retinoic acid is able to induce interferon regulatory...

Retinoic Acid Is Able to Induce Interferon Regulatory Factor 1in Squamous Carcinoma Cells via a STAT-1 IndependentSignalling Pathway1

Zulema A. Percario, Valeria Giandomenico,Gianna Fiorucci, Maria V. Chiantore,Serena Vannucchi, John Hiscott, Elisabetta Affabris,and Giovanna Romeo2

Laboratory of Virology, Istituto Superiore di Sanita [G. F., M. V. C.,S. V., E. A., G. R.], and Istituto Tecnologie Biomediche [G. F., M. V. C.,G. R.], Consiglio Nazionale delle Ricerche, 00161 Rome, Italy;Department of Biology, University of Rome 3, 00146 Rome, Italy[Z. A. P., V. G., E. A.]; and Lady Davis Institute, McGill University,Molecular Oncology Group, Montreal H3T 1E2, Canada [J. H.]

AbstractInterferon regulatory factor 1 (IRF-1) transcriptionfactor binds to DNA sequence elements found in thepromoters of type I IFN and IFN-inducible genes.Transient up-regulation of the IRF-1 gene by virus andIFN treatment causes the consequent induction ofmany IFN-inducible genes involved in cell growthcontrol and apoptosis. We reported recently that IFN-a

and all-trans retinoic Acid (RA) inhibit the cellproliferation of squamous carcinoma cell line ME-180by inducing apoptotic cell death. IRF-1 expressioncorrelates with the IFN-a-induced apoptosisphenomenon and, surprisingly, with the RA-inducedapoptosis phenomenon. To study how these twodifferent ligands cross-talk in the regulation of cellularantitumor responses, the signalling pathways involvedin IRF-1 induction were analyzed in RA and/or IFN-a-treated ME-180 cells. We provide evidence indicatingthat RA-induced IRF-1 gene expression is independentof the STAT-1 activation pathway, despite the presenceof the IFN-g activated sequence element in the genepromoter, but involves nuclear factor-kB activation.Thus, here we first describe the activation of nuclearfactor-kB by both IFN-a and RA in the ME-180 cell line.The induced IRF-1 protein is successively able to bindthe IFN-stimulated responsive element in the promoterof the target gene 2*,5*-oligoadenylate synthetase.

IntroductionIRF-13 was originally identified as a transcription factor thatbinds to DNA sequence elements (IRF-Es) found in the pro-moters of type I IFN and IFN-inducible genes (1–3). The IRF-1gene is virus and IFN inducible (4) and is also induced byother cytokines such as tumor necrosis factor-a, IL-1, IL-6,leukemia inhibitory factor, and prolactin (5–9). These stimulicause the transient up-regulation of the IRF-1 gene andconsequent induction of many IFN-inducible genes. Severalputative binding sites for known transcription factors werefound within the IRF-1-promoter including one IFN-g-acti-vated sequence (GAS; Ref. 10) and one NF-kB site (10, 11).

