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Tumor Biology |
Departments of Radiation Oncology [A. M., N. P., J. L., D. S.], Neurosurgery [D. M. O.], and Pathology and Laboratory Medicine [D. M. O.], University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19004
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VEGF3 is a potent angiogenic factor that has been implicated in the pathogenesis of glioblastomas (reviewed in Ref. 4 ). VEGF is commonly expressed in glioblastomas, as determined by in situ hybridization, but rarely expressed in low-grade gliomas (5 , 6) . Other studies have confirmed this correlation between tumor grade and VEGF expression in gliomas (7 , 8) . Therefore, VEGF is an attractive target for antiangiogenic therapy (reviewed in Ref. 9 ). Numerous studies in animal models have shown that inhibiting VEGF function using neutralizing antibodies (10) , dominant-negative VEGF receptor mutants (11 , 12) , and antisense constructs (13, 14, 15) inhibits growth of glioma cells in vivo and causes regression of blood vessels (16) .
Given the importance of VEGF in the growth of glioblastomas, we were
interested in understanding its regulation in these tumors. Hypoxia,
which is commonly observed in the microenvironment of solid tumors
(17)
, is a well-known stimulus for inducing VEGF
(6)
. Palisading cells in areas next to necrosis within
glioblastomas are thought to up-regulate VEGF secondary to hypoxia
(18)
. Hypoxia increases VEGF mRNA levels by
transactivating the promoter via the transcription factor HIF1. HIF1
consists of an 
subunit that is induced by hypoxia and a ß subunit
that is constitutively present (reviewed in Ref. 19
). HIF1
binds to a specific consensus sequence, 5'-RCGTG-3', which is found
within the HRE in the VEGF promoter (20)
.
However, many glioblastoma cells express high levels of VEGF, even under normoxic conditions. In one study, U87 MG cells were found to express seven times the amount of VEGF mRNA as NIH3T3 fibroblasts or human fetal astrocytes under tissue culture conditions (21) . This suggests that there are factors intrinsic to glioblastomas that lead to VEGF up-regulation independent of the environment. One of these factors could be EGFR, which is overexpressed as a result of gene amplification in 40–50% of malignant human glioma specimens (22, 23, 24) . Many gliomas contain a mutant form of the EGFR known as deltaEGFR or EGFRvIII, which is missing exons 2–7, resulting in an in-frame deletion of 801 bp of the coding sequence of the extracellular domain, rendering the receptor constitutively active (25, 26, 27) .
EGF has been shown to induce VEGF protein secretion in glioblastoma cells. Blockade of EGFR using a neutralizing antibody in A549 squamous carcinoma cells led to a decrease in the level of VEGF (28) . Although the association between EGF and VEGF has been made, relatively little is known about the signaling pathway connecting the two. An extensive body of literature indicates that in nonglioma cell lines, Ras mutations can lead to increased VEGF levels (reviewed in Ref. 29 ). Although glioblastomas do not generally contain Ras mutations (30) , they do have activation of the Ras pathway (31) , presumably because of overexpression of EGFR or other tyrosine kinase growth factor receptors.
In this study, we used a matched pair of human glioblastoma cell lines, U87 MG, the parental line, and U87/T691, a derivative clonal line into which a truncated erbB2/Neu receptor protein containing a premature stop codon at T691 has been introduced. We have shown previously that introduction of the mutant erbB2/Neu protein inhibits kinase activation of EGFR through the formation of nonfunctional heterodimeric receptor complexes (32 , 33) . EGFR-inhibited U87/T691 subclones exhibit a less transformed phenotype by a variety of tumorigenicity assays and also display elevated apoptosis in response to DNA damage (34) . In our studies, we found that the U87/T691 cells contain lower basal levels of VEGF mRNA and protein than the parental U87 MG cells. Using chemical inhibitors and transient transfections with a reporter construct containing the VEGF promoter, we provide evidence that EGFR regulates VEGF at the level of transcription in glioblastoma cells via a pathway involving Ras and PI(3) kinase, which is distinct from the pathway induced by hypoxia. The two pathways can, however, act to increase VEGF mRNA levels in an additive manner.
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MATERIALS AND METHODS |
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Human recombinant EGF was obtained from Sigma and reconstituted in 10
mM acetic acid containing 0.1% BSA at a stock
concentration of 20 µg/ml LY294002 and tyrphostin AG1478 (Alexis
Biochemical) were dissolved in DMSO at stock concentrations of 19 and
31.6 mM, respectively. The stock concentrations of
wortmannin (Sigma) and FTI-277 were 2 and 10 mM,
respectively, both dissolved in DMSO. PD98059 (Alexis Biochemical) was
prepared in DMSO at a stock concentration of 93.6 mM. The
doses of inhibitors used in this study were determined from the
literature and from preliminary experiments in our laboratory.
