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[Cancer Research 60, 3019-3024, June 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

p53 Transdominance But No Gain of Function in Mouse Brain Tumor Model1

Monika E. Hegi2, Michael A. Klein, Daniela Rüedi3, Patrick Chène, Marie-France Hamou and Adriano Aguzzi

Laboratory of Tumor Biology and Genetics, Department of Neurosurgery, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland [M. E. H., M-F. H.], Institute of Neuropathology, University Hospital of Zurich, 8091 Zurich, Switzerland [M. E. H., M. A. K., D. R., A. A.], and Oncology Department, Novartis, 4002 Basel, Switzerland [P. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although p53 mutations in tumors typically result in loss of transactivation of p53 target genes some mutants display gain-of-function activity. The latter has important implications for the design of rational cancer therapy. We previously described a germ-line p53 mutation (deletion of codon 236, Y236{Delta}) associated with a familial brain tumor syndrome. To determine whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236{Delta} or an effect of genetic background we have developed a mouse brain tumor model. Primary neuroectodermal cells deficient for p53 (+/- or -/-) and transduced with Y236{Delta} using a retroviral vector were transplanted into the brain of adult wild-type mice. This neurografting paradigm circumvents the problem of early lethal tumors at extracerebral sites associated with germ-line p53 deficiency. Brain tumors arising in this mouse model were highly invasive, reflecting an important feature of the human disease. Tumors arose from p53+/- cells only when transduced with Y236{Delta}. In keeping with in vitro data showing that Y236{Delta} has dominant-negative activity, these tumors retained the endogenous wild-type p53 allele but accumulated high levels of Y236{Delta}. However, the presence of Y236{Delta} in transplanted p53-/- cells had no effect on the tumor frequency, 15% versus 27% without the mutant. In conclusion, Y236{Delta} is transdominant but exerts no gain-of-function activity mediating a more penetrant tumor phenotype.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the tumor suppressor gene p53 constitute the most common genetic alteration in human tumors (1) . The p53 gene encodes a transcription factor that is involved in many important pathways of tumor suppression such as cell cycle control, induction of apoptosis (reviewed in Ref. 2 ), and inhibition of gene amplification (3) . In the cell, p53 exists at low concentrations in a latent form that becomes activated and accumulates in the nucleus in response to various forms of cellular stress such as DNA damage (4) and hypoxia (5) . Activated p53 can induce cell cycle arrest in G1 mediated by transactivation of the p21 gene (6) or prompt apoptosis through several pathways dependent or independent of its transactivation function (2) . How p53-mediated responses are chosen is not fully understood and is likely to be cell type dependent. In tumors, the p53 gene is usually inactivated by missense mutations in evolutionarily conserved regions (1) . These mutations generally lead to inactivation of transactivation that corrupts important tumor-suppressing functions. Some mutations, however, exhibit cell-type and promoter-dependent differences in their phenotype for functions related to transactivation (7, 8, 9) . If the oligomerization domain remains intact, the mutant can hetero-oligomerize with wild-type p53 and inhibit transactivation of p53-responsive elements in a dominant-negative fashion (10 , 11) . Furthermore, some mutants are known to exhibit some oncogenic properties attributable to gain of function (12 , 13) .

In human low-grade astrocytomas, p53 mutations represent one of the first genetic changes detectable. Forty to 60% of low-grade astrocytomas, most of which eventually progress to the most malignant form, glioblastoma multiforme, carry a mutation in the p53 gene (14) . Furthermore, brain tumors constitute the third most common tumor type, after sarcomas and breast tumors, affecting carriers of a p53 germ-line mutation (15) . Thus, p53 alterations play an important role early in the development of tumors originating from the astrocytic cell lineage. This notion is supported by the fact that loss of wild-type p53 in primary astrocytes accelerates growth and malignant transformation (16) . In contrast, in other human tumor types such as colorectal, lung, and liver cancer, mutations of the p53 gene are associated with later stages of multistep carcinogenesis, suggesting cell type-specific functions.

