Requirement of Erk, but Not JNK, for Arsenite-induced Cell
Transformation*
Chuanshu
Huang,
Wei-Ya
Ma,
Jingxia
Li,
Angela
Goranson, and
Zigang
Dong
From The Hormel Institute, University of Minnesota,
Austin, Minnesota 55912
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ABSTRACT |
Trivalent arsenic (arsenite,
As3+) is a human carcinogen, which is associated with
cancers of skin, lung, liver, and bladder. However, the mechanism by
which arsenite causes cancer is not well understood. In this study, we
found that exposure of Cl 41 cells, a well characterized mouse
epidermal cell model for tumor promotion, to a low concentration of
arsenite (<25 µM) induces cell transformation.
Interestingly, arsenite induces Erk phosphorylation and increased Erk
activity at doses ranging from 0.8 to 200 µM, while
higher doses (more than 50 µM) are required for
activation of JNK. Arsenite-induced Erk activation was markedly
inhibited by introduction of dominant negative Erk2 into cells, while
expression of dominant negative Erk2 did not show inhibition of JNK and
MEK1/2. Furthermore, arsenite-induced cell
transformation was blocked in cells expressing the dominant negative
Erk2. In contrast, overexpression of dominant negative JNK1 was shown
to increase cell transformation even though it inhibits
arsenite-induced JNK activation. Our results not only show that
arsenite induces Erk activation, but also for the first time
demonstrates that activation of Erk, but not JNK, by arsenite is
required for its effects on cell transformation.
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INTRODUCTION |
Arsenite is introduced into the environment during energy
production based on coal, oil shale, and geothermal sources. Once in
the environment, arsenite represents a potential health hazard of
unknown magnitude. Arsenite is associated with increased risks of human
cancer of the skin, respiratory tract, hematopoietic system, and
urinary bladder (1-4). Epidemiological investigations indicated that
long-term arsenic exposure results in promotion of carcinogenesis,
especially in lung and skin via inhalation and ingestion (5). Many
cases of skin cancer have been documented in people exposed to arsenite
through medical or other occupational exposures. It has been reported
that high arsenic levels in drinking water (0.35-1.14 mg/liter)
increased risks of cancer of skin, bladder, kidney, lung, and colon (1,
2, 5, 6). Hence, arsenite is a well documented human carcinogen (5,
7).
Previously, several hypotheses have been proposed to describe the
mechanism of arsenite-induced carcinogenesis (8-14). It has been
suggested that arsenic induces chromosome aberration and sister
chromatid exchange which may be involved in arsenite-induced carcinogenesis (11, 12). Recently, Zhao et al. (13) reported that arsenic may act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression. Additionally, it
was found that arsenite is a potent stimulator of extracellular signal-regulated protein kinase
(Erk)1 and AP-1
transactivational activity and an efficient inducer of c-fos
and c-jun gene expression (10, 14). Induction of c-jun and c-fos by arsenite is associated with
activation of JNK (10). However, the role of JNK activation by arsenite
in cell transformation or tumor promotion is unclear. We have
established cell culture conditions for studying arsenite-induced cell
transformation in this report. Furthermore, our data have shown that
activation of Erk, but not JNK, is required for cell transformation
induced by arsenite.
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MATERIALS AND METHODS |
Plasmids and Reagents--
CMV-neo vector plasmid was
constructed as previously reported (15, 16); dominant negative JNK1
(pcDNA-flag-JNK1 (APF)) was from Dr. Roger J. Davis, Department of
Biochemistry and Molecular Biology, University of Massachusetts Medical
School (17, 18); fetal bovine serum (FBS) and Eagle's minimal
essential medium (MEM) were from Biowhittaker; LipofectAMINE was from
Life Technologies, Inc.; TPA was from Sigma; rabbit polyclonal IgG
against PKC
was from Santa Cruz Biotechnology; EGF was from
Collaborative Research; luciferase assay substrate was from Promega;
and PhosphoPlus MAPK antibody kit, phospho-MEK1/2 antibody,
and p44/42 MAP kinase assay kit were from New England Biolabs.
