Activation of JNK, p38 and ERK mitogen-activated protein kinases by chromium(VI) is mediated through oxidative stress but does not affect cytotoxicity
Show-Mei Chuang,
Geou-Yarh Liou and
Jia-Ling Yang
Molecular Carcinogenesis Laboratory, Department of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan, Republic of China
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Abstract
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In this study we have explored the involvement of oxidative stress in Cr(VI)-induced JNK, p38 and ERK signaling pathways and their effects on Cr(VI) cytotoxicity in human non-small cell lung carcinoma CL3 cells. Exposure to K2Cr2O7 markedly activated JNK and p38 and moderately activated ERK in a dose- (1080 µM) and time-dependent (112 h) manner. The activated p38 decreased markedly and rapidly and the activated JNK decreased gradually when Cr(VI) was removed from the medium. Post-incubation of Cr(VI)-treated cells with H2O2 increased the activities of JNK and p38, but not ERK. Co-administering Cr(VI) with 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, enhanced p38 activation, but did not influence JNK and ERK activation by Cr(VI). Conversely, co-administering Cr(VI) with mannitol, a hydroxyl radical scavenger and a Cr(V) chelator, reduced p38 activation and increased JNK and ERK activation by Cr(VI). These results indicate that p38 activation by Cr(VI) is positively correlated with oxidative stress, while JNK activity can be enhanced by either a quencher (mannitol) or activator (H2O2) of redox reactions in Cr(VI)-exposed CL3 cells. However, both 3AT and mannitol reduced the cytotoxicity of Cr(VI), but H2O2 did not. The JNK activated by Cr(VI) was decreased (~50%) by expression of a kinase-defective form of MKK7 (MKK7A) but not that of MKK4 (MKK4KR), suggesting that activation of JNK by Cr(VI) is mediated through MKK7. SB202190, a specific inhibitor of p38, markedly decreased JNK but did not change ERK activation by Cr(VI). PD98059, a specific inhibitor of ERK kinases MKK1/2, blocked ERK and p38 but did not alter JNK activation by Cr(VI). Neither the specific kinase inhibitors nor expression of MKK7A altered Cr(VI)-induced cytotoxicity. Together, these results suggest that activation of the JNK, p38 and ERK pathways by Cr(VI) is mediated through diverse redox mechanisms, yet their activation does not correlate with Cr(VI) cytotoxicity.
Abbreviations: 3AT, 3-amino-1,2,4-triazole; DCF, dichlorofluorescein; ERK, extracellular signal-regulated kinase; JNK, c-JUN N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; MKP, MAPK phosphotase; PBS, phosphate-buffered saline; ROS, reactive oxygen species.
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Introduction
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Cr(VI) compounds are genotoxic and carcinogenic agents in a variety of experimental systems (13). Industrial exposure to Cr(VI) compounds has also been strongly associated with increased incidence of human lung cancer (13). However, Cr(VI) does not interact directly with macromolecules such as DNA, RNA and proteins (13). The genotoxic and carcinogenic effects of Cr(VI) are associated with its rapidly entering cells and being activated through intracellular reduction pathways (4,5). The cellular components involved in reducing Cr(VI) include ascorbate, glutathione, hydrogen peroxide, cysteine, DT-diaphorase, cytochrome P450 reductases and the mitochondrial electron transport chain (413). Upon metabolic activation of Cr(VI), several reactive species are generated, e.g. Cr(V), Cr(IV), Cr(III), reactive oxygen species (ROS) and other free radicals (47,9,14). Consequently, Cr(VI) induces DNA lesions (9,1520), chromosomal abnormalities (21,22), gene mutations (23,24), signal transduction (25,26) and cell transformation (27) in cultured mammalian cells.
Three groups of mammalian mitogen-activated protein kinases (MAPKs), i.e. the extracellular signal-regulated kinases (ERKs), the c-JUN N-terminal kinases (JNKs) and p38, are activated in response to stimuli such as DNA-damaging agents, cytokines and growth factors (2831). MAPKs are regulated by separate signal transduction pathways that control many aspects of mammalian cellular physiology, including cell growth, differentiation and cell death (2831). One well-known function of MAPKs is to regulate gene expression through phosphorylation of downstream transcription factors (2831). Activation of MAPKs is mediated by distinct MAPK kinases (MKKs), which involves dual phosphorylation on Thr and Tyr within the Thr-Xaa-Tyr motif located in subdomain VIII of MAPKs (2931). ERK is activated by MKK1 and MKK2, while JNK is activated by MKK4 and MKK7 and p38 by MKK3, MKK6 and MKK4 (31). The activities of MAPKs are also negatively regulated by specific MAPK phosphatases (MKPs) (32) and inhibitors (33,34).
Activation of the JNK and p38 pathways is involved in the stress response, growth arrest and apoptosis (2931), whereas the ERK cascade is a critical pathway for mitogenesis and differentiation (28,35). However, the lifespan of activated MAPKs and cell types expressing them can determine diverse gene expression patterns that result in different physiological consequences. For example, transient JNK and p38 induction by tumor necrosis factor
is a survival signal, while persistent activation induces apoptosis (36,37). Transient ERK activation leads to proliferation, while persistent activation mediates growth arrest or differentiation signaling (3840).