Previously, we have described a unique p53 mutant Y236{Delta} that was found in the germ-line of a family with a brain tumor syndrome (17) . In vitro analysis revealed dominant-negative properties of this mutant (18) . The unusual clustering of brain tumors not normally seen in the context of the Li-Fraumeni syndrome raised the possibility that Y236{Delta} selectively predisposes astrocytes to malignant transformation. Here we addressed the question of whether the apparent tropism to the brain reflects a gain-of-function activity of Y236{Delta} or rather an effect of genetic background or environmental factors. To this end we have developed a mouse brain tumor model that avoids the problems posed by extracerebral tumors associated with germ-line p53 deficiency (19) . We have transplanted p53+/- or p53-/- neuroectodermal cells from day 13.5 embryos into the brain of adult wild-type mice. The Y236{Delta} mutant was introduced by retroviral transduction prior to grafting. Here we show that transplantation of p53-/- cells into the brain of wild-type mice leads to tumor formation in 27% of animals after 75 weeks, and that the Y236{Delta} mutation exerts a transdominant effect but not gain-of-function activity in this model system.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Retroviral Vectors and Plasmids.
The ecotropic, replication-defective retroviral vector pLXSN-Y236{Delta} was constructed by cloning the human p53 mutant Y236{Delta} (18) into the BamHI site of the pLXSN vector (20) . The construct was transfected into the GP+E-86 packaging cell line (21) . Clones were picked after selection. The supernatant was filtered (45 µm) and always used fresh. The clone used in the experiments produced a titer of 106 colony-forming units/ml, as determined by titration in NIH-3T3 cells. The retrovirus (pLXSN) without the Y236{Delta} insert displayed a similar titer and was used as a control vector.

For in vitro experiments, the cDNAs for the human p53 mutants Y236{Delta} and R175H/L330A and the mouse wild-type p53 cDNA (Ref. 22 ; kind gift from Moshe Oren) were cloned into a pCite-2a vector (Novagen, Madison, WI) as described earlier (11) .

Donor Mice and Preparation of Neuroectodermal Cells.
p53+/- female mice were mated with p53-/- male mice (15/16 C57BL/6, 1/16 129Sv; Ref. 19 ). On day 13.5 of gestation, the embryos were removed. Both hemispheres of the fetal brain were resected, and special care was taken to remove the leptomeninges. The tail of the embryo was used to determine the p53 genotype. The brains were delivered individually into DMEM, washed with DMEM and PBS at 4°C, incubated in 0.5 ml of DNase/trypsin (0.25% trypsin, 0.1% DNase in PBS; Life Technologies, Inc.; Boehringer Mannheim, Mannheim, Germany) for 15 min on ice, followed by 10 min at 37°C. Neuroectodermal cells were gently triturated with Pasteur pipettes to obtain a mixture of single cells and small cell aggregates. Cells were washed with DMEM containing 10% FCS and then with PBS and plated in 2 ml of fresh, virus-containing supernatant complemented with Polybrene (8 µg/ml) in six-well plates. After 16 h of infection, neuroectodermal cells were harvested, washed, pelleted, and covered with 50 µl of DMEM. Cells were used immediately for transplantation.

Stereotaxic Transplantation.
Eight-week-old adult female C57BL/6 mice were used as transplant recipients. The anesthetized mice were slowly injected with the pelleted neuroectodermal cells derived from one individual embryo into the caudoputamen (coordinates: bregma 0/0, right +2.5 mm, depth 3 mm) using a stereotaxic frame (Narashige, Tokyo, Japan). Cells remaining in the syringe after transplantation were put in culture to test viability, infection efficacy, and expression levels of Y236{Delta}.

