Acquisition of apoptotic resistance in arsenic-induced malignant transformation: role of the JNK signal transduction pathway
Wei Qu1,
Carl D. Bortner2,
Teruaki Sakurai1,
Michael J. Hobson1 and
Michael P. Waalkes1,3
1 Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences, PO Box 12233, Mail Drop F0-09, 111 Alexander Drive, Research Triangle Park, NC 27709, USA and
2 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, 111 Alexander Drive, Research Triangle Park, NC 27709, USA
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Abstract
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This study examined the role of signal transduction and apoptosis in malignant transformation induced by arsenic. Prior study showed that chronic arsenite exposure (500 nM,
18 weeks) induced malignant transformation in rat liver TRL 1215 cells. In the present work, these transformed cells were compared with passage-matched control cells. In addition, TRL 1215 cells were treated subchronically (up to 6 weeks) with arsenic (termed pre-transformed cells) to define events occurring prior to arsenic-induced transformation. Flow cytometry using annexin/FITC revealed that arsenic-induced apoptosis in transformed cells was markedly suppressed in comparison to control or pre-transformed cells. Ro318220, a strong activator of JNK, enhanced arsenite-induced apoptosis in transformed cells. Densitometric analysis of western blots revealed that the ratios of both Bcl-xL/Bax and Bcl-2/Bax were significantly increased (>2.5-fold) in arsenic-transformed cells. Transformed, pre-transformed and control cells were treated with arsenic and levels of phosphorylated extracellular signal-regulated kinases, ERK1/2, JNK1/2 and p38 were determined by western blot analysis. The three mitogen-activated protein kinases (MAPKs) were phosphorylated in a dose-dependent fashion in all cell types. However, the levels of phosphorylated JNK1/2 were markedly decreased in the arsenic-transformed cells, whereas in pre-transformed cells the levels of phosphorylated MAPKs remained the same as in control cells. JNK kinase activity was suppressed in transformed cells whereas Ro318220 enhanced this activity. Thus, during arsenic-induced malignant transformation resistance to apoptosis develops, possibly due to perturbation of the JNK pathway.
Abbreviations: As+3, arsenite the trivalent form of arsenic; EGF, epidermal growth factor; ERK1/2, extracellular signal-regulated kinases1/2; JNK1/2, c-Jun NH2-terminal kinase1/2; MAPKs, mitogen-activated protein kinases; MKP-1, mitogen-activated protein kinase phosphatase-1; PKC, protein kinase C; TRL 1215, a rat liver epithelial cell line; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling.
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Introduction
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Arsenic is an important environmental toxicant and a human carcinogen (1). However, only recently models have emerged to study the potential molecular mechanisms of arsenic carcinogenesis. For instance, our recent work showed that chronic low-level arsenic exposure induces malignant transformation in TRL 1215 cells (2). These transformed cells show up-regulation of a variety of oncogenes and genes associated with rapid cell proliferation (3). Interestingly, a pronounced self-tolerance to arsenic cytolethality occurs concurrently with malignant transformation in these cells (4). The precise basis of this acquired self-tolerance has not been defined.
Despite the adverse effects of arsenicals, they show great promise in the chemotherapy of certain types of human cancer (5). In this regard, arsenic is an effective treatment for acute promyelocytic leukemias (6,7). Arsenic found use in cancer chemotherapy in the 19th and early 20th centuries (8,9). Thus, it appears that arsenic, a known human carcinogen, is also an effective metallo-chemotherapeutic. Therefore, the study of the mechanism of acquired self-tolerance to arsenic could have important implications in both cancer causation and cancer chemotherapy.
Apoptosis is a form of cell death that plays a major role in developmental biology, cellular population dynamics and disease states. Apoptosis typically occurs when cellular genetic damage exceeds repair capacity. The suppression of apoptosis, in the face of significant genetic damage, could facilitate accumulation of aberrant cells and may be a critical step in the pathogenesis of malignancy. On the other hand, arsenic is emerging as an important chemotherapeutic by effectively inducing apoptosis in leukemia cells (6). The apoptotic effects of arsenic are not limited to leukemia cells but occur in a variety of other cell types (10). Thus, it appears that arsenic may activate endogenous signaling pathways for induction of apoptosis.
Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine phosphorylating proteins that mediate the signal transduction pathways from a variety of extracellular signals to regulate the expression of specific genes (11,12). Of the major MAPKs, the extracellular signal-regulated kinases (ERKs) typically transduce growth factor signals that cause cell differentiation or proliferation. In contrast, cytokines and other stress signals activate the c-Jun N-terminal kinase (JNK) and p38 pathways, resulting in stress responses, growth arrest and/or apoptosis (11). These MAPKs are activated by the dual phosphorylation of specific tyrosine and threonine residues, which is performed by regulatory kinases upstream in the signaling cascade. Although activation of JNK and p38 MAPK pathways has been associated with induction of apoptosis (11), the mechanisms involved in the JNK-induced apoptotic response are unclear. Recently, several studies have shown that mitochondria play a central role in inducing apoptosis by releasing various apoptotic factors such as cytochrome c and apoptotic proteases (13,14). Thus, defining the role of mitochondria, including changes in the mitochondrial membrane potential, has become an important focus in the study of apoptotic regulation. Arsenic has been shown to induce MAPK activity and apoptosis in several cell lines (12,15) although any effect on mitochondrial membrane potential has yet to be defined.
Arsenic-induced malignant transformation appears to occur concurrently with the development of tolerance to the acute cytolethal effect of arsenic, at least in some models (2,4). Exactly how such acquired self-tolerance is linked to the carcinogenic transformation process is not clear at present. However, other carcinogenic inorganics can induce cellular apoptotic resistance as an important factor in the acquisition of the malignant phenotype (16).
Thus, the purpose of the present study was to define the role of apoptotic resistance and any concurrent alterations in signal transduction pathways in arsenic-induced malignant transformation using our established in vitro model system (2). In this regard, we found that transformed cells became highly resistant to arsenic-induced apoptosis. This acquired apoptotic resistance in arsenic-transformed cells appeared to arise from the perturbation of JNK1/2 activity. Apoptotic response was restored in the transformed cells by using a JNK activating drug, Ro318220. Acquired apoptotic resistance may have important implications in both arsenic carcinogenesis and pharmacology.
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Materials and methods
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Materials
Sodium arsenite was obtained from Sigma Chemical Company (St Louis, MO). Ro318220 was purchased from Calbiochem (Bad Soden, Germany). The terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling (TUNEL) assay kit was purchased from Boehringer Mannheim (Indianapolis, IN). Annexin V and propidium iodide were purchased from Trevigen (Gaithersburg, MD). Anti-phospho-JNK, anti-phospho-ERK, anti-phospho-p38, anti-phospho-c-Jun (Ser 63) and anti-phospho-ElK1 (Ser 383) antibodies were purchased from New England Biolabs (Beverly, MA). Bcl-xL, Bcl-2 and Bax antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The JC-1 was obtained from Molecular Probes (Eugene, OR).
Cell culture and treatment
The TRL 1215 cell line was originally derived from the liver of 10 day old Fischer F344 rats and cells were cultured as described previously (2). The cells are diploid and normally non-tumorigenic. Control cells were cultured in William's E media containing 10% fetal bovine serum, while 500 nM arsenic (as sodium arsenite) was added to culture medium to induce transformation which occurred at ~18 weeks of exposure. The cells chronically treated with arsenic show extensive evidence of malignant transformation, including the formation of invasive tumors with metastatic potential upon inoculation of these cells into Nude mice (2). Other cells were treated with 500 nM arsenic for up to 6 weeks, a point prior to transformation (2), and are termed `pre-transformed' cells. Previous work shows that cells having undergone only 8 weeks of 500 nM arsenic exposure do not form tumors (2) establishing this time point as clearly prior to transformation. The acute effects of arsenite, including effects on cytotoxicity, apoptosis and mitochondria, were assessed in transformed cells, pre-transformed cells and appropriate passage-matched control cells.
Immunocytochemical detection of apoptosis
Cells grown on 96 well plates were treated with various reagents as indicated, and were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4. DNA strand breaks were identified using a modified TUNEL assay. Briefly, the fixed cells were treated with terminal deoxynucleotidyl transferase, which incorporates fluorescein-tagged nucleotides onto 3-OH termini of fragmented DNA. Cells were then visualized by adding anti-fluorescein antibody conjugated with horseradish peroxidase followed by diaminobenzidine. The positively stained, dark colored nuclei were analyzed under a light microscope.
