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Carcinogenesis |
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, NIH, Morgantown, West Virginia 26505 [P. P. S., S. W., W. T., V. K., J. M., D. H., M. D., V. V., M. I. L.]; Tanabe Seiyaku Company, Kawagishi, Toda 335, Japan [W. T.]; Department of Urology, West Virginia University Medical Center, Morgantown, West Virginia 26505 [N. U.]; and Tako Chuo Hospital, Katori-Gun, Chiba, Japan [F. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although several hypotheses have been proposed, the mechanism
responsible for arsenic carcinogenesis has not been established.
Increasing evidence indicates that arsenic acts on signaling pathways
that regulate cell proliferation rather than causing direct DNA damage
because arsenic exhibits its mutagenic activities only at
concentrations high enough to also produce cell damage
(9)
. Recently, arsenic has been shown to modulate the
mitogen-activated protein kinase cascade in several cell
systems, resulting in the activation of transcription factors, such as
AP-12
(10, 11, 12, 13)
. AP-1 mediates many biological effects of tumor
promoters and is an important regulator of cell growth. The ability of
arsenic to interact with protein thiol groups on key regulatory
proteins and subsequently alters of their activities is likely to
contribute to this effect (10
, 11)
. Accordingly, it has
been demonstrated that arsenic can induce a moderate increase in
keratinocyte cell proliferation, as evidenced by increases in thymidine
incorporation (14)
, cell cycling (15)
,
labeling of the proliferating cell marker Ki-67 (15)
,
ornithine decarboxylase activity (16)
, and expression of
oncogenes and growth factors such as c-fos,
c-jun, c-myc, and transforming growth factor 
(14
, 17)
. Using both in vitro and in
vivo models, the present studies were conducted to determine
whether arsenic produced molecular and histological changes in urinary
bladder epithelium consistent with epigenetic mechanisms for
carcinogenesis, in this case cell proliferation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histology.
Bladders were removed and fixed by immersion in 10% neutral-buffered
formalin and processed for paraffin embedding. Each paraffin block was
step-sectioned and stained with H&E. For scanning electron microscopy,
the specimens were fixed in 2.5% gluteraldehyde, washed in phosphate
buffer, and postfixed in 2% osmium tetroxide. The specimens were
dehydrated with graded alcohol, placed in hexamethyldisalizane, and air
dried. After coating with gold/palladium, the specimens were examined
using a JEOL 6400 (Tokyo, Japan) scanning electron microscope.
For transmission electron microscopy, the samples were fixed as
described for scanning microscopy and embedded in LX-112 epon
(Ladd Research Industry, Burlington, VT). Ultrathin sections
were stained with uranyl acetate and lead citrate and examined by
transmission electron microscopy (JEOL 1220).
For immunohistochemistry, 6-µm sections were prepared from paraffin blocks and placed on microscope slides. Slides were deparaffinized, and the antigen was unmasked using 10 mM sodium citrate buffer (pH 6.0) at 95°C for 5 min. The endogenous peroxidase activity was blocked by peroxidase block (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Slides were treated for 20 min with 1% normal goat serum and incubated overnight at 4°C with a 1:1000 dilution of rabbit antihuman c-jun or c-fos polyclonal antibodies (Santa Cruz Biotechnology Inc.) or a 1:100 dilution of monoclonal anti-PCNA antibody (Vector Laboratories, Inc., Burlingame, CA). After being washed, the samples were incubated for 1 h with a 1:200 dilution of biotinylated goat antirabbit or antimouse IgG antibody, respectively (Vector Laboratories; 1:200) followed by incubation for 1 h with ABC buffer (Vectastain Elite kit, Vector Laboratories, Inc.). Ten mg of diaminobenzidine were dissolved in 20 ml Tris-imidazole buffer (50 mM Trizma, 10 mM imidazole, pH 7.2) containing 3.3 µl of 30% H2O2. Samples were incubated in this buffer for 6 min before rinsing with Tris-imidazole buffer and counterstained with Harris’s hematoxylin (EM Diagnostic Systems, Gibbstown, NJ). Results are presented as the percentage of positively stained nuclei counted in four random areas of the slide.
Arsenic Determination in Tissues.