Cell Culture--
JB6 P+ mouse epidermal cell line,
Cl 41, and its dominant negative Erk2-K52R transfectants, C1 41 DN
MAPK-DN B3 mass1 (19), as well as dominant
negative JNK1 (pcDNA-flag-JNK1 [APF]) transfectant, C141 DN JNK1
mass2, were cultured in monolayers at 37 °C, 5%
CO2 using Eagle's minimal essential medium containing 5%
fetal calf serum, 2 mM L-glutamine, and 25 µg
of gentamicin/ml (20, 21).
Generation of Stable Co-transfectants--
JB6 Cl 41 cells were
cultured in a 6-well plate until they reached 85-90% confluence. We
used 1 µg of CMV-neo vector with or without 12 µg of dominant
negative JNK1 (pcDNA-flag-JNK1 [APF]) plasmid DNA and 15 µl of
LipofectAMINE reagent to transfect each well in the absence of serum.
After 10-12 h, the medium was replaced by 5% FBS MEM. Approximately
30-36 h after the beginning of the transfection the cells were
digested with 0.033% trypsin and cell suspensions were plated into
75-ml culture flasks and cultured for 24-28 days with G418 selection
(300 µg/ml). Stable transfectants were identified by using
phospho-specific antibodies against phosphorylated JNK. Stable
transfected Cl 41 mass1 and Cl 41 DN JNK1 mass2
were established and cultured in G418-free MEM for at least two
passages before each experiment.
Phosphorylation Analysis for Erk and JNK--
Immunoblot
analysis for phosphorylated proteins of Erk and JNK was carried out
using phospho-specific MAPK antibodies against phosphorylated sites of
Erk and JNK as described previously (22, 23). Antibodies were from New
England Biolabs and used according to the manufacturer's
recommendations. PKC
was used as an internal control for sample
protein loaded. Antibody bound proteins were detected by
chemiluminescence (ECL, New England Biolabs).
JNK Activity Assay--
JNK assay was carried out as described
previously (22, 24). Briefly, JB6 C141 cells were starved for 48 h
in 0.1% FBS MEM in 37 °C, 5% CO2 atmosphere incubator.
The cells were washed once with ice-cold phosphate-buffered saline and
exposed to UVC (60 J/m2) or arsenite at the concentration
and times indicated in the figure legends. Then, the cells were washed
once with ice-cold phosphate-buffered saline and lysed in 300 µl of
lysis buffer per sample (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM Na3VO4,
1 mg/ml leupeptin). The lysates were sonicated and centrifuged, and the
supernatant was incubated with 2 µg of N-terminal c-Jun (1-89)
fusion protein bound to glutathione-Sepharose beads overnight at
4 °C. The beads were washed twice with 500 µl of lysis buffer with
phenylmethylsulfonyl fluoride and twice with 500 µl of kinase buffer
(25 mM Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2). The kinase reactions were carried out in the
presence of 100 µM ATP at 30 °C for 30 min. c-Jun
phosphorylation was selectively measured by Western immunoblotting
using a chemiluminescent detection system and specific c-Jun antibodies
against phosphorylation of c-Jun at serine 63.
Erk Activity Assay--
Erk activity was carried out as
described using the protocol of New England Biolabs. In brief, JB6 Cl
41 transfectants were starved for 48 h in 0.1% FBS MEM at
37 °C, 5% CO2 atmosphere incubator. The cells were
exposed to arsenite for the doses and times indicated. The cells were
washed once with ice-cold phosphate-buffered saline and lysed in 300 µl of lysis buffer per sample (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton, 2.5 mM sodium pyrophosphate, 1 mM
-glycerolphosphate, 1 mM Na3VO4,
1 mg/ml leupeptin). The lysates were sonicated and centrifuged, and the supernatant was incubated with phospho-specific p44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody for 4 h at 4 °C; then, incubated with protein A-Sepharose beads overnight
at 4 °C. The beads were washed twice with 500 µl of lysis buffer
with phenylmethylsulfonyl fluoride and twice with 500 µl of kinase
buffer (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2). For determination of Erk-induced phosphorylation of Elk-1 measured by quantitative immunoblotting with phospho-Elk-1 antibody, the kinase reactions were carried out in the presence of 2 µg of Elk-1 fusion protein and 100 µM ATP at 30 °C
for 30 min. Elk-1 phosphorylation is selectively measured by Western immunoblotting using a chemiluminescent detection system and specific antibodies against phosphorylation of Elk-1 at serine 383. For measurement of Erk-induced phosphorylation of Elk-1 by direct assesses
of phosphate incorporation from [
-32P]ATP, the kinase
reactions were carried out in the presence of 2 µg of Elk-1 fusion
protein and 100 µM cold ATP plus 15 µCi of [
-32P]ATP at 30 °C for 30 min. The results were
presented as SDS-polyacrylamide gel electrophoresis autoradiography and
the phospho-Elk-1 bands were cut off and counted.
