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Differential Regulation of Redox Responsive Transcription Factors by the Nephrocarcinogen 2,3,5-Tris(glutathion-S-yl)hydroquinone Thomas J. Weber, ² Qihong Huang, Terrence J. Monks, and Serrine S. Lau* ,‡ Molecular Biosciences, Pacific Northwest National Laboratory, Richland, Washington 99352, and Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712-1074 Received August 25, 2000 2,3,5-Tris(glutathion-S-yl)hydroquinone [TGHQ] is a potent nephrotoxicant and nephro- carcinogen, and induces a spectrum of mutations in human and bacterial cells consistent with those attributed to reactive oxygen species (ROS). Studies were conducted to determine whether the oxidative stress induced by TGHQ in renal proximal tubule epithelial cells (LLC-PK 1 ) modulates transcriptional activities widely implicated in transformation responses, namely 12-O-tetradecanoyl phorbol 13-acetate (TPA) responsive element (TRE)- and nuclear factor kappa B (NF-κB)-binding activity. TGHQ increased TRE- and NF-κB-binding activity in a concentration- and time-dependent manner. Catalase fully inhibited peak TGHQ-mediated TRE- and NF-κB-binding activity. In contrast, although deferoxamine fully inhibited TGHQ- mediated TRE-binding activity, it had only a marginal effect on NF-κB-binding activity. Collectively, these data indicate that TGHQ modulates TRE- and NF-κB-binding activity in an ROS-dependent fashion. Cycloheximide and actinomycin D fully inhibited TGHQ-mediated TRE-binding activity, but in the absence of TGHQ increased NF-κB-binding activity. Although protein kinase C (PKC) is widely implicated in stress response signaling, pretreatment of cells with PKC inhibitors (H-89, calphostin C) did not modulate TGHQ-mediated DNA-binding activities. In contrast, pretreatment of cells with (PD098059), a mitogen activated protein kinase kinase (MEK) inhibitor, markedly reduced TGHQ-mediated TRE-binding activity, but enhanced TGHQ-mediated NF-κB-binding activity. We conclude that TGHQ-mediated TRE- and NF- κB-binding activities are ROS-dependent. Although there is a common requirement for hydrogen peroxide (H 2 O 2 ) in the regulation of these DNA-binding activities, there appears to be divergent regulation after H 2 O 2 generation in renal epithelial cells. Introduction The cellular response to oxygen free radicals is largely influenced by the type and concentration of oxygen radical generated. A single exposure to xanthine/xanthine oxidase predominantly increases smooth muscle cell proliferation, whereas frequent exposures to high levels of xanthine/xanthine oxidase result in cell death (1). Cotreatment studies with superoxide dismutase (SOD) 1 and catalase suggest that xanthine/xanthine oxidase- dependent superoxide anion (O 2 •- ) is mitogenic, while hydrogen peroxide (H 2 O 2 ) is cytotoxic (1). In the presence of transition metal ions, and iron in particular, H 2 O 2 is converted to the extremely reactive hydroxyl radical (OH) which is thought to be the primary toxic species responsible for DNA damage and cell death (2-4). Within this context, we have been investigating the nephrotoxic and nephrocarcinogenic actions of glutathione (GSH) conjugates (quinol-thioethers) of hydroquinone [HQ (5)]. Quinol-thioether-mediated cytotoxicity is markedly re- duced by catalase and deferoxamine, scavengers of H 2 O 2 and Fe 3+ /Fe 2+ , respectively, suggesting that the cytotoxic response is mediated by oxygen free radicals and in particular the OH (4, 6). Reactive oxygen species (ROS) are consistently associ- ated with the regulation of the activator protein-1 (AP- 1) and nuclear factor kappa B (NF-κB) transcription factors (7-9). AP-1 and NF-κB are implicated as causal in transformation responses (10, 11) leading a number of investigators to speculate a role for ROS-dependent regulation of these transcription factors in carcinogenesis. AP-1 is a heterodimeric complex composed of c-jun (c- Jun, JunB, and JunD) and c-fos (c-Fos, Fos B, and Fra- 1) protooncogene family members, as either a Jun:Jun homodimer or Jun:Fos heterodimer that specifically binds to the 12-O-tetradecanoyl phorbol-13-acetate (TPA) re- sponsive element [TRE (12)]. NF-κB DNA-binding activ- ity is associated with at least five different NF- κB family members: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB, and c-Rel (13). The most common NF-κB dimers consist of RelA (p65) and NF-κB1 (p50) or NF- κB2 (p52) subunits (14). H 2 O 2 , but not O 2 •- , is thought to regulate NF-κB DNA-binding activity (9). In contrast, * To whom correspondence should be addressed. Phone: (512) 471- 5190. Fax: (512) 471-5002. E-mail: [email protected]. ² Molecular Biosciences. Center for Molecular and Cellular Toxicology. 1 Abbreviations: AP-1, activator protein-1; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal regulated kinase; FBS, fetal bovine serum; H2O2, hydrogen peroxide; OH, hydroxyl radical; HQ, hydroquinone; MAPK, mitogen activated protein kinase; NF-κB, nuclear factor kappa B; PKC, protein kinase C; ROS, reactive oxygen species; O2 •- , superoxide anion; SOD, superoxide dismutase; TPA, 12- O-tetradecanoyl phorbol-13-acetate; TRE, TPA responsive element; TGHQ, 2,3,5-tris(glutathion-S-yl)hydroquinone. 814 Chem. Res. Toxicol. 2001, 14, 814-821 10.1021/tx000190r CCC: $20.00 © 2001 American Chemical Society Published on Web 06/27/2001

