DNA AND CELL BIOLOGYVolume 11, Number 4, 1992Mary Ann Liebert, Inc., PublishersPp. 269-281

Dioxin Induces Expression of c-fos and c-junProto-Oncogenes and a Large Increase in

Transcription Factor AP-1

ALVARO PUGA,* DANIEL W. NEBERT,* and FRANCE CARRIERt

ABSTRACT

Among environmental pollutants, 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD; dioxin) is one of the mostpotent tumor promoters and teratogens known. The molecular mechanisms responsible for the biological ac-

tivity of TCDD, however, remain largely unknown. In this report, we show that the first observable effectsof TCDD in cultured murine hepatoma cells are a rapid, transient increase in Ca2+ influx and a minor butsignificant elevation of activated, membrane-bound protein kinase C. These changes are then followed by in-duction of the immediate early proto-oncogenes c-fos, jun-B, c-jun, and jun-H, and by large increases inAP-1 transcription factor activity. Induction of these changes by TCDD is delayed compared with that byphorbol esters, although the magnitude of the effects caused by both treatments is similar, and both induc-tion processes can be blocked by staurosporine, a protein kinase C inhibitor. In cultured cells, proto-onco-gene induction by TCDD appears to be independent of the presence of a functional aryl hydrocarbon (Ah)receptor or nuclear translocation protein. These results reveal early events that may lead to the elucidation ofthe molecular basis of TCDD-induced tumor promotion.

INTRODUCTION TCDD is also among the most potent inducers of themammalian CYP1A1 (cytochrome P,450) gene. This gene

TCDD, the prototype congener of a large group of encodes a monooxygenase, aryl hydrocarbon hydroxylasehalogenated polycyclic hydrocarbons, is one of the (Nebert and Gonzalez, 1987; Whitlock, 1987, 1991; Ne-

most potent teratogens and tumor promoters ever tested in bert, 1989; Landers and Bunce, 1991), responsible for theanimal model systems (Pitot et ai, 1980; Knutson and detoxification of many planar polycyclic aromatic hydro-Poland, 1982; Poland and Knutson, 1982; Poland et ai, carbons such as benzo[a]pyrene and 3-methylcholan-1982, 1985; Nebert, 1989; Landers and Bunce, 1991; Whit- threne. Polycyclic hydrocarbons are very strong promuta-lock, 1991). TCDD causes an elevated incidence of hepatic gens and procarcinogens, because the CYP1A1 enzymecarcinoma and other tumors in rodents, and promotes tu- first oxidizes these compounds during metabolism into re-mor formation at one-hundredth the dose of the classical active intermediates that can cause oxidative DNA damagetumor promoter 12-0-tetradecanoylphorbol-13-acetate and form adducts with DNA, starting the mutagenic(TPA) in the skin of hairless mice. Because TCDD is not a events responsible for tumor initiation (see Jerina et ai,mutagen, it has been proposed that it promotes neoplastic 1991 and references therein). Polycyclic hydrocarbons are

expression in cells that have already been initiated (Was- not only substrates for the CYP1A1 enzyme but are alsosom et ai, 1977; Poland and Glover, 1979; Geiger and ligands for the cytosolic aromatic hydrocarbon (Ah) recep-Neal, 1981; Neal et ai, 1982). The molecular basis of this tor. The liganded receptor translocates to the nucleus,activity has not yet been resolved. binds to aromatic /¡ydrocarbon-responsive elements

Laboratory of Developmental Pharmacology, National Institute of Child Health and Human Development, National Institutes ofHealth, Bethesda, MD 20892.

»Present Address: Department of Environmental Health, University of Cincinnati Medical Center, 3223 Eden Avenue, Cincinnati,OH 45267-0056.

tpresent Address: Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD20892.

269

270 PUGA ET AL.

(AhREs) in the 5'-flanking region of the CYP1A1 gene,and acts as a transcriptional activator in a fashion some-what analogous to that of the steroid receptors. Halogena-tion can make polycyclic aromatic compounds very resis-tant to enzymatic oxygénation. For example, the fourchlorines in TCDD prevent the chemical form having any-HC=CH- groups that can be readily oxidized. As a

consequence, the oxidative metabolism of TCDD proceedsat least 30,000 times more slowly than that of benzo[a]-pyrene. The prolonged half-life of TCDD is a major fac-tor, therefore, in explaining its high potency and longduration of action.

Based on genetic evidence in mice, it has generally beenaccepted that TCDD binding to the receptor is an essentialstep required for its toxicity (reviewed in Nebert, 1989;Landers and Bunce, 1991). Many known effects of TCDD,however, do not involve the Ah receptor; these include theinduction of protein kinases (Bombick et ai, 1985, 1988)and phospholipase C (Beebe et ai, 1990), the effects on

plasma membranes and low-density lipoprotein receptor(Bombick et ai, 1984; Matsumura et ai, 1984), and theupregulation of epidermal growth factor (EGF) receptorlevels (Madhukar et ai, 1984), although the latter mayhave a receptor-dependent component (Lin et ai, 1991).Recently, it has been suggested that dioxin toxicity is medi-ated by epoxides and other derivatives of arachidonic acidmetabolism catalyzed by the TCDD-induced CYP1A1 en-

zyme (Rifkind et ai, 1990). In addition, TCDD has beenshown to cause a large elevation of intracellular Ca2* andto induce apoptosis in thymocytes (McConkey et ai, 1988;McConkey and Orrenius, 1989). In humans, there hasbeen insufficient evidence to determine whether TCDD,and halogenated hydrocarbons in general, are teratogenicand carcinogenic. Recent long-term epidemiological stud-ies, however, have established a strong link between expo-sure to high doses of dioxin and certain types of cancers

(Fingerhut et ai, 1991; Manz et ai, 1991).If TCDD is indeed a tumor promoter, it is reasonable to

ask whether it can initiate the signals associated with cellgrowth, differentiation, and development. Particularly rel-evant in this respect is the induction of a group of immedi-ate-early (IE), or "competence," genes whose expression isalso rapidly stimulated by serum, purified growth factors,and phorbol esters such as TPA. Among others, thesegenes encode the transcriptional activator proteins Fos,Jun, and Myc (Greenberg and Ziff, 1984; Angel et ai,1987; Lau and Nathans, 1987; Lamph et ai, 1988; Ryderand Nathans, 1988; Vogt and Bos, 1989; Rivera andGreenberg, 1990). Interaction of FOS with any one on thevarious members of the Jun family (Nakabeppu et ai,1988; Ryder et ai, 1988; Chiu et ai, 1989; Ryder et ai,1989) forms the AP-1 transcription factor (Angel et ai,1988; Chiu et ai, 1988; Curran and Franza, 1988; Kouza-rides and Ziff, 1988; Nakabeppu et ai, 1988; Sassone-Corsi et ai, 1988a,b; Rauscher et ai, 1989; Abate et ai,1990) required for cell proliferation. [Hereafter we shallalso use the AP-1 designation to refer to PEA1, the murinehomologue of AP-1 (Martin et ai, 1988).] Current modelsof eukaryotic development implicate growth factors, hor-mones, neurotransmitters, and a variety of extracellular

ligands in the initiation of the mitogenic signal by a cas-

cade of biochemical events that include: increases of ionfluxes across the membrane; elevation of intracellular pH,Ca2*, and cAMP levels; formation of phosphoinositidemetabolites; and activation of protein kinases (Hendrick-son et ai, 1985; Williams, 1989; Rivera and Greenberg,1990). Transduction of the mitogenic signal to the nucleusactivates the expression of transcription factors such as

AP-1, which, in turn, propagate the signal by controllingthe expression of other genes required for cell prolifera-tion. The involvement of AP-1 in cell proliferation, differ-entiation, and malignant transformation has been shownto occur by two different mechanisms: (i) through muta-tions affecting the coding regions of the v-fos and v-junoncogenes found in transforming retroviruses (Curran etai, 1982; Maki et ai, 1987), and (ii) through deregulatedexpression of normal c-FOS and c-JUN proteins (Miller etai, 1984; Lee et ai, 1988; Raymond et ai, 1989; Schütteet ai, 1989a,b; Yang-Yen et ai, 1990).

In this report we provide evidence that TCDD deregu-lates the expression of the normal c-Fos and c-Jun compo-nents of AP-1. Our results suggest a possible mechanism to

explain the role of TCDD in early events leading to car-

cinogenesis.

MATERIALS AND METHODSCell lines and culture conditions

The mouse Hepa-1 hepatoma cell line (Bernard et ai,1973) was grown to 80% confluence in a-MEM supple-mented with 2 mM glutamine and 10% fetal calf serum.

Before further treatment, the medium was replaced with a-

MEM containing 0.1 % fetal calf serum, and the cells were

maintained for 2 days under these conditions to minimizethe amount of proto-oncogene mRNA and of active AP-1factor present in cultures grown in 10% serum. Thereafter,cultures were treated for periods of time ranging from 15min to 4.5 hr with the following compounds at the indi-cated concentrations and solvents: 15 nM TCDD inDMSO, 150 nM TPA in DMSO, 1 mM dibutyryl cAMP(hereafter referred to simply as cAMP) in water, 100 fiMbenzo[a]pyrene in methanol, or combinations of thesecompounds. We prepared 10,000-fold concentrated stocksof TPA and TCDD, so that the concentration of DMSO inthe treated cultures was 0.01%. The stock of benzo[o]-pyrene was 5 mM, and the methanol concentration in thecultures was 2%. Mock experiments conducted at thesesolvent concentrations showed no effect of solvent in ex-

pression of any of the genes tested. Untreated experimen-tal controls are from cells treated with 0.01% DMSO. Theconcentrations of other reagents used are indicated in thefigures.

