From the Laboratory of Mammalian Molecular Genetics, The Panum Institute 6.5, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
Received for publication, November 1, 2002
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ABSTRACT |
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Malignant astrocytomas are common human primary
brain tumors that result from neoplastic transformation of astroglia or
their progenitors. Here we show that deregulation of the c-Myc pathway in developing astroglia predisposes mice to malignant astrocytomas within 2-3 weeks of age. The genetically engineered murine (GEM) gliomas harbor a molecular signature resembling that of human primary
glioblastoma multiforme, including up-regulation of epidermal growth
factor receptor and Mdm2. The GEM gliomas seem to originate in an
abnormal population of glial fibrillary acidic
protein-expressing cells in the ventricular zone and, analogous
to human glioblastomas, exhibit molecular and morphological
heterogeneity. Levels of connexin 43 in the majority of the tumors are
unaltered from normal tissue, indicating that GEM tumors have retained
the capacity to establish syncytial networks. In line with this,
individual glioma foci are composed of a mixture of actively
proliferating cells expressing c-Myc and proliferating cell nuclear
antigen and less dividing bystander cells that express glial fibrillary
acidic protein and the broad complex tramtrack bric-a-brac/poxvirus and
zinc finger domain protein HOF. A subset of the transgenic mice
harbored, in addition to brain tumors, vestigial cerebellums in which
granule cell migration and radial Bergman glial cell differentiation
were disturbed. These observations argue for a window of vulnerability during astrocyte development where c-Myc overexpression is sufficient to trigger the neoplastic process, presumably by inducing the sustained
growth of early astroglial cells. This is in contrast to most other
transgenic studies in which c-Myc overexpression requires co-operating
transgenes for rapid tumor induction.
Of the tumors that originate in the human brain, astrocyte-derived
gliomas are the most common. They range from slow growing pilocytic
astrocytomas to high grade variants such as anaplastic astrocytomas and
glioblastoma multiforme (1, 2). Astrocytomas are clinically troubling
because they diffusely infiltrate the brain, impeding complete surgical
resection of neoplastic cells, and because lower grade tumors often
progress to higher grade variants. Because the latter respond poorly to
radiotherapies and chemotherapies, malignant astrocytomas are currently
untreatable. Glioblastoma multiforme rank among the most malignant
brain lesions, with a median survival of less than 12 months.
High grade gliomas can be classified into primary and secondary
glioblastomas (3). Primary glioblastomas appear mainly de
novo and are often associated with deregulation of epidermal growth factor receptor
(EGFR)1 expression. Secondary
glioblastomas, on the other hand, develop from the progression of lower
grade tumors that often carry mutations in the p53 gene. In addition to
EGFR and p53 mutations, a number of genetic alterations affecting
interphasing biochemical pathways have been correlated with glioma
pathogenesis. This involves gain-of-function and loss-of-function
mutations in genes regulating p53-mediated apoptosis
(p16ARF and Mdm2), cell cycle arrest (pRb, cdk4, and
p16Ink4a), cell proliferation, and survival (Ras,
mitogen-activated protein kinase, phosphatidylinositol 3-kinase,
and Akt) (4). c-Myc is a basic helix-loop-helix leucine zipper
transcription factor that triggers cell proliferation and apoptosis,
functions that seem to be effectuated mainly through activation of the
cdk4/pRb/E2F pathway (5). c-Myc functions as a sequence-specific
DNA-binding protein in association with its partner Max to facilitate
expression of E-box-containing target genes (6). Moreover, c-Myc has
recently been assigned roles in chromatin remodeling of target
promoters, a process that involves recruitment via the N-terminal
transactivation domain of co-activator complexes including histone
acetylases, ATPase/helicase activities, as well as an
ATP-dependent chromatin remodeling complex (7, 8). In line
with its involvement in a wide range of neoplasms, there is
circumstantial evidence that suggests c-Myc also plays a role in
gliomagenesis, including deregulation of the c-Myc pathway in human
glioblastoma multiforme (9, 10). Although the molecular mechanism(s)
underlying this expression is poorly understood, it may be mediated
by various genetic changes associated with glioma progression
including: increased platelet-derived growth factor receptor
signaling, which is commonly observed during early stages of glioma
pathogenesis (2) and which can trigger deregulated c-Myc expression
through Src tyrosine kinase signaling (11-13), amplification and
rearrangement of the c-Myc locus (14), and inactivation of the c-Myc
pathway antagonist Mxi1 (15, 16). In addition, genetic lesions
affecting the Ras/phosphatidylinositol 3-kinase/Akt pathway, which are
often present in glioblastoma multiforme tumors, can prolong the
half-life of c-Myc and hence contribute to c-Myc accumulation in high
grade gliomas (17).
