From the Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505
Received for publication, December 26, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell cycle checkpoint, a major genomic
surveillance mechanism, is an important step in maintaining genomic
stability and integrity in response to environmental stresses. Using
cells derived from human bronchial epithelial cells, we demonstrate
that NF- It has long been known that environmental and occupational
exposure to arsenic causes a number of human diseases including skin
lesions, peripheral vascular disorders, peripheral neuropathy, liver
injury, and cancers in lung or other organs (1, 2). Paradoxically,
arsenic has also been used for centuries for medicinal purposes, as for
the treatment of syphilis and leukemias (3-5). However, the
mechanistic basis for the carcinogenic or therapeutic effects of
arsenic is still poorly understood. Arsenic is usually considered a
nongenotoxic agent and is assumed to act principally through an
epigenetic effect by interfering with intracellular signaling molecules
that lead to cell cycle progression, DNA repair, ubiquitination,
tubulin polymerization, transcription factor activation, and oncogene
expression (6, 7). Arsenic trioxide (arsenite), rather than arsenic
pentoxide, has been credited with most of the intracellular effects,
although these two forms can interconvert via an intracellular redox
pathway (7). Studies by Cavigelli et al. (6) indicated that
arsenite is far more potent than arsenic pentoxide in stimulating AP-1
transcriptional activity, indicating that arsenite is a more important
carcinogen. It has been speculated that the toxicity of arsenite is due
to its affinity for thiol groups of proteins and possibly from its
induction of oxidative bursts that cause a stress response in the cells.
Both NF- It has been well established that various types of stress including DNA
damage, oxidation and hypoxia, induce cell cycle arrest, allowing time
for DNA repair and thus protecting the organism from the deleterious
consequences of mutation (12, 13). In mammalian cells, the cell cycle
arrest is often dependent upon the expression and functionality of cell
cycle inhibitory proteins such as GADD45 (the growth
arrest- and DNA damage-inducible
protein 45), a protein responsible for the maintenance of
the G2/M checkpoint that prevents improper mitosis (14,
15). Extracellullar stress signals induce rapid expression of GADD45 in
a manner that may be either p53-dependent or
p53-independent (16-18). Both NF- Reagents--
Arsenite was purchased from Aldrich. The
luciferase assay kit was from Promega (Madison, WI). Antibodies against
serine-phosphorylated and nonphosphorylated ERK, JNK, and p38 were from
New England Biolabs (Beverly, MA). ECL Western blotting detection
reagents were from Amersham Pharmacia Biotech. Antibodies
against IKK Cell Transfection--
The human bronchial epithelial cell line,
BEAS-2B, from American Type Culture Collection (ATCC; Manassas,
VA) was cultured in keratinocyte basal medium (Sigma)
supplemented with 30 µg/ml of bovine pituitary extract and 5 ng/ml of
human epidermal growth factor. pCR-FLAG-IKK Kinase Activity Assay--
The IKK activity assay was
performed by the method of Woronicz et al. (21) with minor
modifications. Briefly, BEAS-2B cells, transfected with pCR-IKK Flow Cytometry--
Cells cultured in keratinocyte basal medium
for 24 h were treated with various doses of arsenite for an
additional 2 days in the same medium. To determine the cell cycle
arrest, cells were rinsed with phosphate-buffered saline, trypsinized,
harvested by centrifugation, and resuspended in phosphate-buffered
saline supplemented with 0.4% paraformaldehyde. Approximately
106 cells for each sample were incubated with 20 µg/ml of
propidium iodide (Sigma) per ml, and DNA content was determined using a FACSscan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Western Blotting--
Whole cell extracts were mixed with 3 × SDS-PAGE sample buffer and then subjected to SDS-PAGE in 10 or 16%
gels. The resolved proteins were transferred to a nitrocellulose
membrane. Western blotting was performed as described previously by
using antibodies against IKK NF- Enhanced JNK and ERK Activation by Arsenite in IKK NF-
To verify and extend the observations described above, both IKK JNK Involvement in Arsenite-induced GADD45 Expression--
Having
confirmed that arsenite induced both JNK activation and GADD45
expression (Fig. 2B and Fig. 3B), we wanted to
determine whether activation of MAP kinases was responsible for
arsenite-induced GADD45 expression. We therefore first used two
specific inhibitors for ERK and p38 to investigate the possible
contribution of ERK and p38 to arsenite-induced GADD45 expression.