Urinary bladders from control or arsenic-treated mice were quick-frozen
in acid-free vials and stored at –70°C. The tissue samples were
digested by addition of 6 N HCl at 80°C for 16 h in
a specially designed reaction vessel. Analyses of arsenic tissue levels
were performed by Battelle Marine Sciences Laboratory (Seqium, WA)
using a complex atomic absorption method (19)
. Briefly,
arsenic, MMA, and DMA were converted to volatile arsines by addition of
4% NaBH4 solution. The volatiles were purged
from the sample onto a cooled glass trap packed with 15% OV-3 on
Chromasorb WAW-DMCSO. The trapped arsines were thermally
desorbed to increase boiling points and carried as an inert gas stream
into the quartz furnace of an atomic absorption spectrophotometer. The
first arsine desorbed was AsH3, which represents
total inorganic arsenic in the sample, whereas MMA and DMA were
desorbed and detected several minutes later. To determine the
concentration of arsenite (iAs3+), another
aliquot of sample was placed in the reaction vessel, and Tris buffer
was added. This procedure was repeated to determine only the arsine
produced from arsenite. Quality control was established through
calibration and testing of the hydride generation, purging, and
detection systems.
Cell Cultures.
UROtsa, a SV40 immortalized human urothelium cell line, was obtained
from Dr. G. Petzoldt, (University College, London, United Kingdom). The
cell line does not acquire characteristics of transformed cells
including growth in soft agar or development of tumors in nude mice
(20)
. The cells were grown at
37°C/5%CO2, in RPMI 1640 culture medium
supplemented with 10% FBS and 2 mM L-glutamine
(Life Technologies, Gaithersburg, MD), referred to as complete
medium.
Cell Mitogenesis.
UROtsa cells were seeded in 96-well, flat-bottomed culture plates at a
concentration of 2 x 103
cells/well. The following day (approximately 40% confluent) the
culture medium was replaced with RPMI 1640 medium supplemented with 1%
FBS, and the cells were exposed to different concentrations of sodium
arsenite for 48–72 h. During the last 4 h of incubation,
[3H]thymidine (6.7 Ci/mmol; DuPont New England
Nuclear) was added at a concentration of 0.5 µCi/well. The cells were
detached from the plates by addition of 0.1% trypsin and collected
onto glass-fiber filters using an automated cell harvester (Skatron,
Sterling, VA). Cellular incorporation of
[3H]thymidine was determined by liquid
scintillation counting.
Cell Cycle Analysis.
A flow cytometry, propidium iodide method was used to determine the
cell cycle. Briefly, UROtsa cells in complete medium were seeded at a
concentration of 2 x 106 cells/ml
into Falcon Petri dishes (100/15 mm, Becton Dickinson Labware, Lincoln
Park, NJ). The medium was replaced with RPMI 1640 medium with 1% FBS,
reincubated for 24 h, and treated with sodium arsenite for an
additional 24 h. The cells were removed with 0.1% trypsin, and
the pelleted cells were fixed overnight in 70% ethanol at 4°C. The
cells were stained with 50 mg/ml propidium iodide (Sigma) in the
presence of 100 units/ml RNase A (Sigma) to degrade double-stranded
RNA. Analyses were conducted by flow cytometry (Becton Dickinson
Immunocytometry Systems, FACScan with Quest 1.2 software, Sunnyvale,
CA), and the data were evaluated by Modfit LT 2.0 (Verity Software
House Inc.).
Nuclear Extracts and EMSA.
Nuclear proteins were prepared from aliquots of 1 x 107 cells or frozen samples of bladder tissue
according to the method of Schreiber et al.
(21)
. DNA binding reactions and EMSAs were performed as
described previously (22)
. Briefly, the 5' ends of the
double-stranded oligonucleotides were labeled with
[
32P]ATP (DuPont New England Nuclear) using
6–10 units of T4 polynucleotide kinase (United States Biochemical
Corp./Amersham Pharmacia Biotech, Piscataway, NJ). Binding
reactions (30 µl) were performed on ice for 30 min in reaction
mixtures containing 10 µg of nuclear proteins, 20
mM Tris-HCl (pH 7.8), 100
mM NaCl, 5 mM
MgCl2, 1 mM EDTA, 5
mM DTT, 50 µg/ml BSA, 2 µg of
poly(dI-dC)·poly(dI-dC), 10% glycerol, and approximately 0.1 ng
(2 x 105 cpm) of specified probe.