In this study, we test the hypothesis that overexpression of human
c-Myc 2 in astroglia triggers gliomagenesis. The c-Myc locus consists
of three exons that encode various alternatively translated c-Myc
isoforms, designated c-Myc 1, c-Myc 2, and c-Myc S (18, 19).
Translation of c-Myc 2 and S are initiated from AUG codons in exon II,
whereas c-Myc 1 is initiated from a non-AUG (CUG) codon in exon I. Although both c-Myc 2 and S promote cell proliferation and apoptosis
and have been associated with cellular transformation (19, 20), c-Myc 1 seems to play a separate role in growth arrest control (18). Here we
prepared transgenic mice with a human c-Myc 2- and S-encoding minigene
under transcriptional control of an astrocyte-specific promoter. We
report that transgenic mice overexpressing c-Myc 2 and S proteins
develop early onset genetically engineered murine (GEM) malignant
gliomas that show features of human primary malignant gliomas.
Transgene Construction, Generation, and Screening of
Mice--
To direct transgenic expression of c-Myc 2 and c-Myc S in
astroglia, the myelin basic protein promoter of the myelin basic protein/c-Myc plasmid (21) was removed by digestion with
HindIII and BamHI and replaced with a PCR product
encompassing 2.2 kb of the human GFAP gene transcriptional regulatory
sequences (22). This places the genomic sequence containing exons II
and III of the human c-Myc gene under control of the GFAP promoter. The
transgene fragment was excised from the plasmid backbone by
EcoRI digestion, gel-purified, and microinjected at a
concentration of 6 ng/µl into fertilized mouse eggs as previously
described (23). Transgenic mice were identified by PCR analysis of DNA
samples prepared from tail biopsies using the following PCR program:
one cycle at 96 °C for 5 min; 35 cycles at 96 °C for 30 s,
64 °C for 30 s, and 75 °C for 30 s; and one cycle at
75 °C for 8 min. The primer sets used were:
5'-TACAAGCATGAGCCACCCCAC-3' (GFAP promoter primer, sense) and
5'-GGAGAATCGGACACATCCTCG-3' (human c-Myc primer, antisense) or the
human c-Myc specific primers 5'-CGATTCCTTCTAACAGAAATGTCCTGAG-3' and
5'-AGATTTGGCTCAATGATATATTTGCCAG-3'.
Immunohistochemistry--
In some cases, the mice were given an
intraperitoneal injection of 0.1 mg of BrdUrd/g of mouse weight
(diluted in physiological saline) 3 h before the mice were
sacrificed. The brains and optic nerves were removed from transgenic
and control mice and immediately frozen in liquid N2.
Tissue sections (20 µm) were cut with a cryostat microtome, collected
on gelatinized glass slides, fixed with methanol for 5 min, and either
stained with hematoxylin or subjected to immunohistochemistry. The
fixed sections were preincubated 15 min in phosphate-buffered saline
containing 2% bovine serum albumin to reduce background, and
immunohistochemistry was carried out as previously described using
phosphate-buffered saline with 2% bovine serum albumin as the antibody
diluent (24). The primary antibodies used at 1:100 dilution are mouse
CNPase (Neomarkers), mouse NeuN (Chemicon), mouse GFAP (Chemicon),
mouse VEGF (Santa Cruz), mouse PCNA (Sigma), rabbit GFAP (Sigma),
rabbit c-Myc (Santa Cruz), rabbit cleaved caspase-3 (Cell Signaling
Technology), and rabbit HOF antisera (25). For staining with sheep
bromodeoxyuridine antisera (1:200 dilution; Research Diagnostics), the
fixed sections were first treated for 30 s each with 2 M HCl and 0.1 M borate. Primary antibodies were
detected with tetramethylrhodamine B isothiocyanate-conjugated swine
anti-rabbit and fluorescein isothiocyanate-conjugated goat anti-mouse
(DAKO) and donkey anti-sheep (Research Diagnostics Inc.) at 1:100
dilution. TUNEL labeling was carried out using the in situ
cell death detection kit (Roche Applied Science).
