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J. Biol. Chem., Vol. 277, Issue 16, 14153-14158, April 19, 2002
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From the Lombardi Cancer Center, Department of Oncology, Georgetown
University, Washington, D. C. 20007
Received for publication, December 25, 2001
Glioblastoma multiforme is the most
common highly aggressive human brain cancer, and receptor tyrosine
kinases have been implicated in the progression of this malignancy. We
have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine
kinase receptor for pleiotrophin, a secreted growth factor that is
highly expressed during embryonic brain development and in tumors of
the central nervous system. Here we report on the contribution of
pleiotrophin-ALK signaling to glioblastoma growth. We found ALK
overexpressed in human glioblastoma relative to normal brain and
detected ALK mRNA in glioblastoma cell lines. We reduced the
endogenous ALK in glioblastoma cells by ribozyme targeting and
demonstrated that this prevents pleiotrophin-stimulated phosphorylation
of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK
reduced tumor growth of xenografts in athymic nude mice and prolonged
survival of the animals because of increased apoptosis in the tumors.
These findings directly implicate ALK signaling as a rate-limiting
factor in the growth of glioblastoma multiforme and suggest
potential utility of therapeutic targeting of ALK.
Tumors of glial origin including astrocytomas,
oligodendrogliomas, and ependymomas account for almost 80% of all
primary brain malignancies. Glioblastoma multiforme is both the single
most common glial tumor and the most lethal with a mean survival of only 1 year despite aggressive treatment (1). Although these tumors
exhibit multiple genetic alterations, including loss or mutation of the
tumor suppressors PTEN (2), p53 (3), and INK4a-ARF (4), receptor
tyrosine kinase (RTK)1
signaling seems to play a particularly important role in tumor development and growth. Glioblastomas and glioblastoma cell lines have
been shown to overexpress the tyrosine kinase receptors for epidermal
growth factor (5), platelet-derived growth factor (PDGF) (6),
hepatocyte growth factor (7), nerve growth factor (8), and vascular
endothelial growth factor (9). In addition, these tumors frequently
overexpress the ligands for these RTKs, suggesting a potential role for
autocrine RTK signaling in glioblastoma growth. The importance of RTK
signaling is supported by the finding that the combined activation of
two downstream targets of RTK signaling (Ras and Akt) in neural
progenitor cells induces glioblastoma-like tumors in mice (10).
Pleiotrophin is a secreted heparin-binding growth factor highly
expressed in the developing nervous system and down-regulated in the
adult. Relative to normal brain, pleiotrophin expression is increased
following acute ischemic injury (11) and in tumors (12). This suggests
a potential role of pleiotrophin as a tumor growth factor insofar as
the reactivation of a developmentally regulated signaling pathway may
provide a tumor with a powerful growth signal. In fact, pleiotrophin
expression induces tumor growth and metastasis of NIH3T3 cells (13) and
has a rate-limiting role both as an angiogenic factor (14) and a tumor
growth factor for different tumors including melanoma and
choriocarcinoma (15-17) (reviewed in Ref. 12). In addition,
pleiotrophin activates both the Ras-MAPK and the PI3K-Akt signaling
axes (18), and both pathways are implicated in glial tumorigenesis
(10). Recently we identified ALK, an orphan RTK, as the receptor for
pleiotrophin (19). The ALK tyrosine kinase was originally discovered as
a fusion protein with nucleophosmin due to a t(2;5) translocation (20).
This fusion resulted in constitutive activation of the intracellular
ALK kinase and was shown to induce anaplastic lymphoma (20). More
recently, the full-length ALK receptor has been shown to be highly
expressed in the developing nervous system and down-regulated postnatally (21), very similarly to the expression profile of its
ligand, pleiotrophin (19). Here we report that this tyrosine kinase
receptor is overexpressed in human glioblastoma and is rate-limiting
for the growth of a xenograft model of glioblastoma.
