From the Departments of Microbiology and Immunology
and § Biochemistry and Molecular Pharmacology, Kimmel
Cancer Institute, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Epidermal growth factor receptor (EGF) variant type III (EGFRvIII) is a constitutively active, naturally occurring mutation of the EGF receptor that is found in many types of human tumors. When overexpressed in NIH3T3 fibroblasts, EGFRvIII induces transformation by enhancing cell growth and reducing apoptosis. Analysis of downstream signaling pathways has revealed that extracellular signal-regulated kinase activity is down-regulated, raising doubt as to the significance of this pathway in promoting transformation. We investigated whether the c-Jun N-terminal kinase (JNK) pathway was affected by EGFRvIII. NIH3T3 cells expressing EGFRvIII exhibited a high basal level of JNK activity, which was not present in cells overexpressing the normal EGF receptor. Treatment of cells overexpressing EGFRvIII with inhibitors of the EGF receptor or phosphatidylinositol 3-kinase resulted in the down-regulation of JNK activity. Furthermore, the down-regulation of JNK activity was associated with a loss of properties related to transformation, and there was no evidence for JNK activity in the promotion of apoptosis in these cells. These findings implicate constitutive activation of the JNK pathway in transformation by EGFRvIII.
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INTRODUCTION |
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Activation of the epidermal growth factor (EGF)1 receptor initiates a complex series of events that can enhance tumorigenesis, including cell growth and the promotion of cell survival. The activated receptor initiates a kinase cascade that ultimately activates members of the mitogen-activated protein (MAP) kinase family. These MAP kinases translocate to the nucleus and activate transcription factors, thus influencing the growth responses of the cell. A well defined pathway activated by the EGF receptor is the Ras/extracellular signal-regulated kinase (ERK) pathway (1). Following the assembly of Grb2-Sos-Ras complexes at the cell membrane, a sequential phosphorylation cascade involving Raf, MEK, and then ERK occurs (2). Activation of the ERK family of MAP kinases can then induce cell cycle progression (2).
Several parallel pathways of this kinase cascade have been identified in mammals, including the c-Jun N-terminal kinase (JNK; also called stress-activated protein kinase) pathway (3). The immediate activators of JNK, generically known as MAP kinase kinases, are MKK4 (4-6) and the recently identified MKK7 (7). Although both ERKs and JNKs are activated in a similar manner, they define distinct pathways that respond to different extracellular stimuli. ERKs are preferentially activated in response to mitogens, whereas JNKs are potently activated by cytokines and environmental stresses (1). JNK activity in response to stress is associated with the induction of cell cycle arrest and apoptosis (8-10). Although different stimuli preferentially activate specific MAP kinase pathways, a modest level of cross-activation of different MAP kinase pathways can result from the same stimulus (10-14).
Aside from the use of a particular pathway, the duration of MAP kinase
activity can also determine the physiological response of a cell. For
example, transient induction of JNKs provides a growth enhancement
signal, whereas persistent activity due to UV light or -irradiation
induces apoptosis (10). Consistent with this finding was the
observation that ERK and JNK activation had opposing effects on cell
survival in PC12 cells. Differentiated PC12 cells deprived of nerve
growth factor underwent programmed cell death that was dependent on the
down-regulation of ERK activity and was coupled with increased JNK
activity (8).
Overexpression of the EGF receptor has been implicated in the progression of numerous types of human cancers, including breast carcinomas and astrocytic neoplasms (15, 16). Transfection of the normal EGF receptor into fibroblast cell lines induces ligand-dependent transformation (17), providing a model in which to study the oncogenic effects of the receptor. The addition of EGF to cells overexpressing the normal EGF receptor induces a transient but potent activation of ERK (17, 18). This signaling scheme has been established for many receptor tyrosine kinases and is associated with positive growth responses in cells (19).
