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Carcinogenesisvol.19 no.9 pp.15831589, 1998

Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-B DNAbinding activity and DNA damage in HepG2 cells

Clement A.Musonda and James K.Chipman1

School of Biochemistry, The University of Birmingham, Edgbaston,Birmingham B15 2TT, UK

1To whom correspondence should be addressedEmail: [email protected]

We have investigated the effect of the plant-derivedflavonoid quercetin in relation to potential oxidant andantioxidant activity on nuclear factor B (NF-B) binding

activity and DNA integrity in HepG2 cells. Gel mobilityshift assays using a -32P-labelled NF-B oligonucleotideprobe showed that treatment of HepG2 cells with quercetin(up to 10M, sub-cytotoxic) did not elevate NF-B bindingactivity of nuclear extract protein but did inhibit bindingactivity of an extract from cells treated with the oxidantH2O2. A similar inhibition by quercetin of H2O2-inducedNF-B transcriptional activation was demonstrated usinga cat reporter gene assay. Considering oxidative DNAdamage, using single cell gel electrophoresis (comet) assaywe have demonstrated that quercetin (10 M and below)did not induce DNA strand breaks. However, a markedand statistically significant (P < 0.01 at 10 M) inhibitionof strand breakage produced by H2O2 was detected. The

specific formation of 8-oxo-2-deoxyguanosine (8-oxodG)in calf thymus DNA exposed to either -irradiation or theFenton reaction system was also inhibited (P < 0.01 at 10M) by quercetin in a dose-dependent manner. This wasnot accompanied by formation of 8-oxodG by quercetinitself. The inhibition of 8-oxodG formation by-irradiationwas more potent (IC50 0.05M) than that by the Fentonreaction (IC50 0.5 M), implying that the mechanism ofprotection may be different between the two systems. Theinhibition of both NF-B binding activity and oxidativeDNA damage suggests that its antioxidant potential out-weighs its oxidative potential in a cellular environment,which may contribute to anticarcinogenic and anti-

inflammatory effects.

Introduction

Quercetin, a naturally occuring plant-derived flavonoid, hasbeen at the centre of several genetic toxicity and carcinogenicityinvestigations. Quercetin possesses a wide range of bothpotentially detrimental and protective biological characteristics.For example, it has been reported to inhibit rat mammarycarcinogenesis induced by 7,12-dimethylbenz[a]anthraceneand N-nitrosomethylurea (1,2). In addition, Hertog andHollman recently reviewed the potential health effects of

Abbreviations:8-oxodG, 8-oxo-2-deoxyguanosine; 8-oxoG, 8-oxoguanine;

AP-1, activator protein-1; CAT, chloramphenicol acetyltransferase; DMF,N,N-dimethylformamide; EMSA, electrophoretic mobility shift assay; NF-B,nuclear factor B; PBS, phosphate buffered saline; ROS, reactive oxygenspecies.

Oxford University Press 1583

quercetin (3). However, other reports have shown quercetin tobe mutagenic in the Ames assay (4,5) and carcinogenic at highdoses (6). Moreover, the micronucleus test for genotoxicityprovided contradictory findings (7,8). In vitro quercetin hasthe potential to produce reactive oxygen species (ROS) (9,10),but has also been reported as an antioxidant (11). Consequently,the balance between its reported harmful and potential protect-ive effects on human health, particularly in relation to ROS,remains to be elucidated.

ROS have been reported to play a key role in carcinogenesisby inducing oxidative DNA damage (1214). The abundantoxidative DNA product, 8-oxo-2-deoxyguanosine (8-oxodG),has been reported to be highly mutagenic (14). In addition,ROS are thought to contribute to carcinogenesis throughinterference with signal cascade systems (15,16), includingnuclear factor B (NF-B). NF-B has been associated withcellular proliferation as well as apoptosis and induction of itsactivity has been directly linked with cellular transformation(17,18).