Anchorage-independent Transformation Assay--
JB6 Cl 41 cells
or their transfectants (1 × 104 cells) were exposed
to arsenite and TPA in 1 ml of 0.33% BME agar containing 15% FBS over
3.5 ml of 0.5% BME agar containing 15% FBS in each well of 60-well
plate. The cultures were maintained in 37 °C, 5% CO2
incubator for 4 weeks, then another 3 ml of 0.33% BME agar containing
15% FBS was added to each well and the cultures continued in the 5%
CO2 incubator for another 4 weeks. The TPA- and
arsenite-induced cell colonies were scored at the end of the second and
eighth week after cells were exposed to TPA or arsenite, respectively.
Assay for Cell Proliferation--
Cell proliferation was
determined by [3H]thymidine incorporation assay. For the
study of the influence of expression of dominant negative mutants of
JNK1 or Erk2 on cell proliferation, 5 × 103 of Cl 41 AP-1 mass1, Cl 41 MAPK-DN
B3 mass1, or Cl 41 DN-JNK1 mass2 cells were seeded into each well of a 96-well plate.
After 12 h of culture, the cells were or were not treated with TPA
(10 ng/ml) or EGF (10 ng/ml) for 24 h. Then 0.5 µCi of
[3H]thymidine was added to each well. The cells were
harvested 12 h later, and the incorporation of
[3H]thymidine was detected with a liquid scintillation
counter. The results were presented as counts per minute.
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RESULTS |
Induction of Cell Transformation by Low Concentration of
Arsenite--
Arsenite is a known carcinogen (1-4). Some previous
studies have suggested that arsenite acts as a tumor promoter rather than an initiator (25). However, there is no convenient
anchorage-independent cell transformation model for studying the
molecular mechanism of the tumor promotion effect of arsenite. The
mouse epidermal JB6 cell system is a model to study tumor promotion
in vitro. To study whether arsenite induces JB6 cell
transformation, we exposed JB6 Cl 41 cells to arsenite in soft agar.
Anchorage-independent colonies were observed in the eighth week after
arsenite exposure. However, the transformation rate was lower and the
colonies were smaller than those induced by TPA, which were observed
after 2 weeks of exposure (Fig. 1). Also,
cell transformation can only be observed in cells exposed to low
concentration (25 µM) of arsenite, while no cell
transformation colonies were observed at high concentrations of
arsenite (50-100 µM) (Fig. 1A).

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Fig. 1.
Cell transformation induced by low
concentrations of arsenite. C1 41 cells (104 cells)
were or were not exposed to TPA (10 ng/ml) or different concentrations
of arsenite in 1 ml of 0.33% BME agar containing 15% FBS over 3.5 ml
of 0.5% BME agar containing 15% FBS in each well of 6-well plates.
The cultures were maintained in 37 °C, 5% CO2 incubator
for 4 weeks. Then, another 3 ml of 0.33% BME agar containing 15% FBS
was added to each well and the cultures continued in the 5%
CO2 incubated for another 4 weeks. The TPA- and
arsenite-induced cell colonies were scored at the end of the second and
eighth week after cells were exposed to TPA or arsenite,
respectively.
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Differential Activation of Erk and JNK by
Arsenite--
Previously, we and others have reported that signal
transduction pathways leading to AP-1 activation are required for cell transformation induced by tumor promoters, such as TPA and EGF to occur
(23, 26, 27). It was also reported that arsenite is a potent stimulator
of AP-1 activity and JNK activity (10). To study the molecular basis
for neoplastic transformation activity of arsenite, we examined its
effects on MAP kinase signal transduction pathways in our system. We
found that arsenite could induce activation of both JNK and Erk.
However, the activation of JNK and Erk by arsenite is different. During
the time course and dose-response studies, marked Erk activation could
be observed at 15 min after exposure and at all dosages studied (Figs.
2 and 3).
There was no significant induction of Erk by arsenite after a 30-min
exposure (Fig. 2). In contrast, activation of JNK was only observed at high dosage (>50 µM) and after 60 min of exposure (Figs.