Pretreatment of cells with the ERK inhibitor, PD98059, resulted in the
inhibition of ERK by arsenite in both IKK
Time course studies for both GADD45 induction and JNK activation by
arsenite indicate that JNK activation preceded GADD45 induction by
arsenite. The earliest induction of GADD45 by arsenite appeared at
4 h and peaked at 8 h in both IKK The adverse or beneficial effects of arsenic on humans may
depend upon the manner of exposure and type of cell or tissue exposed. It is known that inhalation of arsenic-containing particles from either
environmental pollutants or occupational sources can lead to
debilitating lung diseases such as cancer (28). The bronchial epithelial cell is one of the first cell types to come in contact with
inhaled matter. Therefore, we used a cell line derived from human
bronchial epithelial cells, BEAS-2B, to investigate the molecular
mechanisms underlying the adverse effects of arsenic. The present study
demonstrates that NF- Transcriptional regulation of genes by NF- The relationship between GADD45 expression and JNK activation has not
been clearly demonstrated. JNK is rapidly activated by exposure of
cells to a variety of stress signals including UV light, Several reports appeared describing the effects of arsenite on the
activation of either NF- The observations on the effects of NF-B and c-Jun N-terminal kinase (JNK) reciprocally regulate
arsenic trioxide (arsenite)-induced, p53-independent expression of
GADD45 protein, a cell cycle checkpoint protein that arrests cells at
the G2/M phase transition. Inhibition of NF-
B
activation by stable expression of a kinase-mutated form of I
B
kinase caused increased and prolonged induction of GADD45 by
arsenite. In contrast, the induction of GADD45 by arsenite was
transient and less potent in cells where the NF-
B activation pathway
was normal. Analysis of the cell cycle profile by flow cytometry
indicated that NF-
B inhibition potentiates arsenite-induced
G2/M cell cycle arrest. Abrogation of JNK activation, on
the other hand, decreased GADD45 expression induced by arsenite,
suggesting a role for JNK activation in GADD45 induction. These results
indicate a molecular mechanism by which NF-
B and JNK may
differentially contribute to cell cycle regulation in response to arsenite.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B and AP-1 are considered stress response transcription
factors that govern the expression of a variety of proinflammatory and
cytotoxic genes (8). NF-
B, a heterodimer composed of two subunits,
p50 and p65, is regulated by specific inhibitors, the I
Bs, which
retain NF-
B in the cytoplasm of nonstimulated cells (9, 10). In
response to stress signals, the I
Bs undergo rapid phosphorylation of
conserved N-terminal serine sites by I
B kinase
(IKK)1 complexes. This
phosphorylation is an essential step required for subsequent
ubiquitination and degradation of I
Bs by SCF-
-TrCP and
proteasome, respectively (11). I
B degradation allows NF-
B dimers
to translocate into nuclei and activate the transcription of target
genes. Unlike NF-
B, AP-1 heterodimers are constitutively localized
within the nuclei. Transactivation of AP-1 is achieved largely through
phosphorylation of its activation domains by c-Jun N-terminal kinases
(JNKs) (8).
B and JNK are well known stress
sensors that can be rapidly activated in response to stress (19, 20).
NF-
B and JNK have also been implicated in cell cycle regulation
under certain circumstances. The objective of the present report is to
investigate the roles of NF-
B and JNK in the expression of GADD45
induced by arsenite in cell lines derived from human bronchial
epithelial cells. We provide evidence in this report that arsenite is
capable of inducing activation of NF-
B and JNK and expression of
GADD45. We demonstrate that interruption of NF-
B activation by
blocking IKK
kinase activity enhances JNK activation and
p53-independent GADD45 expression induced by arsenite. In contrast,
blockage of JNK activation results in decreased GADD45 induction.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
were from Santa Cruz Biotechnology (Santa Cruz, CA) or
Upstate Biotechnology (Lake Placid, NY). Anti-FLAG monoclonal antibody was from Sigma.
and pCR-FLAG-IKK
-KM
(K44A) were gifts from Dr. Hiroyasu Nakano (Juntendo
University, Tokyo, Japan). pcDNA3-FLAG-SEK1-KM was provided
by Dr. Roger Davis (University of Massachusetts, Boston, MA). BEAS-2B
cells were transfected with indicated expression vectors along with a
3×
B-dependent luciferase reporter construct using
LipofectAMINE (Life Technologies, Inc., Rockville, MD) as suggested by
the manufacturer. Single clones of BEAS-2B cells, stably transfected
with the expression vectors for IKK
, IKK
-KM, and luciferase
reporter genes, were isolated in 1 mM G418 for three weeks
and tested by Western blotting and a luciferase activity assay for
expression of the transfected genes. Stably transfected cells were
maintained in regular culture medium supplemented with 250 µM G418. To minimize possible clone variations during the course of selection, several independently derived cell lines expressing control vector, wild-type IKK
, and IKK
-KM with
different expression levels were pooled together, respectively, for the experiments described below.
