Nickel is one of the most abundant transition metals in the earth's crust (1).
It is used in a variety of industrial processes, e.g., nickel refinement, nickel-cadmium
batteries, and electroplating (2). These processes, in addition to the
incineration of nickel-containing wastes and fossil fuels, are responsible for
the majority of nickel aerosols found in both the workplace and the environment
(2). It has been estimated that the average daily exposure to nickel
is between 0.2 and 0.4 µg in both urban and rural environments (2).
Workplace exposure is considerably higher.
The main route for exposure to nickel compounds is by inhalation. Indeed,
a variety of epidemiologic studies have indicated a significant correlation
between the number of respiratory cancers and workplace nickel exposure (3,4).
An effect of nickel compounds in animal models, through inhalation, injection
or ingestion, is to produce tumors (5-7).
The mechanism by which nickel toxicity is exerted has been extensively studied
(8-10). Nickel exposure induces several types of cellular and nuclear
damage (8,11,12). Although nickel is a potent carcinogen, it is generally
not active in mutagenic assays (13-16). This suggests that nickel-induced
toxicity/carcinogenicity may be caused by alterations in gene expression rather
than by direct DNA damage. For example, transcription factors, metallothionein,
and heat shock proteins can be induced by exposure to nickel (17-19).
Nuclear factor 
B
(NF-B)
was first described as a B-cell nuclear factor that binds to immunoglobulin
enhancer and thus was implicated in immune response (20,21). Subsequent
research revealed that NF-B
was not B-cell specific and could bind to specific sites in a variety of gene
promoter/enhancers, e.g., interleukin (IL)-2, IL-6, granulocyte macrophage colony-stimulating
factor, intercellular adhesion molecule-1, and class I major histocompatibility
complex (22-24). Initially the number of inducers of NF-B
was quite small but has since grown substantially, e.g., tumor necrosis factor,
IL-1, ultraviolet radiation, growth factors, free radicals, and viral infection
(22-24). Additionally, there is an increasing body of evidence suggesting
a role for NF-B
in carcinogenesis. For example, NF-B
is implicated in signaling tumor promoter-induced transformation and is activated
by viral transforming proteins (24-26). The importance of NF-B
cannot be overstated, as failure in any of the mechanisms leading to NF-B
activation can have serious consequences for the cell. Studies involving NF-B
are frequently compared with those involving activator protein-1 (AP-1). AP-1
is a transcription factor complex composed of Jun family homodimers or Jun/Fos
heterodimers (27,28). As with NF-B,
AP-1 is activated by a number of different stimuli, including cell stress, cytokines,
growth factors, and neurotransmitters (27-29). Both AP-1 and some of
the gene transcripts regulated by AP-1 are involved in neoplastic transformation
(30-33). As a result we have further investigated the possible involvement
of NF-B
and AP-1 in nickel-induced carcinogenesis.
Materials and Methods
Plasmids and Agents
The cytomegalovirus (CMV)-neo vector plasmid and AP-1-luciferase reporter,
as well as NF-B-luciferase reporter plasmids, were constructed as previously
described (34-36). Anhydrous nickel chloride (NiCl2)
was purchased from Aldrich (Milwaukee, WI, USA); nickel subsulfide (Ni3S2)
was obtained from INCO (Toronto, Canada). Fetal bovine serum (FBS) was obtained
from Life Technologies, Inc. (Carlsbad, CA, USA). Eagle's minimal essential
medium (MEM) and Dulbecco's modified Eagle's medium (DMEM) were both obtained
from BioWhittaker (Walkersville, MD, USA). The luciferase assay substrate was
purchased from Promega (Madison, WI, USA).
Cell Culture
Mouse fibroblasts 3T3 and B82 cells, as well as their NF-B-luciferase
reporter or
AP-1-luciferase reporter stable transfectants, were cultured in DMEM with 10%
FBS, 2 mM l-glutamine, and 25 µg gentamicin/mL (34). Human bronchial
epithelial cell BEAS-2B stable transfectants IB
kinase ß (IKKß) or IKKß dominant negative mutant (IKKß-KM)
were cultured in 10% FBS, 2 mM l-glutamine, and 25 µg gentamicin/mL as
reported by Chen et al. (37) and Huang et al. (38).
