Genotoxic Methylating Agents Modulate Extracellular Signal

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Chem. Res. Toxicol. 2003, 16, 87-94

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Genotoxic Methylating Agents Modulate Extracellular Signal Regulated Kinase Activity through MEK-Dependent, Glutathione-, and DNA Methylation-Independent Mechanisms in Lung Epithelial Cells Anita E. Wichmann,† Nicole M. Thomson,†,‡ Lisa A. Peterson,†,‡ and Elizabeth V. Wattenberg*,† Division of Environmental and Occupational Health and University of Minnesota Cancer Center, School of Public Health, University of Minnesota, Minneapolis, Minnesota, 55455 Received August 17, 2002

Mitogen-activated protein kinases (MAPKs) play a central role in transmitting stress-induced signals stimulated by genotoxic agents. The present study is the first to investigate the mechanisms by which genotoxic alkylating agents modulate MAPKs by directly measuring the effects of methylating agents on MAPK activity, DNA methylation, and intracellular glutathione levels. The effects of acetoxymethylmethylnitrosamine (AMMN), N-nitroso-Nmethylurethane (NMUR), and N-methyl-N-nitrosourea (MNU) on these parameters were compared in a fetal rat lung cell line model (MP48). These compounds were chosen because they methylate DNA via a methanediazonium intermediate and, therefore, should induce similar cellular methylation patterns, although they produce different side products upon decomposition. All three compounds stimulated the activation of the stress-activated MAPKs, c-Jun N-terminal kinase, and p38. In contrast to what has been reported for other methylating agents, these compounds also stimulated the activation of extracellular signal regulated kinase (ERK), a MAPK typically activated by mitogenic agents. O6-methylguanine (O6-mG) is widely considered to be the critical toxic lesion induced by methylating agents, including AMMN, NMUR, and MNU, which form DNA adducts through SN1 reactions. O6-mG does not appear to be a key regulator of MAPK activity by these compounds, however. There is no direct relationship between the levels of O6-mG and the levels of MAPK activation, and formation of O6-mG does not appear to be sufficient to stimulate MAPK activation. The present studies also indicate that depletion of glutathione is not required or sufficient to stimulate MAPK activation by the methylating agents investigated here. The use of a pharmacological inhibitor indicates that these methylating agents activate ERK through a signaling pathway that requires the ERK kinase MEK. Altogether, these data indicate that genotoxic methylating agents activate MAPKs through mechanisms that are likely to involve the alkylation of cellular targets other than DNA.

Introduction Understanding the biochemical and cellular responses to methylating agents is important because this class of genotoxic alkylating agent is prevalent in the environment, as components of tobacco smoke, pesticides, and industrial chemicals, and because these types of alkylating agents are used as cancer chemotherapy agents (1). Research on the mechanisms by which genotoxic compounds contribute to carcinogenesis and toxicity has largely focused on how these agents interact with DNA and cause mutations. Recent studies have shown, however, that methylating agents can also stimulate signal transduction pathways, which can affect cell fate and function (2-7). Therefore, modulation of signaling path* To whom correspondence should be addressed at Division of Environmental and Occupational Health. Phone: (612) 626-0184. Fax: (612) 626-0650. E-mail: [email protected]. † Division of Environmental and Occupational Health. ‡ University of Minnesota Cancer Center.

ways is likely to emerge as another important component of the cellular response to methylating agents. Mitogen-activated protein kinases (MAPKs)1 represent an important class of signaling molecules that are modulated by various types of genotoxic stress. This family of serine/threonine kinases plays a central role in transmitting stress-induced signals to specific cellular targets (8-10). For example, once activated, MAPKs translocate to the nucleus, phosphorylate specific transcription factors, including Elk-1 and c-Jun, and thereby modulate gene expression. Several studies have also implicated MAPKs in the maintenance of cell survival and the stimulation of apoptosis, depending on the signal and cell type (5, 6, 11-19). c-Jun N-terminal kinase 1 Abbreviations: AMMN, acetoxymethylmethylnitrosamine; ERK, extracellular signal regulated kinase; GSH, glutathione; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, ERK kinase; MMS, methyl methane-sulfonate; MNNG, N-methyl-Nnitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; NMUR, N-nitroso-N-methylurethane; O6-mG, O6-methylguanine.