RNA extractions, mRNA detection, andDNA probes

RNA was extracted by the acid guanidium thiocyanatemethod (Chomczynski and Sacchi, 1987). For Northernblots, 20 fig of RNA was analyzed by electrophoresis informaldehyde-agarose gels, and the gels were dried under

TCDD INDUCES c-fos AND c-jun EXPRESSION 271

vacuum at 60°C and processed for in-gel hybridization(Puga et ai, 1990). DNA probes were labeled with [a-"P]-dCTP (3,000 Ci/mmole, Amersham) by nick translation(Maniatis et ai, 1975). The 3'-specific Cypla-1 cDNAprobe represents the 1.2-kb untranslated 3' end of themouse Cypla-1 cDNA clone pMPIFL (Kimura et ai,1984); the c-fos probe was the insert in plasmid pGEM-Fos-3, containing the mouse c-fos cDNA (Halazonetis etai, 1988); the c-myc probe was from the mouse c-myccDNA clone pmMycP7, a derivative of pSV2myc-dhfr(Prochownik and Kukowska, 1986), containing 420 bp ofexon 2; the 0-actin probe was from the chicken (3-actincDNA plasmid pBAl (Cleveland et ai, 1980). Conditionsfor prehybridization, hybridization, and washes were as

described (Puga et ai, 1990).The probes used for SI protection assays were: the 304-

bp Eco Rl-Bgl II fragment of pGemFos-3; the 655-bp Sacl-Sma I fragment of plasmid p465.20 containing the com-

plete coding sequence of mouse jun-B (Lau and Nathans,1987; Ryder et ai, 1988); the 539-bp Eco Kl-Aat II frag-ment of plasmid pJAC.l, containing the coding sequencesof mouse c-jun (Ryder and Nathans, 1988); and the 446-bpEco RI-Äsr Eil fragment of plasmid pXHJ-12.4, contain-ing the coding sequences of mouse jun-D (Ryder et ai,1989). DNA fragments were denatured by boiling and la-beled with [7-32P]ATP at the 5' end using T4 polynucleo-tide kinase. Hybridization reactions contained 0.1-0.2pmole of probe, 20-50 ^g of RNA in 20 id of a buffer con-

taining 0.4 M NaCl, 2 mM EDTA, 40 mM PIPES pH 6.7,and 80% formamide. Reaction mixtures were heated to80°C for 10 min and incubated overnight at 60°C, deter-mined as the optimum hybridization temperature in pre-liminary experiments. For most experiments, a single hy-bridization mixture was used for all four probes. After in-cubation for 16-30 hr, reaction mixtures were diluted10-fold with a buffer containing 0.2 M NaCl, 30 mM so-

dium acetate, pH 4.5, 5 mM ZnS04, 10 ¡ig denatured sal-mon sperm DNA, and 100 units of SI nuclease (BRL).After incubation for 1 hr at room temperature, the reac-

tion was stopped by addition of EDTA to 5 mM and NH4acetate to 2.5 M, and nucleic acids were precipitated with 3volumes of ethanol, using 5 ¡¡.g tRNA as carrier. The pre-cipitate was dissolved in a small volume of sequencingloading buffer and the protected probe was analyzed byelectrophoresis in a denaturing 4% acrylamide-urea gel.After electrophoresis, the gels were dehydrated, dried, andexposed to X-ray film.

Determination of Ca1* influx rates

Unidirectional Ca2* influx rates were determined bymeasuring the initial uptake of 45Ca2* by methods de-scribed (Kojima et ai, 1988). Cells were grown in duplicate24-well dishes in complete a-MEM medium and were madequiescent by incubation for 30 h in medium containing0.1% fetal calf serum. At zero-time, the cells were treatedwith the stimulus, i.e., 15 nMTCDD or 50 \M ATP, thelatter used as a positive control (Duddy et ai, 1989; Joneset ai, 1989). To begin the measurements, the culture me-

dium was replaced with medium containing 11 ¿iCi/ml of

[45Ca]Cl2 (18 mCi/mg Ca2*; Amersham) and the stimulus.Incubation was for 30 sec, 50 sec, and 70 sec, and was ter-minated by quickly washing the cells with 50 mM Tris, pH7.4, 100 mM NaCl, 5 mM CaCl2, and removing them with0.1 ml of 0.5 N NaOH for scintillation counting. Calciuminflux rate was obtained from the slope of the linear re-

gression line of the three points. Control influx rates(100% values) for unstimulated cells were typically in therange of 2-5 pmoles/min per 10,000 cells.

Gel mobility-shift assaysNuclear extracts were prepared by methods described

(Dignam et ai, 1983). DNA-binding reactions were carriedout for 20 min at room temperature in 20 ¡d. of a buffercontaining 20 mM HEPES pH 7.8, 1 mM EDTA, 1 mMDTT, 1 ¡ig of poly(dI-dC)-poly(dI.dC), 10,000 dpm ofDNA probe, and 10% glycerol. Probes were terminally la-beled with polynucleotide kinase in the presence of [y-32P]-ATP. The amount of protein used for the reactions was

determined empirically by titration of each binding activitystudied. Samples were loaded onto nondenaturing 4%polyacrylamide gels; after electrophoresis, the gels were de-hydrated and exposed to X-ray film. The probe used forthe Ah receptor was a 76-bp DNA fragment containing theAhRE3 sequence within nucleotides -1,019 and -943 ofthe Cypla-1 gene (Nebert and Jones, 1989). To confirmthe identity of the band containing the Ah receptor, bind-ing was competed with a 200-fold excess of the 20-mer 5'-CTCTTCTCACGCAACTCCGG-3', of which the centralCACGC is the motif required for Ah receptor binding(Fujisawa-Sehara er ai, 1986, 1987, 1988; Hapgood et al.,1989; Durrin and Whitlock, 1987; Denison et ai, 1988,1989; Neuhold et ai, 1989). The probe used for the detec-tion of AP-1 was the synthetic double-stranded 20-mer 5'-CTAGTGATGAGTCAGCCGGATC-3', in which the cen-

tral TGAGTCA is the canonical AP-1 binding site. Quan-titation was carried out by counting the entire dehydratedgel in an AMBIS scanner.

Computer analysesComputer analyses were done using the WORDSEARCH

algorithm (GCG, Madison, WI). The GenEMBL Acces-sion Numbers of the sequences used were as follows:J00370 and V00727 for mouse c-fos; X57155 for mouse

c-jun; X57154 for mouse jun-B; X57156 for mouse jun-D;and M12345 for mouse c-myc. Other sequences for the hu-man and rat orthologues of some of these genes alsoscored for the presence of the AhRE core sequence.

RESULTSThe steady-state levels of c-fos and jun mRNAsincrease after TCDD treatment

To determine the effect of TCDD on IE proto-oncogeneactivation, we analyzed RNA from mouse hepatoma wild-type cells in Northern blots. As controls we used culturesthat were treated with TPA, cAMP, a combination of

272 PUGA ET AL.

TPA + cAMP, or TCDD + cAMP. TPA and cAMPwere chosen as positive controls because they activate thesignal-transducing, diacylglycerol-dependent protein ki-nase C pathway and the cAMP-dependent protein kinaseA pathway, respectively. TCDD treatment had a dramaticeffect on the accumulation of c-fos mRNA (Fig. 1). After4 hr, the amount of c-fos mRNA present in TCDD-treatedcells was even larger than the amount in controls of TPA-or cAMP-treated cells, although not as high as in cellstreated with both TPA and cAMP. Treatment with TCDDand cAMP did not cause an additional increase over thelevel observed with TCDD treatment alone. Steady-statec-Myc mRNA levels, already high in untreated hepatomacells (Huber and Thorgeirsson, 1987; Zhang et ai, 1987,1988; Duronio et ai, 1990; Olah et ai, 1990; Richards etai, 1990), did not change after TCDD treatment, exceptfor a possible slight decrease. As observed by others(Mechta et ai, 1989), c-Myc mRNA levels decreased sig-nificantly after cAMP treatment (Fig. 1, third panel).TCDD is known to induce CYP1A1 (top row), and the ad-dition of cAMP had no further effect. The levels of /3-actinmRNA, used as a control, were not appreciably changedby any of the treatments (Fig. 1, bottom panel). Thesedata suggest that TCDD is a very potent activator of c-fosgene expression.

