 |
INTRODUCTION |
Vasular Endothelial Growth Factor
(VEGF)1 plays a key role in
tumor angiogenesis. VEGF occurs in at least six isoforms of 121, 145, 165, 183, 189, and 206 amino acids, which are generated from a single
gene by alternative splicing (1). We have reported that
VEGF165 overexpression stimulates angiogenesis in human
ovarian cancer xenografts (2).
Elevated expression of VEGF in tumor cells can be the result of
environmental factors, such as hypoxia, or mutations in oncogenes or
tumor suppressor genes that regulate growth factor signal transduction pathways (3-9). Many stimuli, including hypoxia, growth factors, hormones, and oxidative stressors, can increase VEGF expression in
tumor cells in vitro (6, 10-14). In all mentioned cases, increased VEGF expression can be caused in part by increased VEGF gene
transcription mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) (4-7, 11, 13-15). HIF-1 binds to a
hypoxia-responsive element (HRE) located within the VEGF promoter
(11).
HIF-1 is composed of two subunits, HIF-1
and HIF-1
. The activity
of HIF-1 is regulated mainly by the expression and activity of the
HIF-1
subunit. Although HIF-1
protein is rather stable and
readily detected in the nucleus of most normoxic cells, HIF-1
protein is often hardly detectable because of rapid degradation by the
ubiquitin-proteasome system (16-18). Hypoxia increases the level of
HIF-1
protein by inhibiting its ubiquitination and degradation (19).
Accumulation of HIF-1
protein can also be observed in stimulated
normoxic cells (5, 8, 9, 13-15). HIF-1
is subsequently translocated
to the nucleus, where it can dimerize with HIF-1
to form the HIF-1
complex (20). To be fully active, HIF-1
requires interaction(s) with
coactivators or/and transcription factor(s) (20-23). The activated
protein-1 transcription factor family member c-Jun interacts with
HIF-1
and is suggested to cooperate with HIF-1 in the induction of
VEGF expression by hypoxia (23).
The stabilization and transcriptional activation of the HIF-1
protein involve changes in its phosphorylation state. Activation of the
lipid kinase phosphatidylinositol 3-kinase (PI3K), and/or its
downstream target the protein-serine/threonine kinase Akt, can result
in the phosphorylation and stabilization of HIF-1
under hypoxic and
normoxic conditions (8, 24). In addition, inhibition of PI3K activity
has been shown to reduce the transactivation function of HIF-1
in
hypoxic cells (25). So far, there is no evidence that PI3K and Akt can
phosphorylate HIF-1
directly. Other pathways that regulate HIF-1
phosphorylation involve members of the mitogen-activated protein kinase
(MAPK) family (26). In hypoxic and in stimulated normoxic cells,
p44/p42 MAPK (extracellular signal-regulated kinase (ERK)-1 and ERK-2)
and p38 MAPK enhance the transactivation function of HIF-1
(24, 25,
27). This may occur through direct phosphorylation because these
kinases have been shown to phosphorylate HIF-1
in vitro
(28, 29). In some cases, p44/p42 MAPK and p38 can also influence
HIF-1
protein induction (8, 30). The stress-activated protein
kinases/c-Jun N-terminal kinases (SAPKs/JNKs) do not phosphorylate
HIF-1
in vitro, but may indirectly regulate
HIF-1-mediated transcription of VEGF under hypoxia through
phosphorylating the transcription factor c-Jun (18, 23).
The stabilization and transcriptional activation of HIF-1
may also
involve alterations in its redox state. Evidence is provide that
changes in the levels of reactive oxygen species (ROS) may play a role
in HIF-1
protein induction and HIF-1 transactivation (14, 30-32).
Increased levels of ROS are suggested to mediate PI3K activation under
hypoxia as well as under normoxia (32, 33). The levels of ROS may also
directly or indirectly influence the redox status of cysteine residues
in the transactivation domains of HIF-1
, which can affect
interactions with transcriptional coactivators (21, 22).
In a previous study on the role of oxidative stress in the regulation
of VEGF, we showed that sodium arsenite (NaAsO2) induces HIF-1
protein and VEGF mRNA and protein levels in the human
ovarian cancer cell lines OVCAR-3 and H134 (34). Arsenite induces
oxidative stress by binding to thiol groups of cellular proteins and by increasing the production of ROS. Because the effects of sodium arsenite on HIF-1
protein and VEGF expression are independent of
increased ROS production, we hypothesized that they may be mediated
through binding of arsenite to thiol (SH) groups of the HIF-1
protein itself or of components of signal transduction pathways
involved in HIF-1 or VEGF regulation (34).
Our findings with sodium arsenite may be of clinical relevance. Several
cytotoxic agents in cancer treatment are susceptible of interacting
with thiol groups of cellular proteins. Moreover, arsenic trioxide
(As2O3), another trivalent arsenic compound, has potential as an anticancer agent (35). At low dosages (1-10 µM), arsenic trioxide has a significant cytotoxic effect
on human ovarian cancer cell lines and is suggested to be a useful
agent for the treatment of ovarian cancer (36). Therefore, we now compared the potency of arsenic trioxide with that of sodium arsenite to induce HIF-1
protein and VEGF mRNA and protein levels in the OVCAR-3 human ovarian cancer cell line. We also investigated the role
of the PI3K/Akt pathway and of MAPK family members in sodium arsenite-induced HIF-1
protein accumulation and VEGF expression. Furthermore, we examined whether up-regulation of VEGF mRNA
expression by sodium arsenite was mediated by HIF-1.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Sodium arsenite, arsenic trioxide, wortmannin,
glutathione (GSH), N-acetylcysteine (NAc), and
buthionine-sulfoximine (BSO) were purchased from Sigma. PD98059,
SB203580 and SB202190 were purchased from Calbiochem.
Cell Culture and Cell Treatment--
OVCAR-3 human ovarian
cancer cells and Jnk1+/
Jnk2
/
,
Jnk1
/
Jnk2
/
,
HIF-1
+/+, and
HIF-1
/
mouse fibroblasts were cultured in
Dulbecco's modified Eagle's medium (Invitrogen) supplemented with
10% heat-inactivated fetal calf serum (Sanbio, Uden, The Netherlands),
50 units/ml penicillin (ICN Biochemicals, Zoetermeer, The Netherlands),
and 50 µg/ml streptomycin (ICN Biochemicals). Cells were routinely
cultured in 95% air and 5% CO2 at 37 °C. Hypoxic
conditions were performed by incubation of cells in a tightly sealed
chamber maintained at 1% oxygen, 94% N2, and 5%
CO2 at 37 °C. Immortalized Jnk1+
Jnk2
/
and
Jnk1
/
Jnk2
/
cell
lines were kindly provided by Dr. E. F. Wagner (Research Institute of
Molecular Pathology, Vienna, Austria). These cell lines have been
established independently from individual primary mouse embryo
fibroblasts that were each isolated from a single mouse embryo
following the 3T3 protocol (37, 38). Immortalized HIF-1
+/+ and
HIF-1
/
cell lines were kindly provided by
Dr. G. L. Semenza (Johns Hopkins University School of Medicine,
Baltimore, MD). Both cell lines were established by transfection of
primary mouse embryo fibroblast cultures with an expression vector
encoding simian virus 40 T-antigen as described elsewhere (39).
For treatment of cells with sodium arsenite or arsenic trioxide, cells
were seeded in culture dishes in medium and grown overnight. Thereafter, sodium arsenite or arsenic trioxide was added to the conditioned medium, and cells were incubated further for the time periods as indicated in each experiment. Pretreatment of cells with
wortmannin, PD98059, SB203580, SB202190, GSH, and NAc was performed for
1 h before the addition of sodium arsenite; pretreatment with BSO
was performed for 16 h.