Genotype Analyses by PCR.
The p53 genotype of the embryos and the p53 status of the tumors was determined by PCR as described (23) . The presence of Y236{Delta} in the tumors was assessed using primers 23F (5'-GTG TGG AGT ATT TGG ATG ACA G-3') and 24R (5'-ACT TCA GGT GGC TGG AGT GAG-3') that amplify a 505-bp product spanning exons 6–11 and do not amplify mouse genomic DNA or the mouse p53 pseudogene under the conditions used. The same primers were used for subsequent sequencing to confirm the presence of the mutation. PCR was performed using 2 µl of proteinase K digested tissue as follows. Dissected paraffin-embedded tissue was placed into 50 µl of digestion buffer [50 mM KCl, 10 mM Tris·HCl (pH 9.0), 0.45% NP40, and 0.45% Tween 20] and heated at 75°C for 15 min to release the tissue from the paraffin. Proteinase K (Boehringer Mannheim, Mannheim, Germany) was added to a final concentration of 0.1 mg/ml, and the tissue was digested at 55°C for 5–16 h, followed by heat inactivation of proteinase K (10 min at 85°C). Tissue derived from tails (2 mm) was processed identically (200-µl volume), but without the initial heating step.

Mutation Analysis of Mouse p53 in Tumors.
SSCA4 of the evolutionarily conserved exons 5–8 of the mouse p53 gene was performed using the primers and the method described previously (24) . For exon 5, the following antisense primer sequence was used: (5'-GGA GGA GCC AGG CCA ATG AGA AC-3').

Analysis of Grafts and Tumors.
The mice carrying the transplants were closely monitored for signs of neurological symptoms. Animals were sacrificed when symptomatic or at the end of the experiment 440–480 days after transplantation. Before necropsy, mice were injected twice with BrdUrd.Macroscopically visible brain tumors were divided into three parts that were snap frozen, put in cell culture, and fixed for histology. The brains were fixed for 4–16 h in 4% buffered paraformaldehyde, cut, and embedded in paraffin. Vital transplants or tumors were detected in 174 of 184 (95%) mice analyzed. Five mice had to be killed prematurely (78–340 days after transplantation) because of unrelated causes, and five (2.6%) mice were lost during the observation time of 75 weeks. The sections containing the transplants were stained for expression of GFAP (polyclonal antibody; DAKO, Glostrup, Denmark), a marker for glial cells. Sections of transplants infected with the retroviral vector pLXSN-Y236{Delta} were analyzed immunohistochemically for expression of the human p53 protein using PAb1801 (GENOSYS, Cambridgeshire, United Kingdom) that is specific for human p53 and does not cross-react with murine p53. The tumors were evaluated for expression of the endogenous p53 using the mouse-specific polyclonal antibody CM5. The CM5 serum was preabsorbed with bacterially expressed human p53 (25) and was a generous gift from Dr. Carol Midgley (University of Dundee, Dundee, Scotland). Selected transplants were analyzed for BrdUrd incorporation (anti-BrdUrd antibody; Caltag Lab, San Francisco, CA). Double immunostaining for GFAP and human p53 was performed using the antibodies GP52 (PROGEN, Readysysteme AG, Zurzach, Switzerland) and FITC labeled PAb1801 (Novocastra, Readysysteme AG). Fisher’s exact test was used for statistical analysis of the tumor frequencies.

Immunoprecipitation.
The plasmid (150 ng) encoding the murine wild-type p53 was translated alone or in the presence of the plasmid (50 ng) encoding Y236{Delta}. PAb1801 was used to specifically immunoprecipitate the human p53 as described (18) .

Gel Shift Assay.
The plasmid (50 ng) encoding the murine wild-type p53 was translated alone or in the presence of the plasmids (150 ng) encoding Y236{Delta} or the p53 mutant R175H/L330A, respectively. The proteins were subjected to gel shift assays using the p53 element in the p21 gene and in the presence of PAb421, as described (18) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Y236{Delta} and Cell Proliferation in Neuroectodermal Cell Transplants.
Neuroectodermal cells from 13.5 day p53+/- or p53-/- mouse embryos were infected in vitro with a Y236{Delta}-expressing retrovirus for 16 h without selection and transplanted into the brain of adult wild-type mice. Neuroectodermal cells remaining in the syringe after transplantation were analyzed for expression of Y236{Delta} after 5 days in culture. On average, 30% of the cells expressed Y236{Delta} at a level detectable by immunohistochemistry. In vivo, expression of Y236{Delta} was immunohistochemically detectable in 1–14% of cells from 5 days to 35 weeks after transplantation (n = 19; average, 3.9%; SD, 3.9) independently of the time point of analysis. A similar range of Y236{Delta}-expressing cells was observed in the tumor-free transplants at necropsy (63 weeks). The proliferation rate determined by BrdUrd incorporation was >10% 5 days after transplantation, 1–10% after 2 weeks, 1% after 4 weeks, and undetectable after 12 weeks. In addition, there was no correlation between the p53 genotype of the graft and expression of Y236{Delta}, proliferation rate, or transplant size.

