1 Department of Pharmacology and Cancer Biology, Duke University Medical Center,
Durham, NC 27710, USA
2 Department of Radiation Oncology, Duke University Medical Center, Durham, NC
27710, USA
3 Department of Biostatistics and Bioinformatics, Duke University Medical
Center, Durham, NC 27710, USA
* Author for correspondence (e-mail: rw.reya{at}duke.edu)
Accepted 16 February 2005
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SUMMARY |
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Key words: Medulloblastoma, Brain tumor, Pre-neoplastic, Patched, Hedgehog, Migration, Differentiation, Mouse
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Introduction |
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Approximately 25% of medulloblastoma cases have mutations in components of
the Sonic hedgehog-Patched signaling pathway
(Corcoran and Scott, 2001;
Ellison et al., 2003
). Sonic
hedgehog (Shh) is a potent mitogen for cerebellar granule cell precursors
(GCPs), the cells from which medulloblastoma is believed to arise
(Wechsler-Reya and Scott,
2001
; Wechsler-Reya and Scott,
1999
). Patched functions as an antagonist of Sonic hedgehog
signaling in most tissues (Ingham and
McMahon, 2001
). People with mutations in the patched gene
develop Gorlin's syndrome, a disease characterized by basal cell carcinomas,
skeletal defects and an increased incidence of medulloblastoma
(Hahn et al., 1996
;
Johnson et al., 1996
).
Sporadic medulloblastomas also harbor mutations in patched and other
elements of the Shh pathway (Lam et al.,
1999
; Raffel et al.,
1997
; Taylor et al.,
2002
). Finally, mice heterozygous for mutations in
patched develop cerebellar tumors that resemble human medulloblastoma
(Goodrich et al., 1997
;
Hahn et al., 2000
).
Although patched mutant mice are an important model for
medulloblastoma, the molecular and cellular basis of tumorigenesis in these
mice remains unclear. Homozygous patched knockout mice die during
embryonic development with defects in the nervous system, the heart and other
tissues (Goodrich et al.,
1997). Heterozygotes survive to adulthood, but after 3 months of
age, 14-20% develop medulloblastoma
(Goodrich et al., 1997
;
Wetmore et al., 2000
). The
status of the wild-type patched allele in these tumors is
controversial: some studies have reported expression of wild-type
patched in tumor tissue (Romer et
al., 2004
; Wetmore et al.,
2000
; Zurawel et al.,
2000
), whereas others have suggested that the wild-type allele is
epigenetically silenced (Berman et al.,
2002
). Determining whether patched is lost and
when during tumorigenesis this loss occurs is crucial for
understanding the mechanisms of medulloblastoma formation.
Studies of patched mutant mice suggest that cerebellar
abnormalities precede the appearance of tumors. While one-sixth of these
animals develop medulloblastoma at 3-6 months of age, more than half have
regions of ectopic cells in their cerebella at 4-6 weeks of age
(Corcoran and Scott, 2001;
Goodrich et al., 1997
;
Kim et al., 2003
). These cells
resemble normal GCPs in terms of morphology and location on the surface of the
cerebellum, and have therefore been described as remnants of the external
germinal layer (EGL) from which GCPs originate. However, the presence of these
cells in mice that are destined to develop medulloblastoma raises the
possibility that they may represent a pre-neoplastic stage of tumorigenesis.
Determining whether these cells are merely normal cells that persist into
adulthood, or whether they are partially transformed cells on their way to
becoming tumors has important implications for our understanding of granule
cell development and tumorigenesis.
To gain insight into the early stages of medulloblastoma formation, we have isolated ectopic cerebellar cells from patched mutant mice and studied their molecular and functional characteristics. Our studies demonstrate that these cells share many properties with tumor cells: they express markers of the granule cell lineage, they exhibit activation of Shh target genes and they proliferate extensively in vitro. In addition, we show that these cells (and tumor cells) completely lack expression of the wild-type patched allele, suggesting a mechanism by which these cells maintain active hedgehog signaling. Finally, microarray analysis reveals that these cells have a unique pattern of gene expression that more closely resembles tumor cells than GCPs. Thus, it is likely these cells represent a distinct, pre-neoplastic stage of tumorigenesis. Genes that are differentially expressed at the pre-neoplastic stage include regulators of cell migration, survival and differentiation. Our studies suggest that loss of patched expression and dysregulation of these processes may be crucial early events in the development of medulloblastoma.
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Materials and methods |
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Histological staining
To detect expression of ß-galactosidase in intact cerebellum, tissue
was isolated from adult wild-type or patched+/ mice
(6- to 12-weeks old) and fixed in 4% paraformaldehyde (PFA) at 4°C. After
fixation, tissues were permeabilized in buffer containing 0.01% deoxycholate
and 0.02% IGEPAL CA-630 (both from Sigma, St Louis, MO, USA) for 10 minutes.
Tissues were washed and stained overnight with X-gal reaction mixture
containing 10 mM potassium ferrocyanide, 10 mM potassium ferricyanide, 0.4
mg/ml X-galactoside 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
(X-gal, Sigma) in dimethyl sulfoxide, and 1 mM MgCl2 in
phosphate-buffered saline.
To compare expression of ß-galactosidase and GFP in sections from Math1-GFP/patched+/ mice, cerebella were fixed in 4% PFA, cryoprotected in 25% sucrose, embedded in Tissue Tek-OCT (Sakura Finetek, Torrance, CA, USA) and cryosectioned sagittally at a thickness of 10 µm. One set of sections (for detection of GFP) was post-fixed for 10 minutes in 2% PFA and immediately mounted in Fluoromount G (Southern Biotechnology Associates, Birmingham, AL, USA). Adjacent sections were stained with X-gal as described above, counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA, USA) and mounted in Fluoromount G. Fluorescent (GFP) and bright-field (X-gal) images were acquired using a Nikon TE200 inverted fluorescent microscope and Openlab software (Improvision, Lexington, MA, USA).
For histochemical analysis of neonatal cerebellum, pre-neoplastic lesions and tumors, cerebella were fixed overnight in 10% formalin, transferred to 70% ethanol, paraffin wax-embedded and sectioned at 5 µm. Sections were stained with Hematoxylin and Eosin (Sigma).
