2,3,7,8-Tetrachlorodibenzo-p-Dioxin Counteracts the p53 Response to a Genotoxicant by Upregulating...

TOXICOLOGICAL SCIENCES 115(2), 501–512 (2010)

doi:10.1093/toxsci/kfq082

Advance Access publication March 18, 2010

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Counteracts the p53 Response toa Genotoxicant by Upregulating Expression of the Metastasis Marker

AGR2 in the Hepatocarcinoma Cell Line HepG2

Ariane Ambolet-Camoit,*,† Linh Chi Bui,*,† Stephane Pierre,*,† Aline Chevallier,*,† Alexandre Marchand,*,†,1

Xavier Coumoul,*,† Michele Garlatti,*,† Karine Andreau,§,{ Robert Barouki,*,†,‡ and Martine Aggerbeck*,†,2

*INSERM UMR-S 747, 75006 Paris, France; †Universite Paris Descartes, Centre Universitaire des Saints-Peres, 75006 Paris, France; ‡APHP, Hopital Necker

Enfants Malades, Service de Biochimie Metabolique, 75015 Paris, France; §CNRS EAC 4413, Unite de Biologie Fonctionnelle et Adaptative (BFA), 75013

Paris, France; and {Universite Paris Diderot-Paris 7, 75013 Paris, France

1 Present address: INSERM UMR-S 956, 75013 Paris, France.2 To whom correspondence should be addressed at INSERM UMR-S 747, Toxicologie, Pharmacologie et Signalisation Cellulaire, Centre Universitaire des

Saints-Peres, Universite Paris Descartes, 45 rue des Saints-Peres, 75006 Paris, France. Fax: þ33 1-42-86-38-68. E-mail: [email protected].

Received December 7, 2009; accepted March 5, 2010

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmen-

tal pollutant that binds the aryl hydrocarbon receptor (AhR),

a transcription factor that triggers various biological responses. In

this study, we show that TCDD treatment counteracts the

p53 activation (phosphorylation and acetylation) elicited by

a genotoxic compound, etoposide, in the human hepatocarcinoma

cell line HepG2 and we delineated the mechanisms of this

interaction. Using small interfering RNA knockdown experi-

ments, we found that the newly described metastasis marker,

anterior gradient-2 (AGR2), is involved in this effect. Both AGR2

messenger RNA (mRNA) and protein levels were increased

(sixfold and fourfold, respectively) by TCDD treatment, and this

effect was mediated by the AhR receptor. The half-life of AGR2

mRNA was unchanged by TCDD treatment. Analysis of the

promoter of the AGR2 gene revealed three putative xenobiotic-

responsive elements (XREs) in the proximal 3.5-kb promoter.

Transient transfection of HepG2 cells by the Gaussia luciferase

reporter gene driven by various deleted and mutated fragments of

the promoter indicated that only the most proximal XRE was

active. Binding of the AhR to the endogenous AGR2 promoter

was also triggered by TCDD treatment. These results suggest that

AhR ligands such as TCDD might contribute to tumor pro-

gression by inhibiting p53 regulation (phosphorylation and

acetylation) triggered by genotoxicants via the increased expres-

sion of the metastasis marker AGR2.

Key Words: AGR2; TCDD; AhR; etoposide; p53.

Delineating the effects of combinations of toxicants (cocktail

effects) is one of the most challenging issues in current

toxicology. Since humans and multiple ecosystems are exposed

to hundreds of pollutants, risk assessment should take into

consideration the possible interactions among these chemicals.

This is clearly impractical for all the possible combinations.

However, one feasible approach would be to define the major

signaling pathways activated by toxic compounds and to assess

the interactions among those pathways using representative

activators. In the present study, we focused on the interaction

between a genotoxic compound, etoposide, and a cancer-

promoting nongenotoxic pollutant, 2,3,7,8-tetrachlorodibenzo-

p-dioxin (TCDD), and we found that anterior gradient-2

(AGR2) inhibits the posttranslational modifications of the

p53 protein elicited by etoposide.

TCDD is a ubiquitous chemical in the environment, which

accumulates in animal and human tissues. TCDD has been

classified as a group 1 human carcinogen by the International

Agency for Research on Cancer and as likely to be

carcinogenic to humans by the Environmental Protection

Agency in the United States because it is suspected to play

a role in cancer development (Kaiser, 2000). Most TCDD

effects are mediated by the aryl hydrocarbon receptor (AhR)

(Gu et al., 2000). Upon binding to its ligands, cytoplasmic

AhR, which is bound to several chaperone proteins (Petrulis

and Perdew, 2002; Ramadoss and Perdew, 2005), undergoes

a conformational change that leads to its dissociation from the

chaperone proteins and its translocation into the nucleus

where it dimerizes with its partner Arnt (AhR receptor

nuclear translocator; Swanson, 2002). The heterodimer binds

to xenobiotic-responsive elements (XREs) in the promoters of

target genes resulting in the upregulation of these genes.

TCDD is well known for upregulating the genes encoding the

enzymes involved in the metabolism of xenobiotics, such as

cytochrome P450 (CYP) 1A1 (Xu et al., 2005). However,

a growing body of literature indicates that the AhR controls

the expression of a wide variety of genes that are unrelated to

the metabolism of xenobiotics (reviewed in Kung et al.,2009).

� The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For permissions, please email: [email protected]

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Etoposide is an antineoplastic compound that inhibits

topoisomerase II activity, thus leading to single-strand DNA

breaks and p53 activation. Previous studies have shown that

TCDD attenuates the p53 response to DNA-damaging agents

in HepG2 cells (Paajarvi et al., 2005). In human keratinocytes,

TCDD was shown to attenuate senescence and repress p53

expression leading to immortalization of the cells (Ray and

Swanson, 2004). In AhR�/� fibroblasts transfected with the

AhR under TET-OFF control, AhR activation inhibits E2F1-

induced apoptosis (Marlowe et al., 2008). Although cross talk

between the apoptosis/cell cycle pathways and the AhR

signaling pathway has been described (see Puga et al., 2009

for review), this appears to be a species- or cell type–dependent

phenomena (Marlowe and Puga, 2005) and the molecular

mechanisms are only partially understood.