NF-B has been implicated in the inducible expression ofa wide range of genes in response to oxidative stress (16).Conversely, antioxidants have been reported to inhibit itsactivation (19). NF-B transcription factor has also been

reported to activate the expression of a variety of genesinvolved in inflammatory, immune and acute phase responses(20). The inactive form of NF-B is localized in the cytoplasmand consists of three subunits: DNA binding p50 and p65subunits and an inhibitory subunit, called IB, which is boundto p65 (21,22). IB masks the nuclear localization sequenceand its release initiates activation of NF-B and its subsequenttranslocation to the nucleus, where it can bind to target sitesin DNA (23,24). The classical activated DNA-binding NF-Bis, therefore, a heterodimer consisting of the two subunits p50and p65 (Rel A) (22). Both subunits are members of a largerclass of transcription factors called the Rel family (25).

ROS (as well as other extracellular signals, such as cytokines,viruses and liposaccharides) may activate NF-B via phospho-

rylation of IB by specific kinases (15,26). Phosphorylationof IB renders it susceptible to ubiquitination and subsequentproteolytic degradation by the proteosome (27). There mayalso be other effects of ROS on IB (28).

Hydrogen peroxide (H2O2) has been reported to both enhanceNF-B transcription factor activity in HeLa cells and to induceDNA strand breaks after conversion to the hydroxyl radicalvia the Fenton reaction (12,29,30). In the current study wehave used human-derived HepG2 cells to assess the effects ofquercetin on H2O2-mediated activation of NF-B transcriptionfactor activity as well as on DNA integrity and on 8-oxodGformation in calf thymus DNA exposed to either -irradiationor the Fenton reaction system.

Materials and methodsChemicals

All chemicals, unless otherwise stated, were of the highest quality obtainablefrom either Sigma-Aldrich Chemical Co. (Poole, UK), Promega (Southampton,


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C.A.Musondaand J.K.Chipman

Fig. 1. Effect of quercetin on H2O2-induced NF-B DNA binding activity. (A) Controls including vehicle (0.1% v/v dimethyl sulphoxide) and cold NF-Band AP-1 probes. (B and D) Quantitative analyses of the gels by phosphorimaging analysis. (C) Gel shift mobility assays were with nuclear extracts fromHepG2 cells treated with quercetin and/or H2O2 for 90 min. C.P.M., counts per min of the band shifted by binding of NF-B to the probe. Data are theaverage from two independent experiments. Dimethyl sulfoxide vehicle 0.1% (in the absence of quercetin) did not reduce the effect of H2O2.

UK) or Stratagene (Cambridge, UK). Plasmids pHIV-CAT and pHIV-CATKB, containing NF-B-responsive element consensus sites from the humanimmunodeficiency virus long terminal repeat fused to the cat gene, werekindly provided by Dr Gary J.Nabel (University of Michigan Medical Centre,Ann Arbor, MI).

Cell culture

HepG2 cells (European Collection of Animal Cell Cultures, Salisbury, UK)were routinely grown in Williams E medium supplemented with 10% (v/v)foetal calf serum, 2 mM glutamine, 0.25 mM cysteine, 100 U/ml penicillinand 100 g/ml streptomycin.

Cell extraction and electrophoretic mobility shift assays (EMSAs)

HepG2 cells were washed with phosphate-buffered saline (PBS) and freshserum-free medium containing quercetin (0.1, 1.0 or 10.0 M), and H2O2(150 M) was added. The cells were then incubated at 37C for 90 min.Nuclear extracts were prepared as described previously (31). We have shownpreviously that quercetin is not cytotoxic at these concentrations but is

cytotoxic at 50 M (32). The concentration of H2O2 used has been usedpreviously for activation of NF-B (15,29).

DNA binding reaction mixtures (10 l) contained 10 mM TrisHCl, pH 7.5,50 mM NaCl, 1 mM EDTA and 2 g poly(dIdC). Nuclear extracts (10 g) were

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added prior to [-32P]ATP-labelled NF-B oligonucleotide probe (9.0 pmol)

(Amersham, UK). The reaction mixtures were incubated with the probe

(20 min) followed by analysis of DNA-binding activities by mobility shift assay

(33). Complexes were analysed by non-denaturing 4% PAGE. Quantitativeevaluation of NF-BDNA complex formation was determined by Molecular

Dynamics PhosphorImager analysis.