Determination of apoptosis by flow cytometry
Detection of phosphatidylserine on the outer leaflet of apoptotic cells was performed using annexin V and propidium iodide according to the manufacturer's recommendations. For each sample, 10 000 cells were examined by flow cytometry using a Becton Dickinson FACSort. The percent of apoptotic cells was determined by analysis of the dot plots using CellQuest software.
Cell lysate preparation
After cells were grown to 7080% confluence, they were placed in serum free medium for 24 h and then treated with arsenic for the indicated times and doses. Cells were then washed with ice-cold phosphate-buffered saline, and scraped into cell lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 50 mM pyrophosphate, 1 mM Na3VO4, 1 mM EGTA, 100 mM NaF, 1% Triton X-100, 10% glycerol, 10 mg/ml leupeptin, 10 mg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. The cells were 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 (17). Protein concentration was determined by the method of Bradford (18) using bovine serum albumin as standard.
Western blot analysis
Protein samples (30 mg) derived from the various cell preparations were subjected to SDSpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and probed with various antibodies including antibodies against Bcl-xL, Bcl-2, Bax, phosphorylated JNK1/2, phosphorylated ERK1/2 and phosphorylated p38 MAPK. After incubation with secondary antibodies, immunoblots were visualized with the LumiGlo detection method (New England Biolabs).
JNK and ERK activity assay
The enzymatic activity of JNKs and ERKs were determined by using the JNK and ERK assay kits from New England Biolabs. Briefly, cell extracts containing 250 µg of total protein were incubated overnight at 4°C with either an N-terminal c-Jun (189) fusion protein bound to glutathioneSepharose beads for JNK kinase activity assay or immobilized phospho-44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody for ERK kinase activity assay. The kinase reaction was performed by adding 100 µmol/l ATP to the suspension. Phosphorylation of c-Jun was measured by western blot analysis with a phospho-specific c-Jun antibody that specifically detects Ser63-phosphorylated c-Jun, a site important for c-Jun-dependent transcriptional activity (19). The resulting immunoprecipitate was then incubated with a Elk-1 fusion protein in the presence of ATP and kinase buffer which allows immunoprecipitated active MAP kinase to phosphorylate Elk-1. Phosphorylation of Elk-1 at Ser383 is measured by western blot analyses using a phospho-Elk-1 (Ser383) antibody. Ser383 of Elk-1 is a major phosphorylation site for MAP kinases and is required for Elk-1-dependent transcriptional activity.
Statistical analysis
Student's t-test or ANOVA with subsequent Dunnett's test were used as appropriate. All values are expressed as mean ± SEM of 3 or more replications. Differences were considered significant at P < 0.05.
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Results
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Arsenic transformation produces tolerance to the arsenic-induced apoptosis
Various studies have shown that arsenic can induce apoptosis in several cell lines (5,21). To investigate the role of arsenic in the induction of apoptosis during arsenic-induced malignant transformation, both transformed cells and passage-matched control cells were treated with 5 or 10 µM arsenic (as sodium arsenite) for 24 h. Apoptotic cell nuclei were identified by TUNEL analysis and visualized by light microscope. Acute arsenic treatment induced a high rate of apoptosis in control cells as indicated by numerous TUNEL positive cells (Figure 1
). In contrast, transformed cells were highly resistant to arsenic-induced apoptosis as no TUNEL positive cells were observed.

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Fig. 1. Arsenite (As+3) induces apoptosis in control cells, but not in transformed cells. Transformed and control cells were treated with 5 or 10 µM arsenic (As+3) for 24 h. Apoptotic cell nuclei were identified by TUNEL analysis and visualized by light microscope.
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An additional study compared arsenic-induced apoptosis in transformed and pre-transformed cells. Pre-transformed cells had been exposed to 500 nM arsenic subchronically (up to 6 weeks) and results were compared with appropriate passage-matched control cells. All groups were treated with 5 or 10 µM arsenic for 24 h and apoptosis was determined by flow cytometry using fluorescent-labeled annexin V and propidium iodide. Arsenic-induced apoptosis was markedly reduced in transformed cells compared to appropriate passage-matched control cells (Figure 2
, bottom). On the other hand, arsenic-induced apoptosis was not different from control in the pre-transformed cells (Figure 2
, top). These results indicate that after transformation has occurred, cells acquired a marked resistance to apoptosis induced by arsenic.