We hypothesized that the interaction between the TCDD/

AhR pathway and the p53 pathway could be mediated through

the product of an AhR target gene. Recent high-throughput

analyses of TCDD-regulated gene expression in HepG2 cells

have revealed novel targets for this pollutant, which may

account for some features of its toxicity (Frueh et al., 2001;

Kim et al., 2006; Puga et al., 2000). In particular, the

regulation of several genes involved in cell motility may

account for the toxicity of TCDD observed during develop-

ment and for its effects on genes promoting cancer progression

(Diry et al., 2006). For the purposes of this study, we focused

on the AGR2 gene because previous studies showed that

overexpression of AGR2 attenuates p53 serine phosphorylation

(Pohler et al., 2004). Our microarray analyses have revealed

a 10-fold increase of AGR2 gene expression in HepG2 cells

following 30 h of treatment with 25nM TCDD (Marchand and

Garlatti, unpublished results).

Human AGR2, also designated as hAG2 and as GOB-4 in

the mouse (Komiya et al., 1999), are orthologues of the

secreted Xenopus laevis protein (XAG-2) (Aberger et al.,1998). In this species, the protein is involved in the

differentiation of the mucus-secreting cement gland and the

pattern of anterior neural tissues. In humans, AGR2 expression

is increased in various cancers (Innes et al., 2006; Zhang et al.,2005) and, recently, the protein was suggested to be a potential

oncogene in lung cancer (Fritzsche et al., 2007; Zhu et al.,2007). In addition, overexpression of AGR2 silences p53

activity in ultraviolet (UV)-exposed lung cancer cells (Pohler

et al., 2004). In the present study, we found that TCDD

treatment increased AGR2 protein and messenger RNA

(mRNA) levels and elicited an AhR-mediated activation of

the AGR2 gene promoter. We also provide evidence that

TCDD attenuates p53 phosphorylation and acetylation as well

as caspases-3/7 activation by etoposide and, using small

interfering RNA (siRNA) knockdown experiments, we show

that these effects of TCDD are mediated by the upregulation of

AGR2. Thus, AGR2 appears to be a novel participant involved

in the interaction between the TCDD/AhR pathway and the

etoposide/p53 pathway in HepG2 cells.

MATERIALS AND METHODS

Cell culture and chemicals. The human hepatocarcinoma cell line HepG2,

obtained from the ATCC (# HB-8065), was cultured as described previously

(Bui et al., 2009). TCDD was purchased from Promochem (Molsheim, France).

Benzo(a)pyrene (B(a)P) and etoposide were from Sigma-Aldrich (France).

dimethylsulfoxide (DMSO) 0.15% (Merck) was used as a control. Oligonu-

cleotides were purchased from Operon or Eurogentec (France).

RNA preparation. For most experiments, 0.4 million cells were seeded

into six-well plates and treated, or not, 2 days later with the compounds

indicated in the figures. RNA was prepared using the RNeasy mini kit from

Qiagen (France) as described (Bui et al., 2009).

Quantitative reverse transcriptase-PCR. Reverse transcription was per-

formed using the High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems, France) as described (Bui et al., 2009). Complementary DNAs

(cDNAs) were aliquoted and stored at �80�C. Quantitative PCR was

performed with 40 ng of cDNA as described previously (Bui et al., 2009).

The AGR2 and reference gene RPL13A, a ribosomal gene mRNA, amounts

were measured. The primers were: AGR2 forward 5#-GCCATCAGGA-

GAAAGGTG-3# and reverse 5#-GCCAAAAAGGACACAAAGG-3# and

RPL13A forward 5#-CCTGGAGGAGAAGAGGAAAGAGA-3# and reverse

5#-GAGGACCTCTGTGTATTTGTCAA-3#.

Cellular protein extracts. Proteins were prepared from cells seeded under

the same conditions as for the mRNA experiments. The cells were washed

twice with PBS and scraped into 200 ll of M-Per buffer (Pierce, France)

containing 0.15M NaCl and protease and phosphatase inhibitors (Sigma). The

cellular homogenate was centrifuged for 15 min at 13,400 g at 4�C. The

protein concentration was measured by the bicinchoninic acid method (Pierce)

using bovine serum albumin (fraction V) as a standard. The protein extract

was stored at �80�C.

Western blots. Samples (20–40 lg of extract) and 8 lg of protein ladder

(Fermentas, France) were electrophoresed in a 15 or 12% polyacrylamide, 0.1%

SDS gel. Proteins were transferred onto nitrocellulose Hybond C extra

membranes (GE Healthcare, France). Blots were incubated for 120 min in 0.2%

I-Block solution (Applied Biosystems) and 0.1% Tween-20 in 13 PBS at room

temperature. Primary antibodies (anti-AGR2, Abnova, 1/1000; anti-phospho

p53 [Ser15], Cell Signaling, 1/1000; anti-acetylated p53 [Lys382], Abcam,

1/1000; anti-p53, Cell Signaling, 1/1000 or anti-actin, Abcam, 1/20,000; anti-

AhR [ab2770], Abcam, 1/1000, France) were incubated overnight at 4�C and

rinsed five times (5 min) with 0.1% Tween-20 in 13 PBS at room temperature.