Presently, one of the best-understood systems of signaltransduction is the activation of the so-called STAT proteinsby IFN-a and IFN-g, as well as by other growth factors andcytokines (3, 11, 12). These different stimuli induce tyrosinephosphorylation and the consequent activation of differentcombinations of STAT and STAT-associated proteins in aprocess mediated by the Janus activated kinase family ofprotein tyrosine kinases. The STAT-containing protein com-plexes then participate directly in the induction of gene tran-scription by relocalizing to the nucleus and binding to thepromoters of their cognate target genes. Thus, the IFN-a/b-inducible genes are mainly activated by the phosphorylatedcomplex ISGF-3 consisting of STAT proteins [namelySTAT-1a (p91) or STAT-1b (p84) and STAT-2 (p113)] and theassociated DNA-binding protein p48. ISGF-3 is able to bindISRE. In contrast, IFN-g-inducible genes are activated by aphosphorylated complex, called IFN-g-activated factor,which consists of dimerized STAT-1a (3, 12, 13) able to bindthe GAS element. The STAT-1a homodimer (IFN-g-activatedfactor) is also formed at lower efficiency during IFN-a sig-naling and also contributes to IFN-a signaling. Thus thesetwo complexes, ISGF-3 and GAF, are integral components ofthe system by which IFN stimulation received at the cellsurface is translated into changes in gene transcription in thenucleus. Although ISGF-3 and GAF are responsible for theinitial transmission of the IFN signal to the nucleus, theproper regulation of the broad range of genes induced by theinterferons involves other transcription factors such as IRF-1.IRF-1s bind DNA sequence elements, designated IRF-E (3, 4)and also share homology in their DNA-binding regions withp48, the DNA-binding component of ISGF-3 mentioned ear-Received 8/11/98; revised 12/29/98; accepted 2/8/99.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 Grants from the Consiglio Nazionale delle Ricerche (97.03974.CT04),MURST 40%, and Medical Research Council supported this work.M. V. C. was supported by a fellowship from Fondazione Italiana per LaRicerca sul Cancro (FIRC).2 To whom requests for reprints should be addressed, at Laboratory ofVirology, Istituto Superiore di Sanita, Viale Regina Elena, 299, 00161Rome, Italy. Phone: (39 06) 49903231; Fax: (39 06) 49902082; E-mail:[email protected].

3 The abbreviations used are: IRF-1, IFN regulatory factor 1; ISGF, IFN-stimulated gene factor; ISRE, IFN-stimulated response element; RA, all-trans-retinoic acid; SCC, squamous cell carcinoma; 2-5A, 29,59-oligoad-enylate; RAR, RA receptor; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; STAT, signal transducer and activator of transcription;GAS, IFN-g-activated sequence; GAF, IFN-g-activated factor; NF-kb, Nu-clear factor-kappa B; IL, interleukin; EMSA, electrophoretic mobility shiftassay.

263Vol. 10, 263–270, April 1999 Cell Growth & Differentiation

lier (14). This homology is functionally significant, becauseIRF-1 and ISGF-3 have been shown to bind to overlappingsequences in the promoters of many IFN-a/b-induciblegenes (3).

In addition to regulating the IFN system, IRF-1 manifeststumor-suppressive activities (15, 16), and its inactivation maybe linked to the development of human hematopoietic ma-lignancies (17, 18). IRF-1 is also required for the induction ofapoptosis after DNA damage or culture in low serum infibroblasts carrying an activated c-Ha-ras gene (16). DNAdamage-induced apoptosis is dependent on IRF-1, and twodifferent antioncogenic transcription factors, p53 and IRF-1,are required for apoptotic pathways in T lymphocytes (19,20). Recently, it has been shown that IRF12/2 mouse em-bryonic fibroblasts are deficient in their ability to undergoDNA damage-induced cell cycle arrest (21) mediated bytranscriptional induction of the gene encoding p21 (Waf1,CIP1), a cell cycle inhibitor dependent on both p53 andIRF-1.

We reported recently (22) that IFN-a and RA inhibit cellproliferation of SCC lines by inducing apoptotic cell death.IRF-1 expression correlates with the IFN-a-induced apopto-sis phenomenon and, surprisingly, with the RA-induced ap-optosis phenomenon (22).

RA is a natural metabolite, prototype of retinoids, a groupof vitamin A-related compounds with profound influences oncell growth and differentiation (23). It affects gene expressionby binding RAR and retinoid X receptor, its specific nuclearreceptors, both ligand-activated transcription factors be-longing to the superfamily of nuclear receptors (24).