Differential Regulation of Redox Responsive Transcription Factors by the Nephrocarcinogen 2,3,5-Tris(glutathion-S-yl)hydroquinone

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Page 1: Differential Regulation of Redox Responsive Transcription Factors by the Nephrocarcinogen 2,3,5-Tris(glutathion-S-yl)hydroquinone

Differential Regulation of Redox ResponsiveTranscription Factors by the Nephrocarcinogen

2,3,5-Tris(glutathion-S-yl)hydroquinone

Thomas J. Weber,† Qihong Huang,‡ Terrence J. Monks,‡ and Serrine S. Lau*,‡

Molecular Biosciences, Pacific Northwest National Laboratory, Richland, Washington 99352, andCenter for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology,

College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712-1074

Received August 25, 2000

2,3,5-Tris(glutathion-S-yl)hydroquinone [TGHQ] is a potent nephrotoxicant and nephro-carcinogen, and induces a spectrum of mutations in human and bacterial cells consistent withthose attributed to reactive oxygen species (ROS). Studies were conducted to determine whetherthe oxidative stress induced by TGHQ in renal proximal tubule epithelial cells (LLC-PK1)modulates transcriptional activities widely implicated in transformation responses, namely12-O-tetradecanoyl phorbol 13-acetate (TPA) responsive element (TRE)- and nuclear factorkappa B (NF-κB)-binding activity. TGHQ increased TRE- and NF-κB-binding activity in aconcentration- and time-dependent manner. Catalase fully inhibited peak TGHQ-mediatedTRE- and NF-κB-binding activity. In contrast, although deferoxamine fully inhibited TGHQ-mediated TRE-binding activity, it had only a marginal effect on NF-κB-binding activity.Collectively, these data indicate that TGHQ modulates TRE- and NF-κB-binding activity inan ROS-dependent fashion. Cycloheximide and actinomycin D fully inhibited TGHQ-mediatedTRE-binding activity, but in the absence of TGHQ increased NF-κB-binding activity. Althoughprotein kinase C (PKC) is widely implicated in stress response signaling, pretreatment of cellswith PKC inhibitors (H-89, calphostin C) did not modulate TGHQ-mediated DNA-bindingactivities. In contrast, pretreatment of cells with (PD098059), a mitogen activated protein kinasekinase (MEK) inhibitor, markedly reduced TGHQ-mediated TRE-binding activity, but enhancedTGHQ-mediated NF-κB-binding activity. We conclude that TGHQ-mediated TRE- and NF-κB-binding activities are ROS-dependent. Although there is a common requirement for hydrogenperoxide (H2O2) in the regulation of these DNA-binding activities, there appears to be divergentregulation after H2O2 generation in renal epithelial cells.

Introduction

The cellular response to oxygen free radicals is largelyinfluenced by the type and concentration of oxygenradical generated. A single exposure to xanthine/xanthineoxidase predominantly increases smooth muscle cellproliferation, whereas frequent exposures to high levelsof xanthine/xanthine oxidase result in cell death (1).Cotreatment studies with superoxide dismutase (SOD)1

and catalase suggest that xanthine/xanthine oxidase-dependent superoxide anion (O2

•-) is mitogenic, whilehydrogen peroxide (H2O2) is cytotoxic (1). In the presenceof transition metal ions, and iron in particular, H2O2 isconverted to the extremely reactive hydroxyl radical(•OH) which is thought to be the primary toxic speciesresponsible for DNA damage and cell death (2-4). Withinthis context, we have been investigating the nephrotoxic

and nephrocarcinogenic actions of glutathione (GSH)conjugates (quinol-thioethers) of hydroquinone [HQ (5)].Quinol-thioether-mediated cytotoxicity is markedly re-duced by catalase and deferoxamine, scavengers of H2O2

and Fe3+/Fe2+, respectively, suggesting that the cytotoxicresponse is mediated by oxygen free radicals and inparticular the •OH (4, 6).