Tyrphostin AG1478 at a concentration of 10 µM has been
shown to block EGFR signaling in U87 MG cells (35)
. We
have found that LY294002 and wortmannin inhibit PI(3)
kinase activity,
as measured by phosphorylated AKT at the doses used in this study (data
not shown). We found that PD98059 at a concentration of 25
µM blocked MAP kinase phosphorylation in U87 MG cells
using an antibody against phosphorylated ERK1/2 (Fig. 3F)
.
FTI-277 at a concentration of 10 µM inhibited
Ras farnesylation in these cells (data not shown).
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mRNA Stability Assay.
To measure the half-life of the VEGF message, we added actinomycin D to
dishes of U87 MG cells maintained under normoxic conditions. Samples
were harvested for RNA at intervals thereafter.
Northern Blot Analysis.
Total RNA was isolated with Trizol (Life Technologies), using the
manufacturer’s directions. Ten to 15 µg of RNA were denatured with
formaldehyde and formamide and run on a 0.9% agarose gel containing
formaldehyde. RNA was transferred by capillary action in 20x SSC (1x
SSC is 0.15 M NaCl, 0.15 M sodium citrate, pH
7) to a Duralon-UV membrane (Stratagene) and UV cross-linked prior to
hybridization. Labeling of radioactive probes was performed using
[32P]dCTP and a Prime-It kit (Stratagene) using
the manufacturer’s directions. Hybridization was carried out at
65°C, after which the membranes were washed to a stringency of 0.1x
SSC, 0.1% SDS at 65°C. Autoradiography was carried out at -80°C
with intensifying screens. A 200-bp VEGF cDNA fragment excised with
EcoRI from the pGEMh204 plasmid (Ref. 38
; gift
from Dr. B. Berse, Boston University School of Medicine, Boston, MA)
was used to make radioactive probes for hybridization. To verify equal
loading between lanes, all gels were stained with ethidium bromide.
Furthermore, the membranes were probed with rpL32 (39)
,
which is a ribosomal protein the mRNA level of which we have found to
remain relatively constant under differing oxygen concentrations
(40)
.
Protein Extraction, Western Blot Analysis, and ELISA.
For protein isolation, cells were trypsinized and then pelleted by
centrifugation. The pellets were suspended in PBS and then centrifuged
again. The pellets were then solubilized in 0.3–0.5 ml of 1x sample
lysis buffer (2% SDS, 60 mM TRIS, pH 6.8), Samples were
boiled for 5 min and then passed repeatedly through a 26-gauge needle.
They were centrifuged at 10,000 x g, and the
supernatants were retained. Protein concentrations were determined
using a BCA Protein Assay kit (Pierce).
For Western blotting, equal amounts of total protein were run in each lane of an SDS-PAGE gel (12% acrylamide). Each protein sample was mixed with an equal volume of 2x Laemmli buffer and boiled at 95°C for 5 min before loading onto the gel. After completion of gel electrophoresis, protein was transferred to a Hybond nitrocellulose membrane (Amersham) over 1 h using a blotting apparatus. For detection of the VEGF protein, we used a monoclonal antihuman VEGF antibody (PharMingen) at a dilution of 1:1000, followed by a goat antimouse antibody (Bio-Rad) at a dilution of 1:500. For detection of phosphorylated ERK-1 and ERK-2 kinases, we used the M8159 antibody (Sigma) at a dilution of 1:1000, followed by a goat antimouse antibody (Bio-Rad) at a dilution of 1:500. As a loading control, the blot was reprobed with an anti-ß-actin antibody (Sigma) at a 1:1000 dilution, followed by a goat antimouse antibody (Bio-Rad) at a dilution of 1:500.
VEGF ELISA Assays.
Conditioned media were removed for storage at -80°C. VEGF protein
concentration in the media was determined by ELISA using a commercial
kit (R&D Systems) .
Quantitation of Blots and Data Analysis.
Gels were scanned on an Agfa Arcus II photoscanner using Adobe
Photoshop 4.0. Bands on the gels were quantitated using NIH Image 1.54
software. Curve fitting was performed using Cricketgraph III, version
1.5.1 (Computer Associates). All results shown are representative of at
least two independent, reproducible experiments.
Plasmid Constructs and Transient Transfections.