In addition to overexpression of the normal EGF receptor, many rearrangements of the EGF receptor have been identified in human glial tumors (20, 21). The most common of these spontaneously occurring mutant EGF receptors, known as EGFRvIII, has been studied extensively (20-22). EGFRvIII arises from an in-frame deletion of nucleotides 276-1075 in the normal EGF receptor cDNA (20). The resulting truncated receptor is similar to the normal EGF receptor in its ability to dimerize and autophosphorylate (18, 23). However, EGFRvIII is distinct from the normal receptor in that it does not bind EGF and thus is an unregulated, constitutively active kinase (18, 23).
Expression of EGFRvIII in NIH3T3 cells causes a highly transformed phenotype (17). Despite the fact that this receptor activates MEK, the immediate activator of ERK, there is a low basal level of ERK activity in cells expressing EGFRvIII (17, 18). This implies that the down-regulation of ERK occurs at the MAP kinase level itself, most likely by increased expression of a phosphatase. Down-regulation of the ERK MAP kinase pathway in other types of transformed cells has also been reported (24, 25).
Recently, we found that phosphatidylinositol (PI) 3-kinase is constitutively active in EGFRvIII-transformed cells and that this activity is required for colony formation in soft agar (26). Interestingly, PI 3-kinase has been implicated in the activation of the JNK pathway (27, 28). We have investigated JNK activity in NIH3T3 cells expressing EGFRvIII and have found a high basal activity. Inhibition of EGFRvIII or PI 3-kinase activity down-regulated JNK activity, which was correlated with a loss of transformed morphology. Despite this chronic high level of JNK activity and the low level of ERK activity, these cells have a low rate of apoptosis.
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EXPERIMENTAL PROCEDURES |
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Materials--
Cell culture media and recombinant human EGF were
from Life Technologies, Inc. Tyrphostin AG 1478 (Calbiochem) and LY
294002 (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) were dissolved in Me2SO to a concentration of 20 mM
and stored at 80 °C. [
-32P]ATP,
125I-labeled goat anti-mouse IgG, and
125I-labeled goat anti-rabbit IgG were from NEN Life
Science Products. Unlabeled ATP was from Pharmacia Biotech Inc. The
anti-phosphotyrosine monoclonal antibody PY20 and the anti-pan-ERK
monoclonal antibody were from Transduction Laboratories (Lexington,
KY). The anti-JNK1(C-17) polyclonal antibody was from Santa Cruz (Santa
Cruz Biotechnology, Santa Cruz, CA), and the anti-phospho-p44/p42 MAP
kinase monoclonal antibody was from New England Biolabs Inc. (Beverly,
MA). All other materials were from Fisher unless otherwise
indicated.
Cell Culture--
CO12 20c2/b, HC2 20d2/c, and LTR b2 clones
were generated by transfection of NIH3T3 cells with the cDNA
encoding the normal human EGF receptor, the EGFRvIII cDNA, and
vector only, respectively (17). The NIH3T3 transfectants were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
10% calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml kanamycin, and 350 µg/ml G418. Cells were rinsed with
phosphate-buffered saline and then lysed with 1 ml of cell lysis buffer
(10 mM Na2HPO4, 150 mM
NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.2%
NaH3, 0.004% NaF, 1 mM NaVO4, 25 mM -glycerophosphoric acid, 100 µg/ml
phenylmethanesulfonyl fluoride, and 1 µg/ml each aprotinin and
leupeptin, pH 7.35). Lysates were clarified by centrifugation at
12,000 × g for 10 min at 4 °C. Protein
concentrations were determined using the Bio-Rad DC protein assay.