Protein Extraction and Western Analysis--
Frozen regions of
mouse brains were homogenized on ice in 100 µl of RIPA buffer
containing 450 mM NaCl, 1 mM dithiothreitol, and 0.2% v/v protease inhibitor mixture (Sigma). After 30 min on ice,
the extracts were spun at 14,000 × g for 10 min at
4 °C, and the supernatants were recovered. The protein concentration was measured (Bio-Rad), and 20 µg of each extract was loaded on a
10-20% Tris-glycine gel (BioWhittaker Molecular Applications). Western blots were analyzed using the anti-rabbit or anti-mouse Western
Breeze kit (Invitrogen). The primary antibodies were rabbit EGFR (Santa
Cruz), rabbit Mdm2 (Santa Cruz), rabbit aquaporin-4 (Chemicon), and
rabbit c-Myc (Santa Cruz) (diluted 1:500); mouse VEGF (Santa Cruz),
rabbit E2F1 (Santa Cruz), and rabbit Connexin 43 (Chemicon) (diluted
1:2000); and mouse actin (Sigma), mouse PCNA (Sigma), and rabbit HOF
antisera (25) (diluted 1:5000).
Approximately half of a total of 14 transgenic mice, established
by pronuclear microinjection of a c-Myc 2/S recombinant minigene, developed neurological disabilities (Fig.
1, A and B, and
Table I). The animals were killed at the
time they presented with terminal disease, which in most cases occurred
before 3 weeks of age (Table I). Affected animals harbored markedly
enlarged cerebrums, enlargement of the lateral ventricles, as well as
increased cellularity in the cerebral hemispheres (Fig. 1,
C-H, K, O, and P). The
increment in cellularity involved the neocortex, striatum, and
subventricular zone of all of the affected animals (Table I). The high
cellularity of the tumors as well as the presence of small and large
centers of cell death with or without pseudo-palisading are reminiscent of glioblastoma multiforme (Fig. 1, H, J, and
P). A subset of animals harbored a highly scattered and
widespread tumor cell infiltration in the cerebral cortex without cell
death centers (Fig. 1K). This pattern resembles that of the
extensively infiltrating human malignant glial cell neoplasm
gliomatosis cerebri, which lacks the necrotic centers
characteristic of glioblastoma multiforme. A large circumscribed tumor
was disclosed in one mouse that occupied the dorso-ventral axis of the
diencephalon, i.e. from the thalamus to the optic nerves
(Fig. 1, I and M). This tumor exhibited a high
degree of cellularity, necrotic centers, and microvessels but showed
low to moderate spreading of tumor cells outside the diencephalon. A
subset of animals harbored, in addition to cerebral glioma, a large
intracranial hemorrhage that compressed and atrophied the surrounding
structure (data not shown). Two founder mice, designated Tg-7 and Tg-8,
gave rise to transgenic progeny that became disoriented and ataxic
during the second and third postnatal weeks (Fig. 1N and
Table I). The mice harbored markedly enlarged cerebrums that showed
profiles of diffuse infiltrating malignant glioma lesions with
pseudo-palisading and central cell death centers (Fig. 1, O
and P). The cerebellums of the mice were rudimentary, lacking deep fissures and folia (Fig. 1Q). In line with the
specificity of the GFAP promoter, other organs appeared normal in
affected GFAP/c-Myc transgenic mice.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Establishment of GFAP/c-Myc transgenic mice
and GEM glioma pathology. A, intron/exon structure of
the human c-Myc gene. Exons are shown as open boxes. The
positions of the alternative translational start codons in exons I and
II and the common stop codon in exon III are indicated. B,
structure of the GFAP/c-Myc 2/S minigene. The 2.2-kb transcriptional
regulatory sequence of the human GFAP gene (shaded box) is
positioned upstream of the human c-Myc genomic locus containing exons
II and III. The transcriptional start site is indicated by an
arrow. C-K and M, gross and
histopathological appearance of representative GEM gliomas from
affected F0 transgenic mice, demonstrating the range of
abnormalities observed: enlarged cerebrum (C-E); bulging
out of the temporal cortex (D and E,
asterisks); enlargement of the lateral ventricle
(E and F); diffuse and widespread areas of