For detection of AP-1 DNA-binding activity, an oligonucleotide was
obtained from Santa Cruz Biotechnology containing an AP-1 consensus
sequence, 5'-CGC TTG ATG ACT CAG CCG GAA-3'. Protein-DNA complexes were
separated on a 5% nondenaturing polyacrylamide gel. Gels were
electrophoresed at 125 V in 50 mM Tris-50
mM boric acid/1 mM EDTA,
dried, and autoradiographed overnight. The autoradiograms were scanned
with a computerized laser densitometer (Eagle Eye II image analysis
system, Stratagene, La Jolla, CA). These results were examined using
the One Dscan gel analysis software and the NIH Image 1.54 analysis
software. For characterization of DNA binding activity, nuclear protein
extracts were preincubated for 1 h prior to the addition of
labeled probe with either excess unlabeled oligomers for 30 min or 2
µg/ml of antibodies (Santa Cruz Biotechnology).
Gene Expression Studies.
RNA from cell cultures was extracted using the Rneasy total RNA kit
(Qiagen, Santa Clarita, CA). RNA purity and concentration were assessed
by determining A260/A280 absorption. cDNA was synthesized from 1
µg of RNA. PCR amplification was performed as described previously
using commercially available PCR primers for c-jun,
c-fos, and G3PDH from Clontech Laboratories Inc. (Palo Alto,
CA) and 5-µl aliquots of cDNA, corresponding to 100 ng of RNA
(22)
. RNA concentrations and PCR cycles were titrated to
establish standard curves, to document linearity, and to permit
semiquantitative analysis of signal strength as described previously
(14)
. When appropriate, the specificity of the PCR bands
was confirmed by restriction enzyme analysis of the amplified cDNA,
which generated restriction fragments of the expected size (data not
shown). The PCR products were visualized by UV illumination after
electrophoresis through 2.0% agarose (Ultra-Pure, Sigma) at 60 V for
80 min and staining in Tris borate/EDTA buffer (89 mmol/liter Tris, 89
mmol/liter boric acid, 2.5 mmol/liter EDTA, pH 8.2) containing 0.5
mg/ml ethidium bromide. The gels were analyzed using the Eagle Eye II
image analysis system (Stratagene) and NIH Image 1.54 software. The
area under the curve was normalized for G3PDH content.
Analysis of differential gene expression was performed on UROtsa
cells using the Atlas human cDNA expression array (Clontech, Palo Alto,
CA). Briefly, UROtsa cells were allowed to grow to 70%
confluency and treated with 10 or 50 µM sodium arsenite
or 20 ng/ml PMA for 2 h. Poly(A) RNA was prepared using Oligotex
resin (Qiagen), and 1 µg of mRNA was reverse transcribed from each
test sample with superscript (Perkin-Elmer) in the presence of
[-32P]dATP (NEN Life Science
Products, Boston, MA). The unbound 32P was
removed by gel filtration in Chroma Spin-200 columns (Clontech). The
blots were incubated with 2 x 106
cpm/ml of buffer and hybridized overnight at 68°C to a human cDNA
microarray consisting of 588 human genes under highly regulated
transcriptional control, as indicated in the manufacturer’s
instructions (Clontech). After high stringency washes in 2x
standard saline citrate-SDS, the membranes were exposed and
recorded using a phosphorimaging system and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). Gene expression images were
normalized to housekeeping genes provided in the array. Changes in
expression between control and treatments were analyzed using
ArrayVision program (Imaging Research Inc., Catherines, Ontario,
Canada). Replicate experiments were performed. Only genes that
demonstrated significant and consistent changes in expression between
control and treatment were reported.
Arsenic-induced changes in gene expression were verified by either RT-PCR or RNase protection assay for randomly selected genes. Analysis of RNA samples by RNase protection assay was performed using a commercial kit (RiboQuant kit, PharMingen, San Diego, CA) according to the manufacturer’s instructions. 32P-Labeled antisense RNA probes were produced from commercial DNA templates (in vitro transcription kit, PharMingen). The samples were electrophoresed on a sequencing gel, which was quantitated using a phosphorimaging system and ImageQuant software (Storm, Molecular Dynamics).
Statistical Analysis.
All experiments were replicated and representative findings are shown.