Both IFNs and RA have been shown to suppress thegrowth of certain tumor cells, and a combination of theseagents produces significant additive or synergistic antitumoractivity as well as an enhanced transcriptional induction ofsome IFN-stimulated genes (25–28). To study how these twodifferent ligands cross-talk in the regulation of cellular anti-tumor responses, we have analyzed the signaling pathwaysinvolved in the induction of IRF-1 observed in the ME-180

Fig. 1. A, Reverse transcrip-tion-PCR analysis of the IRF-1mRNA expression in ME-180cells treated or not with 10 mg/mlactinomycin D and/or IFN-a2band/or RA for the indicatedtimes. Total RNA was extracted(1 mg), reverse transcribed, andamplified as described in “Mate-rials and Methods.” The GAPDHmRNA expression was used as acontrol because its stability wasnot modified by actinomycin Dduring the time of treatment. B,analyses of IRF-1 expression inME-180 cells. ME-180 cells weretreated with 1026 M RA and 2000UI/ml IFN-a2b for different times.Total ME-180 cellular proteinswere electrophoresed on 10%acrylamide gel, and immunoblotanalysis was performed usinganti-human IRF-1 polyclonal an-tibody (Santa Cruz Biotechnol-ogy) as described in “Materialsand Methods.”

264 Retinoic Acid Activates IRF-1 Gene Expression

squamous carcinoma cell line treated with RA, comparingthe results with IFN-a induction. Here we provide evidenceindicating that RA-induced IRF-1 gene expression is inde-pendent of the STAT-1 activation pathway, despite the pres-ence of the GAS element in the gene promoter, but involvesNF-kB activation. The induced IRF-1 protein is successivelyable to bind the ISRE sequence in the promoter of the targetgene 29,59-oligoadenylate (2-5A) synthetase.

ResultsRA Induces IRF-1 Gene Expression. The kinetic of induc-tion of IRF-1 mRNA analyzed by RNase protection assay andreported previously (22, 27) indicates a peak at 3 h of RAtreatment and a significant level persisting up to 24–48 h. Toevaluate whether RA treatment could influence IRF-1 mRNAstability, we carried out reverse transcription-PCR analysison RNA samples extracted from ME-180 cells treated withIFN-a2b and RA in the presence or absence of actinomycinD. As expected (Fig. 1A), IRF-1 mRNA accumulated at acomparable level when ME-180 cells were treated with bothIFN and RA. The IRF-1 mRNA decay was determined duringthe 1-, 3-, and 5-h treatments (Lanes 6–8; Refs. 12–14). TheGAPDH mRNA was used as a control because its stabilitywas not modified by actinomycin D during the time of treat-ment (Fig. 1A).

The IFN and RA induction of IRF-1 mRNA level was abol-ished after 3–5 h of IFN/actinomycin treatment as well asafter RA/actinomycin treatment. This shows also in the caseof RA as a transcriptional induction of the IRF-1 gene, re-sponsible for the observed protein accumulation and exclud-ing a reduced degradation rate of the corresponding mRNA.In addition, transient transfection experiments were carriedout on 293 cells by using the pIRF-1 promoter-luc constructtogether with the RSV-lacZ construct as an internal control.Transfected cells were treated for 48 h with RA (1025

M),IFN-b (50 units/ml), and RA 1 IFN-b. Fig. 2A shows thatIFN-b causes a 2.0-fold induction of this construct; RAcauses a 2.4-fold induction. The RA 1 IFN combinationtreatment led to a higher augmentation of reporter activity,up to 4.3-fold induction. Similar results have been obtainedon HepG2 cells (Fig. 2B). Thus, RA could increase the ex-pression of IFN-regulated genes through direct transcrip-tional induction of IRF-1.

RA Induces IRF-1 Protein Expression. Both RA andIFN-a inhibit proliferation of ME-180 cells by inducing a clearapoptosis phenomenon (22). IRF-1 expression correlateswith the IFN-a- and RA-induced apoptosis (22, 27, 29). Thelevels of IRF-1 protein were determined in cells treated with1026

M RA by Western blot analysis using the IRF-1-specificantibody. Fig. 1B shows the presence of a low constitutive

Fig. 2. Effect of RA and IFN on IRF-1 promoter.293 (A) and HepG2 (B) (1.5 3 106 cells) cells wereseeded on 6-cm dishes and transfected with 5 mg ofpIRF-1 luc and 4 mg of RSV-lacZ with calcium-phosphate protocol. Then cells were treated withRA (1025 M), IFN-b (50 units/ml), and their combi-nation. b-Galactosidase and luciferase activity wasmeasured 48 h later as described in “Materials andMethods.” Each experiment included duplicatetransfections. The data shown are the means of foldinductions, normalized with respect to b-galacto-sidase expression.