We observed very little effect of TPA on c-fos mRNAaccumulation, compared with the effect of TCDD (Fig. 1),yet TPA is well-established as a strong inducer of c-fos ex-

pression. One possible explanation for this discrepancy isthat the response to these two drugs follows different ki-netics. To test for this possibility, we analyzed the timecourse of accumulation of c-fos mRNA after treatmentwith TPA and with TCDD. We also analyzed the mRNAsof jun-B, c-jun, and jun-D, the three known members ofthe jun gene family (Angel et ai, 1988; Ryder andNathans, 1988; Ryder et ai, 1988; Ryseck et ai, 1988). Inaddition, we measured mRNA levels in the presence of cy-cloheximide, a protein synthesis inhibitor that increasesboth c-fos and c-jun mRNA stability and prolongs the ele-vation of transcription initiation after stimulation (Green-berg et ai, 1986; Lau and Nathans, 1987; Ryseck et ai,1988).

For the four IE genes tested, the kinetics of the responseto TCDD and to TPA were markedly different. After TPAtreatment, c-fos mRNA accumulation peaked at 30 min,Jun-B and Jun-D between 30 and 60 min, and c-Jun be-tween 60 and 150 min (Fig. 2A), in good agreement withprevious reports (Nathans, 1987; Ryder and Nathans,1988; Ryder et ai, 1988, 1989). In contrast, the TCDD-in-duced expression of c-fos and c-jun was delayed, notshowing a significant increase until 2.5 to 4.5 hr after treat-ment, and Jun-B and Jun-D showed a pattern of sustainedinduction throughout, but with major accumulation alsobetween 2.5 and 4.5 hr after treatment (Fig. 2A). Additionof cycloheximide 1 hr before TCDD resulted in the accu-mulation of all four IE gene transcripts for the five treat-ments used (Fig. 2B). The possible stabilization of mRNAresulting from cycloheximide treatment alone did notcause significant mRNA accumulation, which leads us toinfer that the effect of TCDD takes place at the level of

Q Q.< Q SQ. O <

O I- I- O

a.

2 <

° X+ 2Q. U

P1

c-fos

c-myc

/3-actin

mmm

12 3 4 5 6FIG. 1. TCDD induces accumulation of c-fos transcripts.Wild-type mouse Hepa-1 cells were treated for 4 hr withTCDD, or left untreated (lane 0). Total RNA (20 ¡ig) was

analyzed in formaldehyde-agarose gels, and dried gels were

hybridized with the indicated probes. PI denotes theCypla-1 probe. As established previously, CYP1A1mRNA is undetectable in untreated cells and very abun-dant after TCDD treatment (top panel, lanes 3 and 6); theincrease has been shown to result mostly from transcrip-tional induction (Gonzalez and Nebert, 1985; Neuhold etai, 1986; RayChaudhuri et ai, 1990).

transcription initiation or elongation, and not simply byinterfering with the decay rate of otherwise unstablemRNA species. These results show that TPA stimulationof c-fos and c-jun expression occurs earlier than TCDDstimulation of these same genes, and that the magnitude ofinduction by TCDD is just as great as that by TPA.

Induction requires staurosporine-sensitiveprotein kinase activity

TPA induction is dependent upon protein kinase C,which can be inhibited by staurosporine. Staurosporine is a

serine/threonine protein kinase inhibitor with a markedpreference for protein kinase C (Hidaka et ai, 1984; Smithet ai, 1988; Mahadevan et ai, 1990). To determinewhether induction of the fos and jun proto-oncogenes byTCDD is dependent on protein kinase C activity, we pre-treated wild-type hepatoma cells with staurosporine beforeTCDD treatment. As in previous experiments, we usedTPA and cAMP treatments as positive controls. In addi-tion, we also used benzo[a]pyrene, the classical polycyclic

TCDD INDUCES c-fos AND c-jun EXPRESSION 273

BTPA TCDD CHX

o oin o o in r~-i- n ¡o i- pi

o oW O O to h-v- m <o i- Vi

'«*É0"^^ Jun-B

1

i

c-jun

Jun-D

I12 3 4 5 6 7 8 9 1011 1 2 3 4 5 6

FIG. 2. Induction of IE gene transcription by TCDD. A. Time course of the induction. Wild-type cells were treatedwith TPA or with TCDD for the length of time (in min) indicated above the lanes. Detection of the transcripts was car-ried out by SI nuclease analysis, using 0.2 pmole of each probe and 25 /ig of RNA in one reaction tube for the simultane-ous detection of the members of the jun family and 50 jig of RNA for the detection of c-fos in an individual reaction.B. Effect of protein synthesis inhibition on induction. Cultures were treated for 1 hr with cycloheximide (CHX, 10/ig/ml) prior to addition of the inducers. Treatment was continued for 4 hr, at which time total RNA was extracted andtranscripts were detected by SI nuclease protection. The jun genes were analyzed together in a single reaction using 20 ¡igof RNA and 0.2 pmole of each labeled probe. Pilot experiments indicated that the synergistic effect of TPA and cAMPon c-fos transcription causes a large difference in the optimal exposure times required to visualize all the lanes in the c-/ospanel. The amounts of RNA used in the hybridization reactions for c-fos were adjusted accordingly; they were 12 /ig forthe TPA + cAMP reaction and 50 /ig for all others. Thus, on a comparative basis, the level of mRNA induced by thecombination of TPA + cAMP is approximately four times larger than the level induced by the other treatments.

hydrocarbon inducer of the Cypla-1 gene, that (in contrastto TCDD) is readily metabolized by the CYP1A1 enzyme.As shown in Fig. 3, treatment with staurosporine com-

pletely abolished the accumulation of all mRNAs tested,except for the /3-actin control, suggesting that induction byTCDD, like induction by TPA, is mediated by the proteinkinase C-dependent pathway. When we measured proteinkinase C activity in cultured Hepa-1 hepatoma cells, wefound that TCDD causes a marginal, albeit statistically sig-nificant, activation of membrane-bound protein kinase C.By comparison, TPA effects a doubling of membrane-bound protein kinase C activity, whereas EGF, an inducerof tyrosine kinase used as a negative control, causes no

change (Table 1). These experiments have been repeatedfour times with the same significant effect for TCDD. Twoother protein kinases analyzed, protein kinase A and tyro-sine kinase, were unaffected by TCDD treatment (data notshown). These data suggest that TPA, TCDD and benzo-[a]pyrene induce c-/os and c-jun but not c-myc gene ex-

pression, and that the induction process is blocked by an

inhibitor of protein kinase C.To analyze whether the increase in protein kinase C

could be a consequence of Ca2* signaling (Moolenaar etai, 1986; Chartier and Schiffrin, 1987; Tsunoda, 1991),we measured the rate of Ca2* influx into wild-type and mu-

tant Hepa-1 cells. Figure 4A shows that treatment with

274 PUGA ET AL.

TCDD results in a very rapid increase in the rate of Ca2*influx, which reaches a maximum 2 min following additionof the stimulus and returns to control levels by 6 min. TheTCDD-induced influx reaches maximal values 2.3 timeslarger than those in unstimulated control cells. ATP-in-duced influx, used as a positive control, reaches a lowermaximum between 4 and 5 min after addition. Two mu-

tant cell lines showed different patterns. The nuclear trans-location-deficient c4 line has been shown to exhibit a de-fective Ah receptor «uclear rranslocator (ARNT) protein(Hankinson, 1983; Hankinson et ai, 1985; Hoffinan et al.,

1991). The cl cell line is regarded as "receptorless"; al-though it has < 10% of the wild-type levels of Ah recep-tor, the receptor that does exist functions normally (Han-kinson, 1983; Hankinson et ai, 1985). The c4 line shows a

pattern very similar to that of the wild-type, whereas, incontrast, the "leaky" receptorless mutant cell line c2 is sig-nificantly less responsive to TCDD-induced increases inCa2* influx (Fig. 4B). These data suggest that the TCDD-induced Ca2* influx is independent of the nuclear translo-cation of the Ah receptor, although it may require func-tional ligand-binding receptor activity.

No Inhibitor Staurosp

P1

Jun-B

c-jun

Proto-oncogene induction in receptorless and innuclear translocation-deficient cell lines

Computer analyses revealed that the promoter region ofall the proto-oncogenes tested contains several copies ofthe AhRE core sequence CACGC. This observation raisedthe possibility that their induction by TCDD might followfrom the same molecular mechanism responsible for theinduction of the Cypla-1 gene, i.e., binding of a ligandedAh receptor to regulatory AhRE domains in the genes. Totest this possibility, we examined the effect of TCDD on

IE proto-oncogene expression in the two mutant cell lines,c2 and c4. These cell lines responded differently than thewild type (Fig. 5). CYP1A1 mRNA was not induced byTCDD in the nuclear translocation-defective c4 line, andshowed a low level of inducibility in the receptorless mu-

tant c2 (Fig. 5, lanes 1-14, top panel). These results are inagreement with previous observations (Hankinson, 1983;Hankinson et ai, 1985) and confirm the phenotypic char-acteristics of the cells used. In contrast to wild-type cells,the c4 line shows already derepressed levels of Jun-D, and

Jun-D

c-fos

c-myc

/3-actinmmmgmam^mÊÊgÊSBBSBm

12 3 4 5 6 7 8 9 10

FIG. 3. Proto-oncogene induction in wild-type cells is in-hibited by staurosporine. Cultures were treated for 2 hrwith the drugs indicated above the lanes and control cul-tures were left untreated. Staurosporine (150 nM) wasadded 1 hr before the other drugs, and incubation wascontinued for 2 hr. Transcription of Cypla-1 (PI), c-myc,and /3-actin was analyzed by Northern hybridization using20 fig of total RNA per lane. Transcription of the othergenes was analyzed in a single reaction by SI nuclease pro-tection using 50 fig of total RNA and 0.2 pmole of eachprobe. In the absence of inhibitor, Cypla-1 expression isinduced only by TCDD and by benzo[a]pyrene [B(a)P], asexpected (first panel). jun-B and jun-D are clearly inducedby all the drugs tested (second and fourth panels) and in-duction of c-jun is stronger with TCDD and benzo[a]-pyrene than with TPA or cAMP, the converse being truefor c-fos expression (third and fifth panels). Since sampleswere analyzed 2 hr after addition of the inducers, the ef-fect of TCDD on c-fos mRNA accumulation is not yet de-tectable (see Fig. 2). Derepression of c-myc appears to beinhibited in the presence of cAMP and of benzo[a]pyrene(sixth panel), and the levels of /3-actin are unaltered by thetreatments (seventh panel).

