Oncogene (2004) 23, 1146–1152
& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00
www.nature.com/onc
Notch and Schwann cell transformation
Yiwen Li1,7, Prakash K Rao2,6,7, Rong Wen3, Ying Song3, David Muir4, Peggy Wallace4,5,
Samantha J van Horne2, Gihan I Tennekoon1 and Tom Kadesch*,2
1
Department of Neurology and Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA;
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA; 3Department of
Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA; 4Pediatrics (Neurology Division),
University of Florida College of Medicine, Gainesville, FL 32610, USA; 5Department of Molecular Genetics and Microbiology,
University of Florida College of Medicine, Gainesville, FL 32610, USA
2
Benign plexiform neurofibromas in NF1 patients can
transform spontaneously into malignant peripheral nerve
sheath tumors (MPNSTs). Although mutations in the p53
gene have been found in a subset of MPNSTs and mouse
models support a role for p53 mutations in malignant
conversion, we found that each of three Schwann cell lines
derived from human MPNSTs possessed active p53. One
of the lines expressed the Notch intracellular domain
(NICD), indicative of ongoing Notch signaling. Consistent
with a role in malignancy, NICD was able to transform
primary rat Schwann cells. Transformation was robust –
NICD-transduced cells generated tumors in nude rats –
and was associated with the loss of markers associated
with Schwann cell differentiation. These data suggest that
aberrant Notch signaling may contribute to the conversion
of benign neurofibromas to MPNSTs.
Oncogene (2004) 23, 1146–1152. doi:10.1038/sj.onc.1207068
Keywords: notch; NICD; Schwann; neurofibromatosis;
MPNST; p53
Introduction
Neurofibromatosis type 1 (NF1), also referred to as von
Recklinhausen syndrome, is a common cancer predisposition syndrome caused by loss of the NF1 gene
(Gutmann, 2001; Zhu and Parada, 2002). NF1 is
characterized by the formation of benign neurofibromas, composed primarily of Schwann cells, and plexiform neurofibromas, which can sporadically transform
into malignant peripheral nerve sheath tumors
(MPNSTs; also known as neurofibrosarcomas). NF1
patients also have an increased brain size due to
increased numbers of astrocytes and an increased risk
for developing optic pathway gliomas, pheochromocytomas and myeloid leukemias. The NF1 gene encodes
*Correspondence: T Kadesch, Department of Genetics, University of
Pennsylvania School of Medicine, Philadelphia, PA 19104, USA;
E-mail: [email protected]
6
Current address: Whitehead Institute for Biomedical Research,
Cambridge, MA 02142, USA
7
The first two authors contributed equally to the work.
Received 7 May 2003; revised 28 July 2003; accepted 31 July 2003
neurofibromin, a tumor suppressor that possesses
GTPase-activating protein (GAP) activity. Accordingly,
NF1 loss results in elevated levels of activated Ras. The
conversion of benign neurofibromas to malignant cancer
is not fully understood, but is thought to require one or
more additional genetic events such as the acquisition of
mutations in the p53 and/or Ink4a genes (Menon et al.,
1990; Cichowski et al., 1999; Kourea et al., 1999; Vogel
et al., 1999; Birindelli et al., 2001).
The Notch proteins comprise a family of transmembrane receptors that regulate cell-fate decisions during
metazoan development (Artavanis-Tsakonas et al.,
1999). Mammals express four Notch receptors and at
least six membrane-associated ligands. Notch signaling
within a cell usually requires one or more of the
receptors and expression of a ligand by an adjacent
cell. Notch proteins are found on the cell surface as
proteolyzed heterodimers, and binding of ligand renders
them susceptible to two additional proteolytic cleavages,
culminating in the release of the respective Notch
intracellular domains, NICDs, from the plasma membrane. The released NICDs enter the nucleus and
activate transcription through interactions with Mastermind and the DNA-binding protein CSL (Wu et al.,
2000; Fryer et al., 2002; Jeffries et al., 2002; Wallberg
et al., 2002).