Generation of Stable Transfectants with NF-B-Luciferase
Reporter or AP-1-Luciferase Reporter
3T3 cells were cultured in a 6-well plate until they reached 85-90% confluence.
CMV-neo vector (1 µg) and 15 µL LipofectAMINE reagent, (Gibco BRL,
Rockville, MD, USA) together with 12 µg NF-B-luciferase reporter plasmid
DNA or AP-1-luciferease reporter plasmid DNA, were used to transfect each well
in DMEM in the absence of serum. After 10-12 hr, the medium was replaced
with 10% FBS DMEM. Approximately 30-36 hr after the beginning of the transfection,
the cells were trypsinized with 0.033% trypsin, and cell suspensions were plated
onto 75-mL culture flasks and cultured for 24-28 days with G418 selection
(600 µg/mL). Measuring basal level of luciferase activity identified the
stable transfectants. Stable transfectants 3T3 NF-B mass1 or 3T3 AP-1 mass1
were established and cultured in G418-free MEM for at least two passages before
each experiment.
Assay for NF-B
Activation
Confluent monolayers of 3T3 NF-B
mass1 or IKKß were trypsinized, and 8 
103 viable cells were suspended in 100 µL culture medium in
each well of a 96-well plate. Plates were incubated at 37°C in a humidified
atmosphere containing 5% CO2. Twelve to 24 hr later, cells were starved
by culturing them in 0.1% FBS DMEM for 24 hr. The cells were then exposed to
either Ni3S2 or NiCl2 for NF-B
induction and maintained in culture. The cells were extracted with lysis buffer
at various times, and luciferase activity was measured. The results are expressed
as relative NF-B
activity (35).
Assay for AP-1 Activity
Confluent monolayers of 3T3 AP-1 mass1 or B82 AP-1 mass2 were trypsinized,
and 8
103 viable cells suspended in 100 µL culture medium were added
to each well of a 96-well plate. Plates were incubated at 37°C in a humidified
atmosphere containing 5% CO2. Twelve to 24 hr later, cells were starved
by culturing them in 0.1% FBS DMEM for 24 hr. The cells were then exposed to
either Ni3S2 or NiCl2 for AP-1 induction and
maintained in culture. The cells were extracted with lysis buffer, and luciferase
activity was measured. The results are expressed as relative AP-1 activity (34).
Statistical Analysis
The significance of the difference in the
NF-B and AP-1 activities was determined with the Student t test. The
results are expressed as mean ± SEM.
Western Blot Analysis
Human bronchial epithelial cell line BEAS-2B and its stable transfectant IKKß-KM
were cultured in each well of 6-well plates to 90% confluence. The cells were
exposed to NiCl2 or Ni3S2 and incubated for
different times indicated in the figure legends. The cells were then washed
once with ice-cold phosphate- buffered saline (PBS) and extracted with sodium
dodecyl sulfate (SDS)-sample buffer. The cell extracts were separated on polyacrylamide-SDS
gels, transferred, and probed with one of two antibodies, including rabbit specific
antibody against Cap43 protein or specific antibody against protein kinase C
. The
protein bands specifically bound to primary antibodies were detected using an
anti-rabbit immunoglobulin G (IgG)-AP-linked (Amersham Biosciences, Piscataway,
NJ, USA) as second antibody and an ECF Western blotting system (36).