10.1021/tx0256026 CCC: $25.00 © 2003 American Chemical Society Published on Web 01/01/2003

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(JNK), p38, and extracellular signal regulated kinase (ERK) are three major members of the MAPK family. Agents that induce various types of cellular stress, including heat shock and UV light, predominantly stimulate JNK and p38 activation. Agents that stimulate cell proliferation, such as growth factors, typically stimulate ERK activation. It has been reported that in cell culture systems, the methylating agents methyl methane-sulfonate (MMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) stimulate the activation of JNK and p38, but not ERK (2-7, 11). The mechanisms by which methylating agents modulate signal transduction pathways are not clear. In particular, the role of DNA alkylation in the modulation of signaling pathways stimulated by methylating agents has not been directly explored. For example, it is not known whether methylating agents such as MMS, which form DNA adducts through SN2 reactions, activate MAPKs through the same mechanisms as methylating agents such as MNNG, which form DNA adducts through SN1 reactions. It has been proposed that intracellular levels of glutathione (GSH) play a key role in the regulation of MAPK activity by methylating agents (5). The interpretation of the role of GSH in mediating the activation of MAPKs by methylating agents in these studies is complicated, however, because GSH levels were not directly measured, and therefore direct correlations between GSH levels and MAPK activity could not be established. To investigate the mechanisms by which methylating agents modulate MAPKs, we compared the effects of three different genotoxic methylating agents, acetoxymethylmethylnitrosamine (AMMN), N-nitroso-N-methylurethane (NMUR), and N-methyl-N-nitrosourea (MNU), on JNK, p38, and ERK activity, DNA methylation, and intracellular GSH levels. We chose AMMN, NMUR, and MNU because the chemistry by which these compounds interact with DNA is well characterized (20-23), as are the mechanisms by which specific types of DNA methyl adducts are repaired (24-26). AMMN, NMUR, and MNU all alkylate DNA, and other macromolecules, via a methanediazonium intermediate. Thus, the cellular methylation patterns will be similar for these three compounds. The side products generated during the decomposition of these compounds differ, however. AMMN generates formaldehyde, the decomposition of NMUR releases carbon dioxide, and MNU releases a carbamoylating agent as well as a methylating agent. We hypothesized that if methylation of macromolecules is the key initial signal, then all three compounds should modulate JNK, p38, and ERK in a similar manner. We conducted these studies in an immortalized pre-type II alveolar epithelial cell line derived from fetal rat lung (MP48) (27). MP48 cells have a normal karyotype, and cell proliferation is contact inhibited. We chose this cell line, because in rats, the lung tumors induced by tobacco-specific genotoxic methylating agents appear to originate from type II cells (28). Therefore, MP48 cells are a useful model for studying the mechanisms by which genotoxic methylating agents affect carcinogenesis and toxicity. Our results show that AMMN, NMUR, and MNU activate MAPKs in a similar manner. Surprisingly, and in contrast to what has been reported for MMS and MNNG (5), AMMN, NMUR, and MNU stimulate ERK activation in addition to increasing the activation of JNK and p38. O6-Methylguanine (O6-mG) is widely believed

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to be the critical toxic lesion induced by methylating agents such as AMMN, NMUR, and MNU, which form DNA adducts through SN1 reactions (1, 25). Our data indicate that there is no direct relationship between the levels of O6-mG formation and the levels of MAPK activation. Furthermore, formation of O6-mG does not appear to be sufficient to stimulate MAPK activation. In addition, our results indicate the depletion of GSH is neither required nor sufficient to stimulate MAPK activation in this system. Finally, these studies indicate that all three compounds stimulate ERK activation through a signaling pathway that requires activation of the ERK kinase MEK. Although the side products may play a role in modulating MAPK activity, our results suggest that methylation of intracellular macromolecules other than DNA triggers activation of MAPKs by AMMN, NMUR, and MNU.