TCDD INDUCES c-fos AND c-jun EXPRESSION 275

Table 1. Activation of Protein Kinase Cin Wild-Type Mouse Hepatoma

Hepa-1 Cells

Treatment ActivityTPATCDDEGFNone

26 ± 319 ± 314 ± 213 ± 2

0.0010.0300.654

Membrane frations were prepared as described(Thomas et ai, 1987) and kinase activity was de-termined by phosphorylation of the substratepeptide RFARKGSLRQKNV (House and Kemp,1987), using a kit manufactured by GIBCO-BRL. Activity is expressed in pmoles of "P in-corporated from [7-32P]ATP per min per mg ofmembrane protein. Values shown are the mean± SE of four separate samples each assayed induplicate. Data were analyzed by the unpairedStudent t test; the two-tailed p values shown cor-respond to comparisons with the control group.The p value of the comparison of TPA andTCDD treatments is 0.150, indicating that thedifference between these two groups is not signif-icant.

to a lesser extent, of Jun-B and c-Fos mRNA; the treat-ments caused an increase in mRNA accumulation to levelscomparable to those observed in similarly treated wild-typecells (Fig. 5, lanes 1-7). The behavior of c-jun was some-what different; whereas no c-Jun mRNA could be detectedin untreated c4 cells, none of the treatments increased theaccumulation of this mRNA to the levels found in wild-type cells (compare the c-jun panels in Figs. 3 and 5, lanes1-7). In contrast, in untreated c2 cells, Jun-B, c-Jun, andc-Fos mRNAs are not significantly elevated, and they re-

spond to treatment much like the wild-type cells, whereasJun-D mRNA is already very high and responds to treat-ments in a manner similar to that of c4 cells. It is intrig-uing that the largest increase of c-Fos mRNA accumula-tion in c2 cells occurs not after TPA, but after benzo[a]-pyrene treatment (Fig. 5, lanes 8-14). The c-Myc and 0-ac-tin mRNA levels, and their response to treatments, are

very similar in mutants and wild-type cells.The response to TCDD in the mutant cell lines suggests

that neither a liganded functional Ah receptor nor itstranslocation to the nucleus, are necessary steps for the in-duction of at least c-Fos and Jun-B mRNA. Jun-D mRNAis already derepressed in both mutant lines, and the effectof the Ah receptor on its expression cannot be assessed.The low levels of c-Jun mRNA accumulation in c4 cellsmay be interpreted as an indication of the requirements fora functional ARNT protein in TCDD-induced c-jun ex-

pression. This may not be the case, however, since the ex-

tremely low-level response of this cell line to TPA and thelack of response to cAMP (Fig. 5) suggest that, in additionto the mutation in the Arnt gene, these cells may haveother mutations affecting the regulation of the IE genes.

Un wt c2 c4

2 min

Un wt c2 c4

5 minUn wt c2 c4

20 min

FIG. 4. 45Ca2* influx rates in mouse hepatoma cells stim-ulated with TCDD. A. Kinetics. Rates are plotted at thetime of addition of [45Ca]Cl2. B. Comparison of wild-type and mutant hepatoma cell lines. Duplicate cultures ofthe cell lines (wt, c2, c4) were treated with TCDD as indi-cated above, or left untreated (Un). Three-point rates weredetermined at the times shown. Untreated controls wereconducted for all three cell lines, but the control value at100% is shown only once at each time point (Un), with anerror bar corresponding to the largest of the errors of thethree controls.

TCDD increases the levels of AP-1transcription factor

The presence of elevated c-Fos, c-Jun, Jun-B, andJun-D mRNA may be biologically irrelevant, unless thisaccumulation is followed by a concomitant elevation ofAP-1 activity. To test whether TCDD causes a rise inAP-1, we measured AP-1 DNA-binding activity by gelmobility-shift assays. As a control, we also assayed DNA-binding activity of the Ah receptor. TCDD treatment, withor without concomitant cAMP addition, causes the ap-pearance of an Ah receptor-containing DNA binding activ-ity (Fig. 6A, lanes 5 and 11) whose identity has been wellestablished from the work of this and other laboratories(Fujisawa-Sehara et ai, 1987, 1988; Durrin and Whitlock,1987; Denison et ai, 1988, 1989; Hapgood et ai, 1989;Neuhold et ai, 1989). Competition by an excess of a shortoligonucleotide containing the AhRE3 core sequence con-

276 PUGA ET AL.

P1

Jun-B

c-jun

Jun-D

c-fos

c-myc

ß-actin

12 3 4 5 6 7 8 9 10 11121314

FIG. 5. Proto-oncogene induction in the nuclear translo-cation-defective (c4) and in receptorless (c2) mutant celllines. Experimental conditions were the same as those inFig. 3, except that RNA from the mutant Hepa-1 deriva-tives c4 and c2 was used.

firms the identity of this band (Fig. 6A, lanes 6 and 12),which is not induced by TPA or cAMP. TCDD treatmentalso causes an elevation of the amount of AP-1 transcrip-tion factor complexes in the cells (Fig. 6B). This effect ismuch larger than the effect of cAMP and, kinetically, isslightly delayed relative to the effect of TPA; whereasTPA treatment for 2 hr raises AP-1 by fivefold, this levelrequires 4 hr to be reached after TCDD treatment. Simul-taneous induction with cAMP does not increase signifi-cantly the effect of TPA and has only a minor additional

consequence on the effect of TCDD, raising the AP-1 levelto sixfold over that in untreated cells. Thus, these resultsindicate that TCDD induction of c-Fos and Jun mRNA re-

sults in a major increase in AP-1, to levels comparable tothose elicited by a prototypical tumor promoter.

DISCUSSION

The experiments that we present here provide a possiblemolecular model to explain the role of TCDD in carcino-genesis. In a temporal sense, the sequence of events beginswith the first observable effect of TCDD, i.e., a high,rapid, and transient elevation of the rate of Ca2* influx,which, we believe, initiates a cascade of events ultimatelyleading to cell proliferation. There is indeed a large bodyof evidence linking intracellular Ca2* signaling with growthcontrol (reviewed in Whitaker and Patel, 1990). The actualmechanism of TCDD-induced Ca2* entry is presently un-

known, but is likely to result from agonist-operated open-ing of either receptor- or second messenger-operated chan-nels (Berridge and Irvine, 1989). Activation of protein ki-nase C, the second observable effect of TCDD, takes placewithin 10 min after addition of the drug, and is an essentialstep for TCDD-dependent proto-oncogene induction,since treatment with staurosporine abolishes not only in-duction by TPA but also by TCDD. The next observableeffect of TCDD is the accumulation of IE proto-oncogenemRNAs, first Jun-D within 15 min of treatment, followedby Jun-B, clearly elevated by 60 min, and then c-Jun andc-Fos that are delayed by 2-3 hr. Finally, the levels of thetranscription factor AP-1 become elevated between 2 and 4hr after treatment. The involvement of AP-1 in cell prolif-eration and malignant transformation has been well estab-lished (Curran et ai, 1982; Miller et ai, 1984; Maki et ai,1987; Lee et ai, 1988; Raymond et ai, 1989; Schütte etai, 1989a,b; Yang-Yen et ai, 1990). This temporal schemeis different from that observed in TPA-treated cells, whereall the proto-oncogenes show maximal mRNA levels within30-60 min of treatment. The delay of TCDD relative toTPA treatment is reflected in the rate of AP-1 accumula-tion, although eventually both treatments yield compara-ble levels of functional AP-1 complex.

