Requirement of Erk, but Not JNK, for Arsenite-induced Cell Transformation*

Trivalent arsenic (arsenite, As3+) is a human carcinogen, which is associated with cancers of skin, lung, liver, and bladder. However, the mechanism by which arsenite causes cancer is not well understood. In this study, we found that exposure of Cl 41 cells, a well characterized mouse epidermal cell model for tumor promotion, to a low concentration of arsenite (<25 μm) induces cell transformation. Interestingly, arsenite induces Erk phosphorylation and increased Erk activity at doses ranging from 0.8 to 200 μm, while higher doses (more than 50 μm) are required for activation of JNK. Arsenite-induced Erk activation was markedly inhibited by introduction of dominant negative Erk2 into cells, while expression of dominant negative Erk2 did not show inhibition of JNK and MEK1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing the dominant negative Erk2. In contrast, overexpression of dominant negative JNK1 was shown to increase cell transformation even though it inhibits arsenite-induced JNK activation. Our results not only show that arsenite induces Erk activation, but also for the first time demonstrates that activation of Erk, but not JNK, by arsenite is required for its effects on cell transformation.

Arsenite is introduced into the environment during energy production based on coal, oil shale, and geothermal sources. Once in the environment, arsenite represents a potential health hazard of unknown magnitude. Arsenite is associated with increased risks of human cancer of the skin, respiratory tract, hematopoietic system, and urinary bladder (1)(2)(3)(4). Epidemiological investigations indicated that long-term arsenic exposure results in promotion of carcinogenesis, especially in lung and skin via inhalation and ingestion (5). Many cases of skin cancer have been documented in people exposed to arsenite through medical or other occupational exposures. It has been reported that high arsenic levels in drinking water (0.35-1.14 mg/liter) increased risks of cancer of skin, bladder, kidney, lung, and colon (1,2,5,6). Hence, arsenite is a well documented human carcinogen (5, 7).
Previously, several hypotheses have been proposed to describe the mechanism of arsenite-induced carcinogenesis (8 -14). It has been suggested that arsenic induces chromosome aberration and sister chromatid exchange which may be involved in arsenite-induced carcinogenesis (11,12). Recently, Zhao et al. (13) reported that arsenic may act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression. Additionally, it was found that arsenite is a potent stimulator of extracellular signal-regulated protein kinase (Erk) 1 and AP-1 transactivational activity and an efficient inducer of c-fos and c-jun gene expression (10,14). Induction of c-jun and c-fos by arsenite is associated with activation of JNK (10). However, the role of JNK activation by arsenite in cell transformation or tumor promotion is unclear. We have established cell culture conditions for studying arsenite-induced cell transformation in this report. Furthermore, our data have shown that activation of Erk, but not JNK, is required for cell transformation induced by arsenite.

MATERIALS AND METHODS
Plasmids and Reagents-CMV-neo vector plasmid was constructed as previously reported (15,16); dominant negative JNK1 (pcDNA-flag-JNK1 (APF)) was from Dr. Roger J. Davis, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School (17,18); fetal bovine serum (FBS) and Eagle's minimal essential medium (MEM) were from Biowhittaker; LipofectAMINE was from Life Technologies, Inc.; TPA was from Sigma; rabbit polyclonal IgG against PKC␣ was from Santa Cruz Biotechnology; EGF was from Collaborative Research; luciferase assay substrate was from Promega; and PhosphoPlus MAPK antibody kit, phospho-MEK 1/2 antibody, and p44/42 MAP kinase assay kit were from New England Biolabs.
Generation of Stable Co-transfectants-JB6 Cl 41 cells were cultured in a 6-well plate until they reached 85-90% confluence. We used 1 g of CMV-neo vector with or without 12 g of dominant negative JNK1 (pcDNA-flag-JNK1 [APF]) plasmid DNA and 15 l of LipofectAMINE reagent to transfect each well in the absence of serum. After 10 -12 h, the medium was replaced by 5% FBS MEM. Approximately 30 -36 h after the beginning of the transfection the cells were digested with 0.033% trypsin and cell suspensions were plated into 75-ml culture flasks and cultured for 24 -28 days with G418 selection (300 g/ml). Stable transfectants were identified by using phospho-specific antibodies against phosphorylated JNK. Stable transfected Cl 41 mass 1 and Cl 41 DN JNK1 mass 2 were established and cultured in G418-free MEM for at least two passages before each experiment.