We obtained a portion of the human VEGF promoter (41)
from
Dr. J. Abraham (Scios, Inc., Sunnyvale, CA). A 1.5-kb fragment
of the promoter was excised using SacI and subcloned into
the SacI site in the promoterless pGL3-Basic vector
(Promega) in the proper orientation to make the plasmid
pGL3–1.5kbVEGFprom (Fig. 9B)
. The pGL3–1.5kbVEGFprom
plasmid contains the 47-bp HRE spanning from -985 to -939
(20)
. To delete the HRE, we constructed the plasmid
pGL3–1.2kbVEGFprom by excising a 1.2-bp fragment from the VEGF
promoter with the enzymes PstI and SacI and
subcloning it into the PstI/SacI sites of
pBluescript (Stratagene). The 1.2-kb fragment was then removed using
KpnI and SacI and subcloned into these sites in
pGL3-Basic (Fig. 9B)
.
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Transfections were performed using Fugene (Boehringer Mannheim)
according to the manufacturer’s instructions. Briefly, cells were
split into 60-mm dishes so that 24 h later, they were 
50%
confluent. At this time, each dish was transfected using 6 µl of
Fugene and 2 µg of the reporter plasmid and, to control for
transfection efficiency, 1 µg of pSV-ß-galactosidase (Promega).
Cells were harvested by removing the media, washing twice with PBS, and
directly adding 200 µl of lysis buffer/dish. Of this lysate, 100 µl
was used for luciferase determination, and 5 µl were used for
ß-galactosidase determinations. These determinations were performed
using the LucLite kit (Packard Instrument Company) and the
ß-galactosidase Enzyme Assay System (Promega). Luciferase readings
were performed on a TopCount Microplate Scintillation and Luminescence
Counter (Packard Instrument Co.).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3.8 kb and the
other at 1.4 kb. Under normoxic conditions, U87/T691 cells with
functionally inactivated EGFR signaling expressed both mRNA species;
however, the amount of VEGF mRNA was half that seen in U87 MG cells
(Fig. 1
A, compare Lanes 1 and 3). This
difference was reflected at the protein level as well with U87/T691
cells containing 49% as much VEGF protein as U87 MG cells (Fig. 1B)
. These findings support the notion that EGFR kinase
activation plays a role in maintaining high VEGF levels in U87 MG
cells.
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A, compare Lanes 3 and 4). In
U87/T691 cells, the amount of induction under hypoxia relative to
normoxia was similar to that seen in U87 MG cells, 5.8-fold (2.9/0.5)
versus 4.0-fold.
EGF and Hypoxia Additively Increase VEGF mRNA Levels in U87 MG
Cells.
As an alternate means of showing that VEGF can be regulated by EGF in
these cells, we serum starved U87 MG cells and then restimulated them
with EGF. Fig. 2
shows that stimulation with EGF increased VEGF mRNA levels 2-fold under
normoxic conditions (Lane 2 versus Lane 1). We also
determined the effect of hypoxia in conjunction with EGF stimulation on
VEGF mRNA induction. EGF stimulation and 1% oxygen were both able to
up-regulate VEGF mRNA levels; however, the two stimuli together had an
additive effect in increasing VEGF mRNA levels compared with either
alone (Fig. 2
, Lanes 2–5). The same additive effect was
observed with 5% oxygen and EGF stimulation (Fig. 2
, Lanes 2, 3,
6, and 7).
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50% of the control level (Fig. 3A)
, which confirms that EGFR kinase activity influences VEGF
mRNA regulation. Because the Ras pathway has been shown to be activated
in many glioblastoma cell lines, including U87 MG (31)
, we
blocked Ras activity by using FTI-277, a farnesyltransferase inhibitor
that has been used in other cell lines (46, 47, 48)
. FTI
treatment decreased VEGF mRNA expression to 30% of baseline (Fig. 3B)
.
Because Ras can activate the PI(3)
kinase pathway (reviewed in Ref.
49
), we investigated the consequence of PI(3)
kinase
inhibition. Treatment of U87 MG cells with LY294002, a PI(3)
kinase
inhibitor (50
, 51)
, decreased VEGF mRNA to 20% of the
control level (Fig. 3C)
, as did treatment with wortmannin
(Fig. 3D)
, which inhibits PI(3)
kinase by a different
mechanism (52, 53, 54)
. Another well-characterized signaling
pathway downstream of Ras is the MAP kinase pathway (55)
,
which regulates cell growth in many cell types. However, treatment of
U87 MG cells with the MEK inhibitor PD98059 (56)
had no
effect on VEGF mRNA levels (Fig. 3E)
. We confirmed that the
drug was blocking the MAP kinase pathway at this dose by showing
inhibition of ERK phosphorylation (Fig. 3F)
.
VEGF Protein Levels in U87 MG Cells.