There is no indication that TCDD binds to protein ki-nase C, and thus it is likely that the mode of action ofTCDD is not identical to that of TPA. Several observa-tions support this conclusion. First, TCDD causes a more

discrete elevation of activated protein kinase C than TPA.Second, the delay in induction relative to TPA points tothe possible involvement of one or more intervening stepsbetween the activation of Ca2* influx and the induction ofthese proto-oncogenes. These steps might entail calmodu-lin-dependent protein kinases and Ca2*-ATPases, as de-scribed in other systems (Schönthal et ai, 1991), or otherprotein kinase C-independent pathways of IE gene induc-tion (Stumpo and Blackshear, 1986; Blackshear et ai,1987; Gilman, 1988; Fukumoto et ai, 1990; Graham andGilman, 1991) that may also be sensitive to inhibition bystaurosporine. It is also plausible that increases in Ca2* in-

TCDD INDUCES c-fos AND c-jun EXPRESSION 277

A TPA+ TCDD+Un TPA TCDD cAMP cAMP cAMP

Ah Re

B TPA+ TCDD+TPA TCDD cAMP cAMP cAMP UnI-II-II-II-II-124 242 42424

mi t* *y y

1 2 345 6789 10 11

Fold 5.0 1.6 1.1 3.7 1.7 1.07.0 4.7 2.3 6.3 6.0

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 6. Induction of the Ah receptor and AP-1 DNA-binding activities. Cells were treated for 2 hr and 4 hr with TPA,TCDD, cAMP, TPA + cAMP, or TCDD + cAMP or left untreated. A. Induction of the Ah receptor. Each bindingreaction contained 15 /ig of nuclear protein. To confirm the identity of the band containing the Ah receptor (Ah Re), we

carried out binding reactions in even-numbered lanes (labeled +) that contained a 200-fold excess of the competing 20-mer 5'-CTCTTCTCACGCAACTCCGG-3', as indicated in the Materials and Methods section. In odd-numbered lanes(labeled -), no competitor DNA was added. B. Induction of AP-1. Each binding reaction contained 2 /ig of nuclearprotein. Indicated below each lane is the fold stimulation of AP-1 activity relative to the activity in quiescent untreatedcells (Un). Quantitation was carried out by counting the entire dehydrated gel in an AMBIS scanner.

flux rates and induction of IE genes are not coupled at all,the former being a direct effect of the drug and the latterresulting from cellular mediators induced by the drug or byCYP1A1-dependent metabolism of an endogenous sub-strate.

Many major questions remain open for future work.Paramount among these is the role of the Ah reeptor in IEgene induction. Our experiments using receptorless andnuclear translocation-defective mutant cell lines suggestthat the induction process is independent of the Ah recep-tor, but as indicated before, there are other possible expla-nations for our results. For example, the Ah receptormight not be involved in the earlier events ( < 1-2 hr) of theTCDD response, but might have a function at later times.Better genetic characterization of the available mutant celllines, use of genetically defined inbred mouse strains, andisolation of the Ah receptor itself would be required to ad-dress this question fully.

In conclusion, the results presented herein clearly estab-

lish that planar polycyclic aromatic compounds induce ex-

pression of the c-fos, c-jun, jun-B, and jun-D proto-onco-genes, and the concomitant increase in the AP-1 transcrip-tion factor. Induction of these IE genes is intimately con-

nected with the regulation of cellular proliferation and dif-ferentiation, as well as tumorigenesis. Induction of thesegenes by benzo[a]pyrene, a well-known proven tumor ini-tiator (reviewed in Jerina et ai, 1991), documents the roleof this compound as a tumor promoter as well. We believethat the induction of these genes by TCDD plays a majorrole in the early molecular events surrounding TCDD-in-duced tumor promotion.

ACKNOWLEDGMENTS

We thank Branco Mijaidovic for his helpful advice on

calcium influx analyses, and our colleagues Cindy Bachur-ski, Kathleen Dixon, and Albert Fornace for a critical

278 PUGA ET AL.

reading of the manuscript. We are also indebted to theanonymous reviewer of a previous paper who requestedcontrol experiments that, however unnecessary, led us toinitiate the work presented here. F.C. was supported by a

postdoctoral fellowship from the Human Frontier ScienceProgram Organization.

REFERENCES

ABATE, C, LUK, D., GAGNE, E., ROEDER, R.G., and CUR-RAN, T. (1990). Fos and jun cooperate in transcriptional regu-lation via heterologous activation domains. Mol. Cell. Biol. 10,5532-5535.

ANGEL, P., IMAGAWA, M., CHIU, R., STEIN, B., IMBRA,R.J., RAHMSDORF, H.J., JONAT, C, HERRLICH, P.,and KARIN, M. (1987). Phorbol ester-inducible genes containa common eis element recognized by a TPA-modulated trans-

acting factor. Cell 49, 729-739.ANGEL, P., ALLEGRETTO, E.A., OKINO, S.T., HATTORI,

K., BOYLE, W.J., HUNTER, T., and KARIN, M. (1988).Oncogene jun encodes a sequence-specific rra/is-activator simi-lar to AP-1. Nature 332, 166-171.

BEEBE, L., PARK, S.S., and ANDERSON, L.M. (1990). Differ-ential enzyme induction of mouse liver and lung following a

single low or high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD). J. Biochem. Toxicol. 5, 211-219.

BERNARD, H.P., DARLINGTON, G.J., and RUDDLE, F.H.(1973). Expression of liver phenotypes in cultured mouse hepa-toma cells: Synthesis and secretion of serum albumin. Dev.Biol. 35, 83-96.

BERRIDGE, M.J., and IRVINE, R.F. (1989). Inositol phos-phates and cell signalling. Nature 341, 197-205.

BLACKSHEAR, P.J., STUMPO, D.J., HUANG, J.K., NE-MENOFF, R.A., and SPACH, D.H. (1987). Protein kinaseC-dependent and -independent pathways of proto-oncogene in-duction in human astrocytoma cells. J. Biol. Chem. 262, 7774-7781.

BOMBICK, D.W., MATSUMURA, F., and MADHUKAR, B.V.(1984). TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) causes re-duction in the low density lipoprotein (LDL) receptor activitiesin the hepatic plasma membrane of the guinea pig and rat. Bio-chem. Biophys. Res. Commun. 118, 548-554.

BOMBICK, D.W., MADHUKAR, B.V., BREWSTER, D.W.,and MATSUMURA, F. (1985). TCDD (2,3,7,8-tetrachlorodi-benzo-p-dixoin) causes increases in protein kinases particularlyprotein kinase C in the hepatic plasma membrane of the rat andthe guinea pig. Biochem. Biophys. Res. Comm. 127, 296-302.

BOMBICK, D.W., JANKUN, J., TULLÍS, K., and MATSU-MURA, F. (1988). 2,3,7,8-tetrachlorodibenzo-/7-dioxin causesincreases in expression of c-erb-A and levels of protein-tyrosinekinases in selected tissues of responsive mouse strains. Proc.Nati. Acad. Sei. USA 85, 4128-4132.

CHARTIER, L., and SCHIFFRIN, E.L. (1987). Role of calciumin effects oa atrial natriuretic peptide on aldosterone produc-tion in adrenal glomerulosa cells. Am. J. Physiol. 252,E485-E491.

CHIU, R., BOYLE, W.J., MEEK, J., SMEAL, T., HUNTER,T., and KARIN, M. (1988). The c-Fos protein interacts withc-Jun/AP-1 to stimulate transcription of AP-1 responsivegenes. Cell 54, 541-552.

CHIU, R., ANGEL, P., and KARIN, M. (1989). Jun-B differs inits biological properties from, and is a negative regulator of,c-Jun. Cell 59, 979-986.

CHOMCZYNSKI, P., and SACCHI, N. (1987). Single-step

method of RNA isolation by acid guanidium thiocyanate-phe-nol-chloroform extraction. Anal. Biochem. 162, 156-159.

CLEVELAND, D.W., LOPATA, M.A., MACDONALD, R.J.,COWAN, N.J., RUTTER, W.K., and KIRSCHNER, M.N.(1980). Number and evolutionary conservation of alpha- andbeta-tubulin and cytoplasmic beta- and gamma-actin genesusing specific cloned cDNA probes. Cell 20, 95-105.

CURRAN, T., and FRANZA, B.R., JR. (1988). Fos and Jun:The AP-1 connection. Cell 55, 395-397.

CURRAN, T., PETERS, G., VAN BEVEREN, C, TEICH, N.,and VERMA, I.M. (1982). FBJ murine osteosarcoma virus:Identification and molecular cloning of biologically active pro-viral DNA. J. Virol. 44, 674-682.

DENISON, M.S., FISHER, J.M., and WHITLOCK, J.P. JR.(1988). The DNA recognition site for the dioxin-Ah receptorcomplex. J. Biol. Chem. 263, 17221-17224.

DENISON, M.S., FISHER, J.M., and WHITLOCK, J.P., JR.(1989). Protein-DNA interactions at recognition sites for thedioxin-Ah receptor complex. J. Biol. Chem. 264, 16478-16482.

DIGNAM, J.D., LEBOVITZ, R.M., and ROEDER, R.G.(1983). Accurate transcription initiation by RNA polymerase IIin a soluble extract from isolated mammalian nuclei. NucleicAcids Res. 11, 1475-1489.

DUDDY, S.K., KAAS, G.E.N., and ORRENIUS, S. (1989).Ca2*-mobilizing hormones stimulate Ca2* efflux from hepato-cytes. J. Biol. Chem. 264, 20863-20866.

DURONIO, V., HUBER, B.E., and JACOBS, S. (1990). Partialdown-regulation of protein kinase C reverses the growth inhibi-tory effect of phorbol esters on HepG2 cells. J. Cell Physiol.145, 381-389.

DURRIN, L.K., and WHITLOCK, J.P., JR. (1987). In situ pro-tein-DNA interactions at a dioxin-responsive enhancer associ-ated with the cytochrome P.-450 gene. Mol. Cell. Biol. 7, 3008-3011.

