Opposed arsenite-induced signaling pathways promote cell proliferation or apoptosis in cultured lung cells

Andy T. Y. Lau1,2, Muyao Li4, Ronglin Xie5, Qing-Yu He2,3 and Jen-Fu Chiu1,2,6

1 Institute of Molecular Biology, 2 Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis and 3 Department of Chemistry, The University of Hong Kong, Hong Kong SAR, China, 4 Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405, USA and 5 Department of Cell Biology, University of Massachusetts Medical Center, Worcester, MA 01655, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arsenic is a well-known carcinogen that possibly promotes tumors and the development of various types of cancer in individuals chronically exposed to arsenic in their work or living environment. Many studies have demonstrated the activation of mitogen-activated protein kinase (MAPK) in several cell types by using lethal concentrations of arsenic in the range of 50–500 µM. Since the exposure of humans to arsenic is normally at a much lower level in the workplace or in daily life, it is more relevant to study the effects of arsenic at this lower exposure level. In the present study we aimed at redefining the role of signal transduction pathways in arsenic-induced malignant transformation as well as apoptosis using our established in vitro rat lung epithelial cell model system. Our results indicate a molecular mechanism by which MAPK pathways might differentially contribute to cell growth regulation and cell death in response to different dosages of arsenite. A low level (2 µM) of arsenite stimulated extracellular signal-regulated kinase (ERK) signaling pathway and enhanced cell proliferation, and this arsenite-induced ERK activity was blocked by MEK inhibitor, PD98059. In contrast, a high level (40 µM) of arsenite stimulated the c-Jun N-terminal kinase (JNK) signaling pathway and induced cell apoptosis, and this arsenite-induced JNK activity was blocked by JNK inhibitor II, SP600125. The implications of these findings are that a high concentration of arsenic exposure causes apoptosis, whereas a low concentration of arsenic exposure is carcinogenic and may result in aberrant cell accumulation.

Abbreviations: AP-1, activator protein-1; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GADD45, growth arrest and DNA damage-inducible 45; JNK, c-Jun N-terminal kinase; LEC, lung epithelial cell; MAPK, mitogen-activated protein kinase; NBB, Naphthol blue black; NF-{kappa}B, nuclear factor-kappaB


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is evident that many metals from environmental or industrial sources are human carcinogens (13). Epidemiological studies have established a close association between exposure to arsenic and increased incidences of cancer in arseniasis-endemic areas of the world including Taiwan, Mexico, Chile, Argentina, Thailand, India, Canada and the USA (4,5). Arsenic is carcinogenic to humans, and targets in particular the urinary bladder, liver, kidney, skin, lung, prostate and other internal sites (68). The harmful effects of arsenic were established beyond doubt some decades ago, and the mechanisms of arsenic carcinogenesis have since been under intensive investigation.

Arsenic trioxide displays pleiotropic effects on many biological systems. In addition to acting as a carcinogen, arsenite can also function conversely as a chemotherapeutic agent in the treatment of acute promyelocytic leukemia (9,10). The various effects of arsenite may be mediated through activation of a MAP kinase cascade (11,12). Previous investigations have demonstrated that arsenite activates members of the MAP kinase family, transcription factors such as activator protein-1 (AP-1), and immediate early genes, including c-jun, c-fos and c-myc, which help to regulate the expression of transforming oncoproteins and growth factors (1319).

The mitogen-activated protein kinase (MAPK) pathways transduce signals that lead to diverse cellular responses such as cell growth, differentiation, proliferation, apoptosis and stress responses to environmental stimuli (2025). Each of the three major MAPK pathways consists of three-tiered cascades that induce a pathway composed of phosphorylating proteins that mediate the signal transduction pathways from a variety of extracellular signals to regulate the expression of specific genes (20,26). The extracellular signal-regulated kinase (ERK) pathway typically transduces growth factor signals that lead to cell differentiation or proliferation (23,27), whereas cytokines and stress signals (e.g. ultraviolet irradiation, heat or synthesis inhibitors) activate the c-Jun N-terminal kinase (JNK) and p38 MAPK pathways, resulting in stress responses, growth arrest or apoptosis (7,25,2830). Signaling through the MAPK pathways culminates in the phosphorylation-dependent activation of a variety of transcription factors that modulate cytokine gene expression (3133).