Cr(VI) has been shown to activate ERK in rat H4 hepatoma cells (26). Activation of JNK, p38 and ERK by a very high dose of Cr(VI) (500 µM) has also been reported in human bronchial epithelial cells (41). However, the role of MAPKs in Cr(VI) cytotoxicity remains unclear. Intracellular redox reactions are known to participate in Cr(VI) genotoxicity and carcinogenicity (35). Oxidative stress can also induce MAPK signaling pathways (4244). In this study we therefore investigated the ability of sub-lethal doses of Cr(VI) to activate JNK, p38 and ERK in a human lung adenocarcinoma cell line, CL3, to gain insight into the role of MAPKs in Cr(VI) cytotoxicity. We also explored the effects of H2O2, mannitol [a hydroxyl radical scavenger and a Cr(V) chelator] (45,46) and 3-amino-1,2,4-triazole (3AT; a catalase inhibitor) (47) on Cr(VI)-induced MAPK signaling in order to understand the role of oxidative stress in activation of MAPKs by Cr(VI). The effects of these MAPKs on Cr(VI)-induced cytotoxicity were studied using PD98059, a specific inhibitor of MKK1/2 (the ERK upstream kinases) (48), and SB202190, a specific inhibitor of p38 (49), or by transient transfection assay to block activation of JNK. The results obtained here show that p38 activation by Cr(VI) is positively correlated with oxidative stress, while JNK activity can be enhanced by either a quencher (mannitol) or activator (H2O2) of redox reactions in Cr(VI)-exposed CL3 cells. ERK activated by Cr(VI) was less sensitive than JNK and p38 to the oxidative modulators studied. The activated p38 decreased markedly and rapidly and the activated JNK decreased gradually after Cr(VI) withdrawal, indicating that short-term Cr(VI) exposure transiently activates these kinases. However, activation of these MAPKs did not affect Cr(VI) cytotoxicity.
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Materials and methods
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Cell culture
The CL3 cell line established from a non-small cell lung carcinoma of a 60-year-old male patient in Taiwan (50) was provided by Dr Pan-Chyr Yang (Department of Internal Medicine and Clinical Pathology, National Taiwan University Hospital, Taipei, Taiwan). Cells were cultured in RPMI 1640 medium (Gibco Life Technologies, Grand Island, NY) supplemented with sodium bicarbonate (2.2% w/v), L-glutamine (0.03% w/v), penicillin (100 U/ml), streptomycin (100 µg/ml) and fetal calf serum (10%). Cells were maintained at 37°C in a humidified incubator containing 5% CO2 in air.
Transfection
The expression vector pSR
3-JNKK(K
R) containing a dominant negative form of MKK4 (MKK4KR) was kindly provided by Dr M.Karin (University of California, San Diego, CA) (51). The plasmid containing a dominant negative form of MKK7 (MKK7A) was a gift from Dr J.Han (Scripps Research Institute, La Jolla, CA) (52). Cells (4x105) were plated in a 60 mm Petri dish 1 day before transfection. Plasmids were transfected into CL3 cells by calcium phosphate co-precipitation (53). After incubation for 6 h, the cells were washed with phosphate-buffered saline (PBS), kept in culture for 2 days and then subjected to Cr(VI) treatment.
Treatment
Cells (1x106) in exponential growth were plated 1 day before K2Cr2O7 treatment. Potassium dichromate (Merck, Darmstadt, Germany) was freshly dissolved in MilliQ-purified water (Millipore, Bedford, MA). Cells were treated with K2Cr2O7 for 3 h. In experiments to determine the effect of redox reactions on activation of MAPKs and the cytotoxicity induced by Cr(VI), cells were treated with 80 mM 3AT (Sigma, St Louis, MO) or 80 mM mannitol (Merck) for 2 h and then co-exposed to K2Cr2O7 for 3 h. For H2O2 and Cr(VI) co-treatment, the cells were treated with K2Cr2O7 for 2.5 h before addition of H2O2 for 30 min. To determine the effects of PD98059 (Calbiochem, San Diego, CA) and SB202190 (Calbiochem) on MAPKs and the cytotoxicity induced by Cr(VI), cells were pre-treated with these specific kinase inhibitors for 1 h and then co-exposed to Cr(VI) for 3 h. At the end of treatment the drug-containing medium was removed and the cells were washed twice with PBS.
Cytotoxicity assay
Immediately after treatment cells were trypsinized, diluted and plated at a density of 1001000 cells/60 mm Petri dish in triplicate. The cells were cultured for 1014 days and stained with 1% crystal violet solution. The colony numbers were counted for cytotoxicity determination, calculated as the number of colonies in the treated cells divided by those colonies obtained in the untreated cells.
Cell growth
After treatment cells were cultured in medium containing 10% serum for 03 days before trypsinization. A portion of the cells was mixed with 0.4% trypan blue (Gibco Life Technologies) for 15 min and the number of unstained cells determined using a hemocytometer.