Notch signaling regulates a variety of binary cell fate
choices, including the choice of lymphoid progenitors to
become either B cells or T cells (Allman et al., 2002) and
neural crest-derived progenitors to become either
neurons or glia (Wang and Barres, 2000). Notch
signaling is incompatible with the development of B
cells and neurons, while promoting the development of
T cells and certain types of glia, including Schwann cells
(Morrison et al., 2000), astrocytes (Tanigaki et al.,
2001), radial glia (Gaiano et al., 2000) and Müller glia
(Furukawa et al., 2000). Interestingly, Notch inhibits the
differentiation of oligodendrocytes, a glial cell type
found within the CNS (Wang et al., 1998). In patients
with multiple sclerosis, the inflammatory cytokine TGFb induces expression of the Notch ligand Jagged1 in
actrocytes residing within sclerotic lesions. This is
thought to activate Notch signaling in neighboring
oligodendrocytes and thus block their ability to mature
and to myelinate axons (John et al., 2002).
Transformation of Schwann cells by Notch
Y Li et al
1147
Although Notch signaling typically serves to regulate
cell fate choice, unbridled signaling can also lead to
cancer. The latter outcome is highly cell type specific and
generally correlates with constitutive, ligand-independent expression of NICD. Examples of Notch-induced
neoplasias include T-cell lymphomas (human (Ellisen
et al., 1991) and mouse (Girard et al., 1996; Pear et al.,
1996)), pancreatic cancer (mouse and human (Miyamoto et al., 2003)), mammary epithelial cell tumors (mouse
(Jhappan et al., 1992)), and the transformation – in
collaboration with the Adenovirus E1a protein – of
cultured rat embryonic epithelial cells (Capobianco et al.,
1997). Notch signaling may also be required for Rasmediated transformation of human cells (Weijzen et al.,
2002). While downstream components of the Notch
signaling cascade that govern cell fate choice have been
well described – such as the manner in which Notch
inhibits fly neurogenesis – virtually nothing is known
about the pathways that promote neoplastic transformation. Notch signaling is not universally mitogenic,
and so transformation may be linked to other activities,
such as the inhibition of apoptosis, as has been
described for T lymphocytes (Deftos et al., 1998; Jehn
et al., 1999), or the neutralization of growth-inhibitory
pathways, such as that induced by TGF-b (Rao and
Kadesch, in press). Conceivably, Notch may also
transform cells by stalling differentiation at a point
coincidentally associated with cell proliferation.
We looked for evidence of Notch signaling in
Schwann cell lines derived from human MPNSTs. One
of the three cell lines expressed cleaved Notch (NICD)
and an elevated level of the Notch target Hes-1,
indicating the presence of an active Notch signaling
cascade. In support of an oncogenic role for Notch in
human Schwann cells, we found that forced expression
of NICD induced malignant transformation of rat
Schwann cells.
Results
Notch signaling in Schwann cells derived from a human
MPNST
The conversion of benign plexiform neurofibromas to
malignant peripheral nerve sheath tumors (MPNSTs) in
NF1 patients is likely to involve one or more additional
genetic events. Although mutations in the p53 and Ink4a
genes have been described, these mutations are not
universally present in NF1-derived MPNSTs (Menon
et al., 1990; Kourea et al., 1999; Birindelli et al., 2001).
Also, while mouse models support a strong collaborative relationship between NF1 and p53 mutations,
functional studies of the relevant pathways have not
been carried out in human cells. We therefore examined
the p53 pathway in four Schwann cell lines, one derived
from a benign neurofibroma (NF) (Rutkowski et al.,
2000) and three from MPNSTs (sNF94.3, sNF02.2 and
sNF96.2) (Muir et al., 2001). Cells were treated with
increasing doses of the DNA-damaging agent adriamycin, which induces the expression of p53 and its target
Figure 1 Induction of p53 and p21 in human Schwann cell lines
derived from a benign NF and MPNSTs (sNF94.3, sNF96.2 and
sNF02.2). Cells were treated with the indicated concentrations of
adriamycin for 12 h and extracts were subjected to Western
immunoblot analysis with antibodies directed against the indicated
proteins
p21 in wild-type cells. We found that adriamycin
induced expression of p53 in a dose-dependent manner
in all the four human cell lines (Figure 1, top panel). p21
was also induced in all the four cell lines, but more
efficiently at the lower doses of adriamycin (Figure 1,
lower panel). The lower levels of p21 observed when the
human cells were treated with higher doses of adriamycin were unexpected, but could possibly be due to DNA
damage-induced apoptosis. Although the basal level of
p21 was relatively high in the sNF96.2 line, it was clearly
induced relative to Cdk4. We conclude that the p53
pathway, most likely including the sequestration of
MDM2 by p19ARF, is largely intact in these Schwann cell
lines.
