NADPH oxidase DUOX1 promotes long-termpersistence of oxidative stress after an exposureto irradiationRabii Ameziane-El-Hassania,b,c, Monique Talbota,b, Maria Carolina de Souza Dos Santosa,b,d, Abir Al Ghuzlana,b,d,Dana Hartlb, Jean-Michel Bidarta,b,d, Xavier De Dekene, Françoise Miote, Ibrahima Diallob,d,f, Florent de Vathaireb,d,f,Martin Schlumbergera,b,d, and Corinne Dupuya,b,d,1

aUMR 8200, CNRS, Villejuif F-94805, France; bInstitut Gustave Roussy, Villejuif F-94805, France; cUnité de Biologie et de Recherche Médicale, CentreNational de l’Energie, des Sciences et des Techniques Nucléaires, Rabat M-10001, Morocco; dUniversity Paris-Sud, Orsay F-91400, France; eInstitut deRecherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, 1050 Brussels, Belgium; and fUMR 1018, INSERM,Villejuif F-94805, France

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved March 16, 2015 (received for review November 4, 2014)

Ionizing radiation (IR) causes not only acute tissue damage, butalso late effects in several cell generations after the initial expo-sure. The thyroid gland is one of the most sensitive organs to thecarcinogenic effects of IR, and we have recently highlighted thatan oxidative stress is responsible for the chromosomal rearrange-ments found in radio-induced papillary thyroid carcinoma. Usingboth a human thyroid cell line and primary thyrocytes, we inves-tigated the mechanism by which IR induces the generation of re-active oxygen species (ROS) several days after irradiation. Wefocused on NADPH oxidases, which are specialized ROS-generatingenzymes known as NOX/DUOX. Our results show that IR inducesdelayed NADPH oxidase DUOX1-dependent H2O2 production in adose-dependent manner, which is sustained for several days. Wereport that p38 MAPK, activated after IR, increased DUOX1 viaIL-13 expression, leading to persistent DNA damage and growtharrest. Pretreatment of cells with catalase, a scavenger of H2O2,or DUOX1 down-regulation by siRNA abrogated IR-induced DNAdamage. Analysis of human thyroid tissues showed that DUOX1 iselevated not only in human radio-induced thyroid tumors, but alsoin sporadic thyroid tumors. Taken together, our data reveal a keyrole of DUOX1-dependent H2O2 production in long-term persistentradio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strandbreaks, can cause genomic instability and promote the generationof neoplastic cells through its mutagenic effect.

ionizing radiation | oxidative stress | NADPH oxidase | thyroid |DNA damage

Ionizing radiation (IR) can cause various delayed effects incells, including genomic instability that leads to the accumula-

tion of gene mutations and chromosomal rearrangements, whichare thought to play a pivotal role in radiation-induced carcino-genesis. The persistence of such effects in progeny cells has pro-found implications for long-term health risks, including emergenceof a second malignancy after radiotherapy (1). The thyroid gland isone of the most sensitive organs to the carcinogenetic effects ofIR. The risk of thyroid tumors is maximal for exposure at ayounger age and increases linearly with radiation dose (2). Morethan 90% of these cancers are papillary, presenting a RET/PTCchromosomal rearrangement in 70% of cases. Thus, the thyroidcan serve as a paradigm for analyzing the long-term delayed ef-fects of IR.The mechanism by which radiation exposure is memorized and

leads to delayed DNA breakage remains to be determined. Hyp-oxia and antioxidant therapy reduce the X-ray–induced delayedeffects, suggesting that radio-induced oxidative stress plays a sig-nificant role in determining the susceptibility of irradiated cells togenetic instability (3–5). We recently showed that H2O2 is able tocause RET/PTC1 rearrangement in thyroid cells, indicating that

oxidative stress could be responsible for the RET/PTC rearrange-ment frequently found in radiation-induced thyroid tumors (6).Cells can produce ROS through activation and/or induction of