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Fig. 2. Transformation produces tolerance to the arsenic-induced apoptosis. The various treatment groups and appropriate control cells were treated with 5 or 10 µM arsenic (As+3) for 24 h. Apoptosis was determined by flow cytometry using fluorescent-labeled annexin V and propidium iodide (see Materials and methods). Data are expressed as percent of apoptotic cells. Results are presented as the mean ± SEM, n = 6: (a) indicates a significant (P < 0.05) difference from appropriate untreated control; (b) indicates a significant (P < 0.05) difference from appropriate dosage matched control.
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Effect of acute arsenic treatment on levels of phosphorylated MAPKs in arsenic-transformed cells. The JNK and p38 pathways have been implicated in the regulation of apoptosis induced by various stimuli (11). In addition, several recent papers have suggested that ERK might also be involved in apoptotic signaling (21). Previous studies have demonstrated activation of MAPKs in several cell types using concentrations of arsenic in the range of 300500 µM (12). Thus, to determine which signal pathway may be involved in arsenic-induced apoptosis, transformed and passage-matched control cells were subjected to an acute dose of arsenite (0, 50, 200, 300 or 500 µM) for 30 min. The levels of phosphorylated, ERK1/2, JNK1/2 and p38 were determined by western blot analysis (Figure 3
) and analyzed by scanning densitometry (Table I
). All three MAPKs were phosphorylated in a dose-dependent fashion both in transformed and passage-matched control cells. Interestingly, the level of phosphorylated JNK1/2 was markedly decreased in the transformed cells compared with control cells at each level of arsenic treatment. The levels of phosphorylated ERK1/2 were slightly decreased in transformed cells as compared with control cells but only in the low-dose range. The level of phosphorylated p38 did not show any significant differences between transformed and control cells. In contrast to transformed cells, the levels of phosphorylated ERK1/2, JNK1/2 and p38 in pre-transformed cells remained the same as passage-matched control cells after acute arsenic exposure (data not shown). The membranes used to define phosphorylated forms were stripped and then reprobed with antibodies against JNK, ERK and p38 that recognize both the phosphorylated and non-phosphorylated forms to assess the total amount of the individual MAPK. As shown in Figure 3
, no major differences were observed between transformed and control cells in the levels of total JNK1/2, ERK1/2 and p38. Thus, the decrease in phosphorylated-JNK is probably a decrease in phosphorylation of the native protein in transformed cells rather than a reduction in total cellular JNK protein.

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Fig. 3. Effect of acute arsenic treatment on levels of phosphorylated MAPKs in transformed cells. Control and transformed cells were acutely exposed to various concentrations of arsenic for 30 min. The levels of phosphorylated JNK1/2, ERK1/2 and p38 were determined by western blot analysis. After development, the membranes were stripped and reprobed with regular antibodies against JNK1/2, ERK1/2 and p38 MAPKs. Blots represent a typical result of three independent experiments.
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Table I. Effect of acute arsenic treatment on levels of phosphorylated MAPKs in transformed and passage-matched control cells
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Concentration and time-dependent effects of arsenic treatment on JNK1/2 kinase activity in transformed cells. Dual phosphorylation of JNKs at Thr183/Tyr185 is essential for kinase activity and phosphorylation at this site is an excellent marker of JNK activity (19). To confirm JNK activation, an in vitro kinase assay was performed using a c-Jun N-terminal fusion protein as the substrate. After acute arsenic exposure, JNK activation was markedly reduced in transformed cells in comparison to control cells (Figure 4
). This was true with both the concentrationresponse and time course analysis (Tables II and III
). The levels of phosphorylation of JNK determined by using the phospho-specific antibody (see Figure 3
) were thus consistent with the kinase activity of JNK in cell extracts. Epidermal growth factor (EGF), used as a positive control for stimulating JNK phosphorylation, was much less effective in transformed cells (Figure 4
, top). These results suggest that the activation of the JNK pathway is perturbed in transformed cells, specifically at the point of JNK phosphorylation.