Isolation of granule cell precursors, pre-neoplastic cells and tumor cells
Granule cell precursors (GCPs) were isolated from 7-day-old (P7)
patched+/ mice; pre-neoplastic cells were obtained
from 6-week-old patched mutants; and tumor cells were obtained from
10- to 25-week-old patched mutants displaying physical and behavioral
signs of medulloblastoma. Cells were isolated from each source using a
protocol described in (Wechsler-Reya and
Scott, 1999). Briefly, cerebella were digested in solution
containing 10 U/ml papain (Worthington, Lakewood, NJ, USA) and 250 U/ml DNase
(Sigma), and triturated to obtain a cell suspension. This suspension was
centrifuged through a step gradient of 35% and 65% Percoll (Amersham
Biosciences, Piscataway, NJ, USA), and cells were harvested from the 35%-65%
interface. Cells were resuspended in serum-free culture medium consisting of
Neurobasal containing B27 supplement, sodium pyruvate, L-glutamine and
penicillin/streptomycin (all from Invitrogen, Carlsbad, CA, USA), and counted
on a hemacytometer. Cells used for RNA isolation were centrifuged and flash
frozen in liquid nitrogen. For proliferation assays or immunostaining, cells
were plated on poly-D-lysine (PDL)-coated tissue culture vessels and incubated
in serum-free culture medium.
Flow cytometry and immunofluorescence
To detect ß-galactosidase activity in isolated GCPs, pre-neoplastic
cells and tumor cells, cells purified as described above were stained with
fluorescein di-ß-galactopyranoside (FDG, Marker Gene Technologies,
Eugene, OR, USA) for 2 minutes at 37°C. Cells were washed, incubated for
30 minutes on ice and analyzed on a FACSVantage SE flow cytometer (BD
Biosciences, San Jose, CA). As a control for non-specific FDG staining,
GFP- cells were isolated by fluorescence-activated cell sorting
(FACS) from Math1-GFP/patched+/ mice and stained in
the same manner. These cells are not hedgehog responsive, and therefore
express low levels of the mutant patched allele and low levels of
ß-galactosidase.
To detect expression of surface markers, cells were stained for 1 hour with primary antibodies, washed, stained for 30 minutes with secondary antibodies, and then analyzed by flow cytometry. To detect expression of intracellular markers, cells were plated (1 million cells/well) on PDL-coated coverslips in 24-well plates, and allowed to adhere for 4-6 hours before fixation with 4% PFA. Cells were stained overnight with primary antibodies, washed, stained with secondary antibodies for 2 hours at room temperature, and then mounted in Fluoromount G. Immunofluorescence was detected using a Nikon TE200 inverted microscope and Openlab software.
Antibodies used for flow cytometry and immunofluorescence included the following: nestin and GFAP (both from BD-Pharmingen, San Diego, CA, USA); O4, A2B5, polysialated (PSA)-NCAM and Zic-1 (all from Chemicon, Temecula, CA, USA); TUJ1 (Covance, Berkeley, CA, USA); and 13A4 anti-prominin/CD133 (a generous gift of Wieland Huttner and Denis Corbeil, Max Planck Institute, Dresden, Germany).
Proliferation assays
Cerebellar cells isolated as described above were resuspended in serum-free
medium (Neurobasal + supplements) and transferred to PDL-coated 96-well
plates, at a density of 2x105 cells/well. Cells were pulsed
immediately with tritiated thymidine (methyl-[3H]-Td, Amersham,
Arlington Heights, IL, USA) and cultured for 18 hours. Following culture,
cells were harvested onto filters using a Mach IIIM Manual Harvester 96
(Tomtec, Hamden, CT, USA) and the amount of incorporated radioactivity was
quantitated by liquid scintillation spectrophotometry using a Wallac MicroBeta
microplate scintillation counter (Perkin Elmer, Boston, MA, USA).
RNA isolation and real-time RT-PCR
To isolate total cytoplasmic RNA from GCPs, pre-neoplastic cells and tumor
cells, snap-frozen cell pellets were lysed in buffer containing 0.5% IGEPAL
CA-630, digested with Proteinase K, extracted with phenol:chloroform:isoamyl
alcohol, and precipitated with ethanol. RNA was purified using RNeasy columns
(Qiagen, Valencia, CA, USA) and treated with DNase 1 (DNA-free, Ambion,
Austin, TX, USA) to remove genomic DNA. RNA concentration was determined using
the RiboGreen fluorescent dye (Molecular Probes, Eugene, OR, USA) with a
TD-700 fluorometer (Turner BioSystems, Sunnyvale, CA, USA).
For real-time RT-PCR analysis, first-strand cDNA was synthesized using equivalent amounts of total RNA (0.1-1 µg) in a 20 µl reverse-transcriptase reaction mixture (Invitrogen). Real-time PCR reactions were performed in triplicate using a 25 µl mixture containing iQ SYBR Green Supermix (BioRad, Hercules, CA, USA), water, primers and 1 µl of cDNA. Gene-specific primers were used for: Nmyc, cyclin D1, Gli1, Pax6, Unc5h3 (Unc5c), Atf3, osteopontin (Spp1), Bag3, Foxf2, Klf4 and Neurod1; sequences for these are available upon request. Real-time quantitation was performed using the BIO-RAD iCycler iQ system (BioRad). Serial tenfold dilutions of cDNA were used as a reference for the standard curve calculation. Raw data were normalized based on expression of actin.
For analysis of wild-type and mutant patched expression, cells were isolated from Math1-GFP/patched+/ mice as described above, and then FACS-sorted to obtain pure populations of GFP+ cells. RNA was isolated using the RNAqueous-Micro kit (Ambion), DNase-treated, quantitated, converted to cDNA and subjected to real-time PCR analysis as described above. Primers used to amplify patched were as follows: exons 2-3, 5'-GGC AAG TTT TTG GTT GTG GGT C-3' (forward) and 5'-CCT CTT CTC CTA TCT TCT GAC GGG-3' (reverse); and exons 7-9, 5'-CAT TGG CAG GAG GAG TTG ATT G-3' (forward) and 5'-GCA CCT TTT GAG TGG AGT TTG G-3' (reverse).
Microarray hybridization and analysis
RNA from GCPs, pre-neoplastic cells and tumor cells (isolated as described
above, but not FACS-sorted), and from normal adult cerebellum (not
dissociated), was converted to cDNA using the Superscript Choice cDNA kit
(Invitrogen) and a T7-dT(24) primer (Genset/Proligo, Boulder, CO, USA). cRNA
was generated using a T7-transcription/labeling kit from Enzo Life Sciences
and hybridized to Affymetrix U74Av2 chips (Affymetrix, Santa Clara, CA, USA).
Chips were scanned, and hybridization data were acquired using Affymetrix
Suite 5.0 software. Affymetrix CEL files were normalized and quantified using
Bioconductor software with the gcRMA model to quantify gene expression levels
(Gentleman and Carey, 2002).
Unsupervised principal components analysis (PCA) was used to identify the
relationships among normal adult cerebellum, GCPs, pre-neoplastic cells and
tumor cells based on expression profiles.