or
IKK
-KM, were treated with indicated agents and lysed in a lysis
buffer containing 1% Nonidet P-40, 250 mM sodium
chloride, 50 mM HEPES (pH 7.4), 1 mM
EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, aprotinin (10 µg/ml), and leupeptin
(10 µg/ml). After centrifugation of the lysate at 16,000 × g for 20 min at 4 °C, the supernatant was incubated with
anti-IKK
antibody H-470 or anti-FLAG antibody with rotation for
4 h at 4 °C, followed by the addition of 20 µl of protein
A-agarose and incubation at 4 °C for an additional 2 h. The
immunoprecipitate was collected by centrifugation at 2000 × g and washed three times with lysis buffer and two times with kinase buffer containing 20 mM HEPES (pH 7.4), 20 mM
-glycerophosphate, 1 mM manganes
chloride, 5 mM magnesium chloride, 2 mM sodium
flouride, and 1 mM dithiothreitol. To monitor the kinase
reaction, the immunoprecipitate was incubated in 20 µl of kinase
buffer supplemented with 5 µCi of [
-32P]ATP and 1 µg of glutathione S-transferase-I
B
(1-54)
(CLONTECH, Palo Alto, CA) for 30 min at 30 °C.
The reaction was stopped by addition of SDS sample buffer. The samples
were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE),
which was then transferred onto a nitrocellulose membrane and subjected
to autoradiography.
, FLAG, phospho-specific p53,
phospho-specific JNK, p38, ERK, and anti-rabbit or anti-mouse
IgG-horseradish peroxidase conjugates.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B Is Inhibited in IKK
-KM-Expressing Cells--
IKK
has
been demonstrated to be the major I
B
kinase activated in response
to a variety of stimuli (11). Therefore, inhibition of IKK
by stable
expression of IKK
-KM may impair signal-induced NF-
B activation
with high specificity. Consistent with the original reports by Chu
et al. (22) and Geleziunas et al. (23), stable transfection of wild-type IKK
did not substantially alter basal or
inducible IKK or NF-
B activation compared with the transfection of
control vector (data not shown). Expression of IKK
-KM, however, abolished basal IKK
activity (Fig.
1A). An equal expression of IKK
and IKK
-KM was demonstrated by immunoblotting of the same lysates using anti-FLAG antibody that recognizes FLAG-tagged IKK
or
IKK
-KM (Fig. 1A, bottom panel). Analysis of
NF-
B-dependent reporter gene activity indicates that
arsenite treatment of IKK
-expressing cells induces a
dose-dependent increase of luciferase activity with a peak
at 18 µM arsenite. Higher concentrations of arsenite (more than 20 µM), however, did not increase
NF-
B-dependent luciferase activity further, partially
because of the cytotoxic effect of arsenite at higher doses (data not
shown). No appreciable induction of NF-
B-dependent
luciferase activity by arsenite was observed in IKK
-KM-expressing
cells (Fig. 1B). These results indicate that the IKK
, an
essential component of NF-
B signaling, is defective in IKK
-KM
cells.
View larger version (27K):
[in a new window]
Fig. 1.
NF- B inhibition in
IKK
-KM-expressing cells. A,
BEAS-2B cells were stably transfected with expression vectors for
either IKK
or IKK
-KM, along with a vector for an
NF-
B-dependent luciferase reporter gene. IKK kinase
activity was monitored by an in vitro kinase assay using
total cellular proteins from either IKK
-expressing cells
(lanes 1 and 2, marked with IKK
) or
IKK
-KM-expressing cells (lanes 3 and 4, marked with
IKK
-KM) as described under "Materials and
Methods." Immunoblotting of the same lysates from each transfection
is shown in the lower panel.
p-I
B
indicates phosphorylated I
B
.
B, NF-
B-dependent luciferase assay with both
IKK
cells and IKK
-KM cells treated with various concentrations of
arsenite for 12 h. n = 3.
-KM
Cells--
Genetic interruption studies of the IKK
gene suggest
that the pathway for the activation of JNK is intact in mice deficient in the IKK
gene (24). Consistent with this notion, we found that the
activation of three MAP kinases, ERK, JNK, and p38, was not impaired in
IKK
-KM cells, whereas the pathway for NF-
B activation was blocked
as shown above. We measured the activation of ERK, JNK, and p38 by
arsenite by monitoring the phosphorylation of each of these three MAP
kinases in both IKK
cells and IKK
-KM cells. To our surprise, we
found that IKK
-KM cells exhibited a stronger induction of ERK and
JNK activation by arsenite than did IKK
cells (Fig.