Western Blot Analysis-- Total cell lysate (30 µg) of each sample was subjected to SDS-polyacrylamide gel electrophoresis on 8.5% acrylamide gels for resolution of MAP kinases and 4-20% Tris/glycine gels for resolution of the EGF receptor. Protein was transferred to nitrocellulose filters and blocked for 2 h at room temperature in 100 mM Tris, pH 7.5, 0.9% NaCl, and 0.1% Tween 20 with 5% nonfat dry milk. The anti-phospho-MAP kinase monoclonal antibody was used at a 1:1000 dilution, whereas the anti-phosphotyrosine monoclonal antibody, the anti-pan-ERK monoclonal antibody, and the anti-JNK1 polyclonal antibody were used at 1 µg/ml in the same blocking solution. 125I-Labeled goat anti-mouse IgG (5 × 105 cpm/ml) was used to detect the monoclonal antibodies, and 125I-labeled goat anti-rabbit IgG was used to detect the polyclonal antibodies. Quantitation of signals was performed on a PhosphorImager using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Anchorage-independent Growth Assays-- Soft agar assays were performed as described previously (17) with slight modification. Briefly, cells were fed 1 ml of fresh medium once a week for 3 weeks and treated with 0.1 or 1 µM tyrphostin AG 1478 every 3 days. After 3 weeks of growth, the colonies were counted.
Solid-phase c-Jun Kinase Assay--
GST-c-Jun (amino acids
1-79) was bound to glutathione-Sepharose beads (Pharmacia Biotech
Inc.). JNK was precipitated from 300 µg of whole cell extracts with
GST-c-Jun for 3 h at 4 °C. Precipitates were washed three times
in phosphate-buffered saline containing 1% Nonidet P-40 and 2 mM NaVO4 and rinsed once in kinase reaction
buffer (25 mM Hepes, 25 mM MgCl2, 2 mM dithiothreitol, 0.1 mM NaVO4,
and 25 mM -glycerophosphoric acid). JNK activity was
assayed by resuspending the pellets in 30 µl of kinase reaction buffer containing 5 µCi of [
-32P]ATP and 20 µM unlabeled ATP. Following a 20-min incubation at 30 °C, samples were separated by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose, and kinase activity
was quantitated using the PhosphorImager.
Assay for Apoptosis-- Cells were seeded on coverslips in 6-well dishes and grown in complete medium for 3 days. The cells were then incubated in serum-free DMEM containing either 2 µM tyrphostin AG 1478 or 5 µM LY 294002 for 1 or 3 days and fixed and stained with 4,6-diamidino-2-phenylindole (2 µg/ml) for viewing by fluorescence microscopy. Apoptotic cells were identified by condensed nuclei.
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RESULTS |
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EGFRvIII Constitutively Activates JNK--
Previous work on the
signaling effects of EGFRvIII in NIH3T3 fibroblasts showed that ERK
activity was down-regulated (17, 18), suggesting that signaling though
the Ras/ERK pathway was not required for transformation. We wished to
investigate whether an additional MAP kinase pathway was affected by
the signaling of EGFRvIII. We chose to study the effect of the mutant
receptor on the JNK pathway. JNKs are defined in general by their
ability to phosphorylate two serine residues in the amino-terminal
domain of c-Jun in vitro (29, 30). These enzymes have a high
affinity for this substrate, so a solid-phase kinase assay utilizing
GST-c-Jun (amino acids 1-79) as a substrate for JNK allows measurement
of the kinase activity associated with a cell lysate. We employed this
technique to compare JNK activity in NIH3T3 cells transfected with an
empty expression vector (LTR b2); the normal human EGF receptor (CO12
20c2/b); and the mutant EGF receptor, EGFRvIII (HC2 20d2/c).
Unstimulated LTR b2 and CO12 20c2/b cells exhibited very low levels of
JNK activity that was modestly enhanced by the addition of EGF (100 ng/ml). In contrast, unstimulated HC2 20d2/c cells had a 13-fold higher
JNK activity relative to unstimulated LTR b2 cells (Fig.
1, Kinase Assay), indicating a
high constitutive JNK activity in these cells. The addition of EGF to
HC2 20d2/c cells did not alter the level of JNK activity, which is
consistent with the report that EGFRvIII cannot bind EGF and so is not
influenced by this mitogen (18). Western blot analysis showed that JNK expression was nearly equivalent in all three cell lines (Fig. 1,
JNK), indicating that the increased JNK activity is not
due to the up-regulation of protein expression.