Reactive oxygen species (ROS) are consistently associ-ated with the regulation of the activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) transcriptionfactors (7-9). AP-1 and NF-κB are implicated as causalin transformation responses (10, 11) leading a numberof investigators to speculate a role for ROS-dependentregulation of these transcription factors in carcinogenesis.AP-1 is a heterodimeric complex composed of c-jun (c-Jun, JunB, and JunD) and c-fos (c-Fos, Fos B, and Fra-1) protooncogene family members, as either a Jun:Junhomodimer or Jun:Fos heterodimer that specifically bindsto the 12-O-tetradecanoyl phorbol-13-acetate (TPA) re-sponsive element [TRE (12)]. NF-κB DNA-binding activ-ity is associated with at least five different NF-κB familymembers: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA(p65), RelB, and c-Rel (13). The most common NF-κBdimers consist of RelA (p65) and NF-κB1 (p50) or NF-κB2 (p52) subunits (14). H2O2, but not O2

•-, is thoughtto regulate NF-κB DNA-binding activity (9). In contrast,

* To whom correspondence should be addressed. Phone: (512) 471-5190. Fax: (512) 471-5002. E-mail: [email protected].

† Molecular Biosciences.‡ Center for Molecular and Cellular Toxicology.1 Abbreviations: AP-1, activator protein-1; EMSA, electrophoretic

mobility shift assay; ERK, extracellular signal regulated kinase; FBS,fetal bovine serum; H2O2, hydrogen peroxide; •OH, hydroxyl radical;HQ, hydroquinone; MAPK, mitogen activated protein kinase; NF-κB,nuclear factor kappa B; PKC, protein kinase C; ROS, reactive oxygenspecies; O2

•-, superoxide anion; SOD, superoxide dismutase; TPA, 12-O-tetradecanoyl phorbol-13-acetate; TRE, TPA responsive element;TGHQ, 2,3,5-tris(glutathion-S-yl)hydroquinone.

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the AP-1-dependent induction of gadd153 following H2O2

treatment is inhibited by o-phenanthroline and mannitol,raising the possibility that iron-generated •OH regulatesAP-1-related signal transduction (8). In addition to ROS,a number of growth- and stress-related signal transduc-tion pathways, including the protein kinase C (PKC) andmitogen activated protein kinase (MAPK) pathways,regulate AP-1 and NF-κB activity (15-17). The MAPKcascade is firmly established in the transformationresponse to chemical and polypeptide tumor promoters(11, 18-20), and is typically defined on the basis ofextracellular signal regulated kinase (ERK1/ERK2) ac-tivity (20, 21). PKC represents a group of at least 11different isoforms that transduce signals from a widevariety of stimuli, and are the receptors for tumorpromoting phorbol esters (22).

HQ and 2,3,5-tris(glutathion-S-yl)HQ [TGHQ] arenephrocarcinogens (23, 24). In addition, a single treat-ment of primary renal epithelial cells, isolated from Ekerrats, with TGHQ (100 µM, 4 h) resulted in cell transfor-mation as indicated by the acquisition of anchorage-independent growth (25). The spectrum of mutationsinduced by quinol-thioethers in human and bacterial cellsis consistent with those induced by ROS, indicating thatROS play an important role in HQ and TGHQ-mediatedmutagenicity and carcinogenicity (26). Moreover, HQ-mediated nephrocarcinogenicity may involve a cytotoxicmode of action, and it is well established that HQ-GSHconjugates are potent nephrotoxicants (27). Collectively,these observations raise the possibility that HQ-GSHconjugates mediate the nephrocarcinogenic actions of HQ.

The present studies were conducted to determinewhether TGHQ modulates TRE- and NF-κB-bindingactivity in renal epithelial cells via the generation of anoxidative stress, and to identify signal transductionpathways that regulate these DNA-binding activities.

Materials and Methods

Caution: HQ, TGHQ, and acrylamide are hazardous andshould be handled carefully.

Chemicals. TGHQ was synthesized as previously described(28) and was >99% pure as determined by HPLC. TRE, NF-κB, and AP2 consensus sequences were purchased from Promega(Madison, WI). [γ-32P]-ATP (3000 Ci/mmol) was obtained fromNew England Nuclear (Beverly, MA). Poly d(I-C) was purchasedfrom Boehringer Mannheim (Indianapolis, IN). PD098059 wasfrom Calbiochem (La Jolla, CA). All other chemicals were fromSigma Chemical (St. Louis, MO).

Cell Culture. LLC-PK1 cells were obtained from the Ameri-can Type Culture Collection (CL101) at passage 181. Cells weremaintained in Dulbecco’s Modified Eagle Medium (DMEM; JRHBiosciences; Lenexa, KS) supplemented with 4 g/L D-glucose and10% fetal bovine serum (FBS; Atlanta Biologicals; Norcross, GA)in 5% CO2:95% air at 37 °C. Cells were subcultured bytrypsinization and all experiments were conducted with 5 daypostconfluent cultures at passage levels 187-200.