Cell Culture--
Cell lines were obtained from the American
Type Culture Collection (ATCC, Manassas, VA) and grown in improved
minimum essential medium (Invitrogen) supplemented with 10%
heat-inactivated fetal calf serum.
RNase Protection Assay--
[32P]-UTP-labeled
riboprobes were prepared using linearized DNA templates to generate
antisense transcripts of human ALK (364 nt) and
glyceraldehyde-3-phosphate dehydrogenase (152 nt) or 36B4 (300 nt). 60 µg of total RNA from appropriate cells was mixed with
riboprobes (ALK, 50,000 dpm; glyceraldehyde-3-phosphate dehydrogenase and 36B4, 2,000 dpm) and allowed to hybridize overnight at 50 °C
following denaturation by boiling. The next day unprotected RNA was
digested with RNase A (500 ng/ml) and run on a precast 6% acrylamide
Tris borate/EDTA-urea gel (Invitrogen). The gel was subsequently dried
and left to expose a film at Western Blotting--
5-8 × 104 cells were
plated per well in 6-well plates. Cells were serum-starved for 48 h and treated with growth factors (pleiotrophin generated as in Ref. 22
or PDGF-BB, Upstate Biotechnology, Lake Placid, NY), usually for 5 min.
Where noted, cells were treated with wortmannin or LY294002 (Sigma) for
1 h prior to stimulation with growth factors. Cells were harvested
in lysis buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 40 mM In Situ Hybridization--
For in situ hybridization
of tumor sections, formalin-fixed, paraffin-embedded sections were kept
overnight at 55 °C and for 1 h at 65 °C and were finally
heated in the microwave for 3 min. The sections were deparaffinized by
washes in xylene and decreasing concentrations of ethanol from 100 to
50%. Following treatment with 10 µg/ml proteinase K for 5 min,
slides were incubated in 0.2 M HCl for 8 min and acetylated
in 0.25% acetic anhydride, 0.1 M triethanolamine.
Hybridization with 10 µg/ml digoxigenin riboprobes in hybridization
solution (50% formamide, 10% dextran sulfate, 4× SSC, 1×
Denhardt's solution, 2.5 mg/ml yeast tRNA, and 0.5 mg/ml salmon sperm
DNA) was carried out overnight at 42 °C. Slides were washed for 5 min in 2× SSC, 2× SSC/50% formamide, 1× SSC, 0.5× SSC, and finally
0.1× SSC. Sections were blocked in 2% horse serum in Buffer 1 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl) followed by
overnight incubation with anti-digoxigenin alkaline phosphatase-conjugated antibodies (1:500) at 4 °C. The next day, sections were washed for 5 min in Buffer 1 and Buffer 2 (100 mM Tris-HCl, pH 9.5, 50 mM MgCl2,
100 mM NaCl) and incubated with 4-nitro blue tetrazolium
chloride and 5-bromo-4-chloro-3-indolyl phosphate until color
developed (sense and antisense constructs were incubated for the same
amount of time). The reaction was stopped in 1 mM EDTA, and
after drying sections were mounted with a xylene-based medium. For
in situ hybridization of tumor cell lines, cells were
allowed to attach to glass slides overnight and then were fixed in
methanol:acetic acid (3:1) and 10% formaldehyde. The hybridization and
washes were carried out as with tumor sections, omitting washes of
0.5× and 0.1× SSC. Two pairs of digoxigenin-labeled riboprobes were
generated to correspond to sense and antisense orientations of the ALK
mRNA from nucleotides 1104 to 1341 and from nucleotides 1352 to
1852 (data not shown). Consecutive sections were hybridized with all
probes, and congruency of antisense staining was confirmed.