Grafts consisted mostly of undifferentiated cells 5 days after transplantation, whereas after 4 weeks, the cells were differentiated and well integrated into the surrounding normal brain tissue. Moderate astrogliosis was usually associated with the transplants. The morphology and differentiation pattern (expression of neural and glial markers) were similar to those seen in earlier transplantation experiments (26) and were not genotype dependent.

Transdominant Effect of Y236{Delta} on Brain Tumor Formation.
p53+/- transplants only gave rise to tumors after transduction with Y236{Delta} (Table 1)Citation . Species-specific anti-p53 antibodies showed strong nuclear accumulation of both human and murine p53 in these tumors (Fig. 1Citation , A–C). PCR analysis of tumor DNA confirmed the presence of Y236{Delta} and revealed that the endogenous p53 gene was retained without evidence of mutational inactivation (Fig. 2Citation , Table 2Citation , and data not shown). Taken together, these observations strongly suggest that tumor formation is dependent on transdominant inhibition of the endogenous wild-type p53 by the incoming Y236{Delta}. The number of tumors derived from p53+/- transplants transduced with Y236{Delta} is not significantly different from the controls (3 of 83 versus 0 of 14 and 0 of 19; P > 0.3). This may be attributable to inefficient expression of the mutant in the p53+/- transplants (only 1–14% of cells were immunohistochemically positive for p53). Unlike p53-/- transplants, where by definition all cells lack p53 function, only those cells expressing the mutant are susceptible to dominant-negative inhibition of p53 function in the p53+/- transplants. Thus, the probability of development of a tumor derived from p53+/- transplants transduced with Y236{Delta} is likely to be smaller than for p53-/- transplants simply because the number of cells at risk is smaller. Previously, we showed that Y236{Delta} can form oligomers with human wild-type p53 (18) . To evaluate whether the same is true of murine p53, we cotranslated Y236{Delta} with wild-type murine p53 and immunoprecipitated human p53 with the anti-p53 antibody PAb1801. In keeping with previous studies using wild-type proteins (27, 28, 29) , we found that Y236{Delta} can coprecipitate the murine protein (Fig. 3ACitation ). Furthermore, in a gel shift assay, Y236{Delta} inhibited specific DNA binding by murine wild-type p53 (Fig. 3BCitation ). In contrast, the recessive human p53 mutant R175H/L330A, which cannot form tetramers (11) , does not inhibit DNA binding by murine wild-type p53. These results confirm that Y236{Delta} is also dominant-negative in mice and explain retention of the wild-type allele in the tumors arising from p53+/- grafts.


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  		Table 1 Tumor incidence 75 weeks after transplantation

 