Antibodies--
Rabbit polyclonal antisera directed against
phospho-Akt-1 (Ser473), phospho-p44/p42 MAPK
(Thr202/Tyr204), phospho-SAPK/JNK
(Thr183/Tyr185), phospho-p38 MAPK
(Thr180/Tyr182), phospho-c-Jun
(Ser73), p44/p42 MAPK, SAPK/JNK, p38 MAPK, and a
horseradish peroxidase-coupled anti-rabbit antiserum were purchased
from New England Biolabs. Mouse monoclonal antisera to HIF-1
were
purchased from Novus Biologicals/AbCam (Cambridge, U. K.) and BD
Transduction Laboratories (Alphen a/d Rijn, The Netherlands). The sheep
polyclonal antiserum directed against Akt-1 was from Upstate
Biotechnology Inc. Rabbit polyclonal antisera directed against c-Jun
(H-79) and Raf-1 (C12) were purchased from Santa Cruz Biotechnology.
The mouse monoclonal antiserum against
-actin (C4) was purchased
from Roche Molecular Biochemicals. The polyclonal human antiserum
against topoisomerase I was purchased from TopoGEN (Columbus, OH). The
mouse monoclonal antiserum against human p53 (DO-7) and the horseradish
peroxidase-coupled anti-mouse serum were purchased from DAKO (Glostrup,
Denmark). The horseradish peroxidase-coupled anti-sheep serum was
purchased from Calbiochem.
Preparation of Cell Extracts for Western Blot Analysis and
ELISA--
Cells were washed once with ice-cold phosphate-buffered
saline and lysed by scraping with a rubber policeman in 250 µl of radioimmune precipitation assay buffer (10 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, and 1%
sodium deoxycholate) for Western blotting or in 450 µl of E1A buffer
(50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, and 0.1% Nonidet P-40) for ELISA. Both lysis
buffers were supplemented with 50 mM NaF, 1 mM
Na3VO4, 1.0 mM phenylmethylsulfonyl
fluoride, 0.5 mM trypsin inhibitor, and 0.5 µg/ml
leupeptin. After a 15-min incubation period on ice, the extracts were
clarified by centrifugation at 14,000 rpm for 15 min at 4 °C and
stored at
70 °C. Isolation of nuclear and cytoplasmic protein
fractions from OVCAR-3 cells in the subcellular fractionation
experiment was performed as described previously (40). Protein
concentrations were determined by the Coomassie Plus Protein assay (Pierce).
Western Blotting--
Equal amounts of protein cell extracts
were resolved in SDS-polyacrylamide gels and transferred
electrophoretically onto a polyvinylidene difluoride membrane
(Immobilon). Membranes were blocked for 1 h in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.025% Tween
20) and 5% milk and incubated overnight with antiserum directed
against phospho-Akt-1(1:3,000), phospho-p44/p42 MAPK (1:1,000),
phospho-SAPK/JNK (1:1,000), phospho-p38 (1:1,000), phospho-c-Jun
(1:1,000), HIF-1
(1:500),
-actin (1:2,000), Raf-1 (1:500),
topoisomerase I (1:5,000) or p53 (1:1,000). After washing with TBST,
the membranes were incubated for 1 h with horseradish peroxidase-linked anti-rabbit or anti-mouse antiserum in TBST and 5%
milk. The membranes were washed again with TBST, and proteins were
visualized by enhanced chemiluminescence. To detect both nonphosphorylated and phosphorylated forms of Akt-1, c-Jun, and the
MAPK family members, membranes were stripped for 15 min in strip buffer
(10 mM Tris, pH 8.0, 2% SDS, 10 mM
-mercaptoethanol) at 42 °C and washed with TBST. Subsequently,
the blots were blocked again and incubated for 2 h with Akt-1,
p44/p42 MAPK, SAPK/JNK, or p38-specific antiserum (1:1,000 dilution) or
with a c-Jun-specific antiserum (1:500 dilution) in TBST and 5% milk.
The incubations with horseradish peroxidase-linked anti-rabbit,
anti-mouse, and anti-sheep antisera and the detection of the proteins
were performed as described above.
ELISA--
Equal numbers of cells were plated on 9.6-mm culture
dishes and grown overnight. The conditioned medium of all dishes was collected, pooled, and 450 µl was sampled (time-point
(T) = 0 medium sample). Cells of one dish were
washed and lysed in 450 µl of lysis buffer as described above
(T = 0 lysate sample). The conditioned medium was
divided into 5 volumes. Sodium arsenite was added to 1 volume at a
concentration of 100 µM, and arsenic trioxide was added
to 3 volumes at concentrations of 100, 50, and 10 µM.
Subsequently, conditioned medium with or without sodium arsenite or
arsenic trioxide was again added to culture dishes with cells. Thus, at
T = 0 the amount of VEGF protein in the medium was the
same in each culture dish. Cells were incubated further for the time
periods indicated in each experiment. Thereafter, 450 µl of
conditioned medium was sampled, and cells were lysed. VEGF
concentrations in nondiluted media samples and lysates were determined
in duplicate by ELISA using the reagents and the protocol supplied with
the Quantikine Human VEGF Immunoassay kit (R&D Systems). Differences in
VEGF concentrations in medium and lysates of nontreated versus sodium arsenite- or arsenic trioxide-treated cells
were evaluated using Student's t test for two groups.
p values <0.05 were considered to be significant.
RNase Protection Assay--
Generation of human
-actin and
VEGF165 antisense probes has been described elsewhere (34).
pcDNA3 vectors (Invitrogen) containing a fragment of murine
VEGF164 or
-actin cDNA were used as templates for
the synthesis of murine VEGF164 and
-actin antisense probes. The templates were generated as follows. Total RNA from Jnk1+/
Jnk2
/
mouse
fibroblasts was reversed transcribed using Moloney murine leukemia
virus reverse transcriptase (Invitrogen). Murine VEGF164 cDNA (nucleotides 454-648) was amplified with the forward
primer 5'-ATAACAAGCTTAGCACAGCAGATGTGAATGC-3' and
the reversed primer
5'-GCAACCTCGAGCTTGTCACATCTGCAAGTAC-3'.
Murine
-actin cDNA (nucleotides 630-780) was amplified with the
forward primer
5'-ATAACAAGCTTGCTATGTTGCCCTGGATTTTGAG-3' and the reversed primer 5'-GCAACCTCGAGGGA
AGGAAGGCTGGAAGAGT-3'. Amplification of VEGF164
and
-actin cDNA fragments was performed for 36 cycles at
94 °C, 56 °C, and 72 °C. In addition to VEGF164 or
-actin sequences (underlined), the forward and reverse primers contain restriction sites (bold) for HindIII and
XhoI, respectively. The 216-nucleotide VEGF164
and 172-nucleotide
-actin amplified fragments were extracted with
phenol/chloroform, precipitated, dissolved in water, and digested with
HindIII and XhoI. The digestion mixtures were
subjected to gel electrophoresis, and the VEGF164 and
-actin cDNA fragments were isolated from the agarose gel by use
of the QIAquick gel extraction kit (Qiagen). The isolated VEGF164 and
-actin cDNA fragments were cloned into
the HindIII and XhoI restriction sites of
pcDNA3, and the sequence of the VEGF164 and
-actin
cDNA fragments was verified by sequencing. pcDNA3VEGF164 and pcDNA3
-actin were linearized
with HindIII, and 228-nucleotide VEGF164 and
185-nucleotide
-actin antisense probes were generated with SP6 polymerase.
The RNase protection assay was carried out as described (34).