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Fig. 4. Concentration and time-dependent induction of JNK1/2 kinase activity by arsenic in transformed and passage-matched control cells. Transformed and control cells were treated with arsenic (As+3) for 30 min at the levels indicated for the concentrationresponse assessment (top) or with 300 µM for the time course (bottom). One group of each cell type was also treated with EGF (10 ng/ml) for 5 min. Cell extracts were incubated overnight with c-Jun fusion protein. Phosphorylation of c-Jun at Ser 63 was measured by western blot using Phospho-c-Jun (Ser63) antibody. Blots represent a typical result of three independent experiments.
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Table II. Concentration-dependent induction of JNK1/2 kinase activity by arsenic in transformed and passage-matched control cells
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Table III. Time-dependent induction of JNK1/2 kinase activity by arsenic in transformed and passage-matched control cells
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ERK activation was also assessed by performing an in vitro kinase assay using immobilized phospho-44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody. There were no significant differences in ERK1/2 activity between transformed and control cells (data not shown).
Effect of Ro318220 on activation of JNK1/2 by arsenic. The compound Ro318220, originally characterized as a specific protein kinase C (PKC) inhibitor, has subsequently been found to be a strong activator of JNK (22,23). Thus, studies were designed to determine if Ro318220 could bypass the blockage of JNK activity in transformed cells. Control and transformed cells were pre-treated with Ro318220 followed by arsenic and c-Jun phosphorylation was measured to assess JNK activity. As shown in Figure 5
, JNK activity was decreased in transformed cells compared with control cells regardless of acute arsenic exposure. However, pre-treatment with Ro318220 prior to arsenic effectively bypassed this block in JNK activity, bringing phosphorylation of c-Jun to control levels in transformed cells. Densitometric analysis (Table IV
) clearly showed that JNK1/2 activity was depressed to ~50% of control values in transformed cells acutely exposed to arsenic. However, with Ro318220 pre-treatment before acute arsenic exposure, JNK1/2 activity in transformed cells was restored to control levels. To confirm that the effects of Ro318220 are based on JNK1/2 activation and not due to PKC inhibition, similar experiments were performed with both staurosporine (40 nM) and GF109203 (2.5 mM), two well-known PKC inhibitors (22,23). In contrast to the effects of Ro318220 on JNK1/2 activation, both staurosporine and GF109203 did not affect JNK1/2 activation in either control or transformed cells (data not shown). These results again strongly suggest that the JNK pathway is perturbed during arsenic-induced malignant transformation.

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Fig. 5. Effect of Ro318220 on activation of JNK1/2 by arsenic. Transformed and control cells were pre-treated with 10 µM Ro318220, an activator of JNK, for 30 min, followed by arsenic (As+3, 300 µM) treatment for 30 min. Activity of JNK1/2 was then measured as described in Figure 4 . Blots represent one of three independent experiments.
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Effect of Ro318220 on the apoptotic rate in transformed cells
To determine if JNK activation through Ro318220 could bypass the apoptotic block seen after transformation, transformed and control cells were pre-treated with Ro318220 prior to arsenic (5 or 10 µM). The extent of apoptosis was assessed by flow cytometry (Figure 6
, Table V
). Arsenic was again markedly less effective in inducing apoptosis in transformed cells. The addition of Ro318220 prior to arsenic significantly increased apoptotic rate between 5.2- and 7.8-fold in transformed cells.

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Fig. 6. Effect of Ro318220 on apoptosis by arsenic. Transformed and control cells were pre-treated with 10 µM Ro318220, an activator of JNK, for 30 min, followed by 5 or 10 µM arsenic (As+3) for 24 h. Apoptosis was measured by flow cytometry. Data shown represent a typical result of four independent experiments.