To identify genes that were differentially expressed among GCPs,
pre-neoplastic cells and tumor cells, supervised analysis was carried out. A
gene-by-gene analysis of variance (ANOVA) model with three groups (GCP,
pre-neoplastic, tumor) was used to fit the log2-transformed
intensities. To correct for multiple comparisons, the nominal P-value
was adjusted using the false discovery rate
(Benjamini and Hochberg, 1995).
Genes were considered to be differentially expressed if they satisfied all of
the following criteria: a difference in expression greater than 1.9-fold
between any two groups; a maximum absolute intensity difference larger than 32
units; and an adjusted P-value <0.01. There were 118 genes that
met these criteria. The identities of differentially expressed genes were
verified by integrating data from the Affymetrix and Unigene databases. Gene
functions were determined using information from Gene Ontology, Unigene,
LocusLink and PubMed databases. Clustering was performed with Cluster and
Treeview (Eisen et al., 1998
).
All statistical analysis was performed using R-1.7 software
(Dalgaard, 2002
). Results were
visualized with Spotfire 6.0 (Somerville, MA, USA).
Immunohistological validation of microarray genes
Tissues were processed as described for the X-gal histological staining in
the methods above for P7, 6-week-old, and 10- to 25-week-old patched
mutant mice with tumors. PFA-fixed frozen sections were rehydrated in
Tris-buffered saline and permeabilized with 2% Triton X-100 (Sigma) for 10
minutes. Sections were stained overnight at 4°C with rabbit polyclonal
antibodies specific for Zic3 (Chemicon), Necdin (Upstate, Waltham, MA, USA) or
Hsp105 (Biovision, Mountain View, CA, USA), or with mouse monoclonal
antibodies specific for Pax6 (R&D Systems, Minneapolis, MN, USA). Antibody
staining was detected using the EnVision+ Peroxidase-DAB system (Dako
Cytomation, Carpinteria, CA, USA), as described in the manufacturer's
protocol. Sections were counterstained with Harris Hematoxylin (Sigma) and
mounted using Vectamount (Vector Laboratories).
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Results |
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Pre-neoplastic cells express markers of immature granule cells
Previous studies have suggested that medulloblastoma arises from granule
cell precursors (Buhren et al.,
2000; Kadin et al.,
1970
; Miyata et al.,
1998
). To determine whether pre-neoplastic cells express markers
of the granule cell lineage, we crossed patched+/
mice with Math1-GFP mice (Lumpkin et al.,
2003
). In the developing cerebellum, Math1 is a specific marker
for proliferating granule cell precursors
(Ben-Arie et al., 1996
;
Helms and Johnson, 1998
). As
GCPs cover the surface of the cerebellum during the first 2-3 weeks of life,
the cerebellum of Math1-GFP mice is intensely fluorescent during this period
(Fig. 3A). By 6 weeks of age,
all GCPs have migrated inward and differentiated into mature granule neurons;
thus, GFP is not expressed in the cerebellum of adult Math1-GFP mice
(Fig. 3B). However, in
Math1-GFP/patched+/ mice, regions of ectopic
GFP+ cells are frequently seen in the cerebellum at 6 weeks of age
(Fig. 3C,E). Tumors arising in
these mice are also strongly GFP+
(Fig. 3D). These data suggest
that pre-neoplastic cells and tumor cells express Math1 and are derived from
the granule cell lineage.
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To further examine the phenotype of these cells, we isolated them and
stained them with antibodies specific for various cell types (see Table S1 in
supplementary material). Consistent with the expression of Math1-GFP, a large
percentage of cells in each population expressed neuronal markers (tubulin
ßIII/TuJ1, polysialated NCAM and Zic1)
(Miyata et al., 1998;
Yokota et al., 1996
). A small
number of cells in each population expressed the oligodendrocyte marker O4
(Sommer and Schachner, 1981
),
suggesting that some cells of this lineage co-purify with GCPs in our
isolation procedure. By contrast, very few cells expressed markers of
astrocytes (glial fibrillary acidic protein)
(Bignami et al., 1972
) or
neural stem cells (nestin, CD133/prominin)
(Sawamoto et al., 2001
;
Weigmann et al., 1997
). These
findings support the notion that pre-neoplastic cells are derived from the
granule cell lineage.
Pre-neoplastic cells express elevated levels of hedgehog target genes and proliferate in vitro
In patched heterozygotes, ß-galactosidase activity is a
reporter for expression of the mutant patched allele. Because
patched is a target of the hedgehog pathway
(Goodrich et al., 1996), the
ß-galactosidase activity observed in pre-neoplastic cells
(Fig. 1B,
Fig. 4C) suggests that these
cells have increased activation of the hedgehog pathway. To determine whether
these cells express elevated levels of other hedgehog target genes, we
performed quantitative (real-time) RT-PCR analysis using primers specific for
Gli1, cyclin D1 and Nmyc, genes previously identified as
hedgehog targets in GCPs (Kenney et al.,
2003
; Kenney and Rowitch,
2000
; Oliver et al.,
2003
; Wechsler-Reya and Scott,
1999
). As a reference for the level of mRNA associated with
hedgehog pathway activation, we compared the levels of these genes to the
levels in resting granule cell precursors (cultured for 24 hours in the
absence of Shh). As shown in Fig.
6, freshly isolated GCPs, pre-neoplastic cells and tumor cells all
exhibit significantly elevated levels of Shh target genes when compared with
resting cells (3- to 5-fold for Nmyc, 4- to 9-fold for cyclin
D1, 2000- to 6000-fold for gli1). For each gene, the levels in
GCPs, pre-neoplastic cells and tumor cells were comparable to or higher than
the levels in cells that had been stimulated with Shh for 24 hours (data not
shown). These data suggest that the Shh signaling pathway is activated in
pre-neoplastic cells.
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Pre-neoplastic cells lack expression of wild-type patched
Our studies indicated that GCPs, pre-neoplastic cells and tumor cells all
exhibit hedgehog pathway activation and proliferation when they are isolated.
In GCPs, expression of Shh targets reflects exposure to Shh in vivo prior to
isolation (Kenney and Rowitch,
2000; Wechsler-Reya and Scott,
1999
). In tumor cells, Shh pathway activation has been suggested
to result from silencing of the wild-type patched allele
(Berman et al., 2002
), although
some groups have reported persistent expression of wild-type patched
in tumors (Romer et al., 2004
;
Wetmore et al., 2000
;
Zurawel et al., 2000
). The
status of wild-type patched in pre-neoplastic cells has not been
investigated. Our ability to isolate pre-neoplastic cells and tumor cells to
near-homogeneity allowed us to examine patched expression in these
populations in the absence of contaminating (non-tumor) cells.