Neutral Red Assay. Cell viability was determined using aneutral red assay as previously described (4) with minormodification. Specifically, the volume of extraction solvent usedfor the 60 mm dish was 4 mL. All 60 mm dishes were placed ina wire basket and processed simultaneously to ensure compa-rable handling and extraction times.

TGHQ Treatment. TGHQ was solubilized in distilled waterat a concentration of 10 mg/mL immediately before use andadded directly to culture dishes (0.1% FBS DMEM + 20 mMHepes, pH 7.4; media volume of 3 mL/60 mm dish and 0.5 mL/well of 24 well plate). A distilled water control was used in allcases. Concentrations are expressed as nmol TGHQ/cm2 surface

area to present the data in a normalized fashion. For example,in 24 well plates (0.5 mL/well), 300 µM TGHQ treatment isassociated with an approximate 50% reduction of cell viability.Under these conditions in a 24 well plate, the volume of mediais 0.25 mL/cm2. To maintain the same dose of TGHQ/cm2 in a60 mm dish, a volume of 6 mL is required which is beyond thepractical volume limits of a 60 mm dish. The media volume usedin the 60 mm dish was 3 mL (0.13 mL/cm2).

Deferoxamine Pretreatment. LLC-PK1 cells were pre-treated with 10 mM deferoxamine in 0.1% FBS DMEM for 30min. Prior to TGHQ treatment, monolayers were washed threetimes with PBS to remove residual deferoxamine and minimizenonspecific antioxidant effects of preservatives in the chemicalstock.

Electrophoretic Mobility Shift Assay (EMSA). EMSAswere carried out as described previously (15). The nucleic acidsequence for all the response elements used in this study areavailable from the manufacturer’s catalog (Promega, Madison,WI).

Statistics. Individual comparisons were made using thestudents t-test or ANOVA and Student’s Newman-Keul post hoc.The p ) 0.05 level was accepted as significant.

Results

Comparative Dose Response. To correlate molecu-lar events with cellular response, we investigated TGHQ-mediated cytotoxicity in 24 well plates and 60 mm dishesover a range of concentrations. In this comparison, weused routine culture volumes of 0.5 mL/well in 24 wellplates ()0.25 mL/cm2) and 3 mL/60 mm dish ()0.13 mL/cm2). To maintain equal media volume/cm2 would requirea 6 mL volume in the 60 mm dish which is beyond thepractical volume limits of this dish. This is an importantconsideration since interlaboratory media volumes usedfor routine cell culture vary. For example, many labora-tories use 1 mL media/well in 24 well plates, which wouldrequire a 12 mL volume in a 60 mm dish to maintainequal media volume/cm2, a volume that cannot be achievedin standard 60 mm tissue culture dishes. The significanceof this observation is illustrated in our comparative doseresponse. Treatment of LLC-PK1 cells with TGHQ (100-1000 µM) for 2 h resulted in a concentration-dependentdecrease of cell viability in 24 well plates and 60 mmdishes, as measured using a neutral red assay asdescribed in Materials and Methods (Figure 1). However,the dose of TGHQ required an approximate doubling inthe 60 mm dish to achieve a comparable toxic response(Figure 1A). If the same data are expressed as nmolTGHQ/cm2 (i.e., amount of chemical/cell), the large dif-ference in toxicity is no longer apparent, although slightvariability exists (Figure 1B). The volume of media/dish(24 well ) 0.25 mL/cm2; 60 mm ) 0.13 mL/cm2) isdifferent and may contribute to this slight variability.Within this context, interexperimental variation can beup to 20%. Therefore, the toxic response to TGHQ iscomparable in culture dishes of different size if expressedas nanamoles of TGHQ per squared centimeter, a re-sponse that is not apparent when data are expressed asmolarity. An application of this model to the correlationof DNA-binding activity with gene expression will beillustrated in the discussion section.

TGHQ-induces TRE- and NF-κB-binding activity. Ini-tial studies were conducted to determine the temporalregulation of TRE- and NF-κB-binding activity by TGHQ(62 nmol/cm2) and H2O2. Therefore, we also determinedthe dose-response for H2O2-mediated cytotoxicity in 60mm dishes used for the EMSA. LLC-PK1 cells were

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treated with 0.1-0.4 mM H2O2 for 2 h and cell viabilitydetermined using a neutral red assay as described in theMaterials and Methods. H2O2 treatment resulted in aconcentration-dependent decrease in cell viability (Figure2). A moderately toxic concentration of H2O2 (EC50; 0.27mM) was chosen to determine the effect of H2O2 on TRE-and NF-κB-binding activity.

