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Loss of Net as Repressor Leads to Constitutive Increased c-fos Transcription in Cervical Cancer Cells*

  • Jan van Riggelen
    Affiliations
    Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
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  • Gilles Buchwalter
    Footnotes
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 Rue Laurent Fries, 67404 Illkirch Cedex, France
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  • Ubaldo Soto
    Affiliations
    Department of Biochemistry, School of Medicine, Loma Linda University, Loma Linda, California 92350
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  • Johanna De-Castro Arce
    Affiliations
    Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
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  • Harald zur Hausen
    Affiliations
    Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
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  • Bohdan Wasylyk
    Footnotes
    Affiliations
    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 Rue Laurent Fries, 67404 Illkirch Cedex, France
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  • Frank Rösl
    Correspondence
    To whom correspondence should be addressed: Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Tel.: 49-6221-424900; Fax: 49-6221-424902;
    Affiliations
    Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
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  • Author Footnotes
    ¶ Recipient of an Ministére de la Recherche et de la Technologie (MRT) fellowship.
    ** Supported by the Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer (Equipe labelisée), the Ligue Régionale (Haut-et Bas-Rhin) contre le Cancer, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, and the Hôpital Universitaire de Strasbourg.
    * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 16, 2004DOI:https://doi.org/10.1074/jbc.M409915200
      We have investigated the expression of c-fos in cervical carcinoma cells and in somatic cell hybrids derived therefrom. In malignant cells, c-fos was constitutively expressed even after serum starvation. Dissection of the c-fos promoter showed that expression was mainly controlled by the SRE motif, which was active in malignant cells, but repressed in their non-malignant counterparts. Constitutive SRE activity was not mediated by sustained mitogen-activated protein kinase activity but because of inefficient expression of the ternary complex factor Net, which was either very low or even barely discernible. Chromatin immunoprecipitation assays revealed that Net directly binds to the SRE nucleoprotein complex in non-tumorigenic cells, but not in malignant segregants. Small interfering RNA targeted against Net resulted in enhanced c-fos transcription, clearly illustrating its repressor function. Conversely, stable ectopic expression of Net in malignant cells negatively regulated endogenous c-fos, resulting in a disappearance of the c-Fos protein from the AP-1 transcription complex. These data indicate that loss of Net and constitutive c-fos expression appear to be a key event in the transformation of cervical cancer cells.
      Development of cervical cancer is a multistep process that is initiated by infection with “high risk” human papillomaviruses (HPVs).
      The abbreviations used are: HPV, human papillomavirus; SRE, serum response element; CRE, cAMP response element; TCF, ternary complex factor; SRF, serum response factor; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; AP-1, activator protein-1; JNK, c-Jun N-terminal kinases; ERK, extracellular signal-regulated kinases.
      1The abbreviations used are: HPV, human papillomavirus; SRE, serum response element; CRE, cAMP response element; TCF, ternary complex factor; SRF, serum response factor; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; AP-1, activator protein-1; JNK, c-Jun N-terminal kinases; ERK, extracellular signal-regulated kinases.
      The viral oncoproteins E6 and E7 are indispensable for proliferation of cervical carcinoma cells. They cause unlimited growth, which eventually leads to accumulation of cell damage, chromosomal instability, aneuploidy, and loss of tumor suppressor genes. However, expression of E6/E7 in primary human keratinocytes merely results in immortalization without further progression to tumorigenicity, showing that further events are important (
      • zur Hausen H.
      ). Moreover, HPV-induced cancer can be considered as a recessive genetic trait that can be complemented by somatic cell hybridization with primary human fibroblasts or keratinocytes. A genetically well defined in vitro model is provided by fusion of HPV18-positive HeLa cells with primary human fibroblasts, which results in hybrids that still express HPV18 E6/E7, but are non-tumorigenic in nude mice (
      • Stanbridge E.
      ). Long-term in vitro cultivation generates segregants with morphological features of tumorigenic cells (
      • Lichy J.H.
      • Modi W.S.
      • Seuanez H.N.
      • Howley P.M.
      ).
      The transcription factor AP-1, which is composed of heterodimers between Jun and Fos family members plays a crucial role in various cellular processes such as enhanced proliferation, differentiation, and neoplastic transformation (
      • Eferl R.
      • Wagner E.F.
      ). In the case of HPV-positive cells, there is substantial evidence that AP-1 composition determines their in vivo behavior in nude mice, their sensitivity against growth inhibitory cytokines, as well as their ability to express certain chemokines that are necessary to maintain immunological surveillance of persisting HPV infections (
      • Rösl F.
      • zur Kleine-Lowinski K.
      • Hausen H.
      ). Whereas in asynchronous growing primary human fibroblasts, keratinocytes and HPV16-immortalized human cells AP-1 mainly consists of Jun family members heterodimerized with Fra-1, c-Fos was found to be almost completely absent. Conversely, in cervical carcinoma cell lines such as HeLa, SW756, SiHa, or tumorigenic segregants derived from non-malignant hybrids, increased amounts of c-Fos and low levels of Fra-1 can be discerned, resulting in a prevalent Jun/c-Fos dimerization pattern (
      • Rösl F.