Notch has been implicated in Ras-mediated transformation of human cells (Weijzen et al., 2002). Given that
elevated Ras is elevated as a consequence of mutations
in neurofibromin and therefore almost certainly required in MPNSTs, we examined the Schwann cell lines
for evidence of Notch signaling. A reliable method for
assessing whether or not a cell is undergoing Notch
signaling is to determine if the cells express endogenous
cleaved Notch (NICD). When cell extracts were
analysed by Western immunoblotting using an antibody
specific for endogenous NICD (see Materials and
methods), a positive signal was observed for the
sNF96.2 MPNST Schwann cells, and no signal was
detected for the other cell lines (Figure 2, upper panel).
Given that this antibody recognizes the Notch intracellular domain only after it is cleaved at Val1744, a
positive signal indicates appropriate cleavage by gsecretase and the generation of transcriptionally active
NICD. Consistent with this observation, RT–PCR
analyses showed increased expression of the Notch
target gene Hes-1 in the sNF96.2 cells relative to the
others (Figure 2, center panel). Another Notch target,
Hes-5, was not detected in any of the human cell lines
(data not shown). The high basal level of p21 in the
sNF96.2 cells may also be a consequence of Notch
signaling, since the p21 promoter has been shown to be a
direct Notch target in keratinocytes (Rangarajan et al.,
2001b). The presence or absence of endogenous NICD
did not vary with cell density, suggesting that Notch
activity in these cells did not result from ligand-mediated
activation. We conclude that the sNF96.2 Schwann cell
line was derived from an MPNST undergoing active
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Transformation of Schwann cells by Notch
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Figure 3 NICD-transduced Schwann cells acquire an altered
morphology. The images depict (a) NICD-transduced cells and (b)
MigR1-transduced cells. The regions of high cell density in the
NICD-transduced cells were positive for GFP (not shown)
Figure 2 Analysis of Notch signaling in human Schwann cell lines
derived from a benign NF and MPNSTs. Upper panel: Western
blot for endogenous NICD, Notch and Mre-11 in extracts from the
depicted cell lines. The approximate per cent confluency at the time
of cell harvest is indicated. Lower panel: RNAs for Hes-1 and
HPRT were determined by RT–PCR
Notch signaling, raising the possibility that Notch
contributed to its conversion to malignancy. The
generation of NICD generally requires a g-secretasedependent cleavage event, even if NICD is generated
constitutively (Weng et al., 2003). In support for a
causal role for NICD in the transformed nature of
sNF96.2 cells, treatment with a g-secretase inhibitor
significantly impaired cell proliferation (data not
shown). Whether or not Notch activation is a common
occurrence in MPNSTs will require the generation and
evaluation of additional Schwann cell lines.
NICD induces morphological changes in rat Schwann
cells associated with the extinction of differentiation
markers
To investigate the direct effect of Notch signaling in
mature Schwann cells, we transduced cultured rat cells
with a retrovirus that encodes the intracellular domain
of human Notch1 (NICD) (Ross and Kadesch, 2001) or
a control virus (MigR1) (Pear et al., 1998). Both viruses
express GFP, which was used as a marker for infected
cells. Transient transfections with a Notch-responsive
reporter confirmed the activity of the transduced NICD
(data not shown). Significant morphological changes
were observed in virtually 100% of the NICD-transduced (i.e. GFP-positive) cells. Unlike the parental or
MigR1-transduced cells, which grew as elongated,
spindle-shaped monolayers, the NICD-transduced cells
formed foci of small, rounded cells (a typical example is
shown in Figure 3).