Immunodetection of PTN and ALK--
For
immunohistochemistry, formalin-fixed paraffin-embedded sections were
kept overnight at 55 °C and for 1 h at 65 °C and finally
were heated in the microwave for 3 min. The sections were deparaffinized and incubated with 10 mM citrate buffer (pH
7.4) at 90 °C for 20 min. After incubation with 0.3% hydrogen
peroxide at 4 °C for 20 min and three washes with PBS/0.1% Tween
20, nonspecific binding was blocked with 10% horse or goat serum in
PBS/0.1% Tween 20/2% bovine serum albumin for PTN or ALK staining,
respectively, for 1 h at room temperature. After an additional
three washes slides were incubated with the respective primary
antibodies (monoclonal anti-PTN 4B7 1:20 (23); rabbit anti-ALK-ECD 1:20
(21); rabbit anti-P80 1:50 (Accurate Chemical and Scientific, Westbury,
NY); or goat anti-T18 N-terminal peptide (Santa Cruz Biotechnologies, San Diego, CA)) in PBS/0.1% Tween 20/1% bovine serum albumin at 4 °C in a wet chamber overnight. After washing, bound antibody was
detected using the appropriate biotinylated secondary antibodies and
commercially available detection reagents for visualization (Vector
Labs, Burlingame, CA), and the tissues were counterstained with
hematoxylin. Tumor and normal tissues were provided by the tumor bank
of the Lombardi Cancer Center.
Ribozyme-expressing Cell Lines--
Ribozymes targeted to
hybridize the human ALK mRNA at either
5'-ggactggtcatagcttcc-3' (Rz 1) or
5'-ggaatgtcaattacggct-3' (Rz 2) and cleave the target just
3' to gtc were directionally cloned into pRC/CMV vectors as
described (15). Cells were transfected with these constructs and
selected in 0.5 mg/ml G418 for 2 weeks. Clones were picked from
colonies grown from the initial transfections and expanded in selective medium.
In Vivo Tumorigenicity--
Plated cells were washed in 1×
phosphate-buffered saline and brought into suspension by treatment with
PBS containing 2 mM EDTA. The concentration of cells was
brought to 20 million cells/ml with media supplemented with 10%
fetal calf serum. For each cell line, four (pRC/CMV and Rz1-7) or five
(Rz1-2, Rz2-2, Rz2-3, Rz2-9, and Rz2-10) NU/NU mice were injected
with 0.1 ml of the cell suspension. The tumor area was measured every
3rd day as length × width, and animals were sacrificed when the
total tumor area reached 400 mm2. Each data point
represents 8 to 10 separate tumors, and error bars represent the ± S.E. Tumor growth experiments were repeated twice with reproducible results.
ALK Expression in Human Tumors and Cell
Lines--
Pleiotrophin is expressed at detectable levels in the
normal adult nervous system and overexpressed in brain tumors as well as in other cancers (12). To evaluate the potential contribution of
pleiotrophin signaling, we first studied expression of its receptor,
ALK, in human glioblastoma samples and in tumor cell lines. For the
tumor samples we used in situ hybridization to identify ALK
mRNA and immunohistochemistry to identify the ALK protein, and we
assessed the expression relative to normal brain. Astrocytoma,
glioblastoma multiforme, and oligodendroglioma tumor sections all
showed expression of ALK mRNA (Fig.
1, A-L), whereas normal brain showed no expression of ALK mRNA (Fig. 1,
A-C). Glioblastoma multiforme also showed a
strong staining for ALK protein, whereas the signal was below detection
in normal adjacent brain tissues (Fig. 1, M-P).
This was confirmed in parallel experiments with four additional cases
of glioblastoma (not shown). Anti-ALK antibodies raised against
different antigens, i.e. the extracellular domain (ECD) of
ALK and ECD-derived peptides gave the same distinctive result (not
shown). In cultured cell lines we found ALK mRNA expressed in three
of seven human glioma or glioblastoma lines by RNase protection. A
control for the specificity of the human ALK probe shows no
cross-reaction with rodent ALK mRNA (C6 and SW-13/murine ALK-transfected cells; Fig. 1Q). Rodent 36B4 mRNA is
also not recognized by the human probe in C6 cells. Furthermore, we
found pleiotrophin mRNA expressed in the ALK-positive cell lines
(not shown) supporting a role of pleiotrophin as an autocrine as well as a paracrine stimulator.