To show that the effects we saw at the VEGF mRNA level corresponded to
changes in protein levels, we performed ELISA assays on cell culture
supernatants. As shown in Fig. 4A
, EGF stimulation of serum-starved U87 MG cells led to an
increase in secreted VEGF protein levels (Fig. 4A)
.
Treatment of U87 MG cells with LY294002 resulted in an 50% decrease
in VEGF protein secretion into the cell media (Fig. 4B)
.
These data support the association between EGF stimulation, PI(3)
kinase catalytic activation, and induction of VEGF mRNA and secreted
protein.
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, we found that U87 MG.deltaEGFR cells expressed more VEGF
mRNA parental U87 MG cells under normal tissue cell culture conditions,
and that this difference was even more marked under serum starvation
conditions, consistent with the constitutive signaling induced by
deltaEGFR (33
, 57)
. Treatment of U87 MG.deltaEGFR cells
with LY294002 significantly down-regulated VEGF mRNA, whereas treatment
with PD98059 did not (Fig. 5B)
. Given that deltaEGFR
expression has been found to lead to constitutive activation of the
PI(3)
kinase pathway (58)
, these results lend further
support to the connection between PI(3)
kinase and VEGF up-regulation
in these cells. These observations also suggest that EGFR/PI(3)
kinase
signals regulate the VEGF promoter independently of ERK activation.
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. We plotted the relative VEGF mRNA levels (Fig. 6B)
, from which we calculated that the half-lives of the
3.8- and 1.4-kb species were 4 and <h, respectively, in both cell
lines. The half-life of the upper band is similar to the half-life of
3.8–4.8 h obtained in 293T cells (61)
. Therefore, altered
VEGF mRNA stability does not account for the difference in VEGF levels
in the two cell lines.
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. Thus, differences in VEGF mRNA levels between these two
isogenic glioblastoma cell lines differing with respect to functional
activation of the EGFR kinase can be attributed solely to differences
in activation of the VEGF promoter.
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. In contrast, inhibition of PI(3)
kinase
with either LY94002 or wortmannin significantly decreased VEGF promoter
activity (Fig. 7C)
. As an alternate means of showing the
involvement of the Ras/PI(3)
kinase pathway in VEGF promoter
regulation, we used a dominant-negative Ras construct, pcDNA3/RasN17,
and a dominant-negative p85 construct, pcDNA3/delta p85. Transient
transfection with either construct into U87 MG cells along with the
VEGF promoter construct led to a progressive decrease in promoter
activity with increasing amounts of input plasmid (Fig. 7D)
.
Inhibition of PI(3)
Kinase Does Not Abolish Induction of VEGF mRNA
or the VEGF Promoter by Hypoxia.
Having shown that inhibition of the EGFR/PI(3)
kinase pathway could
lead to decreased VEGF levels under normoxic conditions, we determined
whether this pathway played any role in the induction of VEGF mRNA
under hypoxic conditions. We pretreated U87 MG cells with the EGFR
kinase inhibitor tyrphostin AG1478 (Fig. 8A)
or the PI(3)
kinase inhibitor LY294002 (Fig. 8B)
for 2 h and then subjected them to hypoxia with
0.2% oxygen. The level of VEGF mRNA under normoxic conditions dropped
after incubation with tyrphostin AG1478 (Fig. 8
A, compare
Lanes 3 and 1) and LY294002 (Fig. 8
B,
compare Lanes 3 and 1). However, even
in the presence of tyrphostin AG1478, VEGF mRNA was strongly induced by
hypoxia (Fig. 8
A, compare Lanes 3 and
4). Thus, the hypoxic induction of VEGF mRNA in U87 MG cells
can occur despite pharmacological inhibition of EGFR. These results are
consistent with those shown in Fig. 1
in which U87/T691 cells that have
functional blockade of the EGFR pathway are still inducible for VEGF
mRNA under hypoxic conditions.
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. Because we were interested
specifically in the effect of PI(3)
kinase on VEGF promoter activity,
we transiently transfected the 1.5-kb VEGF promoter reporter construct
into cells, pretreated the cells with LY294002, and then subjected them
to hypoxia. The luciferase levels under normoxia and hypoxia were less
with LY294002 than the corresponding levels in the control set (Fig. 9A)
. However, the induction from normoxia to hypoxia was
similar in the two cases, 2–2.5-fold. These results mirror the
Northern blot results shown in Fig. 8B
. Although LY294002
can decrease VEGF promoter activity by interfering with EGFR/Ras/PI(3)
kinase signaling, it does not completely abolish the hypoxia-induced
rise in VEGF message and promoter activity.