NADPH oxidases, which constitute a family of enzymes knownas NOX/DUOX (7). Unlike other oxidoreductases, NADPHoxidases are “professional” ROS producers, whereas the otherenzymes produce ROS only as by-products along with theirspecific catalytic pathways. ROS produced by NOXs participatein the regulation of many cell functions and have been implicatedin various pathological conditions, including the late side effectsinduced by IR and chemotherapy (8–10). Thyroid cells expressthree of these NADPH oxidases, including two H2O2-generatingsystems located at the apical plasma membrane of the thyroidcells: DUOX2, which is implicated in thyroid hormone bio-synthesis, and DUOX1, whose role in the thyroid is still unknown(11, 12). Furthermore, recently NOX4 was found to be expressedinside these cells (13).Because ROS may contribute to the late effects observed after

radiation exposure, we hypothesized that IR induces a delayedoxidative stress in thyroid cells via the activation and/or in-duction of NADPH oxidase. In the present study, we demon-strate that DUOX1 expression, induced via the IL-13 pathway inresponse to IR, is the primary source of sustained ROS pro-duction that causes persistent DNA damage. We show that p38MAPK activation is required for the increased radio-induced

Significance

Increasing evidence supports the role of chronic oxidativestress in late radiation-induced effects, including malignancyand genetic instability. To date, elevated levels of reactiveoxygen species (ROS) have been considered a cause of per-sistent instability, but until now the mechanism(s) underlyingthe perpetuation of ROS generation in irradiated cells and theirprogeny was undetermined. Cells can produce ROS through ac-tivation and/or induction of NADPH oxidases. The present in-vestigation identifies the DUOX1-based NADPH oxidase as a ROS-generating system induced after irradiation, causing delayed DNAbreakage. Overexpression of DUOX1 in radio-induced thyroidtumors suggests that DUOX1 may contribute to a chronic oxida-tive stress promoting genomic instability and tumorigenesis.

Author contributions: C.D. designed research; R.A.-E.-H., M.T., and M.C.d.S.D.S. performedresearch; A.A.G., D.H., X.D.D., and F.M. contributed new reagents/analytic tools; R.A.-E.-H.,J.-M.B., I.D., F.d.V., M.S., and C.D. analyzed data; and C.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: corinne.dupuy@gustaveroussy.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1420707112/-/DCSupplemental.

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DUOX1 expression. Finally, our analysis of human thyroid tissuesshows that DUOX1 is overexpressed in both radio-induced andsporadic tumors, suggesting that radiation exposure by inducingDUOX1-based oxidative stress might favor a neoplastic processthat can occur naturally. Our findings assign the NADPH oxidaseDUOX1 a previously unidentified role in radio-induced geneticinstability.

ResultsRadiation Exposure Induces Chronic DUOX1-Dependent H2O2 Productionin Human Thyroid Cells. The concentration of extracellular H2O2produced by thyroid cells (HThy-ori) after γ-ray irradiation at 10Gy increased from day 3 up to day 4, and then remained stableuntil day 7 (Fig. 1A). DUOX1 protein level also increased fromday 1 to day 10 after irradiation (Fig. 1B). Strikingly, irradiation(10 Gy) of HThy-ori cells preferentially resulted in the up-reg-ulation of DUOX1 mRNA level (1- to 14-fold) compared notonly with levels of NOX4 and DUOX2 mRNA, two NADPHoxidases expressed in the normal thyroid gland (Fig. 1C), but alsowith the other NOXs (Table S1). The increase in DUOX1mRNA level was dose-dependent (Fig. S1B).DUOX1 needs the maturation factor DUOXA1 to exit the

endoplasmic reticulum and be active on cell surface. The DUOX1/DUOXA1 genes are aligned head-to-head in a compressed genomiclocus on chromosome 15, suggesting that expression of DUOX1oxidase and its maturation factor are coordinated by a commonbidirectional promoter (14). Several alternative splicing variantsof DUOXA1 mRNA have been identified, and the lack of codingexon 6 has been shown to generate inactive forms of DUOXA1 (15).We designed an oligonucleotide primer set in the DUOXA1

mRNA region containing exon 6. Real time quantitative RT-PCR (qRT-PCR) analysis performed at 4 d after a 10-Gy exposureof HThy-ori cells showed that a spliced variant of DUOXA1mRNA encoding an active form was selectively increased in thiscondition. This mRNA variant was up-regulated in a dose-dependent manner (Fig. S1C). Knocking down DUOX1 withspecific siRNA reduced the level of H2O2 produced by irradiatedcells (Fig. 1D), indicating that irradiation induces chronic H2O2

production via DUOX1 up-regulation. Irradiation induced anincrease in cytosolic [Ca2+] in thyroid cells at day 4, consistentwith activation of the calcium-dependent H2O2-generating ac-tivity of DUOX1 (16) (Fig. S1D).