HepG2 cells transfection and cat activity assay

HepG2 cells were transfected at 80% confluency by a liposome-mediated

method (34). Briefly, the HepG2 cells were plated in 100 mm culture plates

(Corning) at a density of 12105 cells/plate and incubated at 37C overnight.

The medium was then replaced with fresh medium (6 ml) containing Tf20

reagent (Promega, UK) and 10 g plasmid pHIV-CATKB (control) or pHIV-

CAT containing the NF-B long terminal repeats (test), and the cells incubated

at 37C for 1 h. Fresh medium (12 ml) containing either quercetin (10 M)

and/or H2O2(150M) was gently added and the plates were incubated further

for 48 h at 37C.

The cells were scraped (rubber policeman) and assayed for CAT activityas described previously (35). In brief, cell extract (100 g protein) obtained

from treated or untreated cells after three cycles of freeze (70C) and thaw

(37C) were mixed with 27 mM acetyl-CoA (Sigma, Poole, UK) in 1 M Tris


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Quercetin inhibits NF-B activity and DNA damage

Fig. 2. Inhibition of NF-B-responsive element-driven CAT activity byquercetin in HepG2 cells. (A) Chromatogram of a representative CAT assaywith cell extracts from control pHIV-CAT KB (lane 5) and test pHIV-CAT(lanes 25) transfectant cells with or without treatment. 3AcCm and 1AcCmrefer to the quantity of acetylated products from chloramphenicol (Cm).Cells were exposed to either quercetin (10 M; lane 2), H2O2 (150 M;lane 1) or both in combination (lane 3) for 48 h and analysed for CATactivity as described in Materials and methods. (B) Histogram of quantifiedCAT activities (average SEM from two independent experiments). pHIV-

CATKB and pHIV-CAT represent plasmids containing the deleted andwild-type NF-B-responsive element consensus sites respectively.

HCl, pH 7.5, followed by addition of 1-deoxy[dichloroacetyl-1-14C]chloram-phenicol (2 l, 57 mCi/mmol; Amersham Life Science, Little Chalfont, UK).The mixture was incubated at 37C for 90 min and 0.6 ml ethyl acetate wasadded to extract the chloramphenicol. The organic layer was dried and takenup in 30 l ethyl acetate, spotted on thin layer silica gel plates (BDH,Lutterworth, UK) and run with chloroform/methanol (95:5 v/v). After autora-diography, spots were quantified by phosphorimaging. Activity of CAT wasmeasured by determining the amount of acetylated chloramphenicol producedfrom 1-deoxy[dichloroacetyl-1-14C]chloramphenicol.

H2O2-induced DNA damage in HepG2 cells

To induce DNA damage, HepG2 cells (initially seeded at 0.81106 cells/flask) grown in 25 cm2 flasks were washed twice in PBS, pH 7.4, and then

treated with Ara C for 30 min to stop DNA polymerase activity beforeexposure to H2O2 (200 M) in the presence or absence of quercetin (0.1, 1.0or 10 M) in serum-free medium for 30 min at 37C in 5% CO 2/95% air.The cells were washed twice with PBS, pH 7.4, and then trypsinized (0.05%

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trypsin in 0.02% EDTA). The cells were gently spun at 200 g for 3 min at

4C and resuspended in low melting point agarose (0.5%) for comet analysis.

Single cell gel electrophoresis (comet) assay

DNA breaks were detected using an adaptation of the method described

previously (36,37). HepG2 cells treated with quercetin or H2O2 were suspendedin 100 l 0.5% (w/v) low melting point agarose in PBS, pH 7.4, at 37C and

immediately pipetted onto frosted glass microscope slides precoated with alayer of 0.5% (w/v) normal melting point agarose similarly prepared in PBS.The agarose was allowed to set for 10 min on ice and then the slides were

immersed in lysing solution (2.5 M NaCl, 100 mM Na 2EDTA, 10 mM Tris,

pH 10.0, with NaOH, 10% v/v dimethyl sulfoxide and 1% v/v Triton X-100)

at 4C for 1 h to remove cellular proteins. Slides were then placed in adouble row in a 260 mm wide horizontal electrophoresis tank containing

electrophoresis buffer (75 mM NaOH and 1 mM Na 2EDTA) for 20 min before

electrophoresis at 25 V for 20 min. The slides were then washed three times

for 5 min each with neutralizing solution (0.4 M TrisHCl, pH 7.5) at 4Cbefore staining with 50 l ethidium bromide (20 g/ml).