To compare the temporal regulation of TRE- and NF-κB-binding activities by these toxicants, the same nuclearextracts were used in parallel in all of our analyses. PeakTGHQ-mediated TRE- and NF-κB-binding activity wasobserved between 2 and 4 h (Figure 3, panels A and B).

NF-κB-binding activity consists of two inducible bandstermed complex 1 and complex 2 (see figure legend).Complex 1 was the major binding activity induced byTGHQ in all the experiments, while the signal forcomplex 2 was more variable. Although several NF-κBcomplexes have been identified (13), it is not knownwhether the complexes observed in our studies representunique heterodimers, or degradation products. Subse-quently, LLC-PK1 cells were treated with H2O2 to deter-mine whether TRE- and NF-κB-binding activities weredirectly regulated by ROS. H2O2 (0.27 mM; 1-5 h)increased TRE- and NF-κB-binding activity in a time-

Figure 1. TGHQ-mediated cytotoxicity in 24 well plates and60 mm culture dishes. LLC-PK1 cells were seeded in therespective culture dish and maintained until 5 day postconflu-ent. Cells were then treated with 100-1000 mM TGHQ in 0.1%FBS DMEM for 2 h. Cell viability measurements were obtainedusing a neutral red assay as described in Materials andMethods. Panel A shows data expressed as molarity and PanelB shows same data expressed as nmol TGHQ/cm2. Valuesrepresent the mean ( SE (n ) 3). (*) Significantly different fromcontrol, p ) 0.05. Similar results were observed in two separateexperiments.

Figure 2. H2O2-mediated cytotoxicity in 60 mm culture dishes.LLC-PK1 cells were seeded in 60 mm culture dish and main-tained until 5 day postconfluent under identical conditions usedfor EMSA analysis. Cells were then treated with 0.1-0.4 mMH2O2 in 0.1% FBS DMEM for 2 h. Cell viability measurementswere obtained using a neutral red assay as described inMaterials and Methods. Values represent the mean ( SE (n )3). (*) Significantly different from control, p ) 0.05. Similarresults were observed in two separate experiments.

Figure 3. Temporal regulation of TRE- and NF-κB-bindingactivity by TGHQ and H2O2. (A) TRE-binding activity in LLC-PK1 cells treated with 62 nmol TGHQ/cm2 (O) and 0.27 mMH2O2 (b) for 1-5 h. (B) NF-κB-binding activity in cells treatedwith 62 nmol TGHQ/cm2 (circle) and 0.27 mM H2O2 (square)for 1-5 h. Two inducible NF-κB-binding complexes were ob-served and designated complex 1 (solid symbol) and complex 2(open symbol). Similar results were observed in two separateexperiments.

Figure 4. Concentration-dependent regulation of TRE- andNF-κB-binding activity by TGHQ. LLC-PK1 cells were treatedwith 25-126 nmol TGHQ/cm2 for 3 h and processed formeasurements of TRE (A)- and NF-κB (B)-binding activity in astandard EMSA as described in Materials and Methods. Similarresults were observed in two separate experiments.

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dependent fashion (Figure 3, panels A and B). ControlTRE- and NF-κB-binding activities in 5 day postconfluentLLC-PK1 cells did not change over time, consistent withour previous reports (15).

TGHQ-Induced TRE- and NF-KB-Binding Activi-ties Are ROS-Dependent. A differential dose-responsefor peak TRE- and NF-κB-binding activity was observedin LLC-PK1 cells treated with 25-126 nmol TGHQ/cm2

(Figure 4). Maximal TRE-binding activity 3 h followingchemical treatment was observed between 25 and 76nmol TGHQ/cm2, whereas maximal NF-κB-binding activ-ity was observed between 76 and 126 nmol TGHQ/cm2.To determine whether TGHQ-mediated TRE- and NF-κB-binding activities were ROS dependent, LLC-PK1 cellswere treated with TGHQ in the presence or absence ofcatalase (H2O2 scavenger) and deferoxamine (Fe2+/Fe3+

chelator). Catalase (10 units/mL) as a cotreatment fullyinhibited peak TGHQ (76 nmol/cm2)-mediated TRE- andNF-κB-binding activities indicating a requirement forH2O2 in these responses (Figure 5). Pretreatment withthe iron chelator deferoxamine (10 mM, 30 min) fullyinhibited inducible TRE-binding activity suggesting thatthis response is dependent upon iron-mediated •OHgeneration. Deferoxamine had a lesser effect on NF-κB-binding activity, suggesting this response is regulated byboth H2O2 and the •OH (Figure 5), consistent with anumber of reports indicating that H2O2 is the primaryROS regulating NF-κB (9, 29). In contrast, peak TPA (10ng/mL, 1 h)-mediated TRE- and NF-κB-binding activitieswere not inhibited by catalase or deferoxamine (Figure5), arguing against nonspecific inhibition of inducibleDNA-binding activities by either of these treatments. Thecatalase used for these experiments is from an anti-oxidant-free stock, and therefore inhibition of TGHQ-

mediated DNA-binding activities by catalase cannot beattributed to a nonspecific antioxidant effect. Specificityfor the binding reaction was confirmed by addition ofunlabeled target DNA, which competitively eliminatedthe inducible band, and with unlabeled nontarget DNAwhich was without effect.