Whole cell extract preparation
Cells were lysed in a buffer containing 20 mM HEPES, pH 7.6, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF, 0.5 µg/ml leupeptin, 1 µg/ml aprotinin and 100 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride. The cell lysate was rotated at 4°C for 30 min, centrifuged at 10 000 r.p.m. for 10 min and the precipitates discarded. Protein concentrations were determined with the BCA protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as the standard.
Kinase activity assay
The JNK activity assay was performed using GSTcJun(179) as substrate according to the procedure described by Hibi et al. (54), with modifications. Briefly, 100 µg of proteins prepared from whole cell extract were mixed with 5 µg of GSTcJun(179) and 20 µl of glutathioneSepharose 4B suspension (Amersham Pharmacia Biotech, Arlington Heights, IL) at 4°C for 3 h and then centrifuged. After three washes in HEPES binding buffer (20 mM HEPES, pH 7.6, 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA and 0.05% Triton X-100) and one wash in kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 2 mM DTT and 0.1 mM Na3VO4), the pelleted beads were resuspended in 30 µl of kinase buffer containing 20 µM ATP and 2 µCi [
-32P]ATP (6000 Ci/mmol; Amersham, Pharmacia Biotech). The reaction was performed at room temperature for 30 min and terminated by washing with HEPES binding buffer. Phosphorylated proteins were eluted with 30 µl of 1.5x Laemmli sample buffer, boiled for 5 min and resolved on a 10% SDSpolyacrylamide gel. The gel was dried and autoradiograghed. The relative radioactivity was quantitated using a computing densitometer equipped with the ImagQuant analysis program (Molecular Dynamics, Sunnyvale, CA).
Western blot analysis
Cellular protein (50 µg) was loaded onto 10% SDSpolyacrylamide gels. The protein bands were then transferred electrophoretically to PVDF membranes (NEN Life Science Products, Boston, MA). Membranes were probed with primary antibody, followed by a horseradish peroxidase-conjugated second antibody (Bio-Rad, Hercules, CA). Phospho-specific antibodies for p38 (no. 9211) and ERK (no. 9101) were purchased from New England Biolabs (Beverly, MA). Anti-JNK1 antibody (G151-666) was purchased from Pharmingen (San Diego, CA). Anti-ERK2 (C-14) and anti-p38 (C-20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody reaction was detected using the enhanced chemiluminescence detection procedure according to the manufacturer's recommendations (Amersham, Pharmacia Biotech).
Fluorescence measurement
After Cr(VI) treatment 1x106 cells were suspended in PBS and incubated with 80 µM 2',7'-dichlorofluorescein diacetate (Estman Kodak) for 30 min in the dark. The cells were then centrifuged and washed once with cold PBS. The reaction of intracellular peroxides (55) and Cr(V) (46) with 2',7'-dichlorofluorescein would result in dichlorofluorescein (DCF), the intensity of which was detected with a fluorescence spectrophotometer using excitation and emission wavelengths of 502 and 523 nm, respectively.
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Results
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Growth rate and cytotoxicity of Cr(VI)-treated CL3 cells
Human lung adenocarcinoma CL3 cells in exponential growth were cultured in serum-free medium for 18 h before they were exposed to 540 µM K2Cr2O7 [equivalent to 1080 µM Cr(VI)] in serum-free medium for 3 h. The cells were then washed with PBS and kept in culture in complete medium for 03 days and the number of viable cells determined by trypan blue exclusion assay. The Cr(VI)-treated cells did not grow on the first day after treatment, but those exposed to 1020 µM Cr(VI) proliferated 23 days after treatment (Figure 1A
). Cells exposed to high Cr(VI) doses (4080 µM) hardly proliferated (Figure 1A
). The cytotoxicity of Cr(VI)-treated cells was also determined by colony-forming ability assay and the relative survival of cells calculated. Approximately 70 and 25% of cells survived after being exposed to 10 and 30 µM Cr(VI), respectively (Figure 1B
).

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Fig. 1. Growth rate and cytotoxicity of CL3 cells treated with Cr(VI). (A) Cells (1x105) were grown in complete medium for 36 h, followed by sustenance in serum-free RMPI medium for 18 h, and then exposed to K2Cr2O7 for 3 h. At the end of treatment the cells were washed with PBS, fed with complete medium and cultured for another 03 days. The numbers of viable cells were determined by trypan blue exclusion assay. (B) Cells (1x106) were plated and cultured overnight, treated with Cr(VI) for 3 h in serum-free medium and subjected to a colony-forming ability assay. The replating efficiency of untreated control cells was 51.3 ± 4.0 (n = 6). Results were obtained from three (A) or between four and eight (B) experiments and the bars in the curves denote SEM.
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Activation of JNK, p38 and ERK by Cr(VI)
CL3 cells were treated with 1080 µM Cr(VI) in serum-free medium for 3 h and a whole cell extract prepared to examine activation of JNK, p38 and ERK. The kinase activity of JNK was determined using [
-32P]ATP and GSTcJUN(179), a specific JNK substrate (54). Activation of p38 or ERK was assayed using phospho-specific antibodies. Figure 2
shows that Cr(VI) markedly induced JNK activity in a dose-dependent manner; 10 and 80 µM Cr(VI) activated to 4- and 69-fold the untreated level, respectively. Low doses of Cr(VI) (1020 µM) did not significantly activate p38, while 3080 µM Cr(VI) could activate p38 to 8- to 23-fold the untreated level (Figure 2
). Activation of ERK was moderately increased by Cr(VI); 1030 µM Cr(VI) induced 2-fold and 4080 µM Cr(VI) induced 4-fold the untreated level (Figure 2
). Western blot analyses showed that the protein levels of ERK, JNK and p38 in Cr(VI)-treated cells were the same as in untreated cells (data not shown).