In this study, we investigated the effects of arsenite on cell growth as well as its signal transduction pathways in rat lung epithelial cell line (LEC). Most recent studies have used lethal concentrations of arsenic in the range of 50–500 µM (15,17,26,34). Since stress responses to different levels of arsenic may employ different streams of cellular signal transduction systems, the effect of arsenite on MAPK signaling pathways has been re-examined in this study. The purpose of this study is to examine the signal transduction pathways induced by arsenic in our established in vitro rat LEC model system (13,35) with low and high levels of sodium arsenite (2 and 40 µM), respectively. We report here that a low level of arsenite stimulated ERK signaling pathway and enhanced cell proliferation. On the contrary, a high level of arsenite stimulated the JNK signaling pathway and induced cell apoptosis.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Materials
Sodium arsenite was purchased from Sigma (St Louis, MO). PD98059 and SP600125 were from Calbiochem (San Diego, CA). All other general chemicals were supplied from Amersham Biosciences (Sweden, Uppsala) and Sigma (St Louis, MO). Antibodies used for western blot were purchased from Sigma (St Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA), Upstate Biotechnology (Lake Placid, NY) and Zymed Laboratories (San Francisco, CA).

Cell culture
A rat LEC line was isolated and characterized by Li et al. (13). Cells were routinely grown at 37°C in 95% air/5% CO2 using F-12 nutrient supplemented with 2 mmol/l glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco BRL, Grand Isle, NY) and 10% newborn bovine serum (JRH Bioscience, Lenexa, KS).

Toxicity determination
Cell viability was measured by Naphthol blue black (NBB) staining assay (36). Rat LEC were plated in 96-well plates at 2 x 104/well in complete media and incubated overnight. The medium was then changed and the cells were treated with various amounts of sodium arsenite for the indicated time. At the end of the experiment, the media were removed and the cells were fixed in 10% formalin for 5–10 min and stained with NBB solution (0.05% NBB in 9% acetic acid with 0.1 M sodium acetate) for 30 min at room temperature. The wells were washed three times with H2O to remove the free dye. The attached dyes were eluted with 150 µl of 50 mM NaOH. The optical densities at 595 nm were measured by using a Model E1 310 Autoplate reader (Bio-Tek Instruments, Winooski, VT).

Flow cytometric cell cycle analysis
Cell cycle distribution was determined by using flow cytometry as described (37). Cells were cultivated for 48 h in serum-free medium and then either left untreated or treated with 2 or 40 µM of sodium arsenite for 24 h. At the end of the experiment, cells were harvested, resuspended in Vindelov's reagent and analyzed with a FACStar Plus flow cytometer. For each sample, at least 9000 independent events were analyzed, providing a solid statistical basis for determining the percentage of cells in each cell cycle phase using the Lysis II software program.

Assessment of DNA synthesis
LEC cells were seeded in 24-well plates at 2 x 104/well in complete media and allowed to grow for an additional 24 h. Following a 24 h starvation period in serum-free media, cells were then grown in media with various concentrations of sodium arsenite. Eighteen hours later, cells were labeled for 2 h with 1 µCi/ml [3H]thymidine ([3H]TdR) and washed several times with PBS. Trichloroacetic acid (TCA) was added to a final concentration of 5%, and the radioactivity contained in the acid precipitable material was collected onto glass fiber filters and washed with pre-cooled 5% TCA and absolute ethanol. The filters were dried at 50°C for 2 h. Incorporated radioactivity was counted in a liquid scintillation spectrometer using a scintillation cocktail of 4 g ppo and 0.4 g popop dissolved in 1 l of toluene. The cultures were performed in triplicate.