increased cell proliferation (F and G,
asterisks); center of cell death with pseudo-palisading
(H, arrow); a well circumscribed tumor localized
to the diencephalon (I, asterisk); areas of
necrosis observed in a large tumor (J, arrow);
scattered GEM glioma cell infiltration in the cerebrum without cell
death centers (K); and optic nerve glioma (M,
arrow). L, optic nerve from a normal mouse
(arrow). N-Q, brain histopathology in the Tg-7
F1 transgenic mice, showing ataxia and balance difficulties
(N); enlargement of the cerebrum and atrophy of the
cerebellum compared with a normal brain (O); infiltrating
areas of increased cellularity in the cerebrum together with
pseudo-palisading (P); and the presence of a vestigial
cerebellum (Q). LV, lateral ventricle;
Cer, cerebellum.
Malignant gliomas in GFAP/c-Myc transgenic mice
Transgenic animals harbored a spectrum of abnormal brain cells ranging
from an increased cellular density around the lateral ventricles to
diffuse infiltration of neoplastic cells in the cerebral cortex. To
reveal the cellular origin of these cell populations, we investigated
whether or not they expressed the astrocytic intermediate filament
protein GFAP. As shown in Fig. 2
(A-F), the abnormal cell populations in both the neocortex
and periventricular zone express GFAP, indicating that they originate
in cells of the astroglial lineage. In line with a glial cell origin,
there was no expression of the neuronal differentiation marker NeuN in
these cells (Fig. 2G). There were numerous hypertrophic
GFAP-positive astrocytes located in the vicinity of tumor foci, and
processes from some of these cells appeared to be in direct physical
contact with tumor cells (Fig. 2H). Whether these cells
represent normal reactive astroglia that develop in response to brain
pathology or they constitute a subpopulation of diffusely proliferating
neoplastic cells is not clear at the present. Moreover, GFAP-expressing
cells also infiltrated the white matter of affected brains in
association with a severe hypomyelination (Fig. 2,
I-L).
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A characteristic feature of human malignant gliomas is that they are
often composed of a mixture of proliferating, dying, and metabolically
stressed cells that express angiogenic factors. To determine whether or
not the GEM gliomas show some of these characteristics, we first
investigated the pattern of BrdUrd labeling in brains of affected
transgenic mice following administration of BrdUrd 3 h before
killing. This short time frame ensures that only actively dividing cell
populations are labeled. Using BrdUrd labeling and immunohistochemical
localization, numerous BrdUrd-positive cells were revealed in both
diffuse and circumscribed glioma lesions (Fig.
3, A-D). On the contrary,
only a few positive nuclei were disclosed in age-matched nontransgenic
control animals, and they appeared mainly in regions of postnatal
neurogenesis such as the subgranular zone of the dentate gyrus (data
not shown). In line with a high tumor grade, there was also
morphological evidence of regressive processes in the GEM gliomas,
including circumscribed and small centers of cell death
(Fig. 1). Thus, to reveal whether apoptosis plays a role in the
continued loss of cells in the tumors, we used molecular markers of
programmed cell death including TUNEL labeling and staining with an
anti-cleaved caspase 3 antibody. Numerous TUNEL-positive nuclei were
scattered around large necrotic centers in the GEM gliomas (Fig. 3,
E and F). As mentioned above, some GEM gliomas
showed a widespread diffuse infiltration of the cerebral cortex in the
absence of cell death centers. These gliomas, however, were
TUNEL-positive and harbored cleaved caspase 3-expressing apoptotic
cells scattered within tumor foci (Fig. 3, G-J). Expression of the vascular endothelial cell growth factor (VEGF) is up-regulated in human malignant glioma cells located at the periphery of necrotic centers (26, 27). In line with this, VEGF expression was also up-regulated in GEM glioma cells surrounding cell death areas (Figs. 3,
K and L, and
4).