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Fig. 2. Dose-dependent activation of JNK, p38 and ERK by Cr(VI) in CL3 cells. Cells were cultured in serum-free medium for 18 h and treated with Cr(VI) for 3 h. The cells were washed with PBS twice and the whole cell extracts were isolated for JNK activity assay using GSTcJUN(179) as substrate. The same cell extract obtained from Cr(VI)-treated CL3 cells was examined for the levels of the dual phosphorylated forms of p38 and ERK using phospho-specific antibodies. The relative radioactivity was quantitated using a computing densitometer equipped with the ImagQuant analysis program. Both p42 and p44 phospho-ERK were quantitated. The relative activities of JNK, p38 and ERK averaged from between four and six experiments are shown under the autoradiographs.
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CL3 cells were exposed to 30 µM Cr(VI) in serum-free medium for various times (112 h) to determine the persistence of these activated MAPK signals. Figure 3
shows that Cr(VI) markedly activated JNK and p38 in a time-dependent manner. However, the kinetics of their activation were different, i.e. Cr(VI) activated p38 faster than JNK (Figure 3
). Cr(VI) moderately activated ERK independent of the exposure time (Figure 3
). To further investigate the stability of Cr(VI)-activated JNK and p38, cells were treated with 3050 µM Cr(VI) for 3 h, washed with PBS, cultured for a further 0, 1 or 8 h in complete medium and then assayed for activation of JNK, p38 and ERK. Figure 4
shows that Cr(VI)-activated p38 decreased markedly and rapidly, while Cr(VI)-activated JNK decreased gradually when the metal was removed from the medium. Conversely, the elevated ERK activity in Cr(VI)-treated cells increased during the recovery period, which may be due to the effect of serum (Figure 4
).

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Fig. 3. Time-dependent activation of JNK, p38 and ERK by Cr(VI) in CL3 cells. Cells were treated with 30 µM Cr(VI) in serum-free medium for 112 h. The whole cell extracts were isolated for determination of the activities of JNK, p38 and ERK as described in Figure 2 . Data averaged from three experiments are shown under the autoradiographs.
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Fig. 4. Duration of activation of JNK, p38 and ERK by Cr(VI) in CL3 cells. Cells were treated with 30 or 50 µM Cr(VI) in serum-free medium for 3 h, washed with PBS and cultured continually in RPMI-1640 medium containing 10% serum for various times as indicated. The whole cell extracts were isolated for determination of the activities of JNK, p38 and ERK as described in Figure 2 . Data averaged from three experiments are shown under the autoradiographs.
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Effect of H2O2, mannitol and 3AT on the activation of JNK, p38 and ERK by Cr(VI)
Intracellular redox reactions have been implicated in Cr(VI) genotoxicity (35). To explore the role of oxidative stress in Cr(VI)-induced MAPK signals, we treated CL3 cells with Cr(VI) for 2.5 h before addition of H2O2 for 30 min. Figure 5A
shows that 50500 µM H2O2 (30 min exposure) markedly induced JNK and p38 activities in a dose-dependent manner, and moderately activated ERK. This result indicates that Cr(VI) and H2O2 have similar induction patterns for these MAPKs. Co-administration Cr(VI) with H2O2 increased activation of JNK and p38 but slightly decreased activation of ERK (Figure 5A
). This result suggests that JNK and p38 but not ERK induced by Cr(VI) may be further enhanced by increasing the oxidative stress in cells.

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Fig. 5. Effects of H2O2, mannitol and 3AT on the activation of JNK, p38 and ERK in Cr(VI)-treated cells. (A) CL3 cells were treated with Cr(VI) for 3 h or hydrogen peroxide for 30 min. In the combined treatment, serum-starved CL3 cells were treated with Cr(VI) for 2.5 h before addition of hydrogen peroxide for 30 min. (B) CL3 cells were treated with D-mannitol (80 mM, DM) or 3AT (80 mM) for 2 h and then co-incubated with Cr(VI) for another 3 h. The whole cell extracts were isolated for determination of the activities of JNK, p38 and ERK as described in Figure 2 . Data averaged from between four and six experiments are shown under the autoradiographs.