Quantification of the comet assay

Ethidium bromide stained slides were examined with a Zeiss Axiovert 10fluorescence microscope (Zeiss, Germany). One hundred comets on each slide

were quantified with computerized image analysis using Komet 3.0 (Kinetic

Imaging Ltd, Liverpool, UK) according to the relative intensity of fluorescencein the tail and results were grouped according to the criteria as described

previously (38).

Exposure of DNA to FeCl2/H2O2 (Fenton reaction system)

The protection against 8-oxodG formation in calf thymus DNA (reported to

be highly susceptible to treatment with H2O2) (39) was investigated in this

part of the study. Calf thymus DNA was solubilized in ultra high quality

water to a concentration of 500 g/ml. Reaction mixtures (made up to 1.0 mlwith PBS) containing DNA (125 g), FeCl2 (250 M), H2O2 (100 M) and

different concentrations of quercetin (0.1, 1.0, 10 or 100 M) in 0.01% (v/v)

dimethylformamide (DMF) were incubated for 15 min at 37C. DMF was used

as the vehicle solvent for quercetin in reaction mixtures. This concentration of

DMF was found in our laboratory not to affect H2O2-induced 8-oxodG

formation (A.Harper, unpublished data). The reaction was terminated by

adding 100 l 0.3 M sodium acetate and 4.0 ml ice-cold ethanol. The DNA

was precipitated at 20C overnight followed by centrifugation (1000 g;

15 min). The DNA pellets were dissolved in 5 mM sodium citrate, 20 mMsodium chloride buffer, pH 6.5, with HCl and centrifuged (2000 g, 10 min at

4C). The supernatant was used for 8-oxodG analysis.

Exposure of DNA to -irradiation

The solutions containing DNA (125 g in 1 ml) and different concentrations

of quercetin dissolved in DMF (0.1%) were irradiated at 4C at a dose rate

of 2.34 Gy/min for 9 min from a 60Co source. DNA was then precipitated as

described above.

Determination of 8-oxodG and deoxyguanosine (dG)

The amount of 8-oxodG and dG was measured using HPLC with electrochem-

ical and UV detection, respectively (40). Briefly, DNA (50 g) in 100 l

5 mM sodium citrate, 20 mM sodium chloride, pH 6.5, was heated to 95C

for ~4 min. The solution was cooled on ice and incubated with 20 mM sodium

acetate buffer, pH 4.8, containing 0.1 mM ZnCl2 and 5 U nuclease P1 at

45C for 30 min, followed by incubation with 90l 50 mM TrisHCl, pH 7.4,

and 3 U alkaline phosphatase at 37C for 1 h. The samples were centrifuged

as described above. The DNA hydrolysates (50 l) were injected onto a

reverse phase C18HPLC column. The mobile phase contained 12.5 mM citric

acid, 25 mM sodium acetate, 30 mM NaOH and 10 mM acetic acid, pH 5.1,

with a flow rate of 1 ml/min. A Pye Unicam LC-UV detector was used for

quantification of dG and a LC-4B amperometric detector (Bioanalytical

System, Luton, UK) with temperature control was used for detection of

8-oxodG.

The amounts of 8-oxodG and dG were calculated from the peak area based

on their corresponding standards (40) and the results expressed as the ratio

of 8-oxodG compared with dG.

Statistical analysis

The per cent tail DNA has been suggested as the most appropriate parameter

for quantifying DNA strand breaks and comet data have been described as

not following a normal distribution (38,41), thus, the comet data were analysed

using a non-parametric test (MannWhitney) as well as a parametric test(ANOVA) using Minitab software. The latter test was also used for assessment

of 8-oxodG. Results were expressed as means SEM and significance was

accepted at P 0.05.


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Fig. 3. Effect of quercetin on 8-oxodG formation by -irradiation (A) and the Fenton reaction (B). Values represent the means

SDs of three separateexperiments (analysed in duplicate). Results are expressed as per cent of the control which contained the vehicle (0.01% v/v DMF). The background level of8-oxodG was 0.001% of dG. Positive controls gave 0.005% (-irradiation) and 0.015% (Fenton reaction). Solvent (0.01% v/v DMF) alone did notsignificantly elevate the control value (90% of control).