Signals were visualized, and quantification was carried out as previously

described using actin to normalize the signals (Bui et al., 2009).

mRNA stability. HepG2 cells were treated with either the vehicle (0.15%

DMSO) or 25nM TCDD for 30 h. 5,6-Dichloro-1b-ribofuranosyl benzimid-

azole (DRB; Sigma-Aldrich) (10lM) was then added to the cells, and mRNA

was prepared at various times after this addition.

Plasmids and transient transfections. Fragments of the AGR2 gene

promoter were obtained by PCR and subcloned into the TOPO TA cloning

vector (Invitrogen, France). Plasmids were prepared (QIAprep Spin Miniprep

Kit; Qiagen), and after DNA digestion, the promoter fragment was cloned into

the pGLuc vector (Biolabs, Ozyme, France). Mutagenesis was performed using

the Quick Change XL site-directed mutagenesis kit from Stratagene (France).

The PCR products were purified (Mini-Elute PCR purification kit; Qiagen) and

cloned in the pGLuc vector as described above. Mutations were verified by

DNA sequencing (GATC, France).

HepG2 cells (0.4 million cells per well in six-well plates) were transiently

transfected in duplicate or triplicate by the lipofectamine-2000 reagent, using

1 lg DNA per well, according to the manufacturer’s instructions (Invitrogen).

Sixteen hours later, fresh medium containing the compounds was added to the

cells for 30 h. The Gaussia luciferase activity was assayed with the Biolabs kit

according to the manufacturer’s instructions in a Berthold luminometer.

502 AMBOLET-CAMOIT ET AL.


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Chromatin immunoprecipitation assay. HepG2 cells were seeded into

150 mm–diameter dishes 48 h before being processed for chromatin

immunoprecipitation (ChIP; ~20 3 106 cells per dish). Treatments with TCDD

(25nM) were performed for 15 and 45 min in Dulbecco’s Modified Eagle

Medium without phenol red and supplemented with 3% charcoal-treated calf

serum. The cells were rinsed with 37�C PBS, and nuclear proteins were cross-

linked to DNA for 10 min at 37�C by adding formaldehyde to a final concentration

of 1%. Cross-linking was stopped by adding glycine to a final concentration of

0.125M for 5 min on ice. The cells were collected in ice-cold PBS supplemented

with complete protease inhibitor tablets (Cat. 1873580; Roche, Mannheim,

Germany) and pelleted. Pellets were lysed in 500 ll of lysis buffer (1% SDS;

10mM EDTA; 50mM Tris-HCl, pH 8.1; and protease inhibitors) for 10 min on

ice. The solution was sonicated 10 times for 30 s each at the high position with

a Dismembrator 300 (Fisher). The soluble chromatin was then centrifuged at

13,400 g for 15 min at 4�C and then diluted 1:10 in ChIP dilution buffer (0.01%

SDS; 1.1% Triton X-100; 1.2mM EDTA; 16.7mM Tris-HCl, pH 8.1; 167mM

NaCl; and protease inhibitors). An aliquot of soluble chromatin was set aside as

the input fraction. Diluted chromatin was precleared with protein A/G Plus

agarose (Cat Sc-2003; Santa Cruz) at 4�C for 2 h with rotation. Protein A/G

agarose beads were pelleted, the supernatant was transferred to a new tube, and

incubated with 5 lg of anti-AhR antibody (Cat SA-210; BioMol) or 5 lg of

normal rabbit IgG (negative control, Cat Sc-2027; Santa Cruz) for immunopre-

cipitation overnight at 4�C with rotation. After immunoprecipitation, fresh

protein A/G agarose beads were added and incubated for 1 h at 4�C with rotation.

Agarose beads were pelleted and washed sequentially for 5 min with the

following buffers: once with buffer I (0.1% SDS; 1% Triton X-100; 2mM EDTA;

200mM Tris-HCl, pH 8.1; 150mM NaCl; and protease inhibitors), twice with

buffer II (0.1% SDS; 1% Triton X-100; 2mM EDTA; 200mM Tris-HCl, pH 8.1;

500mM NaCl; and protease inhibitors), and twice with buffer III (0.25M LiCl; 1%

nonidet-P40; 1% deoxycholate; 1mM EDTA; and 10mM Tris-HCl, pH 8.1).

Protein-DNA complexes were eluted by adding 400ll of elution buffer (1% SDS,

0.1M NaHCO3, and protease inhibitors) with incubation for 20 min at room

temperature with rotation (input samples were included). The cross-linking was

reversed by the addition of NaCl to a final concentration of 0.2M and overnight

incubation at 65�C. The DNA were purified using a PCR purification kit (Cat

28106; Qiagen) and eluted in 25ll. ChIP DNA were subjected to quantitative PCR

using the specific primers covering the region between�807 and�651 bp of the

human AGR2 promoter region (forward primer: 5#-AAATGGTCTTGAGGG-

GAAAAA-3# and reverse primer 5#-GCAACTTCCCTCCACAGAGT-3#). PCR

cycles were proceeded as follows: Taq activation (15 min), denaturation (15 s,

95�C), and annealing and extension (1 min, 60�C). The data were presented

according to the classical comparative Ct method except that we choose to present

the values obtained with the mock antibody instead of using them as a reference

(compared to classical mRNA expression studies). In each condition (Input, AhR

ChIP, and Mock ChIP), an untreated sample was used as a control. The amount of

the AGR2 target gene in TCDD-treated cells, relative to the control sample, was

measured by the equation 2�DCt, where DCt ¼ (Ct TCDD sample � Ct control

sample). The equation thus represents the expression of the target gene in the

TCDD-treated sample, relative to its expression in the control sample.

Nuclear extract preparation and electrophoretic mobility shift assay.