Phosphorylation Analysis for Erk and JNK-Immunoblot analysis for phosphorylated proteins of Erk and JNK was carried out using phospho-specific MAPK antibodies against phosphorylated sites of Erk and JNK as described previously (22,23). Antibodies were from New England Biolabs and used according to the manufacturer's recommendations. PKC␣ was used as an internal control for sample protein loaded. Antibody bound proteins were detected by chemiluminescence (ECL, New England Biolabs).
JNK Activity Assay-JNK assay was carried out as described previ-* This work was supported by National Institutes of Health Grant CA74916 and The Hormel Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  ously (22,24). Briefly, JB6 C141 cells were starved for 48 h in 0.1% FBS MEM in 37°C, 5% CO 2 atmosphere incubator. The cells were washed once with ice-cold phosphate-buffered saline and exposed to UVC (60 J/m 2 ) or arsenite at the concentration and times indicated in the figure legends. Then, the cells were washed once with ice-cold phosphatebuffered saline and lysed in 300 l of lysis buffer per sample (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1 mg/ml leupeptin). The lysates were sonicated and centrifuged, and the supernatant was incubated with 2 g of N-terminal c-Jun (1-89) fusion protein bound to glutathione-Sepharose beads overnight at 4°C. The beads were washed twice with 500 l of lysis buffer with phenylmethylsulfonyl fluoride and twice with 500 l of kinase buffer (25 mM Tris, pH 7.5, 5 mM ␤-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ). The kinase reactions were carried out in the presence of 100 M ATP at 30°C for 30 min. c-Jun phosphorylation was selectively measured by Western immunoblotting using a chemiluminescent detection system and specific c-Jun antibodies against phosphorylation of c-Jun at serine 63.
Erk Activity Assay-Erk activity was carried out as described using the protocol of New England Biolabs. In brief, JB6 Cl 41 transfectants were starved for 48 h in 0.1% FBS MEM at 37°C, 5% CO 2 atmosphere incubator. The cells were exposed to arsenite for the doses and times indicated. The cells were washed once with ice-cold phosphate-buffered saline and lysed in 300 l of lysis buffer per sample (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerolphosphate, 1 mM Na 3 VO 4 , 1 mg/ml leupeptin). The lysates were sonicated and centrifuged, and the super-natant was incubated with phospho-specific p44/42 MAP kinase (Thr 202 /Tyr 204 ) monoclonal antibody for 4 h at 4°C; then, incubated with protein A-Sepharose beads overnight at 4°C. The beads were washed twice with 500 l of lysis buffer with phenylmethylsulfonyl fluoride and twice with 500 l of kinase buffer (25 mM Tris, pH 7.5, 5 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ). For determination of Erk-induced phosphorylation of Elk-1 measured by quantitative immunoblotting with phospho-Elk-1 antibody, the kinase reactions were carried out in the presence of 2 g of Elk-1 fusion protein and 100 M ATP at 30°C for 30 min. Elk-1 phosphorylation is selectively measured by Western immunoblotting using a chemiluminescent detection system and specific antibodies against phosphorylation of Elk-1 at serine 383. For measurement of Erk-induced phosphorylation of Elk-1 by direct assesses of phosphate incorporation from [␥-32 P]ATP, the kinase reactions were carried out in the presence of 2 g of Elk-1 fusion protein and 100 M cold ATP plus 15 Ci of [␥-32 P]ATP at 30°C for 30 min. The results were presented as SDS-polyacrylamide gel electrophoresis autoradiography and the phospho-Elk-1 bands were cut off and counted.
Anchorage-independent Transformation Assay-JB6 Cl 41 cells or their transfectants (1 ϫ 10 4 cells) were exposed to arsenite and TPA in 1 ml of 0.33% BME agar containing 15% FBS over 3.5 ml of 0.5% BME agar containing 15% FBS in each well of 60-well plate. The cultures were maintained in 37°C, 5% CO 2 incubator for 4 weeks, then another 3 ml of 0.33% BME agar containing 15% FBS was added to each well and the cultures continued in the 5% CO 2 incubator for another 4 weeks. The TPA-and arsenite-induced cell colonies were scored at the end of the second and eighth week after cells were exposed to TPA or arsenite, respectively.