TGHQ-Induced TRE-Binding Activity Is Tran-scriptionally Dependent. TRE- and NF-κB-bindingactivity in the EMSA can represent activation of existingprotein or the transcriptional upregulation of AP-1 andNF-κB. To determine the role of transcription and

Figure 5. Effect of catalase and deferoxamine on peak TGHQ- and TPA-mediated TRE- and NF-κB-binding activity. LLC-PK1 cellswere treated with TGHQ (76 nmol/cm2; 3 h) or TPA (10 ng/mL; 1 h) in the presence and absence of catalase (10 U/ml cotreatment)and deferoxamine (10 mM; 30 min pretreatment) and processed for measurements of TRE (A)- and NF-κB (B)-binding activity.Specificity for the binding reaction was confirmed by addition of excess unlabeled target DNA which competitively eliminated theinducible bands, and by addition of excess unlabeled nontarget DNA which was without effect. Similar results were observed in twoseparate experiments.

Figure 6. Effect of cycloheximide and actinomycin D on peakTGHQ-mediated TRE (top panel)- and NF-κB (bottom panel)-binding activity. LLC-PK1 cells were pretreated for 10 min with10 µg/mL actinomycin D and 50 µg/mL cycloheximide, subse-quently treated with 76 nmol TGHQ/cm2 for 3 h, and processedfor measurements of TRE (A)- and NF-κB (B)-binding activityin a standard EMSA as described in Materials and Methods.Similar results were observed in two separate experiments.

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translation in this response, LLC-PK1 cells were pre-treated with actinomycin D (10 µg/mL) or cycloheximide(50 µg/mL) for 10 min as previously described (30),subsequently treated with 76 nmol TGHQ/cm2 for 3 h,and TRE- and NF-κB-binding activity determined. TGHQ-mediated TRE-binding activity was fully inhibited byactinomycin D and cycloheximide, but not DMSO, sug-gesting this response is transcriptionally dependent(Figure 6). The transcriptional-dependence suggests thatthe TRE-binding response does not represent the direct

activation of existing AP-1 by the •OH (Figure 5).Actinomycin D and cycloheximide alone increased NF-κB-binding activity. Thus, the dependence of the NF-κB-binding response to TGHQ on transcription/translationcould not be determined from these studies. The increaseof NF-κB-binding activity by actinomycin D and cyclo-heximide is observed in other epithelial cell types (31).

We have previously shown that prostanoid- and phor-bol ester-induced TRE-binding activity are markedlyreduced by PKC inhibitors (H-89; calphostin C) in LLC-PK1 cells (15, 32). LLC-PK1 cells were pretreated withH-89 (20 µM) and calphostin C (100 nM) for 30 min,subsequently treated with 76 nmol of TGHQ/cm2 for 3 hand TRE- and NF-κB-binding activity determined. H-89and calphostin C pretreatment did not inhibit TGHQ-mediated TRE- and NF-κB-binding activities (Figure 7),suggesting these responses are not dependent on H-89-and calphostin C-sensitive PKC isoforms.

Modulation of TGHQ-Induced TRE- and NF-KB-Binding Activity by a MEK Inhibitor. TRE- and NF-κB-binding activity are also regulated by the MAPKsignal transduction pathway, which is typically definedon the basis of extracellular signal regulated kinase(ERK1/ERK2) activity (33-35). PD098059 inhibits MEKactivity, an upstream regulator of the ERKs. To deter-mine whether the TRE- and NF-κB-binding response toTGHQ was dependent on the MAPK pathway, LLC-PK1

cells were pretreated for 60 min with 50 µM PD098059as described (36), subsequently treated with 12-63 nmolTGHQ/cm2 for 3 h, and TRE- and NF-κB-binding activitydetermined. TGHQ-mediated TRE-binding activity wasmarkedly reduced by PD098059 (Figure 8). In contrast,PD098059 pretreatment did not inhibit NF-κB-binding

Figure 7. TGHQ-mediated TRE- and NF-κB-binding activityis not sensitive to PKC inhibitors. LLC-PK1 cells were pretreatedfor 30 min with 20 µM H-89 and 100 nM calphostin C,subsequently treated with 76 nmol TGHQ/cm2 for 3 h, andprocessed for measurements of TRE (top panel)- and NF-κB(bottom panel)-binding activity in a standard EMSA as de-scribed in Materials and Methods. Similar results were observedin two separate experiments.