2, A and
B), whereas both IKK
cells and IKK
-KM cells showed a
similar induction of p38 activation by arsenite (Fig.
2C).
View larger version (60K):
[in a new window]
Fig. 2.
Enhanced ERK and JNK activation in
IKK -KM cells in response to arsenite.
Both IKK
cells and IKK
-KM cells were treated with various
concentrations of arsenite for 12 h. Total cellular proteins were
used for the determination of ERK (A), JNK (B),
and p38 (C) activation. Phosphorylation of ERK, JNK, and p38
was indicated as p-ERK, p-JNK, and
p-p38, respectively. Nonphosphorylated total proteins of
ERK, JNK, and p38 were also determined as internal controls.
B Inhibition Potentiated GADD45 Induction by
Arsenite--
Arsenite has been reported to suppress cell growth in
certain cell types (3, 4). This growth inhibitory effect of arsenite may be due to either the induction of cell apoptosis or the activation of cell cycle checkpoints. Cell cycle checkpoints exist at the G1/S and G2/M transitions that are regulated in
response to a variety of stress signals. GADD45 has been shown to be an
essential component of the G2/M cell cycle checkpoint
induced by UV light or methyl methanesulfonate (15). To determine
whether arsenite is capable of inducing cell cycle arrest, we measured
expression of GADD45 in both IKK
cells and IKK
-KM cells. As
depicted in Fig. 3A, arsenite
induced GADD45 expression in a dose-dependent manner.
Compared with the response in IKK
cells, arsenite induced a more
pronounced expression of GADD45 in IKK
-KM cells where NF-
B
activation was defective (Fig. 3A, top arrow).
Previous studies suggested that the induction of GADD45 in response to
-radiation is p53-dependent (25). The cell line we used
was functionally p53-deficient (26). Furthermore, as indicated in Fig.
3A, arsenite failed to induce notable changes in the
phosphorylation of Ser15 and Ser20 sites on p53
in either IKK
cells or IKK
-KM cells (Fig. 3A, middle and bottom arrows). In a parallel
experiment, we observed that chromate induced a strong phosphorylation
of Ser15 and Ser20 sites on p53 in a
dose-dependent manner in IKK
-KM cells (data not shown).
The phosphorylation of N-terminal Ser15, Ser20,
and possibly Ser6 of p53 has been shown to reduce the
interaction between p53 and MDM2 and thereby protect p53 from
degradation by proteasome (27). Thus, these results suggest that GADD45
induction by arsenite is independent of p53.
View larger version (61K):
[in a new window]
Fig. 3.
Inhibition of NF- B
potentiated arsenite-induced, p53-independent GADD45 expression.
A, IKK
cells and IKK
-KM cells were treated with
different concentrations of arsenite for 12 h. Total cellular
proteins were subjected to immunoblotting for the detection of GADD45
protein using 16% SDS-PAGE gels (top panel).
N.S., nonspecific bands. The same cellular proteins were
also used to determine the status of phosphorylation of
Ser15 (middle panel) and Ser20
(bottom panel) sites on the p53 protein using 10% SDS-PAGE
gels. B, IKK
cells (upper row) and IKK
-KM
cells (lower row) were either untreated or treated with
various concentrations of arsenite as indicated. Cell cycle profile was
determined 48 h after exposure of the cells to arsenite.
2N and 4N DNA contents correspond to cells in
G1 and G2/M, respectively.
cells and IKK
-KM cells were treated with different doses of arsenite
and examined for cell cycle arrest by flow cytometric analysis. In the
absence of arsenite treatment, the majority of both IKK
cells and
IKK
-KM cells were in G1 phase (Fig. 3B). 48 h after arsenite treatment, IKK
-KM cells showed a marked
increase in cells arrested in G2/M phase and a
corresponding decrease in the number of cells in G1 phase,
in a dose-dependent manner. Although IKK
cells exhibited
a similar but less potent dose-dependent increase of cells
in G2/M phase in response to arsenite, the change in
G1 cells is marginal, suggesting that, in contrast to
IKK
-KM cells, most of the IKK
cells are able to exit from
G2/M phase and enter the G1 phase.
cells (data not shown) and
IKK
-KM cells (Fig. 4B).
The same treatment, however, had no effect on arsenite-induced GADD45
expression (Fig. 4A, lanes 3 and 9).