We analyzed patched expression in GCPs, pre-neoplastic cells and tumor cells by real-time RT-PCR. To distinguish between wild-type and mutant patched transcripts, we used two sets of primers: one derived from exons 7-9, which are present in both the wild-type and mutant alleles, and another derived from exons 2-3, which can only amplify sequences present in the wild-type allele (see Fig. 7A). As shown in Fig. 7B, transcripts containing exons 7-9 were comparably expressed in GCPs, pre-neoplastic cells and tumor cells. By contrast, transcripts containing exons 2-3 (wild-type patched) were found in GCPs but were absent from the majority of pre-neoplastic cells and tumor cells (Fig. 7C). Similar results were seen using a pair of primers within exon 2. Overall, loss of wild-type patched was observed in 6 out of 7 pre-neoplastic samples and 13 out of 13 tumor samples, but was never seen in GCPs or in normal adult cerebellum from patched+/ mice (see Fig. S1 in supplementary material). These results suggest that the Shh pathway activation and proliferation seen in pre-neoplastic and tumor cells results from de-repression of the pathway due to loss of patched. Moreover, they indicate that loss of patched occurs at an early stage of tumorigenesis, well before cells have committed to becoming full-blown tumors.
|
To determine how closely related these samples were in terms of gene expression, we performed unsupervised principal components analysis (PCA). When the analysis included normal adult cerebellum (Fig. 8A), GCPs, pre-neoplastic cells and tumor cells appeared highly similar to one another and quite distinct from adult cerebellum. This is not surprising, as adult cerebellum consists of postmitotic neurons and glia of various lineages whereas GCPs, pre-neoplastic cells and tumor cells are all proliferating cells derived from the granule cell lineage. By contrast, when GCPs, pre-neoplastic cells and tumor cells were compared directly to one another, each population exhibited a unique pattern of gene expression (Fig. 8B). Hierarchical clustering of the samples (Fig. 8C) supported this conclusion, and indicated that pre-neoplastic cells and tumor cells resemble one another more closely than either population resembles GCPs. Thus, GCPs, pre-neoplastic cells and tumor cells are readily distinguishable at a molecular level.
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Discussion |
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Pre-neoplastic cells in patched mutant mice were first described
by Goodrich et al. as `regions of increased X-gal staining on the surface of
the cerebellum' (Goodrich et al.,
1997). These cells have been suggested to represent either a
persistent external germinal layer or an early stage of tumorigenesis
(Corcoran and Scott, 2001
;
Goodrich et al., 1997
;
Kim et al., 2003
). The
distinction is important: if these cells are essentially normal GCPs (except
for their persistence into adulthood), their relevance for understanding
tumorigenesis may be limited. However, if they are partially transformed and
need only acquire a small number of additional changes to become tumors, then
studying their properties may shed light on the earliest changes required in
medulloblastoma. Isolation of these cells has allowed us to study their
molecular and functional characteristics in detail. Although pre-neoplastic
cells resemble GCPs in terms of location, lineage and hedgehog pathway
activation, they differ significantly from GCPs in terms of gene expression,
including loss of patched. In these respects, pre-neoplastic cells
are much more similar to tumor cells. Based on these observations, it is
likely that they represent an early stage of tumorigenesis.
In this context, it is worth noting that persistent EGL has been observed
in other mutant mice. For example, animals with mutations in brain-derived
neurotrophic factor (BDNF), matrix metalloprotease 9 (MMP9), retinoid-related
orphan receptor alpha (ROR/staggerer) or the peroxisome assembly gene
PEX2 show delayed GCP migration and persistence of the EGL
(Borghesani et al., 2002
;
Faust, 2003
;
Messer and Hatch, 1984
;
Vaillant et al., 2003
).
Moreover, astrotactin 1 null mice exhibit ectopic accumulations of GCPs that
superficially resemble the foci observed in patched heterozygotes
(Adams et al., 2002
). However,
none of these animals develop medulloblastoma, suggesting that persistence of
the EGL alone is not sufficient for tumorigenesis. The fact that the ectopic
cells in patched mutant mice more closely resemble tumor cells than
GCPs and occur in animals that develop tumors supports the use
of the term `pre-neoplastic' to describe these cells.
Pre-neoplastic cells arise from proliferating granule cell precursors
The fact that pre-neoplastic cells and tumor cells express markers of the
granule cell lineage is consistent with the notion that they are derived from
granule cell precursors. The same has been postulated for human
medulloblastomas, particularly those with a desmoplastic appearance
(Buhren et al., 2000;
Kadin et al., 1970
;
Miyata et al., 1998
).
Desmoplastic tumors which represent 20-30% of human medulloblastomas
frequently harbor mutations in the Shh pathway, and have a gene
expression profile that resembles normal GCPs
(Pomeroy et al., 2002
).
Interestingly, recent studies have demonstrated that some human
medulloblastomas (including desmoplastic tumors) express markers of neural
stem cells (Hemmati et al.,
2003
; Singh et al.,
2003
; Singh et al.,
2004
). This could mean that they are derived from neural stem
cells, or that they have acquired stem cell markers as a consequence of
transformation (Oliver and Wechsler-Reya,
2004
). Notably, few of the cells that we isolate from the
patched mutant mice express markers of neural stem cells (nestin,
CD133/prominin). Thus, tumorigenesis in the patched mutant mice does
not appear to involve acquisition of a neural stem cell phenotype. Whether
these cells exhibit other properties of neural stem cells such as
self-renewal or the capacity to differentiate into neurons and glia
remains to be determined.
Loss of patched and activation of the hedgehog pathway in pre-neoplastic cells
Our studies indicate that GCPs, pre-neoplastic cells and tumor cells
express elevated levels of hedgehog target genes and proliferate in culture.
In the case of GCPs, these responses probably reflect exposure to Shh in vivo
shortly before isolation (Wechsler-Reya
and Scott, 1999). In the case of pre-neoplastic cells and tumor
cells, de-repression of the hedgehog pathway could result from loss of
patched expression. Although some studies have suggested that
wild-type patched continues to be expressed in tumors
(Romer et al., 2004
;
Wetmore et al., 2000
;
Zurawel et al., 2000
), others
have suggested that the wild-type patched locus in tumor cells may be
silenced (Berman et al., 2002
).
Our studies support the latter view, demonstrating a striking loss of
wild-type patched expression at both the pre-neoplastic and tumor
stages.