Figure 8. Inhibition of TGHQ-mediated TRE-binding activity by PD098059. LLC-PK1 cells were pretreated for 60 min with 50 µMPD098059, subsequently treated with 12-63 nmol TGHQ/cm2 for 3 h, and processed for measurements of TRE-binding activity ina standard EMSA as described in Materials and Methods. Groups shown represent TRE-binding activity with (O) or without (9)PD098059 pretreatment. Similar results were observed in two separate experiments.

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activity from the same nuclear extracts used for the TRE-binding analysis (Figure 9). Qualitatively, PD098059pretreatment appeared to enhance TGHQ-mediated NF-κB-binding activity, suggesting MEK-related signalingmay antagonize this DNA-binding response.

Discussion

We have investigated the regulation of redox respon-sive transcription factors, that are widely implicated incarcinogenesis, by TGHQ a redox active nephrocarcino-genic metabolite of HQ (23-25). TGHQ-mediated TRE-and NF-κB-binding activities are fully inhibited by cata-lase (Figure 5) and are therefore ROS-dependent. Inprevious studies, we have demonstrated that deferox-amine inhibits quinol-thioether-mediated cytotoxicity (6,37), DNA single strand breaks (37), and gadd153 expres-sion, a molecular marker of genotoxic stress (6). Defer-oxamine fully inhibits TGHQ-mediated TRE-bindingactivity (Figure 5) suggesting this response is •OH-dependent. Deferoxamine had a lesser effect on TGHQ-mediated NF-κB-binding activity (Figure 5), suggestingthe NF-κB response is regulated by both H2O2 and the•OH. Because TRE-binding activity is transcriptionallydependent (Figure 6) and the temporal regulation of TRE-and NF-κB-binding activity are comparable (Figure 3),the •OH-dependent regulation of these DNA-binding acti-vities is likely secondary to the genotoxic stress response.

A correlation between increasing H2O2-mediated cyto-toxicity and decreasing TRE-binding activity has beenobserved in cultured neuronal cells (38) and AP-1 DNA-binding activity is inhibited by high ROS concentrationsvia oxidation of a critical cysteine residue (39). Quinol-

thioethers are redox active and are electrophiles thatcovalently bind and inactivate cellular macromolecules(40). Thus, the reduction of TGHQ-mediated TRE-bindingactivity at progressively higher concentrations may berelated to deregulated gene expression due to overtcytotoxicity (compare Figures 1 and 4), the generationof high concentrations of ROS, or the direct inhibition ofAP-1 via covalent binding of TGHQ-derived reactiveelectrophilic metabolites. The activation of NF-κB byH2O2 is well established (9, 29) and high ROS concentra-tions also inhibit NF-κB DNA-binding activity, presum-ably via oxidation of a sensitive thiol. NF-κB-bindingactivity can be restored by treatment of nuclear extractswith reducing agents such as 2-mercaptoethanol (41).NF-κB-binding activity was increased in a dose-depend-ent fashion and therefore, appears to be a direct responseto quinone-thioether challenge. However, this observationmust be interpreted with caution since reducing agentwas present in the extraction buffer. A functional NF-κB-luciferase assay is required to better understand thecorrelation between inducible NF-κB-binding activity andNF-κB-dependent transcriptional activity under theseexperimental conditons.

AP-1 is highly responsive to genotoxic stress (42), andquinol-thioether-mediated DNA damage is observed within15 min of exposure (4). Therefore, genotoxic stressprecedes the TRE-binding response (Figure 3). As notedabove, deferoxamine inhibits many of the cellular re-sponses to TGHQ, including the expression of gadd153,a molecular marker of genotoxic stress (43-45). Ofinterest, a role for the •OH in the AP-1-dependentregulation of gadd153 has been reported (8), and our

Figure 9. Increased TGHQ-mediated NF-κB-binding activity by PD098059. LLC-PK1 cells were pretreated for 60 min with 50 µMPD098059, subsequently treated with 12-63 nmol TGHQ/cm2 for 3 h, and processed for measurements of NF-κB-binding activity ina standard EMSA as described in Materials and Methods. Groups shown represent NF-κB-binding activity with (open) or without(solid) PD098059 pretreatment for complex 1 (square) and complex 2 (circle). Similar results were observed in two separateexperiments.

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findings are consistent with a •OH/AP-1-dependent regu-lation of gadd153. In support of this statement, TGHQ-mediated TRE-binding activity (Figure 3) and gadd153gene expression (6) exhibit comparable temporal- andconcentration-dependent regulation (50-500 µM TGHQgroups shown in ref 6 are equivalent to 8-80 nmolTGHQ/cm2). Collectively, these observations form a plau-sible model where •OH-dependent genotoxic stress re-sults in the transcriptional upregulation of AP-1, whichin turn, regulates gadd153 expression. As described inthe Results, the concentration-dependent correlate forTRE-binding activity and gadd153 expression would notbe apparent if the data were expressed as molarity.