Pleiotrophin Signal Transduction in Glioblastoma Cells via
PI3-Kinase--
We utilized the U87MG cell line to study the
contribution of the pleiotrophin-ALK axis to the malignant phenotype of
glioblastoma. U87MG cells are a well characterized model system to
study tumorigenesis and signaling in glioblastoma (see Refs. 24 and 25)
and to express different receptor tyrosine kinases and their ligands such as epidermal growth factor receptor (26) and PDGF receptor (6). Furthermore, U87MG cells contain a mutated and inactive form of
the tumor suppressor PTEN ( Ribozyme-mediated Reduction of Endogenous ALK Specifically Reduces
Pleiotrophin Signaling--
To assess whether pleiotrophin signaling
through ALK is rate-limiting for the malignant phenotype of U87MG
cells, we generated derivative cell lines in which the endogenous ALK
is reduced by constitutive expression of ALK mRNA targeted
ribozymes. We have previously applied this approach to evaluate the
contribution of different gene products to the malignant phenotype
(15-17, 30) (reviewed in Ref. 31), and we initially screened different
ALK-targeted ribozymes for efficacy. From this we selected two ribozyme
expression vectors that target sequences in the 5' and 3' ends of the
ALK mRNA (Rz1 and Rz2, respectively) and generated a panel of
stably transfected U87MG cell lines with high, medium, and low residual ALK mRNA levels (Fig. 3, A
and B). Real-time quantitative PCR (not shown) as well as
in situ hybridization of pRC/CMV and Rz1-7 cells confirmed
high and low residual ALK mRNA levels, respectively (see Fig.
3C). In these cell lines the ability of pleiotrophin to
stimulate Akt phosphorylation was reduced in parallel with the reduced
endogenous ALK mRNA (Fig. 3, D and F). In
contrast, Akt phosphorylation induced through an independent tyrosine
kinase receptor (PDGF-R) that is expressed in U87MG cells (6) was unaffected by the reduction of ALK (Fig. 3, E and
F). From these results we concluded that the ability of
pleiotrophin to induce Akt phosphorylation is strictly dependent on
ALK.
Reduction of ALK, Xenograft Tumor Growth, and Animal
Survival--
Interestingly, the derivative U87MG cells with different
residual levels of ALK showed no significant difference in
proliferation rate or colony formation in soft agar (not shown). This
suggests that under in vitro growth conditions, ALK is not a
rate-limiting factor. To determine whether the reduction of
pleiotrophin-ALK signaling affects the in vivo tumor growth
of the U87MG cells, we next grew the different cell lines as tumor
xenografts in nude mice. The control cells with the highest residual
levels of ALK formed rapidly growing tumors (Fig.
4A) at a rate that was
indistinguishable from wild-type cells (not shown). In contrast, tumor
growth of the ALK-depleted cells was significantly reduced (Fig.
4A). As with the responsiveness to pleiotrophin induction of
Akt phosphorylation, the tumor size of the xenografts grown from the
different cell lines was directly dependent on the level of ALK
expression in a gene dose-dependent manner (Fig.
4B). This gene dose effect was observed with clonal and
mass-transfected cell lines (not shown). Finally, the difference in
tumor growth also resulted in a shift of the survival curve; although
all of the control mice died by the 20th day after injection, most mice
injected with the ALK-depleted cells survived at least twice as long
(Fig. 4C), and the median survival time correlated with the
residual ALK levels. The increased survival time of mice
injected with the ALK-depleted cells Rz1-2 and Rz1-7 was
statistically significant compared with those mice injected with the
control cells pRC/CMV (p = 0.0027 and p = 0.0058, respectively).