One critical feature of our studies is the use of primers that distinguish
between wild-type and mutant patched transcripts. In most studies
that have reported continued patched expression in tumors from
patched mutant mice, the data are based on northern analysis or in
situ hybridization using probes that recognize both wild-type and mutant
alleles. Although the mutant allele is clearly expressed in tumors, it is
non-functional and would not be expected to contribute to the behavior of
tumor cells. Our studies are also distinct in that we have separated
pre-neoplastic and tumor cells from normal tissue. This is important because
several cell types in the normal adult cerebellum express patched
(Goodrich et al., 1997;
Traiffort et al., 1999
), and
these cells can contribute significantly to RNA isolated from intact tumor
tissue. By FACS-sorting cells that express Math1-GFP, we have eliminated these
contaminating populations and analyzed expression of patched
specifically in pre-neoplastic cells and tumor cells.
The mechanism by which patched is lost in pre-neoplastic cells and
tumor cells remains unclear. Although studies of medulloblastoma cell lines
derived from patched+/p53-/- mice have
demonstrated that the wild-type patched allele can be silenced by
methylation (Berman et al.,
2002), we have found no evidence for this in primary tumor cells
from patched+/ mice. Extensive sequencing of the
four major CpG islands within and upstream of the patched promoter
revealed no methylation in GCPs, pre-neoplastic cells or tumor cells (data not
shown). Although methylation (or some other chromatin modification) could be
present elsewhere in the patched gene, it is also possible that loss
of patched expression results from mutations or deletions in the
patched gene, or from loss of a signaling molecule or transcription
factor that regulates patched expression. Mutational analysis of the
patched locus, and studies of the transcription factors bound to the
promoter, may help resolve this issue.
Regardless of the mechanism, our observation of decreased wild-type patched expression in both pre-neoplastic cells and tumor cells indicates that loss of patched is an early (and perhaps initiating) event in tumorigenesis. The fact that only a subset of pre-neoplastic cells develop into tumors implies that other changes besides loss of patched are required for the transition from the pre-neoplastic stage to more malignant stages of medulloblastoma. Whether these changes result from mutations or epigenetic events within pre-neoplastic cells themselves, or whether they arise from changes in the surrounding microenvironment, is an important question for future study.
Identification of genes associated with medulloblastoma progression
GCPs, pre-neoplastic cells and tumor cells resemble one another in many
ways. And yet, GCPs are present in all of patched mutant mice (at the
neonatal stage), pre-neoplastic cells are present in over half of these
animals (at 6 weeks), and tumors occur in only 15-20%. Thus, these populations
must differ from one another. To identify intrinsic differences between GCPs,
pre-neoplastic cells and tumor cells, we analyzed gene expression using
microarrays. These studies revealed that the three populations are remarkably
similar when compared with normal adult cerebellum, but show significant
differences when compared directly to one another. Notably, because all three
populations exhibit hedgehog pathway activation and proliferation,
differentially expressed genes include few hedgehog targets or components of
the cell cycle machinery. Rather, major differences are detected in genes
associated with cell migration, survival and differentiation.
Our approach differs from several recent studies of gene expression
analysis in medulloblastoma (Boon et al.,
2003; Kho et al.,
2004
; Lee et al.,
2003
; MacDonald et al.,
2001
; Park et al.,
2003
; Pomeroy et al.,
2002
; Wechsler-Reya,
2003
). In most cases, these investigators compared medulloblastoma
with other brain tumors or with normal adult cerebellum. For example, Pomeroy
et al. found that medulloblastoma has a distinct gene expression profile
compared with other pediatric brain tumors
(Pomeroy et al., 2002
). In
particular, desmoplastic medulloblastomas (which often harbor mutations in the
hedgehog pathway) have elevated expression of hedgehog target genes such as
Nmyc, patched, gli1 and IGF2 (insulin-like growth factor 2).
Similarly, Boon et al. used serial analysis of gene expression (SAGE) to
compare human medulloblastoma and normal brain, and found increased expression
of hedgehog targets and cell cycle regulators such as Nmyc and thymidylate
synthase (Boon et al., 2003
;
Oliver et al., 2003
). These
findings are consistent with the fact that medulloblastoma cells are highly
proliferative cells that exhibit hedgehog pathway activation, whereas normal
brain consists primarily of post-mitotic neurons in which the hedgehog pathway
is inactive. In contrast to these studies, we compared medulloblastoma cells
with their cells of origin, proliferating GCPs. This allowed us to control for
proliferation and hedgehog pathway activation and, instead, to identify other
genes that may play an important role in tumor progression.
Similar to our studies, Lee et al. analyzed gene expression in
medulloblastoma and in neonatal cerebellum
(Lee et al., 2003). Both
studies found that gene expression in medulloblastoma was much more similar to
neonatal cerebellum than to adult cerebellum. Yet there were also key
differences between these studies. In particular, Lee et al. found that
medulloblastoma is characterized by increased expression of genes associated
with the granule cell lineage, hedgehog pathway activation and proliferation
(math1, srebp, hexokinase, Nmyc, cyclin D1, sfrp1), whereas we did
not detect elevated expression of these genes. One important distinction
between the two studies is the use of intact tissues versus dissociated cells.
Lee et al. compared intact tumor tissue (which consists largely of GCP-like
cells) and intact neonatal cerebellum (which contains not only GCPs but also
significant numbers of post-mitotic granule cells and other cell types). By
contrast, we compared purified populations of GCPs, pre-neoplastic cells and
tumor cells, all of which are highly enriched for proliferating GCP-like
cells. As a result, genes associated with cell lineage and degree of hedgehog
pathway activity were not differentially expressed in our screen.
Dysregulation of migration, differentiation and apoptosis in medulloblastoma
Our study is unique in that we compared gene expression in similar
populations of cells at three different stages of tumor progression. This
allowed us to identify genes that distinguish these stages. Interestingly,
many of these genes were regulators of migration, survival and
differentiation, processes that have been studied in the context of normal
granule cell development but were not previously known to play a role in
medulloblastoma. Most prominent among these genes were regulators of cell
migration. These included transcription factors (Pax6), surface receptors
(Unc5h3), secreted proteins (PAF acetylhydrolase) and ECM molecules (Collagen)
that have been implicated in granule cell migration
(Engelkamp et al., 1999;
Fishman and Hatten, 1993
;
Przyborski et al., 1998
;
Tokuoka et al., 2003
).
Notably, the majority of the genes we identified have been found to promote
migration, and were downregulated in pre-neoplastic cells. Decreased
expression of these genes during the early stages of tumorigenesis is
interesting because pre-neoplastic cells clearly exhibit aberrant migration:
whereas GCPs migrate inward during normal development, pre-neoplastic cells
(and tumor cells) remain stuck on the surface of the cerebellum. A similar
location has been noted for human medulloblastomas, which often spread through
the meninges but rarely invade the inner layers of the cerebellum
(Koeller and Rushing,
2003
).