Hybridization of total RNA to a 136-nucleotide human
-actin antisense probe gives rise to a protected fragment of 130 nucleotide, whereas hybridization to the 185-nucleotide murine
-actin antisense probe is expected to result in the protection of a fragment of 151 nucleotides. It should be noted that hybridization of total RNA to the
301-nucleotide human VEGF165 antisense probe and the 228-nucleotide murine VEGF164 can give rise to fragments of
different sizes because of protection by VEGF mRNAs of different
isoforms (1, 34, 41, 42). Protection by human VEGF165
mRNA and murine VEGF164 mRNA is most efficient,
giving rise to protected fragments of 252 and 195 nucleotides,
respectively. Therefore, effects on VEGF mRNA levels were monitored
by assessing the mRNA levels of human VEGF165 and
murine VEGF164.
Plasmid Constructs--
The pGL3 promoter vector was purchased
from Promega. This vector contains the SV40 basal promoter upstream of
luciferase coding sequences and the SV40 late polyadenylation signal.
5xHREpGL3 contains five copies of a HIF-1 consensus sequence of the
human VEGF promoter and was cloned from the reporter plasmid
5HRE/hCMVmp (43). 5HRE/hCMVmp was digested with KpnI and
BglII, and the KpnI-BglII fragment was
ligated into the KpnI-BglII-digested
pGL3-promoter vector. 5xjun2pGL3 contains five copies of the
jun2 element of the human c-jun promoter (44) and
was constructed by ligation of the PvuII-BglII
fragment of the 5xjun2-tata-luciferase reporter construct (45) in the
SmaI-BglII-digested pGL3 promoter vector.
Transient Transfection and Luciferase Assay--
OVCAR-3 cells
were seeded on six-well culture plates and grown overnight in medium.
Transfection of OVCAR-3 cells with luciferase reporter constructs was
performed in duplicate wells by the calcium-phosphate precipitation
method (46). After a 6-h incubation period with the precipitate, the
cells were washed and refed with culture medium. 40 h later, cells
were left untreated or exposed to sodium arsenite or hypoxia for 6 h. Cells were washed with phosphate-buffered saline and lysed in 250 µl of lysis reagent (25 mM Tris phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM CDTA, 10% glycerol,
1% Triton X-100). Protein concentrations were determined by the
Coomassie Plus Protein assay and
-galactosidase activity was
assessed as described by Sambrook et al. (47). Luciferase
activity was determined by mixing 30 µl of cell extract with 100 µl
of luciferase assay reagent (Promega) and subsequent measurement of
relative light units for 10 s in a luminometer (Lumat LB 9507, EG&G Berthold, Bad Wildbad, Germany). The mean relative luciferase
activity was calculated as the mean luciferase activity (relative light
units)/
-galactosidase (A420/µg of
protein/min) of duplicate extracts.
 |
RESULTS |
High Concentrations of Arsenic Compounds Induce HIF-1
Protein
and VEGF Expression in OVCAR-3 Cells--
We compared the effects of
different concentrations of arsenic trioxide with those of sodium
arsenite on HIF-1
protein, VEGF mRNA, and VEGF protein
expression in OVCAR-3 cells (Fig. 1). As can be seen in Fig. 1A, high concentrations of arsenic
trioxide (100 and 50 µM) could indeed induce HIF-1
protein after 4 and 8 h in OVCAR-3 cells, but to a much lesser
extent than an equimolar concentration of sodium arsenite (100 µM). We also tested the effect of 8- and 24-h exposure
periods to 10 µM arsenic trioxide. This treatment did not
induce detectable changes in HIF-1
protein expression in OVCAR-3
cells.

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Fig. 1.
Effect of sodium arsenite and trivalent
arsenic on the levels of HIF-1 protein,
VEGF165 mRNA, and VEGF165 protein in
OVCAR-3 cells. OVCAR-3 cells were exposed to 100 µM
sodium arsenite or to 100, 50, or 10 µM arsenic trioxide
for the indicated time periods. After collection of conditioned medium,
cells were lysed, and protein and total RNA were extracted.
A, HIF-1 protein levels were assessed by subjecting 100 µg of protein to SDS-gel electrophoresis (7.5% gel) followed by
Western blotting with a HIF-1 -directed antiserum. An unidentified
protein that is nonspecifically recognized by the HIF-1 -directed
antiserum or by the secondary horseradish peroxidase-linked antiserum
is indicated as NS. B, VEGF165
mRNA levels were assessed by the RNase protection assay. Total RNA
was hybridized to human VEGF165 and -actin antisense
probes. t-RNA (lane T) was hybridized as a negative
control. The 252- and 130-nucleotide fragments protected by the
mRNAs of VEGF165 and -actin and the full-length
probes are indicated. The histogram indicates the relative
VEGF165 mRNA expression in sodium arsenite or arsenic
trioxide-treated OVCAR-3 cells in which nontreated controls
(T = 0) were set at 100%. C,
VEGF165 protein concentrations in conditioned media
(left panel) and lysates (right panel) of
sodium arsenite or arsenic trioxide-treated OVCAR-3 cells were
determined by ELISA. As a control, VEGF protein concentrations were
also measured in conditioned media and lysates of nontreated cells. The
histograms represent the mean ± S.D. of duplicate samples in a
representative experiment. Significant differences in mean VEGF
concentrations in medium and lysates of nontreated versus
sodium arsenite or arsenic trioxide-treated cells after individual
incubation periods are indicated by an asterisk
(p < 0.05). Results of sodium arsenite and arsenic
trioxide as shown in A, B, and C were
assessed in parallel in one experiment that was representative of three
independent ones giving comparable results.
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|
The effects of arsenic trioxide on VEGF mRNA expression were
assessed by the RNase protection assay (Fig. 1B). As
determined by measurement of the signal intensities of the
252-nucleotide VEGF165- and 130-nucleotide
-actin-protected fragments, VEGF165 mRNA levels in
OVCAR-3 cells were increased 3- and 5-fold after 4 and 8 h of
exposure to 100 µM arsenic trioxide, respectively. A 2- and 3-fold increase in VEGF165 mRNA expression was
observed after 4 and 8 h of exposure to 50 µM
arsenic trioxide. An 8-h exposure period to low concentrations of
arsenic trioxide (10 µM) did not influence the level of
VEGF165 mRNA, whereas a weak elevation (1.5-fold) was
observed after 24 h of exposure. In agreement with the effects on
HIF-1
protein, arsenic trioxide at 100 µM was a less
potent inducer of VEGF165 mRNA than sodium arsenite. 100 µM sodium arsenite increased the level of
VEGF165 mRNA up to 6- and 7-fold after 4 and 8 h
of exposure.
We next assessed whether induction of VEGF mRNA levels by arsenic
trioxide resulted in an increased production of VEGF protein. OVCAR-3
cells were grown overnight, and arsenic trioxide and sodium arsenite
were added to the conditioned medium. VEGF concentrations in the
conditioned medium or in cell lysates of treated and nontreated cells
were measured by ELISA. As can be seen in Fig. 1C, VEGF protein levels in the conditioned medium were increased significantly after 8 h of exposure to 100 and 50 µM of arsenic
trioxide compared with the levels in the medium of control cells
(p < 0.05). In the lysates, increased production of
VEGF was evident after 4 h of incubation and was more pronounced
after 8 h (p < 0.05). As expected, a higher
increase in VEGF protein concentrations in conditioned medium and
lysate was observed after treatment with 100 µM sodium
arsenite. Note that a weak elevation of VEGF protein levels was
detected in conditioned medium and lysate of cells that were exposed to
10 µM arsenic trioxide for 24 h. These results
clearly show that arsenic trioxide induces HIF-1
protein accumulation and VEGF expression in OVCAR-3 cells, albeit less potently
than sodium arsenite. In addition, these results indicate that long
exposure to a low, clinically relevant concentration of arsenic
trioxide can weakly increase VEGF165 mRNA and protein expression in the absence of a detectable change in the level of
HIF-1
protein expression.