VEGF Promoter Lacking the HRE Is Still Responsive to EGF and PI(3)
Kinase Inhibition.
In all of the previously discussed experiments using the VEGF promoter,
we used the plasmid pGL3–1.5kbVEGFprom, which contains 1.5 kb of the
VEGF promoter including the HRE, which contains a HIF-1 binding site
(Fig. 9B)
. To separate effects mediated by the HRE, we
created another luciferase reporter vector, pGL3–1.2kbVEGFprom, in
which the HRE sequence was deleted (see "Materials and Methods" and
Fig. 9B
). When this reporter was transfected into U87 MG
cells, its activity was still induced in response to EGF (Fig. 9C)
and down-regulated in response to PI(3)
kinase
inhibition (Fig. 9D)
. These experiments show that EGF
stimulation and PI(3)
kinase responsiveness of the VEGF promoter can
occur independently of the HRE.
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DISCUSSION |
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We showed that blockade of EGFR in U87 MG cells led to a down-regulation of VEGF mRNA levels, not through alteration in message stability but through decreased transcription at the level of the VEGF promoter. This is an important distinction because VEGF has been shown to be regulated via changes in RNA stability in some situations such as hypoxia (59 , 60) . To better define the signaling pathway leading to transcription of the VEGF promoter, we used several different pharmacological agents. Treatment with the EGFR inhibitor tyrphostin AG1728 led to a significant decrease in promoter activity, even greater than that seen at the mRNA level. The reason for this is unclear but may relate to factors that independently regulate VEGF mRNA stability.
Treatment of U87 MG cells with a FTI led to a decrease in VEGF mRNA levels as well as in VEGF promoter activity. FTIs have been used to inhibit Ras function in various systems (reviewed in Ref. 62 ) and have been shown to decrease the level of VEGF in H-ras-transformed intestinal cells to levels seen in non-ras-transformed cells (63) . The action of FTIs is not specific for Ras because these drugs will inhibit the function of any protein that requires farnesylation to be active. However, we found there was a progressive decrease in VEGF promoter activity with increasing amounts of transfected RasN17 plasmid, providing specific evidence for the involvement of the Ras pathway in VEGF signaling in glioblastoma cells.
Both the MAP kinase pathway and the PI(3) kinase pathway are known to be downstream of Ras. We have demonstrated that in U87 MG cells, EGF stimulation can activate both the MAP kinase pathway (64) and the PI(3) kinase pathway (65) . In the current study, we have shown that the MAP kinase pathway is not involved in the regulation of VEGF mRNA in these glioblastoma cells by using the specific MEK inhibitor PD98059 (56) . This is in direct contrast to results obtained by other investigators using rodent fibroblasts in which transfection with elements from the Ras/MAP kinase pathway resulted in up-regulation of VEGF expression (66 , 67) , and treatment with the MEK inhibitor PD98059 led to a decrease in VEGF levels (68) .
However, in U87 MG human glioblastoma cells, we found that the PI(3)
kinase inhibitors LY294002 and wortmannin both decreased VEGF mRNA
levels. Conversely, in U87 MG cells transfected with the mutant vIII
EGFR, which is known to constitutively activate PI(3)
kinase, VEGF mRNA
was increased relative to parental U87 MG cells. Therefore, in human
glioblastoma cells, our data argue that VEGF mRNA levels are regulated
by the PI(3)
kinase pathway, not the MAP kinase pathway. We believe
that the difference between our results and those cited above
(66, 67, 68)
using rodent fibroblasts are attributable to
cell-type differences. In support of the importance of cell context,
VEGF regulation was similarly found to be dependent on PI(3)
kinase but
independent of MAP kinase in ras-transformed intestinal
epithelial cells (68)
. PI(3)
kinase has also been
implicated in VEGF regulation in Ras transformed endothelial cells
(69)
, as well as in its regulation by platelet-derived
growth factor in human umbilical vein endothelial cells
(70)
. Zundel et al. (71)
reported
in human glioblastoma cells that the tumor suppressor gene
PTEN regulates PI(3)
kinase induced VEGF expression in a
HIF1-dependent fashion. Zhong et al. (72)
found that EGF could up-regulate the level of HIF1 protein in some
prostate carcinoma cells via a PI(3)
kinase-dependent pathway. However,
we found that a reporter construct in which the HRE containing the HIF1
binding site was deleted was still up-regulated by EGF and
down-regulated by inhibition of PI(3)
kinase. Therefore, our results
indicate that there also exists a pathway involving PI(3)
kinase, but
independent of HIF1, by which EGF can transactivate the VEGF
promoter.