Results
Effect on Induction of NF-B
in Mouse Fibroblast 3T3 Cells by Nickel Compounds
Figure 1. Induction of NF-kB activity by nickel
compounds in mouse fibroblast 3T3 cells. 3T3 NF-kB mass1 cells (8 ¥
103) were seeded into each well of a 96-well plate. After being cultured
at 37C overnight, the cells were starved for 12 hr by replacing the medium
with 0.1% FBS DMEM. The cells were then treated as follows: (A) 1 µg/cm2
Ni3S2 or 1 µM NiCl2 for 48 hr. (B) For a time-course study, the cells
were exposed to 1 µg/cm2 Ni3S2 for various times as indicated. (C)
For a dose–response study, the cells were exposed to different concentrations
of Ni3S2 as indicated for 48 hr. The luciferase activity was then measured
and the results are presented as NF-kB–dependent transcription activity
relative to control. Each bar indicates the mean and standard deviation
of four repeat assay wells. Asterisk (*) indicates a significant increase
from control (p < 0.05). |
To determine the effects of nickel on NF-B activation in mouse fibroblast
cells, we incubated mouse fibroblast 3T3 cells with either Ni3S2
(2 µg/cm2) or NiCl2 (1 mM) and monitored the effect
on NF-B-dependent transcriptional activation. Shown in Figure 1A is the relative
NF-B activity in 3T3 cells after treatment with either Ni3S2
or NiCl2. It can be seen that Ni3S2 is a potent
activator of NF-B and induces an approximately 12-fold increase in NF-B relative
to untreated cells. NiCl2 treatment also produces an increase in
NF-B activity (~6-fold), although not quite as pronounced as with Ni3S2.
Figure 1B shows the time course for maximal NF-B activation upon Ni3S2
treatment. The results indicate a gradual increase in relative NF-B activity
over a period of 24 hr (5-fold increase). Activation increases to a maximum
after 48 hr (12-fold) before decreasing again after 72 hr (3-fold). A dose-response
study of the effect of Ni3S2 treatment indicates that
induction of NF-B activity is concentration dependent, as shown in Figure 1C.
The most effective dose range of Ni3S2 treatment on 3T3
cells was 1.0-2.0 µg/cm2. These results indicate that NF-B
is involved in the cellular response to nickel compounds.
Effect on Induction of NF-B
in Human Bronchial Epithelial BEAS-2B Cells by Nickel Compounds
A variety of epidemiologic studies indicated that nickel exposure is correlated
with an increase in the incidence of respiratory cancers (3-7).
To understand the involvement of NF-B activation in the response of the respiratory
system to nickel compounds, we tested the effect of Ni3S2
and NiCl2 on NF-B activity in human bronchial epithelial BEAS-2B
cells. As shown in Figure 2A, treatment of cells with either Ni3S2
or NiCl2 also leads to an increase in NF-B activity. The increase
in NF-B activity upon Ni3S2 treatment is approximately
4.5-fold relative to that in the control, whereas NiCl2 treatment
leads to an approximately 2.7-fold increase in activity. This induction was
also observed in the dose response of NF-B activity to Ni3S2
(Figure 2B). These results, taken together with the results from 3T3 cells,
indicate that the induction of NF-B is involved in the response of the cell
to nickel compounds.
Figure 2. Induction of NF-B
activity by nickel compounds in human bronchial epithelial BEAS-2B cells.
BEAS-2B IKKß transformed cells (8
103) were seeded into each well of a 96-well plate. After being
cultured at 37°C overnight, the cells were starved for 12 hr by replacing
the medium with 0.1% FBS DMEM. The cells were then treated as follows: (A)
1 µg/cm2 Ni3S2 or 1 mM NiCl2
for 36 hr. (B) For a dose-response study, the cells were exposed
to different concentrations of Ni3S2 as indicated
for 36 hr. The luciferase activity was then measured and the results are
presented as NF-B-dependent
transcription activity relative to control. Each bar indicates the mean
and standard deviation of four repeat assay wells. Asterisk (*) indicates
a significant increase from control (p < 0.05). |
Absence of Induction of AP-1 Activity with Nickel Compounds
To test whether the response of cells to Ni3S2 and NiCl2
involves AP-1, we also generated stable AP-1-luciferase 3T3 transfectants. As
shown in Figure 3, treatment of cells with Ni3S2 or NiCl2
did not show any induction of AP-1 activity in 3T3 cells, whereas NF-B activation
was observed. In contrast, ultraviolet-C (UVC) radiation resulted in increases
in both NF-B and AP-1 activity (Figure 3). These results indicated that NF-B
but not AP-1 was involved in the response of cells to nickel compounds.