Experimental Procedures Caution: AMMN, NMUR, and MNU are mutagenic as well as carcinogenic in laboratory animals. Chemicals and Reagents. Waymouth’s Medium, fetal bovine serum (FBS), protein A agarose beads, Lipofectamine 2000, and horseradish peroxidase-conjugated goat anti-mouse antibody were purchased from Invitrogen Life Technologies (Carlsbad, CA). Glutathione-agarose beads, anisomycin, phorbol 12-myristate 13-acetate (TPA), leupeptin, and aprotinin were purchased from Sigma Chemical Company (St. Louis, MO). AMMN, NMUR, and MNU were purchased from the National Cancer Institute Chemical Repository (Midwest Research Institute, Kansas City, MO). MMS was purchased from Acros Organics/Fisher Scientific (Pittsburgh, PA). PD 98059 was purchased from Calbiochem (La Jolla, CA). Anti-JNK and antiERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ERK and anti-phospho-p38 were purchased from Cell Signaling (Beverly, MA). [γ-32P]ATP was purchased from NEN Life Science Products (Boston, MA) and ICN Biomedicals (Costa Mesa, CA). Hybond ECL nitrocellulose membrane and the ECL detection system were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Immobilon-P PVDF membrane was purchased from Millipore (Bedford, MA). pGEX-2T-GST-c-Jun (1-79 aa) was the gift of Dr. Daniel Mueller (Department of Medicine, University of Minnesota). pGEX-GST-6His-Elk (308-428 aa) was the gift of Dr. Robert A. Hipskind (Institut de Genetique Moleculaire de Montepellier CNRS, Montepellier, France). GST fusion proteins were expressed and isolated as previously described (29). Cell Culture. MP48 cells were the gift of Dr. David Ingbar (Department of Medicine, University of Minnesota). This is an immortalized pre-type II alveolar epithelial cell line derived from fetal rat lung described in ref 27. MP48 cells were grown in a gassed (5% CO2), humidified incubator at 37 °C in Waymouth’s medium supplemented with 10% FBS. Cytotoxicity Assay. Cytotoxicity was determined using the Cell Titer 96 Non-Radioactive Cell Proliferation Assay according to the protocol recommended by Promega (Madison, WI). Briefly, cells were plated at a density of 2 × 104 cells/well in 96-well plates 1 day prior to each experiment. Confluent cells were incubated for 2 h in Waymouth’s/10% FBS in the presence or absence of the appropriate agents. The cells were washed twice with warm (37 °C) phosphate-buffered saline (PBS) and incubated in medium for 24 h. Then, 15 µL of dye solution was added to each well. After a 2-hour incubation, 100 µL of stop solution was added. The plate was incubated either for 1 h at 37 °C or overnight at room temperature to allow for solubilization. Absorbance was read at 570 and 630 nm using a 96-well plate reader. LC50s were calculated using published methods described in ref 30.