Similarly, the p38 inhibitor, SB203580, also failed to inhibit the
levels of GADD45 induced by arsenite (Fig. 4A, lanes
4 and 10). Both inhibitors by themselves had no effect
on GADD45 expression (Fig. 4A, lanes 5,
6, 11, and 12). Because there is no
specific pharmacological inhibitor available for JNK, we next performed
transient transfection of IKK
-KM cells with a dominant negative
mutant of SEK1 (SEK1-KM) to determine whether JNK activation
contributed to arsenite-induced GADD45 expression. Compared with empty
vector (pcDNA) transfection (Fig. 4C, upper
panel, lanes 1-5), SEK1-KM transfection partially reduced JNK activation by arsenite (Fig. 4C, lower
arrow) and caused an appreciable suppression of GADD45 expression
induced by arsenite (Fig. 4C, upper arrow,
lanes 6-10).
View larger version (34K):
[in a new window]
Fig. 4.
Effects of MAP kinase inhibitors on
arsenite-induced GADD45 expression. A, cells stably
expressing IKK or IKK
-KM were left untreated or were pretreated
with 100 µM ERK inhibitor, PD98059 (PD), or 20 µM p38 inhibitor, SB203580 (SB), for 2 h,
followed by incubation with 10 µM arsenite
(As) for an additional 12 h. The expression level of
GADD45 was determined by immunoblotting. N.S., nonspecific
bands. B, the effectiveness of the ERK inhibitor, PD98059,
on arsenite-induced ERK activation was determined in IKK
-KM cells.
C, IKK
-KM cells were transiently transfected with an
empty vector, pcDNA, or a vector for SEK1-KM to block JNK
activation. After 48 h, cells were treated with arsenite as
indicated for an addition 12 h. The expression of GADD45
(upper panel) and activation of JNK (lower panel)
were determined. N.S., nonspecific bands. D, time
course studies of GADD45 induction (top panel) and JNK
activation (middle panel) by 10 µM arsenite.
The nonphosphorylated total JNK protein was determined as an internal
control (bottom panel). N.S., nonspecific
bands.
cells and IKK
-KM cells (Fig. 4D, top arrow, lanes 3,
4, 8, and 9). After a 24-h treatment
of cells with arsenite, GADD45 expression declined but was still
prominent in IKK
-KM cells (Fig. 4D, lane 10),
whereas only a trace amount of GADD45 induction by arsenite was
observed at this time point in IKK
cells (Fig. 4D,
lane 5). The activation of JNK by arsenite, on the other
hand, was seen as early as 1 h, at a time where no appreciable
GADD45 induction was observed (Fig. 4D, middle
and top arrows). Again, an increase in JNK activation by
arsenite was observed in IKK
-KM cells at these time points (Fig.
4D, compare lanes 2 and 3 with
lanes 7 and 8).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B and JNK are reciprocal regulators for
arsenite-induced, p53-independent expression of GADD45, a
G2/M cell cycle checkpoint protein. Following inhibition of
NF-
B by stable expression of IKK
-KM, arsenite induced a prolonged increase in GADD45 expression (Fig. 3, A and B).
On the other hand, in IKK
-expressing cells where the NF-
B
activation pathway is normal, arsenite induced a transient and less
potent expression of GADD45 (Fig. 3, A and B).
These results suggest that NF-
B activation may be unfavorable for
the induction of cell cycle checkpoint proteins that maintain genomic
integrity. GADD45 has been considered a p53 target gene whose
transcription/expression is dependent on the activation of p53 (16,
29). Several p53 binding sites have been identified in the regions of
the promoter, intron 1, intron 2, and intron 3 of GADD45 genes (30).
However, the cells used in the present report were previously shown to be functionally p53-deficient (26, 31). Furthermore, the fact that
arsenite neither induced N-terminal phosphorylation of p53 protein as
reported in the present studies nor induced p53-dependent reporter gene activity as demonstrated by Huang et al. (32) suggests that the induction of GADD45 by arsenite is through a p53-independent pathway.
B has been described
extensively (9, 10). However, only a few recent reports have
demonstrated a nontranscriptional or repressive transcriptional regulation of NF-
B on cellular genes. In differentiating myocytes, Guttridge et al. (33) demonstrated that NF-
B activated by
tumor necrosis factor
down-regulated MyoD mRNA at a
post-transcriptional level. In rat L6 muscle cells, studies by Du
et al. (34) indicated a negative transcriptional regulation
by NF-
B on a gene encoding proteasome C3 subunit. It is unclear
whether the negative regulation of NF-
B on GADD45 observed in the
present studies is similar to that seen with MyoD or proteasome C3
subunit. Analysis of GADD45 gene revealed several consensus
B sites
or
B-like sites in the promoter and intron
regions.2 We are currently
investigating whether these NF-
B binding sites contribute to
the down-regulation of GADD45 induced by arsenite by generating GADD45
gene reporter constructs with various deletion mutants.