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To further investigate the role of oxidative stress in induction of MAPKs by Cr(VI), CL3 cells were treated with 80 mM mannitol, a hydroxyl radical scavenger and Cr(V) chelator (45,46), or 80 mM 3AT, a catalase inhibitor (47), for 2 h, followed by incubation with 30 µM Cr(VI) for 3 h in serum-free medium. A whole cell extract was prepared to examine activation of JNK, p38 and ERK. Figure 5B
shows that 3AT co-administration enhanced p38 activation but did not alter JNK and ERK activation by Cr(VI). In contrast, mannitol reduced p38 activation and increased JNK and ERK activation by Cr(VI) (Figure 5B
). 3AT alone could enhance the activity of ERK (Figure 5B
). Western blot analyses showed that the protein levels of ERK, p38 and JNK in 3AT-, mannitol- and/or Cr(VI)-treated cells were the same as in untreated cells (data not shown). These results suggest that p38 and JNK activation by Cr(VI) is mediated differently through oxidative stress in CL3 cells, while ERK is less sensitive.
Effect of H2O2, mannitol and 3AT on the DCF fluorescence level induced by Cr(VI)
The 2',7'-dichlorofluorescein fluorescent probe has been frequently used to measure intracellular peroxides. The intracellular formation of Cr(V) has also been reported to react with this fluorescent probe giving rise to DCF fluorescence (46). The levels of intracellular peroxides and Cr(V) are both considered as indicators of intracellular oxidative stress. Using the 2',7'-dichlorofluorescein fluorescent probe we have measured the intracellular oxidative stress levels of cells exposed to Cr(VI) in the presence or absence of H2O2, mannitol or 3AT. Figure 6
shows that DCF fluorescence levels in Cr(VI)-treated cells were significantly induced in a dose-dependent manner (10 µM, P < 0.05; 3050 µM, P < 0.01). Cells exposed to 3AT (80 mM) alone also had significantly higher levels of DCF fluorescence (P < 0.01, Figure 6
). Mannitol (80 mM) or H2O2 (500 µM) alone slightly reduced or enhanced DCF fluorescence above the background level in cells (Figure 6
). Both 3AT and H2O2 enhanced, while mannitol reduced, DCF fluorescence in Cr(VI)-treated cells, but these effects were insignificant (Figure 6
). The results indicate that Cr(VI) induces intracellular oxidative stress in CL3 cells.

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Fig. 6. Effects of H2O2, mannitol and 3AT on the induction of DCF fluorescence intensities in Cr(VI)-treated cells. CL3 cells were treated with Cr(VI) in the presence or absence of H2O2, D-mannitol (DM) or 3AT as described in Figure 5 . After treatment, cells were washed and trypsinized and 1 000 000 cells were incubated with 80 µM 2',7'-dichlorofluorescein diacetate in PBS for 30 min at 37°C in the dark. The cells were then centrifuged, washed with cold PBS and the intracellular DCF fluorescence intensity measured as described in Materials and methods. Results were obtained from four experiments and the bar represents SEM. *P < 0.05 and **P < 0.01 using Student's t-test for comparison between Cr(VI)- and 3AT-treated cells and the untreated control.
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Effect of H2O2, mannitol and 3AT on the cytotoxicity and growth rate of Cr(VI)-treated cells
Cells were co-exposed to Cr(VI) with either H2O2, mannitol or 3AT. The cytotoxicity and growth rate were determined by colony-forming ability assay and trypan blue exclusion assay, respectively. As shown in Figure 7A
, ~45% of the cells survived when they were exposed to 200 µM H2O2, whereas 80 mM mannitol or 80 mM 3AT did not cause significant cytotoxicity. Co-administering either mannitol or 3AT with Cr(VI) augmented the cytotoxicity induced by 3050 µM Cr(VI), whereas co-administering H2O2 did not alter the Cr(VI)-induced cytotoxicity (Figure 7A
). The number of viable cells determined 3 days after exposure to 30 µM Cr(VI) was 23% of the untreated population (Figure 7B
). Co-administering mannitol (P < 0.05) or 3AT (P < 0.01) further decreased the number of viable cells in the Cr(VI)-treated population (Figure 7B
). H2O2 markedly reduced the number of viable cells (P < 0.001) and did not further decrease the number of viable cells in the presence of Cr(VI) (Figure 7B
).

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Fig. 7. Effects of H2O2, mannitol and 3AT on the cytotoxicity and growth rate of CL3 cells treated with Cr(VI). CL3 cells were treated with Cr(VI) in the presence or absence of H2O2, D-mannitol (DM) or 3AT as described in Figure 5 . (A) Cytotoxicity was determined by colony-forming ability assay. The relative survival of cells was calculated from between three and five independent experiments and the bars represent SEM. (B) Cells (2x105) were grown in complete medium for 18 h and then exposed to Cr(VI) and/or ROS modulators. At the end of treatment the cells were washed with PBS, fed with complete medium and cultured for another 3 days. The numbers of viable cells were determined by trypan blue exclusion assay. a**, P < 0.01 using Student's t-test for comparison between Cr(VI)- and H2O2-treated cells and the untreated cells; b* and b**, P < 0.05 and P < 0.01 using Student's t-test for comparison between Cr(VI)-treated cells and those exposed to the same doses of Cr(VI) in the presence of mannitol or 3AT.