Radiation Induces IL-13 in Human Thyroid Cells. To identify themolecular mechanism underlying chronic DUOX1 expressionafter irradiation, we analyzed the gene expression profile ofimmune-related genes in HThy-ori cells at different intervalsafter γ-ray exposure at 10 Gy. Among the cytokine genes ana-lyzed, IL-1β, IL-13, IL-6, IL-8, and TNF-α were found to be up-regulated after irradiation in a time-dependent manner (Fig. S1E and F). The induction of IL-13 mRNA was correlated with anincreased level of IL-13 protein in HThy-ori cells assayed byWestern blot analysis at days 4 and 7 after irradiation (Fig. 2A).A neutralizing IL-13 monoclonal antibody (clone 32116) thatblocks the binding of IL-13 to its receptor abrogated radiation-induced H2O2 production (Fig. 2B). In addition, IL-13 down-regulation by RNA interference resulted in a significant reductionof DUOX1 mRNA level (Fig. S1G). Our data indicate that IL-13regulates the radiation-induced increase in DUOX1 expression.

p38 MAPK Regulates DUOX1 Expression. To define the upstreammechanisms that regulate IL-13–induced DUOX1 expression inirradiated cells, we tested the effect of pharmacologic inhibitorsof NF-KB and canonical mitogen-associated protein kinase(MAPK) pathways. Of these inhibitors, only SB203580 (SB), whichspecifically inhibits p38 MAPK, attenuated the induction ofDUOX1 mRNA expression at day 4 after irradiation (Fig. S1H).Because these data supported a role for p38 MAPK in radi-

ation-induced DUOX1 expression, we analyzed the time courselevels of total and phosphorylated p38 MAPK and its downstreamtarget HSP27 after radiation exposure (10 Gy) (17). Western blotanalysis showed that p38 MAPK and HSP27 phosphorylation rosesubstantially at 24 h and remained elevated until day 10 (Fig. 2C).Importantly, the kinetics of p38 MAPK activation closely paralleledchanges in DUOX1 expression. Knocking down p38 MAPK withspecific siRNA affected radiation-induced DUOX1 mRNA ex-pression (Fig. 2D). Treatment of thyroid cells with the p38 MAPKinhibitor SB decreased radio-induced H2O2 production (Fig. S2A),and p38 MAPK down-regulation by RNA interference also affectedthe increased expression of IL-13 protein at day 4 (Fig. S2B). Takentogether, these data indicate that p38 MAPK activation plays akey role in radiation-induced up-regulation of DUOX1 via IL-13.

DUOX1 Is Involved in Radio-Induced DNA Damage. A high radiationdose generates persistent DNA damage foci, leading to prolongedDNA damage response activation for several days (18). One of thefirst proteins to respond to DNA double-strand breaks (DSBs) isAtaxia Telangiectasia Mutated (ATM), a member of the phos-phoinosityl-3 kinase-like kinase (PIKK) family. ATM substratesinclude H2AX, a nucleosomal histone variant, and p53-bindingprotein 1 (53BP1). At day 4 postirradiation, phosphorylated formof H2AX (γH2AX) and 53BP1 localized to DSBs, forming char-acteristic foci (Fig. 2E). Immunoblot analysis of the time-dependentstimulation of γH2AX in nonirradiated and irradiated cells showedthat it increased starting at day 2 after irradiation (Fig. 2F).ATM also phosphorylates the DDR kinase checkpoint kinase

2 (Chk2), which promotes growth arrest. Kinetic analysis of Chk2phosphorylation after 10-Gy irradiation showed an increase at1 d after irradiation that was sustained for 10 d (Fig. S2C). Weperformed a cell cycle profile analysis of HThy-ori cells afterirradiation. As shown in Fig. 3A, irradiated cells exhibited anincreased percentage of cells in G2/M phase, indicating a G2/Marrest. Many cells showed aberrantly enhanced DNA content(>4n), likely reflecting partial re-replication of the genomeuncoupled from cell division. This was accompanied by up-regulation of p21, a cell-cycle inhibitor (Fig. S2D).

Fig. 1. Radiation increases oxidative stress and DUOX1 expression in theHThy-ori3.1 cell line. (A) Radiation exposure-induced extracellular H2O2

production at the indicated days. (B) Time induction of DUOX1 protein fromwhole-cell lysates of irradiated cells analyzed by Western blot analysis.Vinculin served as a loading control. (C) Comparative expression of DUOX1,DUOX2, and NOX4 genes in irradiated HThy-ori cells, analyzed by real-timeqRT-PCR. (D) (Upper) Effect of siDUOX1 vs. siControl on extracellular H2O2

production activity measured at day 4 post-IR. (Lower) DUOX1 protein ex-pression of the corresponding experiment from particulate fractions. Valuesare mean ± SE. **P < 0.01.