Visualization of apoptotic cells by confocal microscope
2 x 106 treated cells were prefixed in 3.7% paraformaldehyde in 1x PBS, pH 7.2 at room temperature for 15 min, followed by 5% acetic acid–95% ethanol fixation at -20°C for 20 min. Fixed cells were stained in 50 µg/ml propidium iodide (1x PBS, pH 7.2) containing 100 µg/ml DNase-free RNase A for 30 min at 37°C. Cells were then mounted on slides and examined for apoptotic cells with a Bio-Rad MRC-1000 Confocal Scanning Laser Microscope.

Quantification of apoptotic death
DNA fragmentation was measured to quantify apoptotic cells with the Cell Death ELISA method (Boehringer-Mannheim, Indianapolis, IN). Assays were carried out according to the manufacturer's instructions. To normalize the data with equal cell numbers, additional plates were seeded and treated exactly as above, and the cell numbers were measured by NBB staining assay as described above.

Cell lysate preparation and western blot analysis
After cells were grown to 70–80% confluence, they were placed in serum-free medium for 24 h and then treated with sodium arsenite for the indicated times and doses. Cells were then washed with ice-cold PBS, and scraped into cell lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM Na3VO4, 50 mM pyrophosphate, 2 mM EDTA, 100 mM NaF, 1% Triton X-100, 10% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. The cells were vortexed briefly and incubated in lysis buffer for 30 min on ice and centrifuged at 15 000 g for 15 min. The supernatant was designated as the cell lysate and used for western blot analysis (38). For gel mobility shift assay for AP-1, nuclear proteins from lung cells exposed to sodium arsenite were isolated and used as mentioned (39). Protein concentration was determined by the Bradford method, using bovine serum albumin as standard (40). Equal amounts of proteins from each set of experiments were fractionated on SDS–polyacrylamide gel and subjected to western blot analysis as described previously (39). Proteins were transferred onto PVDF membranes, and the membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween 20 and probed with various antibodies, including phospho-ERK1/2, phospho-JNK1/2, ERK1/2, JNK1/2, Bcl-2, Bax, growth arrest and DNA damage-inducible 45 (GADD45) and ß-actin. After incubation with secondary antibodies, immunoblots were visualized with the ECL detection kit (Amersham Biosciences, Sweden, Uppsala). For reprobing the membrane with another antibody, the membrane was stripped in 2% SDS, 0.1% 2-mercaptoethanol and 50 mM Tris–Cl, pH 6.8 and boiled at 50°C for 30 min.

Statistical analysis
Statistical analysis was performed using two-tailed Student's t-test, and P < 0.05 was considered significant. Data are expressed as the mean ± SD of triplicate samples, and the reproducibility was confirmed in three separate experiments.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytotoxicity of arsenite in LEC cells
Since human exposure to arsenic is normally at a much lower level than that studied in previous experiments, we deliberately used low arsenic conditions in this study in order to achieve results directly relevant to arsenic toxicity in humans. To conduct our experiments, we first determined the cytotoxicity of sodium arsenite in LEC cells by NBB staining assay. Arsenite showed a typical sigmoidal curve in toxicity versus concentration, and the LC50 were determined to be 9 µM (Figure 1).



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Fig. 1. Dose-dependent cytotoxicity of arsenite on LEC cells. LEC cells were plated in 96-well plates at 2 x 104/well and incubated overnight in an incubator. On the following day the cells were treated with different doses of arsenite. After 48 h, the number of cells was measured by NBB staining assay. Data are mean values from three trials.