ALK Levels, Mitotic Index, and Apoptosis in
Tumors--
In light of the fact that pleiotrophin signaling through
ALK activates a pathway known to promote cell survival, namely
PI3-kinase and Akt, we decided to investigate the rate of apoptosis in
the xenograft tumor specimens and compare this with the mitotic index in the same samples. Using sections from size-matched tumors, we
examined hematoxylin and eosin- and TUNEL-stained sections for mitotic
and apoptotic cells, respectively (Fig.
5A). We found no significant
difference in the number of mitotic figures but a striking difference
in the number of TUNEL positive cells. In fact, the number of TUNEL
positive cells correlated directly with the reduction of ALK levels
(Fig. 5B). Recent studies suggested a role of PTEN in tumor
angiogenesis (25), and thus we also assessed whether the reduction of
ALK in the U87MG cells influenced the in vitro production of
endothelial cell growth factors as well as the extent of tumor
angiogenesis in vivo. Neither the endothelial cell growth
stimulatory activity present in the supernatants of the different U87MG
cell lines nor the extent of tumor angiogenesis measured in the tumor
samples was significantly affected by the reduction of ALK (not
shown). Overall, these findings suggest that the
pleiotrophin-ALK signaling provides an essential survival signal that
is rate-limiting for tumor growth of U87MG cells.
We recently identified ALK, an orphan RTK, as the receptor for the
growth factor pleiotrophin (19). Previous studies have shown that a
t(2;5) translocation can generate a fusion protein of the intracellular
ALK kinase domain with nucleophosmin and that this constitutively
active kinase can induce anaplastic lymphoma (20). In contrast with
this limited role of the nucleophosmin/ALK fusion protein for lymphoid
neoplasms, the full-length ALK receptor has been shown to be expressed
in the developing nervous system and by virtue of its expression
pattern is implicated with nervous system physiology (21). We now
report that one of the highly malignant tumors of the brain,
glioblastoma, overexpresses ALK, and we demonstrate that the
pleiotrophin-ALK pathway is essential for tumor growth of one of the
commonly used model systems, U87MG cells.
Furthermore, our initial studies on the expression of ALK in
cancers of epithelial origin showed ALK expression in one-third of
breast cancer cell lines (7 of 18) and in a little over one-half of
breast cancer tissues from patients (8 of 13). Still, the expression levels of ALK in breast cancers relative to normal tissues were not as
prominent as in glioblastoma, and we thus focused the studies reported
here on this latter tumor type. Interestingly, depletion of ALK from
Hs578T breast cancer cells that express high levels of the ALK receptor
as well as pleiotrophin significantly inhibited tumor growth in
animals2 very similar to the
data reported here for the U87MG cells. This suggests a potentially
rate-limiting role of ALK for tumor types other than glioblastoma that
utilize this growth factor receptor pathway.
As outlined above, the major pathway affected by the pleiotrophin-ALK
interaction is apoptosis, and this begs the question as to whether the
activity of apoptosis-inducing cytotoxic drugs might cooperate with
this pathway. To test this hypothesis we studied the efficacy of
cisplatinum, an alkylating agent used to treat glioblastoma, in the
U87MG cell panel. Indeed, cells with reduced residual levels of
endogenous ALK become more sensitive to cisplatinum-induced
apoptosis.2 This suggests the potential for a
positive interaction between ALK tyrosine kinase-targeted and
conventional cytotoxic therapies.
In the current paper, we have utilized ribozyme targeting of ALK to
generate a panel of cells with a range of residual endogenous expression of this receptor tyrosine kinase. The residual receptor levels correlated with 1) pleiotrophin induction of Akt
phosphorylation, 2) tumor growth, 3) median survival, and 4) apoptosis
in a gene dose-dependent manner. Finally, the tumor cells
became more sensitive to a cytotoxic agent, cisplatinum, upon the
reduction of ALK.2 This suggests to us that drugs that
specifically inhibit the pleiotrophin-stimulated ALK kinase
activity hold the promise to be efficacious against tumors that utilize
this growth stimulatory axis.