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Effect of suppression of Cr(VI)-activated JNK by a kinase-defective MKK7 on Cr(VI) cytotoxicity
Both MKK4 and MKK7 are reported to be upstream activators of JNK in response to various stimuli (5660). To determine the pathway of JNK activation by Cr(VI), we transfected CL3 cells with a kinase-defective form of MKK4 (MKK4KR) or MKK7 (MKK7A). Cells were transfected with 10 µg of vectors, allowed 2 days for expression and then exposed to Cr(VI) for 3 h in serum-free medium. Figure 8A
shows that the JNK activity stimulated by 30 µM Cr(VI) was 50% suppressed by expression of MKK7A in CL3 cells. Yet expression of MKK4KR did not significantly alter Cr(VI)-induced JNK activity (Figure 8A
). This result is in contrast to the mechanism of JNK activation observed in cells irradiated with UV light (254 nm) (Figure 8A
). The results suggest that JNK activation by Cr(VI) is mediated through MKK7 but not MKK4 in CL3 cells. However, transient expression of MKK7A did not significantly alter the cytotoxicity induced by 30 µM Cr(VI) (Figure 8B
).

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Fig. 8. JNK activation by Cr(VI) in CL3 cells is mediated through MKK7, which does not affect Cr(VI) cytotoxicity. Cells (4x105) were plated in a 60 mm dish the day before transfection. Vectors containing MKK7A or MKK4KR (10 µg each) were co-precipitated with calcium phosphate and then transfected into CL3 cells. After incubation for 6 h, the cells were washed with PBS and kept in culture for 2 days. The cells were then exposed to 30 µM Cr(VI) in serum-free medium for 3 h or irradiated with UV light (40 J/m2, 254 nm) as a positive control. The fluence rate of UV light was measured with a UVX radiometer (UVP Inc., CA). (A) Whole cell extracts were isolated immediately after Cr(VI) treatment or 30 min after UV irradiation. The activity of JNK activity was determined as described in Figure 2 . The numbers shown under the autoradiographs are the relative activities of JNK averaged from between three and five experiments. (B) Cytotoxicity was determined by colony-forming ability assay. The relative survival of cells was calculated from three experiments and the bars represent SEM.
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Effects of PD98059 and SB202190 on Cr(VI) cytotoxicity
PD98059 specifically binds to MKK1/2, ERK upstream kinases, and thereby inhibits ERK phosphorylation and activation, but does not inhibit activation of many other Ser/Thr protein kinases, including JNK and MAPK-activated protein kinase 2, a specific p38 downstream kinase (48). The pyridinylimidazole inhibitors of p38, such as SB202190 and SB203580, were found to have no effect on the activity of many other protein kinases, including other MAPK family members (49,61,62). To examine whether ERK and p38 play a role in Cr(VI) cytotoxicity, CL3 cells were exposed to Cr(VI) in the presence of PD98059 and SB202190. Figure 9
shows that SB202190 (10 µM) significantly decreased JNK activity but did not influence ERK activation by Cr(VI). Because these pyridinylimidazole inhibitors bind to the ATP pocket of p38 and block its intrinsic ATPase activity without affecting its phosphorylation sites, the phospho-specific anti-p38 antibody is not suitable for analysis of activation of p38 pathways (49,61). On the other hand, PD98059 (50 µM) reduced Cr(VI)-activated ERK to endogenous levels and decreased Cr(VI)-activated p38 by ~60%, but did not significantly alter Cr(VI)-activated JNK (Figure 9
). The results indicate that ERK activation by Cr(VI) may be necessary for p38 activation. Nonetheless, co-administering SB202190 or PD98059 did not influence the cytotoxicity of Cr(VI)-treated cells (Figure 10
). The growth rate of Cr(VI)-treated cells was also not affected by these kinase inhibitors (data not shown).

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Fig. 9. Effects of SB202190 and PD98059 on the activities of JNK, ERK and p38 induced by Cr(VI) in CL3 cells. PD98059 (50 µM) or SB202190 (10 µM) was added to cells 1 h before Cr(VI) treatment for 3 h. Whole cell extracts were isolated for determination of the activities of JNK, p38 and ERK as described in Figure 2 . The induced levels of ERK, JNK and p38 activity were quantified from at least three experiments. *P < 0.05 using Student's t-test for the comparison between Cr(VI)-treated cells and those exposed to the same doses of Cr(VI) in the presence of PD98059 or SB202190. ns, not significant.
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Fig. 10. Effect of the SB202190 and PD98059 on the cytotoxicity of Cr(VI)-treated CL3 cells. PD98059 (50 µM) or SB202190 (10 µM) was co-administrated with Cr(VI) as described in Figure 9 . Cytotoxicity was determined by colony-forming ability assay. The relative survival of cells was calculated from four independent experiments and bars represent SEM.
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Discussion
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Cr(VI) genotoxicity and carcinogenicity are highly associated with the generation of reactive species, including Cr(V), Cr(IV), Cr(III) and free radicals, during its intracellular reduction (49). These reactive species may affect signal transduction pathways that could play epigenetic roles in Cr(VI) carcinogenicity. Cr(VI) at a very high dose (500 µM) can activate JNK, p38 and ERK in human bronchial epithelial cells (41). However, the results of such a super-lethal dose cannot be correlated with carcinogenesis. In this study we have demonstrated that Cr(VI) at sub-lethal doses can activate JNK, p38 and ERK in human lung adenocarcinoma CL3 cells. JNK and p38 could be persistently activated provided that Cr(VI) was constantly present in the culture medium. However, activated p38 decreased rapidly and activated JNK reduced gradually after Cr(VI) withdrawal. The duration of activation of JNK and p38 may be correlated with the half-life of the reactive species generated through intracellular reduction of Cr(VI), which may be diminished after Cr(VI) withdrawal. Furthermore, H2O2 activated MAPKs to similar levels as those activated by Cr(VI) and Cr(VI) elevated the amounts of DCF fluorescence in CL3 cells, suggesting that redox reactions may be involved in activation of these MAPKs.