Assay for Cell Proliferation-Cell proliferation was determined by FIG. 1. Cell transformation induced by low concentrations of arsenite. C1 41 cells (10 4 cells) were or were not exposed to TPA (10 ng/ml) or different concentrations of arsenite in 1 ml of 0.33% BME agar containing 15% FBS over 3.5 ml of 0.5% BME agar containing 15% FBS in each well of 6-well plates. The cultures were maintained in 37°C, 5% CO 2 incubator for 4 weeks. Then, another 3 ml of 0.33% BME agar containing 15% FBS was added to each well and the cultures continued in the 5% CO 2 incubated for another 4 weeks. The TPA-and arseniteinduced cell colonies were scored at the end of the second and eighth week after cells were exposed to TPA or arsenite, respectively.

FIG. 2. Time course of activation of Erk and JNK by arsenite.
A, for assay of phosphorylated Erk and JNK, 8 ϫ 10 4 of JB6 Cl 41 cells were seeded into each well of the 6-well plates. After culturing at 37°C for 24 h, the cells were starved for 48 h by replacing medium with 0.1% FBS MEM. Four h before cells were exposed to TPA or arsenite, the medium was changed to serum-free MEM. Then, the cells were or were not exposed to TPA (10 ng/ml) or arsenite (200 M). The cells were extracted at different time points and phosphorylated proteins of Erk and JNK as well as internal control protein kinase C (PKC) ␣ were determined as described previously (22,23). B, assays of JNK activity. JB6 C1 41 cells were cultured in monolayers in 100-mm diameter dishes to 90% confluency. The cells were starved by changing the medium with 0.1% FBS MEM medium for 48 h. The cells were or were not exposed to 200 M arsenite for the times indicated. The cells were harvested and JNK activity was measured as described previously (24).

Induction of Cell Transformation by Low Concentration of
Arsenite-Arsenite is a known carcinogen (1)(2)(3)(4). Some previous studies have suggested that arsenite acts as a tumor promoter rather than an initiator (25). However, there is no convenient anchorage-independent cell transformation model for studying the molecular mechanism of the tumor promotion effect of arsenite. The mouse epidermal JB6 cell system is a model to study tumor promotion in vitro. To study whether arsenite induces JB6 cell transformation, we exposed JB6 Cl 41 cells to arsenite in soft agar. Anchorage-independent colonies were observed in the eighth week after arsenite exposure. However, the transformation rate was lower and the colonies were smaller than those induced by TPA, which were observed after 2 weeks of exposure (Fig. 1). Also, cell transformation can only be observed in cells exposed to low concentration (25 M) of arsenite, while no cell transformation colonies were observed at high concentrations of arsenite (50 -100 M) (Fig. 1A).
Differential Activation of Erk and JNK by Arsenite-Previously, we and others have reported that signal transduction pathways leading to AP-1 activation are required for cell transformation induced by tumor promoters, such as TPA and EGF to occur (23,26,27). It was also reported that arsenite is a potent stimulator of AP-1 activity and JNK activity (10). To study the molecular basis for neoplastic transformation activity of arsenite, we examined its effects on MAP kinase signal transduction pathways in our system. We found that arsenite could induce activation of both JNK and Erk. However, the activation of JNK and Erk by arsenite is different. During the time course and dose-response studies, marked Erk activation could be observed at 15 min after exposure and at all dosages studied (Figs. 2 and 3). There was no significant induction of Erk by arsenite after a 30-min exposure (Fig. 2). In contrast, activation of JNK was only observed at high dosage (Ͼ50 M) Four h before cells were exposed to TPA or arsenite, the medium was changed to serumfree MEM. Then, the cells were or were not exposed to TPA (10 ng/ml) or UVC (60 J/m 2 ) or different concentrations of arsenite as indicated. The cells were extracted at the time points as indicated. The phosphorylated proteins of ERK (A) and JNK (B) as well as internal control protein kinase C (PKC) ␣ were determined as described in the phosphospecific antibody kit (New England Biolabs). C, assay of JNK activity. JB6 C1 41 cells were cultured in monolayers in 100-mm diameter dishes to 90% confluency. The cells were starved by changing the medium with 0.1% FBS MEM medium for 48 h. Then, the cells were or were not exposed to UVC (60 J/m 2 ), TPA (10 ng/ml) for 30 min or different concentrations of arsenite for 120 min. The cells were harvested and JNK activity was measured as described previously (24).

FIG. 4.