      • Das B.C.
      • Lengert M.
      • zur Geletneky K.
      • Hausen H.
      ,
      • Soto U.
      • Das B.C.
      • Lengert M.
      • zur Finzer P.
      • Hausen H.
      • Rösl F.
      ,
      • Soto U.
      • Denk C.
      • Finzer P.
      • zur Hutter K.J.
      • Hausen H.
      • Rösl F.
      ,
      • Talora C.
      • Sgroi D.C.
      • Crum C.P.
      • Dotto G.P.
      ). Moreover, in view of the fact that ectopic expression of c-fos induces tumorigenicity in non-malignant hybrids by changing the Jun/Fra-1 ratio in favor of Jun/c-Fos (
      • Soto U.
      • Das B.C.
      • Lengert M.
      • zur Finzer P.
      • Hausen H.
      • Rösl F.
      ), it is reasonable to assume that c-Fos regulation plays a fundamental role in the conversion of HPV-positive cells toward malignancy (
      • zur Hausen H.
      ).
      c-fos transcription is tightly controlled in normal cells and inappropriate expression contributes to various phenotypic changes (
      • Eferl R.
      • Wagner E.F.
      ). c-fos transcription is normally very low but is transiently induced in response to a wide range of extracellular stimuli, including serum, growth factors, cytokines, and tumor promoters (
      • Angel P.
      • Karin M.
      ). Impaired serum inducibility of c-fos is a feature of senescent human fibroblasts (
      • Seshadri T.
      • Campisi J.
      ), whereas its overexpression is a marker for progression of skin tumors and in general related to tumorigenicity (
      • Angel P.
      • Szabowski A.
      • Schorpp-Kistner M.
      ). Transient induction of c-fos expression is mainly mediated by four promoter elements: the sis-inducible element, the serum response element (SRE), the cAMP response element (CRE), and the c-fos AP-1 site (
      • Treisman R.
      ,
      • Gonzales M.
      • Bowden G.T.
      ). The majority of the signals regulate the SRE (
      • Price M.A.
      • Cruzalegui F.H.
      • Treisman R.
      ), which is constitutively occupied by the serum response factor (SRF) and a ternary complex factor (TCF) (
      • Herrera R.E.
      • Shaw P.E.
      • Nordheim A.
      ). Three members of the TCF family have been identified: Elk-1 (
      • Shaw P.E.
      • Schroter H.
      • Nordheim A.
      ,
      • Hipskind R.A.
      • Rao V.N.
      • Mueller C.G.
      • Reddy E.S.
      • Nordheim A.
      ), Sap1 (
      • Dalton S.
      • Treisman R.
      ), and Sap2/Erp/Net (
      • Giovane A.
      • Pintzas A.
      • Maira S.M.
      • Sobieszczuk P.
      • Wasylyk B.
      ,
      • Lopez M.
      • Oettgen P.
      • Akbarali Y.
      • Dendorfer U.
      • Libermann T.A.
      ). Net and to a lesser extent Elk-1 are repressors and all three TCFs are activators when phosphorylated by mitogen-activated protein kinases (MAPKs) (
      • Buchwalter G.
      • Gross C.
      • Wasylyk B.
      ). The SRE is inactive or repressive in the absence of positive signaling (
      • Giovane A.
      • Sobieszczuk P.
      • Ayadi A.
      • Maira S.M.
      • Wasylyk B.
      ,
      • Li Q.J.
      • Yang S.H.
      • Maeda Y.
      • Sladek F.M.
      • Sharrocks A.D.
      • Martins-Green M.
      ) but can be induced by two pathways: TCF phosphorylation by MAPKs (
      • Whitmarsh A.J.
      • Shore P.
      • Sharrocks A.D.
      • Davis R.J.
      ,
      • Cahill M.A.
      • Janknecht R.
      • Nordheim A.
      ) and Rho-actin signaling to SRF (
      • Hill C.S.
      • Wynne J.
      • Treisman R.
      ,
      • Miralles F.
      • Posern G.
      • Zaromytidou A.I.
      • Treisman R.
      ). Moreover, SRE-mediated c-fos transcription is modulated by the exchange of TCFs and the recruitment of co-regulatory complexes such as histone acetylases (
      • Nissen L.J.
      • Gelly J.C.
      • Hipskind R.A.
      ,
      • Li Q.J.
      • Yang S.H.
      • Maeda Y.
      • Sladek F.M.
      • Sharrocks A.D.
      • Martins-Green M.
      ) or histone deacetylases (
      • Yang S.H.
      • Sharrocks A.D.
      ). Less is known about the transcriptional repression processes that controls basal activity and the transient and self-terminating expression pattern of the c-fos gene (
      • Cahill M.A.
      ).