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For studying the JNK signal transduction pathway, we used 300 µM arsenic to treat cells for 30 min. This treatment is commonly used in other studies with arsenic and the JNK pathway (12) and the higher level is designed to give a clear, observable response. For studying apoptosis, we generally used 5 or 10 µM arsenic to treat cells for 24 h to better simulate an environmental exposure. However, when the treatment protocol used to assess JNK pathway activity (300 µM, 30 min) was similarly used to assess apoptosis, the results showed the same pattern as when lower concentrations of arsenic (5 or 10 µM) were used for longer periods (24 h), as arsenic-induced apoptosis was again significantly perturbed in transformed cells and the addition of Ro318220 prior to arsenic significantly enhanced apoptotic rate in transformed cells (data not shown). Using the TUNEL assay, which relies on the morphological characteristics of apoptosis, it was also found that pre-treatment with Ro318220 prior to arsenic (300 µM arsenic for 30 min) again overcame the resistance to apoptosis in transformed cells (data not shown). Thus, the perturbation of JNK1/2 activation is correlated with apoptotic resistance in transformed cells regardless of the precise arsenic treatment protocol used to induce apoptosis.
Expression of pro- and anti-apoptotic proteins
Bcl-xL and Bcl-2 are important anti-apoptotic proteins and Bax is an important pro-apoptotic protein. It has been reported that multiple signal transduction pathways including JNK are capable of modifying Bcl-xL and Bcl-2 family members to reset susceptibility to apoptosis (14,24). Thus, the levels of Bcl-xL, Bcl-2 and Bax proteins in control and arsenic-transformed cells were determined by western blot analysis (Figure 7
). The levels of both Bcl-xL and Bcl-2 protein were substantially increased (~1.5-fold) whereas the level of Bax protein was markedly decreased (~1.5-fold) in the transformed cells. The densitometry analysis showed that the ratios of both Bcl-xL/Bax and Bcl-2/Bax, which are though to be important critical determinants in dictating cellular apoptosis, were significantly increased (>2.5-fold) in transformed cells, a finding indicative of reduced apoptosis.

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Fig. 7. Effect of transformation on the ratios of Bcl-xL/Bax and Bcl-2/Bax. Western blot analysis was performed on cellular lysates prepared from control and arsenic-transformed cells using specific Bcl-xL, Bcl-2 and Bax antibodies as detailed in the Materials and methods section. (A) Lysates prepared from control or arsenic-transformed cells immunoblotted with the Bcl-xL, Bcl-2 and Bax antibodies. Data shown represent a typical result of four independent experiments. (B) The levels of Bcl-xL and Bax protein were analyzed by scanning densitometry and used to calculate the ratio of Bcl-xL/Bax. (C) The levels of Bcl-2 and Bax protein were analyzed by scanning densitometry and used to calculate the ratio of Bcl-2/Bax. An asterisk (*) indicates a significant (P < 0.05) difference between control and transformed cells. Data represent the mean ± SEM of four independent experiments.
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Discussion
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Although inorganic arsenic is clearly carcinogenic in humans, it is unclear if it acts through genotoxic or epigenetic mechanisms. An important epigenetic mechanism could be the inappropriate activation or inactivation of signal transduction pathways resulting in aberrant cell accumulation. In this regard, we reported previously that chronic low-level arsenic exposure induces malignant transformation in a rat liver epithelial cell line (2) and that self-tolerance to the cytolethal effects of arsenic occurs concurrently with malignant transformation (4). So it is quite possible that these transformed cells may show alterations in signal transduction pathways that participate in the control of cell population dynamics. Further study with these transformed cells indicates they are hyperproliferative and show over-expression of a variety of genes associated with cell proliferation, including oncogenes (3). In fact, the present study demonstrates that after malignant transformation has occurred, these arsenic-transformed cells acquire a marked resistance to arsenic-induced apoptosis. Thus, the acquisition of apoptotic resistance leading to aberrant cell accumulation may be an important event in arsenic carcinogenesis, at least in this model. In addition, signal transduction pathways that participate in apoptosis are perturbed in these cells, fortifying the concept that apoptotic control mechanisms are disrupted as cells become transformed through arsenic exposure.