Dissociation of MAPK Activity from O6-mG Levels Kinase Assays. Cells were plated at a density of 6 × 105 cells/60-mm plate 1 day prior to each experiment. Confluent cells were incubated in Waymouth’s/10% FBS in the presence or absence of the appropriate agents, washed twice with ice-cold PBS, and harvested in the appropriate lysis buffer. JNK lysis buffer contained 25 mM Hepes, pH 7.5; 0.1% Triton X-100; 300 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA, pH 8.0; 20 mM β-glycerolphosphate; 1 mM Na3VO4; 0.5 mM DTT; 1 mM PMSF; 10 µg/mL aprotinin and 10 µg/mL leupeptin. ERK lysis buffer contained 200 mM Tris-Cl, pH 7.4; 1% Triton X-100; 10% glycerol; 137 mM NaCl; 1.5 mM MgCl2; 1 mM EGTA; 20 mM β-glycerol-phosphate; 1 mM Na3VO4; 50 mM NaF; 1 mM PMSF; 10 µg/mL aprotinin; and 10 µg/mL leupeptin. Immunocomplex assays for JNK and ERK activity were then conducted as previously described (31). The levels of the substrates used in the kinase reactions were monitored by staining the gels with Coomassie Brilliant Blue. Data were only used from gels in which the substrate levels are the same in each reaction. Phosphorylation was detected by autoradiography and quantified using a Bio-Rad Fluor-S MultiImager and Bio-Rad Quantity One software. Western Blot Analysis. Ten to twenty micrograms of protein from kinase assay cell lysates was resolved on a 10% SDS-PAGE mini-gel and transferred to either nitrocellulose or PVDF membranes. Blots were blocked in a Tris-buffered saline solution containing 5% dry nonfat milk and 0.1% Tween-20 for 1 h at room-temperature and incubated with primary antibody overnight at 4 °C. Blots were then washed and incubated with a horseradish peroxidase coupled goat anti-rabbit secondary antibody for 1 h at room temperature. Specific antibody binding was visualized using the ECL detection system. Western blot analysis of total ERK, p38, and MEK levels indicates that AMMN, NMUR, and MNU do not cause an increase in total ERK, p38, or MEK protein levels under the conditions of these studies. Measurement of O6-mG and 7-Methylguanine Levels in MP48 DNA. For each sample, cells were grown until confluent in three 150 mm plates. The cells were incubated in Waymouth’s/10% FBS in the presence or absence of the appropriate agents, washed twice with PBS, trypsinized, resuspended in media, pooled, and counted. Cells were pelleted by centrifugation and then resuspended in PBS at a concentration of 1 × 107 cells/ mL. DNA was isolated using the NucleoBond Nucleic Acid Purification Kit (Clontech, Palo Alto, CA) according to the protocol for Genomic DNA Purification from Blood/Cell Culture with the following modifications: the incubation with proteinase K was conducted at room temperature for 45 min, and the precipitated DNA was spooled onto a hooked pasteur pipet, transferred to a new vial, washed first with 70% ethanol, washed again with 100% ethanol, and dried under nitrogen gas. Levels of 7-mG, O6-mG, and guanine were determined by HPLC analysis with fluorescence detection (Waters 470 scanning fluorescence detector with SAT/IN enhancement; Waters Corp., Milford, MA) and separated on a single Partisil 10 SCX column (Phenomenex, Torrance, CA). Neutral thermal hydrolysates were eluted with 100 mM ammonium phosphate buffer, pH 2.0 (1 mL/min). Mild acid hydrolysates were eluted with the same buffer plus 10% methanol (1 mL/min). Standard curves were constructed for each analysis to determine adduct concentration. The amount of 7-mG and O6-mG were related to the guanine concentration of each sample. Measurements of Intracellular GSH Levels. GSH levels were measured using the ApoGSH Glutathione Detection Kit from BioVision Research Products (Mountain View, CA). Cells were plated at (4-5) × 105 cells/well in 6-well plates and grown until confluent the following day. Confluent cells were treated in Waymouth’s/10% FBS in the presence or absence of the appropriate agents. BioVision’s protocol was followed with the following modification: cells were washed twice with ice-cold PBS and lysed directly in the plate in 100 µL of BioVision’s lysis buffer.

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Figure 1. Concentration-response curves for AMMN, NMUR, and MNU-induced cytotoxicity. MP48 cells were incubated for 2 h with various concentrations of AMMN (closed squares), NMUR (closed circles), or MNU (open circles). The cells were washed and then incubated for 24 h in complete media. Cell viability was determined as described in the Experimental Procedures. 100% represents cell viability in cells incubated as described above, but without addition of any methylating agent. Each point represents the mean ( standard error of at least three independent experiments. For individual experiments cell viability was determined in triplicate for each concentration. The LC50s were calculated by the method described in ref 30.

Results and Discussion To investigate the activation of MAPKs by AMMN, NMUR, and MNU, we compared their effects on JNK, p38, and ERK activity at the concentration at which each methylating agent induces a common cellular response, a 50% decrease in cell viability (the LC50). To determine the LC50 for each compound, the cells were incubated for 2 h with various concentrations of AMMN, NMUR, or MNU, washed and cell viability determined 24 h later, as described in the Experimental Procedures. The LC50s for the compounds differed by over 2 orders of magnitude (Figure 1). The LC50 for AMMN was 0.02 mM, the LC50 for NMUR was 0.3 mM, and the LC50 for MNU was 8 mM. The large differences between the LC50s could be due to differences in the delivery of the reactive methylating agent to cellular targets. For example, MNU spontaneously decomposes in aqueous solutions (32). Therefore, delivery of methanediazonium ions to cellular targets will depend, in part, on how rapidly MNU decomposes in the extracellular media. The release of methanediazonium ions from AMMN and NMUR requires ester hydrolysis, which is catalyzed by intracellular esterases (33). The delivery of methanediazonium ions produced from AMMN and NMUR will depend, in part, on the levels of esterases available in the cell. We measured 7-methylguanine (7-mG) as a dosimeter to reflect the delivery of methanediazonium ions to DNA (Figure 2A). We also measured O6-mG because this adduct is widely believed to be the critical toxic lesion induced by methylating agents (Figure 2B) (1, 25). The cells were incubated at the LC50s for each compound and 7-mG and O6-mG measured at various times over a 2 h time course (Figure 2). The kinetics of 7-mG and O6-mG formation were similar, suggesting that DNA repair is saturated under the conditions of these experiments. Figure 2 shows that DNA methylation levels are not directly proportional to the concentration of the parent compound in the media. DNA adduct levels did not