      In the present study we have unraveled the mechanism underlying deregulated c-fos expression in cervical carcinoma cells. Tumorigenic cells express relatively low levels of net and high levels of c-fos. Re-introduction of Net, in either a transient or a stable fashion, suppresses c-fos promoter-directed transcription and triggers the disappearance of the c-Fos protein from the AP-1 complex. In contrast, non-tumorigenic HPV-positive cells express relatively high levels of net and low levels of c-fos. Within these cells, Net can be detected bound to the endogenous c-fos promoter and decreasing Net levels with siRNA increases c-fos expression. These results support the notion that deregulation of Net, and in turn of c-fos expression, is an important event in the multistep progression to cervical cancer, the second leading cancer in women worldwide.

      EXPERIMENTAL PROCEDURES

      Cell Lines and Somatic Cell Hybrids—The cervical carcinoma cell lines HeLa, SiHa, CaSki, SW756, human lung fibroblasts (IMR-90), the non-tumorigenic somatic cell hybrid (“444”) between HeLa and IMR-90, and the tumorigenic segregant (“CGL3”) (
      • Stanbridge E.
      ) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% FCS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Stable CGL3-Net clones were grown in the same medium containing 1.2 mg/ml G418 (Invitrogen).
      Cell Treatment—Subconfluent cells were starved for 24 h in DMEM without FCS followed by incubation with 10% FCS for 1–8 h. Asynchronously growing cells were incubated with 500 units/ml TNF-α (Strathman Biotech GmbH, Hannover, Germany) for 15 min. For U0126 (Calbiochem), 10 or 20 μm cells were incubated in the culture medium for 5 h. For Latrunculin B, 0.5 or 2 μm cells were incubated in the culture medium for 5 h (
      • Tsakiridis T.
      • Bergman A.
      • Somwar R.
      • Taha C.
      • Aktories K.
      • Cruz T.F.
      • Klip A.
      • Downey G.P.
      ).
      Plasmid Constructs—pc-fos-luc, containing the –400/+50 region of the human c-fos promoter, was kindly provided by G. T. Bowden (
      • Gonzales M.
      • Bowden G.T.
      ). The pSRE5-Luc contains 5 tandem copies of the c-fos SRE, and the pCRE4-Luc, four copies of the CRE-binding sequence fused to the TATA minimal promoter (Stratagene). pCI-neo and the SV40 early promoter driven Renilla luciferase construct (pRL-SV40) were purchased from Promega. pTL2-hNet encodes the human Net cDNA driven by the SV40 early promoter (
      • Giovane A.
      • Sobieszczuk P.
      • Ayadi A.
      • Maira S.M.
      • Wasylyk B.
      ). The corresponding empty vector construct (pTL2) was derived from pTL2-hNet by SmaI excision of the 1.4-kilobase pair cDNA insert.
      Transient and Stable Transfections—The Effectene™ transfection reagent (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions. For reporter gene assays, 0.7 μg of firefly luciferase reporter plasmid was transiently co-transfected with various constructs. Transfection efficiencies were normalized with pRL-SV40. Stable CGL3-Net cell lines were generated by co-transfection of CGL3 cells with pTL2-hNet and pCI-neo. Clones were isolated by G418 selection and maintained in DMEM with 10% FCS, 1% penicillin/streptomycin, and 1.2 mg/ml G418.
      Luciferase Reporter Assays—Cell extracts were prepared 24 h after transfection. Firefly luciferase activity was measured in 25 mm glycylglycine, 15 mm Mg2SO4, 5 mm ATP, pH 7.8, and 7 μg/ml luciferin with a luminometer (Lumat 9501, Berthold). Renilla luciferase activity was examined using the Renilla Luciferase Assay System (Promega) according to the manufacturer's instructions. Experiments were done in triplicate and normalized luciferase activity is shown as x-fold increase relative to the basal activity.
      RNA Interference—Net targeted siRNAs (Dharmacon, Inc.) were designed according to published criteria (
      • Reynolds A.
      • Leake D.
      • Boese Q.
      • Scaringe S.
      • Marshall W.S.
      • Khvorova A.
      ). Sequences used were 5′-ACGAUGGUGAAUUCAAGCU-3′ (Net1) and the 5′-CUCGUCCUUCACCAUUAAU-3′ (Net2). The siRNA sequence targeting the GL2 luciferase was already described (
      • Elbashir S.M.
      • Harborth J.
      • Lendeckel W.
      • Yalcin A.
      • Weber K.
      • Tuschl T.
      ). 3 μg of the siRNA duplexes were transfected into 444 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's guidelines. Cells were harvested 48 h after transfection and total RNA was extracted with RNASolv (Omega Biotek). 1 μg of total RNA was used for first-strand cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). 10% of the first strand reaction was used for PCR to analyze the expression of net (5′-AGACCAAGTCTCCATCTCTTC-3′ and 5′-GACTAAGGCTGCTCCAGAAAT-3′), c-fos (5′-AGTGGAACCTGTCAAGAGCAT-3′ and 5′-GCTCCCAGTCTGCTGCATAGA-3′), and 28 S (5′-GGCGGCCAAGCGTTCATAGC-3′ and 5′-GCCAAGCACATACACCAAAT-3′). PCR conditions were 94 °C for 5 min, 94 °C for 30 s; 61 °C for 30 s, 72 °C for 30 s (25 cycles for net, 25 cycles for c-fos, and 15 cycles for 28 S) and 72 °C for 5 min.