*
This work was supported by National Institutes of Health
Grants CA71585 and SPORE (to. A. W.) and from the Breast Cancer
Program of the United States Department of Defense (to C. P. and
A. W.).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.
Published, JBC Papers in Press, January 23, 2002, DOI 10.1074/jbc.M112354200
2
C. Powers, A. Aigner, G. E. Stoica, K. McDonnell, and A. Wellstein, unpublished data.
The abbreviations used are:
RTK, receptor
tyrosine kinase;
ALK, anaplastic lymphoma kinase;
ECD, extracellular
domain;
PDGF, platelet-derived growth factor;
nt, nucleotides;
PBS, phosphate-buffered saline;
PI3-kinase, phosphatidylinositol 3-kinase;
MAPK, mitogen-activated protein kinase;
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end-labeling;
CMV, cytomegalovirus;
PTN, pleiotrophin.
Pleiotrophin Signaling through Anaplastic Lymphoma Kinase Is
Rate-limiting for Glioblastoma Growth*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C. Expected sizes of protected
fragments are 237 nt (ALK), 104 nt (glyceraldehyde-3-phosphate dehydrogenase), and 220 nt (36B4).
-glycerophosphate, 0.25% sodium
deoxycholate, 1% Nonidet P-40, 50 mM sodium fluoride, 20 mM sodium pyrophosphate, 1 mM EGTA, 1 mM sodium orthovanadate, 1 µg/ml aprotin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, and 100 µg/ml Pefabloc), rocked at
4 °C for 20 min, and cleared by centrifugation at 4 °C. Protein
was quantitated with the BCA protein assay (Pierce), and 10 µg was
subjected to PAGE on precast 4-20% or 10% gels (Invitrogen). Gels
were transferred to polyvinylidene difluoride membranes and blocked in
5% milk in 1× PBS-T (phosphate-buffered saline, pH 7.5, with 0.05%
Tween 20) at room temperature for 1 h. Primary antibody (either
anti-phosphoAkt or anti-Akt, Cell Signaling, Beverly, MA) was used at
1:1000 dilution in 5% milk in 1× PBS-T and incubated overnight at
4 °C. Secondary, peroxidase-conjugated anti-rabbit antibody
(Amersham Biosciences) was used at 1:2000 dilution, and ECL (Amersham
Biosciences) was used according to the manufacturer's instructions.
After visualizing, blots were stripped (62.5 mM Tris-HCl,
pH 6.7, 100 mM 2-mercaptoethanol, 2% (w/v) SDS) for 30 min
at 42 °C, re-blocked, and subjected to subsequent Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (85K):
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Fig. 1.
ALK expression in human glial tumor
tissues and cell lines. A-L, detection of
ALK mRNA in human glial tumors by in situ hybridization.
Hematoxylin and eosin (H&E) (A, D,
G, and J), antisense probe for ALK mRNA
(B, E, H, and K) and the
corresponding sense control probe (C, F,
I, and L) staining human astrocytoma
(A-F), glioblastoma
(G-I), and oligodendroglioma
(J-L) tumors are shown.
M-P, detection of ALK protein in human
glioblastoma tumor samples by immunohistochemistry. Adjacent normal
brain is negative for ALK mRNA and protein
(A-C and M-N).
Space bar, 20 µm. Q, detection of human ALK
mRNA in cell lines by RNase protection. Three of seven human glial
cell lines (U87MG, U118MG, and U138MG) are positive for ALK mRNA.
C6 rat glioma and SW-13/mALK express rodent ALK mRNA and should not
show cross-reactivity with the human ALK probe. 36B4 is a human
gene-specific loading control. ACT, Astrocytoma;
GBM, glioblastoma multiforme; and ODG,
oligodendroglioma.
exon 3) (27, 28), and expression of an
active form of PTEN suppresses tumorigenicity and tumor angiogenesis
(24, 25). The lipid phosphatase PTEN controls signaling pathways that
involve PI3-kinase activity (28), and we thus initially examined
whether pleiotrophin signals through PI3-kinase in the U87MG cells. For
this we monitored phosphorylation of the downstream target molecule
Akt, an anti-apoptotic effector of PI3-kinase signaling that is
activated by phosphorylation (28, 29). Pleiotrophin induced Akt
phosphorylation at serine 473 in a dose-dependent manner
and reached saturation at a very low concentration of the ligand (<1
ng/ml) after 5 min of treatment (Fig.