 
Arsenite induces LEC cell proliferation and apoptosis
To gain further evidence for the ability of a low level of arsenite exposure to influence cell proliferation, cells were examined by fluorescence-activated cell sorting analysis for changes in cell cycle distribution following treatment with 2 or 40 µM arsenite. Cells were first subjected to 48 h of serum starvation to enrich the cells at G1 phase. They were then either left untreated or treated with 2 or 40 µM sodium arsenite and examined 24 h later. In the absence of arsenite treatment, ~10% of cells were in the S phase (Figure 2A). A low concentration (2 µM) of arsenite led to a significant increase in the number of cells in the S phase (18%), indicative of increased DNA synthesis (Figure 2B). In contrast, a higher level (40 µM) of arsenite stimulated cell apoptosis. The number of cell deaths increased from ~5.6% in control cells to 31% in 40 µM arsenite treated cells (Figure 2C). To further confirm whether 40 µM sodium arsenite induces LEC cell apoptosis, the cells were examined by confocal microscopy and the amount of DNA fragmentation was determined by Cell Death ELISA. A high concentration (40 µM) of arsenite induced nuclear chromatin morphological changes (Figure 3A, right panel) in the treated cells, in contrast with the untreated control cells (Figure 3A, left panel), and produced DNA fragmentation in a dose-dependent manner (Figure 3B). Both results are consistent with flow cytometric data.



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Fig. 2. Flow cytometric analysis on the cell cycle distribution of LEC cells after treatment with arsenite. LEC cells were serum starved for 48 h and then treated with (B) 2 or (C) 40 µM arsenite for 24 h. (A) Control cells without treatment of arsenite. Letters on the diagrams: A, Sub-G1 phase (apoptotic phase); B, G0/G1 phase; C, S phase and D, G2/M phase.

 


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Fig. 3. Arsenite induced LEC cell apoptosis. (A) Arsenite induced nuclear chromatin morphological changes in LEC cells after treatment with 40 µM of sodium arsenite for 24 h. Cells were then stained with the fluorochrome propidium iodide and visualized under a confocal microscope (right panel). Control cells without treatment of sodium arsenite were shown on left panel (Magnification: 600x). (B) LEC cells were plated in 24-well plates at 1 x 105/well and incubated for 24 h in an incubator, after which cells were treated without or with 5, 10, 20 or 40 µM sodium arsenite, respectively, for 24 h. Cytoplasmic extracts from one set of cells were used to detect fragmented DNA as indicator of apoptosis by a Cell Death ELISA method. Another set of cells was counted for the numbers using NBB staining assay. The optical density obtained from the DNA fragmentation assay was normalized to equal number of cells and presented as relative to the control.

 
Stimulation of DNA synthesis and AP-1 activity by arsenite
To confirm that the proportion of cells entering S phase is greater in cells growing in lower level of arsenite (2 µM) (Figure 2B) than in the control untreated cells (Figure 2A), the effects of arsenite exposure for 18 h on [3H]TdR incorporation into LEC cells were studied. There was an initial stimulation of incorporation of [3H]TdR into LEC cells at very low concentrations (1–2 µM) of arsenite exposure, followed by an inhibition of incorporation of [3H]TdR into the cells as the concentrations of arsenite were raised (Table I). Several studies (41) have provided evidence for tumor promoter-induced cell transformation by stimulation of DNA synthesis and enhancement of AP-1 activity. To investigate whether a low concentration (2 µM) of arsenite would stimulate AP-1 activity, the nuclear extract (5 µg) of LEC cells was used to carry out gel mobility shift assay for AP-1. We used the AP-1 binding site (5'-AGCATGAGTCAGACACCTCTGGC-3') as oligonucleotide probe. Arsenite clearly increased the binding activity of the nuclear extract to the oligonucleotide probe of AP-1 after treatment for 2 h, with a peak at 4 h post-treatment (Figure 4).


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Table I. DNA synthesis in LEC cells treated with arsenite for 18 h

 


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Fig. 4. Arsenite induced AP-1 activity. Induction of AP-1 binding activity in LEC cells exposed to 2 µM sodium arsenite for 0, 1, 2, 4 or 8 h. Nuclear protein (5 µg) from cells treated with arsenite for various time periods was used to analyze for DNA binding activity using a 32P-end-labeled, double-stranded, oligonucleotide consensus sequence for AP-1. Protein–DNA complexes were resolved by non-denaturing gel electrophoresis and visualized by autoradiography.