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We next investigated the pattern of c-Myc expression in various GEM
gliomas. As shown in Fig. 4, expression of the c-Myc transgene was
revealed in tumor biopsies from c-Myc 2/S transgenic mice but not in
brain biopsies from control brains. We consistently detected two c-Myc
specific bands of ~64 and 52 kDa in Western blots. The 64-kDa band is
consistent with the molecular mass of c-Myc 2, and the 52-kDa band is
consistent with that of c-Myc S. The c-Myc S isoform is 100 amino acids
shorter than c-Myc 2, corresponding to a difference of about 11 kDa.
Notably, the c-Myc S isoform seems to predominate in a well
circumscribed glioma, whereas a more equal distribution of the two
c-Myc isoforms is seen in diffuse infiltrating tumors (Fig. 4). PCNA is
a component of DNA polymerase and is widely used as a nuclear
marker of continuously dividing tumor cell populations (28). As
expected for actively dividing malignant cells, there was a strong
correlation between the pattern of PCNA expression and that of c-Myc in
the tumors (Fig. 4). Because expression of a number of signaling
molecules is enforced in human malignant gliomas, we next determined
whether this was also the case in the early onset GEM gliomas.
Overexpression and/or amplification of EGFR occur in many (~40% of
cases) human primary glioblastomas but less often in lower grade
gliomas and secondary glioblastomas (3). Hence, EGFR overexpression is characteristic of de novo malignant lesions and presumably
responsible for activation of the Ras pathway in these tumors, because
activating p21 Ras mutations are uncommon in gliomas (29). As shown in Fig. 4, we found EGFR overexpression in tumor biopsies from various GEM
gliomas. Moreover, the p53-negative regulator Mdm2 is also overexpressed in many human primary glioblastomas (3). Mdm2 has been
noted to bind to p53 to both inhibit transcription and to target p53
for degradation resulting in an increased resistance of tumor cells to
programmed cell death (30). In line with a primary malignancy, a subset
of GEM gliomas showed a marked increase in Mdm2 expression (Fig. 4).
Thus, the c-Myc-expressing GEM gliomas seem to harbor a molecular
signature resembling that of human primary glioblastomas. Cerebral
edema occurs in association with most malignant brain tumors including
gliomas (31). Affected GFAP/c-Myc transgenic mice showed features of
brain edema, including swelling of the brain, demyelination of white
matter tracts, as well as reactive glioses (Figs. 1, D and
O, and 2, H-J). Hence, we investigated the
pattern of expression of the astroglial water channel protein
aquaporin-4 in GEM gliomas because this protein has been claimed to be
up-regulated in human edematous malignant gliomas (32). We found a
moderate increase in aquaporin-4 expression in diffuse infiltrating GEM
tumors (Fig. 4). In contrast, there was no discernable change in
expression of the cell proliferation-associated transcription factor
E2F1 in the tumors (Fig. 4).
We next investigated the pattern of connexin 43 (Cx 43) expression in
different GEM gliomas, because this astroglial gap junction protein has
been noted to play important roles in glioma cell invasion as well as
in the establishment of astroglial tumor syncytia (33, 34). Moreover, a
variable expression of Cx 43 has been disclosed in human glioblastoma
multiforme tumors (33, 35-37). In line with a role of the protein in
astroglioma cell spreading and invasion, there was a marked lack of
Cx 43 expression in a well circumscribed GEM glioma as
opposed to diffuse infiltrating lesions (Fig. 4). The fact that the
latter tumors express the astroglial gap junction protein Cx 43 (Fig.