Comparison study of Erk activation by two different methods. JB6 Cl 41 cells were seeded into 100-mm dishes (A) or 6-well plates (B) and cultured in 37°C, 5% CO 2 incubator until 80 -90% confluent. The cells were starved for 48 h replacing medium with 0.1% FBS MEM. Four h before cells were exposed to arsenite, the medium was changed to serum-free medium. Then the cells were treated with different concentrations of arsenite for 15 min. A, the cells were extracted with lysis buffer. A monoclonal phospho-specific antibody against Erk is used to selectively immunoprecipitate active Erk from cell lysates. The immunoprecipitated proteins were then incubated with 2 g of Elk-1 fusion protein in the presence of kinase buffer and cold 100 M ATP plus 15 Ci of [␥-32 P]ATP. The results were presented as SDS-polyacrylamide gel electrophoresis autoradiography and the phospho-Elk-1 bands were cut off and counted; B, the cells were extracted with SDS sample buffer, and Erk as well as phospho-Erk were determined as described in the PhosphoPlus MAPK antibody kit (New England Biolabs). and after 60 min of exposure (Figs. 2 and 3). These results indicated that dosages of arsenite for activation of Erk, but not JNK, are consistent with those for cell transformation. Therefore, Erk activation may be involved in arsenite-induced cell transformation. Interestingly, we also observed phosphorylated Erk-like bands in cells treated with arsenite for 15 min when we used antiphospho-JNK antibody (Fig. 2A). These bands were very consistent with the Erk activation when we used the anti-phosphorylated Erk antibody (Fig. 2A). The reason for this may be due to cross-reaction of antiphospho-JNK antibody with phosphorylation of Erk ( Fig. 2A). To directly measure the Erk activity induced by arsenite, we assessed the Erk activity by measuring phosphate incorporation from [␥-32 P]ATP. The results showed that exposure of cells to arsenite caused markedly an increase of phosphate incorporation from [␥-32 P]ATP to the Erk substrate Elk-1 (Fig. 4A). These increases appear to be in a dose-dependent manner (Fig. 4A). The maximus induction of Erk activity by arsenite is similar to these induced by 10 ng/ml of TPA or EGF (Fig. 4A). We have compared dose-responses between Erk phosphorylation and Erk-induced phosphorylation of Elk-1 by direct measurement of phosphate incorporation from [␥-32 P]ATP. Results from both methods are generally correlated. However, the method by using [␥-32 P]ATP is more sensitive than that by directly measuring Erk phosphorylation (Fig. 4, A and B).
No Inhibition of Arsenite-induced Cell Transformation by Expression of Dominant Negative JNK1-To rule out a role of JNK activation in arsenite-induced cell transformation, we established a dominant negative JNK1 stable transfectant, Cl 41 DN JNK1 mass 2 . The dominant negative JNK1 mutant (APF) is the double point mutation that changes the phosphorylation sites Thr 183 and Tyr 185 to Ala and Phe, respectively (18,19). This mutation blocks JNK activation. The stable transfectant was generated by "mass culture selection" of pooled clones as described previously (23). To determine whether dominant negative JNK1 have blocking effects on JNK activation, we compared the JNK phosphorylation induced by arsenite between dominant negative transfectant Cl 41 DN-JNK1 mass 2 2 were seeded into each well of 6-well plates. After culturing at 37°C for 24 h, the cells were starved for 48 h by replacing medium with 0.1% FBS MEM. Four h before cells were exposed to arsenite, the medium was changed to serum-free MEM. Then, A, for JNK activation, the cells were or were not exposed to arsenite (200 M) for time as indicated; B, for Erk activation, the cells were exposed to different concentrations of arsenite for 15 min. The cells were extracted and phosphorylated JNK and Erk proteins were determined as described in the PhosphoPlus MAPK antibody kit (New England Biolabs).

FIG. 6. Cell transformation induced by arsenite is specifically blocked by introduction of dominant negative
Erk2, but not dominant negative JNK1. C1 41 CMV-neu mass 1 , C1 41 DN-JNK1 mass 2 , or C1 41 MAPK DN B 3 mass 1 were or were not exposed to different concentrations of arsenite in 1 ml of 0.33% BME agar containing 15% FBS over 3.5 ml of 0.5% BME agar containing 15% FBS in each well of 6-well plates. The cultures were maintained in 37°C, 5% CO 2 incubator for 4 weeks. Then, another 3 ml of 0.33% BME agar containing 15% FBS was added to each well and the cultures were continued in 5% CO 2 for another 4 weeks. The arsenite-induced cell colonies were scored at the eighth week after cells were exposed to arsenite. phorylation was impaired by introduction of dominant negative JNK1, while there were no significant effects on arsenite-induced Erk phosphorylation (Fig. 5). Expression of dominant negative JNK1 was shown to increase the cell transformation rate by arsenite (Fig. 6). Taken together with the results regarding the difference between the dose-response curves for JNK activation (Fig. 3, B and C) and the transformation (Fig. 1) at different arsenite concentrations, we ruled out the possible role of arsenite-induced JNK activation in cell transformation.