Induction of HIF-1
Protein and VEGF Expression by Sodium
Arsenite Is Not Mediated through PI3K--
Because it has been
suggested that sodium arsenite can activate PI3K in some cell types, we
investigated the role of this pathway in sodium arsenite-induced
HIF-1
protein accumulation and VEGF expression in OVCAR-3 cells
(48). PI3K-mediated activation of Akt-1, the most frequently studied
isoform of Akt, involves phosphorylation on its amino acid residues
Ser473 and Thr308. We first monitored the
phosphorylation of Akt-1 upon sodium arsenite treatment in OVCAR-3
cells by Western blotting with an antiserum recognizing
Ser473-phosphorylated Akt-1. As can be seen in Fig.
2A, OVCAR-3 cells showed a
relatively high basal level of phospho-Akt-1. After 15 min of exposure
to 100 µM sodium arsenite, Akt-1 phosphorylation on
Ser473 was increased, which was sustained until at least
4 h of exposure. After 8 h, the levels of phospho-Akt-1 were
decreased to basal. The same results were obtained with an antiserum
directed against Thr308 (data not shown). The levels of
total Akt-1 were unchanged upon sodium arsenite treatment for up to
8 h. We examined further the involvement of PI3K in sodium
arsenite-induced Akt-1 phosphorylation by assessing the effects of the
PI3K inhibitor wortmannin. Fig. 2B shows that pretreatment
with 100 nM wortmannin completely blocked induction of
Akt-1 phosphorylation by sodium arsenite. Wortmannin alone did not
significantly affect the basal level of phospho-Akt-1. Again, the total
levels of Akt-1 protein remained constant. These results suggest that
sodium arsenite indeed activates the PI3K/Akt-1 pathway in OVCAR-3
cells.

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Fig. 2.
Effect of wortmannin on sodium
arsenite-induced Akt-1 phosphorylation, HIF-1
protein accumulation, and VEGF expression in OVCAR-3 cells.
OVCAR-3 cells were exposed to 100 µM sodium arsenite in
the absence or presence of 100 or 500 nM wortmannin or to
100 or 500 nM wortmannin alone. Wortmannin was added 1 h before the addition of sodium arsenite. After the indicated periods
of exposure to sodium arsenite, cells were lysed, and protein and total
RNA were extracted. A and B, the phosphorylation
of Akt-1 was monitored by subjecting 100 µg of protein to SDS-gel
electrophoresis (8% gel) followed by Western blotting with an
antiserum specific to Ser(P)473. The blots were stripped
and reprobed with a total Akt-1 antiserum. C, HIF-1
protein levels were examined by subjecting 100 µg of protein to
SDS-gel electrophoresis (7.5% gel) followed by Western blotting with a
HIF-1 -directed antiserum. D, VEGF165 mRNA
levels were analyzed by RNase protection as in B.
|
|
We next investigated the effect of wortmannin on sodium
arsenite-induced HIF-1
protein accumulation by analyzing HIF-1
protein levels in the extracts of Fig. 2A by Western blot.
In sharp contrast to the phosphorylation of Akt-1, induction of
HIF-1
protein by sodium arsenite was completely unaffected by 100 nM wortmannin (Fig. 2C). Pretreatment with
wortmannin concentrations as high as 500 nM only weakly
inhibited HIF-1
protein accumulation. Analysis of total RNA by the
RNase protection assay in parallel samples revealed that neither sodium
arsenite-induced VEGF165 mRNA levels nor the basal
VEGF165 mRNA levels in OVCAR-3 cells were influenced by
pretreatment with 100 and 500 nM concentrations of
wortmannin (Fig. 2D). These results strongly suggest that
induction of HIF-1
protein and VEGF expression by sodium arsenite is
independent of the PI3K/Akt-1 pathway in OVCAR-3 cells.
Sodium Arsenite activates p44/p42 MAPK, SAPK/JNK, and p38 with
Different Kinetics in OVCAR-3 Cells--
Depending on the cell type,
sodium arsenite can activate different MAPK family members (49), which
may contribute to HIF-1
protein accumulation and VEGF expression.
Therefore, we first monitored the activity of p44/p42 MAPK, SAPK/JNK,
and p38 upon sodium arsenite treatment in OVCAR-3 cells by detection of
their phosphorylated, activated forms on Western blots.
As can be seen in Fig. 3, nonstimulated
OVCAR-3 cells showed strong phosphorylation of p42/p44 MAPK. This is
consistent with a previous study reporting a high basal activity of
p44/p42 MAPK in these cells (50). A weak increase in the
phosphorylation of p44/p42 MAPK was observed as early as 5 min after
the addition of 100 µM sodium arsenite and remained
detectable until at least 1 h of exposure. The phosphorylation of
both kinases returned to basal levels after 2 h and was even
reduced to lower levels after 4 and 8 h. Sodium arsenite also
induced the activity of JNK1, JNK2, and p38, albeit with different
kinetics. A strong increase in the phosphorylation of JNK1, JNK2, and
p38 was observed after 1 h of sodium arsenite treatment, and
increased phosphorylation of all three kinases was prolonged until at
least 8 h of treatment. Changes in phosphorylation of p42/p44
MAPK, JNK1, JNK2, and p38 were not accompanied by alterations in the
levels of these proteins. These data indicate that sodium arsenite
activates p44/p42 MAPK, JNK1, JNK2, as well as p38 with different
kinetics in OVCAR-3 cells. Activation of p44/p42 MAPK occurred rapidly
but was only moderate and transient. In contrast, activation of
JNK1, JNK2, and p38 was slower but more potent and sustained.

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Fig. 3.
Effect of sodium arsenite on the
phosphorylation of MAPK family members in OVCAR-3 cells. OVCAR-3
cells were exposed to 100 µM sodium arsenite for the
indicated time periods. 25 µg of protein was subjected to SDS-gel
electrophoresis (10% gel) and Western blotting. Phosphorylation of
p44/p42 MAPK, JNK1/2 (p46, p54), and p38 was detected using
phospho-antisera. The blots were stripped and reprobed with antisera
recognizing both nonphosphorylated and phosphorylated forms of p44/p42
MAPK, JNK1/2 (p46, p54), and p38.
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Sodium Arsenite-induced HIF-1
Protein Accumulation and VEGF
Expression Are Attenuated by Inhibitors of p38 MAPK Activity--
To
examine further the involvement of p44/p42 MAPK and p38 in the
induction of HIF-1
protein and VEGF expression by sodium arsenite,
we analyzed the effect of the kinase inhibitors PD98059, SB202190, and
SB203580 in OVCAR-3 cells. PD98059 is a specific inhibitor of MAPK/ERK
kinase (MEK)-1 and MEK-2, which phosphorylate and activate p44/p42
MAPK. SB202190 and SB203580 directly inhibit the activity of p38 by
binding to its ATP binding domain.
Fig. 4, A-C, shows that
induction of HIF-1
protein and VEGF165 mRNA
expression after 4 and 8 h of exposure to 100 µM
sodium arsenite was slightly, but not significantly, reduced by
pretreatment with high concentrations of PD98059 (50 µM).
The basal level of HIF-1
protein and VEGF165 mRNA
was also hardly affected by this agent. PD98058 was functional in
inhibiting p44/p42 MAPK activity at the concentrations used, as
demonstrated by the reduced level of phospho-p44/p42 MAPK in the
presence and absence of sodium arsenite (Fig. 4D). In fact,
phospho-p44/p42 MAPK levels were even below basal and hardly detectable
after 4 and 8 h of sodium arsenite and PD98059. These data
indicate that p44/p42 MAPK does not regulate induction of HIF-1
protein and VEGF expression in OVCAR-3 cells.

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Fig. 4.