4) indicates that they have the capacity to establish syncytial tumor
networks between GEM glioma cells and astroglia (33, 34). Consistent
with the presence of tumor syncytia, only a subpopulation of cells
within individual GEM tumor foci seems to express the c-Myc transgene (Fig. 5, A-D). Moreover,
these c-Myc-expressing cells appear to constitute an actively dividing
subpopulation because they co-express the PCNA antigen (Fig. 5,
E-H). Thus, diffuse infiltrating GEM tumors seem to
comprise both actively dividing true neoplastic cells as well as a less
proliferating bystander cell population.
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We next investigated the possibility that the less dividing cell
population of the GEM tumors express the novel astroglial marker HOF.
The HOF gene encodes a nuclear broad complex tramtrack bric-a-brac/poxvirus and zinc finger domain factor expressed in both
developing and mature astrocytes as well as in reactive astroglia during brain pathology (25). As shown in Fig.
6 (A-C), levels of HOF
expression are up-regulated in some but not all GEM gliomas. Using
BrdUrd labeling and immunohistochemical localization, we were unable to
detect either BrdUrd and HOF (Fig. 6, D-G) or
PCNA and HOF (Fig. 6, H and I) double-labeled
cells in diffuse GEM lesions. Thus, HOF is unlikely to be expressed in
actively dividing GEM tumor cells. Moreover, HOF is
expressed in cells with both large and small sized nuclei (Fig.
6I). Co-immunolocalization experiments show that HOF
expression in GEM gliomas is often confined to cells that show robust
expression of the astroglial differentiation marker GFAP (Fig. 6,
J and K). Taken together, these data suggest that
diffuse infiltrating GEM tumors, like human malignant gliomas, are a
mixed cell population comprising tumorous and less malignant reactive
elements.
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As shown above, affected F1 or F2 transgenic
mice of the Tg-7 and Tg-8 strains harbor sausage-shaped cerebellar
rudiments (Fig. 1, O and Q). The deficiency in
these animals seems to affect the developmental transformation of
vestigial cerebellums into a mature structure with distinct internal
granule cell and molecular layers (Fig.
7, A, E,
I, M, and Q). During normal cerebellar
development, granule neuron precursors in the external granule cell
layer migrate inward through the molecular layer along the processes of
radial Bergman glia to settle in the internal granule cell layer (38). Because radial Bergmann glia are thought to be essential for granule cell migration as well as morphological transformation of the cerebellum during development (39), disruption of the differentiated functions of these cells may in part explain the cerebellar phenotype in the animals. Moreover, the GFAP promoter has been noted to be active
in radial Bergman glia in some transgenic mouse models (40, 41). In
normal cerebellums, the GFAP-positive radial processes of Bergman glia
extend from a subpial position across the molecular layer to terminate
in the Purkinje cell layer at the border of the internal granule cell
layer (Fig. 7, B-D). In line with a proposed defect in
Bergman glia development, the rudimentary cerebellums show a pronounced
lack of radial glia in the subpial region (Fig. 7, F,
N, and R). Because mature cerebellar granule cells, as opposed to immature cells, express the neuronal
differentiation marker NeuN (25), we performed NeuN
immunohistochemistry to investigate whether differentiated granule
neurons were present in the vestigial cerebellums in the absence of a
distinct internal granule cell layer. Consistent with a Bergman
glia-mediated migration failure of granule cells, numerous
NeuN-positive granule cells were scattered randomly in subpial
locations (Fig. 7, G, H, O, P, S, and T). On the contrary, only
few NeuN-positive cells were located in cell-dense regions at the
center of vestigial cerebellums (Fig. 7, I and K)
where GFAP-expressing glial cells prevail (Fig. 7, J
and L).