Statistical significance was determined by one-way ANOVA. When the
F value was significant, the means were compared using
Fisher’s post hoc analysis. In all statistical comparisons,
P < 0 0.05 was used to indicate a
significant difference.
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RESULTS |
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. The urinary bladder epithelium from mice receiving
control water was composed of differentiated, 1–2-cell-thick
transitional cells, whereas the bladders from animals on arsenic
drinking water had 6–8-cell-thick transitional cells consistent with
mild hyperplasia. There was no microscopic evidence of inflammation or
necrosis. Hyperplastic urothelial cells did not form papillary
structures but progressed toward the lumen of the bladder. Occasional
squamous metaplasia without keratinization was observed in some
hyperplastic areas. There was no evidence of microcrystalluria,
calculi, or amorphous precipitates in any of the tissues examined.
Amorphous urinary precipitates that contain calcium phosphate can
result from administration of high doses of some organic sodium salts
and lead to bladder tumors in some animal species (23)
.
Ultrastructural changes caused by arsenic in the uroepithelium,
analyzed by transmission electron microscopy, revealed the presence of
pleomorphic projections thought to be microvilli formation on the
luminal surface (Fig. 1B)
. Although not definitive, previous
morphological studies have suggested that the occurrence of microvilli
reflects a higher metabolic activity associated with epithelial cell
proliferation (24)
. Scanning electron microscopic
examination of bladders from control mice revealed flat, polygonal
cells with protruding microridges on the luminal surfaces (Fig. 1C)
. In contrast, bladders from arsenic treated animals had
slightly raised, variable size cells with distinct cell borders,
lending a cobblestone appearance. Although there was a slight
(approximately 20–25%) decrease in water intake in arsenic-treated
mice, the paired water group showed no effects on bladder histology,
serum albumin levels, or hematocrits, indicating that pathological
changes were caused by arsenic.
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. The increased percentage of PCNA-positive cells after 4 weeks of
exposure to arsenic exposure was similar to that observed at 16 weeks
(data not shown).
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. In contrast to urine, quantitative chemical analyses demonstrated
that trivalent arsenite was the predominant form of arsenic contained
within bladder tissue, with over 10 µg/g of the parent compound
present compared to 4.5 µg/g of DMA and only trace amounts of the
other metabolites.
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. Increased DNA binding activity was not obtained when a mutated AP-1
probe was substituted for the consensus AP-1 probe, establishing the
specificity of the arsenic-induced AP-1 complex. The subunits
constituting the AP-1 complex activated by arsenic was determined
immunochemically using antibodies against known members of the family.
The complex was abrogated using antibodies broadly reactive with
members of c-fos or c-jun family and was supershifted by antibodies
specific for the c-jun p39 and c-fos p62 subunit. The AP-1 complex was
only slightly supershifted by antibody against JunD and unaltered by
antibodies specific for JunB and Fra1, indicating that the complex is
composed primarily of c-jun p39/c-fos p62 heterodimers. Consistent with
these observations, immunohistochemical examination using polyclonal
antibodies revealed an increase in c-jun and c-fos immunostaining in
the nuclei of bladder epithelium from exposed mice indicating that the
increase in AP-1 activities was associated with an increase in
immunoreactive proteins (Fig. 5)
. The percentage of cells that stained positive for c-jun and c-fos
proteins from bladders of arsenic treated mice was 38 ± 8 and 76 ± 12%, respectively, compared to
approximately 2 ± 1% in control mice. The ability of
arsenic to induce AP-1 activation in vivo was confirmed
using TRE-luciferase reporter transgenic mice, in which sodium arsenite
exposure resulted in a greater than 2-fold increase in AP-1 activity in
bladder tissue, as measured by luciferase activity. PMA, a strong
activator of AP-1 when applied to the skin, induced a 3-fold increase
in bladder AP-1 activity after intrabladder installation (20 mg/ml;
2 h).
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. A significant increase in c-fos expression occurred within
1 h of arsenite treatment, followed by a rapid decline, whereas
c-jun expression was also up-regulated, with a peak response
at 3 h. These time points and slight differences in maximum
expression are consistent with published data (11
, 12) .