265Cell Growth & Differentiation

expression of IRF-1 protein in untreated control cells. RAtreatment significantly increased this basal level, startingfrom 5 h. The signal appeared highly enhanced later on (24h), persisting up to 80 h. An internal control from cells treatedfor 2 h with 2000 IU/ml IFN-a2b was also included.

RA Does Not Activate the STAT-1 Pathway. Because aputative GAS element (30) was identified within the IRF-1promoter and IRF-1 was induced by both IFN-a and RA, theSTAT-1 activation was analyzed in cell extracts from ME-180cells treated with RA or IFN-a2b as indicated. By immuno-precipitation using anti-p91 followed by immunoblotting us-ing anti-phosphotyrosine-specific antibodies (see “Materialsand Methods”), STAT-1a (p91) was phosphorylated by 10min after IFN-a treatment (Fig. 3) and remained activated upto 1 h (data not shown). After RA treatment, phosphorylationof STAT-1 was not observed and specifically STAT-1 was notactivated during the interval of IRF-1 induction. The anti-phosphotyrosine blots were reanalyzed, after stripping, withanti-STAT-1 antibody. p91 was present at a similar level afterall treatment conditions (Fig. 3). A clear increase in p84 at latetimes of treatment (16–48 h) was observed.

Next, EMSA experiments were performed using an oligo-nucleotide representing the GAS element of the IRF-1 pro-moter and ME-180 whole-cell extracts to analyze transcrip-tion factor binding to this region at different times after IFN-a

and RA treatment. After IFN-a2b treatment, a GAF/GAScomplex appeared at 10 min and was maintained up to 2 h(Fig. 4). The specificity of complex formation was confirmedby a competition experiment performed in the presence of200-fold molar excess of the oligonucleotide (see Fig. 4);anti-STAT-1 treatment supershifted the observed complex(data not shown). No GAF/GAS complex was observed in cellextracts treated with RA, in agreement with negative resultsof phosphorylation activation of the GAF componentSTAT-1.

RA Activates NF-kB. The IRF-1 promoter also containsbinding sequences for the NF-kB transcription factor (10, 11)

which, like IRFs, regulates cell growth-controlled gene ex-pression. To determine whether RA-induced IRF-1 gene ac-tivation may be mediated by NF-kB binding to its targetsequence in the promoter of the IRF-1 gene, gel shift assayswere performed using an oligonucleotide representing theNF-kB binding site in the IRF-1 promoter and ME-180 whole-cell extracts treated with RA and IFN-a. In the extracts ofboth IFN-a and RA-treated ME-180 cells, two specific induc-ible complexes were detected (Fig. 5a). Supershift of themajor complex was observed in RA and IFN-a2b cell extractsby treatment with NF-kB p50 antibody (Fig. 5b, Lanes 4 and8), whereas the upper complex was abolished by treatmentwith NF-kB p65 antibody (Lanes 3 and 7). These resultsindicate that IFN-a activates NF-kB binding to its targetsequence in the promoter of the IRF-1 gene, thus resulting inthe observed high inducibility of the gene. RA induction ofIRF-1 gene expression could be mediated at least in part byNF-kB.

RA-induced IRF-1 Binds ISRE Sequence. To testwhether the induced IRF-1 was functional in activating IFN-inducible genes involved in regulating cell growth, gel shiftassays were performed using an ISRE fragment from 2-5Asynthetase promoter as a probe. As shown in Fig. 6a, RAtreatment induced a specific protein-DNA complex from 6 to48 h. The same complex was induced by IFN-a at 5 h aftertreatment and was efficiently competed by a 200-fold molarexcess of cold hE-IRS oligonucleotide. When the extractswere preincubated with anti-IRF-1 antibodies, the ISRE com-plex was abolished (Fig. 6b, Lanes 4 and 10), thus indicatingthe presence of the induced IRF-1 in the RA- and IFN-induced complex.