      Cell Extracts and Western Blot Analysis—Nuclear extracts for electrophoretic mobility shift assays (EMSA) and Western blot analysis were prepared as described previously (
      • Bachmann A.
      • Hanke B.
      • Zawatzky R.
      • Soto U.
      • zur van Riggelen J.
      • Hausen H.
      • Rosl F.
      ). Protein concentration was determined by the Bradford method (Bio-Rad) using bovine serum albumin as a standard. Extracts were separated in 10% SDS-PAGE, electrotransferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) and probed with the following antibodies: c-Fos (catalog number 06-341, Upstate Biotechnology), c-Jun (sc-1694 X), Fra-1 (sc-605 X), and SRF (sc-335 X) from Santa Cruz Biotechnology; phospho-Ser103 SRF (number 4261), Elk-1 (number 9182), phospho-Ser383 Elk-1 (number 9181) from New England Biolabs (Frankfurt, Germany), and polyclonal anti-Net sera number 2005 (
      • Giovane A.
      • Sobieszczuk P.
      • Ayadi A.
      • Maira S.M.
      • Wasylyk B.
      ). For MAPK analyses, total cellular extracts were prepared using a lysis buffer containing 50 mm Tris, 2% SDS, 10% glycerol, 0.74 m β-mercaptoethanol, sonicated on ice using a Sonifier 250 (Branson Ultrasonics, Geneva, Switzerland), and finally heated for 5 min at 99 °C. Protein concentrations were determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad). Antibodies used for detection of ERK1/2 (number 9102), phospho-Thr202/Tyr204 ERK1/2 (number 9101S), c-Jun N-terminal kinases (JNK) (number 9252), phospho-Thr183/Tyr185 JNK (number 9251S), p38-MAPK (number 9212), and phospho-Thr180/Tyr182 p38 MAPK (number 9211S) were obtained from New England Biolabs. For Western blot analysis to monitor Net after siRNA delivery, total cellular extracts were prepared using a lysis buffer containing 0.4 m KCl, 20 mm Tris-HCl, pH 7.5, 20% glycerol, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, and the proteinase inhibitor mixture Complete (Roche Diagnostics, Penzberg, Germany). Cellular lysis was performed by 3 cycles of freezing/thawing and finally heating for 10 min at 99 °C. Membranes were incubated overnight in Tris-buffered saline supplemented with 5% skim milk powder (Roth) and 0.1% Tween 20 (Sigma). The bands were visualized with horseradish peroxidase-conjugated anti-rabbit IgG or an anti-mouse IgG (Promega) using the ECL (enhanced chemiluminescence) detection system (PerkinElmer Life Sciences, Inc). Loading was confirmed by re-incubating the membranes with a monoclonal actin-specific antibody (ICN Biomedicals, Costa Mesa, CA). For re-incubation with additional antibodies, the filters were stripped for 5 min in 0.2 m NaOH.
      RNA Analysis and Reverse Transcriptase (RT)-PCR—Cytoplasmic RNA was isolated with the RNeasy Kit (Qiagen) according to the manufacturer's instructions. To check RNA quality, 5 μg of RNA was separated on 1% agarose gels in the presence of ethidium bromide under non-denaturing conditions (
      • Rösl F.
      • zur Dürst M.
      • Hausen H.
      ). For RT 1–3 μg of RNA was mixed with 0.2 μg of random primers (Roche), heated at 70 °C for 10 min, and chilled on ice. The mixture was supplemented with reaction buffer (50 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2), 10 mm dithiothreitol, 500 μm deoxynucleoside triphosphate mixture (Roche) and incubated at 25 °C for 10 min. 100 Units of reverse transcriptase SuperScript II (Invitrogen) was added, the reaction was incubated at 42 °C for 50 min, heated to 70 °C for 15 min, and chilled on ice. PCR was performed in a total volume of 50 μl containing 10 mm Tris-HCl, pH 8.3, 200 μm deoxynucleoside triphosphate mixture (Roche Diagnostics), 500 nm forward and reverse primers, 5 units of Taq polymerase (Invitrogen), and 1–3 μl of RT product, using an MJ Research PTC-200 thermal cycler. The conditions were: c-fos, primers 5′-AACTTCATTCCCACGGTCAC-3′ and 5′-CCTTCTCCTTCAGCAGGTTG-3′, 35 cycles of 30 s at 94 °C, 45 s at 55 °C, and 30 s at 72 °C (the last extension time was 10 min) and rapid cooled to 4 °C; fra-1, primers 5′-GCGCCTAGGCCTTGTATCTCCCTTTCCCC-3′ and 5′-CCGCTCGAGGCGAGGAGGGTTGGAGAGCC-3′, 35 cycles and annealing temperature of 65 °C; net, primers 5′-AACTACGACAAGCTGAGCAGAGC-3′ and 5′-AGCGGTCTCGGATGTGGAAGG-3′, annealing temperature of 55 °C (
      • Giovane A.