EMSAs were conducted to determine whether the increase in expression of
these genes was associated with AP-1 binding activity. UROtsa cells
exposed to a 5 or 50 µM concentration of sodium
arsenite showed a dose-response increase in AP-1 DNA binding activity
(Fig. 7)
. PMA was used as a positive control. Both MMA and DMA induced AP-1 DNA
binding activity in UROtsa cells, but they were less potent than the
parent form. DNA binding was specific for AP-1, because preincubation
of the nuclear extracts with excess cold AP-1 inhibited the response,
whereas preincubation with mutated AP-1 had no effect on binding
activity. Consistent with findings in urinary bladders from mice
exposed to arsenic, the DNA binding complex was composed primarily of
c-jun and c-fos subunits, because the complex was supershifted by
antibodies to c-fos (Fig. 7
, dotted arrow) and
abrogated by antibodies to c-jun. Arsenic-induced AP-1 DNA binding was
neither inhibited nor supershifted by antibodies against fos B,
fra 1, fra 2, jun B, or jun D (data not shown).
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, exposure to iAs3+ for 72 h induced a
significant, albeit modest, increase in thymidine uptake in UROtsa
cells. Additionally, cell cycle analysis of UROtsa cells with propidium
iodide-stained nuclei revealed that arsenic induced an increase in the
number of cells in S-phase and a concomitant decrease in
G0/G1.
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; Fig. 8
). The microarray analysis was conducted in triplicate, and only genes
modulated by arsenic more than 2-fold in all three replicates are
described. This percentage of genes expressed is similar to that
altered in EVV304 cells treated with homocysteine (29)
and
HepG2 hepatoma cells treated with ß-mercaptoethanol
(30)
, both of which are thiol-containing agents that
affect cell growth. Among the genes that are associated with cell
growth that were consistently increased by greater than 2-fold at 10
µM sodium arsenite were c-fos, Pig 7,
Rho 8 and EGR-1. A number of genes associated
with stress responses were also increased, including GADD45
and GADD153. Several genes were up-regulated only at the
high arsenic concentration. Most of these were also associated with
mitogenic activity and cell cycle regulation, such as heparin-binding
EGF-like growth factor, bone morphogenic protein 2, and
Waf-1. Arsenic also increased the expression of BCL-2 binding component
and BCL-2 binding athanogene 1 genes, the expression of which was
involved in inhibition of apoptosis. The expression of several genes
from this panel was down-regulated by arsenic (Table 2)
. PMA was
included in the array studies as a positive control for evaluation of
gene expression (not all of the genes induced by PMA are not listed in
Table 2
). To help establish the reliability of the cDNA microarray
system in identification of differentially expressed genes, randomly
selected genes modulated by arsenic in UROtsa cells were
analyzed by an RNase protection assay (Fig. 8D)
. Consistent
with the results from cDNA microarray, arsenic induced an increase in
GADD45 (4-fold), c-fos (9-fold), and
WAF-1 (2-fold) but did not affect p53 expression.
The arsenic-induced up-regulation of other genes, such as EGR-1,
GADD153, and Pig7 was confirmed by RT-PCR (data not
shown).
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DISCUSSION |
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The metabolism of arsenic follows similar pathways in both humans and
laboratory animals; in these pathways, pentavalent arsenic is
first reduced to the trivalent form, which is subsequently methylated
to MMA acid and then to DMA acid (25)
. The majority of
evidence supports that the inorganic forms, particularly trivalent
arsenite, are the primary toxic metabolites associated with arsenic
(33)
, although several reports have suggested a role for
methylated metabolites. For example, exposure of rats to high doses of
DMA for 32 weeks enhanced diethylnitrosamine-induced urinary bladder
tumors (34)
, whereas exposure for 97–104 weeks produced
bladder tumors without diethylnitrosamine initiation (35)
.
In this respect, Arnold et al. (36)
observed
urothelial toxicity and hyperplasia in rats administered DMA in the
feed at 100 ppm. The present studies demonstrate that, despite the
predominance of DMA in the urine, sodium arsenite accumulated in
bladder tissue. This would suggest that tissues that have high affinity
for sodium arsenite or fail to metabolize it represent the most
susceptible targets. Furthermore, in studies with UROtsa cells, sodium
arsenite was a more effective inducer of cell growth and AP-1
activation than the methylated forms, although the methylated forms
still possess significant activity (Fig. 7)
. Consistent with these
observations, in collaboration with M. Styblo et al. of the
University of North
Carolina,3
it was revealed that both keratinocytes and UROtsa cells are extremely
poor methylators of arsenic when compared to other cells types, such as
hepatocytes or HeLa cells.