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A genetically encoded highly specific fluorescent probe hasbeen developed for detecting H2O2 inside living cells (19). Thisbiosensor, known as Hyper, has been shown to have submicromolaraffinity to H2O2 and to be insensitive to other oxidants. Using themammalian expression vector encoding nuclear-targeted Hyper,we observed changes in the fluorescence of Hyper in nuclei atday 4 postirradiation (Fig. 3B). In addition, we targeted a yellowfluorescent protein-based redox sensor (rxYFP) to the nucleus ofthyroid cells and monitored the nuclear redox changes in re-sponse to H2O2 by analyzing both reduced and oxidized forms ofrxYFP after 30 min. In the presence of 12.5 μM H2O2, which isthe dose of H2O2 produced by DUOX1 (Fig. 1D), nucleus-rxYFP became oxidized, confirming changes in the nuclear redoxenvironment in response to extracellular H2O2 (Fig. 3C).Exogenous H2O2 induces DNA damage in thyroid cells (6, 20).

To establish a link between H2O2-generating NADPH oxidaseDUOX1 activation and DNA damage observed several daysafter irradiation, we performed RNA interference experiments(Fig. 3D and Fig. S2E). ATM depletion prevented phosphoryla-tion of its substrate H2AX. Knockdown of DUOX1, DUOXA1,IL-13, or p38 MAPK significantly reduced the level of γH2AX andthe number of 53BP1 nuclear foci (Fig. S2F). Inhibition of bothp38 and IL-13 affected IL-13 expression more significantly, pro-viding better protection against DNA damage (Fig. S3A). Incontrast, depletion of p22phox, the NOX functional partner (except

for NOX5), had no effect on the expression of γH2AX andphospho-p38 MAPK (Fig. S3B). Finally, treatment of cells withcatalase, a scavenger of H2O2, protected DNA from DSBs in-duced at postirradiation (Fig. 3D). Taken together, these dataindicate involvement of DUOX1-dependent H2O2 generation indelayed radio-induced DNA damage. Overexpression of bothDUOX1 and DUOXA1 in thyroid cells at a level producing ex-tracellular concentration of H2O2 comparable to that measured atpostirradiation also increased expression of γH2AX (Fig. S3C).

Extracellular H2O2 Reproduces the Effect of Irradiation on DUOX1.Preincubation of HThy-ori cells for 4 h before irradiation ortreatment of cells with catalase at days 2 and 3 postirradiationsignificantly decreased the level of γH2AX analyzed at day 4postirradiation (Fig. 4A). This was related to a significant decreasein DUOX1 mRNA level (Fig. 4B). Conversely, treatment of cellswith H2O2 induced a dose-dependent increase of DUOX1 ex-pression at day 4 after treatment, which was associated with in-creases in both H2AX and p38 MAPK phosphorylation (Fig. 4C).

DUOX1 Mediates Radiation-Induced H2O2 Production in Primary HumanThyrocytes. After radiation exposure, primary human thyrocytesincreased H2O2 production at day 4. This effect was independentof serum (Fig. S3D). DUOX1 mRNA was found to be selectivelyup-regulated after irradiation, and this was correlated with anincrease in DUOX1 protein level (Fig. 5A, Fig. S3E, and TableS2). DUOX1 inactivation led to a significant reduction in radia-tion-induced H2O2 production (Fig. 5B). p38 MAPK and IL-13were activated and up-regulated, respectively, several days after10-Gy irradiation, and their depletion also affected the radio-induced expression of DUOX1 (Fig. 5 C and D).p38 MAPK down-regulation by RNA interference was able to

counteract the selective up-regulation of DUOXA1 (+ exon 6)mRNA expression by irradiation (Fig. S4A). H2O2 reproducedthe effect of irradiation on DUOX1 expression in human thy-rocytes (Fig. S4B), and its degradation by the catalase preventedup-regulation of DUOX1 (Fig. 5D). Interestingly, DUOX1