It has long been unclear how failure to migrate inward is related to tumorigenesis. One possibility is that pre-neoplastic cells and tumor cells are unable to migrate because they are proliferating or locked in a precursor state. Alternatively, the inability to migrate could be an intrinsic defect that plays an essential role in tumorigenesis. For example, by losing the ability to migrate, cells may remain trapped in an environment (e.g. close to the pial surface) that facilitates their continued growth and survival. Our finding that genes involved in migration are downregulated in pre-neoplastic cells strongly supports the notion of an intrinsic defect. However, further experiments will be necessary to determine whether changes in migration-associated genes are necessary for the development of medulloblastoma.
Genes that regulate differentiation may also be crucial in determining the course of tumorigenesis. Importantly, we found that pre-neoplastic cells have reduced expression of genes associated with granule cell differentiation (e.g. Zic3 and Neurod1). This is consistent with the fact that these cells continue to proliferate and retain expression of GCP markers, whereas normal GCPs can exit the cell cycle and differentiate into mature granule neurons. Whether the inability to differentiate is a cause or a consequence of transformation remains to be determined, but the identification of specific transcription factors that may regulate this process will allow us to test the significance of differentiation directly. Further studies perturbing the expression of these factors in vivo are underway.
Finally, our data suggest that altered stress responses may contribute to
tumor formation in patched mutant mice. Heat-shock proteins,
co-chaperones (Hsp60, Hsp105, Dnajb1, Dnajb10 and Bag3) and the transcription
factor Atf3 all function to protect cells from stress-induced apoptosis and
necrosis (Hatayama et al.,
2001; Nakagomi et al.,
2003
; Takayama et al.,
2003
). The increased expression of these genes in pre-neoplastic
cells and tumor cells may reflect an increased ability to survive under stress
conditions, which may be crucial for the early stages of tumorigenesis. In
fact, several lines of evidence suggest that dysregulation of apoptosis or
stress responses may be important for medulloblastoma formation. For example,
crossing patched mutant mice with homozygous p53 knockout mice leads
to a dramatic increase in tumor incidence: whereas only 14-20% of
patched heterozygotes develop medulloblastoma, more than 95% of
patched+//p53-/- mice develop tumors
(Wetmore et al., 2001
).
Similarly, animals that lack both p53 and PARP1, an enzyme that promotes cell
death in response to DNA damage, develop medulloblastoma
(Tong et al., 2003
). Notably,
no mutations in p53 have been found in tumors from patched
heterozygotes (Wetmore et al.,
2001
), suggesting that in these animals some other element of the
apoptotic machinery may need to be inactivated for tumors to form. The genes
we have found to be upregulated in pre-neoplastic cells may provide some
important clues to the mechanisms by which apoptosis is subverted in
medulloblastoma.
The identification of a pre-neoplastic stage in murine medulloblastoma
raises the possibility that a similar stage exists in the human disease. If
so, the genes we have identified as markers of pre-neoplastic cells may be
useful for early detection of medulloblastoma, particularly in people with an
inherited susceptibility to the disease
(Gorlin, 1987;
Hamilton et al., 1995
).
Detection of medulloblastoma at early stages may increase the effectiveness of
conventional medulloblastoma therapy. In addition, if the genes we have
identified play a causal role in tumor progression, they may serve as valuable
targets for therapy. Recent studies suggest that pharmacologic antagonists of
the hedgehog pathway may be effective at inhibiting growth of medulloblastoma
cells in vitro and in vivo (Berman et al.,
2002
; Romer et al.,
2004
). Therefore, identification of other pathways that are
crucial for tumorigenesis may open up new avenues for treatment of this
devastating disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2425/DC1
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REFERENCES |
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---|
Adams, N. C., Tomoda, T., Cooper, M., Dietz, G. and Hatten, M. E. (2002). Mice that lack astrotactin have slowed neuronal migration. Development 129,965 -972.[Medline]
Ben-Arie, N., McCall, A. E., Berkman, S., Eichele, G., Bellen, H. J. and Zoghbi, H. Y. (1996). Evolutionary conservation of sequence and expression of the bHLH protein Atonal suggests a conserved role in neurogenesis. Hum. Mol. Genet. 5,1207 -1216.[CrossRef][Medline]
Benjamini, Y. and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57,289 -300.
Berman, D. M., Karhadkar, S. S., Hallahan, A. R., Pritchard, J.
I., Eberhart, C. G., Watkins, D. N., Chen, J. K., Cooper, M. K., Taipale, J.,
Olson, J. M. et al. (2002). Medulloblastoma growth inhibition
by hedgehog pathway blockade. Science
297,1559
-1561.
Bignami, A., Eng, L. F., Dahl, D. and Uyeda, C. T. (1972). Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43,429 -435.[CrossRef][Medline]
Boon, K., Edwards, J. B., Siu, I. M., Olschner, D., Eberhart, C. G., Marra, M. A., Strausberg, R. L. and Riggins, G. J. (2003). Comparison of medulloblastoma and normal neural transcriptomes identifies a restricted set of activated genes. Oncogene 22,7687 -7694.[CrossRef][Medline]
Borghesani, P. R., Peyrin, J. M., Klein, R., Rubin, J., Carter, A. R., Schwartz, P. M., Luster, A., Corfas, G. and Segal, R. A. (2002). BDNF stimulates migration of cerebellar granule cells. Development 129,1435 -1442.[Medline]
Buhren, J., Christoph, A. H., Buslei, R., Albrecht, S., Wiestler, O. D. and Pietsch, T. (2000). Expression of the neurotrophin receptor p75NTR in medulloblastomas is correlated with distinct histological and clinical features: evidence for a medulloblastoma subtype derived from the external granule cell layer. J. Neuropathol. Exp. Neurol. 59,229 -240.[Medline]
Corcoran, R. B. and Scott, M. P. (2001). A mouse model for medulloblastoma and basal cell nevus syndrome. J. Neurooncol. 53,307 -318.[CrossRef][Medline]
Dalgaard, P. (2002). Introductory statistics with R. New York: Springer Verlag.
Eisen, M. B., Spellman, P. T., Brown, P. O. and Botstein, D.
(1998). Cluster analysis and display of genome-wide expression
patterns. Proc. Natl. Acad. Sci. USA
95,14863
-14868.
Ellison, D. W., Clifford, S. C., Gajjar, A. and Gilbertson, R. J. (2003). What's new in neuro-oncology? Recent advances in medulloblastoma. Eur. J. Pediatr. Neurol. 7, 53-66.[CrossRef][Medline]
Engelkamp, D., Rashbass, P., Seawright, A. and van Heyningen,
V. (1999). Role of Pax6 in development of the cerebellar
system. Development 126,3585
-3596.