We found that hypoxia could interact with EGF stimulation to increase
VEGF levels in an additive manner. Hypoxic induction of VEGF still
occurred when the EGFR/PI(3)
kinase pathway was inhibited. U87/T691
cells with defective EGFR kinase function still up-regulated VEGF upon
exposure to hypoxia. Likewise, pretreatment of U87 MG cells with the
EGFR inhibitor tyrphostin AG1478 or the PI(3)
kinase inhibitor LY294002
failed to completely abolish the induction of VEGF by hypoxia. We found
that hypoxia in the presence of an intact EGFR/PI(3)
kinase pathway
resulted in higher absolute levels of VEGF mRNA than hypoxia with EGFR
or PI(3)
kinase inactivation. However, the fold-induction of the VEGF
mRNA from normoxia to hypoxia was similar, regardless of whether the
EGFR/PI(3)
kinase pathway was intact. This indicates that the induction
of VEGF by hypoxia can occur despite PI(3)
kinase inhibition. At first
glance, these results appear to be at odds with recent results showing
that PI(3)
kinase can regulate HIF1 activity (71
, 72)
.
However, Zhong et al. (72)
found that HIF1
levels under hypoxic conditions were only partly inhibited by PI(3)
kinase inhibitors at doses that completely inhibited expression under
normoxic conditions. Therefore, it is likely that even if PI(3)
kinase
plays a role in the induction of HIF1 by hypoxia, other signaling
mechanisms are involved as well.
These findings open the way for defining the cis-acting
element(s) in the VEGF promoter, other than the HRE and
trans-acting factors downstream of the PI(3)
kinase pathway,
that are involved in transactivation in response to EGF. To date, there
have only been a few transcription factors other than HIF1 that have
been shown to be regulated by PI(3)
kinase signaling, including the
forkhead family members (73)
and nuclear factor-
B
(74)
. Further work on defining the signal transduction
pathways downstream of EGF might have potential clinical applications
in suggesting ways of decreasing VEGF levels in glioblastomas. For
example, our data suggest that therapeutic approaches that target EGFR
in glioblastomas may also have the added benefit of reducing tumor
angiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Department of Radiation
Oncology, University of Pennsylvania, Philadelphia, PA, and by grants
(to D.M.O.) from the Veterans Administration Merit Review Program and
The Brain Tumor Society. 
2 To whom requests for reprints should be
addressed, at Room 195, John Morgan Building, University of
Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia,
Pennsylvania 19104. Phone: (215) 614-0078; Fax: (215) 898-0090;
E-mail: maity{at}mail.med.upenn.edu
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; HIF1, hypoxia inducible factor 1; EGFR,
epidermal growth factor receptor; PI(3) kinase, phosphatidylinositol
3'-kinase; HRE, hypoxia-responsive element; MAP, mitogen-activated
protein kinase; ERK, extracellular signal-regulated kinase; FTI,
farnesyltransferase inhibitor.
Received 2/24/00. Accepted 8/17/00.
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S. A. Lang, D. Klein, C. Moser, A. Gaumann, G. Glockzin, M. H. Dahlke, W. Dietmaier, U. Bolder, H. J. Schlitt, E. K. Geissler, et al. Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo Mol. Cancer Ther., March 1, 2007; 6(3): 1123 - 1132. [Abstract] [Full Text] [PDF]
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M. Aghi, K. S. Cohen, R. J. Klein, D. T. Scadden, and E. A. Chiocca Tumor Stromal-Derived Factor-1 Recruits Vascular Progenitors to Mitotic Neovasculature, where Microenvironment Influences Their Differentiated Phenotypes. Cancer Res., September 15, 2006; 66(18): 9054 - 9064. [Abstract] [Full Text] [PDF]
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N. Pore, Z. Jiang, A. Gupta, G. Cerniglia, G. D. Kao, and A. Maity EGFR Tyrosine Kinase Inhibitors Decrease VEGF Expression by Both Hypoxia-Inducible Factor (HIF)-1-Independent and HIF-1-Dependent Mechanisms. Cancer Res., March 15, 2006; 66(6): 3197 - 3204. [Abstract] [Full Text] [PDF]
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M. Aghi, P. Gaviani, J. W. Henson, T. T. Batchelor, D. N. Louis, and F. G. Barker II Magnetic Resonance Imaging Characteristics Predict Epidermal Growth Factor Receptor Amplification Status in Glioblastoma Clin. Cancer Res., December 15, 2005; 11(24): 8600 - 8605. [Abstract] [Full Text] [PDF]