      • Sobieszczuk P.
      • Ayadi A.
      • Maira S.M.
      • Wasylyk B.
      ); GAPDH internal control, primers 5′-TGGATATTGTTGCCATCAATGACC-3′ and 5′-GATGGCATGGACTGTGGTCATG-3′ (
      • Griffiths D.J.
      • Venables P.J.
      • Weiss R.A.
      • Boyd M.T.
      ), 35 cycles and annealing temperature of 65 °C. The PCR products were analyzed with 1–2% agarose gels containing ethidium bromide.
      EMSA—The AP-1 consensus sequence 5′-CGCTTGATGACTCAGCCGGAA-3′ from the human collagenase promoter (
      • Lee W.
      • Mitchell P.
      • Tjian R.
      ) was used. Oligonucleotides were made with an Applied Biosystems (Foster City, CA) synthesizer and purified by high performance liquid chromatography. The annealed oligonucleotides were labeled with 3000 Ci/mmol [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs), and purified with a 15% polyacrylamide gel. Binding reactions were performed in 20 μl containing 10% glycerol, 12 mm HEPES, pH 7.9, 4 mm Tris-HCl, pH 7.9, 60 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 0.6 mg/ml bovine serum albumin, 2 μg of poly(dI-dC), and 2 μg of nuclear extract. After 5 min, 10,000 cpm of the probe was added and incubation was continued for 30 min at room temperature. For supershift assays, 2 μg of c-Fos antibody (sc-52 X, Santa Cruz Biotechnology) was added and further incubated for 1 h at 4 °C. The complexes were resolved with 5.5% non-denaturing polyacrylamide gels (29:1 cross-linking ratio). The gels were dried and exposed to x-ray films (Amersham Biosciences) or analyzed using PhosphorImager Storm 820 and ImageQuant software (Amersham Biosciences).
      Chromatin Immunoprecipitation (ChIP) Assay—The ChIP Assay Kit (number 17–295) from Upstate Cell Signaling Solutions (Lake Placid, NY) was used according to the manufacturer's instructions. Subconfluent cells were cross-linked with 1% formaldehyde for 10 min at 37 °C, washed, and scraped in ice-cold PBS containing the proteinase inhibitor mixture Complete (Roche Diagnostics). After lysis of 1 × 106 cells, the DNA was sheared into 200–1000-bp fragments by sonification (4 times for 10 s, Sonifier 250, Branson Ultrasonics, Geneva, Switzerland) and immunoprecipitated with 4 μl of polyclonal anti-Net sera numbers 2005 or 2007 (
      • Giovane A.
      • Sobieszczuk P.
      • Ayadi A.
      • Maira S.M.
      • Wasylyk B.
      ) or anti-SRF (sc-335X, Santa Cruz Biotechnology). The co-precipitated DNA was analyzed by semi-quantitative PCR as described above. The conditions for PCR and the primers were as followed: c-fos SRE (position –300) 5′-GAGCAGTTCCCGTCAATCC-3′ and 5′-CCCCAAGATGAGGGGTTT-3′, 35 cycles and annealing temperature of 57 °C. c-fos promoter (position –600), 5′-CAAACGCAGGAACAGTGCTA-3′ and 5′-GAAGGAATGCGCCCCCTAC-3′, 35 cycles and annealing temperature of 57 °C. GAPDH was used as an additional control sequence located on a heterologous chromosome (
      • Benham F.J.
      • Povey S.
      ). The PCR products were analyzed on 1.5% agarose gels in the presence of ethidium bromide.

      RESULTS

      Inverse Correlation of c-fos and fra-1 Expression in Tumorigenic and Non-tumorigenic HPV-positive Cell Lines—To monitor the regulation of the transcription factor AP-1 in cells differing in their capability to form tumors in immunocompromised animals, we used HPV 18-positive HeLa cells and derived somatic cell hybrids as an experimental model (
      • Stanbridge E.
      ). This allows the examination of AP-1 in a cellular environment harboring identical viral transcription cassettes (
      • Rösl F.
      • zur Dürst M.
      • Hausen H.
      ), but where the oncoproteins are expressed in a different genetic background (
      • Bachmann A.
      • Hanke B.
      • Zawatzky R.
      • Soto U.
      • zur van Riggelen J.
      • Hausen H.
      • Rosl F.
      ). As depicted in Fig. 1, asynchronously growing tumorigenic (HeLa, CGL3) and non-tumorigenic hybrids (444) varied considerably in their c-Fos and Fra-1 content. Both proteins can be resolved as broad bands by SDS-PAGE because of differential post-translational phosphorylation (
      • Okazaki K.
      • Sagata N.