Faust, P. L. (2003). Abnormal cerebellar histogenesis in PEX2 Zellweger mice reflects multiple neuronal defects induced by peroxisome deficiency. J. Comp. Neurol. 461,394 -413.[CrossRef][Medline]
Fishman, R. B. and Hatten, M. E. (1993). Multiple receptor systems promote CNS neural migration. J. Neurosci. 13,3485 -3495.[Abstract]
Gentleman, R. and Carey, V. (2002). Bioconductor. R. News 2,11 -16.
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. P. (1996). Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev. 10,301 -312.[Abstract]
Goodrich, L. V., Milenkovic, L., Higgins, K. M. and Scott, M.
P. (1997). Altered neural cell fates and medulloblastoma in
mouse patched mutants. Science
277,1109
-1113.
Gorlin, R. J. (1987). Nevoid basal-cell carcinoma syndrome. Medicine 66, 98-113.[Medline]
Hahn, H., Wicking, C., Zaphiropoulous, P. G., Gailani, M. R., Shanley, S., Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A. B., Gillies, S. et al. (1996). Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85,841 -851.[CrossRef][Medline]
Hahn, H., Wojnowski, L., Specht, K., Kappler, R., Calzada-Wack,
J., Potter, D., Zimmer, A., Muller, U., Samson, E. and Quintanilla-Martinez,
L. (2000). Patched target Igf2 is indispensable for the
formation of medulloblastoma and rhabdomyosarcoma. J. Biol.
Chem. 275,28341
-28344.
Hamilton, S. R., Liu, B., Parsons, R. E., Papadopoulos, N., Jen,
J., Powell, S. M., Krush, A. J., Berk, T., Cohen, Z., Tetu, B. et al.
(1995). The molecular basis of Turcot's syndrome. New
Engl. J. Med. 332,839
-347.
Hatayama, T., Yamagishi, N., Minobe, E. and Sakai, K. (2001). Role of hsp105 in protection against stress-induced apoptosis in neuronal PC12 cells. Biochem. Biophys. Res. Commun. 288,528 -534.[CrossRef][Medline]
Helms, A. W. and Johnson, J. E. (1998).
Progenitors of dorsal commissural interneurons are defined by MATH1
expression. Development
125,919
-928.
Hemmati, H. D., Nakano, I., Lazareff, J. A., Masterman-Smith,
M., Geschwind, D. H., Bronner-Fraser, M. and Kornblum, H. I.
(2003). Cancerous stem cells can arise from pediatric brain
tumors. Proc. Natl. Acad. Sci. USA
100,15178
-15183.
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signaling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas, J. M., Quinn, A. G., Myers, R. M., Cox, D. R., Epstein, E. H., Jr et al. (1996). Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272,1668 -1671.[Abstract]
Kadin, M. E., Rubinstein, L. J. and Nelson, J. S. (1970). Neonatal cerebellar medulloblastoma originating from the fetal external granular layer. J. Neuropathol. Exp. Neurol. 29,583 -600.[Medline]
Kenney, A. M. and Rowitch, D. H. (2000). Sonic
hedgehog promotes G(1) cyclin expression and sustained cell cycle progression
in mammalian neuronal precursors. Mol. Cell. Biol.
20,9055
-9067.
Kenney, A. M., Cole, M. D. and Rowitch, D. H.
(2003). Nmyc upregulation by sonic hedgehog signaling promotes
proliferation in developing cerebellar granule neuron precursors.
Development 130,15
-28.
Kho, A. T., Zhao, Q., Cai, Z., Butte, A. J., Kim, J. Y.,
Pomeroy, S. L., Rowitch, D. H. and Kohane, I. S. (2004).
Conserved mechanisms across development and tumorigenesis revealed by a mouse
development perspective of human cancers. Genes Dev.
18,629
-440.
Kim, J. Y., Nelson, A. L., Algon, S. A., Graves, O., Sturla, L. M., Goumnerova, L. C., Rowitch, D. H., Segal, R. A. and Pomeroy, S. L. (2003). Medulloblastoma tumorigenesis diverges from cerebellar granule cell differentiation in patched heterozygous mice. Dev. Biol. 263,50 -66.[CrossRef][Medline]
Koeller, K. K. and Rushing, E. J. (2003). From
the archives of the AFIP: medulloblastoma: a comprehensive review with
radiologic-pathologic correlation. Radiographics
23,1613
-1637.
Lam, C. W., Xie, J., To, K. F., Ng, H. K., Lee, K. C., Yuen, N. W., Lim, P. L., Chan, L. Y., Tong, S. F. and McCormick, F. (1999). A frequent activated smoothened mutation in sporadic basal cell carcinomas. Oncogene 18,833 -836.[CrossRef][Medline]
Lee, Y., Miller, H. L., Jensen, P., Hernan, R., Connelly, M.,
Wetmore, C., Zindy, F., Roussel, M. F., Curran, T., Gilbertson, R. J. et
al. (2003). A molecular fingerprint for medulloblastoma.
Cancer Res. 63,5428
-5437.
Lumpkin, E. A., Collisson, T., Parab, P., Omer-Abdalla, A., Haeberle, H., Chen, P., Doetzlhofer, A., White, P., Groves, A., Segil, N. et al. (2003). Math1-driven GFP expression in the developing nervous system of transgenic mice. Gene Expr. Patterns 3, 389-395.[CrossRef][Medline]
MacDonald, T. J., Brown, K. M., LaFleur, B., Peterson, K., Lawlor, C., Chen, Y., Packer, R. J., Cogen, P. and Stephan, D. A. (2001). Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat. Genet. 29,143 -152.[CrossRef][Medline]
Messer, A. and Hatch, K. (1984). Persistence of cerebellar thymidine kinase in staggerer and hypothyroid mutants. J. Neurogenet. 1,239 -248.[Medline]
Miyata, H., Ikawa, E. and Ohama, E. (1998). Medulloblastoma in an adult suggestive of external granule cells as its origin: a histological and immunohistochemical study. Brain Pathol. 15,31 -35.
Nakagomi, S., Suzuki, Y., Namikawa, K., Kiryu-Seo, S. and
Kiyama, H. (2003). Expression of the activating transcription
factor 3 prevents c-Jun N-terminal kinase-induced neuronal death by promoting
heat shock protein 27 expression and Akt activation. J.
Neurosci. 23,5187
-5196.