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  		Fig. 1. Accumulation of Y236{Delta} and murine p53 in tumor cells. A, the glioblastoma-like tumor developed from a p53+/- transplant transduced with Y236{Delta} (25 weeks after transplantation; Ag8431, see Fig. 2Citation for molecular analysis). The tumor mass takes up most of one hemisphere and exhibits multiple sites of hemorrhage. Most neoplastic cells display predominantly nuclear accumulation of Y236{Delta} protein, as detected with the human p53-specific PAb1801 ({alpha}hup53). Simultaneously, endogenous p53 is expressed in a high fraction of the tumor cells visualized with the mouse p53-specific antibody CM5 ({alpha}mp53). Histology (HE, H&E staining) revealed a highly malignant tumor with necrosis, mitotic figures, and multinucleated giant cells. GFAP, the marker for glial cells, is expressed to a variable degree throughout the tumor. B, controls demonstrating species specificity of the CM5 antibody ({alpha}mp53). A human tumor stained with PAb1801 ({alpha}hu53) shows typical nuclear staining (left), whereas CM5 is negative ({alpha}mp53, middle) in the same tumor. Detection of mouse p53 with CM5 ({alpha}mp53, right) in an adenocarcinoma derived from a p53 transgenic mouse (Ref. 41 ; a kind gift of R. Wiseman, National Institute of Environmental Health Sciences). C, diffusely infiltrating tumor is shown arising from neuroectodermal transplant. The highly invasive tumor arose from a p53+/- transplant accumulating high amounts of Y236{Delta} ({alpha}hup53; 75 weeks after transplantation). Anti-p53 staining shows neoplastic cells migrating along fiber tracts and invading the whole brain. This tumor displays nuclear accumulation of endogenous p53 in most neoplastic cells ({alpha}mp53). Histology (HE, H&E staining) revealed a malignant tumor with polymorphic cell nuclei and high mitotic activity. Cells are grouped in characteristic PNET rosettes. D, the coronal section of this brain (68 weeks after transplantation) displays a p53-/- transplant located in the ventricle (*). Transplant-derived cells expressing Y236{Delta} ({alpha}hup53) are infiltrating both hemispheres. BrdUrd (BrdU) incorporation suggests that these cells are involved in a neoplastic process.

 


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  		Fig. 2. p53 status of the induced brain tumors. A, the presence of the endogenous p53 alleles was determined in the tumors (137 bp) using primers specific for either the p53 knock-out (k/o) allele or the wild-type (wt) allele. Simultaneous amplification of a 238-bp fragment of the fibroblast growth factor 7 gene served as an internal PCR control (ctr). The genotype of the transplant in animal Ag8461 and Ag8482 was p53-/- and therefore lacks a wild-type p53 allele, as confirmed in the respective microdissected tumors (T). Only a very faint wild-type band is visible, which might be attributable to contaminating normal tissue from the host mouse (N, normal brain from same paraffin section). The presence of the p53 k/o allele in the tumors unequivocally indicates that these originate from the transplants. The transplant in mouse Ag8431 was originally p53+/-, and the respective tumor has retained the single intact p53 allele, as verified by SSCA (data not shown; see Fig. 1ACitation for histology). B, the presence of the introduced human Y236{Delta} is confirmed in the tumor using specific primers for a 505-bp fragment of human p53. The sequence of this fragment was verified by direct sequencing (not shown). pos ctr, positive control; neg ctr, negative control.

 

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  		Table 2 Tumor type, genotype, and p53 expression

 


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  		Fig. 3. Y236{Delta} oligomerizes with the murine wild-type p53 and exerts a transdominant effect. A, the murine p53 protein (mp53) was in vitro synthesized alone or together with Y236{Delta}. The proteins were immunoprecipitated with human p53-specific PAb1801, denatured, and loaded on a 10% SDS-PAGE. Lanes IVT, translation mixtures before immunoprecipitation. Lanes IP show that mouse p53 (mp53) is precipitated in the presence of Y236{Delta}, suggesting complex formation. Band with *, alternative translation product. Right, molecular weight (in thousands). B, Murine p53 (mp53) was in vitro synthesized alone or together with Y236{Delta} or the recessive mutant R175H/L330A, respectively. The specific DNA binding activity of the translation mixtures was evaluated in the presence of the activating antibody PAb421. Y236{Delta} inhibits wild-type mouse p53 binding to the p53-responsive element in the p21 promoter. In contrast, the presence of R175H/L330A, which has a mutated oligomerization domain, had no effect (11) .

 
No Effect of Y236{Delta} on Tumor Formation in p53-null Transplants.
Tumors arose from 27% (10 of 37) of p53-/- transplants after mock infection or transduction with the control vector (pLXSN; Table 1Citation ). In contrast with what would be expected for a gain-of-function mutant, the tumor frequency was actually lower after introduction of Y236{Delta} into the p53-/- transplants (15%; 5 of 33; P = 0.18). Y236{Delta} was expressed in three of these five tumors (Fig. 1DCitation and Table 2Citation ). In the other two brains, ~10% of the residual normal transplanted cells expressed the human p53 mutant, but the tumors were negative. This suggests that there was no biological selection for expression of Y236{Delta} in cells lacking wild-type p53. There was also no evidence for tumor induction by retroviral insertional mutagenesis, because introduction of the control vector did not increase the tumor frequency relative to mock-infected p53-/- cells (Table 1)Citation .