HepG2 cells (3 million/10-cm dish) were treated for 90 min with 25nM TCDD

or 0.15% DMSO. Cells were washed, scraped on ice into PBS, 1mM EDTA,

and centrifuged for 5 min at 1500 3 g at 4�C. The pellet was resuspended in

250 ll of 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes)

(pH 7.9), 50mM NaCl, 0.5M sucrose, 0.1mM EDTA, 0.5% Triton, 1mM DTT,

and 1/100 phosphatase and protease inhibitors, incubated on ice for 5 min,

centrifuged for 10 min at 150 3 g at 4�C. The nuclear pellet was resuspended

in 500 ll of 10mM Hepes (pH 7.9), 10mM KCl, 0.1mM EDTA, 0.1mM

EGTA, 0.5% Triton, 1mM DTT, and phosphatase and protease inhibitors and

centrifuged for 5 min at 150 3 g at 4�C. The pellet was resuspended in 500 ll

of 10mM Hepes (pH 7.9), 500mM NaCl, 0.1mM EDTA, 0.1mM EGTA, 0.5%

Triton, 1mM DTT, and phosphatase and protease inhibitors and mixed for

15 min at 4�C. The solution was centrifuged for 15 min at 14,000 3 g at 4�Cand the supernatant containing nuclear proteins was stored at �80�C.

The electrophoretic mobility shift assay was performed as described

(Gouedard et al., 2004). Briefly, synthetic double-stranded DNA probe

(forward strand: 5#-GGCTTCCTGCTTGCGTGGCAGGAAAGT-3#) was la-

beled with (a-32P) dCTP (PerkinElmer). The nuclear extract (15 lg) was

incubated for 20 min with a 50-fold excess of nonlabeled probe or 3 lg anti-

AhR antibody (Ab63636; Abcam) at room temperature, and the labeled probe

(corresponding to 100,000 cpm) was added for an additional 20-min

incubation. DNA-protein complexes were separated under nondenaturing

conditions on a 6% polyacrylamide gel, detected, and quantified.

AhR and AGR2 gene silencing. HepG2 cells (0.4 million cells per well in

six-well plates) were transfected with 5nM of either control siRNA or two

different siAhR (Qiagen) using HiPerfect transfection reagent according to the

manufacturer’s protocol (Qiagen) as described (Bui et al., 2009). After 12 h, the

medium was replaced by fresh medium, and 16 h later, the cells were treated, or

not, with 25nM TCDD for an additional 30 h. Control siRNA was a custom

GFP siRNA (ref 1027020), siAhR1 was the fluorescent Hs_AHR_5_HP

validated siRNA (ref SI02780148) and siAhR2 was the Hs_AHR_6_HP

validated siRNA (ref SI03043971).

For siAGR2 experiments, HepG2 cells were transfected using the ‘‘Fast-

Forward Protocol’’ (Qiagen). HepG2 cells were seeded and transfected

simultaneously with 5nM of either control siRNA (ref SR-CL000-005;

Eurogentec) or siAGR2 (ref SI00293447; Qiagen), using HiPerfect transfection

reagent. After 16 h, the cells were treated with either 0.15% DMSO or 25nM

TCDD. Twenty-four hours later, the cells were treated, or not, with either 15 or

75lM etoposide and DMSO or TCDD for an additional 24 h.

Caspases-3/7 activities assay. HepG2 cells (5000 cells per well in 96-well

plates) were seeded and transfected, or not, with control siRNA or siAGR2 in

triplicate. Cells were treated as described above in ‘‘AhR and AGR2 gene

silencing.’’ After a 48-h treatment, 100 ll of fresh medium and 100 ll of

Caspase-Glo 3/7 reagent (Promega) were added to each well. After 90-min

incubation at room temperature, the luminescence was measured in a plate-

reading luminometer (ENVision 2101 Multi-label Reader; PerkinElmer).

XTT assay. HepG2 cells (10,000 cells per well in 96-well plates) were

seeded and treated, or not, for 24 h by 25nM TCDD prior to the treatment for an

additional 24 or 48 h by various concentrations of etoposide or DMSO. Cell

viability was measured by the XTT test following the manufacturer’s

instructions (In Cytotox XTT, Xenometrix; Biogenic, France).

Statistical analysis. The data (mean ± SEM) were obtained from at least

three different experiments. A Kruskal-Wallis test (nonparametric comparison

of k independent series) was followed by an ANOVA test (parametric

comparison of k independent series, Fisher’s exact test). A value of p < 0.05 or

less was accepted as being statistically significant.

RESULTS

Interaction between TCDD and Etoposide Treatments

Treatment of HepG2 cells with etoposide led to the activation

of p53 as demonstrated by an increased phosphorylation of serine

15 (Fig. 1A) and by an increased acetylation of lysine

382 (Fig. 1B). Under these experimental conditions, the total

amount of p53 was increased by etoposide treatment as already

described by Paajarvi et al. (2005) (Fig. 1A). p53 activation may

initiate either apoptosis or cell cycle arrest and, ultimately,

senescence, depending on the severity of the damage (Kruse and

Gu, 2009). We, therefore, investigated the regulation of the

effector caspases-3/7, following the same treatment. As shown in

Figure 1C, etoposide treatment increased the caspases-3/7

activities. When combined with etoposide, TCDD prevented,

TCDD-REGULATED AGR2 INHIBITS ETOPOSIDE EFFECT 503


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partially, the increases in p53 phosphorylation and acetylation

and caspases-3/7 activities by the genotoxic agent, whereas

TCDD alone had no effect (Fig. 1). These experiments show that

the distinct signaling pathways activated by these two toxicants

interact at the level of the p53 protein hub. We next evaluated the

effect of a combination of etoposide and TCDD on the viability/

proliferation of the cells as compared to each compound alone,

by measuring the activity of the mitochondrial succinate

dehydrogenase system. As shown in Figure 1D, TCDD does

not modify the enzyme activity as compared to the vehicle

DMSO. Treatment of the cells for 24 and 48 h with 30 and 75lM

etoposide led to a statistically significant decrease of the enzyme

activity. Pretreatment of the cells for 24 h with TCDD prior to

etoposide treatment partially prevented this decrease (Fig. 1D).