Oliver, T. G. and Wechsler-Reya, R. J. (2004). Getting at the root and stem of brain tumors. Neuron 42,885 -888.[CrossRef][Medline]
Oliver, T. G., Grasfeder, L. L., Carroll, A. L., Kaiser, C.,
Gillingham, C. L., Lin, S. M., Wickramasinghe, R., Scott, M. P. and
Wechsler-Reya, R. J. (2003). Transcriptional profiling of the
Sonic hedgehog response: a critical role for N-myc in proliferation of
neuronal precursors. Proc. Natl. Acad. Sci. USA
100,7331
-7336.
Park, P. C., Taylor, M. D., Mainprize, T. G., Becker, L. E., Ho, M., Dura, W. T., Squire, J. and Rutka, J. T. (2003). Transcriptional profiling of medulloblastoma in children. J. Neurosurg. 99,534 -541.[Medline]
Pomeroy, S. L., Tamayo, P., Gaasenbeek, M., Sturla, L. M., Angelo, M., McLaughlin, M. E., Kim, J. Y., Goumnerova, L. C., Black, P. M., Lau, C. et al. (2002). Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 415,436 -442.[CrossRef][Medline]
Przyborski, S. A., Knowles, B. B. and Ackerman, S. L.
(1998). Embryonic phenotype of Unc5h3 mutant mice suggests
chemorepulsion during the formation of the rostral cerebellar boundary.
Development 125,41
-50.
Raffel, C., Jenkins, R. B., Frederick, L., Hebrink, D., Alderete, B., Fults, D. W. and James, C. D. (1997). Sporadic medulloblastomas contain PTCH mutations. Cancer Res. 57,842 -845.[Abstract]
Romer, J. T., Kimura, H., Magdaleno, S., Sasai, K., Fuller, C., Baines, H., Connelly, M., Stewart, C. F., Gould, S., Rubin, L. L. et al. (2004). Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/)p53(/) mice. Cancer Cell 6,229 -240.[CrossRef][Medline]
Sawamoto, K., Nakao, N., Kakishita, K., Ogawa, Y., Toyama, Y.,
Yamamoto, A., Yamaguchi, M., Mori, K., Goldman, S. A., Itakura, T. et al.
(2001). Generation of dopaminergic neurons in the adult brain
from mesencephalic precursor cells labeled with a nestin-GFP transgene.
J. Neurosci. 21,3895
-3903.
Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins,
C., Squire, J. and Dirks, P. B. (2003). Identification of a
cancer stem cell in human brain tumors. Cancer Res.
63,5821
-5828.
Singh, S. K., Clarke, I. D., Hide, T. and Dirks, P. B. (2004). Cancer stem cells in nervous system tumors. Oncogene 23,7267 -7273.[CrossRef][Medline]
Sommer, I. and Schachner, M. (1981). Monoclonal antibodies (O1 to O4) to oligodendrocyte surfaces: An immunocytological study in the central nervous system. Dev. Biol. 83,311 -327.[CrossRef][Medline]
Takayama, S., Reed, J. C. and Homma, S. (2003). Heat-shock proteins as regulators of apoptosis. Oncogene 22,9041 -9047.[CrossRef][Medline]
Taylor, M. D., Liu, L., Raffel, C., Hui, C. C., Mainprize, T. G., Zhang, X., Agatep, R., Chiappa, S., Gao, L., Lowrance, A. et al. (2002). Mutations in SUFU predispose to medulloblastoma. Nat. Genet. 31,306 -310.[CrossRef][Medline]
Tokuoka, S. M., Ishii, S., Kawamura, N., Satoh, M., Shimada, A., Sasaki, S., Hirotsune, S., Wynshaw-Boris, A. and Shimizu, T. (2003). Involvement of platelet-activating factor and LIS1 in neuronal migration. Eur. J. Neurosci. 18,563 -570.[CrossRef][Medline]
Tong, W. M., Ohgaki, H., Huang, H., Granier, C., Kleihues, P.
and Wang, Z. Q. (2003). Null mutation of DNA strand
break-binding molecule poly(ADP-ribose) polymerase causes medulloblastomas in
p53(/) mice. Am. J. Pathol.
162,343
-352.
Traiffort, E., Charytoniuk, D., Watroba, L., Faure, H., Sales, N. and Ruat, M. (1999). Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system. Eur. J. Neurosci. 11,3199 -3214.[CrossRef][Medline]
Vaillant, C., Meissirel, C., Mutin, M., Belin, M. F., Lund, L. R. and Thomasset, N. (2003). MMP-9 deficiency affects axonal outgrowth, migration, and apoptosis in the developing cerebellum. Mol. Cell. Neurosci. 24,395 -408.[CrossRef][Medline]
Wechsler-Reya, R. J. (2003). Analysis of Gene
Expression in the Normal and Malignant Cerebellum. Recent Prog.
Horm. Res. 58,249
-261.
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22,103 -114.[CrossRef][Medline]
Wechsler-Reya, R. and Scott, M. P. (2001). The developmental biology of brain tumors. Annu. Rev. Neurosci. 24,385 -428.[CrossRef][Medline]
Weigmann, A., Corbeil, D., Hellwig, A. and Huttner, W. B.
(1997). Prominin, a novel microvilli-specific polytopic membrane
protein of the apical surface of epithelial cells, is targeted to plasmalemmal
protrusions of non-epithelial cells. Proc. Natl. Acad. Sci.
USA 94,12425
-12430.
Wetmore, C., Eberhart, D. E. and Curran, T.
(2000). The normal patched allele is expressed in
medulloblastomas from mice with heterozygous germ-line mutation of patched.
Cancer Res. 60,2239
-2246.
Wetmore, C., Eberhart, D. E. and Curran, T.
(2001). Loss of p53 but not ARF accelerates medulloblastoma in
mice heterozygous for patched. Cancer Res.
61,513
-516.
Yokota, N., Aruga, J., Takai, S., Yamada, K., Hamazaki, M., Iwase, T., Sugimura, H. and Mikoshiba, K. (1996). Predominant expression of human zic in cerebellar granule cell lineage and medulloblastoma. Cancer Res. 56,377 -383.[Abstract]
Zakhary, R., Keles, G. E., Aldape, K. and Berger, M. S. (2001). Medulloblastoma and primitive neuroectodermal tumors. In Brain Tumors: An Encyclopedic Approach (ed. A. H. Kaye and E. R. Law), pp. 605-615. London: Churchill Livingstone.
Zurawel, R. H., Allen, C., Wechsler-Reya, R., Scott, M. P. and Raffel, C. (2000). Evidence that haploinsufficiency of Ptch leads to medulloblastoma in mice. Genes Chromosomes Cancer 28,77 -81.[CrossRef][Medline]