 
Arsenite stimulates different MAPK pathways
To determine whether arsenite exposure affects the activity of specific factors involved in signaling, we used antibodies against phospho-ERK1/2 and phospho-JNK1/2 to examine the effect of arsenite on MAPK pathways by western blot. The immunoblot data revealed that JNK activity was very weakly stimulated or nearly unchanged after cell exposure to 2 µM sodium arsenite (Figure 5A). A common feature of known tumor promoters is their ability to perturb the ERK signaling pathway. For example, phorbol ester, okadaic acid and butylated hydroxytoluene hydroperoxide all lead to the activation of ERK. Our data show that a low concentration (2 µM) of arsenite can activate ERK, suggesting that arsenite at low concentrations may act as a tumor promoter in arsenic-induced carcinogenesis (Figure 5A). In addition, several recent papers have also suggested that ERK might be involved in cell proliferation signaling (31).



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Fig. 5. The effects of arsenite exposure on MAPK activities in LEC cells. LEC cells were serum starved for 24 h and then treated with (A) 2 or (B) 40 µM of sodium arsenite, cells were lysed at various time points, and protein extracts were subjected to SDS–PAGE followed by immunobloting using antibodies against phospho-ERK1/2 and phospho-JNK1/2 (Upstate Biotechnology), respectively. After development, the membranes were stripped and reprobed with regular antibodies against ERK1/2 (Upstate Biotechnology) and JNK1/2 (Santa Cruz Biotechnology) MAPKs. The intensities of the bands of phosphorylated ERK1/2 and JNK1/2 were quantified and plotted as a relative amount, setting 1 for control (no treatment of arsenite). The data are representative of three independent experiments. *Significantly different from controls (P < 0.05).

 
The JNK pathway has been implicated in the regulation of apoptosis induced by various stimuli (20). As 40 µM sodium arsenite induces LEC cell apoptosis, we therefore determined which signaling pathway might be involved in arsenic-induced LEC cell apoptosis. In contrast to arsenite exposure at a low level, 40 µM sodium arsenite enhanced the JNK signal transduction pathway, as indicated by the increase in activated JNK (Figure 5B).

Inhibition of arsenite-induced ERK activity by MEK inhibitor, PD98059
To provide further support for the hypothesis that a low level of arsenite exposure induces the ERK pathway and promotes cell proliferation, LEC cells were left untreated or pre-treated with 100 µM PD98059 for 30 min before exposure to 2 µM arsenite for 24 h and then subjected to NBB staining assay for determination of viability. A total of 2 µM of arsenite treatment promoted cell proliferation and increased the cell viability (Figure 6). In the presence of PD98059 and 2 µM of arsenite treatment, the increase in viability was blocked and closed to the control level. This suggests that cell proliferation correlates to arsenite-induced ERK activity.



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Fig. 6. Arsenite-induced ERK activity correlates to cell proliferation. LEC cells were left untreated or pre-treated with 100 µM PD98059 for 30 min before exposure to 2 µM arsenite for 24 h and then subjected to NBB staining assay for determination of viability. The percentage of viability was plotted as 100% for control (no treatment of arsenite). Data are mean values from three trials. *A significant difference (P < 0.05) from the uninhibited controls.

 
Expression of pro- and anti-apoptotic proteins
Bcl-2 is an important anti-apoptotic protein, and Bax an important pro-apoptotic protein. It has been reported that the JNK and other signaling pathways are capable of modifying Bax and Bcl-2 family members to reset susceptibility to apoptosis (42,43). The levels of Bcl-2 and Bax proteins in control and arsenite treated cells were determined by western blot analysis. The level of Bcl-2 protein was significantly decreased whereas the level of Bax protein was markedly increased in the cells treated with 40 µM arsenite (Figure 7). The ratio of Bcl-2/Bax, which is thought to be an important determinant in dictating apoptosis, was significantly decreased (9.3-fold) in cells treated with 40 µM arsenite compared with the control cells, a finding indicative of an increase in induced apoptosis (Figure 7). On the contrary, the ratio of Bcl-2/Bax increased 1.5-fold in cells treated with 2 µM arsenite compared with the controls, implying that the cells are more reluctant to committing apoptosis, a phenomenon resembling transformed cells (Figure 7).