2 and 3). These results indicated that dosages of arsenite for
activation of Erk, but not JNK, are consistent with those for cell
transformation. Therefore, Erk activation may be involved in
arsenite-induced cell transformation. Interestingly, we also observed
phosphorylated Erk-like bands in cells treated with arsenite for 15 min
when we used antiphospho-JNK antibody (Fig. 2A). These bands
were very consistent with the Erk activation when we used the
anti-phosphorylated Erk antibody (Fig. 2A). The reason for
this may be due to cross-reaction of antiphospho-JNK antibody with
phosphorylation of Erk (Fig. 2A). To directly measure the
Erk activity induced by arsenite, we assessed the Erk activity by
measuring phosphate incorporation from [
-32P]ATP. The
results showed that exposure of cells to arsenite caused markedly an
increase of phosphate incorporation from [
-32P]ATP to
the Erk substrate Elk-1 (Fig.
4A). These increases appear to
be in a dose-dependent manner (Fig. 4A). The
maximus induction of Erk activity by arsenite is similar to these
induced by 10 ng/ml of TPA or EGF (Fig. 4A). We have
compared dose-responses between Erk phosphorylation and Erk-induced
phosphorylation of Elk-1 by direct measurement of phosphate
incorporation from [
-32P]ATP. Results from both
methods are generally correlated. However, the method by using
[
-32P]ATP is more sensitive than that by directly
measuring Erk phosphorylation (Fig. 4, A and
B).

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Fig. 2.
Time course of activation of Erk and JNK by
arsenite. A, for assay of phosphorylated Erk and JNK,
8 × 104 of JB6 Cl 41 cells were seeded into each well
of the 6-well plates. After culturing at 37 °C for 24 h, the
cells were starved for 48 h by replacing medium with 0.1% FBS
MEM. Four h before cells were exposed to TPA or arsenite, the medium
was changed to serum-free MEM. Then, the cells were or were not exposed
to TPA (10 ng/ml) or arsenite (200 µM). The cells were
extracted at different time points and phosphorylated proteins of Erk
and JNK as well as internal control protein kinase C (PKC)
were determined as described previously (22, 23). B,
assays of JNK activity. JB6 C1 41 cells were cultured in monolayers in
100-mm diameter dishes to 90% confluency. The cells were starved by
changing the medium with 0.1% FBS MEM medium for 48 h. The cells
were or were not exposed to 200 µM arsenite for the times
indicated. The cells were harvested and JNK activity was measured as
described previously (24).
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Fig. 3.
Differential dose-response of activation of
Erk and JNK by arsenite. JB6 Cl 41 cells (8 × 104 cells) were seeded into each well of 6-well plates.
After culturing at 37 °C for 24 h, the cells were starved for
48 h by replacing medium with 0.1% FBS MEM. Four h before cells
were exposed to TPA or arsenite, the medium was changed to serum-free
MEM. Then, the cells were or were not exposed to TPA (10 ng/ml) or UVC
(60 J/m2) or different concentrations of arsenite as
indicated. The cells were extracted at the time points as indicated.
The phosphorylated proteins of ERK (A) and JNK
(B) as well as internal control protein kinase C
(PKC) were determined as described in the
phosphospecific antibody kit (New England Biolabs). C, assay
of JNK activity. JB6 C1 41 cells were cultured in monolayers in 100-mm
diameter dishes to 90% confluency. The cells were starved by changing
the medium with 0.1% FBS MEM medium for 48 h. Then, the cells
were or were not exposed to UVC (60 J/m2), TPA (10 ng/ml)
for 30 min or different concentrations of arsenite for 120 min. The
cells were harvested and JNK activity was measured as described
previously (24).
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Fig. 4.
Comparison study of Erk activation by two
different methods. JB6 Cl 41 cells were seeded into 100-mm dishes
(A) or 6-well plates (B) and cultured in
37 °C, 5% CO2 incubator until 80-90% confluent. The
cells were starved for 48 h replacing medium with 0.1% FBS MEM.