Inhibition of Erk Activation Blocks Arsenite-induced Cell Transformation-The results described above revealed that Erk activation by arsenite may be involved in its cell transformation. To test this possibility, we used dominant negative Erk2-K52R stable transfectant, Cl 41 MAPK-DN B 3 mass 1 (19). We found that overexpression of dominant negative Erk2 blocks arsenite-induced Erk activation and cell transformation (Figs. 6 and 7A), while there is no marked influence on arsenite-induced phosphorylations of JNK or MEK 1/2 (Fig. 7, B and  C). However, the cell proliferation of the C1 41 MAPK DN B 3 mass 1 cells are not significantly different from those of C1 41 AP-1 mass 1 cells and Cl 41 DN-JNK 1 mass 2 (Table I). This data is consistent with previous findings that the cell transformation is dissociated from mitogenesis in JB6 cells (15,50). These data also indicate that lack of cell transformation of C1 41 MAPK DN B 3 mass 1 cells in response to arsenite is not due to inhibition of cell growth by transfection of dominant negative Erk2. Our results demonstrate that Erk activation, but not JNK activation, is required for arsenite-induced cell transformation. DISCUSSION This study investigated the arsenite-induced signal transduction pathway and its role in arsenite-induced cell transformation. Exposure of JB6 Cl 41 cells to low concentrations (Ͻ25 M) of arsenite lead to cell anchorage-independent growth, while there are no cell transformation colonies at a high concentration (100 M) of arsenite. In contrast, Erk activation could be seen at all dosages studied, whereas JNK activation could only be observed at high doses of arsenite. Furthermore, introduction of dominant negative Erk2-K52R into cells blocks Erk activation as well as cell transformation induced by arsenite, while it does not block JNK activation and MEK 1/2 activation. In addition, overexpression of dominant negative JNK1 increases arsenite-induced cell transformation even though it blocks arsenite-induced JNK activation. These results demonstrate that arsenite induces Erk activation and for the first time provides strong evidence that Erk activation, but not JNK activation, is required for arsenite-induced cell transformation.
Arsenic is the first metal to be identified as a human carcinogen (28). Arsenic can exist in trivalent and pentavalent forms and in organic or inorganic compounds (7,28). Both inorganic and organic forms are absorbed by human and animal skin (7). Autoradiographic animal studies show that following chronic exposure, arsenic accumulates in the skin and hair (7). It is known that long-term arsenic exposure can result in carcinogenesis (5). Many cases of skin cancer have been reported among people exposed to arsenic through medical or occupational exposures to trivalent arsenic compounds (7). Epidemiological studies in areas of high arsenic in drinking water are associated with increased risk of cancer of skin, bladder, kidney, lung, and colon (6). Recently, Zhao et al. (13) reported that chronic exposure of culture cells to low concentrations of arsenic lead to 70% of cells exhibiting morphological changes indicative of transformation at 18 weeks of exposure (13). However, in animal experiments, arsenic exposure shows no reliable evidence of its carcinogenicity (29). One possible explanation for the positive carcinogenicity of arsenic compounds in humans and the negative carcinogenicity in experimental ani-mals is that arsenite may be a tumor promoter, but not an initiator of carcinogenesis (10,25). This explanation is supported by the observation that arsenic acts with other agents to alter or enhance other biological effects, which are potentially involved in progression of carcinogenesis (30 -33). For example, arsenite accumulation in skin increases the sensitivity of skin to ultraviolet (UV) light and sequentially increased its carcinogenic effect (34,35). Smoking may synergistically interact with arsenic (30,31).