Tumor Types and Tumor Invasion.
If all cell types evolving from the neuroectoderm were equally susceptible to p53-linked tumor formation, a variety of tumor types would be expected in this mouse model. In the transplants, Y236{Delta} was expressed in various neuroectodermal cell types including astrocytes, as determined by double staining of the transplants for GFAP and human p53 (data not shown). However, only two major classes of neoplasm developed (Table 2)Citation , tumors of astrocytic origin (two glioblastomas and one anaplastic astrocytoma), and PNETs (14) . One tumor displayed features of an ependymoma. One mouse exhibited a pituitary adenoma, which was considered spontaneous and of endogenous origin because the tumor lacked the expected p53 knock-out allele. The tumor latency ranged from 25 to 75 weeks and was neither related to the genotype of the transplant nor to the tumor type.

A remarkable feature of most tumors (15 of 18 neoplasms) in this study was their highly invasive nature. Fig. 1 and DCitation shows tumor cells migrating along fiber tracts throughout the brain.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Transdominance But No Gain-of-Function Activity of Y236{Delta}.
The absence of both wild-type p53 alleles or the presence of the human p53 mutant Y236{Delta} in p53+/- transplants conferred susceptibility to development of highly invasive brain tumors, whereas reduced gene dosage (p53+/-) alone had no effect. The dominant-negative effect of Y236{Delta} is supported by the fact that both the human mutant and the endogenous wild-type p53 protein accumulated in neoplastic cells (Fig. 1Citation , A and C). From these observations, we conclude that Y236{Delta} inactivated the mouse p53 protein by sequestration in inactive heterotetramers, in accordance with in vitro data showing transdominance of Y236{Delta} over murine wild-type p53 (Fig. 3Citation ). This extends our previous demonstration of the dominant-negative effect of Y236{Delta} over human wild-type p53 in vitro and a dose-dependent transdominance of Y236{Delta} over human wild-type p53 upon transfection into LoVo cells (18) . If Y236{Delta} were to exert a gain-of-function effect on brain tumor penetrance, we would expect transduction of the Y236{Delta} mutant into p53-/- cells to increase the tumor yield. In fact, the reverse was true; 15% of mice receiving p53-/- cells transduced with Y236{Delta} developed tumors, compared with 27% of mice receiving parental p53-/- cells (P > 0.1). To detect a gain-of-function effect significant at the 5% level would require a >3-fold increase in the numbers of tumors in the mutant-infected group (17 versus the observed 5 tumors in 33 mice). This lack of effect on tumor formation of Y236{Delta} in the absence of wild-type p53 corroborates observations in p53-/- mice expressing a p53 mutant transgene (30) . We conclude that Y236{Delta} does not exert a gain-of-function effect, selectively predisposing to brain tumor formation in this model. It follows that the observed clustering of brain tumors in the family with the Y236{Delta} germ-line mutation (17) is more likely attributable to an effect of genetic background or environmental factors. We cannot exclude a priori that the pathogenesis of brain tumors in mice and humans is so different as to render this type of analysis meaningless. Nevertheless, we believe that our transplantation model is well suited to test the hypothetical gain-of-function activity of further p53 germ-line mutations postulated to confer a higher penetrance of the cancer phenotype (31) .