Arsenite has a strong affinity for thiol groups in macromolecules, and it has been proposed that arsenic modulates cellular phosphorylation events either through binding to c-Jun-N-terminal kinase phosphatases, resulting in prolonged c-Jun-N-terminal kinase and p38 activation (11) , or through binding to cysteine residues in the EGF receptor, resulting in activation of extracellular signal-related protein kinase (10 , 12) . Alternatively, arsenic can inhibit glutathione reductase and diminish cellular levels of reduced glutathione (37) , resulting in an altered cellular redox state. Oxidative stress has been associated with activation of genes involved in stress and proliferative responses, such as GADD153 and c-myc, respectively (38 , 39 , 40) , probably through activation of oxidant-sensitive transcription factors (41) . These events have in common the ability to activate AP-1. Studies involving a variety of cell culture and animal model systems have established the importance of AP-1 activation in preneoplastic-to-neoplastic transformation. In this report, AP-1 is a critical mediator of tumor promotion because it alters gene expression in response to classical tumor promoters such as UV radiation and TPA. Similar to these properties of well-characterized tumor promoters, iAs3+-induced mitogen-activated protein kinase signaling pathways have been related to increased AP-1-DNA binding in several cell systems, including HeLa cells, PC-12 cells, and keratinocytes (10 , 11 , 17) .
The present studies provide evidence that arsenite transactivates AP-1, and this is accompanied by uroepithelial proliferation. This was demonstrated in vitro using a human bladder epithelial cell line, as well as in vivo after exposure of normal and TRE-luciferase reporter transgenic mice. Characterization of the arsenic-induced AP-1 DNA binding complex demonstrated that the complex consisted of c-Jun/c-Fos heterodimers; this is a common heterodimer responsible for regulating cell growth (26) . Arsenic induction of AP-1 DNA binding activity is accompanied by up-regulation of c-fos and c-jun nuclear proteins in bladder epithelial cells. Of particular relevance to the present studies is a report that c-jun expression is as a concomitant factor associated with urinary bladder transitional carcinomas (27 , 28) . Because arsenic has been linked mainly to development of typical transitional carcinomas, without any unique clinical or histopathological features (42) , it can be expected that genes induced by arsenic are typical of molecules involved in bladder carcinogenesis.
The results obtained from the present studies prompted us to
examine for changes in specific gene expression using cDNA microarrays,
on the assumption that arsenic would alter the expression of genes that
regulate cell growth and arrest. We elected to examine UROtsa cells
treated for 2 h with 10 or 50 µM
iAs3+. Gene expression is a dynamic process, and
the profile presented would be influenced by the specific culture
conditions, time points examined, and concentration of the test
chemical. Nonetheless, it can be assumed a general profile of gene
expression might emerge that would provide some insights into the
mechanism(s) by which arsenic exerts its effects. The cDNA microarrays
revealed consistent activation of 16 genes by the higher concentration
of arsenic, 7 of which were also induced by the lower concentration
tested. Furthermore, decreased expression was observed in six genes at
both the high and low concentrations. In addition to previously
reported early immediate genes modulated by arsenic, including
c-fos and c-myc (14
, 17
, 38)
, the
cDNA microarray demonstrated a strong induction of the
EGR-1 gene. EGR-1, which encodes zinc
finger DNA binding transcription factors, has been related to cell
proliferation induced by mitogens such as EGF, nerve growth
factor, or serum (43)
. Recently, overexpression of
EGR-1 has been associated with human prostate cancer and correlated
with the pathomorphological stage of malignancy (44)
.
Functional EGR-1 binding sites are found in the promoter domains of a
large number of genes involved in cell growth, including transforming
growth factor , insulin growth factor II, c-myc,
thymidine kinase, and cyclin D (see Ref. 44
and references
therein). We also observed that arsenic, in contrast to PMA,
induced genes implicated in response to cellular stress and growth
arrest, such as GADD153 and GADD45. Activation of
these genes is an integral part of endoplasmic reticulum stress and is
associated with the activation of CAAT/enhancer binding protein
and the modulation of pathways leading to cell death or cell
regeneration (39)
. Arsenic also altered genes that encode
antiapoptotic proteins, such as BCL-2 binding protein and BAG-1, repair
associated protein, and proteins involved in cytoskeleton
reorganization. Although the precise role of these genes in
arsenic-induced malignancies, if any, needs to be defined, the present
studies suggest that arsenic, through AP-1, activates genes of which
the products are involved in neoplastic cell proliferation and arrest.