Fig. 2. IL-13 and p38 MAPK regulate radiation-induced DUOX1 expression.(A) Immunoblot detection of IL-13 protein in nonirradiated and irradiatedHThy-ori3.1 cells at days 4 and 7. (B) HThy-ori3.1 cells were incubated withincreasing concentrations of IL-13–neutralizing antibody from day 2 afterγ-irradiation (10 Gy), and the H2O2-generating activity was measured at day4. (C) Increase in p38 MAPK phosphorylation after irradiation. Cells wereirradiated, and whole-cell lysates were collected at the indicated days.Downstream target of p38 MAPK (Hsp27) was also analyzed by Western blotanalysis. (D) p38 MAPK knockdown with interference RNA decrease the levelof DUOX1 mRNA expression in irradiated HThy-ori3.1 cells at day 4, as an-alyzed by real-time qRT-PCR. (E) Persistent DNA damage foci. HThy-ori3.1cells were untreated or irradiated (10 Gy), then fixed and stained for 53BP1(green), γH2AX (red), and DNA (DAPI; blue) at day 4. (F) Time course analysisof γH2AX in HThy-ori3.1 cells untreated or irradiated (10 Gy). Whole-celllysates were collected at the indicated days thereafter. Vinculin served as aloading control. Values are mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. DUOX1 is involved in delayed radio-induced DNA damage. (A) Cellcycle phase distribution in untreated and irradiated (10 Gy) cells analyzed atday 4. The percentage of cells was determined by flow cytometry. Values aremean ± SE from three independent measurements. (B) Confocal analysis ofH2O2-dependent fluorescence in HThy-ori3.1 cells expressing pHyper-nuc at4 d after irradiation. (C) Redox Western blot of nuc-rxYFP. (D) Immunoblotfor γH2AX in untreated and irradiated (10 Gy) cells. The cells were trans-fected with different siRNAs against DUOX1, IL-13, p38 MAPK, or ATM 2 dbefore being collected at day 4 for analysis. Catalase (250 U/mL) was addedin the cell medium 2 d before cells were collected for Western blot analysis.γH2AX was quantified by densitometry. Values are mean ± SE. *P < 0.05;**P < 0.01; ***P < 0.001.

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inactivation itself affected the level of p38 MAPK phosphoryla-tion analyzed at day 7 postirradiation, indicating that DUOX1contributes to long-term maintenance of the radiation-inducedeffect (Fig. S4C). The DNA damage response was activated inhuman thyrocytes at day 4. γH2AX and 53BP1 formed charac-teristic foci in the nucleus of irradiated cells, suggesting thepresence of DNA DSBs (Fig. S4D). This was associated with cellgrowth arrest, as evidenced by activation of Chk2 via an increasein its phosphorylated form, increase in G2/M phase arrest, andup-regulation of p21 (Fig. 5E and Fig. S4 E and F). The DNAdamage response was also activated when the thyrocytes weretreated with H2O2 (Fig. S4G).Immunofluorescence analysis showed that DUOX1 was ex-

pressed both at the plasma membrane and in the perinuclearcompartment (Fig. 6A). A significant reduction in radio-inducedDNA damage detected by γH2AX was observed after DUOX1inactivation (Fig. 6B). This was confirmed by immunofluores-cence analysis showing that DUOX1-inactivated cells displayed50% reduction of DNA-damage foci (Fig. 6 C and D). Takentogether, these data indicate that DUOX1 is involved in radio-induced DSBs in primary thyroid cells.

DUOX1 Is Increased in Thyroid Cancers. Human exposure to IR is astrong risk factor for the development of thyroid tumors. Toinvestigate whether our findings in human thyrocytes are rele-vant to radio-induced human thyroid tumors, we analyzed theexpression of DUOX1 and IL-13 in 20 thyroid tumor tissuesfrom patients with a history of radiation exposure during child-hood (Table S3). DUOX1 and IL-13 mRNA levels were mea-sured in normal (n = 6) and tumor tissues by real-time qRT-PCR, and the DUOX1 gene expression level was significantlyhigher in radio-induced thyroid tumors than in normal thyroidtissues (Fig. 7). In sporadic thyroid tumors (Table S4), the in-crease in DUOX1 level was of borderline significance. Expressionof both DUOX1 and IL-13 was detected by immunohistochemistryin normal thyrocytes, but clear overexpression of both proteins wasobserved in sporadic and radio-induced thyroid tumors (Fig. S5).