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DISCUSSION |
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Our results show that deregulation of the c-Myc pathway in astroglia predisposes mice to early onset gliomas. Consistent with an early astroglial origin of tumor cells is the observation that an abnormal population of GFAP-expressing cells were manifest following the onset of gliogenesis in the postnatal subventricular zone of the lateral ventricles (42). The early astroglial onset of the GEM gliomas may be explained by a window of vulnerability during astrocyte development where deregulation of the c-Myc pathway effectively sustains cell proliferation. Perhaps the continued presence of active c-Myc/Max complexes in developing astroglia prevents induction of terminal differentiation by opposing (e.g. Mad/Max) complexes of the Myc/Mad/Max network (6, 43). In the absence of counter-balanced apoptosis, the increase in cellularity that results from sustained Myc/Max-mediated proliferation of astroglial precursors may be instrumental in the short latency of GEM gliomas in this model. Deregulation of the c-Myc pathway has also been noted to trigger genomic instability (44-47). This instability coupled with an increased cellularity may accelerate the process of neoplastic progression of c-Myc-overexpressing astroglia, resulting in induction of co-operating oncogenes, including those involved in cell survival and proliferation. Furthermore, it has been suggested that human primary glioblastomas originate in primitive glial precursor cells as opposed to secondary glioblastomas that may arise from dedifferentiation of low grade gliomas (4). In addition to early astroglial origin, the molecular signature of the GEM tumors resembles that of human primary glioblastoma multiforme, including EGFR and Mdm2 up-regulation.
In contrast to this study our previous efforts to establish a transgenic mouse model for oligodendrocytic gliomas using the same human c-Myc transgene but under control of a myelin gene promoter (myelin basic protein/c-Myc) were less successful (21, 24). Although some of the transgenic mice harbored abnormal cellular profiles in the striatum and neocortex in association with BrdUrd positive cells, there is no evidence of an increased cellularity and/or GEM gliomas in these mice (21).2 On the contrary, affected myelin basic protein/c-Myc mice developed a lethal programmed cell death disorder characterized by a widespread and scattered oligodendrocyte loss and pronounced hypomyelination of white matter tracts. Deregulation of the c-Myc pathway in developing oligodendroglia seems to be compliant with the ability of these cells to differentiate but not with the cell cycle arrest required for myelin formation. Although the foci of diffuse GEM gliomas in GFAP/c-Myc mice harbor numerous apoptotic cells, it remains to be determined whether this is primarily a result of aberrant cell cycle activity in differentiating astroglia and/or a consequence of metabolic stress.
The 150-amino acid N-terminal transactivation domain of human c-Myc 2 comprises two conserved Myc homology boxes, designated MBI (amino acids 45-65) and MBII (amino acids 128-144). The MBI box plays a role in stabilization of c-Myc 2 through phosphorylations of Thr58 and Ser62 (5). The MBII subregion interacts with TRRAP, which in turn functions as a co-activator that recruits histone acetylase activities and hence links c-Myc to nucleosome remodeling processes (8, 43, 48). c-Myc S proteins arise from a leaky ribosome scanning of the c-Myc 2 transcript, resulting in translation initiation at two closely spaced internal methionines (Met101 and Met110) located upstream of the MBII subregion (19). We found that the majority of GEM tumors expressed comparable levels of c-Myc 2 and c-Myc S, with the exception of a highly circumscribed malignant tumor that harbored a severalfold increase in c-Myc S. The synthesis of comparable levels of the two c-Myc isoforms has been noted in some hematopoietic tumor cell lines (19). Although c-Myc S has retained many of the biological activities of c-Myc 2, including E-box binding via heterodimerization with Max, triggering of cell proliferation, and apoptosis (19, 20), it remains to be determined whether the N-terminal transactivation domain-truncated c-Myc S isoforms have oncogenic potential in vivo.