Pretreatment of cell extract with both anti-p48 and anti-p91 antibodies as well as anti-STAT-2 failed to affect thecomplex formation (Fig. 6b, Lanes 5–7 and 11–13), indicatingthe absence of STATs in the observed complex. On the otherhand, a specific RA induction of the p48 protein belonging tothe IRF family is observed in our system (Fig. 7).

DiscussionWe reported earlier (27) that RA as well as IFN-a2b inhibitproliferation of ME-180 cells in a dose- and time-dependent

Fig. 3. Activation of STAT-1 in ME-180 cells. Tyrosine phosphorylation(pTyr) and protein quantitation of STAT-1 protein in ME-180 cells treatedwith IFN-a2b (2000 IU/ml) and RA (1026 M) for the indicated times wereexamined. Proteins from whole-cell lysates were immunoprecipitated andimmunoblotted as described in “Materials and Methods.”

Fig. 4. EMSA on ME-180 cells treated with IFN-a2b (2000 IU/ml) and RA(1026 M) for the indicated times. Assays on whole-cell extracts incubatedwith labeled oligonucleotide representing the GAS element found in theIRF-1 promoter were performed as described in “Materials and Methods.”


manner. A markedly increased growth-inhibitory effect wasobserved when a combination treatment was carried out.ME-180 cells underwent massive apoptotic cell death aftertreatment with either RA or with IFN, both agents beingcapable of inducing a significant level of programmed celldeath (22). The phenomenon was much more extensivewhen the two drugs were given together.

Increased expression of the IRF-1, which manifests tumor-suppressive (19), apoptosis-inducing, and cell cycle arrestactivities in different cell types (22, 31), correlated with bothRA- and IFN-a-induced apoptosis. In fact, RA per se acti-vated IRF-1 gene expression in ME-180 cells. In particular,the induction of IRF-1 mRNA by 1 h after RA treatment, withlevels increasing at 3 and 5 h and persisting up to 24–48 h(see also Refs. 22 and 27) correlates with the kinetics ofIRF-1 protein accumulation (Fig. 1B). In addition, the signalappears strongly enhanced at later times (48 and 80 h). Thelate high level of IRF-1 protein correlates with the maximuminhibitory effect of cell proliferation and apoptosis observedin ME-180 cells treated with RA.

In addition, SiHa, a cell line derived from squamous cervixcarcinoma but resistant to RA-induced inhibition of prolifer-

ation and apoptosis, did not show RA-induced IRF-1 geneexpression (22). Nonetheless, SiHa cells increase IRF-1 ex-pression after IFN-a treatment (22) and respond to IFN-a

growth inhibition and induction of apoptosis. These resultsstrengthen the involvement of IRF-1 in the mechanism of RAand IFN-a growth inhibition and apoptosis. Expression ofIRF-1 may be one of the molecular mechanisms by which IFNand RA cross-talk in the regulation of cellular antitumor re-sponses and form the basis for the synergistic actions of RAand IFNs in SCC.

In support of this idea, the RA-induced growth inhibition ofacute promyelocytic leukemia cells correlated with IRF-1expression (32, 33), although RA activation of the STATpathway remains controversial. Gianni et al. (32) showedactivation of the STAT pathway by RA, whereas Matikainenet al. (33) did not. In addition, RA enhancement of IFN growthsuppression in a breast tumor cell line has been described(26) via a transcriptional enhancement of IFN-inducible geneexpression based on the RA-dependent up-regulation of thelevel of STAT-1.

Fig. 5. EMSA on ME-180 cells treated with IFN-a2b (2000 IU/ml) and RA(1026 M) for the indicated times. A), assays on whole-cell extracts, incu-bated with labeled oligonucleotide representing the NF-kB binding sitefound in the IRF-1 promoter, were performed as described in “Materialsand Methods.” B), assays on whole-cell extracts, incubated with labeledoligonucleotide representing the NF-kB binding site found in the IRF-1promoter, were performed as described in “Materials and Methods.”Preincubations with specific anti NF-kB p65 or p50 antibodies wereperformed to demonstrate the presence of these subunits in the observedcomplexes (see “Materials and Methods”).