Effect of kinase inhibitors on sodium
arsenite-induced HIF-1 protein and VEGF
mRNA levels in OVCAR-3 cells. OVCAR-3 cells were exposed to
100 µM sodium arsenite in the absence or presence of the
kinase inhibitors PD98059 (50 µM), SB202190 (20 µM), and SB203580 (20 and 5 µM) or to the
individual kinase inhibitors alone. The kinase inhibitors were added
1 h before the addition of sodium arsenite. After the indicated
periods of exposure to sodium arsenite, cells were lysed, and protein
and RNA were extracted. A, HIF-1 protein levels were
assessed as in Fig. 2C. B, VEGF165
mRNA levels were determined as in Fig. 1B. C,
the relative VEGF165 mRNA expression was determined by
signal intensity measurements of VEGF165- and
-actin-protected fragments on autoradiographs and subsequent
normalization of VEGF165 values against -actin internal
control values. The values in the bar graph are the
means ± S.D. of the relative VEGF165 mRNA
expression of three independent experiments in which nontreated
controls were set at 100%. Significant differences between the
relative VEGF165 mRNA expression after incubation with
sodium arsenite and that of control cells are indicated by an
asterisk (p < 0.05). D,
phosphorylation of p44/p42 MAPK was examined as described in Fig. 3.
E, phosphorylation of c-Jun was monitored by sub- jecting 100 µg of protein to SDS-gel electrophoresis (10% gel)
followed by Western blotting with an antiserum specific to
Ser(P)73. The blot was stripped and reprobed with an
antiserum recognizing both nonphosphorylated and phosphorylated forms
of c-Jun.
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Pretreatment with the p38 inhibitors SB202190 and SB203580 could
potently inhibit induction of HIF-1
protein and VEGF mRNA expression by sodium arsenite (Fig. 4, A-C). As assessed at
4 and 8 h after the addition of 100 µM sodium
arsenite, both effects were almost completely attenuated by a 20 µM concentration of each inhibitor. Significant
inhibition of HIF-1
protein accumulation and VEGF mRNA
expression was also observed by pretreatment with 5 µM
SB202190 (data not shown) and SB203580 (Fig. 4, A-C).
Treatment with the two inhibitors alone did not significantly influence the basal level of HIF-1
protein and VEGF165 mRNA in
OVCAR-3.
In some cell types, high concentrations (20-100 µM) of
SB202190 or/and SB203580 can inhibit the activity of JNKs (51).
Activated JNKs are known to enhance the activity of the transcription
factor c-Jun through the phosphorylation of two serine residues at
positions 63 and 73 (26). To examine whether JNK activity was
influenced by SB203580 in sodium arsenite-treated OVCAR-3 cells, we
analyzed the phosphorylation of c-Jun on Ser73 and the
total level of c-Jun by Western blotting. Fig. 4E shows that
phosphorylated c-Jun proteins were not detectable in nonstimulated OVCAR-3 cells. After 2 h of sodium arsenite treatment,
phosphorylation of c-Jun protein was clearly observed and was increased
further until at least 8 h of exposure. Consistent with findings
that phosphorylation on Ser63 and Ser73
decreases the electrophoretic mobility of the c-Jun protein (52), slower migrating forms of c-Jun were detectable after sodium arsenite treatment. The total level of c-Jun protein was also increased by
sodium arsenite. Pretreatment with 20 µM SB203580 did not
prevent the phosphorylation of c-Jun on Ser73 in OVCAR-3
cells upon exposure to sodium arsenite (Fig. 4E). Hyperphosphorylated, retarded c-Jun protein band(s) were clearly detectable. The observed decrease in the level of phospho-c-Jun protein
is more likely the result of an inhibitory effect of SB203580 on sodium
arsenite-induced c-Jun protein expression instead of inhibition of
c-Jun phosphorylation. Pretreatment with 5 µM SB203580 did also not affect c-Jun phosphorylation and had only a minor effect
on c-Jun protein levels. These results support that attenuation of
sodium arsenite-induced HIF-1
protein accumulation and VEGF mRNA
expression by SB203580 is specific and exclude the possibility that
this is caused by a toxic effect of this inhibitor on OVCAR-3 cells. In
addition, these data indicate that SB203580 does not influence JNK
activity in sodium arsenite-treated OVCAR-3 cells at the concentrations
used. Therefore, our results with SB203580 and SB202190 strongly
suggest that induction of HIF-1
protein and VEGF expression by
sodium arsenite is mediated by p38 in OVCAR-3 cells.
Activation of p38 MAPK and Induction of HIF-1
Protein by Sodium
Arsenite Are Co-modulated by the Level of Intracellular GSH--
We
have shown previously that the level of intracellular GSH is critical
for induction of HIF-1
protein and VEGF expression by sodium
arsenite in OVCAR-3 cells (34). Pretreatment with agents that elevate
intracellular GSH levels, such as GSH and NAc (a precursor of GSH),
attenuate sodium arsenite-induced HIF-1
accumulation and VEGF
expression, whereas depletion of intracellular GSH by pretreatment with
BSO potentiates both effects (34). The latter agent causes GSH
depletion by inhibiting
-glutamylcysteine synthetase, an enzyme
involved in the synthesis of GSH (53).
In light of our findings with the p38 inhibitors that p38 may be an
upstream regulator of HIF-1
protein and VEGF expression, we assessed
whether activation of p38 by sodium arsenite was also modulated by
exposure to GSH, NAc, and BSO prior to the addition of sodium arsenite
in OVCAR-3 cells. As a control, the level of the HIF-1
protein was
analyzed in parallel in the same extracts. Fig.
5A shows that pretreatment
with 10 and 20 mM GSH and NAc almost completely attenuated
sodium arsenite-induced phosphorylation of p38 in OVCAR-3 cells.
Differences in the signal of the phospho-p38 band were not caused by
changes in the total level of p38 because these remained almost
constant under all circumstances. The basal level of p38
phosphorylation appeared not to be influenced by pretreatment with 20 mM concentrations of the antioxidants alone. In agreement
with our previous findings (34), GSH and NAc pretreatment also
attenuated HIF-1
protein induction, whereas a minor effect was
observed on the basal level of HIF-1
protein.

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Fig. 5.
Effect of GSH, NAc, and BSO pretreatment on
induction of p38 phosphorylation by sodium arsenite in OVCAR-3
cells. A, OVCAR-3 cells were exposed to 100 µM sodium arsenite in the absence or presence of 10 or 20 mM GSH or 10 or 20 mM NAc. GSH and NAc were
added 1 h before the addition of sodium arsenite. In addition,
cells were exposed to 20 mM GSH and 20 mM NAc
alone. After an 8-h exposure to sodium arsenite, whole cell extracts
were prepared. HIF-1 protein levels and phosphorylation of p38 were
assessed by subjecting 100 µg of protein to SDS-gel electrophoresis
(7.5% gel) followed by Western blotting with a HIF-1-directed
antiserum or with a phospho-p38 antiserum. For assessing total protein
levels of p38, the blot was stripped and reprobed with an antiserum
recognizing both nonphosphorylated and phosphorylated forms of p38.
B, OVCAR-3 cells were exposed to 30 µM sodium
arsenite in the absence or presence of 500 µM BSO. BSO
was added 16 h before the addition of sodium arsenite. Cells were
also exposed to 500 µM BSO alone. After 4, 6, and 8 h of exposure to sodium arsenite, whole cell extracts were prepared.
HIF-1 protein levels and the phosphorylation of p38 were assessed as
in A.