The resistance to apoptosis observed in arsenic-transformed cells may play a role in acquired self-tolerance by allowing cells that would normally be eliminated by fully functioning apoptotic pathways to survive. It would be expected that such survival could lead to accumulation of genetically damaged or altered cells. This has several potential implications for arsenic-induced malignancies. For instance, it could allow damaged or altered cells to inappropriately escape apoptosis and potentially proliferate, thus providing for initiating events in carcinogenesis. When the basal or induced rate of apoptosis is perturbed in arsenic-transformed cells, spontaneously genetically damaged or altered cells would be more likely to escape from apoptosis and proliferate, thus enhancing the potential for tumor initiation and/or progression. Therefore, even if perturbed apoptosis occurs only in a numerically small number of cells, this blockage of apoptosis in arsenic-transformed cells could hold important implications for carcinogenesis. The reduced basal rate in arsenic-transformed cells may be a response to a chronic apoptotic stimulus (arsenic) in these cells, although this is speculative. Other reports have also associated acquired apoptotic resistance with malignant cellular transformation in in vitro systems (2527). On the other hand, perturbed apoptosis could also enhance the potential for a malignant cell population to expand, thus impacting tumor progression. In this regard, other inorganic carcinogens can effect tumor progression. For example, repeated injections of rats with cadmium, although not altering incidence, clearly enhances the malignant progression of tumors resulting at the site of injection, as assessed both by regional invasiveness and distant metastasis (28). In addition, exposure of tumorigenic myoblast to cadmium in vitro enhances tumor progression when cells are inoculated into nude mice (29). Cadmium-transformed human prostate epithelial cells also show apoptotic resistance and form highly aggressive tumors after inoculation into nude mice (16). These effects on tumor progression would be consistent with the proposed imbalance between proliferation and apoptosis that would lead to a selective growth advantage from an apoptotic block in pre-malignant or malignant tumor cells (30). In fact, cadmium can also be an effective inhibitor of chemically induced apoptosis (31). Accumulating data with a variety of inhibitors of apoptosis other than cadmium are, in fact, consistent with a role of apoptotic suppression in tumor progression (32). Therefore, acquired resistance to apoptosis, as seen in the present study with arsenic transformation, may have important implications for both the initiation of arsenic-induced malignancies and eventual tumor progression, although further research will be required to establish these conclusions.
The activation of apoptosis is regulated by many different signals that originate from both intracellular and the extracellular sites. It has been reported that activation of JNK is associated with the induction of apoptosis (11). However, the mechanisms involved in JNK-induced apoptosis are presently not known. Interestingly, recent studies have suggested that mitochondria play a central role in inducing apoptosis (13,14). Apoptotic stimuli can cause the generation of intracellular signals, which result in mitochondria depolarization and appearance of permeability transition pores through which several mitochondrial proteins translocate to the cytoplasm. The release of various pro-apoptotic factors from mitochondria, such as cytochrome c and apoptosis-inducing factor (AIF), has been reported to be critical for apoptosis (13,33). Previous studies have shown that JNK is activated by genotoxic stress and functions upstream to the induction of apoptosis in response to DNA damage (34). A recent study indicates that genotoxic stress induces translocation of SAPK/JNK to mitochondria (14). Furthermore, Tournier et al. (35) have reported that mitochondria are influenced by pro-apoptotic signal transduction through the JNK pathway and the absence of JNK caused a defect in the mitochondrial death-signaling pathway, including the failure to release cytochrome c. The present results clearly demonstrate that JNK activation is perturbed in transformed cells and that Ro318220, a strong activator of JNK, circumvents apoptotic resistance seen in these same cells. Thus, arsenite-induced resistance to apoptosis appears to be involved with the specific suppression of the JNK pathway, but this may not be the only cause of this resistance. Moreover, it has been reported that SAPK/JNK associates with the anti-apoptotic Bcl-xL and Bcl-2 proteins in mitochondria (14,24). Bcl-2 family members provide the most fundamental regulatory mechanism for apoptosis and a major site of their action is the mitochondria. Translocation of SAPK/JNK to mitochondria is functionally important for interactions with Bcl-xL in the apoptotic response (14). In this regard, the anti-apoptotic protein Bcl-2 has been reported to be phosphorylated and inactivated by JNK (35). The ratio of Bcl-2 to Bax dictates the susceptibility of cells to undergo apoptosis (36). The present study showed that the ratios of both Bcl-xL/Bax and Bcl-2/Bax are significantly increased >2.5-fold, suggesting that in transformed cells apoptosis is perturbed, thus providing a selective survival advantage for transformed cells and possibly contributing to tumorigenesis. Taken together, it appears that chronic arsenic exposure may be acting indirectly to induce events associated with initiation or progression of the carcinogenic process by altering certain signal transduction pathways and associated apoptotic response.