It is hypothesized that these events serve as a prerequisite for
arsenic-induced carcinogenesis by increasing the growth rate of
initiated or mutated cells.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Toxicology and Molecular Biology Branch, National
Institute for Occupational Safety and Health, NIH, 1095
Willowdale Road, Morgantown, WV 26505. Phone: (304) 285-6156; Fax:
(304) 285-6038; E-mail: phs9{at}cdc.gov 
2 The abbreviations used are: AP, activating
protein; DMA, dimethylarsenic; EGF, epidermal growth factor;
EGR, early growth response; EMSA, electrophoretic mobility shift assay;
FBS, fetal bovine serum; G3PDH, glyceraldehyde 3-phosphate
dehydrogenase; MMA, monomethylarsenic; PCNA, proliferating cell nuclear
antigen; PMA, phorbol myristate acetate; RT, reverse
transcription; TRE, TPA responsive element; TPA,
12-O-tetradecaanoylphorbol-13-acetate.
3 M. Styblo et al., unpublished
data.
Received 12/ 2/99. Accepted 5/ 3/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A. S. Andrew, V. Bernardo, L. A. Warnke, J. C. Davey, T. Hampton, R. A. Mason, J. E. Thorpe, M. A. Ihnat, and J. W. Hamilton Exposure to Arsenic at Levels Found in U.S. Drinking Water Modifies Expression in the Mouse Lung Toxicol. Sci., November 1, 2007; 100(1): 75 - 87. [Abstract] [Full Text] [PDF]
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S. Somji, X. D. Zhou, S. H. Garrett, M. A. Sens, and D. A. Sens Urothelial Cells Malignantly Transformed by Exposure to Cadmium (Cd+2) and Arsenite (As+3) Have Increased Resistance to Cd+2 and As+3-Induced Cell Death Toxicol. Sci., December 1, 2006; 94(2): 293 - 301. [Abstract] [Full Text] [PDF]
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 W. Ouyang, J. Li, Q. Ma, and C. Huang Essential roles of PI-3K/Akt/IKK{beta}/NF{kappa}B pathway in cyclin D1 induction by arsenite in JB6 Cl41 cells Carcinogenesis, April 1, 2006; 27(4): 864 - 873. [Abstract] [Full Text] [PDF]
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 A. M. Bode and Z. Dong Inducible Covalent Posttranslational Modification of Histone H3 Sci. Signal., April 26, 2005; 2005(281): re4 - re4. [Abstract] [Full Text] [PDF]
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L.-H. Yih, S.-W. Hsueh, W.-S. Luu, T. H. Chiu, and T.-C. Lee Arsenite induces prominent mitotic arrest via inhibition of G2 checkpoint activation in CGL-2 cells Carcinogenesis, January 1, 2005; 26(1): 53 - 63. [Abstract] [Full Text] [PDF]
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J. Liu, Y. Xie, J. M. Ward, B. A. Diwan, and M. P. Waalkes Toxicogenomic Analysis of Aberrant Gene Expression in Liver Tumors and Nontumorous Livers of Adult Mice Exposed in utero to Inorganic Arsenic Toxicol. Sci., February 1, 2004; 77(2): 249 - 257. [Abstract] [Full Text] [PDF]
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Xing Cui, Song Li, A. Shraim, Y. Kobayashi, T. Hayakawa, S. Kanno, M. Yamamoto, and S. Hirano Subchronic Exposure to Arsenic Through Drinking Water Alters Expression of Cancer-Related Genes in Rat Liver Toxicol Pathol, January 1, 2004; 32(1): 64 - 72. [Abstract] [PDF]
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P. B. Tchounwou, A. K. Patlolla, and J. A. Centeno Invited Reviews: Carcinogenic and Systemic Health Effects Associated with Arsenic Exposure--A Critical Review Toxicol Pathol, October 1, 2003; 31(6): 575 - 588. [Abstract] [PDF]
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 J. Li, M. Gorospe, J. Barnes, and Y. Liu Tumor Promoter Arsenite Stimulates Histone H3 Phosphoacetylation of Proto-oncogenes c-fos and c-jun Chromatin in Human Diploid Fibroblasts J. Biol. Chem., April 4, 2003; 278(15): 13183 - 13191. [Abstract] [Full Text] [PDF]