-radiation,
and toxic metals (35-37). A yeast two-hybrid screen indicated that
GADD45 interacts with MEKK4, an MAPK kinase kinase activating JNK and
p38, suggesting a requirement of GADD45 for JNK activation (38). This
notion, however, was not supported by two follow-up studies using
embryonic fibroblasts derived from gadd45-null mice or cells in
which the GADD45 expression was diminished (39, 40). Treatment of
gadd45+/+ and gadd45
/
cells with ultraviolet C, hydrogen
peroxide, and other stress inducers revealed no deficiency in JNK
activation in gadd45
/
cells (39). In our studies, we noted that JNK
activation by arsenite preceded arsenite-induced GADD45 expression. JNK
activation was apparent as early as 1 h after arsenite
stimulation, a time point where no appreciable induction of GADD45 was
seen (Fig. 3B). Similarly, dose-response studies suggest
that a slightly higher dose of arsenite is required for GADD45
induction (Fig. 2B) than that for JNK induction (Fig.
3A). Finally, inhibition of JNK activation partially reduced GADD45 expression induced by arsenite (Fig. 4C). Therefore,
it is likely that JNK activation is an upstream, rather than a
downstream, event in GADD45 induction by arsenite.
B or JNK during the preparation of this
manuscript. Using BEAS-2B cells, the same cell line used for stable
transfection of IKK
or IKK
-KM described in the present studies,
Roussel and Barchowsky (41) reported that 500 µM arsenite inhibited tumor necrosis factor-induced NF-
B activation by directly blocking IKK activity. We found that lower concentrations of arsenite, from 5 to 20 µM, were capable of activating NF-
B in a
dose-dependent manner, whereas higher concentrations of
arsenite, more than 40 µM, inhibited NF-
B activation
as indicated by the NF-
B-dependent reporter gene assay
(Fig. 1B). This inhibitory effect of arsenite on NF-
B at
higher concentrations is largely because of its cytotoxic effects in
our experimental system.3 In
HeLa cells and HEK293 cells, arsenite has been shown to be able to
bind to cysteine 179 of IKK
and inhibit IKK activity induced
by tumor necrosis factor
, interleukin 1, and phorbol 12-myristate
13-acetate (42). Therefore, the observed activation of NF-
B by
arsenite in the present report may indicate an alternative mechanism of
NF-
B activation that is possibly independent of IKK. In bladder
epithelial cells, Simeonova et al. (43) noted that 5 to 50 µM arsenite activated AP-1 DNA binding activity and GADD45 gene expression, indicating an involvement of JNK or other MAP
kinases in the induction of GADD45 by arsenite. The upstream signaling
molecules leading to activation of JNK and IKK in response to arsenite
remain to be defined. It has been demonstrated that p21-activated
kinase is required for arsenite-induced JNK activation (44). It would
be interesting to determine whether p21-activated kinase is also
involved in arsenite-induced IKK activation.
B and JNK on arsenite-induced,
p53-independent GADD45 expression not only provide mechanistic clues
concerning the effects of arsenic but may also aid in developing new
strategies for the therapeutic use of arsenic in certain types of
leukemias. In most tissues or cells, where the activation pathway of
NF-
B is normal, arsenite may be carcinogenic because of the activation of NF-
B that may prevent induction of cell cycle
checkpoint proteins that maintain genomic stability. The therapeutic
use of arsenite in certain diseases, such as leukemias, may require strategies for the simultaneous inhibition of NF-
B. Such a
combination may potentiate the anticancer effects of arsenite by
increasing the induction of checkpoint proteins that either arrest cell
cycle progression or facilitate cell apoptosis.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Hiroyasu Nakano
(Juntendo University, Tokyo, Japan) for providing
pCR-FLAG-IKK and pCR-FLAG-IKK
-KM (K44A)-expressing vectors, to
Dr. Roger Davis (University of Massachusetts, Boston, MA) for the
gift of pcDNA-SEK1-KM vector, and to Dr. Chuanshu Huang (NIOSH)
for sharing anti-phospho-specific p53 antibodies.
![]() |
FOOTNOTES |
---|
* 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.
Supported by a Career Development award under a cooperative
agreement from the Centers for Disease Control and Prevention through
the Association of Teachers of Preventive Medicine. To whom
correspondence should be addressed: PPRB of NIOSH, 1095 Willowdale Rd,
Morgantown, WV 26505. Tel: 304-285-6021; E-mail: lfd3@cdc.gov.