FINGERHUT, M.A., HALPERIN, W.E., MARLOW, D.A.,PIACITELLI, L.A., HONCHAR, P.A., SWEENEY, M.H.,GREIFE, A.L., DILL, P.A., STEENLAND, K., andSURUDA, A.J. (1991). Cancer mortality in workers exposed to2,3,7,8-tetrachlorodibenzo-/j-dioxin. N. Engl. J. Med. 324,212-218.

FUJISAWA-SEHARA, A., SOGAWA, K., NISHII, C, andFUJII-KURIYAMA, Y. (1986). Regulatory DNA elements lo-calized remotely upstream from the drug-metabolizing cyto-chrome P-450c gene. Nucleic Acids Res. 14, 1465-1477.

FUJISAWA-SEHARA, A.A, SOGAWA, K., YAMANE, M.,and FUJII-KURIYAMA, Y. (1987). Characterization of xeno-biotic responsive elements upstream from the durg-metaboliz-ing cytochrome P-450c gene: A similarity to glucocorticoid reg-ulatory elements. Nucleic Acids Res. 15, 4179-4191.

FUJISAWA-SEHARA, A., YAMANE, M., and FUJII-KURI-YAMA, Y. (1988). A DNA-binding factor specific for xenobi-otic-responsive elements of P-450c gene exists as a cryptic formin cytoplasm: Its possible translocation to nucleus. Proc. Nati.Acad. Sei. USA 85, 5859-5863.

FUKUMOTO, Y., KAIBUCHI, K., OKU, N., HORI, Y., andTAKAI, Y. (1990). Activation of the c-fos serum-response ele-ment by the activated c-Ha-ras protein in a manner indepen-dent of protein kinase C and cAMP-dependent protein kinase.J. Biol. Chem. 265, 774-780.

GEIGER, L.E., and NEAL, R.A. (1981). Mutagenicity testing of2,3,7,8-tetrachlordibenzo-p-dioxin in histidine auxotrophs ofSalmonella typhimurium. Toxicol. Appl. Pharmacol. 59, 125-129.

GILMAN, M.Z. (1988). The c-fos serum response element re-

sponds to protein kinase C-dependent and -independent signalsbut not to cyclic AMP. Genes Dev. 2, 394-402.

TCDD INDUCES c-fos AND c-jun EXPRESSION 279

GONZALEZ, F.J., and NEBERT, D.W. (1985). Autoregulationplus upstream positive and negative control regions associatedwith transcriptional activation of the mouse P,-450 gene. Nu-cleic Acids Res. 13, 7269-7288.

GRAHAM, R., and GILMAN, M. (1991). Distinct protein tar-

gets for signals acting at the c-fos serum response element. Sci-ence 251, 189-192.

GREENBERG, M.E., and ZIFF, E.B. (1984). Stimulation of 3T3cells induces transcription of the c-fos proto-oncogene. Nature311, 433-438.

GREENBERG, M.E., HERMANOWSKI, A.L., and ZIFF, E.B.(1986). Effect of protein synthesis inhibitors on growth factoractivation of c-fos, c-myc, and actin gene transcription. Mol.Cell. Biol. 7, 1217-1225.

HALAZONETIS, T.D., GEORGOPOULOS, K., GREEN-BERG, M.E., and LEDER, P. (1988). c-Jun dimerizes with it-self and with c-Fos, forming complexes of different DNA bind-ing affinities. Cell 55, 917-924.

HANKINSON, O. (1983). Dominant and recessive aryl hydrocar-bon hydroxylase-deficient mutants of mouse hepatoma lineHepa-1 and assignment of recessive mutants to three comple-mentation groups. Somat. Cell Genet. 9, 497-514.

HANKINSON, O., ANDERSEN, R.D., BIRREN, B., SAN-DER, F., NEGISHI, M., and NEBERT, D.W. (1985). Muta-tions affecting the regulation of transcription of the cyto-chrome P,-450 gene in the mouse Hepa-1 cell line. J. Biol.Chem. 260, 1790-1795.

HAPGOOD, J., CUTHILL, S., DENIS, M., POELLINGER,L., and GUSTAFSSON, J.A. (1989). Specific protein-DNA in-teractions at a xenobiotic-responsive element: Copurification ofdioxin receptor and DNA-binding activity. Proc. Nati. Acad.Sei. USA 86, 60-64.

HENDRICKSON, S.L., COCHRAN, B.H., REFFEL, A.C., andSTILES, CD. (1985). In Mediators in Cell Growth and Differ-entiation. R.J. Ford and A.L. Maizel, eds. (Raven, New York)pp. 71-85.

HIDAKA, H., INAGAKI, M., KAWAMOTO, S., and SASAKI,Y. (1984). Isoquinolinesulfonamides, novel and potent inhibi-tors of cyclic nucleotide dependent protein kinase and proteinkinase C. Biochemistry 23, 5036-5041.

HOFFMAN, E.C., REYES, H., CHU, F.F., SANDER, F.,CONLEY, L.H., BROOKS, B.A., and HANKINSON, O.(1991). Cloning of a factor required for activity of the Ah (di-oxin) receptor. Science 252, 954-958.

HOUSE, C, and KEMP, B.E. (1987). Protein kinase C containsa pseudosubstrate prototope in its regulatory domain. Science238, 1726-1729.

HUBER, B.E., and THORGEIRSSON, S.S. (1987). Analysis ofc-myc expression in a human hepatoma cell line. Cancer Res47, 3414-3420.

JERINA, D.M., CHADHA, A., CHEH, A.M., SCHURDAK,M.E., WOOD, A.W., and SAYER, J.M. (1991). Covalentbonding of bay-region diol epoxides to nucleic acids. Adv. Exp.Med. Biol. 283, 533-553.

JONES, D.P., McCONKEY, D.J., NICOTERA, P., and OR-RENIUS, S. (1989). Calcium-activated DNA fragmentation inrat liver nuclei. J. Biol. Chem. 264, 6398-6403.

KIMURA, S., GONZALEZ, F.J., and NEBERT, D.W. (1984).The murine Ah locus. Comparison of the complete cytochromeP,-450 and P2-450 cDNA nucleotide and amino acid sequences.J. Biol. Chem. 259, 10705-10713.

KNUTSON, J.C., and POLAND, A. (1982). Response of murineepidermis to 2,3,7,8-tetrachlorodibenzo-p-dioxin: Interactionof the Ah and hr loci. Cell 30, 225-234.

KOJIMA, I., MATSUNAGA, H., KUROKAWA, K., OGATA,E., and NISHIMOTO, I. (1988). Calcium influx: An intracellu-

lar message of the mitogenic actium of insulin-like growthfactor-I. J. Biol. Chem. 263, 16561-16567.

KOUZARIDES, T., and ZIFF, E. (1988). The role of the leucinezipper in the fos-jun interaction. Nature 336, 646-651.

LAMPH, W.W., WAMSLEY, P., SASSONE-CORSI, P., andVERMA, I.M. (1988). Induction of proto-oncogene JUN/AP-1 by serum and TPA. Nature 334, 629-631.

LANDERS, J.P., and BUNCE, N.J. (1991). The Ah receptor andthe mechanism of dioxin toxicity. Biochem. J. 276, 273-287.

LAU, L.F., and NATHANS, D. (1987). Expression of a set ofgrowth-related immediate early genes in BALB/c 3T3 cells: Co-ordinate regulation with c-fos or c-m^c- Proc. Nati. Acad. Sei.USA 84, 1182-1186.

LEE, W., LIN, C, and CURRAN, T. (1988). Activation of thetransforming potential of the human fos proto-oncogene re-

quires message stabilization and results in increased amounts ofpartially modified fos protein. Mol. Cell. Biol. 8, 5521-5527.

LIN, F.H., CLARK, G., BIRNBAUM, L.S., LUCIER, G.W.,and GOLDSTEIN, J.A. (1991). Influence of the Ah locus on

the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the he-patic epidermal growth factor receptor. Mol. Pharmacol. 39,307-313.

MADHUKAR, B.V., BREWSTER, D.W., and MATSUMURA,F. (1984). Effects of in v/'vo-administered 2,3,7,8-tetrachlorodi-benzo-p-dioxin on receptor binding of epidermal growth factorin the hepatic plasma membrane of rat, guinea pig, mouse, andhamster. Proc. Nati. Acad. Sei. USA 81, 7407-7411.

MAHADEVAN, L.C., WILLS, A.J., HIRST, E.A., RATHJEN,P.D., and HEATH, J.K. (1990). 2-Aminopurine abolishesEGF- and TPA-stimulated pp33 phosphorylation and c-fos in-duction without affecting the activation of protein kinase C.Oncogene 5, 327-335.

MAKI, Y., BOS, T., DAVIS, C, STARBUCK, M., and VOGT,P.K. (1987). Avian sarcoma virus 17 carries the jun oncogene.Proc. Nati. Acad. Sei. USA 84, 2848-2852.

MANIATIS, T., JEFFREY, A., and KLEID, D.G. (1975). Nu-cleotide sequence of the rightward operator of phage lambda.Proc. Nati. Acad. Sei. USA 72, 1184-1188.