Fig. 6. EMSA on ME-180 cells treated with IFN-a2b (2000 IU/ml) and RA(1026 M) for the indicated times. A), an assay on whole-cell extracts,incubated with labeled oligonucleotide representing ISRE fragment of2-5A synthetase gene promoter, was performed as described in “Mate-rials and Methods.” B), assay on whole-cell extracts, incubated withlabeled oligonucleotide representing ISRE fragment of 2-5A synthetasegene promoter, was performed as described in “Materials and Methods.”Preincubations with the indicated specific antibodies were performed todemonstrate the presence of these proteins in the observed complexes(see “Materials and Methods”).


The present study sheds light on the signalling pathwaysinvolved in the induction of IRF-1 observed in ME-180 cellstreated with RA. We have provided evidence that RA doesnot activate the STAT-1 pathway because no tyrosine phos-phorylation of this protein was observed in this condition oftreatment, whereas a clear rapid induction of tyrosine phos-phorylation of STAT-1 was observed after IFN-a treatment.Also, no formation of GAF/GAS complex was observed inRA-treated cell extracts, when EMSA experiments using theGAS element of IRF-1 promoter as a probe was performed.This result shows that RA does not activate the STAT-1signaling pathway in these conditions. As expected, a spe-cific IFN-a-induced complex was observed. This complexwas supershifted by anti-STAT-1 antibody (data not shown).In addition, the RA augmentation of the STAT-1a level de-scribed in breast tumor cells MCF-7 (26) and suggested tocontrol cell growth is not observable in our system (Fig. 3).The STAT-1a level in ME-180 squamous carcinoma cells isnot affected by RA but is significantly increased by IFN-a

treatment.4

We also observed that NF-kB transcription factor is in-volved in the RA induction as well as the IFN-a induction ofIRF-1 gene expression in SCC. It has been reported thatNF-kB-regulated genes encode proteins involved in the rapidresponse to pathogens or stress, including the acute-phaseproteins, cytokines, and cellular adhesion molecules (34).NF-kB also plays a critical role in T-cell activation. In acti-vated monocytes and macrophages, genes such as thoseencoding granulocyte-colony stimulating factor, macroph-age-colony stimulating factor and granulocyte/macrophage-colony stimulating factor; the inflammatory cytokines IFN-b,tumor necrosis factor-a, IL-1, and IL-6; receptors for tissuefactor and IL-2 a-chain; the chemotactic protein MCP-1/JE

and nitric oxide synthase are highly induced as a result ofregulatory control by NF-kB (34–36). Once NF-kB DNA-binding activity is activated, numerous target genes are se-lectively regulated by the transcriptional activation potentialof different homo- and heterodimer combinations. Also, vari-ations in the NF-kB consensus sequence to which the sub-units bind and cooperativity between different transcriptionfactor families and NF-kB/Rel contribute to the specificity ofgene activation (37, 38).

In our study, an important finding has been that IRF-1induction by RA in ME-180 cells probably involves the NF-kBsystem by both homo- and heterodimer activation (p50, p50)(p50, p65). The eventual functional interaction betweenNF-kB and additional transcription factor activation remainsto be seen. In fact, the presence of a putative RAR-bindingsite in the promoter of the IRF-1 gene has been suggested(33). In our hands, EMSA experiments performed using theindicated (33) putative RAR-binding site in the IRF-1 pro-moter as a labeled probe (2455 to 2440) showed no specificRA-induced shift complex (data not shown). NF-kB activa-tion is not observed in the study by Matikainen et al. (33)performed on APL cells, where it was reported that RA in-duces IRF-1 expression without activating both NF-kB andSTAT pathways, and alternative mechanisms have been sug-gested. In addition we observed RA induction of IRF-1 pro-moter, containing both GAS and NF-kB consensus sites aswell as the putative RAR element, linked to a luciferasereported construct in 293 and HepG2 cells.