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Figure 2. 7-mG and O6-mG levels in MP48 cells incubated with AMMN, NMUR, or MNU. MP48 cells were incubated for 15, 30, 60, or 120 min with 0.02 mM AMMN (closed squares), 0.3 mM NMUR (closed circles), or 8 mM MNU (open circles). (A) 7-mG and (B) O6-mG levels were measured as described in the Experimental Procedures. The data points shown are the mean ( standard deviation of either two or three replicates.

increase after 60 min of exposure to AMMN. This suggests that under the conditions of these studies, hydrolysis of AMMN, and therefore the production of methanediazonium ions, is complete by 60 min. NMURinduced DNA adduct levels continued to rise through 120 min of exposure, which indicates that hydrolysis of NMUR is not complete over the time course shown. By 120 min, the levels of 7-mG and O6-mG in cells exposed to NMUR were at least 2-fold higher than in cells exposed to AMMN. The DNA adduct levels were higher in cells exposed to MNU than in cells exposed to either AMMN or NMUR at each of the time points shown. The levels of 7-mG and O6-mG did not increase after 60 min of exposure to MNU, which indicates that by 60 min the production of methanediazonium ions by the decomposition of MNU is complete. 7-mG and O6-mG levels in cells exposed to MNU were approximately 3-fold higher than DNA adduct levels in NMUR -treated cells at 15, 30, and 60 min. By 120 min, the levels of O6-mG were approximately 2-fold higher than the levels in cells exposed to NMUR and over 4-fold higher than in cells exposed to AMMN. Altogether, these data demonstrate that the concentration of the compounds in the media does not accurately reflect intracellular methylation levels, and that the delivery of methanediazonium ions to the nucleus differs significantly depending on the compound. The observation that DNA methylation levels differ at the LC50s for AMMN, NMUR, and MNU also suggests

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that under the conditions of these studies, DNA methylation is not the sole determinant of cell death. It is important to note that these studies were conducted on cells that were undergoing minimal cell division. Under these conditions, O6-mG is probably not the critical toxic lesion. The cytotoxicity induced by O6-mG is widely believed to be dependent on futile mismatch repair (25). It has also been suggested that MNU-induced cytotoxicity in dividing cells is due, at least in part, to the depletion of NAD and ATP caused by excessive DNA damageinduced poly(ADP-ribosyl)ation (34). Accordingly, 3-aminobenzamide, which inhibits poly(ADP-ribose)polymerase, blocked MNU-induced cell death in replicating fibroblasts (34). Our preliminary results indicate that 3-aminobenzamide does not prevent MNU-induced cytotoxicity in our system (data not shown), suggesting that the cells are not dying due to depletion of ATP by poly(ADP-ribosyl)ation. Another minor methyl adduct, 3-methyladenine, is also involved in the toxic effects of methylating agents (35, 36). While we did not measure this adduct, the levels of 3-methyladenine will be proportional to the levels of 7-mG and O6-mG (20). Therefore, because there was no correlation between toxicity and levels of DNA methylation, it is unlikely that 3-methyladenine is responsible for toxicity under these conditions. Interestingly, cells treated with MNU tolerate a higher level of methylation than cells treated with AMMN or NMUR (Figure 1 and Figure 2). One possible explanation for this observation is that the side products produced upon decomposition of AMMN, NMUR, and MNU modulate cell death pathways. Preliminary results suggest that cell death in these studies occurs through apoptosis (data not shown). Studies by others showed that the alkylating/carbamoylating agent 1,3-bis(2-chloroethyl)1-nitrosourea inhibits apoptosis through carbamoylation and inhibition of caspases (37). A similar mechanism may explain the ability of MNU-treated cells to tolerate higher levels of methylation. It is also possible that the formaldehyde produced by the decomposition of AMMN contributes to the toxicity of this compound. The mechanisms by which methylating agents induce cell death in nondividing cells, and the contribution of side products to cytotoxicity, are important questions that require further investigation. Because modulation of MAPK activity is a central response to various types of cellular stress, we determined whether AMMN, NMUR, and MNU activate MAPKs (38). When cells were incubated at the LC50s of the methylating agents, only NMUR and MNU stimulated the activation of MAPKs (Figure 3). NMUR and MNU increased JNK and p38 activity to similar levels (Figure 3, panels A and B). Surprisingly, NMUR and MNU also stimulated the activation of ERK (Figure 3D). AMMN did not induce a detectable increase in JNK, p38, or ERK activity even when cells were incubated at the LC50 for AMMN for up to 4 h (Figure 3, panels A, B, and C, and data not shown). Concentrations of AMMN above the LC50 did increase JNK, p38, and ERK activation, however (Figure 4). Altogether, these data indicate that at the LC50 determined in these studies NMUR and MNU, but not AMMN, stimulate signals that activate MAPKs. There is no apparent correlation between the levels of DNA methylation and the levels of MAPK activation. For example, incubation of cells with 0.05 mM AMMN for 30 min produced higher levels of 7-mG and O6-mG (4350