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Continuous Stimulation of the Normal EGF Receptor Does Not Result
in Elevated Levels of JNK Activity--
Since the constitutive
activity of EGFRvIII resulted in a high basal level of JNK activity, we
wanted to determine if the normal EGF receptor could produce the same
effect when continuously stimulated with ligand. Quiescent CO12 20c2/b
cells were stimulated with EGF (100 ng/ml) and assayed at certain
intervals for the activation of the EGF receptor, JNK, and ERK. CO12
20c2/b cells stimulated with EGF showed a maximal activation of the EGF
receptor at 5 min, which slowly declined to basal levels by 24 h
of stimulation (Fig. 2,
pTyr). ERK induction coincided with EGF receptor
activation, which was maximal at 5 min of ligand stimulation and
declined to undetectable levels by 24 h (Fig. 2,
Active
MAPK). Maximal JNK activity was detected after 5 min of EGF
stimulation, but was greatly reduced by 1 h (Fig. 2, Kinase
Assay). JNK activity was above basal levels up to 48 h of EGF
stimulation, but was still well below that seen in HC2 20d2/c cells
(Figs. 1 and 2).
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Inhibition of EGFRvIII Down-regulates JNK, and Receptor
Reactivation Leads to Transient ERK but Sustained JNK
Activation--
Other studies (11, 12) and our data have shown that
the normal EGF receptor does not strongly activate JNK. We wished to
determine if the constitutive JNK activity associated with the HC2
20d2/c cell line was an effect of signaling from the mutant receptor.
To examine this, we utilized a specific inhibitor of the EGF receptor,
tyrphostin AG 1478. Previous work has shown that HC2 20d2/c cells
maintained in 2 µM tyrphostin AG 1478 for 3 days and then
serum-starved for 10 h showed a nearly complete loss of tyrosine
phosphorylation of EGFRvIII (26). Tyrphostin AG 1478 inhibited ERK
activation of EGF-treated, but not platelet-derived growth
factor-treated, CO12 20c2/b cells, indicating the specificity of the
inhibitor for the EGF receptor (26). When HC2 20d2/c cells were treated
with tyrphostin AG 1478, there was a reduction in JNK activity in
association with inhibition of receptor activity (Fig.
3A, pTyr and
Kinase Assay, 0 time point).
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Transformed Morphology of HC2 20d2/c Cells Parallels JNK Activity-- We examined the morphology of these cells after tyrphostin AG 1478 treatment and then following release. Cells treated as described above showed a reversion to the normal planar appearance of fibroblasts. Following release from tyrphostin AG 1478, HC2 20d2/c cells displayed a similar morphology for the first 12 h of release (Fig. 3B). However, between 12 and 24 h of release, these cells regained their transformed appearance and resembled untreated HC2 20d2/c cells, which coincided with the reactivation of JNK.
HC2 20d2/c Cells Treated with Tyrphostin AG 1478 Lose the Ability to Grow in Soft Agar-- The reversion of the transformed morphology of HC2 20d2/c cells treated with tyrphostin AG 1478 suggests a reduction or loss of transformation potential. To further analyze this, anchorage-independent growth studies were performed on HC2 20d2/c and CO12 20c2/b cells treated with tyrphostin AG 1478. Both cell lines formed colonies in soft agar when grown in complete medium (Fig. 3C, CM). Treatment of HC2 20d2/c cells with 0.1 µM tyrphostin AG 1478 resulted in a 37% reduction in colony formation, whereas treatment with 1.0 µM tyrphostin AG 1478 resulted in a 94% inhibition of colony growth. Tyrphostin AG 1478 treatment of CO12 20c2/b cells yielded the same profile seen in tyrphostin AG 1478-treated HC2 20d2/c cells. Colony formation of CO12 20c2/b cells in the presence of 0.1 µM tyrphostin AG 1478 was reduced by 64% and by 92% with 1.0 µM tyrphostin AG 1478 (Fig. 3C). These data further suggest that the relationship between the constitutive activities of EGFRvIII and JNK is critical for the transformation state of cells expressing this mutant EGF receptor.