We next examined the NICD-transduced cells for
changes in gene expression. When viral transduction
efficiencies were high, we were able to analyse a highly
enriched population without purification. As judged by
Oncogene
Figure 4 NICD-transduced Schwann cells express low levels of
differentiation markers. (a) MigR1- or NICD-transduced cell
extracts were analysed by Western for Notch and the potential
Notch-responsive genes HES-1, HES-5 and ErbB2 (left panel), and
for the Schwann cell markers P0, P75 and GFAP (right panel). (b)
MigR1- or NICD-transduced cells were analysed by RT–PCR for
the Schwann cell markers P0, Sox-10 and ErbB3
Western blotting, the NICD-transduced cells expressed
NICD (primers were designed to detect human Notch)
and higher levels of Hes-5, a known Notch/CSL target
gene (Figure 4a) (de la Pompa et al., 1997; Beatus et al.,
1999). The protein corresponding to another Notch
target, Hes-1 (Jarriault et al., 1995), was detected in the
parental and MigR1-transduced cells, and was not
significantly increased in the presence of NICD. In this
regard, the rat cells differ from the human cells which
express NICD and Hes-1, but not Hes-5. We speculate
that transcription factors other than NICD influence
expression of the Hes genes. Interestingly, the NICDtransduced cells lost expression of several Schwann cellspecific markers. Western immunoblot analysis showed
that the level of the myelin protein P0 was dramatically
reduced in the presence of NICD, as were p75 NGF
receptor and GFAP (Figure 4b). RT–PCR analyses also
showed a decreased level of P0 mRNA as well as
message corresponding to ErbB3. RNA corresponding
to Sox-10, a glia-promoting transcription factor that
promotes the expression of the myelin P0 and ErbB3
Transformation of Schwann cells by Notch
Y Li et al
1149
genes (Stolt et al., 2002), was also reduced in NICDtransduced cells (Figure 4c). We considered two
explanations for these results: either NICD caused the
Schwann cells to actively de-differentiate, or NICD
expression is stable only in a subpopulation of
undifferentiated Schwann cells present in the original
pool of cells. We favor the former explanation, since we
do not observe an appreciable fraction of cells with the
morphology of NICD-transduced cells in the absence of
NICD retroviral transduction.
NICD transforms rat Schwann cells
The morphology of the NICD-transduced Schwann cells
was reminiscent of transformed cells that are not contact
inhibited. Indeed, NICD-transduced Schwann cells, but
not parental or MigR1-transduced cells, were able to
form colonies in soft agar (data not shown). An analysis
of specific cell cycle markers (Figure 5a) showed
dramatically elevated levels of the Cyclins A, D1 and
D2 in the NICD-transduced cells. Cdk2 levels were
slightly higher, while the level of Cdk4 was unchanged.
Levels of the Cdk inhibitory proteins p15INK4b and p27
were not affected by NICD. The level of phosphorylated
ERK, diagnostic of increased Ras-MAPK signaling,
was also elevated in the NICD-transduced cells. Finally,
induction of p53 and its target p21 by adriamycin was
unaffected by NICD (Figure 5b). Overall, these data
indicate that NICD has appreciable effects on the
Figure 6 Tumors generated by NICD-transduced Schwann cells
in nude rats. Animals were injected subcutaneously with NICDtransduced Schwann cells. Tumors developed at each of the
injection sites and reached 2–2.5/cm a month after injection (not
shown). (a, b) Pathological sections revealed dense transformed
Schwann cells in the tumors with numerous blood vessels.
Hematoxylin and eosin stain. Original magnification: 100 (a)
and 400 (b)
Figure 5 NICD-transduced Schwann cells express altered levels of
cell cycle-regulatory proteins. Extracts from (a) MigR1- or NICDtransduced rat Schwann cells or (b) MigR1- or NICD-transduced
rat Schwann cells treated with the indicated concentrations of
adriamycin for 12 h were subjected to Western immunoblot
analyses using antibodies directed against the indicated proteins
critical components of the cell cycle in Schwann cells,
consistent with cellular transformation.
To determine if the aforementioned changes involved
true oncogenic transformation, we assessed the ability of
NICD-transduced cells to generate tumors in rats.