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Fig. 7. The effects of arsenite exposure on expressions of Bcl-2 and Bax proteins. LEC cells were serum starved for 24 h and left untreated or treated with 2 or 40 µM sodium arsenite for 24 h. Total cellular proteins were subjected to western blot analysis for the detection of Bcl-2 and Bax proteins using anti-Bcl-2 and anti-Bax antibodies (Santa Cruz Biotechnology). The same blot was also re-probed with a monoclonal anti-ß-actin antibody (Sigma) to monitor the loading difference. The data are representative of three independent experiments.

 
Inhibition of arsenite-induced JNK activity by JNK inhibitor II, SP600125
To show that a high level of arsenite exposure induces the JNK pathway and leads to apoptosis, LEC cells were left untreated or pre-treated with 20 µM SP600125 for 30 min before exposure to high levels (20, 30 or 40 µM) of arsenite for 24 h. They were then subjected to western blot analysis for the levels of Bax and NBB staining assay for determination of viability. High levels of arsenite (20–40 µM) treatment stimulated Bax in a dose-dependent manner, compared with the controls (Figure 8A). This stimulation of Bax can be blocked by SP600125. In the NBB staining assay, similar results to western blot can be observed. High levels of arsenite (e.g. 20 µM) treatment induced apoptosis and decreased the cell viability compared with the control cells (Figure 8B). In the presence of SP600125 and arsenite treatment, the decrease in viability can be partially blocked and cells can be partially rescued from apoptosis. The fact that cell viability cannot be fully recovered by JNK inhibitor may be due to the high toxicity of arsenite at high levels, so that the inhibitor cannot rescue all the cells from committing apoptosis. In fact we discovered that once the cells were treated with high levels of arsenite for 24 h, they were unable to recover, and finally committed apoptosis even though they were washed free of arsenite and allowed to recover (unpublished data). These results suggest that cell apoptosis correlates to arsenite-induced JNK activity.



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Fig. 8. Arsenite-induced JNK activity correlates to cell apoptosis. LEC cells were serum starved for 24 h and then left untreated or pre-treated with 20 µM SP600125 for 30 min before exposure to high levels (20, 30 or 40 µM) of arsenite for 24 h. They were then subjected to (A) western blot analysis to detect the levels of Bax. The same blot was also re-probed with a monoclonal anti-ß-actin antibody to monitor the loading difference. The data are representative of three independent experiments performed by the authors. In (B), NBB staining assay was performed for determination of viability in LEC cells treated with a high level (20 µM) of arsenite for 24 h essentially the same as in (A), but without serum starvation before treatment. The percentage of viability was plotted as 100% for control (no treatment of arsenite). Data are mean values from three trials. *A significant difference (P < 0.05) from the uninhibited controls.

 
Arsenite induces GADD45 expression
The induction of apoptosis by higher levels of arsenite may be due to the activation of cell cycle checkpoints. GADD45 is an essential component at the G2/M cell cycle checkpoint induced by UV light or methanesulfonate (44). Recently, GADD45 has also been shown to be induced by treatment of arsenite in human bronchial epithelial cells, BEAS-2B (45). To find out whether high levels of arsenite would also induce GADD45 in LEC cells, we measured the expression of GADD45 by western blot in LEC cells after treatment with various amounts of sodium arsenite. Arsenite induced GADD45 expression only at high concentrations but not at low concentrations, compared with the controls (Figure 9). The induction of GADD45 expression was in a dose-dependent manner.