In order to determine the mechanism of this cross talk, we

analyzed the gene targets of TCDD as revealed by our

microarray data and we found that the AGR2 gene, which has

previously been shown to counteract p53 activation (Pohler

et al., 2004), was one of the putative targets of TCDD in

HepG2 cells (Marchand et al., 2005; Marchand and Garlatti,

unpublished data). We, therefore, investigated the regulation of

the AGR2 gene by TCDD in order to establish its role in the

etoposide-TCDD interaction.

TCDD Increases AGR2 mRNA and Protein Expression inHepG2 Cells

HepG2 cells were treated with vehicle alone (0.15% DMSO)

or with 25nM TCDD within the concentration range typically

used for experiments with human cells (5–100nM). RNA levels

were monitored by quantitative real-time PCR (qRT-PCR). As

shown in Figure 2A, a sixfold increase in RNA levels was

found after 30 h of TCDD treatment. The increase in the

amount of mRNA first became significant at 8 h and remained

stable between 30 and 72 h (Fig. 2A). A 60-fold increase of the

FIG. 1. Interaction between etoposide and TCDD on p53 phosphorylation and acetylation and caspase-3/7 activities in HepG2 cells. (A) HepG2 cells were

pretreated with either 0.15% DMSO or 25nM TCCD for 24 h and incubated, or not, with 15lM etoposide for an additional 24 h. Western blot analysis was

performed on protein extracts, and p53 was identified with antibodies directed against total p53 or phospho p53 (Ser15). Intensities were normalized by comparison

to the actin signal (three separate experiments). There was a statistically significant difference (Fisher’s exact test) in p53 phosphorylation when the cells were treated

with etoposide plus TCDD as compared to etoposide alone (*p < 0.05). (B) HepG2 cells were pretreated with either 0.15% DMSO or 25nM TCCD for 24 h and

incubated, or not, with 75lM etoposide for an additional 24 h. Western blot analysis was performed on protein extracts and p53 was identified with an antibody

directed against acetylated-p53 (Lys382). Intensities were normalized by comparison to the actin signal (four separate experiments). There was a statistically

significant difference (Fisher’s exact test) in p53 acetylation when the cells were treated with etoposide plus TCDD as compared to etoposide alone (**p < 0.001 and

*p < 0.05). (C) HepG2 cells were pretreated with either 0.15% DMSO or 25nM TCDD for 24 h and incubated, or not, with 75lM etoposide for an additional 24 h.

There was a statistically significant increase (Fisher’s exact test) in caspases-3/7 activities following etoposide treatment as compared to DMSO as well as

a significant decrease following treatment with etoposide plus TCDD as compared to etoposide alone (*p < 0.05, **p < 0.001, and ***p < 0.0001). (D) HepG2 cells

were pretreated or not for 24 h with 25nM TCDD and treated for either 24 or 48 h with 15, 30, or 75lM etoposide. Mitochondrial succinate dehydrogenase activity

was measured (three separate experiments in triplicate). There was a significant decrease in enzyme activity following treatment with 30 and 75lM etoposide

(****p < 0.0001, ***p < 0.001, and **p < 0.01). Pretreatment with TCDD partially prevented the decrease in enzyme activity by etoposide (*p < 0.05).



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amount of mRNA for the classical AhR target gene, CYP1A1,

was found at 4 h and a 200-fold increase at 12 h (data not

shown).

The amount of AGR2 protein after treatment, as measured

by Western blot analysis, also increased in cellular extracts of

HepG2 cells treated for 8–72 h with 25nM TCDD as compared

to the vehicle. A typical Western blot is shown in Figure 2B.

Quantification showed a statistically significant threefold to

fourfold increase of the AGR2 protein level in cellular extracts

following 24–72 h of TCDD exposure (Fig. 2C).

TCDD Regulation of AGR2 Involves the AhR

Since most TCDD effects are mediated by its receptor, AhR,

we investigated the effects of another ligand of the AhR. As

shown in Figure 3A, benzo(a)pyrene was as potent as TCDD in

increasing AGR2 protein levels after 30 h of treatment

(fourfold increase).

To further assess the role of the AhR in the regulation of

AGR2 gene expression by TCDD, we used targeted gene

silencing. We verified that the AhR protein expression was

inhibited with specific AhR-targeted siRNA (Fig. 3B, right).

Figure 3B (left) shows the effect of AhR siRNA on the increase

in AGR2 mRNA levels elicited by TCDD. Transfection of the

control siRNA did not modify the upregulation of AGR2

expression by TCDD. However, a statistically significant 75%

decrease of the TCDD response was observed when AhR-

targeted siRNAs were transfected. Together, these results

clearly demonstrate the involvement of the AhR in the

regulation of AGR2 by TCDD.

AGR2 mRNA Stability Is not Modified by TCDD Treatment

In order to discriminate between an effect of TCDD on

AGR2 mRNA transcription or stability, we measured the

AGR2 half-life in HepG2 cells treated, or not, by TCDD. For

this purpose, after a 30-h pretreatment by TCDD, the cells were

treated with DRB, an RNA polymerase II inhibitor. The

amount of AGR2 mRNA was measured as a function of time.

In both control and TCDD-treated HepG2 cells, mRNA levels

declined with half-lives of 20 and 26 h, respectively, with no

statistical difference between the two groups (Fig. 4). In cells

treated with either DMSO or TCDD in the absence of DRB, the

mRNA level remained unchanged (data not shown). Hence,

TCDD did not alter mRNA stability and its effect was likely

transcriptional.