Astroglia function as syncytial networks in which numerous cells are connected by gap junctional pores and where small secondary messengers such as inositol 1,4,5-trisphosphate, cAMP, ATP, and ions are shared by the cytoplasm of groups of cells (49). Moreover, astroglial syncytia also establish gap junctional communication with neurons and oligodendroglia and play pivotal roles in supplying the former with essential energy metabolites as well as in glutamate recycling at synaptic terminals (50). The main gap junctional protein of astroglia, Cx 43, is expressed in many human malignant gliomas (33, 35-37). The establishment of Cx 43-mediated syncytial tumor networks with astroglia has been noted to be important for glioma cell invasion in vivo (34). Consistent with this finding, we observed a pronounced lack of Cx 43 protein in a well circumscribed GEM glioma that exhibited little tumor cell spreading. On the contrary, Cx 43 protein was present in biopsies from diffuse infiltrating GEM lesions. The survival of metabolically active glioma cells depends on an efficient supply of energy metabolites. Assuming that GEM gliomas establish functional gap junctions with bystander astroglia, this could serve as an important route for energy metabolite supply in diffuse infiltrating tumor cells in the absence of adequate vascularization. Furthermore, the foci of diffuse infiltrating GEM tumor cells appear morphologically similar, although they comprise cells with distinct molecular signatures. Our results show that the diffuse GEM lesions are a mixed cell population comprising actively proliferating elements that co-express c-Myc and PCNA and a subpopulation of less dividing bystander cells that co-express HOF and GFAP. A complicated problem in neuro-oncology is the difficulty of distinguishing true astrocytic tumor cells from reactive glia. Furthermore, although reactive astroglia rarely divide, GFAP is not a reliable marker for nondividing cells because actively dividing astroglia have been noted to occasionally express the intermediate filament protein (51). The finding that HOF is a marker of cells that are not actively dividing in diffuse infiltrating GEM lesions suggests that the protein in association with GFAP could serve as a marker of nontumorous bystander cells in human malignant gliomas. However, whether HOF is a definitive marker for nondividing reactive elements in diffuse infiltrating gliomas including those in humans is under investigation. Our preliminary results indicate that the pattern of HOF expression in human grade IV malignant gliomas resembles that disclosed in GEM gliomas.3
In addition to GEM gliomas, a subset of the transgenic mice also developed a deficiency affecting morphogenesis of the cerebellum. The deficiency correlates with a pronounced lack of radial Bergman glia in the molecular layer, resulting in impaired granule cell migration. Hence, we surmise that sustained c-Myc expression in Bergmann glia blocks the differentiation of these cells into an ordered array of radial rails traversing the molecular layer. In the absence of this glial rail, migrating granule neurons born in the external granular cell layer settle and differentiate prematurely into NeuN-expressing granule neurons in the presumptive molecular layer. In addition, some affected transgenic mice seem to harbor a migration deficiency of pyramidal neurons in the archicortex in which the morphology of Ammon's horn of the hippocampus is abnormal, consisting of a wide band of noncompacted pyramidal cells (data not shown). However, the exact mechanisms of these phenotypes as well as the role played by the Myc pathway remain to be determined.
In summary, we have shown that developing astroglia are highly
vulnerable to deregulation of the c-Myc pathway leading to formation of
early onset GEM tumors that show features of human primary malignant
glioblastoma multiforme. Based on these findings, we surmise that
sustained c-Myc activity in primitive glial cell precursors is
instrumental in the development of at least a subset of human primary
malignant gliomas, although this remains to be determined. An
interesting feature of this model is the molecular heterogeneity of the
GEM tumors comprising both an actively dividing cell population and a
less dividing bystander population that expresses the novel astroglial
marker HOF in association with GFAP. Furthermore, the ability to obtain
affected transgenic offspring through heterozygous crosses of the Tg-8
line greatly facilitates ongoing studies on the biology of these tumors.
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ACKNOWLEDGEMENTS |
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We thank Heidi Nielsen for expert technical assistance and Michael Brenner for plasmid DNA.
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FOOTNOTES |
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* This work was supported by grants from the Danish Cancer Society, the Lundbeck Foundation, Løvens Kemiske Fabriks Foundation, the Michelsen Foundation, and the NOVO-Nordisk Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
45-35327722; Fax: 45-35327701; E-mail: naj@imbg.ku.dk.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M211195200
2 F. Lihme and N. A. Jensen, unpublished data.
3 L. Rask and N. A. Jensen, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor receptor; GEM, genetically engineered murine; GFAP, glial fibrillary acidic protein; BrdUrd, bromodeoxyuridine; CNPase, 2',3'-cyclic nucleotide 3'-phosphodiesterase; VEGF, vascular endothelial cell growth factor; PCNA, proliferating cell nuclear antigen; TUNEL, TdT-mediated dUTP nick end labeling; DAPI, 4,6-diamidino-2-phenylindole; Cx 43, connexin 43.
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