Results

Effect of quercetin on H2O2-induced NF-B binding activity

A gel shift assay showed inhibition of H 2O2-induced NF-Bactivation by quercetin in HepG2 cells. In accord with otherreports (15), a major specific shifted band of probe DNA wasobserved on addition of control cell extracts (Figure 1A, lanes1 and 2). Specificity was confirmed by loss of the band onaddition of excess cold NF-B but not by addition of anequivalent amount of cold activator protein-1 (AP-1) probe(Figure 1A, lanes 3 and 4, respectively). Quantification of theintensity of the shifted band was achieved by phosphorimaging(Figure 1B and D). H2O2 (150 M) elevated NF-B activity(Figure 1C and D, lane 4) compared with controls and quercetininhibited H2O2-induced NF-B activity in HepG2 cells (lanes14).

The effect of quercetin on NF-B-responsive element-drivenCAT activity in HepG2 cells was also investigated. H2O2produced approximately a doubling of the response seen incontrol cells transfected with plasmid pHIV-CAT KB (lackingthe NFB-responsive element) (Figure 2) but in the presence

of quercetin (10M) the effect of H2O2was reduced by ~50%.An identical pattern of response was seen in two independentexperiments.

Effect of quercetin on calf thymus DNA exposed to the Fentonreaction system and -irradiation

Conversion of dG to 8-oxodG in intact cells by H2O2 wasfound not to be at a sufficient level to investigate protectiveeffects of quercetin. Quercetin inhibited both Fenton reaction-and -irradiation-induced 8-oxodG formation in a dose-depend-ent manner. The inhibition of -irradiation-induced 8-oxodGformation was more potent (IC50 0.05M) than that by theFenton reaction (IC50 0.5 M) (Figure 3). Quercetin inhibited90% of-irradiation-induced 8-oxodG formation, while only~60% of Fenton reaction-induced 8-oxodG formation wasinhibited under the conditions employed. Inhibition was statist-ically significant (P at least 0.05) (ANOVA).

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Effect of quercetin on DNA strand breakage in HepG2 cells

We also investigated the inhibitory effect of quercetin inHepG2 cells on H2O2-induced DNA damage using the singlecell electrophoresis (comet) assay. Quercetin (10 M) inhibitedH2O2-induced DNA damage (Figures 4 and 5) and did not itselfproduce DNA damage. Statistical significance was indicated byboth parametric and non-parametric tests (P 0.05, values

for the latter are given). However, at a cytotoxic concentrationof quercetin (100M) DNA damage was apparent (P 0.01)(Figure 6).

Discussion

Although a number of studies have suggested the potentialgenotoxicity of quercetin (4244), a few investigations havealso reported various potential beneficial effects of quercetin,especially regarding oxidative damage. Sahu and Washington(45) and Rahman et al. (10) indicate the potential for highconcentrations of quercetin to produce ROS in the presenceof metal ions and this might contribute to mutagenicity.However, Sahu (46) also recognized the dual role of quercetin

as a pro-oxidant and an antioxidant depending on its environ-ment (47). ROS are known to cause oxidative DNA damage(12,13), which is considered to play an important role incarcinogenesis and ageing (14,48). An abundant DNA oxida-tion product is 8-oxodG, which can cause misreading (49),can activate certain oncogenes such as H-ras and K-ras (50)and has been associated with cancer of several organs (forexample, see 5153). Quercetin did not elevate 8-oxodG incalf thymus DNA nor did it cause the formation of DNAstrand breaks in HepG2 cells at sub-cytotoxic concentrations.The latter technique can detect oxidative DNA damage in cells(54), such as that produced by H2O2, as demonstrated here.These data argue against an ability of quercetin to produceROS under the conditions employed. The results agree withthose of Duthie et al.(55), who found quercetin-induced DNAstrand breaks in various cell types occurred only at 100 Mquercetin or above (concentrations which were cytotoxic in