TCDD-Induced Activation of AGR2 Is Mediated at theTranscriptional Level

To elucidate the transcriptional nature of the TCDD effect,

transfection experiments were performed following promoter

sequence analysis. Three putative XREs were found in the

proximal 4000-bp promoter by MatInspector analysis. Dele-

tions of the AGR2 gene promoter were introduced into a basic

expression vector upstream of the Gaussia luciferase, a reporter

FIG. 2. TCDD increased AGR2 mRNA and protein levels in HepG2

cells. HepG2 cells were treated for various times with either 25nM TCDD

or the vehicle (0.15% DMSO). (A) The AGR2 mRNA levels measured by

qRT-PCR were normalized to the level obtained by treatment with the

vehicle alone (4–19 experiments performed in duplicate). There was

a significant increase (Fisher’s exact test) in AGR2 mRNA levels after

treatment with TCDD for 8–72 h (*p < 0.05 and ***p < 0.0001).

(B) Proteins were analyzed by Western blot (anti-AGR2 monoclonal

antibody). A typical Western blot is shown. (C) Quantification of the

amount of AGR2 protein. Intensities were normalized by comparison to the

actin signal (3–10 separate experiments). There was a significant increase in

AGR2 protein (Fisher’s exact test) following TCDD treatment for 24–72 h

(*p < 0.02 and ***p < 0.0001).



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enzyme secreted into the medium of the cell culture. These

constructs were transiently transfected into HepG2 cells to

analyze the putative functional XRE(s) involved in the

regulation of the AGR2 gene by AhR agonists. The cells were

treated with TCDD or the vehicle, and the luciferase activity

released into the culture medium was measured. As expected,

TCDD had no effect on the empty plasmid and the basal

activity was low (0.3 arbitrary units). In contrast, all the AGR2

constructs tested displayed a higher basal activity (2–4 arbitrary

units) and a twofold to fivefold increase following treatment

with 25nM TCDD was observed for the constructs tested

(Fig. 5A).

In order to determine which XREs were responsible for the

induction by TCDD of the luciferase activity, XREs 1 and

2 were mutated. As shown in Figure 5A, the mutation of the

proximal XRE (XRE1) completely abolished the effect of

TCDD on the regulation of the AGR2 promoter, whereas the

mutation in XRE2 did not modify the reporter activity.

Deletion of XRE3 did not modify the regulation of the reporter

gene by dioxin. ChIP experiments demonstrated that treatment

for 45 min with TCDD stimulated AhR recruitment to the

region of the endogenous AGR2 gene promoter that encom-

passes XRE1 (Fig. 5B, left). Moreover, electrophoretic

mobility shift assay experiments revealed a specific band when

a probe encompassing XRE1 was incubated with nuclear

extracts prepared from TCDD-treated HepG2 cells. This band

was displaced by competition with a 50-fold excess of cold

probe and was partially decreased in intensity when an anti-

AhR antibody was used (Fig. 5B, right).

A dose-response curve was performed to determine the

responsiveness of the longest AGR2 construct (pGLuc

AGR2 �3401/þ8) to TCDD. A significant increase of the

Gaussia luciferase activity was obtained at a concentration as

low as 1nM TCDD and up to 50nM TCDD (Fig. 5C). Another

AhR ligand, benzo(a)pyrene (5lM), was as effective as TCDD

(fourfold increase) in increasing the reporter activity of this

construct (Fig. 5C).

TCDD Attenuation of Etoposide Action Is Mediated by AGR2

As shown in Figure 1A, etoposide treatment induced

phosphorylation of Ser15 and acetylation of Lys382 in p53.

Pretreatment with TCDD partially prevented these modifica-

tions of p53 by etoposide. In order to determine whether AGR2

was involved in the TCDD effect, HepG2 cells were

transfected with either control siRNA or AGR2 siRNA prior

to the treatments. AGR2 siRNA transfection significantly

decreased AGR2 mRNA levels of both untreated or TCDD-

treated cells (75–80% decrease, data not shown). AGR2 siRNA

transfection decreased markedly the amount of AGR2 protein

FIG. 4. Half-life of AGR2 mRNA in HepG2 cells. HepG2 cells were

pretreated for 30 h with either 25nM TCDD or the vehicle (0.15% DMSO).

Then, the cells were treated, or not, with 10lM DRB and mRNA was isolated

as a function of time. The AGR2 mRNA level was assessed by qRT-PCR. The

statistical analysis (Fisher’s exact test) showed no statistically significant

difference in the half-life of AGR2 mRNA in cells treated, or not, by TCDD in

the presence of DRB (six experiments performed in duplicate).

FIG. 3. AGR2 regulation by TCDD involves the AhR. (A) HepG2 cells

were treated for 30 h with either 25nM TCDD, 5lM benzo(a)pyrene (BaP), or

the vehicle (0.15% DMSO). Proteins were analyzed by Western blot (anti-

AGR2 monoclonal antibody). (A) A typical Western blot is shown. Intensities

were normalized by comparison to the actin signal (three separate experiments).

There was a significant increase (Fisher’s exact test) in AGR2 protein following

treatment with TCDD and B(a)P (**p < 0.001). (B) Left, siRNAs (control or

AhR) were transiently transfected into HepG2 cells using the HiPerfect reagent.

After 16 h, the cells were treated, or not, with 25nM TCDD for 30 h. The

AGR2 mRNA level was measured by qRT-PCR. The mean fold increase ±

SEM as compared to the control for four independent experiments performed

in duplicate is shown. Statistical analysis (Fisher’s exact test) showed that

the TCDD-induced increase in AGR2 mRNA was significantly decreased with

the two siAhR RNAs as compared to the control siRNA (*p < 0.05,

***p < 0.0001). Right, Western blot of the amount of AhR protein in HepG2

cells transfected with either control siRNA or AhR siRNA and treated or not

with 25nM TCDD is shown. Actin is shown as a control.