Four h before cells were exposed to arsenite, the medium was changed to
serum-free medium. Then the cells were treated with different
concentrations of arsenite for 15 min. A, the cells were
extracted with lysis buffer. A monoclonal phospho-specific antibody
against Erk is used to selectively immunoprecipitate active Erk from
cell lysates. The immunoprecipitated proteins were then incubated with
2 µg of Elk-1 fusion protein in the presence of kinase buffer and
cold 100 µM ATP plus 15 µCi of
[ -32P]ATP. The results were presented as
SDS-polyacrylamide gel electrophoresis autoradiography and the
phospho-Elk-1 bands were cut off and counted; B, the cells
were extracted with SDS sample buffer, and Erk as well as phospho-Erk
were determined as described in the PhosphoPlus MAPK antibody kit (New
England Biolabs).
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No Inhibition of Arsenite-induced Cell Transformation by Expression
of Dominant Negative JNK1--
To rule out a role of JNK activation in
arsenite-induced cell transformation, we established a dominant
negative JNK1 stable transfectant, Cl 41 DN JNK1 mass2. The
dominant negative JNK1 mutant (APF) is the double point mutation that
changes the phosphorylation sites Thr183 and
Tyr185 to Ala and Phe, respectively (18, 19). This mutation
blocks JNK activation. The stable transfectant was generated by "mass culture selection" of pooled clones as described previously (23). To
determine whether dominant negative JNK1 have blocking effects on JNK
activation, we compared the JNK phosphorylation induced by arsenite
between dominant negative transfectant Cl 41 DN-JNK1 mass2
and the control transfectant Cl 41 CMV-neo mass1. The
results show that arsenite-induced JNK phosphorylation was impaired by introduction of dominant negative JNK1, while there were no significant effects on arsenite-induced Erk phosphorylation (Fig.
5). Expression of dominant negative JNK1
was shown to increase the cell transformation rate by arsenite (Fig.
6). Taken together with the results
regarding the difference between the dose-response curves for JNK
activation (Fig. 3, B and C) and the
transformation (Fig. 1) at different arsenite concentrations, we ruled
out the possible role of arsenite-induced JNK activation in cell
transformation.

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Fig. 5.
Introduction of dominant negative JNK1
specifically inhibits arsenite-induced activation of JNK. 8 × 104 JB6 C1 41 CMV-neu mass1 or C1 41 DN-JNK1
mass2 were seeded into each well of 6-well plates. After
culturing at 37 °C for 24 h, the cells were starved for 48 h by replacing medium with 0.1% FBS MEM. Four h before cells were
exposed to arsenite, the medium was changed to serum-free MEM. Then,
A, for JNK activation, the cells were or were not exposed to
arsenite (200 µM) for time as indicated; B,
for Erk activation, the cells were exposed to different concentrations
of arsenite for 15 min. The cells were extracted and phosphorylated JNK
and Erk proteins were determined as described in the PhosphoPlus MAPK
antibody kit (New England Biolabs).
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Fig. 6.
Cell transformation induced by arsenite is
specifically blocked by introduction of dominant negative Erk2, but not
dominant negative JNK1. C1 41 CMV-neu mass1, C1 41 DN-JNK1 mass2, or C1 41 MAPK DN B3
mass1 were or were not exposed to different concentrations
of arsenite in 1 ml of 0.33% BME agar containing 15% FBS over 3.5 ml
of 0.5% BME agar containing 15% FBS in each well of 6-well plates.
The cultures were maintained in 37 °C, 5% CO2 incubator
for 4 weeks. Then, another 3 ml of 0.33% BME agar containing 15% FBS
was added to each well and the cultures were continued in 5%
CO2 for another 4 weeks. The arsenite-induced cell colonies
were scored at the eighth week after cells were exposed to
arsenite.
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Inhibition of Erk Activation Blocks Arsenite-induced Cell
Transformation--
The results described above revealed that Erk
activation by arsenite may be involved in its cell transformation. To
test this possibility, we used dominant negative Erk2-K52R stable
transfectant, Cl 41 MAPK-DN B3 mass1 (19). We
found that overexpression of dominant negative Erk2 blocks
arsenite-induced Erk activation and cell transformation (Figs. 6 and
7A), while there is no marked influence on arsenite-induced phosphorylations of JNK or
MEK1/2 (Fig. 7, B and C). However,
the cell proliferation of the C1 41 MAPK DN B3
mass1 cells are not significantly different from those of
C1 41 AP-1 mass1 cells and Cl 41 DN-JNK1
mass2 (Table I). This data is
consistent with previous findings that the cell transformation is
dissociated from mitogenesis in JB6 cells (15, 50). These data also
indicate that lack of cell transformation of C1 41 MAPK DN
B3 mass1 cells in response to arsenite is not
due to inhibition of cell growth by transfection of dominant negative
Erk2. Our results demonstrate that Erk activation, but not JNK
activation, is required for arsenite-induced cell transformation.