Dissociation of MAPK Activity from O6-mG Levels

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Figure 5. Time courses of AMMN-stimulated JNK and ERK activation, and MNU-stimulated p38 activation. Cells were incubated for the indicated times with 0.05 mM AMMN and assayed for (A) JNK activation or (B) ERK activation as described in the legend for Figure 3. (C) Cells were incubated for the indicated times with 8 mM MNU and assayed for p38 phosphorylation as described in the legend for Figure 3. The data are representative of at least two independent experiments. Figure 3. Effects of AMMN, NMUR, and MNU on JNK, p38, and ERK activity when cells are incubated at the LC50. Cells were incubated for 60 min in the absence (control) or presence of 0.02 mM AMMN, 0.3 mM NMUR, or 8 mM MNU, and then assayed for (A) JNK activation or (B) p38 activation as described in the Experimental Procedures. JNK activity was monitored using an immunocomplex assay. Phosphorylation of GST-c-Jun indicates JNK activation. p38 activity was monitored using Western blot analysis and an antibody that detects the phosphorylated, active form of p38 (phospho-p38). Cells were incubated for the indicated times in the absence or presence of (C) 162 nM TPA or 0.02 mM AMMN, or (D) 0.3 mM NMUR or 8 mM MNU, and then assayed for ERK activation as described in the Experimental Procedures. ERK activity was monitored using Western blot analysis and an antibody that detects the phosphorylated, active forms of ERK1 and ERK2 (pERK). Quantification of GST-c-Jun phosphorylation and p38 phosphorylation was conducted using a Bio-Rad Fluor-S MultiImager. The data are representative of at least two independent experiments.

Figure 4. Concentration-response for AMMN-stimulated activation of JNK, p38 and ERK. Cells were incubated for 1 h with the indicated concentrations of AMMN and then assayed for (A) JNK, (B) p38, and (C) ERK activation, as described in the legend to Figure 3. The data are representative of at least two independent experiments.

( 60 and 410 ( 19 pmol/µmol of guanine, respectively) than incubation of cells for 60 min with 0.3 mM NMUR (3300 ( 78 and 317 ( 35 pmol/µmol of guanine, respectively). Yet 0.05 mM AMMN does not stimulate detectable JNK or ERK activation by 30 min (Figure 5, panels A and B), whereas 0.3 mM NMUR stimulates robust JNK and ERK activation by 60 min (Figure 3). In addition,

Figure 6. Effect of GSH depletion on MAPK activation. Cells were incubated for 24 h in the absence (C) or presence of 1 mM DL-buthionine-S,R-sulfoximine (B), and assayed for JNK, p38, or ERK activation. Cells were incubated with anisomycin (A) as a positive control for JNK and p38 activation or TPA (T) as a positive control for ERK activation. The data are representative of at least two independent experiments.