Constitutive JNK Activity Is Dependent on PI 3-Kinase in HC2
20d2/c Cells--
Our laboratory has recently found that
one of the effects of chronic signaling from EGFRvIII is constitutive
PI 3-kinase activity (26). Recently, it was shown that constitutively
active forms of PI 3-kinase activate the JNK pathway (27, 28). This led us to ask if the high level of PI 3-kinase activity was responsible for
the high basal level of JNK activity in HC2 20d2/c cells. HC2 20d2/c
and CO12 20c2/b clones were treated with LY 294002, a specific
inhibitor of PI 3-kinase activity, in the same manner as described for
tyrphostin AG 1478. The inhibition of PI 3-kinase activity in HC2
20d2/c cells resulted in a corresponding reduction in JNK activity
without affecting ERK activity (Fig.
4A). The addition of LY 294002 to HC2 20d2/c cells resulted in a partial morphological reversion and
growth inhibition (Fig. 4B). In a previous publication (26),
we also found that treatment of HC2 20d2/c cells with 2 and 5 µM LY 294002 caused 71 and 99% reductions in soft agar
colony efficiency. Interestingly, the reduction of JNK activity induced
in LY 294002-treated HC2 20d2/c cells was nearly equivalent to that
seen in tyrphostin AG 1478-treated HC2 20d2/c cells, indicating that
the high basal level of JNK activity was dependent on the up-regulated
PI 3-kinase activity. JNK and ERK activation levels were also
determined in LY 294002-treated CO12 20c2/b cells. EGF-stimulated CO12
20c2/b cells showed similar levels of ERK activation regardless of
treatment with the PI 3-kinase inhibitor (Fig. 4A,
Active MAPK). However, EGF-stimulated CO12 20c2/b cells
showed a decrease in JNK activation when compared with cells treated
with LY 294002 (Fig. 4A, Kinase Assay).
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JNK Activity Does Not Correlate with the Induction of Apoptosis in Cells Expressing EGFRvIII-- In PC12 cells, prolonged JNK activity and a loss of ERK activity correlate with the induction of apoptosis (8). HC2 20d2/c cells possess the same enzymatic profile, but, in contrast, are actively growing. Since this is in the presence of serum, we examined HC2 20d2/c cells following 1 and 3 days of serum starvation for the induction of apoptosis and compared the results with LTR b2 cells. Cells were assayed for the presence of nuclear condensation and JNK activity. A small percentage of apoptotic cells was noted in the LTR b2 cell line following 1 day of serum starvation, whereas there was negligible apoptosis in the HC2 20d2/c cell line (Fig. 5, bar graphs). The low basal JNK activity in LTR b2 cells was further decreased after 1 day of serum starvation, whereas the robust activity in HC2 20d2/c cells remained unaltered (Fig. 5, Kinase Assay). After 3 days of serum starvation, there was a large increase in the number of apoptotic cells in the LTR b2 cell line, but only a slight increase in the HC2 20d2/c cell line. JNK activity in LTR b2 cells remained extremely low, whereas there was actually a 77% decrease in HC2 20d2/c cells. Since EGFRvIII and PI 3-kinase contribute to the enhanced growth rate in HC2 20d2/c cells, we examined the effect of tyrphostin AG 1478 and LY 294002 on apoptosis. After 1 day of treatment, tyrphostin AG 1478 showed a small effect on LTR b2 cells, but by 3 days, there was a high percentage of apoptotic cells in both the LTR b2 and HC2 20d2/c cell lines (Fig. 5, bar graphs). LY 294002 also increased the incidence of apoptosis in both cell lines, although not to the same extent. There was no JNK activity in LTR b2 cells in the presence of either inhibitor, whereas there was a 60% decrease in HC2 20d2/c cells (Fig. 5, Kinase Assay). Taken together, these data suggest that EGFRvIII and PI 3-kinase contribute to cell survival, but that JNK activity does not contribute to cell death.