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Fig. 7.
Overexpression of dominant negative Erk2
blocks arsenite-induced activation of Erk, but not JNK or
MEK1/2. JB6 C1 41 AP-1 mass1 or C1 41 MAPK-DN B3 mass1 cells were seeded into 100-mm
dishes (A) or 6-well plates (B and C).
After culturing at 37 °C for 24 h, the cells were starved for
48 h by replacing medium with 0.1% FBS MEM. Four h before cells
were exposed to arsenite, the medium was changed to serum-free MEM.
Then, A, the cells were treated with 200 µM
arsenite for the times as indicted. The cells were extracted and Erk
activity was determined as described under "Materials and Methods."
B, the cells were treated with 100 µM arsenite
for times as indicated. The cells were extracted with SDS sample buffer
and the phospho-JNK, phospho-MEK1/2, JNK, as well as
MEK1/2, were measured as described in previous reports (22,
41).
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DISCUSSION |
This study investigated the arsenite-induced signal transduction
pathway and its role in arsenite-induced cell transformation. Exposure
of JB6 Cl 41 cells to low concentrations (<25 µM) of arsenite lead to cell anchorage-independent growth, while there are no
cell transformation colonies at a high concentration (100 µM) of arsenite. In contrast, Erk activation could be
seen at all dosages studied, whereas JNK activation could only be
observed at high doses of arsenite. Furthermore, introduction of
dominant negative Erk2-K52R into cells blocks Erk activation as well as cell transformation induced by arsenite, while it does not block JNK
activation and MEK1/2 activation. In addition,
overexpression of dominant negative JNK1 increases arsenite-induced
cell transformation even though it blocks arsenite-induced JNK
activation. These results demonstrate that arsenite induces Erk
activation and for the first time provides strong evidence that Erk
activation, but not JNK activation, is required for arsenite-induced
cell transformation.
Arsenic is the first metal to be identified as a human carcinogen (28).
Arsenic can exist in trivalent and pentavalent forms and in organic or
inorganic compounds (7, 28). Both inorganic and organic forms are
absorbed by human and animal skin (7). Autoradiographic animal studies
show that following chronic exposure, arsenic accumulates in the skin
and hair (7). It is known that long-term arsenic exposure can result in
carcinogenesis (5). Many cases of skin cancer have been reported among
people exposed to arsenic through medical or occupational exposures to
trivalent arsenic compounds (7). Epidemiological studies in areas of high arsenic in drinking water are associated with increased risk of
cancer of skin, bladder, kidney, lung, and colon (6). Recently, Zhao
et al. (13) reported that chronic exposure of culture cells to low concentrations of arsenic lead to 70% of cells exhibiting morphological changes indicative of transformation at 18 weeks of
exposure (13). However, in animal experiments, arsenic exposure shows
no reliable evidence of its carcinogenicity (29). One possible
explanation for the positive carcinogenicity of arsenic compounds in
humans and the negative carcinogenicity in experimental animals is that
arsenite may be a tumor promoter, but not an initiator of
carcinogenesis (10, 25). This explanation is supported by the
observation that arsenic acts with other agents to alter or enhance
other biological effects, which are potentially involved in progression
of carcinogenesis (30-33). For example, arsenite accumulation in skin
increases the sensitivity of skin to ultraviolet (UV) light and
sequentially increased its carcinogenic effect (34, 35). Smoking may
synergistically interact with arsenic (30, 31).
The JB6 Cl 41 cell is a post-initiated mouse epidermal cell line and
represents an excellent in vitro model for studying tumor promotion (15, 36). In JB6 Cl 41 cells, different tumor promoters such
as TPA, EGF, and tumor necrosis factor-
induce the formation of
large, tumorigenic anchorage-independent colonies in soft agar at a
high frequency (36). In this study, we exposed Cl 41 cells to arsenite
and found that anchorage-independent growth of cells could be observed
at low doses (<25 µM). However, the transformation activity of arsenite is weaker than the positive control TPA. Arsenite-induced cell transformation required longer exposure times,
transformation frequency was lower, and the colonies were smaller than
TPA-induced colonies. Our results not only support the hypothesis that
arsenite is a tumor promoter rather than an initiator of
carcinogenesis, but also provide a useful cell culture model to study
the mechanisms of arsenite-induced neoplastic transformation.