Roughly one million cells, representing pools of either
NICD-transduced or MigR1-transduced cells, were
injected subcutaneously at three spots on the nape of
nude rats. Tumors developed in three of three rats
injected with NICD-transduced Schwann cells (at all
three spots), but not in rats injected with control
Schwann cells (data not shown). The tumors reached a
diameter of 0.5–1 cm after 2 weeks, and 2–2.5 cm after 1
month. Fluorescence microscopy confirmed that the
tumor cells were all GFP positive (data not shown). The
tumors were highly vascular and the cells had a small,
round morphology, reminiscent of a primitive neuroectodermal tumor (Figures 6a and b). These results
indicate that expression of NICD in Schwann cells can
induce transformation and generate cells capable of
growing as tumor masses.
Oncogene
Transformation of Schwann cells by Notch
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1150
Discussion
In humans, activated Notch has been implicated as a
causal agent in T-cell lymphoma (Notch1; Ellisen et al.,
1991) and pancreatic cancer (Miyamoto et al., 2003).
Notch has also been shown to induce or to accelerate the
development of T cell (Girard et al., 1996; Pear et al.,
1996) and mammary epithelial neoplasias in mice
(Jhappan et al., 1992). In most cases, normal regulation
of Notch is disrupted – through chromosomal translocation, provirus insertion or viral transduction – leading
to constitutive expression of NICD. Despite these
examples, Notch’s ability to act as an oncogene is
highly cell type specific and poorly understood. Retroviral transduction of hematopoietic precursors in mice
results only in T-cell tumors; B-cell development is
blocked and other cell types are relatively unaffected
(Pui et al., 1999). Somewhat conflicting results indicate
that Notch proteins cooperate with Papilloma virus
genes to transform epithelial cells (Rangarajan et al.,
2001a), but that Notch1, specifically, must be downregulated for sustained expression of the viral oncogenes
(Talora et al., 2002). The specific deletion of Notch1 in
mouse skin results in an increased frequency of tumors,
implicating Notch1 as a tumor suppressor in that
context (Nicolas et al., 2003). Similarly, abrogating
Notch activity in NIH 3T3 cells promotes FGFdependent cellular transformation (Small et al., 2003).
However, since FGF acts as a mitogen for rat Schwann
cells (Davis and Stroobant, 1990), it is unlikely that
Notch acts as a general antagonist of FGF signaling.
While certain aspects of Notch’s oncogenic effects can
be mimicked in cultured cells, the mechanisms underlying these effects, including their cell type specificity,
are largely unknown. Salivary gland tumors carrying the
t(11;19)9q21;p13) translocation express a novel fusion
protein containing the Notch coactivator MAML2
(Tonon et al., 2003). Although this strongly implicates
the Notch pathway in salivary gland tumorigenesis, the
MECT1–MAML2 fusion protein activates gene expression independently of CSL, thereby suggesting the
existence of a noncanonical signaling pathway for these
Notch-induced tumors.
Here we show that Notch can transform primary
Schwann cells in vitro. The only other example of
Notch-induced transformation of cultured cells involves
rat embryo kidney (RKE) cells, but that requires
cooperation with the adenovirus E1A gene (Capobianco
et al., 1997). The reasons underlying the ability of Notch
to function alone to transform Schwann cells are not
known, but may be related in part to the observed
increase in cyclin expression, a result that was also
reported in the RKE system (Ronchini and Capobianco,
2001). Despite this apparent disruption in cell cycle
control, Notch does not appear to be acting as a
mitogen per se. Our transformed cells do not grow more
rapidly than the parental cells, and they will overtake a
dish containing untransduced cells only if the cells are
allowed to grow beyond confluency, conditions that
favor cells whose growth is not subject to contact
inhibition (SJv, unpublished observations). A somewhat
Oncogene
related alteration in growth characteristics has been
described for Notch4-transduced mammary epithelial
cells, which acquire an invasive phenotype when grown
on collagen gels (Soriano et al., 2000). This particular
alteration has been proposed to be important for
Notch4’s ability to induce mammary tumors in mice.
Transformation by NICD is associated with the loss
of Schwann cell differentiation markers. Perhaps, the
most significant of these is Sox10, a transcription factor
expressed in neural crest cells and necessary for the
terminal stages of glial cell development (Britsch et al.,
2001; Stolt et al., 2002). However, this raises a paradox.