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To examine the effect of GSH depletion on p38 phosphorylation, OVCAR-3
cells were pretreated with 500 µM BSO for 16 h
before the addition of a lower, suboptimal concentration of sodium
arsenite (30 µM). Because sodium arsenite reduces
intracellular GSH levels by itself, the effect of BSO is more
pronounced when OVCAR-3 cells are exposed to a suboptimal concentration
of this agent. Fig. 5B shows that pretreatment with BSO
clearly potentiated the phosphorylation of p38 after 4, 6, and 8 h
of exposure to sodium arsenite, without affecting the total levels of
this protein. Pretreatment with 500 µM BSO alone for a
period of 24 h hardly affected the basal levels of p38
phosphorylation. It should be mentioned that potentiation of p38
activity in the presence of BSO was already observed after 4 h,
whereas potentiation of HIF-1
protein accumulation was not detected
until 6 h of exposure to sodium arsenite. Thus, the p38 response
in GSH-depleted OVCAR-3 cells upon sodium arsenite treatment precedes
the response of HIF-1
. This result is consistent with the role of
p38 as an upstream regulator of HIF-1
protein induction by sodium
arsenite. In addition, the above data further support that regulation
of HIF-1
accumulation and VEGF expression by intracellular GSH in
sodium arsenite-treated OVCAR-3 cells involves modulation of p38 activity.
JNK1 and JNK2 Are Not Essential for Induction of HIF-1
Protein
and VEGF164 mRNA Expression by Sodium Arsenite in Mouse
Fibroblast Cell Lines--
Unfortunately, examination of the role of
JNKs in sodium arsenite-treated OVCAR-3 cells is complicated by the
lack of pharmacological agents that specifically inhibit their
activity. Therefore, we have examined whether sodium arsenite can
affect HIF-1
protein and VEGF mRNA levels in two mouse
fibroblast cell lines that were deficient of Jnk1 and
Jnk2. We also analyzed a mouse fibroblast cell line in which
one of the four Jnk allelles, a Jnk1
allele, was wild type. The lack of JNK activity in the two
Jnk1
/
Jnk2
/
fibroblast cell lines was confirmed by Western blotting demonstrating the absence of Ser73-phosphorylated c-Jun proteins in
extracts of sodium arsenite-treated cells (Fig.
6A). In contrast,
phosphorylation of c-Jun was potently induced in sodium
arsenite-treated Jnk1+/
Jnk2
/
cells (Fig. 6A). As can be
seen in Fig. 6B, a clear induction of HIF-1
protein was
observed in the two Jnk1
/
Jnk2
/
cell lines after 4 and 8 h of
exposure to 100 µM sodium arsenite. The increase in the
level of HIF-1
protein upon sodium arsenite treatment was more
pronounced than that observed in Jnk1+/
Jnk2
/
fibroblasts. In all three cell lines,
the levels of HIF-1
protein were highest after 4 h of treatment
with sodium arsenite.

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Fig. 6.
Effect of sodium arsenite on
HIF-1 protein and/or VEGF164
mRNA levels in Jnk1-
Jnk2-deficient mouse fibroblast cell lines.
Jnk1+/ Jnk2 / and
Jnk1 / Jnk2 / mouse
fibroblast cell lines were treated with 100 µM sodium
arsenite for the indicated time periods before cells were lysed and
protein and total RNA were extracted. A, c-Jun
phosphorylation and HIF-1 protein levels were assessed as in Figs.
4E and 2C, respectively. B,
VEGF164 mRNA levels were examined by the RNase
protection assay. Total RNA was hybridized to murine
VEGF164 and -actin antisense probes. t-RNA
(lane T) was hybridized as a negative control. The
195-nucleotide fragment protected by the mRNAs of
VEGF164 is indicated. Hybridization of -actin mRNAs
to the -actin antisense probe resulted in the protection of three
different fragments, including the expected fragment of 151 nucleotides
(indicated by an arrow, see text below). The
full-length VEGF164 and -actin antisense probes are
shown in the two leftmost lanes. The histogram indicates the
relative VEGF165 mRNA expression in sodium
arsenite-treated cells in which nontreated control cells
(T = 0) were set at 100%. Quantification was performed
by signal intensity measurements of VEGF164 and -actin
(151-nucleotide)-protected fragments on the autoradiograph of the RNase
protection experiment shown and subsequent standardization of
VEGF164 values against -actin internal control values.
The experiments in A and B are representative of
three independent ones with similar results.
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The effect of sodium arsenite on the levels of VEGF164
mRNA in these cell lines was determined in parallel by the RNase
protection assay. As can be seen in Fig. 6B, a
VEGF164-protected fragment with the expected size of 195 nucleotides and a low signal intensity was detected in nontreated
Jnk1
/
Jnk2
/
as
well as in Jnk1+/
Jnk2
/
fibroblasts. Protection of a
151-nucleotide
-actin fragment was also observed in all three cell
lines. The VEGF164- and
-actin-protected fragments were
not detected after hybridization of the antisense probes to control
tRNA. In addition, two other protected fragments were visualized which
were smaller in size. The protection of these fragments was specific
and, although not easily explained, may also be caused by hybridization
by
-actin mRNAs. The levels of VEGF164 mRNA in
the two Jnk1
/
Jnk2
/
cell lines were increased 3-4-fold
after 4 h of treatment with 100 µM sodium arsenite.
In Jnk1+/
Jnk2
/
fibroblasts, induction of VEGF mRNA expression by sodium arsenite was also detectable, but was less pronounced (2-fold). Elevated levels
of VEGF164 mRNA were still observed after 8 h of
exposure to arsenite in all three cell lines. In summary, our results
in mouse fibroblast cell lines demonstrate that induction of HIF-1
protein and VEGF mRNA levels by sodium arsenite is not restricted to human ovarian cancer cells. More importantly, the data suggest that JNK1 and JNK2 are not essential for these effects.
HIF-1
Protein Is Translocated to the Nucleus in Sodium
Arsenite-treated OVCAR-3 Cells--
So far, it is unclear whether
induction of HIF-1
protein by sodium arsenite leads to the formation
of functional HIF-1. Because the translocation of HIF-1
to the
nucleus is an important regulatory event in the activation of HIF-1, we
first examined the subcellular localization of HIF-1
in nontreated
and sodium arsenite-treated OVCAR-3 cells. Because hypoxia has been
shown to induce efficient nuclear translocation of HIF-1
in several
cell types (20), we assessed the subcellular distribution of HIF-1
in hypoxic OVCAR-3 cells as a control. Cytoplasmic and nuclear protein
fractions were extracted from nontreated OVCAR-3 cells and from cells
exposed to 100 µM sodium arsenite or hypoxia (1%
O2) for 6 h, and identical amounts of protein extract
were subjected to SDS-PAGE and Western blotting. In addition, whole
cell extracts were prepared and analyzed in parallel. Fig.
7 shows that a low level of HIF-1
protein expression was detected in whole cell extracts of nontreated
OVCAR-3 cells and that the level of HIF-1
protein was potently
induced upon exposure to sodium arsenite as well as to hypoxia (34). A
low level of HIF-1
protein was observed in the nuclear fraction but not in the cytoplasmic fraction of nontreated OVCAR-3 cells. Much higher levels of HIF-1
were observed in the nuclear fractions as
well as in the cytoplasmic fractions of OVCAR-3 cells that were exposed
to sodium arsenite and hypoxia. As a control for the fractionation
procedure, we also analyzed the levels of proteins with a known
subcellular localization (Fig. 7). As assessed in whole cell extracts,
the levels of the cytoplasmic proteins Raf-1 and
-actin the nuclear
proteins topoisomerase I and p53 (mutant conformation) were unaffected
by treatment with sodium arsenite and hypoxia. Raf-1 and
-actin were
indeed present primarily in the cytoplasmic fractions, whereas
topoisomerase I and p53 were detected mainly in the nuclear fractions.
These control tests confirmed that the fractionation procedure was
valid. Altogether, these data strongly indicate that HIF-1
is
transported to the nucleus in nontreated OVCAR-3 cells and that
HIF-1
protein accumulates in the nucleus and in the cytoplasm of
OVCAR-3 cells upon exposure to sodium arsenite and hypoxia.