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P. H. Thaker, S. Yazici, M. B. Nilsson, K. Yokoi, R. Z. Tsan, J. He, S.-J. Kim, I. J. Fidler, and A. K. Sood Antivascular Therapy for Orthotopic Human Ovarian Carcinoma through Blockade of the Vascular Endothelial Growth Factor and Epidermal Growth Factor Receptors Clin. Cancer Res., July 1, 2005; 11(13): 4923 - 4933. [Abstract] [Full Text] [PDF]
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P. Sini, L. Wyder, C. Schnell, T. O'Reilly, A. Littlewood, R. Brandt, N. E. Hynes, and J. Wood The Antitumor and Antiangiogenic Activity of Vascular Endothelial Growth Factor Receptor Inhibition Is Potentiated by ErbB1 Blockade Clin. Cancer Res., June 15, 2005; 11(12): 4521 - 4532. [Abstract] [Full Text] [PDF]
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D. Vallbohmer, W. Zhang, M. Gordon, D. Y. Yang, J. Yun, O. A. Press, K. E. Rhodes, A. E. Sherrod, S. Iqbal, K. D. Danenberg, et al. Molecular Determinants of Cetuximab Efficacy J. Clin. Oncol., May 20, 2005; 23(15): 3536 - 3544. [Abstract] [Full Text] [PDF]
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B. P. Schneider and K. D. Miller Angiogenesis of Breast Cancer J. Clin. Oncol., March 10, 2005; 23(8): 1782 - 1790. [Full Text] [PDF]
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D. J. Hicklin and L. M. Ellis Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis J. Clin. Oncol., February 10, 2005; 23(5): 1011 - 1027. [Abstract] [Full Text] [PDF]
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P. Traxler, P. R. Allegrini, R. Brandt, J. Brueggen, R. Cozens, D. Fabbro, K. Grosios, H. A. Lane, P. McSheehy, J. Mestan, et al. AEE788: A Dual Family Epidermal Growth Factor Receptor/ErbB2 and Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor with Antitumor and Antiangiogenic Activity Cancer Res., July 15, 2004; 64(14): 4931 - 4941. [Abstract] [Full Text] [PDF]
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A. Zetser, Y. Bashenko, H.-Q. Miao, I. Vlodavsky, and N. Ilan Heparanase Affects Adhesive and Tumorigenic Potential of Human Glioma Cells Cancer Res., November 15, 2003; 63(22): 7733 - 7741. [Abstract] [Full Text] [PDF]
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 J. van den Boom, M. Wolter, R. Kuick, D. E. Misek, A. S. Youkilis, D. S. Wechsler, C. Sommer, G. Reifenberger, and S. M. Hanash Characterization of Gene Expression Profiles Associated with Glioma Progression Using Oligonucleotide-Based Microarray Analysis and Real-Time Reverse Transcription-Polymerase Chain Reaction Am. J. Pathol., September 1, 2003; 163(3): 1033 - 1043. [Abstract] [Full Text] [PDF]
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R. J. Gilbertson, D. A. Hill, R. Hernan, M. Kocak, R. Geyer, J. Olson, A. Gajjar, L. Rush, R. L. Hamilton, S. D. Finkelstein, et al. ERBB1 Is Amplified and Overexpressed in High-grade Diffusely Infiltrative Pediatric Brain Stem Glioma Clin. Cancer Res., September 1, 2003; 9(10): 3620 - 3624. [Abstract] [Full Text] [PDF]
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 V. A. Shatrov, V. V. Sumbayev, J. Zhou, and B. Brune Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) accumulation via redox-dependent mechanisms Blood, June 15, 2003; 101(12): 4847 - 4849. [Abstract] [Full Text] [PDF]
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T. Abe, K. Terada, H. Wakimoto, R. Inoue, E. Tyminski, R. Bookstein, J. P. Basilion, and E. A. Chiocca PTEN Decreases in Vivo Vascularization of Experimental Gliomas in Spite of Proangiogenic Stimuli Cancer Res., May 1, 2003; 63(9): 2300 - 2305. [Abstract] [Full Text] [PDF]
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K. D. Burroughs, J. Oh, J. C. Barrett, and R. P. DiAugustine Phosphatidylinositol 3-Kinase and Mek1/2 Are Necessary for Insulin-Like Growth Factor-I-Induced Vascular Endothelial Growth Factor Synthesis in Prostate Epithelial Cells: A Role for Hypoxia-Inducible Factor-1? Mol. Cancer Res., February 1, 2003; 1(4): 312 - 322. [Abstract] [Full Text] [PDF]
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 K. D. Miller, C. J. Sweeney, and G. W. Sledge The Snark is a Boojum: the continuing problem of drug resistance in the antiangiogenic era Ann. Onc., January 1, 2003; 14(1): 20 - 28. [Abstract] [Full Text] [PDF]