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FIG. 5. TCDD increases AGR2 gene promoter activity. (A) AGR2 gene promoter constructs (wild type or mutated in the XRE) in front of the Gaussia luciferase

were transiently transfected into HepG2 cells, which were treated, or not, with 25nM TCDD for 30 h. The reporter activity was expressed in arbitrary units. There

was a statistically significant (Fisher’s exact test) difference in reporter activity between treated and untreated cells (**p < 0.001, ***p < 0.0001, 3–19 experiments

for each construct). (B) ChIP experiment was carried out on the AGR2 promoter XRE1 region. HepG2 cells were treated, or not, with 25nM TCDD for 45 min.



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as compared to control siRNA in cells treated with etoposide

alone or combined with TCDD (Fig. 6A). In AGR2-silenced

cells, the inhibitory effect of TCDD on p53 phosphorylation

(serine 15) and acetylation (lysine 382) was abolished, whereas

control siRNA treated cells were still responsive to TCDD

(Figs. 6A and 6B). This indicates that the attenuation of p53

activation by TCDD was dependent, at least in part, on AGR2

expression. We also investigated the role of AGR2 expression

on the effector caspases-3/7 activities. As shown in Figure 6C,

when the cells were transfected by the control siRNA,

pretreatment by TCDD led to a statistically significant 50%

decrease of the effect of etoposide on caspases-3/7 activities.

The negative effect of TCDD on etoposide activation of

caspases-3/7 was considerably reduced in the presence of

AGR2 siRNA.

DISCUSSION

In this paper, we report a cross talk mechanism between

etoposide, a genotoxicant, which is a potent activator of the

p53 pathway, and TCDD, a tumor promoter, which activates

the AhR pathway in the hepatocyte-derived cell line HepG2. In

particular, pretreatment of HepG2 cells by TCDD partially

counteracts the activation of p53 induced by etoposide.

A protective effect of TCDD on the decrease in activity of

mitochondrial succinate dehydrogenase elicited by etoposide

was observed, suggesting a prosurvival/proliferative role for

TCDD in HepG2 cells. The finding that the human AGR2

silences p53 response to DNA damage (Pohler et al., 2004) and

our transcriptomic results showing that TCDD regulates AGR2

mRNA levels in the same cellular model (Marchand and

Garlatti, personal data) led us to hypothesize that this new

TCDD target could be partially responsible for some of the

effects of TCDD on p53 regulation, as proposed in Figure 7.

Therefore, we characterized the positive regulation, by TCDD,

of the gene encoding the human AGR2 and showed that TCDD

attenuates etoposide-mediated p53 phosphorylation and acet-

ylation through AGR2 gene regulation.

Induction of AGR2 by TCDD could have several con-

sequences. One hypothesis is that induction of AGR2 by

TCDD, which is a recognized carcinogen or other AhR ligands

could promote tumor formation. Indeed, recently, AGR2 was

shown to be overexpressed in several human cancers—prostate,

pancreas, lung, and breast cancer (Fritzsche et al., 2006, 2007;

Innes et al., 2006; Missiaglia et al., 2004; Ramachandran et al.,2008; Sitek et al., 2009; Thompson and Weigel, 1998; Wang

et al., 2008; Zhang et al., 2005, 2006). AGR2 also has been

proposed as a novel putative metastasis marker in breast,

prostate, and colorectal cancer (Fletcher et al., 2003; Liu et al.,2005; Smirnov et al., 2005; Thompson and Weigel, 1998), and

it is associated with poor survival of prostate cancer patients

(Zhang et al., 2007b). Stable transfection of lung carcinoma

H1299 cells by a vector expressing AGR2 was shown to

enhance colony formation, an assay that measures the ability of

a gene to influence cell survival. In another study, the

esophageal adenocarcinoma SEG-1 cell line displayed 82%

fewer colonies in soft agar and smaller tumor xenografts

when the AGR2 gene was silenced by short hairpin RNA as

compared to wild-type cells (Wang et al., 2008). Furthermore,

AGR2-expressing NIH3T3 cells showed enhanced foci forma-

tion in soft agar and were able to grow as tumors in nude mice

(Wang et al., 2008). Altogether, both in vivo and in vitrostudies suggest that the expression of this new TCDD target

correlates with a cancerous phenotype.

The data described here indicate that etoposide and TCDD

elicit opposite effects on p53 regulation. The induction of AGR2

by TCDD suggests that the AGR2 gene product may account for

the cross talk between these toxicants, which belong to two

distinct classes. Indeed, the increase in caspases-3/7 activities

and p53 phosphorylation and acetylation by etoposide is partially

blunted by TCDD treatment, and this effect appears to be

correlated with AGR2 induction. Our preliminary experiments

also indicate that constitutive overexpression of AGR2 in HepG2

cells inhibits both phosphorylation (Ser15) and acetylation

(Lys382) of p53 in cells treated with etoposide (Ambolet-Camoit

and Aggerbeck, unpublished results). Thus, the combination of

two toxicants with distinct mechanisms of action could produce

additional harmful effects that result from decreased adaptive

pathways. It will now be of interest to study the effect of other

pollutant combinations on p53 activity and AGR2 induction.

TCDD exposure leads to the activation of several target

genes involved in cell plasticity (Bui et al., 2009; Diry et al.,2006). Other results have shown that the AhR is involved

in cell-cell or cell-substratum adhesion; thus, this receptor

might play a role in cell migration (Barouki et al., 2007;

Carvajal-Gonzalez et al., 2009) and could have a major impact

on cell adhesion and matrix metabolism (Kung et al., 2009).