To further explore whether the effects of Ni3S2 and
NiCl2 on AP-1 activity are cell specific, we tested the effect of
nickel compounds on AP-1 activity in fibroblast B82 cells. As with the 3T3 cells,
treatment of B82 cells with either Ni3S2 or NiCl2
did not lead to an increase in AP-1 activity (Figure 4). Again, UVC radiation
treatment resulted in an increase in AP-1, indicating that the absence of induction
of AP-1 transcriptional activation toward Ni3S2 and NiCl2
treatment was not cell-type specific. This was consistent with our previous
findings in C141 cells (39).
|
Figure 4. No induction of
AP-1 activity by nickel compounds in mouse fibroblast B82 cells. B82 AP-1
mass2 (8
103) were seeded into each well of a 96-well plate. After being
cultured at 37°C overnight, the cells were starved for 12 hr by replacing
the medium with 0.1% FBS DMEM. The cells were then treated with Ni3S2
(2 µg/cm2), NiCl2 (1 mM), or UVC radiation
(30 J/cm2) for 36 hr. The luciferase activity was then measured
and the results are presented as relative AP-1 activity. Each bar indicates
the mean and standard deviation of four repeat assay wells. Asterisk (*)
indicates a significant increase from control (p < 0.05).
|
Induction of Cap43 in BEAS-2B Stable Transfectants
Both Ni3S2 and NiCl2 induce a novel gene,
Cap43, which is also induced by hypoxia and the calcium ionophore A23187
(40,41). Recently it was found that Cap43 was expressed only in
cancer cells, not in normal cells (42). The mechanism by which nickel
acts is not well understood. To determine whether NF-B activation by nickel
is involved in nickel-induced Cap43 expression, we compared Cap43
expression between human bronchial epithelial BEAS-2B cells and their stable
transfectant IKKß-KM cells. The results showed that an overexpression
of a IKKß-KM did not affect nickel-induced Cap43 expression (Figure
5). This suggests that the signal transduction pathway leading to NF-B activation
by nickel compounds does not involve Cap43 expression by nickel.
|
Figure 5. Induction of Cap43
protein expression by Ni3S2 (A) or NiCl2 (B) in both BEAS-2B and IKKb-KM
cells. Subconfluent (90%) monolayers of BEAS-2B and IKKb-KM in 6-well
plates were subjected to either (A) Ni3S2 (2 µg/cm2) or (B) NiCl2
(1 mM) and cultured for time points as indicated. The cells were then
washed once with ice-cold PBS and extracted with SDS-sample buffer. The
cell extracts were separated on polyacrylamide-SDS gels, transferred,
and probed with rabbit polyclone antibodies against Cap43. The Cap43 protein
band specifically bound to the primary antibody was detected using an
antirabbit IgG-AP-linked as second antibody and an ECF Western blotting
system (38). PKC was used as internal control of protein loaded.
|
Discussion
In this study we investigated the effect of Ni3S2 and
NiCl2 on the transcription factor NF-B and AP-1 in various cell
culture models. NF-B activation by nickel compounds was found in mouse fibroblasts
(3T3) and human bronchoepithelial cells (BEAS-2B), whereas nickel treatment
did not induce any activation of AP-1 in the same cells. Furthermore, NF-B
activation by nickel compounds was not required for Cap43 expression,
as overexpression of the IKKß-KM had no effect on Cap43 expression.
Our results indicate that both insoluble Ni3S2 and soluble
NiCl2 are effective inducers of NF-B activation in mouse fibroblast
3T3 cells and human bronchoepithelial BEAS-2B cells. As has been shown previously,
insoluble Ni3S2 appears to be more effective in potentiating
a biochemical response than NiCl2 (43). The effect of Ni3S2
is both time and dose dependent. The maximum effect on NF-B activation by Ni3S2
takes place after 48-hr exposure. The most effective dose is 1.0-2.0 µg/cm2,
although the lower dose of 0.5 µg/cm2 is still very effective
in inducing an increase in NF-B activity. Ni3S2 was toxic
to cultured hamster lung fibroblasts at 0.5 µg/cm2 (15),
whereas in our system cytotoxicity does not appear to be a factor until after
greater than 48-hr exposures and doses above 2.0 µg/cm2. This
observation is supported by data that
NF-B activity would increase relative to that of the control (Figure 1B). The
reason for this difference may be due to cell-type specificity.