      ). Similar to normal human lung fibroblasts (IMR-90), nuclear extracts obtained from 444 cells showed a complete absence of the c-Fos protein and only a marginal detection of c-fos-specific RNA after semiquantitative RT-PCR. In contrast, fra-1 was highly expressed both on mRNA and protein levels. Examining malignant HeLa and CGL3 cells, both inherently express more c-fos mRNA and corresponding protein than their non-malignant counterparts.
      Serum Inducibility of Fos Expression in Non-malignant and Malignant Cells—To determine whether increased mRNA and protein levels in tumorigenic cells remain constitutive after serum depletion, cells were starved for 24 h 1 day after seeding. Expression was monitored by Western blot and RT-PCR analyses (Fig. 2A). Considering both parental HeLa and CGL3 cells, starvation was not accompanied with any changes of c-fos expression. However, when the cells were incubated for 1 h with fresh serum, c-fos could be induced. On the other hand, in asynchronized as well as in serum-deprived non-malignant cells, c-fos expression was barely detectable, whereas serum addition led to a striking accumulation of c-Fos both on protein and RNA level. Next we examined binding affinity and composition by EMSAs. As shown in Fig. 2B, serum starvation of non-malignant cells resulted in a substantial reduction of AP-1 binding affinity, whereby AP-1 completely lacked c-Fos. Nonetheless, 1 h of serum stimulation was sufficient to incorporate c-Fos into AP-1, leading to the appearance of lower migrating bands after antibody addition. In malignant counterparts, omission of serum did not reveal any reduction of binding. Here, AP-1 inherently contained c-Fos that was further elevated in its ratio upon serum stimulation. In kinetic experiments (Fig. 2C), c-Fos was initially undetectable in serum-starved non-tumorigenic cells, but appeared within 1 h after serum addition, increased up to 5 h and returned to baseline level ∼8 h after supplementation. However, within CGL3 cells, stimulation resulted in a prolonged increase of c-Fos, even after several hours. Hence, only in non-tumorigenic cells, c-Fos behaves as a typical immediately early gene product, where the extent and the temporal range of its transcription are tightly controlled (
      • Rivera V.M.
      • Greenberg M.E.
      ).
      Figure thumbnail gr3
      Fig. 3Analysis of c-fos promoter activity. A, luciferase activity in HeLa, 444, and CGL3 cells using a c-fos promoter (–400/+50 region) luciferase reporter (left), a 5 × SRE-luciferase construct (middle), or a 4 × CRE-luciferase vector (right). A co-transfected SV40-driven Renilla luciferase construct was used for normalization. Ordinate, normalized luciferase activity relative to 444. All experiments were done in triplicate. B, schematic overview of the cis-regulatory elements of the c-fos promoter and the regulatory element-specific luciferase reporters. SIE, sis-inducible element; FAP-1, c-Fos AP-1-like sequence; TATA, TATA-like sequences.
      Figure thumbnail gr1
      Fig. 1Inverse correlation of c-fos and fra-1 expression. IMR-90, HeLa, 444, and CGL3 were harvested 1 day after seeding. Upper part, RNA quality monitored by agarose gel electrophoresis, and amounts of c-fos, fra-1, and GAPDH were estimated by semi-quantitative RT-PCR. 28 S and 18 S ribosomal RNAs and sizes in bp are indicated. Lower part, Western blots of nuclear extracts (20 μg/lane) using 10% SDS-PAGE gels and antibodies against c-Fos, Fra-1, and actin as loading control. Molecular masses are indicated in kDa.
      Figure thumbnail gr2
      Fig. 2Constitutive, but still inducible c-fos expression in tumorigenic cell lines. A, c-fos expression in IMR-90, 444, HeLa, and CGL3 cell lines cultivated for 24 h in DMEM containing 10% FCS (untreated), for 24 h in DMEM without FCS (starved) or for 1 h with 10% FCS (1 h serum) after 24 h of depletion. RT-PCR and Western blot analyses were carried out as indicated in the legend to . B, EMSAs using 32P-labeled AP-1 consensus oligonucleotides and 2 μg of a c-Fos antibody for the “supershifts.” The AP-1 and retarded complexes are marked by arrows. Serum omission and re-addition was performed as above. C, kinetics of c-Fos induction. 444 and CGL3 cells, starved of FCS for 24 h, were stimulated with 10% FCS for 1–8 h. Western blot analyses were performed using 40 μg of nuclear protein extract per lane and antibodies were raised against c-Fos and actin as loading control.
      The SRE Mainly Contributes to an Increased c-fos Promoter Activity in Tumorigenic Cells—To test whether c-fos is differentially regulated at the level of transcriptional initiation, transient transfection assays were performed. Comparing the relative luciferase activity, where the absolute counts for the 444 hybrids were arbitrarily set as 1, the c-fos promoter was found to be 2–4-fold more active in malignant than in non-tumorigenic cells (Fig. 3). In another set of experiments, we analyzed the SRE and the CRE, known to be critical in spatial and temporal gene response (
      • Wang Y.
      • Prywes R.
      ,
      • Gonzales M.
      • Bowden G.T.