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DISCUSSION |
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Expression of mutant forms of the EGF receptor has been implicated in the progression of many types of human tumors (20, 21). EGFRvIII is the most frequently occurring natural alteration associated with this receptor (20-22). We studied NIH3T3 fibroblasts transfected with EGFRvIII to elucidate the signaling mechanisms responsible for transformation in these cells. Work in this and other laboratories showed that activation of the normal EGF receptor resulted in a transient but potent ERK induction, whereas EGFRvIII generated minimal ERK induction (17, 18). This low level of ERK activity raised questions as to the significance of the ERK pathway in promoting transformation. Analysis of the JNK pathway showed that EGFRvIII-transfected cells displayed a high constitutive level of JNK activity, which was not present in cells overexpressing the normal EGF receptor. Although stimulation of the EGF receptor can transiently activate JNK, the constitutive activation of JNK seen in EGFRvIII-transfected NIH3T3 cells may define a novel mechanism associated with transformation by this receptor.
We have further defined the pathway by identifying PI 3-kinase as the upstream activator of JNK. Interestingly, EGFRvIII was recently shown to constitutively activate PI 3-kinase, and this activity was essential for the transforming potential of the mutant receptor (26). Inhibition of PI 3-kinase activity associated with HC2 20d2/c cells resulted in a reduction in JNK activity and partial morphological reversion of the transformed appearance of HC2 20d2/c cells. The ability of PI 3-kinase to influence both cell morphology and JNK activity could be explained by its interaction with members of the Rho family of proteins (31, 32), which are known to regulate the actin cytoskeleton (33, 34) and to activate the JNK pathway (35-38). Taken together, we speculate that the constitutive PI 3-kinase activity induced by EGFRvIII expression is responsible for the high basal level of JNK activity and that this contributes to the transformed phenotype induced by EGFRvIII.
The low level of ERK activation exhibited by cells expressing the mutant receptor has been suggested to be regulated by expression of a phosphatase (17). Interestingly, in NIH3T3 fibroblasts, expression of MAP kinase phosphatase-1, a phosphatase specific for ERKs, is induced by activation of the JNK pathway (39). The constitutive JNK activity associated with EGFRvIII-transfected cells may induce constant expression of MAP kinase phosphatase-1, resulting in the low basal ERK activity exhibited in these cells. We are currently determining if this is the mechanism responsible for down-regulating ERK activation in HC2 20d2/c cells and if such down-regulation is essential for the transformation potential of the mutant receptor.
In support of our data implicating continuous JNK activity with a growth advantage in EGFRvIII transfectants, recent studies found that the JNK pathway was required for transformation by both the Trp-Met (40) and Bcr-Abl (41) oncoproteins. Like EGFRvIII, the Trp-Met oncoprotein is a constitutively active receptor tyrosine kinase that is not influenced by ligand (42). Dominant-negatives forms of Grb2 transformed into cells expressing the Trp-Met protein resulted in a reverted morphology that was associated with both down-regulated PI 3-kinase and JNK activities (40). It is interesting to speculate that preferential activation of the JNK pathway may be a characteristic of unregulated kinases with high transformation potential, such as seen in fibroblasts expressing EGFRvIII, Trp-Met, or Bcr-Abl.
Analysis of EGFRvIII in NIH3T3 cells has provided a model in which the transforming effects of this mutant receptor can be elucidated. We have shown that the transformed state of EGFRvIII-expressing cells is associated with the preferential and constitutive activation of the JNK pathway. These findings also demonstrate that cells are capable of sustained JNK activity and low ERK activity without the induction of apoptosis. In light of our findings and recent reports by others (40, 41) implicating the JNK pathway in the promotion of transformation, it is becoming clear that certain oncoproteins and/or cell types actually utilize JNK and not ERK for transformation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA51093 and CA69495 and Training Grant 5-T32-DK07705-04 and by a grant from the American Cancer Society.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.: 215-503-4650; Fax: 215-923-4498; E-mail: a_wong{at}lac.jci.tju.edu.
1 The abbreviations used are: EGF, epidermal growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; EGFRvIII, EGF receptor variant type III; PI, phosphatidylinositol; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; MEK, MAP kinase kinase.
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REFERENCES |
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