2A). The phosphorylation
signal was maintained for at least 1 h (Fig. 2B). The
rapid response suggested to us that Akt phosphorylation is not caused
by a secondary effect of pleiotrophin signaling but rather is due to a
close link between the activated receptor of pleiotrophin and
PI3-kinase. Pleiotrophin-induced Akt phosphorylation was inhibited by
pretreatment of U87MG cells with the inhibitors LY294002 and
wortmannin, confirming that this effect is indeed mediated by
PI3-kinase (Fig. 2C). Interestingly, the MAPK pathway in
U87MG cells is activated constitutively, and no increase in phosphorylation was observed after treatment with pleiotrophin (not
shown). This suggested to us that the glioblastoma cells predominantly
use the PI3-kinase pathway for pleiotrophin-ALK signaling in contrast
to epithelial cells in which ALK stimulation by pleiotrophin results in
activation of both the MAPK and PI3-kinase pathways (18, 19).
View larger version (43K):
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Fig. 2.
Pleiotrophin-induced Akt phosphorylation in
U87MG cells. Representative immunoblots of the effects of
pleiotrophin on Akt phosphorylation (on serine 473) in serum-starved
wild-type U87MG cells. A, dose-response of pleiotrophin on
Akt phosphorylation. B, time course following treatment with
2 ng/ml pleiotrophin. C, inhibition by LY294002 and
wortmannin of Akt phosphorylation in response to treatment with 1 ng/ml
pleiotrophin. Akt protein blots are shown in the respective lower
panels.
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Fig. 3.
Effect of ALK depletion by ribozymes on
pleiotrophin signaling. A and B, detection
of ALK mRNA by RNase protection assay in empty vector (pRC/CMV) and
different ribozyme transfected U87MG cells. Autoradiogram
(A) and quantitation by PhosphorImager analysis
(B) are shown relative to control
(pRC/CMV). Results are representative of two
independent experiments. C, in situ hybridization
of pRC/CMV and Rz1-7 cells confirming high and low expression of ALK
mRNA, respectively. Dose-response of pleiotrophin (D)
and PDGF-BB (E) on pRC/CMV cells (squares),
Rz1-7 cells (triangles), and Rz2-2 cells
(circles) is shown. F, comparison of the effect
of pleiotrophin (0.5 ng/ml, open circles) or PDGF-BB (20 ng/ml, closed circles) as a function of ALK levels in the
different cell lines from B. Densitometric analyses of
results of the immunoblots are presented as the mean of three
independent experiments ± S.E. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 4.
Xenograft tumor growth. A,
size of pRC/CMV (squares), Rz1-2 (diamonds),
Rz1-7 (triangles), and Rz2-9 (circles)
xenograft tumors as a function of time after tumor cell inoculation.
B, tumor size at day 13 of different cell lines (see Fig.
3B) as a function of residual ALK levels. C,
survival curves of mice from A. Results are representative
of two independent experiments. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 5.
Analysis of mitosis and apoptosis in tumor
xenografts. A, high power (400×), hematoxylin and
eosin (H&E) (left column), and TUNEL
(right column) stained sections from size-matched
pRC/CMV (upper row) and Rz1-2
(lower row) xenograft tumors. B, number of
mitotic figures (open circles) and TUNEL positive cells
(closed circles) as a function of relative ALK levels.
Results are presented as the mean of 20 high power (400×) fields ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Oncology, Georgetown University, 3970 Reservoir Rd., Washington,
D. C. 20007. Tel.: 202-687-3672; Fax: 202-687-4821; E-mail:
wellstea@georgetown.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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