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Fig. 9. GADD45 protein levels in LEC cells treated with sodium arsenite. LEC cells were serum starved for 24 h and then treated with various amounts of sodium arsenite for 10 h. Total cellular proteins were subjected to western blot analysis for the detection of GADD45 protein using anti-GADD45 antibody (Zymed Laboratories). The intensity of the bands was quantified and plotted as a relative amount, setting 1 for control (no treatment of arsenite). The data are representative of three independent experiments.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Environmental and occupational exposure to arsenic is associated with an increased risk of skin, urinary bladder, liver, kidney and respiratory tract cancers (68). The carcinogenicity of arsenic can be caused by either genotoxic or epigenetic mechanisms. Arsenic is usually assumed to act principally via an epigenetic effect. An important epigenetic mechanism could be the inappropriate activation or inactivation of signal transduction pathways leading to the activation of transcription factors, which in turn modulates the gene expression (1,2,1317,46,47). In the present study, we investigated the effects of arsenite on cell growth and its signal transduction pathways in a rat LEC line. Our results indicated that a molecular mechanism by which MAPK pathways might differentially contribute to cell growth regulation and cell death depends on the dosage of arsenite being exposed.

Using flow cytometric analysis, we demonstrated that 2 µM of arsenite exposure for 24 h led to a significant increase in the number of cells in the S phase and DNA synthesis as compared with that of control. Increased cell proliferation following arsenic exposure has also been demonstrated in skin (48) and urinary bladder (49). Thus, a low level of arsenite does appear to enhance cell proliferation, consistent with its role as a tumor promoter. On the other hand, high levels of arsenite cause apoptosis, as evidenced by the appearance of cells accumulated in the sub G1 phase, nuclear chromatin morphological changes and DNA fragmentation in a dose-dependent manner from 10–40 µM of arsenite treatment.

AP-1 transactivation has been shown to be required for tumor promoter-induced cell transformation (41). AP-1 binding activity can be induced either by increases in expression of AP-1 genes, in the phosphorylation of existing c-Jun proteins, or both. The transactivation function of c-Jun can be stimulated through phosphorylation at serines 63 and 73 by a single type of protein kinase, termed JNK (also called stress-activated protein kinase or SAPK) (50), which exists in several isoforms (23). The JNKs are members of the MAPK family and, like ERK1 (p44) and ERK2 (p42), are activated through phosphorylation at conserved Thr and Tyr residues (23). By using gel mobility shift assay, we showed that the low level of arsenite exposure enhanced AP-1 binding activity in LEC cells, and this may be mediated by increase in c-jun and c-fos gene expression levels (13). Our data, as well as results reported by Chen et al. (17), indicate that the low level of arsenite exposure induces cell proliferation by the over- expression of cell proliferation-associated genes including oncogenes, such as c-jun and c-fos.

Previous studies have demonstrated the activation of MAPKs in several cell types by arsenic. For example, the JB6 mouse epidermal cell line exposed to low doses of arsenite demonstrated ERK stimulation associated with cell transformation (46). ERK subtypes, members of the MAPK family, were recognized as key transducers in the signaling cascade mediating the expression of cell growth-related gene (31). In the present study, we demonstrated that a low concentration of arsenite induced ERK activation but not JNK activation. A study conducted with PC12 cells, used commonly to explore MAPK activation, has demonstrated that arsenite treatment activates ERK in the EGFR-dependent pathway, and the interaction of arsenite with EGFR vicinal thiols has been proposed as a triggering mechanism in this event (17). Arsenic might activate the EGFR–ERK pathway to induce gene expression and mitogenicity in urinary bladder epithelium. EGFR has been extensively studied as an integral part of human urinary bladder carcinogenesis (51). However, these authors used a much higher level (400 µM) of arsenite in their experiments (17). Whether the activation of ERK by 2 µM arsenite is also through the EGFR-dependent pathway will be further investigated. Our results demonstrated that a low level (2 µM) of arsenite induced cell proliferation in LEC cells, and this induction appears to be mediated by activation of MAP kinase family members, ERKs. This arsenite-induced ERK activity, which promotes cell proliferation, can be blocked by MEK inhibitor, PD98059, as reflected by the percentage of viability from NBB staining assay. The results suggest that cell proliferation correlates to arsenite-induced ERK activity. On the contrary, a higher level (40 µM) of arsenite caused LEC cell apoptosis, and it appears to be mediated by the JNK signaling pathway. This arsenite-induced JNK activity, which causes cell apoptosis, can be blocked by JNK inhibitor II, SP600125, as evidenced by the levels of Bax from western blot analysis and the percentage of viability from NBB staining assay, respectively. Both data suggest that cell apoptosis correlates to arsenite-induced JNK activity. Our data demonstrated that ERK and JNK MAPKs could be differentially activated by arsenite. These results are consistent with the data reported by Huang et al. (46,52).