H2O2 enhanced Cr(VI) activation of JNK and p38 but slightly decreased ERK activation, suggesting that JNK and p38 activated by Cr(VI) may be further enhanced by increasing intracellular oxidative stress. 3AT enhanced p38 activation but did not significantly alter the activities of JNK and ERK in Cr(VI)-treated cells. On the other hand, mannitol decreased p38 activation and increased the activities of JNK and ERK in Cr(VI)-treated cells. It has been reported that 3AT blocks catalase activity and increases the levels of intracellular peroxides, whereas mannitol quenches Cr-mediated redox reactions by functioning as a hydroxyl radical scavenger and a Cr(V) chelator (45,46). The results obtained here indicate that p38 activation by Cr(VI) is positively correlated with intracellular oxidative stress, while JNK activity can be enhanced by either an increase or decrease in redox reactions in Cr(VI)-exposed CL3 cells. Alternatively, Cr(V)mannitol may activate JNK and ERK but not p38. In addition, ERK activity was only moderately affected by Cr(VI) and H2O2, suggesting that the ERK pathway is less sensitive to oxidative stress than the JNK and p38 pathways in CL3 cells. However, the levels of ERK activation by stress may also be affected by the background activity of ERK in different cell lines, e.g. Cr(VI) markedly and persistently activates ERK in rat H4 hepatoma cells that contain low basal levels of ERK activity (26), while CL3 cells possess high basal levels of ERK activity.
The generation of Cr(V), Cr(IV), Cr(III) and free radicals during Cr(VI) intracellular reduction can result in attacks on cellular macromolecules and subsequently result in various types of DNA damage, including strand breaks, DNAprotein crosslinks, DNADNA crosslinks, CrDNA adducts and base modifications (9,1520). It is known that DNAprotein or DNADNA crosslinks are poorly repaired in comparison with DNA strand breaks generated in Cr(VI)-treated mammalian cells (16,63). Therefore, DNA crosslinks are much more toxic lesions than DNA strand breaks. The present study has shown that Cr(VI) cytotoxicity is not affected by expression of kinase-defective MKK7A, which can suppress Cr(VI)-induced JNK, or by co-treatment with the p38-specific inhibitor SB202190, which can also suppress Cr(VI)-induced JNK, or PD98059, which decreases both Cr(VI)-activated ERK and p38. The p38 and JNK signals transiently induced by Cr(VI) may not be correlated with DNAprotein or DNADNA crosslinks, which are the major causative agents of cytotoxicity. Rather, the transient induction of these stress-activated MAPKs may be related to DNA strand breaks. Also, H2O2 did not alter Cr(VI) cytotoxicity while 3AT and mannitol enhanced it, suggesting that the numbers of lethal lesions (crosslinks) are increased by 3AT and mannitol but not by H2O2.
Cr(VI) did not induce high levels of apoptosis in CL3 cells (at 50 µM induction was only 2-fold above the background level using the annexin Vfluorescein isothiocyanate binding assay; data not shown), although apoptosis can be markedly induced by cadmium chloride, which is highly associated with persistent activation of JNK and p38 in the same cell (64). JNK and p38 signaling could transmit physiological consequences other than apoptosis in different cell types. For example, JNK signaling is necessary for embryo development (6567) and has been implicated in protection against alkylating agents in 3T3 cells (68). Additionally, JNK1 is selectively activated by the apoptosis inhibitor protein hILP for protection against ICE-induced apoptosis (69). Activation of p38 by interleukin-2 or interleukin-7 is required to transmit the mitogenic signal in T cells (70). The p38 pathway activated by phorbol esters may contribute to a more invasive phenotype in a human squamous cell carcinoma cell line (71). Moreover, isoforms of MAPKs may possess opposing functions, e.g. while p38
induces apoptosis, p38ß inhibits it (72). All the above evidence indicates that activation of JNK or p38 can be a protective mechanism to support cell survival and may contribute to carcinogenesis. The epigenetic role of activation of JNK and p38 by Cr(VI) in Cr(VI) carcinogenesis, although Cr(VI) does not affect cytotoxicity or apoptosis, deserves further study.