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Fig. 4. Representative comet images of HepG2 cells following treatment with different concentrations of quercetin (1100 M) and/or H2O2 (200 M).(A) Control; (B and C) treated with quercetin (1 and 10 M, respectively); (D) 100 M quercetin (cytotoxic); ( E) quercetin and H2O2 (10 and 200 M,respectively); (F) H2O2 alone (200 M). The cell population profiles are shown in Figure 5.

our study). The results are also in accord with the conclusionsof Nakayama (56). Puppo (57) noted the likelihood thatreductants in the cell would preclude the activity of quercetinin redox cycling of iron, thus providing a plausible explanation

for the apparent lack of DNA oxidation in HepG2 cells.In contrast with the lack of damage, an antioxidant influence

of quercetin was apparent. Quercetin inhibited both -irradi-ation- and Fenton reaction-induced 8-oxodG formation in calfthymus DNA in a concentration-dependent manner. It alsosuppressed H2O2-mediated DNA strand breakage in HepG2cells. Inhibition of irradiation-induced 8-oxodG formation wasmore potent than that induced by the Fenton reaction system(IC50 values of 0.05 and 0.5 M, respectively), implying thatdifferent mechanisms may be involved. These findings agreewith our recent report of an antioxidant influence on dichloro-fluorescin oxidation in HepG2 cells (32). Supportive evidencefor quercetin as an antioxidant in cells comes from studies ofits ability to scavenge O2

and OH based on the action ofhydroxyl groups in the aromatic B ring (5861). However,iron chelation (11,62) may contribute to the antioxidant effectin the case of our studies using Fenton chemistry, but not with

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damage induced by irradiation. These effects have recentlybeen discussed by Paganga et al. (63). A further possibility isthat quercetin binding to DNA (64) may mask oxidation targetsites. This protective effect against DNA oxidation may

contribute to the reported cancer chemopreventative effect ofquercetin (1).

The balance between oxidative and antioxidative influencesof quercetin was also assessed by studying the effect on NF-B transcription factor. The involvement of ROS in activationof NF-B is now well established and accepted (28,65,66) andthis has been strongly supported by the observations thatantioxidants inhibit NF-B activity (15,29). We found thatquercetin did not enhance the level of NF-B transcriptionfactor as assessed in nuclear extracts by gel shift mobilityassays and by the use of a transient transfection and reportergene assay for NF-B activation in HepG2 cells. Again, incontrast, quercetin inhibited H2O2-mediated NF-B activity.Enhanced NF-B DNA binding activity is linked to chronicinflammatory diseases (20) and thus its inhibition by quercetinmay relate to the anti-inflammatory effect of the flavonoid(67,68). However, it is important to note that prolonged


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Fig. 5. Inhibition by quercetin of H2O2-mediated DNA damage (cometassay) in HepG2 cells. Grade of damage (% DNA in tail) was categorizedas follows: none, 5%; low, 520%; medium, 2040%; high, 4095%;total, 95% with respect to DNA. Cells were treated with H 2O2 (200 M)or quercetin (10 M) (or both) and compared with controls (containing0.1% v/v dimethyl sulfoxide vehicle). Two hundred cells were scored intotal (two separate experiments). Using mean per cent tail DNA values,H2O2-treated cells were significantly different from control (P 0.001) andquercetin gave a significant inhibition (P 0.01) of the H2O2-inducedeffect.

Fig. 6. The effect of quercetin on DNA damage (comet assay) in HepG2cells. One hundred cells were scored per concentration in each of twoseparate experiments and mean values were used to categorize damage asindicated in Figure 5. The level of damage to DNA (as in Figure 5) wasassessed by the per cent of DNA in the comet tail.

inhibition of its activity may be detrimental, since it is involvedin the regulation of immune and defence genes (20).

Acknowledgements

We are indebted to Drs Tanya Franklin and Aristides G.Eliopoulos for thehelp with the CAT assay as well as Ms Jane Davies for her excellent technical

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assistance. We thank Dr Gary Nabel for providing the plasmids containingNF-B response elements. This work is part of the doctoral thesis ofC.A.M. and was supported by an Association of Commonwealth UniversitiesScholarship.

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Received on February 25, 1998; revised on May 7, 1998; accepted on

May 7, 1998

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