Recently we have also reported that chronic low-level arsenite exposure induces malignant transformation in a rat LEC line (35). Using a proteomic approach, several differentially expressed proteins were identified in lung cells after transformation by a treatment of 1.5 µM arsenite for 12 weeks. The expressions of these proteins could potentially be important for transformation induced by arsenite. Some of these proteins were found to be present in mitochondria and participate in mitochodrial respiratory chain and ATP production (35). Results from our proteomic study also suggested that the expression of pro-apoptotic protein Bax was suppressed in arsenite-induced transformed cells. In the present study, we also found that the ratio of Bcl-2/Bax increased in LEC cells treated with a low level of arsenite, which induces cell proliferation and might eventually lead to cell transformation. On the contrary, higher levels of arsenite treatment decreased the ratio of Bcl-2/Bax and caused cell death.

We have shown that arsenite suppressed LEC cell growth at a higher concentration. This growth inhibitory effect of arsenite may be due either to the induction of cell apoptosis, or the activation of cell cycle checkpoints, or both. A recent study has shown that nuclear factor-kappaB (NF-{kappa}B) and JNK are reciprocal regulators for arsenite-induced cell cycle and expression of GADD45 (45). GADD45 has also been shown in recent studies to be an essential component at the G2/M cell cycle checkpoint induced by UV light, methanesulfonate (44), or by treatment of arsenite in human bronchial epithelial cell line, BEAS-2B (45). The data from these studies are consistent with our own results. Previously, we found that a low concentration (5 µM) of arsenite was able to activate NF-{kappa}B in a dose-dependent manner (13). In this paper we also showed that a low level of arsenite did not induce JNK activity and GADD45 expression. However, a high concentration of arsenite increased JNK activity and GADD45 expression. Based on our results, we conclude that, depending on the dosage, arsenite can lead either to cell proliferation or to apoptosis, through the ERK or JNK pathways, respectively (Figure 10). These results also suggest that a low level of arsenite exposure is carcinogenic, because the stimulation of cell proliferation thereby induced may prevent induction of cell cycle checkpoint proteins that maintain genomic stability and result in aberrant cell accumulation.



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Fig. 10. A model of arsenic action based on our data is presented. Outcome depends on the dosage of arsenite exposed, which can either lead to cell proliferation through the ERK pathway or apoptosis through the JNK pathway.

 

    Notes
 
6 To whom correspondence should be addressed Email: jfchiu{at}hkucc.hku.hk Back


    Acknowledgments
 
We thank Drs A.Barchowsky and D.Wilmshurst for reviewing the manuscript, Yuan Zhou for technical assistance and Celia N.L.Lau for critical reading of the manuscript. This investigation was partially supported by seed funding from the University of Hong Kong (No. 10204004/39815/43700/301/01 and 10204007/38181/25200/301/01), RGC grant (HKU7395/03 M) and the Areas of Excellence scheme of the Hong Kong University Grants Committee.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 15, 2003; revised August 16, 2003; accepted September 12, 2003.





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