Gene-specific qPCR analysis with primers spanning the AhR-binding region in the AGR2 gene promoter was performed on genomic DNA immunoprecipitated with

either anti-AhR or mock IgG antibody and an equal amount of input DNA as described in the ‘‘Materials and Methods’’ section. The nontreated sample was used

as control for each condition (***p < 0.0001, three experiments) (left). The electrophoretic mobility shift assay shows the binding of nuclear extracts prepared

from TCDD-treated HepG2 cells (25nM, 90 min) to a probe encompassing the XRE1 (lane 2), after competition with an anti-AhR antibody (lane 3) and after

competition with a 503 nonlabeled probe (lane 4). Lane 1 corresponds to the free probe (right). (C) The longest wild-type construct (�3401/þ8 AGR2) transiently

transfected into HepG2 cells was treated with 0.15% DMSO or increasing TCDD concentrations for 30 h or with 5lM B(a)P. The reporter activity is expressed

in arbitrary units. There was a statistically significant (Fisher’s exact test) difference in reporter activity between treated and untreated cells at concentrations above

1nM TCDD (**p < 0.001, ***p < 0.0001, three to five experiments).



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FIG. 6. TCDD attenuates the phosphorylation and the acetylation of p53 and the caspases-3/7 activities elicited by etoposide via AGR2. HepG2 cells

transfected by either control siRNA or siRNA directed against AGR2 were pretreated with either 0.15% DMSO or 25nM TCCD for 24 h and incubated, or not,

with 15 or 75lM etoposide for an additional 24 h. (A) Protein extracts were analyzed by Western blot. Phosphorylated and acetylated p53 and AGR2 were

measured with specific antibodies. (B) Intensities on Western blots were normalized as compared to the actin signal (three to five separate experiments). There

was a statistically significant decrease in p53 levels when the cells tranfected by the control siRNA were treated with etoposide and TCDD as compared to

etoposide alone (*p < 0.04 and p < 0.02 for phosphorylated and acetylated p53, respectively, Fisher’s exact test), whereas no difference was observed when the

cells were transfected by AGR2 siRNA. (C) HepG2 cells transfected by either control siRNA or siRNA directed against AGR2 were pretreated with either 0.15%

DMSO or 25nM TCCD for 24 h and incubated, or not, with 75lM etoposide for an additional 24 h. Caspases-3/7 activities were measured as described in the

‘‘Materials and Methods’’ section. There was a statistically significant increase (Fisher’s exact test) in activity following etoposide and etoposide plus TCDD

treatments as compared to DMSO. However, the increase with the mixture of etoposide plus TCDD was significantly less than with etoposide alone when control

siRNA was used (Fisher’s exact test), whereas with AGR2 siRNA there was no difference between the mixture of etoposide plus TCDD and etoposide alone

(**p < 0.001 and ***p < 0.0001).



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These new roles of the AhR suggest that it could have key

functions in human cancer progression. Recent experiments

have pointed out that AhR activation inhibits apoptosis

triggered by UV light (Chopra et al., 2009; Park and

Matsumura, 2006) or by the E2F1 regulator of the cell cycle

(Marlowe et al., 2008) or by diethylnitrosamine (Paajarvi et al.,2005). Culture-induced senescence in keratinocytes is also

suppressed by TCDD or by cigarette smoke (Ray and

Swanson, 2009; Zhang et al., 2007a). Our finding that AGR2

is a novel target of the AhR that downregulates p53 and

caspases-3/7 activities elicited by a genotoxicant is another

argument for the possible involvement of AhR in tumor

progression. This hypothesis is strengthened by the fact that

AGR2 expression is increased in several human cancers and is

correlated with poor survival in breast cancer patients

(Barraclough et al., 2009).

It is noteworthy that AGR2 has been shown to be increased

by different stress conditions, such as serum or oxygen

depletion (Zweitzig et al., 2007) or allergen exposure in

asthma (Di Valentin et al., 2009). In addition, AGR2

expression is increased in the esophageal Barrett’s epithelium,

a tissue that is subjected to various stresses such as acid-bile

reflux, heatshock, and oxidative damage (Pohler et al., 2004).

Our study shows that AGR2 is also induced during xenobiotic

stress. Thus, AGR2 appears to be a target frequently activated

under stressful cellular conditions, and it is possible that the

combination of several stresses leading to AGR2 induction

may result in additional toxic effects. Although little is known

about AGR2 function in mammals, a protein disulfide

isomerase (PDI) function is suspected, based on sequence

homology (Persson et al., 2005), and AGR2 could have

a unique role in intestinal mucus production by forming

disulfide bonds with mucin 2 (Park et al., 2009). The fact that

AGR2 is probably a member of the PDI chaperone family

correlates with the fact that its expression is increased by

a variety of stress conditions, which require chaperone activity

in the endoplasmic reticulum. However, other still unknown

functions of AGR2 may also be important, in addition to the

fine-tuning of p53 activity. Since AGR2 expression is

correlated with cancer aggressiveness, it might constitute

a worthwhile therapeutic target in the future if more extensive

knowledge of its activity and of its regulation by hormones and

chemicals is obtained.

FUNDING

AFSSET (Agence Francxaise de Securite Sanitaire de

l’Environnement et du Travail [ES-2005-003]); ANR (Agence

Nationale de la Recherche [06SETS26, Oncopop]); ARC

(Association pour la Recherche sur le Cancer [3927]);

INSERM (Institut National de la Sante et de la Recherche

Medicale); Universite Paris Descartes; Ministere de l’Enseigne-

ment Superieur et de la Recherche (Bourse doctorale, Ariane

Ambolet-Camoit, Aline Chevallier); Fondation pour la

recherche Medicale (bourse post-doctorale, Lin-Chi Bui);

Ligue contre le Cancer (bourse post-doctorale, Lin-Chi Bui);

Region Ile de France (Bourse doctorale, Stephane Pierre).

ACKNOWLEDGMENTS

We thank Dr L. P. Aggerbeck for critical reading of the

manuscript.

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