This result suggests that RA induces the expression ofIFN-regulated genes and increases their IFN-controlled ex-pression (see Fig. 2) via the direct transcriptional induction ofIRF-1 (see also Ref. 39).

IRF-1 transcription factor is able to recognize DNA se-quence elements in the promoter of various genes involvedin regulating cell proliferation (3, 40). Of interest, we foundthat the RA-induced IRF-1 binds to the hE-IRS consensussequence of the 2-5A synthetase promoter containing theISRE element in a gel mobility shift assay. This observationcorrelates with the slight induction of the gene expressioninduced by RA treatment in our system (data not shown).

Because the presence of an IFN-a-induced STAT-1-p48complex binding the ISRE with high specificity and capableof activating transcription has been described, and becausep48 belongs to the IRF family (41), we investigated the pres-ence of STAT-1 or p48 in the observed ISRE/IRF1 complex.Pretreatment of cell extract with both anti-p48 and anti-p91antibodies failed to affect the complex formation (Fig. 6),indicating the absence of these proteins in the observedcomplex. On the other hand, RA (as well as IFN-a) is able toincrease the expression level of the p48 (Fig. 7) protein.

In view of the results reported here, the important next stepis to analyze in SCC the IRF-1 involvement in the regulationof expression of apoptosis- and cell cycle-related genes thatmay mediate the induction of apoptosis by RA.

Materials and MethodsCell Culture. The human epidermoid carcinoma cell line ME-180, iso-lated from an omental metastasis of a rapidly spreading cervical carci-noma, was maintained in McCoy’s 5a medium supplemented with 10%4 Unpublished results.

Fig. 7. p48 immunoblot analysis. The assay was performed on ME-180total cellular proteins electrophoresed on 10% acrylamide gel by usinganti-human p48 polyclonal antibody (United Biomedical, Inc.) as de-scribed in “Materials and Methods.”


fetal bovine serum, previously inactivated at 56°C for 20 min. This cell linewas obtained from the American Type Culture Collection (Rockville, MD).Cells were grown to ;85–90% of confluence in a humidified atmosphereof 5% CO2 at 37°C. RA (Sigma Chemical Co., St. Louis, MO) was addedto the medium from a stock solution of 1022 M in DMSO to a finalconcentration of 1026 M or 1025 M, as described. Cells treated with thesame volume of DMSO were used as a control in all the experimentsperformed. 293 cells were grown in DMEM supplemented with 10% FBSand HepG2 in DMEM-F12 plus 10% fetal bovine serum. RecombinantIFN-a2b (INTRON A; 2 3 108 IU/mg of protein; Shering Corp., Milan, Italy)was added to the medium from a stock solution of 106 IU/ml. Humanrecombinant IFN-b (Rebif; 3 3 108 IU/mg of protein; ARES-SERONO) wasadded to the medium from a stock solution of 104 IU/ml to the finalconcentration.

Western Blot and Immunoprecipitation Analyses. Whole-cell ly-sates were prepared in lysis buffer [0.5% NP40, 1% Triton X-100, 50 mM

Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.25% sodiumdeoxycholate, 0.5 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 1mg/ml leupeptin, 1 mg/ml pepstatin, 20 mM NaF, and 1 mM sodium or-thovanadate were freshly added to the buffer before each use], electro-phoresed on a 7 or 10% SDS-polyacrylamide gel as indicated, andtransferred to nitrocellulose for 60 min at 100V with a Bio-Rad transblot.Western blot detection was performed with the indicated antibodies anddeveloped with reagents for ECL (Amersham). Protein concentration wasdetermined by the Bio-Rad Protein Assay. p91 tyrosine phosphorylationwas analyzed on proteins from whole-cell lysates immunoprecipitatedwith specific anti-p91 antibody, electrophoresed on 7% SDS-polyacryl-amide gel, and blotted with 4G10 (UBI) and PY20 (ICN) mixed monoclonalantiphosphotyrosine antibodies. All other antibodies were from SantaCruz Biotechnology.