How can Notch signaling be required in vertebrate
gliogenesis, as has been described for multiple glial cell
types including Schwann cells, yet repress expression of
a gene necessary for Schwann cell development? This
cannot be attributed to the high-level expression of
activated Notch in our experiments – similar retroviral
vectors promote the formation of both Müller glia and
radial glia in the CNS (Furukawa et al., 2000; Gaiano
et al., 2000). It is more likely that the precise target cell
determines the ultimate consequences of Notch activity,
and we have targeted NICD to differentiated Schwann
cells, as opposed to undifferentiated precursors. Moreover, Notch signaling inhibits the formation of oligodendrocytes, indicating that the effect of Notch on
gliogenesis is not universal (Wang et al., 1998). Notch
signaling has also been correlated with the inability of
oligodendrocytes to mature and myelinate nerves within
the CNS of patients with multiple sclerosis (John et al.,
2002). Our studies with Schwann cells raise the
possibility that inappropriate Notch signaling within
the CNS may also initiate demyelination by causing
oligodendrocytes to dedifferentiate.
The ability of NICD to transform Schwann cells is
interesting, and by itself provides a useful in vitro model
system to study Notch-mediated oncogenesis. However,
we have also provided evidence that activated Notch is
induced in a human NF1 MPNST. Although the present
sample size is small due to the difficulty in purifying and
culturing Schwann cells from MPNSTs, the identification of a cell line that expresses activated Notch suggests
that at least one case of malignant transformation is
associated with constitutive Notch signaling. Such
signaling could be due to a chromosomal translocation
involving one of the four Notch genes (leading to ligandindependent NICD expression; Ellisen et al., 1991) or to
high-level expression of a Notch ligand that induces
signaling in neighboring cells (despite the presence of
NICD in subconfluent cultures). It will be very
important to learn the frequency with which Notch is
activated in MPNST and, depending on the underlying
cause, if Notch inhibition might provide an effective
treatment.
Materials and methods
Schwann cell lines
Rat Schwann cells were isolated from the sciatic nerves of 2day-old rat pups using the method described by Brockes et al.
Transformation of Schwann cells by Notch
Y Li et al
1151
specificity of this antibody is such that it does not recognize the
Notch intracellular domain unless it has been appropriately
cleaved. Antibodies against b-tubulin and p53 were from
Sigma (St Louis, MO, USA) and Novacastra Laboratories,
Ltd (New Castle, UK), respectively. Antibodies against Cyclin
D1, Cyclin D2, CDK2, CDK4, p21, p27, Cdk4, Jagged-1,
Notch1, GFAP and Erb B2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against MRE11
was from Novus Biologicals (Littleton, CO, USA). Rabbit
anti-P0 antibody was provided by Dr Bruce Trapp (Cleveland
Clinic, OH, USA). Rabbit anti HES-1 and HES-5 antibodies
were from Chemicon International (Temecula, CA, USA).
Rabbit antibody against p75 was obtained from Promega
(Madison, WI, USA).
(1979). The cells were maintained in DMEM supplemented
with 10% heat-inactivated fetal bovine serum. Rat Schwann
cells were expanded using 2 mM forskolin and 25 ng/ml
recombinant glial growth factor. At 1 week prior to the
analyses of cells, both forskolin and glial growth factor were
removed. Passage-matched Schwann cells were infected with
the parental, GFP-expressing MigR1 retrovirus (Pear et al.,
1998), or the NICD- and GFP-expressing retrovirus (Ross and
Kadesch, 2001). When needed, transduced cells were purified
by FACS using GFP as a marker.