by 60 min the cells exposed to MNU have substantially higher levels of DNA methylation than cells exposed to NMUR (Figure 2), yet the levels of JNK and ERK activation are similar (Figure 3, panels A and D). Likewise, MNU does not activate p38 by 15 min (Figure 5C), although the levels of DNA methylation at this time point are higher than the levels of DNA methylation observed in cells incubated with NMUR by 60 min (Figure 2), a time point by which NMUR induces robust p38 activation (Figure 3B). Altogether, these data suggest that DNA methylation is not sufficient to trigger MAPK activation and furthermore does not play an obvious role in modulating the magnitude of MAPK activity. Studies from other laboratories concluded that the intracellular levels of GSH play a key role in the activation of JNK and p38 by MMS (5). We used three complementary approaches to investigate whether a decrease in GSH levels mediates the activation of MAPKs by AMMN, NMUR and MNU in MP48 cells. First, we determined if depletion of GSH alone stimulates the activation of MAPKs. DL-Buthionine-S,R-sulfoximine (BSO) depletes GSH by inhibiting γ-glutamylcysteine synthetase (39). Incubation of the cells for 24 h with BSO depleted GSH to approximately 60% of control levels (Figure 8A) but did not stimulate JNK, p38, or ERK activity (Figure 6). Second, we assessed the effect of GSH depletion by BSO on the activation of MAPKs by AMMN, NMUR, and MNU. Figure 7 shows that preincubation

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Figure 7. Effect of GSH depletion on activation of MAPKs by AMMN, NMUR, and MNU. Cells were incubated for 24 h in the absence (-) or presence (+) of 1 mM BSO and then incubated for one more hour in the presence of BSO and 0.02 mM AMMN, 0.3 mM NMUR, 8 mM MNU, or no alkylating agent (C). Cell lysates were assayed for (A) JNK, (B) p38, or (C) ERK activity as described in the legend for Figure 3. The data are representative of at least two independent experiments.

Figure 8. Depletion of GSH is not required or sufficient to activate MAPKs. (A) Cells were incubated for 24 h with 1 mM BSO or incubated for 1 h with 0.02 mM AMMN, 0.05 mM AMMN, 0.3 mM NMUR, 8 mM MNU, or 1 mM MMS, and GSH levels measured as described in the Experimental Procedures. GSH levels are expressed as percent control where 100% is the level of GSH in cells incubated in the absence of BSO or alkylating agents. (B) Cells were incubated for the indicated times with 1 mM MMS and assayed for JNK or p38 activation as described in the legend to Figure 3. ERK activation was monitored using an immunocomplex assay described in the Experimental Procedures. Phosphorylation of the substrate GST-Elk-1 indicates ERK activation. The data are representative of at least two independent experiments.

of the cells with BSO does not increase the activation of JNK, p38, or ERK by the methylating agents. Third, we considered whether any of the compounds deplete GSH in a manner that correlates with their ability to activate MAPKs. Incubating cells for 1 h with 0.05 mM AMMN, conditions that trigger the activation of JNK, p38, and ERK (Figure 4), did not cause a detectable decrease in

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Figure 9. AMMN, NMUR, and MNU stimulate ERK activation through a MEK-dependent pathway. (A) Cells were incubated for the indicated times with either 0.05 mM AMMN, 0.3 mM NMUR, or 8 mM MNU, lysed, and assayed for MEK activation by Western blot analysis and an antibody that binds to the phosphorylated, active form of MEK (pMEK). (B) Cells were incubated for 1 h in the absence (-) or presence (+) of 50 µM PD 98059 (PD) and then incubated for either 2 h with 0.05 mM AMMN, 1 h with 0.3 mM NMUR, or 2 h with 8 mM MNU. Cells were lysed and assayed for ERK activation by using an immunocomplex assay described in the Experimental Procedures. Phosphorylation of the substrate GST-Elk-1 indicates ERK activation. The data are representative of at least two independent experiments.

GSH (Figure 8A). This indicates that GSH depletion is not required for activation of MAPKs by AMMN. After a 1 h incubation, MNU depletes GSH to levels below those depleted by NMUR, yet the two compounds activate JNK, p38, and ERK to a similar extent at this time point (Figure 3). These data indicate that there is no direct correlation between the levels of GSH in the cells and the levels of MAPK activation. Finally, after a 1 h incubation, MMS depletes GSH to levels similar to the levels in MNU-treated cells and below those detected in NMUR-treated cells, yet a 1 h incubation with MMS does not stimulate detectable activation of JNK, p38, or ERK (Figure 8B). Altogether, these data indicate that depletion of GSH is not required or sufficient to trigger the activation of MAPKs by the alkylating agents used in these studies. ERK is activated by the dual phosphorylation of specific tyrosine and threonine residues (40). Dephosphorylation of either the tyrosine residue or the threonine residue is sufficient to inactivate ERK (41). Growth factors, such as epidermal growth factor, activate ERK through the Ras/Raf/MEK/ERK cascade by activating the GTPase Ras, which activates the protein kinase Raf, which phosphorylates and activates the ERK kinase MEK, which phosphorylates and activates ERK (40). Some stress-inducing agents, such as heat shock and arsenite appear to increase MAPK activity through the inhibition of phosphatases (42, 43). Our results indicate that the methylating agents used in these studies activate ERK, at least in part, by stimulating the activation of a protein kinase cascade. All three compounds stimulate the activation of MEK (Figure 9A) with kinetics that correspond to their activation of ERK (Figure 3D and Figure 5B). Preincubation of the cells with PD 98059, a specific inhibitor of MEK (44), blocked the ability of all three compounds to activate ERK (Figure 9B). Altogether, these data indicate that all three methylating agents activate ERK through a mechanism that requires the activation of the upstream ERK kinase MEK. We are currently investigating whether