AP-1 and NF-κB are regulated by a number of signaltransduction pathways, including those coupled to ERKactivation (33-35) and PKC (15). TGHQ-mediated TRE-binding activity is markedly reduced by a MEK inhibitor(Figure 8), suggesting an important role for the MAPKcascade in the TRE-binding response to TGHQ, consist-ent with our previous findings that quinol-thioethersincrease ERK activity in LLC-PK1 cells.2 TRE-bindingactivity was not fully inhibited by PD098059, suggestingthis response may be regulated by additional MEK-independent signaling events, consistent with the obser-vation that interventions targeting both the genotoxic-and ERK-stress response pathways dramatically reduceinducible TRE-binding activity (Figures 5 and 8). Incontrast, NF-κB-binding activity from the same nuclearextracts was not inhibited by PD098059 (Figure 9). Thisobservation suggests that the NF-κB response is notmediated by MEK-related signaling.

Many “nongenotoxic” nephrotoxicants induce tumorsin the kidney of mice and rats, suggesting a specific effecton a “preinitiated” population of cells (46, 47). Cytotoxicmodes of chemical-induced carcinogenesis have also beenimplicated for toxicants such as chloroform (48). There-fore, there is precedence for a number of mechanisms bywhich quinol-thioethers may induce tumors or modulatecarcinogenic processes. In particular, quinol-thioether-mediated cytotoxicity is associated with a prominentoxidative stress (4), and ROSs are widely implicated incarcinogenic processes (49). Acute oxidative injury isthought to produce cell death and a compensatoryincrease in cell proliferation, resulting in the clonalexpansion of preinitiated cells. Alternatively, a sublethalconcentration of ROS could result in damage to DNAwhich, if mis-repaired, would lead to the generation ofnewly initiated cells. In this context, TGHQ is mutagenicin a manner consistent with the participation of the •OHin DNA damage (26). Chronic oxidative injury maycontribute to carcinogenesis through the modulation ofgrowth-related signal transduction pathways, such asthose associated with AP-1 and NF-κB, which are acti-vated by ROS and implicated as causal in transformationresponses (8, 9, 11, 16, 18, 29). Our data indicate thatquinol-thioether-mediated TRE- and NF-κB-binding ac-tivities precedes overt cytotoxicity (Figures 1 and 4) and,therefore, may contribute to the carcinogenic propertiesof this compound.

The induction of TRE- and NF-κB-binding activity isalso associated with the regulation of cell death andsurvival in mammalian cells. NF-κB activation is associ-

ated with apoptosis in rat striatum following kainic acidtreatment (50). In contrast, the activation of AP-1 andNF-κB by H2O2 in oligodendrocytes is believed to promotecell survival, while the formation of •OH promotes celldeath (51). Prostanoid-mediated activation of AP-1 andNF-κB induces protection against TGHQ-induced cyto-toxicity in LLC-PK1 cells (6, 15, 32), suggesting thesetranscription factors promote survival in renal epithelialcells. Promotion of cell survival by AP-1 and/or NF-κBwould have plausible implications in carcinogenic proc-esses and could result in the fixation of mutationsinduced by quinol-thioethers (26). However, it is impor-tant to note that the signal transduction pathwaysregulating TRE- and NF-κB-binding activity by pros-tanoids and TGHQ are clearly different. Prostanoid-mediated DNA-binding activities are inhibited by H-89and calphostin C (15), while these PKC inhibitors haveno effect on TGHQ-mediated DNA-binding activities(Figure 7). Therefore, additional studies are required todetermine whether PKC-independent activation of TRE-and NF-κB-binding activity will also promote cell survivalin renal epithelial cells.

In summary, we have shown that TGHQ-mediatedTRE- and NF-κB-binding activities are ROS-dependentin LLC-PK1 cells. Peak TGHQ-induced TRE-bindingactivity is increased in a •OH-dependent fashion, tran-scriptionally dependent, precedes overt cytotoxicity, andis markedly reduced by a MEK inhibitor. Peak TGHQ-induced NF-κB-binding activity appears to be regulatedby both H2O2 and the •OH, and is not reduced by a MEKinhibitor. The differential effects of deferoxamine andPD098059 on TGHQ-mediated TRE- and NF-κB-bindingactivity provide evidence that these transcription factorsare regulated by different signaling pathways after H2O2

generation. Collectively, our findings support an ROS-dependent effect of TGHQ on signal transduction eventsimplicated in tumor promotion.

Acknowledgment. This work was supported by agrant from the Department of Energy (DE-AC06-76RLO1830) to Dr. Thomas Weber and by a grant from theNational Institute of General Medical Sciences (GM39338) to Dr. Serrine Lau. This work was also supportedby grants from the National Institute of EnvironmentalHealth Sciences (T32 ES 07247 and ES 07884).

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