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N. Pore, S. Liu, D. A. Haas-Kogan, D. M. O'Rourke, and A. Maity PTEN Mutation and Epidermal Growth Factor Receptor Activation Regulate Vascular Endothelial Growth Factor (VEGF) mRNA Expression in Human Glioblastoma Cells by Transactivating the Proximal VEGF Promoter Cancer Res., January 1, 2003; 63(1): 236 - 241. [Abstract] [Full Text] [PDF]
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J. S. Eshleman, B. L. Carlson, A. C. Mladek, B. D. Kastner, K. L. Shide, and J. N. Sarkaria Inhibition of the Mammalian Target of Rapamycin Sensitizes U87 Xenografts to Fractionated Radiation Therapy Cancer Res., December 15, 2002; 62(24): 7291 - 7297. [Abstract] [Full Text] [PDF]
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M. Mayerhofer, P. Valent, W. R. Sperr, J. D. Griffin, and C. Sillaber BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1alpha , through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin Blood, November 15, 2002; 100(10): 3767 - 3775. [Abstract] [Full Text] [PDF]
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S. A. Woods, C. J. McGlade, and A. Guha Phosphatidylinositol 3'-kinase and MAPK/ERK kinase 1/2 differentially regulate expression of vascular endothelial growth factor in human malignant astrocytoma cells Neuro-oncol, October 1, 2002; 4(4): 242 - 252. [Abstract] [PDF]
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S. Kaluz, M. Kaluzova, A. Chrastina, P. L. Olive, S. Pastorekova, J. Pastorek, M. I. Lerman, and E. J. Stanbridge Lowered Oxygen Tension Induces Expression of the Hypoxia Marker MN/Carbonic Anhydrase IX in the Absence of Hypoxia-inducible Factor 1{alpha} Stabilization: A Role for Phosphatidylinositol 3'-Kinase Cancer Res., August 1, 2002; 62(15): 4469 - 4477. [Abstract] [Full Text] [PDF]
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C. Treins, S. Giorgetti-Peraldi, J. Murdaca, G. L. Semenza, and E. Van Obberghen Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway J. Biol. Chem., July 26, 2002; 277(31): 27975 - 27981. [Abstract] [Full Text] [PDF]
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C. B. Knobbe, A. Merlo, and G. Reifenberger Pten signaling in gliomas Neuro-oncol, July 1, 2002; 4(3): 196 - 211. [Abstract] [PDF]
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Y. Liang, X.-Y. Li, E. J. Rebar, P. Li, Y. Zhou, B. Chen, A. P. Wolffe, and C. C. Case Activation of Vascular Endothelial Growth Factor A Transcription in Tumorigenic Glioblastoma Cell Lines by an Enhancer with Cell Type-specific DNase I Accessibility J. Biol. Chem., May 24, 2002; 277(22): 20087 - 20094. [Abstract] [Full Text] [PDF]
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M. Alvarez-Tejado, A. Alfranca, J. Aragones, A. Vara, M. O. Landazuri, and L. del Peso Lack of Evidence for the Involvement of the Phosphoinositide 3-Kinase/Akt Pathway in the Activation of Hypoxia-inducible Factors by Low Oxygen Tension J. Biol. Chem., April 12, 2002; 277(16): 13508 - 13517. [Abstract] [Full Text] [PDF]
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S. D. Averbuch Lung Cancer Prevention: Retinoids and the Epidermal Growth Factor Receptor--A Phoenix Rising? Clin. Cancer Res., January 1, 2002; 8(1): 1 - 3. [Full Text] [PDF]
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A. Viloria-Petit, T. Crombet, S. Jothy, D. Hicklin, P. Bohlen, J. M. Schlaeppi, J. Rak, and R. S. Kerbel Acquired Resistance to the Antitumor Effect of Epidermal Growth Factor Receptor-blocking Antibodies in Vivo: A Role for Altered Tumor Angiogenesis Cancer Res., July 1, 2001; 61(13): 5090 - 5101. [Abstract] [Full Text] [PDF]
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S. Murono, H. Inoue, T. Tanabe, I. Joab, T. Yoshizaki, M. Furukawa, and J. S. Pagano Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells PNAS, May 24, 2001; (2001) 121016998. [Abstract] [Full Text] [PDF]
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S. Murono, H. Inoue, T. Tanabe, I. Joab, T. Yoshizaki, M. Furukawa, and J. S. Pagano Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells PNAS, June 5, 2001; 98(12): 6905 - 6910. [Abstract] [Full Text] [PDF]
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and 
, U87 MG cells; • and 
,
U87/T691 cells. 