The mitogen-activated protein kinase phosphotases (MKPs) are responsible for inactivation of MAPKs in various cells (17,37). Mitogen-activated protein kinase phosphotase-1 (MKP-1) is the most prominent and best-characterized member of the MKP family and has been shown to inactivate JNK in vitro and in vivo (17,38). Various stresses that activate JNK can induce MKP (39). Thus, the duration of JNK activation can be regulated by MKP through a feedback mechanism (17). Ro318220 is structurally related to staurosporine, an agent which acts as ATP-competitive inhibitor of PKC (22). However, Ro318220, in addition to potently inhibiting PKC, also inhibits MKP-1 expression, activates JNK and induces c-Jun expression in a PKC-independent manner (22). Therefore, Ro318220, which was originally characterized as a PKC inhibitor, has subsequently been identified as an activator of JNK1/2 via inhibition of MKP-1 expression (22,23). Furthermore, at least in some instances, induction of MKP-1 appears to be responsible for prevention of apoptosis through reducing activation of JNK (17). The present study demonstrates that arsenite-induced resistance to apoptosis in transformed cells appears to be involved in the specific perturbation of the JNK pathway. Ro318220 exposure stimulated JNK1/2 activity and circumvented the apoptotic block in these cells. Therefore, chronic arsenite exposure may induce the over-expression of MKP-1 in transformed cells, which could be responsible for blockage of apoptosis by perturbation of the JNK pathway. Experiments attempting to test this hypothesis are currently under way.
Apoptosis is clearly an important mechanism for the elimination of abnormal cells and probably plays an important role in the prevention of tumor development and growth. Recent evidence indicates that besides being carcinogenic, arsenic can also act as a chemotherapeutic by inducing apoptosis specifically within certain tumor cells (6). In particular, arsenic treatment is very effective against acute promyelocytic leukemia (6,7). As arsenic-induced apopotosis is thought to be its main mode of action as a cancer chemotherapeutic, the acquired perturbation of apoptosis with chronic arsenic exposure may well decrease the metalloid's efficacy as a chemotherapeutic. Therefore, the acquired tolerance and apoptotic resistance seen with chronic arsenite exposure in the present study could potentially impact the role of arsenic as a chemotherapeutic, although there are no clinical reports of acquired arsenic tolerance. On the other hand, there are many reports that apoptotic resistance is associated with drug resistance in tumor cells. In addition, we have found these arsenic transformed cells to be cross-tolerant to common cancer chemotherapeutics such as cis-platnium, adriamycin, vinblastin and actinomycin D (40). So acquired self-tolerance to arsenic with chronic exposure may impact arsenic pharmacology, although further study will be required to precisely define this impact.
Recently, we have demonstrated that arsenic uptake is the same in transformed cells as compared with control cells (40). Efflux of arsenic is increased by up-regulation of genes encoding for efflux pumps in transformed cells but only after extensive metabolism, and in all likelihood, a long residence time (40). Thus, both control and arsenic-transformed cells would see the same levels of arsenic, although transformed cells would be more efficient in eventually ridding themselves of the metalloid (40). This makes biokinetic differences in cellular arsenic exposure unlikely as a complete source of the observed differences in apoptotic response seen in the present study between arsenic-transformed and control cells.
In summary, after arsenic-induced malignant transformation has occurred, transformed cells appear to acquire resistance to apoptosis. This resistance to apoptosis appears linked to a perturbation of the JNK pathway, increased expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, as well as reduced expression of pro-apoptotic protein Bax, although these may not be the only causes. This acquired self-resistance to apoptosis may play an important role in arsenic-induced tumor initiation and progression and may also impact the pharmacological efficacy of arsenic in cancer chemotherapy.
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Notes
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3 To whom correspondence should be addressedEmail: Waalkes{at}niehs.nih.gov 
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Acknowledgments
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The authors thank Drs Larry K.Keefer, Jie Liu and William E.Achanzar for critical review of this manuscript. The technical assistance of Tammy Dawson is also acknowledged.
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Received April 24, 2001;
revised September 18, 2001;
accepted September 28, 2001.