Transcription factors, such as AP-1, NF
B, and nuclear factor of
activated T cells, are major mediators involved in cell proliferation, differentiation, and transformation (7, 15, 37, 38). Cell
transformation is a complex process which involves many transcription factors and signaling pathways (38-47). A growing body of evidence indicates that the activation of signal transduction pathways leads to
increased transcription factor activity, such as AP-1, NF
B, which
seems to be required for tumor promoter-induced cell transformation
(23, 38, 39, 45-49). It was reported that arsenite could stimulate
AP-1 activation and expression of c-jun and c-fos
in HeLa cells (10). In JB6 cells, we also found that treatment of cells
with arsenite caused activation of AP-1 activity (data not shown). It
is known that the signaling leading to AP-1 activation is mediated
through three mitogen-activated protein (MAP) kinase pathways,
including Erk, JNK, and p38 kinase (10, 18, 22). We, therefore,
investigated the possible role of MAP kinases involved in
arsenite-induced cell transformation in JB6 cells. Exposure of Cl 41 cells to arsenite not only activates JNK, which is consistent with
results from previous reports (10), but also induces Erk activation.
The results from time course studies show that Erk activation only
occurred at very early exposure, while JNK activation occurred much
later. It should be noted that Erk activation could be seen at all
doses studied, whereas JNK activation was only observed at high doses
(>50 µM). The dosages for induction of cell
transformation are correlated with that for activation of Erk, but not
for JNK, revealing that Erk activation may be involved in
arsenite-induced cell transformation. This was supported by our
findings that dominant negative Erk2 dramatically inhibits
arsenite-induced cell transformation (Fig. 5), while dominant negative
JNK increased the cell transformation (Fig. 5) even though it blocks
JNK activation by arsenite (Fig. 4A). The reason for
induction of Erk, but not cell transformation by high doses of
arsenite, may be due to induction of apoptosis at these doses. Our
current results indicate that arsenite at dosages more than 100 µM strongly induces apoptosis of JB6 Cl 41 cells (data
not shown). Our results for Erk activation by arsenite are different
from data reported by Cavigelli et al. (10) in which arsenite did not show any induction of Erk. The explanation for this
may be either that different cell lines were used in these studies or
the time point for Erk activation was missed by Cavigelli et
al. (10). During the preparation of this paper, Ludwig et al. (14) reported that arsenite induces Erk activation in the human embryonic kidney cell line HEK292 and this Erk activation appears
to be late phase and dependent on activation of p38 kinase. However, in
JB6 cells, we found that Erk activation occurs very early, while p38
kinase activation was observed later than 60 min after cell exposure to
arsenite. Our recent studies indicated that expression of dominant
negative p38 kinase in Cl 41 cells could not block arsenite-induced Erk
activation, while it blocks p38 kinase activation (data not shown).
Therefore, Erk activation is not dependent on p38 kinase activation in
JB6 cells. The reason for this may be due to the difference of cell
lines used. Taken together, our results strongly suggest that arsenite
induces Erk activation and Erk activation induced by arsenite is
required for its cell transformation to occur. The biological role of
JNK activation induced by arsenite is under current study.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Douglas Bibus for critical
reading, Dr. Roger S. Davis for the generous gift of dominant negative
JNK1 plasmids, and Jeanne A. Ruble for secretarial assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant CA74916 and The Hormel Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: The Hormel Institute,
University of Minnesota, 801 16th Ave. NE, Austin, MN
55912. Tel.: 507-437-9640; Fax: 507-437-9606; E-mail:
zgdong{at}smig.net.
 |
ABBREVIATIONS |
The abbreviations used are:
Erk, extracellular signal-regulated protein kinases;
AP-1, activated
protein-1;
BME, basal medium Eagle;
CMV, cytomegalovirus;
EGF, epidermal growth factor;
FBS, fetal bovine serum;
JNK, c-Jun N-terminal
kinases;
MAPK, mitogen-activated protein kinases;
MEM, minimal
essential medium;
TPA, 12-O-tetradecanoylphorbol-13-acetate.
 |
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