DiscussionA growing body of evidence appears to support the concept thatchronic oxidative stress might drive the progression of radiation-induced late effects (4). A radiation-induced increase in ROSgeneration and/or an oxidative stress has been observed in vivo(21). Our results reveal that both DUOX1 and its maturationfactor DUOXA1 are up-regulated several days after irradiation

in human thyrocytes, supporting the role of DUOX1-basedNADPH oxidase in a chronic oxidative stress. DUOX1 has beenoriginally identified in the thyroid gland (12). Like its counter-part DUOX2, it was first characterized as active only at theapical cell surface of thyrocytes, where it produces H2O2 in theextracellular colloid space. Because exposure of thyroid cells toexogenous H2O2 can induce DNA breaks (20) and produce RET/PTC1 rearrangement (6), it was conceivable that extracellularH2O2 produced by DUOX could be implicated in DNA damage.Although H2O2 is relatively stable and has a high membrane-dif-fusible capacity, a paracrine effect of H2O2 most likely explainsH2O2 accumulation in the nucleus (6) (Fig. 3B), which was recentlyshown to be associated with nuclear redox changes (22) (Fig. 3C).The generation of DNA damage leads to the accumulation of

characteristic foci of DNA damage response (DDR) factors. Atlow radiation doses, these foci disappear within hours, indicatingthe presence of repairable DNA lesions. In contrast, at higherradiation doses, a few clearly detectable DDR foci persist formany days, especially DNA DSBs (18). Importantly, siRNA-mediated abrogation of both DUOX1 expression and DUOXA1expression resulted in a significant decrease in postirradiationDNA damage in thyroid cells (Figs. 3D and 6 B–D, and Fig. S2 Eand F), identifying for the first time, to our knowledge, a key roleof DUOX1-dependent H2O2 production in persistent radio-induced DNA damage and, consequently, in DDR signaling.Although DNA damage can arise through the direct in-

teraction of oxidants with genomic DNA, it also can be gener-ated by oxidation of DNA precursors in the nucleotide pool (23)or by an imbalance in the dNTP pools caused by inhibition ofenzymes involved in nucleotide synthesis by ROS (24). Chronicdepletion or imbalance in the nucleotide pool inflicted by radio-induced H2O2 production may lead to replication stress with

Fig. 4. H2O2 mediates the radiation effect. (A) Effect of pretreatmentor treatment with 250 U/mL catalase on γH2AX expression analyzed at day4 post-IR. (B) Decreased level of DUOX1 mRNA expression at day 4 aftertreatment of irradiated cells with catalase, as analyzed by real-time qRT-PCR.(C) Dose-dependent increases in DUOX1, γH2AX, and phospho-p38 MAPKexpression treated with H2O2 and analyzed after 4 d.

Fig. 5. DUOX1 is involved in radio-induced H2O2 production in humanprimary thyrocytes. (A) Time course analysis of DUOX1 protein expression inirradiated human thyrocytes. Nonirradiated cells served as a negative con-trol. Vinculin served as a loading control. (B) Effect of siDUOX1 vs. siControlon the extracellular H2O2 production activity of untreated and irradiatedthyrocytes. DUOX1 protein expression of the corresponding experiment isshown. (C) Immunoblot for IL-13 and for p38 MAPK phosphorylation inuntreated and irradiated (10 Gy) thyrocytes. Whole-cell lysates were col-lected at the indicated days after irradiation. (D) Immunoblot for DUOX1 inuntreated and irradiated (10 Gy) cells. The cells were transfected with dif-ferent siRNAs against DUOX1, IL-13, p38 MAPK, or SB (p38 MAPK inhibitor).Catalase (250 U/mL) was added in the cell medium 2 d before cells werecollected for Western blot analysis. Protein expression was quantified bydensitometry. The mean ± SE value from three independent measurementsis reported. (E) Induction of p21 at day 4 (10 Gy) in human thyrocytes ana-lyzed by Western blot analysis. β-actin served as a loading control. Values aremean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001.