§ To whom requests for reprints should be addressed: PPRB of NIOSH, 1095 Willowdale Rd, Morgantown, WV 26505. Tel.: 304-285-6158; Fax: 304-285-5938; E-mail: Xshi@cdc.gov.
Published, JBC Papers in Press, January 9, 2000, DOI 10.1074/jbc.M011682200
2 Chen et al., unpublished observations.
3 Chen et al., manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IKK, IB kinase;
JNK(s), c-Jun N-terminal kinase(s);
ERK, extracellular
signal-regulated kinase;
IKK
-KM, kinase-mutated form of IKK
;
PAGE, polyacrylamide gel electrophoresis;
MAP, mitogen-activated
protein;
SEK1-KM, kinase-mutated form of SEK1.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gebel, T. (2000) Toxicology 144, 155-162[CrossRef][Medline] [Order article via Infotrieve] |
2. | Kayajanian, G. M. (2000) Ecotoxicol. Environ. Saf. 45, 195-197[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Zhu, X. H.,
Shen, Y. L.,
Jing, Y. K.,
Cai, X.,
Jia, P. M.,
Huang, Y.,
Tang, W.,
Shi, G. Y.,
Sun, Y. P.,
Dai, J.,
Wang, Z. Y.,
Chen, S. J.,
Zhang, T. D.,
Waxman, S.,
Chen, Z.,
and Chen, G. Q.
(1999)
J. Natl. Cancer Inst.
91,
772-778 |
4. | Akao, Y., Yamada, H., and Nakagawa, Y. (2000) Leuk. Lymphoma 37, 53-63[Medline] [Order article via Infotrieve] |
5. |
Estrov, Z.,
Manna, S. K.,
Harris, D.,
Van, Q.,
Estey, E. H.,
Kantarjian, H. M.,
Talpaz, M.,
and Aggarwal, B. B.
(1999)
Blood
94,
2844-2853 |
6. | Cavigelli, M., Li, W. W., Lin, A., Su, B., Yoshioka, K., and Karin, M. (1996) EMBO J. 15, 6269-6279[Abstract] |
7. | Hayes, R. B. (1997) Cancer Causes Control 8, 371-385[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Karin, M.,
and Delhase, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9067-9069 |
9. | Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-683[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Chen, F.,
Castranova, V.,
Shi, X.,
and Demers, L. M.
(1999)
Clin. Chem.
45,
7-17 |
11. | Karin, M., and Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, 621-663[CrossRef][Medline] [Order article via Infotrieve] |
12. | Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1999) Nature 401, 616-620[CrossRef][Medline] [Order article via Infotrieve] |
13. | Piwnica-Worms, H. (1999) Nature 401, 535[CrossRef][Medline] [Order article via Infotrieve] |
14. | Fornace, A. J., Jr., Alamo, I., Jr., and Hollander, M. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8800-8804[Abstract] |
15. |
Wang, X. W.,
Zhan, Q.,
Coursen, J. D.,
Khan, M. A.,
Kontny, H. U., Yu, L.,
Hollander, M. C.,
O'Connor, P. M.,
Fornace, A. J., Jr.,
and Harris, C. C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3706-3711 |
16. |
Yu, J.,
Zhang, L.,
Hwang, P. M.,
Rago, C.,
Kinzler, K. W.,
and Vogelstein, B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14517-14522 |
17. |
O'Reilly, M. A.,
Staversky, R. J.,
Watkins, R. H.,
Maniscalco, W. M.,
and Keng, P. C.
(2000)
Am. J. Physiol.
278,
L552-L559 |
18. | Jin, S., Zhao, H., Fan, F., Blanck, P., Fan, W., Colchagie, A. B., Fornace, A. J., Jr., and Zhan, Q. (2000) Oncogene 19, 4050-4057[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Manna, S. K.,
Zhang, H. J.,
Yan, T.,
Oberley, L. W.,
and Aggarwal, B. B.
(1998)
J. Biol. Chem.
273,
13245-13254 |
20. | Ip, Y. T., and Davis, R. J. (1998) Curr. Opin. Cell Biol. 10, 205-219[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Woronicz, J. D.,
Gao, X.,
Cao, Z.,
Rothe, M.,
and Goeddel, D. V.
(1997)
Science
278,
866-869 |
22. | Chu, W. M., Ostertag, D., Li, Z. W., Chang, L., Chen, Y., Hu, Y., Williams, B., Perrault, J., and Karin, M. (1999) Immunity 11, 721-731[Medline] [Order article via Infotrieve] |
23. |
Geleziunas, R.,
Ferrell, S.,
Lin, X.,
Mu, Y.,
Cunningham, E. T., Jr.,
Grant, M.,
Connelly, M. A.,
Hambor, J. E.,
Marcu, K. B.,
and Greene, W. C.