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Fig. 7.
Determination of the localization of
HIF-1 in nontreated and hypoxia- or sodium
arsenite-treated OVCAR-3 cells by biochemical subcellular
fractionation. OVCAR-3 cells were either not treated or exposed to
1% oxygen (hypoxia) or 100 µM sodium arsenite under
normoxia for 6 h. Cytoplasmic and nuclear fractions as well as
whole cell extracts were prepared, and 100 µg of protein was
subjected to SDS-gel electrophoresis (7.5% gel) and Western blotting
with antisera directed against HIF-1 , -actin, Raf-1,
topoisomerase I, and p53.
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Induction of HIF-1
Protein by Sodium Arsenite May Not Lead to
the Formation of Functional HIF-1 in OVCAR-3 Cells--
To examine
further the functional activity of HIF-1
upon sodium arsenite
treatment, OVCAR-3 cells were transiently transfected with a 5xHREpGL3
luciferase reporter gene construct containing five HRE binding sites
from the human VEGF promoter in front of the SV40 minimal promoter. The
empty pGL3 promoter construct, containing only the SV40 minimal
promoter in front of the luciferase gene, was transfected as a negative
control. We also transfected a 5xjun2pGL3 reporter construct, which
contains five copies of the jun2 element from the
c-jun promoter in front of the SV40 minimal promoter. The
jun2 element binds c-Jun/ATF-2 heterodimers and is suggested
to mediate induction of c-jun gene expression by agents that
induce different types of cellular stress, including sodium arsenite
(54, 55). As mentioned earlier, c-Jun is a substrate of JNKs, whereas
ATF-2 is phosphorylated and activated by JNKs as well as by p38 (26).
Because c-jun mRNA levels were elevated in sodium
arsenite-treated OVCAR-3 cells (data not shown), we transfected the
5xjun2pGL3 reporter construct as a positive control.
As can be seen in Fig. 8, the 5xHREpGL3
construct was not induced by 50 and 100 µM sodium
arsenite as measured after 6 h of exposure. To exclude the
possibility that the 5xHREpGL3 construct was not functional,
transfected OVCAR-3 cells were exposed for the same period of 6 h
to hypoxia in parallel. Note that HIF-1
protein expression was
strongly induced after 6 h of exposure to hypoxia as well as after
6 h of exposure to 100 µM sodium arsenite (Fig. 7).
In agreement with findings in other cell types, the expression of the
5xHREpGL3 reporter gene was significantly greater (7-fold) than that of
the parental pGL3 construct in hypoxic OVCAR-3 cells. This suggests
that the 5xHREpGL3 construct is functional in mediating transcriptional
responses through HIF-1. The 5xjun2pGL3 construct was induced ~2-fold
after treatment with 50 and 100 µM sodium arsenite. This
activation is actually 3-fold when normalized for the activity of the
parental pGL3 construct under the same conditions. Although the
kinetics of activation of c-Jun/ATF-2 and HIF-1 upon sodium arsenite
treatment may differ, our findings with the 5xjun2pGL3 construct
suggest that the assay conditions used allow detection of increased
expression of pGL3 reporter constructs in arsenite-treated OVCAR-3.
Altogether, these data indicate that induction of HIF-1
protein by
sodium arsenite may not lead to the formation of functional HIF-1
in OVCAR-3 cells.

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Fig. 8.
Induction of HIF-1
protein by sodium arsenite does not result in HIF-1-mediated
transcriptional activation. OVCAR-3 cells were transiently
transfected in duplicate wells of six-well culture plates with 0.5 µg
of the indicated pGL3-luciferase reporter constructs together with 0.25 µg of pCMVLacZ and 2.0 µg of pUC19 carrier DNA (per well). pCMVLacZ
was cotransfected to correct for a variable transfection efficiency.
40 h after transfection, cells were left untreated or exposed to
50 or 100 µM sodium arsenite or 1% oxygen (hypoxia) for
an additional period of 6 h. The relative luciferase activity in
the histogram represents the mean luciferase
(RUL)/ -galactosidase ratio/µg of total protein/min ± S.D. of duplicate extracts. Three independent experiments were
performed with similar outcome. The results of one of them are
shown.
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HIF-1
Is Not Essential for Induction of VEGF Expression by
Sodium Arsenite in Mouse Fibroblasts--
To examine further the role
of HIF-1 in sodium arsenite-induced VEGF mRNA expression, we have
studied the effect of this agent on VEGF mRNA expression in a wild
type and a HIF-1
-deficient mouse fibroblast cell line (39, 56). As
shown in the RNase protection assay in Fig.
9, unstimulated HIF-1
+/+
as well as HIF-1
/
fibroblasts express a very low
level of VEGF164 mRNA. After 4 h of exposure to
sodium arsenite, VEGF164 mRNA levels were elevated ~2.5-fold in HIF-1
+/+ cells. In
HIF-1
/
cells, the increase in VEGF164
mRNA at this time point was even higher, 3.5-fold. In both cell
types, the
-actin mRNA levels were not altered upon sodium
arsenite treatment. These findings in mouse fibroblasts strongly
suggest that HIF-1(
) does not play a role in the up-regulation of
VEGF mRNA expression by sodium arsenite and support our suggestive
data in OVCAR-3 cells that induction of HIF-1
protein by sodium
arsenite does not result in the generation of functional HIF-1.

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Fig. 9.
Effect of sodium arsenite on
VEGF164 mRNA levels in
HIF-1 +/+ and
HIF-1 /
mouse fibroblast cell lines.
HIF-1 +/+ and
HIF-1 / mouse fibroblast cell lines were
treated with 100 µM sodium arsenite for the indicated
time periods before cells were lysed and total RNA was extracted.
VEGF164 mRNA levels were examined by the RNase
protection assay as in Fig. 6B.
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DISCUSSION |
We have demonstrated previously that sodium arsenite induces
HIF-1
protein as well as VEGF mRNA and protein expression in the
human ovarian cancer cell lines OVCAR-3 and H134 (34). Here, we show
that 50 and 100 µM arsenic trioxide, an experimental
anticancer agent, exerts the same effects in OVCAR-3 cells. Although
treatment of OVCAR-3 cells with 10 µM arsenic trioxide
for a relatively long period of exposure (24 h) did not influence
HIF-1
protein levels, a weak elevation was observed in the
expression of VEGF165 mRNA (1.5-fold). The levels of
VEGF protein in conditioned medium and in cell lysate were increased 3- and 2-fold, respectively. These findings suggest that at low,
clinically achievable concentrations of arsenic trioxide, ovarian
cancer cells may respond with up-regulation of VEGF protein.
The finding that arsenic trioxide induces HIF-1
protein and VEGF
expression was not unexpected because different arsenic compounds
containing trivalent arsenic have been shown to exert similar
biological effects (49). At least part of these effect are believed to
be caused by the ability of trivalent arsenic to bind to SH groups of
important cellular (signaling) proteins. Both sodium arsenite and
arsenic trioxide can activate PI3K and/or the MAPK family members
p44/p42 MAPK, SAPKs/JNKs, and p38 (48, 49). We demonstrated that these
kinases were indeed activated by sodium arsenite in OVCAR-3 cells.
Our experiments with the PI3K inhibitor wortmannin and the p44/p42
MAPK inhibitor PD98059 strongly suggest that sodium arsenite-induced
HIF-1
protein and VEGF mRNA expression are independent of
PI3K/Akt-1 and p44/p42 MAPK. Interestingly, phenylarsine oxide, another
trivalent arsenic compound, can also induce HIF-1
protein through a
PI3K/Akt-1-independent mechanism (57).