MANZ, A., BERGER, J., DWYER, J.H., FLESCH-JANYS,D., NAGEL, S., and WALTSGOTT, H. (1991). Cancer mor-

tality among workers in chemical plant contaminated with di-oxin. The Lancet 338, 959-964.

MARTIN, M.E., PIETTE, J., YANIV, M., TAN, W.-J., andFOLK, W. (1988). Activation of the polyomavirus enhancer bya murine activator protein (API) homolog and two contiguousproteins. Proc. Nati. Acad. Sei. USA 85, 5839-5843.

MATSUMURA, F., BREWSTER, D.W., MADHUKAR, B.V.,and BOMBICK, D.W. (1984). Alteration of rat hepatic plasmamembrane functions by 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD). Arch. Environ. Contam. Toxicol. 13, 509-515.

McCONKEY, D.J., and ORRENIUS, S. (1989). 2,3,7,8-tetra-chlorodibenzo-p-dioxin kills glucocoticoid-sensitive thymocytesin vivo. Biochem. Biophys. Res. Commun. 160, 1103-1008.

McCONKEY, D.J., HARTZELL, P., DUDDY, S.K.,HAKANSSON, H., and ORRENIUS, S. (1988). 2,3,7,8-Tetra-chlorodibenzo-p-dioxin kills immature thymocytes by Ca2*-me-diated endonuclease activation. Science 242, 256-259.

MECHTA, F., PIETTE, J., HIRAI, S.-L, and YANIV, M.(1989). Stimulation of protein kinase C or protein kinase A me-

diated signal transduction pathways shows three modes of re-

sponse among serum inducible genes. The New Biologist 1,297-304.

MILLER, A., CURRAN, T., and VERMA, I.M. (1984). c-fosprotein can induce cellular transformation: A novel mechanismof activation of a cellular oncogene. Cell 36, 51-60.

MOOLENAAR, W.H., DEFIZE, L.H.K., and DE LAAT, S.W.

280 PUGA ET AL.

(1986). Ion signalling by growth factor receptors. J. Exp. Biol.124, 359-373.

NAKABEPPU, Y., RYDER, K., and NATHANS, D. (1988).DNA binding activities of three murine Jun proteins: Stimula-tion by Fos. Cell 55, 907-915.

NEAL, R.A., OLSON, J.R., GASIEWICZ, T.A., and GEIGER,L.E. (1982). The toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mammalian systems. Drug Metab. Rev. 13, 355-385.

NEBERT, D.W. (1989). The Ah locus: Genetic differences in tox-icity, cancer, mutation, and birth defects. Crit. Rev. Toxicol.20, 153-174.

NEBERT, D.W., and GONZALEZ, F.J. (1987). P450 genes:Structure, evolution and regulation. Annu. Rev. Biochem. 56,945-993.

NEBERT, D.W., and JONES, J.E. (1989). Regulation of themammalian cytochrome P,450 (CYP1A1) gene. Int. J. Bio-chem. 21, 243-252.

NEUHOLD, L.A., GONZALEZ, F.J., JAISWAL, A.K., andNEBERT, D.W. (1986). Dioxin-inducible enhancer region up-stream from the mouse P, gene and interaction with a heterolo-gous enhancer. DNA 5, 403-411.

NEUHOLD, L.A., SHIRAYOSHI, Y., OZATO, K., JONES,J.E., and NEBERT, D.W. (1989). Regulation of mouse

Cypla-1 gene expression by dioxin: Requirement of two as-act-ing elements during induction. Mol. Cell. Biol. 9, 2378-2386.

OLAH, E., KOTE, Z., NATSUMEDA, Y., YAMAJI, Y.,JARAI, G., LAPIS, E., FINANCSEK, I., and WEBER, G.(1990). Down-regulation of c-myc and c-Ha-ras gene expressionby tiazofurin in rat hepatoma cells. Cancer Biochem. Biophys.11, 107-117.

PITOT, H.C., GOLDSWORTHY, T., CAMPBELL, H.A., andPOLAND, A. (1980). Quantitative evaluation of the promo-tion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcino-genesis from diethylnitrosamine. Cancer Res. 40, 3616-3620.

POLAND, A., and GLOVER, E. (1979). An estimate of the max-

imum in vivo covalent binding of 2,3,7,8-tetrachlorodibenzo-z>-dioxin to rat liver protein, ribosomal RNA, and DNA. CancerRes. 39, 3341-3344.

POLAND, A., and KNUTSON, J.C. (1982). 2,3,7,8-Tetrachloro-dibenzo-p-dioxin and related halogenated aromatic hydrocar-bons: Examination of the mechanisms of toxicity. Annu. Rev.Pharmacol. Toxicol. 22, 517-554.

POLAND, A., PALEN, D., and GLOVER, E. (1982). Tumourpromotion by TCDD in skin of HRS/J hairless mice. Nature300, 271-273.

POLAND, A., KNUTSON, J., and GLOVER, E. (1985). Studieson the mechanism of action of halogenated aromatic hydrocar-bons. Clin. Physiol. Biochem. 3, 147-154.

PROCHOWNIK, E.V., and KUKOWSKA, J. (1986). Deregu-lated expression of c-myc by murine erythroleukaemia cells pre-vents differentiation. Nature 322, 848-850.

PUGA, A., RAYCHAUDHURI, B., SALATA, K., ZHANG,Y.-H., and NEBERT, D.W. (1990). Stable expression of mouseCypla-1 and human CYP1A-2 cDNAs transfected into mouse

hepatoma cells lacking detectable P450 enzyme activity. DNAand Cell Biol. 9, 425-436.

RAUSCHER, F.J., HI, VOULALAS, P.J., FRANZA, B.R.,JR., and CURRAN, T. (1989). Fos and Jun bind cooperativelyto the AP-1 site: Reconstitution in vitro. Genes Dev. 2,1687-1699.

RAYCHAUDHURI, B., NEBERT, D.W., and PUGA, A.(1990). The murine Cypla-1 gene negatively autoregulates itsown transcription and that of other members of the aromatichydrocarbon-responsive [Ah] gene battery. Mol. Endocrinol. 4,1773-1781.

RAYMOND, V., ATWATER, J., and VERMA, I.M. (1989). Re-moval of an mRNA destabilizing element correlates with the in-creased oncogenicity of proto-oncogene c-fos. Oncogene Res.5, 1-12.

RICHARDS, CA., SHORT, S.A., THORGEIRSSON, S.S., andHUBER, B.E. (1990). Characterization of a transformingN-ras gene in the human hepatoma cell line Hep G2: Addi-tional evidence for the importance of c-myc and ras coopera-tion in hepatocarcinogenesis. Cancer Res. 50, 1521-1527.

RIFKIND, A.B., GANNON, M., and GROSS, S.S. (1990).Arachidonic acid metabolism by dioxin-induced cytochromeP-450: A new hypothesis on the role of P-450 in dioxin toxicity.Biochem. Biophys. Res. Commun. 172, 1180-1188.

RIVERA, V.M., and GREENBERG, M.E. (1990). Growth fac-tor-induced gene expression: The ups and downs of c-fos regu-lation. The New Biologist 2, 751-758.

RYDER, K., and NATHANS, D. (1988). Induction of proto-oncogene c-jun by serum growth factors. Proc. Nati. Acad.Sei. USA 85, 8464-8467.

RYDER, K., LAU, L.F., and NATHANS, D. (1988). A gene ac-tivated by growth factors is related to oncogene \-jun. Proc.Nati. Acad. Sei. USA 85, 1487-1491.

RYDER, K., LANAHAN, A., PEREZ-ALBUERNE, E., andNATHANS, D. (1989). jun-D: A third member of the jun genefamily. Proc. Nati. Acad. Sei. USA 86, 1500-1503.

RYSECK, R.P., HIRAI, S.I., YANIV, M., and BRAVO, R.(1988). Transcriptional activation of c-jun during the G0/G1transition in mouse fibroblasts. Nature 334, 535-537.

SAFE, S., ASTROFF, B., HARRIS, M., ZACHAREWSKI, T.,DICKERSON, R., ROMKES, M., and BIEGEL, L. (1991).2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related com-

pounds as antioestrogens: Characterization and mechanism ofaction. Pharmacol. Toxicol. 69, 400-409.

SASSONE-CORSI, P., LAMPH, W.W., KAMPS, M., andVERMA, I.M. (1988a). /os-Associated cellular p39 is related tonuclear transcription factor AP-1. Cell 54, 553-560.

SASSONE-CORSI, P., RANSONE, L.J., LAMPH, W.W., andVERMA, I.M. (1988b). Direct interaction between fos and junnuclear oncoproteins: Role of the 'leucine zipper' domain.Nature 336, 692-695.

SCHONTHAL, A., SUGARMAN, J., BROWN, J.H., HAN-LEY, M.R., and FERAMISCO, J.R. (1991). Regulation ofc-fos and c-jun protooncogene expression by the Ca2* inhibitorthapsigargin. Proc. Nati. Acad. Sei. USA 88, 7096-7100.

SCHÜTTE, J., MINNA, J., and BIRRER, M. (1989a). Deregu-lated expression of human c-jun transforms primary rat em-

bryo cells in cooperation with an activated c-Ha-ras gene andtransforms Rat-la cells as a single gene. Proc. Nati. Acad. Sei.USA 86, 2257-2261.