Dissociation of MAPK Activity from O6-mG Levels

these methylating agents directly activate MEK, or whether they activate MEK through activation of Ras and Raf. The observation that AMMN, NMUR, and MNU stimulate sustained ERK phosphorylation suggests that they may also modulate ERK activity by inhibiting ERK phosphatases. To our knowledge, this is the first demonstration that methylating agents that form DNA adducts through SN1 reactions activate ERK. Several laboratories have demonstrated that MMS and MNNG activate JNK and p38 (2-7). One possible explanation for our results is that AMMN, NMUR, and MNU stimulate signaling pathways that are not stimulated by MMS and MNNG. The observation that MMS stimulates ERK activation in our system (Figure 8B) suggests that this is unlikely to fully explain our results. Another likely possibility is that lung epithelial cells express different modulators of ERK than the cell types used by other investigators. Lung is the target of alkylating agents present in tobacco smoke, which suggests that the MP48 lung epithelial cell line may be a particularly useful model for investigating the mechanisms of action of methylating agents. Genotoxic agents that cause different types of DNA damage have different effects on MAPK activation. UV light activates ERK, JNK, and p38 (5, 7); the cancer chemotherapy drug cisplatin activates JNK and p38 (45), but does not activate ERK (46); and the potent mutagen ethylnitrosourea does not activate ERK or JNK (5). Whether methylation stimulates signals that are not triggered by ethylation is not clear, because levels of cellular alkylation were not measured in these studies. Studies from different laboratories present conflicting results regarding the role of DNA damage in triggering MAPK activation (5, 47). In most cases, DNA damage was not directly measured, therefore, complicating the interpretation of the role of DNA damage in triggering signal transduction pathways. Further studies are required to investigate the role of JNK, p38, and ERK activation in the effects of the methylating agents on cell fate. The observation that AMMN does not stimulate MAPK activity at the LC50 suggests that activation of JNK, p38, or ERK is not required for AMMN-induced cell death at this concentration. Preliminary studies, which used pharmacological inhibitors, indicate that inhibition of either ERK or p38 alone is not sufficient to affect cell death induced by AMMN, NMUR, or MNU. The role of JNK in mediating the effects of the methylating agents on cell fate is currently being explored. The studies presented here are the first to directly assess the association between DNA methylation and MAPK activation. Our studies indicate that DNA methylation does not play a key role in modulating MAPK activity in this system. Our results also suggest that caution should be taken in drawing a link between DNA damage and activation of signaling pathways, in part because the concentration of the genotoxic agent in the media does not necessarily directly reflect the amount of DNA damage induced in the cell (Figure 1 and Figure 2). The observation that all three methylating agents have similar effects on MAPKs indicates that methylation via their common methanediazonium intermediate does play a key role in modulating MAPK activity, however. We are currently investigating how alkylation of macromolecules other than DNA contribute to the modulation of signal transduction pathways.

Chem. Res. Toxicol., Vol. 16, No. 1, 2003 93

Acknowledgment. This work was supported by National Institutes of Health Activities to Promote Research Collaborations Supplements to CA-59887 (L.A.P.) and to CA-72498 (E.V.W.), National Institutes of Health Grants CA-59887 (L.A.P.) and CA-72498 (E.V.W.), and seed grants from the University of Minnesota Academic Health Center (L.A.P. and E.V.W.) and the University of Minnesota School of Public Health and Minnesota Medical Foundation Faculty Research Grant Program (E.V.W.). We thank Teresa Kurtz for contributing preliminary results, Nicholette Zeliadt for technical assistance, and Dr. Laura Mauro for insightful discussions.

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