Recently, it was suggested that the transcription factor c-Jun
cooperates with HIF-1 in the activation of VEGF and that JNKs mediate
this activation by phosphorylating c-Jun on Ser63 and
Ser73 (23). We showed induction of HIF-1
protein and
VEGF165 mRNA in immortalized
Jnk1+/
Jnk2
/
and
Jnk1
/
Jnk2
/
mouse
fibroblast cell lines by sodium arsenite, which did not involve c-Jun
phosphorylation on Ser63 and Ser73. Thus, our
data provide strong evidence that JNK activity is not essential for the
effects of sodium arsenite on HIF-1
and VEGF.
The p38 MAPK inhibitors SB202190 and SB203580 completely abolished
induction of HIF-1
protein and VEGF165 mRNA
expression by sodium arsenite in OVCAR-3 cells. A role of p38 as an
upstream activator in the effects of sodium arsenite on HIF-1
and
VEGF was supported by additional observations. First, activation of p38
was observed after 1 h of exposure to sodium arsenite and clearly
preceded the induction of HIF-1
protein and VEGF165
mRNA levels (observed after 4 and 2 h, respectively) (34).
Second, we found p38 kinase activation as well as HIF-1
protein and
VEGF165 mRNA expression to be co-modulated by the level
of intracellular GSH (34). Elevation of intracellular GSH by
pretreatment with GSH or NAc (a precursor for GSH) inhibited the
effects of sodium arsenite on HIF-1
protein and VEGF165
mRNA expression in OVCAR-3 cells, whereas depletion of
intracellular GSH by BSO showed the reverse (34). We demonstrated here
that pretreatment with GSH, NAc, and BSO influenced the effects of
sodium arsenite on p38 activity in the same manner. Thus, p38 may act
as a mediator in the modulation of HIF-1
accumulation and VEGF
expression by intracellular GSH. Third, we have transient cotransfected
OVCAR-3 cells with an expression vectors containing either a puromycin
resistance gene or a gene encoding an unphosphorylatable
dominant-negative mutant of p38 (DNp38). As assessed by real time
quantitative PCR, induction of VEGF165 mRNA by sodium
arsenite after 3 days of selection with puromycin was partly inhibited
(20-40%) upon cotransfection of DNp38 in three independent
experiments. The partial inhibition is likely the result of suboptimal
selection of transfected cells with puromycin. Upon stable
cotransfection, 19 puromycin-resistant OVCAR-3 clones did not show
detectable expression of DNp38, indicating that overexpression of DNp38
may inhibit OVCAR-3 cell growth. For further studies, the generation of
OVCAR-3 cells with inducible expression of DNp38 may be required.
Although a role for p38 in the regulation of HIF-1
protein levels
under normoxia has been suggested in some studies, this kinase has
mainly been implicated in the regulation of HIF-1
transactivation
(25, 29). The mechanism by which p38 exerts its effect on HIF-1
,
however, is still unclear. We have provided evidence that sodium
arsenite induces the level of HIF-1
protein by inhibiting its
degradation (34). The degradation of HIF-1
is controlled by a small
domain of 200 amino acids (amino acids 401-603), called the
oxygen-dependent degradation domain (17). Interestingly,
this oxygen-dependent degradation domain contains three
serine residues (at positions 581, 589, and 594) which may be putative
targets for direct phosphorylation by p38. The
oxygen-dependent degradation domain mediates the
interaction of HIF-1
with the tumor suppressor protein von Hipple
Lindau (pVHL) under normoxic conditions (58). pVHL is part of a
multiprotein complex possessing associated E3 ubiquitin-ligase activity
and is thought to target the degradation of HIF-1
(58, 59).
Phosphorylation of Ser581, Ser589, or
Ser594 by p38 may disrupt or prevent the formation of the
HIF-1
·pVHL complex. It is, however, questionable whether HIF-1
is a direct target for p38 in sodium arsenite-treated OVCAR-3 cells.
Activation of p38 already occurs after 1 h of sodium arsenite
treatment, whereas HIF-1
protein accumulation is not detectable
until 4 h (34). This suggests that p38 may regulate the HIF-1
protein indirectly, through phosphorylation of other factors. A
possible candidate is FK506 binding protein-rapamycin-associated
protein (FRAP), an effector of Akt, which may be activated by sodium
arsenite in a p38-dependent manner (60). FRAP has been
suggested to mediate hypoxia- and growth factor-induced HIF-1
protein expression (6).
The finding that up-regulation of HIF-1
protein as well as VEGF
mRNA by arsenite is blocked by p38 kinase inhibitors would support
that induction of VEGF expression is mediated by HIF-1. Elevation of
VEGF mRNA expression in sodium arsenite-treated OVCAR-3 cells,
however, was detected at earlier time points (after 2 h) than
HIF-1
protein stabilization (after 4 h) (34). Moreover, exposure of OVCAR-3 cells to sodium arsenite did not result in transcriptional activation of a 5xHRE-dependent reporter
gene in a transient transfection assay. Furthermore, treatment of
OVCAR-3 with low concentrations of arsenic trioxide also up-regulated VEGF mRNA expression in the absence of a detectable change in the
level of HIF-1
protein. Taken together, these data indicate that
up-regulation of VEGF expression by arsenite and arsenic trioxide is
mediated by other factors than HIF-1. This is supported by experiments
in HIF-1
+/+ and HIF-1
/
mouse
fibroblast cell lines, demonstrating that elevation of VEGF mRNA
levels upon sodium arsenite treatment is even slightly higher in
HIF-1
/
cells than in HIF-1
+/+ cells.
It is unclear why HIF-1 is not functional in sodium arsenite-treated
OVCAR-3 cells. Our results from subcellular fractionation experiments
showing that HIF-1
protein is clearly detectable in nuclear cell
fractions of sodium arsenite-treated OVCAR-3 cells suggest that
HIF-1
protein is properly translocated to the nucleus. It remains to
be explored whether HIF-1
can dimerize to HIF-1
and whether this
complex is able to bind to DNA and to transcriptional coactivators.
Several studies suggest that the DNA binding activity of HIF-1 and its
interactions with transcriptional coactivators is dependent on its
redox state (21, 22, 61). Addition of sulfhydryl-reactive agents to
extracts of hypoxic cells can inhibit HIF-1 DNA binding (61). These
agents can also inhibit the binding of HIF-1
to coactivator(s) (21).
Possibly, arsenic agents inhibit the binding of HIF-1 to DNA or to
coactivators through direct interaction with cysteine residues. The
former hypothesis is supported by the finding that HIF-1
protein
induction by phenylarsine oxide does not result in the formation of an
active HIF-1 DNA-binding complex (57). In agreement with our results on
sodium arsenite, this compound did also not provoke
HRE-dependent reporter gene activation (57).
Recently, evidence was provided that VEGF mRNA can be stabilized in
response to p38 activation under normoxic conditions (62). Experiments
with the transcription inhibitor actinomycin D suggested, however, that
induction of VEGF mRNA levels by sodium arsenite is at the
transcriptional level (34). Besides the HRE, the VEGF promoter contains
binding sites for several other transcription factors that can mediate
transcriptional activation of VEGF in a HIF-1-independent manner under
normoxic conditions (63, 64). As a first attempt to identify the
transcription factors that mediate sodium arsenite-induced VEGF
expression in OVCAR-3 cells, we performed transient transfection assays
with luciferase reporter constructs containing the full-length VEGF
promoter (
2274/+379) (11). Although the expression of this reporter
construct was inducible by hypoxia in OVCAR-3 cells, we have never
observed any activation of this reporter by sodium arsenite. Additional experiments are required to establish whether the effect of sodium arsenite on VEGF expression is the result of transcriptional activation or of mRNA stabilization. Further investigation of the mechanism that mediates sodium arsenite-induced VEGF expression and of the exact
role of p38 in this effect will provide important knowledge on the
regulation of VEGF expression under conditions of oxidative stress.