SCHÜTTE, J., VIALLET, J., NAU, M., SEGAL, S., FE-DORKO, J., and MINNA, J. (1989b). jun-B inhibits and c-fosstimulates the transforming and rra/w-activating activities ofc-jun. Cell 59, 987-997.

SMITH, CD., GLICKMAN, J.F., and CHANG, K.J. (1988).The antiproliferative effects of staurosporine are not exclu-sively mediated by inhibition of protein kinase C. Biochem.Biophys. Res. Commun. 156, 1250-1256.

STUMPO, D.J., and BLACKSHEAR, P.J. (1986). Insulin andgrowth factor effects on c-fos expression in normal and proteinkinase C-deficient 3T3-L1 fibroblasts and adiopocytes. Proc.Nati. Acad. Sei. USA 83, 9453-9457.

THOMAS, T.P., GOPALAKRISHNA, R., and ANDERSON,W.B. (1987). Hormone- and tumor promoter-induced activa-tion or membrane association of protein kinase C in intactcells. Methods Enzymol. 141, 399-411.

TSUNODA, Y. (1991). Oscillatory Ca2* signaling and its cellular

TCDD INDUCES c-fos AND c-jun EXPRESSION 281

function. The New Biologist 3, 3-17.TZUKERMAN, M., ZHANG, X.-K., and PFAHL, M. (1991).

Inhibition of estrogen receptor activity by the tumor promoter12-0-tetradecanylphorbol-13-acetate: A molecular analysis.Mol. Endocrinol. 5, 1983-1992.

VOGT, P.K., and BOS, T.J. (1989). The oncogene jun and nu-

clear signalling. Trends Biol. Sei. 14, 172-175.WASSOM, J.S., HUFF, J.E., and LOPRIENO, N. (1977). A re-

view of the genetic toxicology of chlorinated dibenzo-p-dioxins.Mutat. Res. 47, 141-160.

WHITAKER, M., and PATEL, R. (1990). Calcium and cell cyclecontrol. Development 108, 525-542.

WHITLOCK, J.P., JR. (1987). The regulation of cytochromeP-450 gene expression. Pharmacol. Rev. 39, 147-161.

WHITLOCK, J.P., JR. (1991). Genetic and molecular aspects of2,3,7,8-tetrachlorodibenzo-p-dioxin action. Annu. Rev. Phar-macol. Toxicol. 30, 251-277.

WILLIAMS, L.T. (1989). Signal transduction by the platelet-de-rived growth factor receptor. Science 243, 1564-1570.

YANG-YEN, H.-F., CHIU, R., and KARIN, M. (1990). Eleva-tion of AP-1 activity during F9 cell differentiation is due to in-creased c-jun transcription. The New Biologist 2, 351-361.

ZHANG, X.K., HUANG, D.P., CHIU, D.K., and CHIU, J.F.(1987). The expression of oncogenes in human developing liverand hepatomas. Biochem. Biophys. Res. Commun. 142, 932-938.

ZHANG, X.K., WANG, Z., LEE, A., HUANG, D.P., andCHIU, J.F. (1988). Differential expression of cellular onco-

genes during rat liver development. Cancer Lett. 41, 147-155.

Address reprint requests to:Dr. Alvaro Puga

Department of Environmental HealthUniversity of Cincinnati Medical Center

3223 Eden AvenueCincinnati, OH 45267-0056

Received for publication December 17, 1991, and in revised formJanuary 13, 1992.

NOTE ADDED IN PROOF

TCDD and related compounds are known to exhibit a

broad spectrum of Ah-receptor-dependent anti-estrogenicresponses in the intact rat and in human MCF-7 cell cul-tures (Safe et ai, 1991), although the mechanism is notunderstood. The recent finding that estrogen receptor ac-

tivity can be inhibited by the AP-1 components c-JUN andc-FOS in MCF-7 cells (Tzukerman et ai, 1991), combinedwith our demonstration in the present report of TCDD-in-duced large increases in AP-1 transcription factor activity,provides a possible explanation to the anti-estrogenic ac-tion of TCDD.

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1129-1142. [CrossRef]46. Anthony F. Trombino, Richard I. Near, Raymond A. Matulka, Shi Yang, Laurie J. Hafer, Paul A. Toselli, Dong W. Kim,

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47. T Hsu. 2000. Activator protein 1 (AP-1)– and nuclear factor κB (NF-κB)–dependent transcriptional events in carcinogenesis.Free Radical Biology and Medicine 28:9, 1338-1348. [CrossRef]

48. M Schwarz. 2000. Ah receptor ligands and tumor promotion: survival of neoplastic cells. Toxicology Letters 112-113:1-3,69-77. [CrossRef]

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52. W Zhao, K.S Ramos. 1998. Modulation of Hepatocyte Gene Expression by the Carcinogen Benzo[a]pyrene. Toxicology inVitro 12:4, 395-402. [CrossRef]

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55. Andrei A. Panteleyev, Renate Thiel, Reinhard Wanner, Juan Zhang, Viadmir S. Roumak, Ralf Paus, Diether Neubert,Beate M. Henz, Thomas Rosenbach. 1997. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Affects Keratin 1 and Keratin17 Gene Expression and Differentially Induces Keratinization in Hairless Mouse Skin. Journal of Investigative Dermatology108:3, 330-335. [CrossRef]

56. L Zhang. 1997. Benzo(a)pyrene, but Not 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Alters Cell Proliferation and c-Mycand Growth Factor Expression in Human Placental Choriocarcinoma JEG-3 Cells. Biochemical and Biophysical ResearchCommunications 231:1, 117-120. [CrossRef]

57. Elena Ainbinder, Svetlana Bergelson, Ron Pinkus, Violet Daniel. 1997. Regulatory Mechanisms Involved in Activator-Protein-1 (AP-1)-Mediated Activation of Glutathione-S-Transferase Gene Expression by Chemical Agents. EuropeanJournal of Biochemistry 243:1-2, 49-57. [CrossRef]

58. Thomas A. Gasiewicz. 1997. Exposure to dioxin and dioxin-like compounds as a potential factor in developmental disabilities.Mental Retardation and Developmental Disabilities Research Reviews 3:3, 230-238. [CrossRef]

59. Michael Sjögren, Lars Ehrenberg, Ulf Rannug. 1996. Relevance of different biological assays in assessing initiatingand promoting properties of polycyclic aromatic hydrocarbons with respect to carcinogenic potency. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 358:1, 97-112. [CrossRef]

60. VASILIS VASILIOU, CHRISTINE A. KOZAK, RONALD LINDAHL, DANIEL W. NEBERT. 1996. MouseMicrosomal Class 3 Aldehyde Dehydrogenase: AHD3 cDNA Sequence, Inducibility by Dioxin and Clofibrate, and GeneticMapping. DNA and Cell Biology 15:3, 235-245. [Abstract] [Full Text PDF] [Full Text PDF with Links]

61. John Whysner, Gary M. Williams. 1996. 2,3,7,8-Tetrachlorodibenzo-p-dioxin mechanistic data and risk assessment: Generegulation, cytotoxicity, enhanced cell proliferation, and tumor promotion. Pharmacology & Therapeutics 71:1-2, 193-223.[CrossRef]

62. M Andersen. 1995. Negative selection in hepatic tumor promotion in relation to cancer risk assessment. Toxicology 102:1-2,223-237. [CrossRef]

63. K Ramos. 1995. Growth-related signaling as a target of toxic insult in vascular smooth muscle cells: Implications inatherogenesis. Life Sciences 57:7, 627-635. [CrossRef]

64. Nancy E. Zorn, Diane H. Russell, Arthur R. Buckley, Marie D. Sauro. 1995. Alterations in splenocyte protein kinase C(PKC) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vivo. Toxicology Letters 78:2, 93-100. [CrossRef]

65. R. Mehta. 1995. The potential for the use of cell proliferation and oncogene expression as intermediate markers during livercarcinogenesis. Cancer Letters 93:1, 85-102. [CrossRef]

66. P. Fernandez-Salguero, T. Pineau, D. Hilbert, T. McPhail, S. Lee, S. Kimura, D. Nebert, S. Rudikoff, J. Ward, F. Gonzalez.1995. Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268:5211,722-726. [CrossRef]

67. Jae-Ho Yang, Johng S. Rhim. 1995. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: molecular mechanism of carcinogenesis and itsimplication in human in vitro model. Critical Reviews in Oncology/Hematology 18:2, 111-127. [CrossRef]

68. Stephen H. Safe. 1995. Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacology & Therapeutics 67:2, 247-281. [CrossRef]

69. Anil K. Jaiswal. 1994. Antioxidant response element. Biochemical Pharmacology 48:3, 439-444. [CrossRef]70. DANIEL W. NEBERT, ALVARO PUGA, VASILIS VASILIOU. 1993. Role of the Ah Receptor and the Dioxin-Inducible

[Ah] Gene Battery in Toxicity, Cancer, and Signal Transduction. Annals of the New York Academy of Sciences 685:1Immunomodulat, 624-640. [CrossRef]

71. Violet Daniel. 1993. Glutathione S-Transferases: Gene Structure and Regulation of Expression. Critical Reviews inBiochemistry and Molecular Biology 28:3, 173-207. [CrossRef]