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generation of DNA strand breaks and genomic instability (25).Thus, further studies are needed to determine how DUOX1induces DNA damage.Persistent changes precede the establishment of senescence-

associated phenotypes, including growth arrest (26–28) andsenescence-associated secretory phenotype (SASP), with potentautocrine and paracrine activities (18, 29). In the present study,persistent DNA damage foci in thyroid cells were associated withincreased expression of cytokines, including IL-13. Importantly,this cytokine, which has been implicated in the development oflate effects of radiation (30), mediates radio-induced DUOX1expression in thyroid cells. Until recently, the Th2 cytokines IL-4and IL-13 were considered to selectively up-regulate DUOX1(31, 32); however, recent data show that human thyrocytes ex-posed to IL-4 or IL-13 present a selective up-regulation ofDUOX2 and DUOXA2 genes (33), indicating that, dependingon cell context, these cytokines regulate both DUOX genes.In human fibroblasts, p38 MAPK induces the senescence

growth arrest and cytokine secretion in response to X-ray ex-posure (34). In this case, p38 MAPK phosphorylation increasedonly slightly over the 24 h after X-ray irradiation (10 Gy) andreached a peak after several days. We also observed a delayedp38 MAPK response to irradiation in human thyroid cells, whichcontrolled both IL-13 and DUOX1 expression (Fig. 5D and Fig.S2B). A feedback loop involving ROS in permanent growth ar-rest has been described previously (35). Knockdown of DUOX1expression affected p38 activation, IL-13 expression, and DNA

damage, confirming the involvement of DUOX1-derived H2O2

in the maintenance of DDR in irradiated thyroid cells (Figs. S2Band S4C). Catalase not only protected cells from the toxic effectsof H2O2, but also suppressed radio-induced DUOX1 expression(Figs. 4 A and B and 5D). Conversely, H2O2 induced a delayedincrease in DUOX1 expression (Fig. 4C), indicating that H2O2

produced during irradiation through water radiolysis may me-diate part of the radiation effect.Partially transformed and tumorigenic cells systematically and

spontaneously emerge from senescent cultures (36). Senescence-associated ROS may be a cause of both senescence, through theirdeleterious effects, and of the emergence of pretumoral cells,through their mutagenicity. Thus, DNA-damaged thyroid cellsresulting from radio-induced DUOX1-dependent H2O2 genera-tion could emerge from senescence and propagate chromosomeabnormalities and mutations that lead to tumorigenesis. This mayexplain why radio-induced thyroid cancers overexpress DUOX1.In conclusion, our findings demonstrate that chronic H2O2

production in human thyroid cells in response to irradiationexposure is mediated by DUOX1 (Fig. S6). The p38 MAPKpathway is involved in IL-13–induced DUOX1 expression. Thus,DUOX1 as a major source of radio-induced H2O2 may causesubstantial DNA damage in progeny of irradiated cells and theirneighboring bystanders and, consequently, participate in the ini-tiation and development of thyroid tumors. Therefore, DUOX1might constitute a potential target for specific inhibitors to miti-gate the side effects of radiotherapy.

Materials and MethodsCell Culture and Treatments with Inhibitors and Cytokines. HThy-ori cells andprimary human thyroid cells were cultured as described previously (13);details are provided in SI Materials and Methods. At 2 d after irradiation,thyroid cells were treated for 24 h with different doses of inhibitors. In-hibitors of the MAPK pathway (U0126, SB, InSolution JNK inhibitor II) andNFκB pathway (IKK Inhibitor II) were purchased from Calbiochem.

In Vitro Irradiation. Exponentially growing HThy-ori cells were plated at 24 hbefore irradiation at 2.5 × 105/well in six-well plates in 10% (vol/vol) serum-containing medium. Immediately before irradiation, cell culture mediumwas replaced by fresh culture medium, and cells were exposed to a singledose of γ-irradiation from a generator operating at 200 KV and 15 mA at adose rate of 3 Gy/min. At 24 h after irradiation, cells were cultured in 3%(vol/vol) serum-containing medium at different times before being har-vested. Human primary thyrocytes were first plated in a 75-cm2 flask. Whencells reached 70% of confluence, culture medium was replaced by 0.2% FCSculture medium without TSH and insulin. One week later, thyrocytes wereplated at 2.5 × 105/well in six-well plates in the same medium. After 24 h,cells were irradiated as described above and harvested at different times.

Fig. 6. DUOX1 is involved in persistent radio-induced DNA damage in hu-man thyrocytes. (A) Localization of membrane protein (lamin A/C; red) andDUOX1 (green) analyzed by immunofluorescence in untreated and irradi-ated (10 Gy) thyrocytes. The merged red and green channels show colocal-ization in yellow. Thyrocytes were transfected with siRNA against DUOX1 atdays 2 and 4 post-IR and analyzed at day 7. (B) Affect of inhibition of DUOX1on irradiation-induced γH2AX expression. DUOX1 and γH2AX were visual-ized by Western blot analysis in thyrocytes at day 7. (C and D) Quantificationof the number of merged γH2AX/53BP1 foci in nuclei of untreated and ir-radiated thyrocytes at days 4 and 7. Thyrocytes were transfected with siRNAagainst DUOX1 as described above. Cells were fixed and stained for 53BP1foci (green) and γH2AX foci (red) at days 4 and 7 post-IR.

