaneously in only 14% to 30% of Ptc+/- mice [14,16]. The incomplete penetrance of Ptc on tumor formation in mice, coupled with the low frequency of PTC mutations in human tumors, suggests that additional molecules are involved. One of these is insulin-like growth factor 2. Tumor formation is completely eliminated in mice that are Igf2-/-/Ptc+/- double mutants . Loss of p53 accelerates medulloblastoma formation in Ptc+/- mice, possibly by increasing genomic instability . However, P53 gene mutations are rarely found in medulloblastomas. Dysregulation of MYC may be another factor promoting PNET formation by sustaining the growth of undifferentiated, CNS progenitor cells or by facilitating leptomeningeal spread of tumor cells. Our experimental observation, that MYC promotes colony formation in soft agar more efficiently in Ptc+/- cells, suggests that MYC overexpression and Ptc haploinsufficiency may cooperate to enhance tumor cell growth in human PNETs.
In these studies, we have investigated the in vivo effects of several genetic changes found in human PNETs but we have not generated tumors. The cell-of-origin for cerebral PNETs remains unknown. Strong evidence points to a neuronal precursor in the external granular layer of the cerebellum as the cell-of-origin for medulloblastomas . It is possible that in vivo gene transfer of MYC or CTNNB1 to a cell type other than nestin-expressing CNS progenitors might give results different from those reported here for the Ntv-a mouse line. Furthermore, our gene transfer experiments were carried out in postnatal mice. It is possible that the nestin+ cells present in the cerebrum at that developmental time point may not give rise to PNETs. We do know that these cells are capable of transformation into gliomas with appropriate genetic alterations [22,23].
The study reported here demonstrates the utility of the RCAS-TVA system for testing the individual contributions of oncogenes and tumor-suppressor genes to CNS progenitor cell growth. Although MYC dysregulation recapitulates the leptomeningeal pattern of PNET growth, further research will be needed to identify the molecular defects required to transform progenitor cells completely and cause PNETs to form inside the brain parenchyma.
The authors thank Heidi Hahn (Technical University of Munich, Neuherberg, Germany) and Andreas Zimmer (University of Bonn, Bonn, Germany) for the Ptc+/- mouse line, and Harold Varmus (Memorial Sloan-Kettering Cancer Center, New York, USA), in whose laboratory the RCAS-MYC and RCAS-β-catenin vectors were constructed.
1This work was supported by a grant from the Pediatric Brain Tumor Foundation of the United States.