The JB6 Cl 41 cell is a post-initiated mouse epidermal cell line and represents an excellent in vitro model for studying FIG. 7. Overexpression of dominant negative Erk2 blocks arsenite-induced activation of Erk, but not JNK or MEK 1/2 . JB6 C1 41 AP-1 mass 1 or C1 41 MAPK-DN B 3 mass 1 cells were seeded into 100-mm dishes (A) or 6-well plates (B and C). After culturing at 37°C for 24 h, the cells were starved for 48 h by replacing medium with 0.1% FBS MEM. Four h before cells were exposed to arsenite, the medium was changed to serum-free MEM. Then, A, the cells were treated with 200 M arsenite for the times as indicted. The cells were extracted and Erk activity was determined as described under "Materials and Methods." B, the cells were treated with 100 M arsenite for times as indicated. The cells were extracted with SDS sample buffer and the phospho-JNK, phospho-MEK 1/2 , JNK, as well as MEK 1/2 , were measured as described in previous reports (22,41).
tumor promotion (15,36). In JB6 Cl 41 cells, different tumor promoters such as TPA, EGF, and tumor necrosis factor-␣ induce the formation of large, tumorigenic anchorage-independent colonies in soft agar at a high frequency (36). In this study, we exposed Cl 41 cells to arsenite and found that anchorage-independent growth of cells could be observed at low doses (Ͻ25 M). However, the transformation activity of arsenite is weaker than the positive control TPA. Arsenite-induced cell transformation required longer exposure times, transformation frequency was lower, and the colonies were smaller than TPA-induced colonies. Our results not only support the hypothesis that arsenite is a tumor promoter rather than an initiator of carcinogenesis, but also provide a useful cell culture model to study the mechanisms of arsenite-induced neoplastic transformation.
Transcription factors, such as AP-1, NFB, and nuclear factor of activated T cells, are major mediators involved in cell proliferation, differentiation, and transformation (7,15,37,38). Cell transformation is a complex process which involves many transcription factors and signaling pathways (38 -47). A growing body of evidence indicates that the activation of signal transduction pathways leads to increased transcription factor activity, such as AP-1, NFB, which seems to be required for tumor promoter-induced cell transformation (23,38,39,(45)(46)(47)(48)(49). It was reported that arsenite could stimulate AP-1 activation and expression of c-jun and c-fos in HeLa cells (10). In JB6 cells, we also found that treatment of cells with arsenite caused activation of AP-1 activity (data not shown). It is known that the signaling leading to AP-1 activation is mediated through three mitogen-activated protein (MAP) kinase pathways, including Erk, JNK, and p38 kinase (10,18,22). We, therefore, investigated the possible role of MAP kinases involved in arsenite-induced cell transformation in JB6 cells. Exposure of Cl 41 cells to arsenite not only activates JNK, which is consistent with results from previous reports (10), but also induces Erk activation. The results from time course studies show that Erk activation only occurred at very early exposure, while JNK activation occurred much later. It should be noted that Erk activation could be seen at all doses studied, whereas JNK activation was only observed at high doses (Ͼ50 M). The dosages for induction of cell transformation are correlated with that for activation of Erk, but not for JNK, revealing that Erk activation may be involved in arsenite-induced cell transformation. This was supported by our findings that dominant negative Erk2 dramatically inhibits arsenite-induced cell transformation (Fig. 5), while dominant negative JNK increased the cell transformation (Fig. 5) even though it blocks JNK activation by arsenite (Fig. 4A). The reason for induction of Erk, but not cell transformation by high doses of arsenite, may be due to induction of apoptosis at these doses. Our current results indicate that arsenite at dosages more than 100 M strongly induces apoptosis of JB6 Cl 41 cells (data not shown). Our results for Erk activation by arsenite are different from data reported by Cavigelli et al. (10) in which arsenite did not show any induction of Erk. The explanation for this may be either that different cell lines were used in these studies or the time point for Erk activation was missed by Cavigelli et al. (10).
During the preparation of this paper, Ludwig et al. (14) reported that arsenite induces Erk activation in the human embryonic kidney cell line HEK292 and this Erk activation appears to be late phase and dependent on activation of p38 kinase. However, in JB6 cells, we found that Erk activation occurs very early, while p38 kinase activation was observed later than 60 min after cell exposure to arsenite. Our recent studies indicated that expression of dominant negative p38 kinase in Cl 41 cells could not block arsenite-induced Erk activation, while it blocks p38 kinase activation (data not shown). Therefore, Erk activation is not dependent on p38 kinase activation in JB6 cells. The reason for this may be due to the difference of cell lines used. Taken together, our results strongly suggest that arsenite induces Erk activation and Erk activation induced by arsenite is required for its cell transformation to occur. The biological role of JNK activation induced by arsenite is under current study. a Cell proliferation was done as described under "Materials and Methods." The results were presented as the average and standard deviation of counts per minute for assays of triplicate wells.