RT-PCR Analysis of IRF-1 mRNA. Total cellular RNA prepared (42)from ME-180 cells treated or not with 10 mg/ml of actinomycin D (Sigma)and/or IFN-a2b (2000 IU/ml) and/or RA (1026 M) for the indicated timeswas reverse transcribed and subsequently amplified. The cycler programconsisted of an initial denaturation of 3 min at 95°C, followed by 30 cyclesof denaturation for 40 s at 94°C, primer annealing for 40 s at 62°C, andextension for 1 min at 72°C. A negative control-lacking template RNA orreverse transcriptase was included in each experiment. The PCR productswere run on 3% agarose gel. The sequences for the GAPDH primers were:sense, CCATGGAGAAGGCTGGGG; and antisense, CAAAGTTGTCATG-GATGACC. The sequences for the IRF-1 primers were: sense, CCA-GAGAAAAGAAAGAAAGTCG; and antisense, CACATGGCGACAGT-GCTGG.

Oligonucleotides. For gel shift competitions and experiments, thefollowing double-stranded oligonucleotides were used (the top strand isshown. Consensus sequence is underlined.): GAS element, 59-GATC-GATTTCCCCGAAAT-39 (43); hE-IRS, 59-CTCCTCCCTTCTGAGGAAAC-GAAACCAACAGCAGTCCAAG-39 (44); and NF-kB motif, GGGCCGGC-CAGGGCTGGGGAATCCCGCTAAGTGTTTGGAT (30).

DNA Electrophoretic Mobility Shift Assay (EMSA). To measure theassociation between DNA-binding proteins and different DNA sequences,the double-stranded oligonucleotides (10 pmol) described above wereend-labeled with [g-32P]ATP (30 mCi; 6000 Ci/mmol NEN) by T4 polynu-cleotide kinase (Biolabs). The labeled oligonucleotide probes (10,000–20,000 cpm) were incubated for 30 min at 4°C and 20 min at roomtemperature in a final volume of 20 ml containing 20 mg of cell extractproteins [in 20 mM HEPES (pH 7.9), 50 mM NaCl, 10 mM EDTA, 2 mM EGTA,0.5% NP40, 0.5 mM DTT, 10 mM sodium molybdate, 100 mM NaF, 10mg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride] in a bindingbuffer containing 75 mM KCl, 20 mM Tris-HCl (pH 7.5), 13% (v/v) glycerol,1 mM DTT, 1 mg of bovine serum albumin, and 2 mg of poly(dI)-poly(dC)(Pharmacia). Cold competitors were added in 200-fold molar excess of theradiolabeled probe. For antibody treatments, 1–2 mg of specific antibodywere added to 20 mg of cell extract proteins. The analysis of DNA-proteincomplexes (45) was carried out on 5% polyacrylamide gels in 25 mM

Tris-borate (pH 8.2), 0.5 mM EDTA.Transient Transfection Assay. 293 and HepG2 cells (1.5 3 106 cells/

6-cm dish) were seeded and transfected with 5 mg of pIRF-1 Iuc and 4 mgof RSV-LacZ using the calcium-phosphate method. The 1.3-kb IRF-1promoter fragment subcloned in promoterless luciferase reporter genevector was described previously (11). RSV-lacZ construct was obtainedfrom A. Levi (Consiglio Nazionale delle Ricerche, Rome, Italy). The trans-

fected cells were treated as described in the Fig. 2 legend. Cells wereharvested 48 h later. Relative luciferase activity (Promega luciferase assaysystem) was measured with a luminometer and normalized by b-galac-tosidase activity.

AcknowledgmentsWe are grateful to Francesca Lancillotti for her contribution to the “Dis-cussion” of this work. We thank Roberto Orsatti and Stefania Mochi fortechnical assistance, Roberto Gilardi for preparing drawings, and SabrinaTocchio for editorial assistance.

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