The results showing that Ni3S2 and NiCl2
potentiate NF-B
but not AP-1 activity in different cell culture models were intriguing. NF-B
has been the focus of considerable research since its discovery in 1986 (20,
21). NF-B
is a member of the NF-B/Rel
family and exists in an inactive form in cells through formation of a complex
with IB
(22,44-51). Phosphorylation of IB
leads to ubiquitination of the cytoplasmic NF-B
complex and subsequent degradation of the complex to produce the active form
of NF-B
(52-54). NF-B
is then translocated to the nucleus from the cytoplasm, where it induces gene
activation. Considerable evidence has been presented to implicate NF-B
activation with tumor promotion in cell models (23,25,55). For example,
both v-Rel and p52/Lyt-10, members of the NF-B
family, and Bcl-3, an IB
family member, are potentially oncogenic (24). In addition, it was shown
separately that the c-myc oncogene promoter implicated in Burkitt lymphoma
is activated by NF-B
(56) and that NF-B
positively regulates the expression of the translocated c-myc gene in
Burkitt lymphoma. Additionally, overexpressed IB
in 3T3 cells blocked the ability of ras alleles to induce focus formation,
again suggesting a role for NF-B
(57). Furthermore, overexpressed IB
crossed with v-Rel transgenic mice induced a delay in death from leukemia (23).
AP-1 is a transcription factor complex composed of members of the Jun and
Fos families of proteins (27,28). Both AP-1 and NF-B are activated by
similar stimuli, including growth factors, cytokines, and UVC radiation, leading
to altered gene expression (27-29). Like NF-B, AP-1 has been implicated
in tumor promotion in different cell models (27,30,31,58-65). AP-1
activity was also elevated in mouse epidermal JB6 cells, indicating various
stages of tumor promotion (59). Furthermore, tumor promotion could be
inhibited by the use of several types of AP-1 inhibitors (38,60-63,66).
In light of the important roles that both NF-B and AP-1 play in tumor promotion
by many chemicals, we wished to investigate the signal transduction pathways
involved in the carcinogenic properties of nickel. The results indicate that
Ni3S2 and NiCl2 specifically induce NF-B activity
but not AP-1 activity in mouse fibroblast 3T3 cells. The specificity of Ni3S2
and NiCl2 for NF-B activity is further supported by the time-course
and dose-response studies, as well as by the observation that UVC stimulates
both NF-B and AP-1 in 3T3 cells. A comparison with fibroblast B82 cells also
showed that AP-1 activity was increased by UVC exposure but not by Ni3S2
or NiCl2.
Cap43 has been reported to be specifically induced by nickel compounds
in a variety of cell lines (40,41). Although the function of the Cap43
protein is not well understood, it does appear to be induced in response to
an increase in intracellular concentration of Ca2+ (41). The
complete mechanism of signal transduction leading to Cap43 expression
has yet to be elucidated, but it has been shown that nickel induces HIF-1 and
that this, in turn, activates Cap43 transcription (67). Our current investigation
using BEAS-2B and IKKß-KM indicates that overexpression of IKKß-KM
did not block Cap43 induction in response to both Ni3S2
and NiCl2. Our results suggest that induction of Cap43 does
not involve signals arising from the NF-B pathway.
To summarize the results, Ni3S2 and NiCl2
activate NF-B in both mouse fibroblast 3T3 cells and human bronchoepithelial
BEAS-2B cells. In addition, AP-1 activity is unaffected by nickel treatment
in mouse 3T3, human bronchoepithelial BEAS-2B, and mouse C141 cells, which indicates
that the response to nickel must involve a signal transduction pathway that
terminates with NF-B rather than AP-1. Also, NF-B activation by nickel compounds
is not required for Cap43 expression.
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Last Updated: October 17, 2002