p53 Alterations in Initiation and Progression of Brain Tumors.
Our mouse model, in addition, provides some insights into the initiating and promoting potential of p53 alterations in the formation of brain tumors. We have shown that the presence of the dominant-negative Y236{Delta} mutant in p53+/- neuroectodermal cells or the absence of p53 conferred brain tumor susceptibility, whereas reduced gene dosage alone (p53+/-) was not sufficient to predispose to brain tumor formation within the observation period of 75 weeks. Obligatory inactivation of both p53 alleles was also reported in chemically induced brain tumors of p53-deficient mice (32) . Furthermore, the inactivation of only one p53 allele (p53+/-) in the GFAP-v-src transgenic mouse, another mouse model for malignant astrocytoma, did not confer higher susceptibility to development of brain tumors or reduced latency (33) . These experimental observations emphasize the importance of transdominant inhibition of the normal p53 protein as a consequence of a single mutational event at early stages of carcinogenesis as opposed to the necessary two hits comprising deletion of both p53 alleles. This notion might be particularly important in attempts modeling astrocytic brain tumors, because p53 mutations represent one of the first genetic alterations detected in human low-grade astrocytomas. Furthermore, homozygous loss of both p53 alleles is a rare event in human gliomas (34) .

The tumor latency in this study ranged from 25 to 75 weeks (median, 38). This exceeds the natural life span of p53-/- mice, which may explain the low frequency of brain tumors observed in those mice (19) . The long tumor latency suggests that loss of p53 function is not sufficient for tumor induction but requires additional genetic alterations. This is probably facilitated by genetic instability attributable to loss of p53 function (16 , 35) .

Cell Type Specificity of p53-associated Tumor Formation.
Because of the pluripotent differentiation potential of neuroectodermal cells, the neurotransplantation procedure allows for assessment of possible cell type-specific effects of the introduced gene. When expressing cooperating viral and cellular oncogenes in rat neurografts, we found development of neuroectodermal tumors, the histopathological features of which were dependent on the oncogenes expressed (36) . In the mouse transplants, Y236{Delta} was expressed in various neuroectodermal cell types including astrocytes. Two major classes of brain tumors developed (Table 2)Citation : tumors of astrocytic origin, and PNETs. The same two tumor types are observed in Li-Fraumeni syndrome and in association with p53 mutation in sporadic human brain tumors (15 , 37, 38, 39) . In contrast, most other brain tumor types rarely contain p53 mutations (40) . Thus, this mouse brain tumor model reflects the tumor spectrum associated with p53 mutation in humans.

Model for Highly Invasive Brain Tumors.
Most rodent brain tumor models produce well-delineated lesions with little or no infiltration of surrounding normal brain tissue. A very striking finding of this study was the diffuse invasiveness of 15 of the 18 brain tumors (Fig. 1 and DCitation ). Widespread invasion is a characteristic feature of human astrocytic tumors and PNETs and represents a major problem in surgical management, because the presence of single tumor cells at sites distant from the origin of the tumors, often makes it impossible to remove the tumors completely. The extensive invasion of tumors after transplantation of cells with altered p53 suggests that this neuroectodermal grafting model recapitulates the human disease better than existing approaches and may facilitate identification of genetic determinants of brain tumor spread.


   ACKNOWLEDGMENTS
 
We are indebted to L. Donehower for providing the p53-deficient mice. We thank M. König, B. Pfister, L. Vlk, and B. Odermatt for histology; N. Wey and H. Nef for artwork; and W. Seelentag for help with statistical analysis. We appreciated the helpful discussions with J. Hainfellner, P. Kleihues, R. Janzer, and N. de Tribolet, and we thank R. Iggo for critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This project was supported by grants of the Swiss National Foundation (to M. E. H., A. A., and E. G. V. M.), the Swiss Cancer League (to M. E. H.), the Swiss Cancer League/Cancer League of the Canton of Aargau (to A. A.), and the Deutsche Forschungsgemeinschaft (to M. A. K.). Back

2 To whom requests for reprints should be addressed, at Laboratory of Tumor Biology and Genetics, Department of Neurosurgery, Centre Hospitalier Universitaire Vaudois, BH19-110, 1011 Lausanne, Switzerland. Phone: 41-(21) 314 2582; Fax: 41-(21) 314 2587; E-mail: Monika.Hegi{at}chuv.hospvd.ch Back

3 Present address: Catalys AG, 8304 Wallisellen, Switzerland. Back

4 The abbreviations used are: SSCA, single-strand conformation analysis; BrdUrd, 5-bromodeoxyuridne; GFAP, glial fibrillary acidic protein; PNET, primitive neuroectodermal tumor. Back

Received 10/29/99. Accepted 3/30/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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