      ). Whereas CRE-controlled reporter plasmids did not reveal any notable differences, SRE-driven luciferase constructs showed a 5-fold higher activity in HeLa cells and an about 7-fold higher activity in tumorigenic segregants when compared with 444 cells. This suggests that the SRE acts as the major cis-regulatory sequence in normal cells, mediating both induction and successive repression of the gene.
      Extracellular Signal-regulated Kinases (ERK) 1/2, JNK, and p38 MAPKs Are Not Responsible for Constitutive SRE Activity in Tumorigenic Cells—Because the SRE is activated by MAP kinases (
      • Whitmarsh A.J.
      • Shore P.
      • Sharrocks A.D.
      • Davis R.J.
      ), we next examined whether tumorigenic cells have enhanced activities of three major types of MAPKs, the ERK, JNK, and p38 MAPK. TNF-α was used to stimulate these pathways (
      • Yang S.H.
      • Sharrocks A.D.
      • Whitmarsh A.J.
      ). As shown in Fig. 4A, there was no detectable activated JNK or p38 MAPK in tumorigenic or non-tumorigenic cells, but phosphorylation was induced after TNF-α application. However, as depicted for ERK1/2, little but still visible phosphorylation in untreated HeLa and CGL3 cells could be discerned. Differential phosphorylation could be not attributed to quantitative changes of the proteins, because control incubations with non-phosphorylation-specific antibodies revealed that the net amount of the corresponding proteins was the same. The higher pre-existing phosphorylation of ERK1/2 in 444 cells that have lower SRE activity reinforces the notion that increased MAP kinase activity does not account for the greater SRE activity in tumorigenic cell lines.
      Figure thumbnail gr4
      Fig. 4Activities of MAP kinase pathways. A, Western blots of total cellular extracts (20 μg/lane). HeLa, 444, and CGL3 cells were cultivated for 24 h in DMEM, 10% FCS (untreated), or stimulated for 15 min with TNF-α (500 units/ml). Filters were first incubated with antibodies recognizing the phosphorylated forms of the ERK1/2, JNK, or p38, or total non-phosphorylated ERK1/2, JNK, or p38. B, effect of ERK1/2-inhibition on c-fos expression. 444 and CGL3 cells were cultivated for 24 h in DMEM including 10% FCS (untreated) and incubated for 5 h with 10 or 20 μm of the MEK1/2-inhibitor U0126. Upper part, semi-quantitative RT-PCR for c-fos and GAPDH using cytoplasmic RNA. Lower part, Western blot analysis of phosphorylated and non-phosphorylated ERK1/2. The sizes of the PCR products and proteins are indicated.
      In response to extracellular stimuli, ERK1/2 are normally activated by phosphorylation by the dual-specific MAPK kinases MEK1/2, which in turn activates c-fos transcription via phosphorylation of TCF family members at the SRE (
      • Price M.A.
      • Cruzalegui F.H.
      • Treisman R.
      ). Hence, one can anticipate that inhibition of ERK1/2 by the MEK1/2-inhibitor U0126 should prevent induction of SRE-mediated c-fos transcription (
      • Li Q.J.
      • Yang S.H.
      • Maeda Y.
      • Sladek F.M.
      • Sharrocks A.D.
      • Martins-Green M.
      ). As shown in Fig. 4B, U0126 treatment resulted in a dose-dependent decline of ERK1/2 phosphorylation, whereas the amount of the protein remained constant. Inspecting the steady state level of the c-fos mRNA, no difference was detectable. One can therefore conclude that constitutive c-fos transcription in tumorigenic cells was not depending on ERK1/2 or JNK/p38-MAPKs, even though the gene was still sensitive to serum stimulation. This raised the question, whether phosphorylation or the expression of SRE-activator proteins were elevated in a MAPK-independent manner.
      Expression and Phosphorylation Levels of the SRE-activator Proteins Elk-1 and SRF—In response to stimulation, c-fos SRE is activated by phosphorylation of TCFs such as Elk-1 via the Ras-ERK pathway (
      • Gille H.
      • Kortenjann M.
      • Strahl T.
      • Shaw P.E.
      ). In addition, c-fos can be also induced via Rho-actin signaling, targeting SRF in a TCF-independent manner (
      • Hill C.S.
      • Wynne J.
      • Treisman R.
      ,
      • Miralles F.
      • Posern G.
      • Zaromytidou A.I.
      • Treisman R.
      ). To investigate the potential contribution of these two signaling routes, we examined the basal phosphorylation levels of both Elk-1 and SRF. Treatment with TNF-α again served as a positive control for a temporal defined MAPK activation. Western blot analyses (Fig. 5A) revealed similar basal levels of Elk-1 and SRF phosphorylation in tumorigenic and non-tumorigenic cells, which could be further stimulated by TNF-α. In addition, inhibition of Rho-acting signaling by Latrunculin B (
      • Miralles F.
      • Posern G.
      • Zaromytidou A.I.
      • Treisman R.