(1998)
Mol. Cell. Biol.
18,
5157-5165 |
24. |
Li, Q.,
Van Antwerp, D.,
Mercurio, F.,
Lee, K. F.,
and Verma, I. M.
(1999)
Science
284,
321-325 |
25. | Kastan, M. B., Zhan, Q., el-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992) Cell 71, 587-597[Medline] [Order article via Infotrieve] |
26. | Lehman, T. A., Modali, R., Boukamp, P., Stanek, J., Bennett, W. P., Welsh, J. A., Metcalf, R. A., Stampfer, M. R., Fusenig, N., Rogan, E. M., et al.. (1993) Carcinogenesis 14, 833-839[Abstract] |
27. |
Ashcroft, M.,
Kubbutat, M. H.,
and Vousden, K. H.
(1999)
Mol. Cell. Biol.
19,
1751-1758 |
28. | Lubin, J. H., Pottern, L. M., Stone, B. J., and Fraumeni, J. F., Jr. (2000) Am. J. Epidemiol. 151, 554-565[Abstract] |
29. |
Zhao, R.,
Gish, K.,
Murphy, M.,
Yin, Y.,
Notterman, D.,
Hoffman, W. H.,
Tom, E.,
Mack, D. H.,
and Levine, A. J.
(2000)
Genes Dev.
14,
981-993 |
30. | Zhan, Q., Carrier, F., and Fornace, A. J., Jr. (1993) Mol. Cell. Biol. 13, 4242-4250[Abstract] |
31. | Gerwin, B. I., Spillare, E., Forrester, K., Lehman, T. A., Kispert, J., Welsh, J. A., Pfeifer, A. M., Lechner, J. F., Baker, S. J., Vogelstein, B., et al.. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2759-2763[Abstract] |
32. |
Huang, C.,
Ma, W. Y.,
Li, J.,
and Dong, Z.
(1999)
Cancer Res.
59,
3053-3058 |
33. |
Guttridge, D. C.,
Mayo, M. W.,
Madrid, L. V.,
Wang, C. Y.,
and Baldwin, A. S., Jr.
(2000)
Science
289,
2363-2366 |
34. |
Du, J.,
Mitch, W. E.,
Wang, X.,
and Price, S. R.
(2000)
J. Biol. Chem.
275,
19661-19666 |
35. |
Dickens, M.,
Rogers, J. S.,
Cavanagh, J.,
Raitano, A.,
Xia, Z.,
Halpern, J. R.,
Greenberg, M. E.,
Sawyers, C. L.,
and Davis, R. J.
(1997)
Science
277,
693-696 |
36. |
Chen, F.,
Demers, L. M.,
Vallyathan, V.,
Ding, M.,
Lu, Y.,
Castranova, V.,
and Shi, X.
(1999)
J. Biol. Chem.
274,
20307-20312 |
37. | Samet, J. M., Graves, L. M., Quay, J., Dailey, L. A., Devlin, R. B., Ghio, A. J., Wu, W., Bromberg, P. A., and Reed, W. (1998) Am. J. Physiol. 275, L551-L558[Medline] [Order article via Infotrieve] |
38. | Takekawa, M., and Saito, H. (1998) Cell 95, 521-530[Medline] [Order article via Infotrieve] |
39. |
Shaulian, E.,
and Karin, M.
(1999)
J. Biol. Chem.
274,
29595-29598 |
40. |
Wang, X.,
Gorospe, M.,
and Holbrook, N. J.
(1999)
J. Biol. Chem.
274,
29599-29602 |
41. | Roussel, R. R., and Barchowsky, A. (2000) Arch. Biochem. Biophys. 377, 204-212[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Kapahi, P.,
Takahashi, T.,
Natoli, G.,
Adams, S. R.,
Chen, Y.,
Tsien, R. Y.,
and Karin, M.
(2000)
J. Biol. Chem.
275,
36062-36066 |
43. |
Simeonova, P. P.,
Wang, S.,
Toriuma, W.,
Kommineni, V.,
Matheson, J.,
Unimye, N.,
Kayama, F.,
Harki, D.,
Ding, M.,
Vallyathan, V.,
and Luster, M. I.
(2000)
Cancer Res.
60,
3445-3453 |
44. | Porter, A. C., Fanger, G. R., and Vaillancourt, R. R. (1999) Oncogene 18, 7794-7802[CrossRef][Medline] [Order article via Infotrieve] |