Although, the pyridinylimidazole inhibitors of p38 have been reported to have no effect on the activity of other MAPK family members (49,61,62), SB202190 was found to significantly decrease JNK activity induced by Cr(VI). SB202190 and SB203580 also inhibited JNK activity induced by UV light in CL3 cells (G.Y.Liou, unpublished data). Moreover, SB203580 has been shown to inhibit JNK2 activity (73,74). In contrast, specific inhibition of the ERK pathway by PD98059 could reduce Cr(VI) activation of p38 by ~60%, suggesting that cross-talk exists between the ERK and p38 pathways. Cross-talk between ERK and stress-activated p38 and JNK has been reported. For example, ERK activation by arsenite (500 µM) is highly dependent on the MKK6/p38 pathway (75). Inhibition of ERK activation by PD98059 can potentiate JNK1 activation induced by ionizing radiation (76,77). However, cross-talk between MAPKs does not occur in CL3 cells exposed to a carcinogenic cadmium compound (64). These results indicate that the interactions between MAPK signal pathways are variable and may be stimulus- and cell type-dependent. This phenomenon could also be associated with the physiological regulation systems of MAPKs, e.g. activation of the JNK pathway in NIH 3T3 fibroblasts results in MKP-1 gene expression, which can inactivate ERK (78). However, Cr(VI) did not affect the total amount of MKP-1 in CL3 cells (S.M.Chuang, unpublished data), suggesting that MKP-1 is not involved in the regulation of MAPK activities in Cr(VI)-treated cells.
Full activation of MAPKs requires specific upstream kinases (MKKs) in response to different stimuli. The JNK upstream activators thus far identified are MKK4 and MKK7 (29,59,60). We have demonstrated here that the JNK activity stimulated by Cr(VI) is suppressed by expression of a dominant negative form of MKK7. In contrast, expression of a dominant negative form of MKK4 did not alter JNK activation by Cr(VI). The results suggest that JNK activation by Cr(VI) is at least mediated in part through its upstream activator MKK7 and is independent of MKK4 in CL3 cells. As a comparison we have shown that dominant negative MKK4 was more effective than dominant negative MKK7 in blocking JNK activity induced by UV light (Figure 8
). These results are consistent with JNK activation by various stimuli being mediated differentially through MKK4 and MKK7, e.g. tumor necrosis factor
and interleukin-1 activate MKK7 much more than MKK4, whereas, anisomycin and UV light activate MKK4 more than MKK7 (56,58). Nevertheless, kinase-defective MKK7A could not completely block JNK activation by Cr(VI). Putative JNK upstream activators other than MKK7 and MKK4 may be induced by Cr(VI). Such an uncharacterized JNK upstream activator has been observed in cells exposed to hyperosmolarity (58). Additionally, Cr(VI) may affect the scaffold or adapter proteins that specify distinct signal cascades (79).
Negative control mechanisms, such as specific MKPs (32) or MAPK inhibitors (33,34), may be involved in regulating activation of MAPKs. MKP-1, encoded by an immediate early gene, is induced by environmental stress and growth factors to down-regulate JNK, ERK and p38 activities in the nucleus (32). The cytosolic M3/M6 MKP is highly specific for JNK and p38 inactivation (80,81), while association of MKP-3 with ERK in the cytosol markedly stimulates ERK dephosphorylation and inactivation (82,83). Cr(VI) may enhance the activities of MKPs to down-regulate activation of p38, JNK and ERK. Furthermore, thioredoxin and glutathione S-transferase
were recently identified as direct physiological inhibitors of apoptosis signal-regulating kinase 1 (a MAPK kinase kinase) and JNK, respectively (33,34). Both thioredoxin and glutathione S-transferase play important roles in detoxification of agents causing redox damage and the balance of cellular redox levels (84,85). Exposure of cells to Cr(VI) could generate oxidative stress, which may transmit signals that separate these antioxidants from stress-activated signal molecules, thereby promoting activation of JNK and p38. After Cr(VI) withdrawal specific MKPs could dephosphorylate and inactivate JNK and p38, which subsequently associate with the inhibitory antioxidants to terminate stress-activated signal cascades. Further investigation of negative regulation systems affected by Cr(VI) should shed light on the detailed mechanisms of the activation of MAPKs.
In summary, Cr(VI) markedly induces the JNK and p38 pathways and moderately activates the ERK pathway in CL3 cells. Activation of p38 is positively correlated with oxidative stress, while JNK can be enhanced by an increase or decrease in redox reactions and ERK is less sensitive to oxidative stress in Cr(VI)-treated CL3 cells. However, activation of these MAPKs does not significantly alter Cr(VI) cytotoxicity. This may be attributed to the fact that: (i) the short duration of activation of these kinases by Cr(VI) may not affect the levels of lethal lesions, such as DNA crosslinks; (ii) cross-talk between MAPKs may obstruct observation of the effect of individual kinases; (iii) Cr(VI) may activate other signal transduction pathways to counteract the effects of MAPKs. The present study has demonstrated that stress-activated signals are markedly induced by sub-lethal doses of Cr(VI), suggesting an epigenetic role in Cr(VI) carcinogenesis.
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Notes
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1 To whom correspondence should be addressed Email: jlyang{at}life.nthu.edu.tw 
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Acknowledgments
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The authors would like to thank Dr P.-C.Yang for providing the CL3 cells and Drs M.Karin and J.Han for the expression vectors. This work was supported by the National Science Council, Republic of China under contract no. NSC88-2311-B007-028.
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Received November 30, 1999;
revised March 24, 2000;
accepted April 13, 2000.