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Z. He, W.-Y. Ma, G. Liu, Y. Zhang, A. M. Bode, and Z. Dong Arsenite-induced Phosphorylation of Histone H3 at Serine 10 Is Mediated by Akt1, Extracellular Signal-regulated Kinase 2, and p90 Ribosomal S6 Kinase 2 but Not Mitogen- and Stress-activated Protein Kinase 1 J. Biol. Chem., March 14, 2003; 278(12): 10588 - 10593. [Abstract] [Full Text] [PDF]
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E. I. Salim, H. Wanibuchi, K. Morimura, M. Wei, M. Mitsuhashi, K. Yoshida, G. Endo, and S. Fukushima Carcinogenicity of dimethylarsinic acid in p53 heterozygous knockout and wild-type C57BL/6J mice Carcinogenesis, February 1, 2003; 24(2): 335 - 342. [Abstract] [Full Text] [PDF]
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M. Wei, H. Wanibuchi, K. Morimura, S. Iwai, K. Yoshida, G. Endo, D. Nakae, and S. Fukushima Carcinogenicity of dimethylarsinic acid in male F344 rats and genetic alterations in induced urinary bladder tumors Carcinogenesis, August 1, 2002; 23(8): 1387 - 1397. [Abstract] [Full Text] [PDF]
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 W. H. Miller Jr., H. M. Schipper, J. S. Lee, J. Singer, and S. Waxman Mechanisms of Action of Arsenic Trioxide Cancer Res., July 15, 2002; 62(14): 3893 - 3903. [Abstract] [Full Text] [PDF]
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 M. R. Saban, N.-B. Nguyen, T. G. Hammond, and R. Saban Gene Expression Profiling of Mouse Bladder Inflammatory Responses to LPS, Substance P, and Antigen-Stimulation Am. J. Pathol., June 1, 2002; 160(6): 2095 - 2110. [Abstract] [Full Text] [PDF]
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L.-H. Yih, K. Peck, and T.-C. Lee Changes in gene expression profiles of human fibroblasts in response to sodium arsenite treatment Carcinogenesis, May 1, 2002; 23(5): 867 - 876. [Abstract] [Full Text] [PDF]
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 W. H. Miller Jr. Molecular Targets of Arsenic Trioxide in Malignant Cells Oncologist, April 1, 2002; 7(90001): 14 - 19. [Abstract] [Full Text] [PDF]
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J. B. Wijeweera, A. J. Gandolfi, A. Parrish, and R. C. Lantz Sodium Arsenite Enhances AP-1 and NF{{kappa}} B DNA Binding and Induces Stress Protein Expression in Precision-Cut Rat Lung Slices Toxicol. Sci., June 1, 2001; 61(2): 283 - 294. [Abstract] [Full Text] [PDF]
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P. P. Simeonova, S. Wang, M. L. Kashon, C. Kommineni, E. Crecelius, and M. I. Luster Quantitative Relationship between Arsenic Exposure and AP-1 Activity in Mouse Urinary Bladder Epithelium Toxicol. Sci., April 1, 2001; 60(2): 279 - 284. [Abstract] [Full Text] [PDF]
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F. Chen, Y. Lu, Z. Zhang, V. Vallyathan, M. Ding, V. Castranova, and X. Shi Opposite Effect of NF-kappa B and c-Jun N-terminal Kinase on p53-independent GADD45 Induction by Arsenite J. Biol. Chem., March 30, 2001; 276(14): 11414 - 11419. [Abstract] [Full Text] [PDF]
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P. P. Simeonova, S. Wang, T. Hulderman, and M. I. Luster c-Src-dependent Activation of the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway by Arsenic. ROLE IN CARCINOGENESIS J. Biol. Chem., January 18, 2002; 277(4): 2945 - 2950. [Abstract] [Full Text] [PDF]
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) and tissue (
) were
collected, and metabolites were determined as described in "Materials
and Methods." Each value represents mean a of 2–4 animals. The
control levels of arsenic in the bladder issue and urine were similar
to the assay detection limits (3 ng/g or ng/ml), except that urinary
concentrations of DMA were 88 ± 15 ng/ml.