Fig. 7. Comparative expression of DUOX1 (A) and IL-13 (B) genes in humanthyroid tissues analyzed by real-time qRT-PCR. Data are expressed as mRNArelative expression levels, determined as x-fold of calibrator correspondingto a pool of thyroid tissue samples. RI, radio-induced thyroid cancers (n = 20;Table S3); SP, sporadic thyroid cancers (n = 9; Table S4); normal tissues, n = 6.

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ROS Detection. Extracellular H2O2 generation was quantified by the AmplexRed/HRP assay (Molecular Probes, Invitrogen), which detects the accumula-tion of a fluorescent oxidized product. The H2O2 released was quantified(nmol H2O2/h/10

5 cells) using standard calibration curves. For nuclear ROSdetection, pHyPer-transfected cells were plated onto glass-bottom dishes,and the fluorescence (excitation/emission: 488/530 nm) was examined at day4 post-IR under a Zeiss LSM 510 confocal microscope.

Western Blot Analysis. Particulate cell fractions were solubilized in 20 mMTris·HCl pH 7.8 containing 135 mM NaCl, 1 mMMgCl2, 2.7 mM KCl, 1% TritonX100, 10% (vol/vol) glycerol, and a mixture of protease and phosphataseinhibitors (Calbiochem). Immunodetection was performed as describedpreviously (13) using as primary antibody either anti-DUOX (1/1,000) (12) ora rabbit polyclonal anti-DUOX1 antiserum (1/1,000) raised against residues988–1011 (Eurogentec) that does not detect DUOX2 (Fig. S1A). Immune com-plexes were detected with an AP-coupled anti-rabbit IgG antibody (1/4,500;Promega) or HRP-coupled anti-rabbit antibody (1/15,000; Southern Biotech).

For total cell lysates, cells were solubilized in 100 mM Tris·HCl pH 7.0containing 2.5% (wt/vol) SDS, 1 mM EDTA, 1 mM EGTA, 4 M urea, and a mixtureof phosphatase and protease inhibitors (Calbiochem). Primary antibodies werep-p38 MAPK Thr18 0/tyr182 XP rabbit, p38αMAP kinase, p-Chk2 (Thr-68),ChK2, p-HSP27 (ser 82), HSP27, p-ATM (ser1981), and ATM (all from CellSignaling Technology); γH2AX Ser-139 mouse and IL-13 (both from Milli-pore); H2AX and Vinculin (both from Abcam); p21 (sc-397; Santa Cruz Bio-technology); and actin (Sigma-Aldrich).

Transfection of siRNAs. Cells were transfected at 60% confluence at 2 d afterradiation exposure with siRNA against DUOX1 (stealth RNAi duplexHSS182411), scrambled siRNA control (Invitrogen), or siRNA against IL-13,p38 MAPK, and ATM (smart pool, from Dharmacon) using INTERFERINtransfection reagent (Polyplus-Transfection) according to the manufacturer’sprotocol. Cells harvested on day 7 after irradiation were transfected twice, atday 2 and day 4.

Tissue Samples and Immunohistochemistry. Thirty-five frozen tissue specimenswere obtained from the Institut Gustave Roussy (Tables S3 and S4). Histo-pathological diagnosis was performed according to World Health Organi-zation guidelines. The series comprises 20 radio-induced thyroid tumors,9 sporadic tumors, and 6 normal tissues. Sporadic tumors were matchedby histology and TNM classification. Immunohistochemistry is described inSI Materials and Methods.

Statistical Analysis. Statistical analyses were performed using GraphPad Instatsoftware for ANOVA and the Student t test, with the level of significance setat P < 0.05.

ACKNOWLEDGMENTS. We are indebted to Fanny Prenoix for her excellenttechnical assistance and Didier Méthivier (Unité U848 INSERM) and YannLécluse (PFIC of Gustave Roussy) for their assistance with flow cytometry.This work was supported by grants from Electricité de France, Associationpour la Recherche sur le Cancer, Institut National du Cancer, and ProgrammesInternationaux de Coopération Scientifique: CNRS-France/CNRST-Maroc andPHC Volubilis/Toubkal.

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