The human cell lines sNF94.3, sNF96.2 and sNF02.2 were
established using methods described previously (Muir et al.,
2001). All specimens included in this study were obtained in
accordance with the protocols approved by the University of
Florida IRB. Originative tumors were characterized as
malignant peripheral nerve sheath tumors (neurofibrosarcoma) by histopathology, and were obtained from patients
meeting the NF1 diagnostic criteria. Tumor pieces were
minced and dissociated for 3–5 h with dispase (1.25 U/ml;
Collaborative Research) and collagenase (300 U/ml; type XI,
Sigma) in L15 medium containing 10% calf serum and
antibiotics. The digested tissue was dispersed by trituration
and strained through a 30-mesh nylon screen. Collected cells
were seeded on laminin-coated dishes and grown in DMEM
containing 10% fetal bovine serum, 5% calf serum, glial
growth factor-2 (25 ng/ml) and antibiotics. Cultures were
subsequently grown and rapidly expanded without laminin
and glial growth factor-2. The three immortal cell lines had
spindle-shaped morphology and were immunopositive for S100 and faintly for p75 (low-affinity nerve growth factor
receptor), indicating Schwann cell lineage. They were subsequently grown in DMEM containing 10% heat-inactivated
fetal bovine serum.
Total RNA (2 mg) was used to obtain cDNA by reverse
transcription using RETROscript first-strand synthesis kit
(Ambion, Inc., Austin, TX, USA), and then subjected to PCR
using 0.5 U/ml Amplitaq (Applied Biosytems, Foster City, CA,
USA), as per the manufacturer’s instructions, using the
following conditions: 90 s at 941C, 45 s at 941C, 45 s at 591C,
40 s at 721C (30 cycles) and 60 s at 721C. Primer pairs were as
follows: HES-1: 50 -CAGCCAGTGTCAACACGACAC-30
and
50 -TCGTTCATGCACTCGCTGAG-30 ;
P0:
50 GCCCTGCTCTTCTCTTCTTT-30
and
50 -CCAACAC
CACCCCATACCTA-30 ; Sox-10: 50 -GAGGAGGTGGGCG
and
50 -AGCTCTGTCTTTGGGG
TTGGGCTCTTC-30
0
0
TGGTTGGA-3 ; ErbB3: 5 -GCGTTGCCAGTTGTCCCCA
TAA-30
and
50 -AGCGTCTCATAGCCCTTTTGTT-30 ;
0
HPRT: 5 -GTTGGATACAGGCCAGACTTTGTTG-30 and
50 -TGGGGACGCAGCAACTGACATTTCT-30 .
Western immunoblotting
Tumorigenicity assays
Cells were washed and then lysed in RIPA buffer (50 mM Tris
pH 8.0, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS,
1 mM PNPP, 20 nM calyculin A, 1 mM Na Vanadate, 1 mM
phenylmethylsulfonylfluoride, 1 mg/ml leupeptin, 10 mg/ml
aprotinin). Lysates were collected, clarified by centrifugation
for 15 min at 41C, resolved by PAGE and transferred to
nitrocellulose membranes. Typically, the membranes were then
incubated with the primary antibody at 41C overnight,
followed by horseradish peroxidase-conjugated secondary
antibody (1 : 10 000 dilution, Amersham Pharmacia Biotech,
Cleveland, OH, USA) at room temperature for 1 h. Bands
were visualized by an enhanced chemiluminescence (ECL)
detection system (Amersham Pharmacia Biotech) or LumilightPlus (Roche, Indianapolis, IN, USA). Adriamycin was from
Bedford Laboratories (Bedford, OH, USA). Rabbit antibody
against cleaved Notch1/NICD (val1744) was obtained from
Cell Signaling Technology (Beverly, MA, USA). Note that the
Tumorigenicity assays were performed by injecting 106 cells (in
100 ml PBS) subcutaneously into nude rats. The cells were
injected at three sites on the nape of 8–9-week-old female
Athymic nude rats. The rats were monitored twice weekly and
killed after 30 days (when a tumor of 2–2.5 cm diameter was
apparent). Tumor tissue was fixed in PBS containing 4%
paraformaldehyde and embedded in paraffin. Tissue sections
were stained with hematoxylin/eosin for the examination of
tumor structure by light microscopy.
RT–PCR
Acknowledgements
The first two authors, YL and PKR, contributed equally. We
would like to thank Dr Shara Kabak for ERK blot (Figure 5).
This work was supported by grants RO1 GM58228 (to TK),
R01 EY12727 (to RW), R01 NS21700 (to GT) from the
National Institutes of Health and RG2780 (to GT) from the
National Multiple Sclerosis Society.
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