      ) did not reveal any effect of c-fos expression in CGL3 cells (Fig. 5B), also excluding this pathway being responsible for enhanced c-fos transcription. Taken together, these data argue against the concept that increased SRE activity was because of constitutive hyperphosphorylation of Elk-1 or SRF.
      Figure thumbnail gr5
      Fig. 5Analysis of Elk-1 and SRF. A, Western blot analysis of phosphoserine 383 Elk-1 (P-Elk-1), phosphoserine 103 SRF (P-SRF), and non-phosphorylated Elk-1 and SRF. The cells were cultivated for 24 h in DMEM including 10% FCS (untreated) and stimulated for 15 min with TNF-α (500 units/ml), as indicated. Nuclear proteins (20 μg/lane) were separated by 10% SDS-PAGE. Actin was the loading control. B, semiquantitative RT-PCR of c-fos and GAPDH in CGL3 cells after 5 h treatment with 0.5 and 2 μm latrunculin B. The positions of the 28 S and 18 S rRNAs as well as the sizes of the PCR products are indicated.
      Diminished Net Expression in Tumorigenic Cells: Loss of Repressor Function at the c-fos Promoter—Because none of the classical activators were found to be involved in differential c-fos expression, we reasoned that the loss of a repressor at the SRE might be responsible for constitutive transcription in tumorigenic cells. Within the TCF family, two members can principally act as repressors. In the absence of MAP kinase signaling, Net and to lesser extent Elk-1, have been demonstrated to repress transcription (
      • Giovane A.
      • Sobieszczuk P.
      • Ayadi A.
      • Maira S.M.
      • Wasylyk B.
      ,
      • Yang S.H.
      • Vickers E.
      • Brehm A.
      • Kouzarides T.
      • Sharrocks A.D.
      ). Because there were no quantitative and qualitative differences of Elk-1, Net expression was monitored. Interestingly, high levels of Net were only detected in normal human fibroblasts (IMR-90) and in non-tumorigenic hybrids (444), whereas Net was significantly diminished in HeLa cells and almost absent in tumorigenic segregants both on RNA and protein levels (Fig. 6A). Moreover, as depicted in Fig. 6B, we also found an inverse correlation of Net and c-fos transcription in other cervical carcinoma cells. Both HPV16-positive SiHa as well as HPV18-positive SW756 cells exhibited low levels of Net, whereas c-fos transcription was highly up-regulated. Exceptional for cervical carcinoma cells, c-fos was weakly transcribed in HPV16-positive CaSki cells (
      • Soto U.
      • Denk C.
      • Finzer P.
      • zur Hutter K.J.
      • Hausen H.
      • Rösl F.
      ). Notably, examining these cells by RT-PCR and Western blot analysis, a high amount of Net could be discerned (Fig. 6B). Because it has been postulated that Net significantly contributes to the repression of basal c-fos SRE activity (
      • Buchwalter G.
      • Gross C.
      • Wasylyk B.
      ), these data may provide the first direct mechanistic link between its diminished expression and increased c-fos transcription in a highly clinical relevant form of human cancer.
      Figure thumbnail gr6
      Fig. 6Semi-quantitative RT-PCR and Western blot analysis of Net expression. IMR-90, HeLa, 444, CGL3, CaSki, SiHa, and SW756 cells were harvested 1 day after seeding. A, upper part: RNA quality monitored by agarose gel electrophoresis. The amounts of net and GAPDH were estimated by semiquantitative RT-PCR. 28 S and 18 S ribosomal RNAs and sizes in bp are indicated. Lower part: Western blots of nuclear extracts (40 μg/lane) using 10% SDS-PAGE gels and antibodies against Net and actin. Molecular masses are indicated in kDa. B, upper part: semiquantitative RT-PCR for net, c-fos, and GAPDH. Lower part: Western blot as described in panel A.
      In Vivo Binding of Net to the c-fos SRE: Loss of Repressor Function in Tumorigenic Cells—To demonstrate that Net is differentially recruited at the c-fos SRE nucleoprotein complex in vivo, ChIPs were carried out (Fig. 7). Formaldehyde cross-linked protein-DNA complexes were immunoprecipitated using two different polyclonal antibodies against Net. Co-precipitated DNA fragments were amplified using specific primers surrounding the area of the c-fos SRE. The specificity of the ChIP reaction was confirmed using a randomly selected DNA sequence of the c-fos upstream region (position –600) and for a coding stretch within the GAPDH gene. Equal amounts of sheared genomic DNA were used as template. SRF was found to be constitutively associated with the SRE in both cell lines. In contrast, Net binding at the c-fos SRE occurred exclusively in 444 cells, but was absent in CGL3. These data suggest that the loss of Net leads to increased c-fos transcription in malignant HPV-positive cells.
      Figure thumbnail gr7
      Fig. 7ChIP detection of Net bound to the c-fos SRE. A, two different polyclonal antibodies recognizing Net (IP: anti-Net 05 and 07) and a polyclonal antibody directed against SRF (IP: anti-SRF) were used. The co-precipitated DNA fragments were analyzed by PCR using specific primers for the c-fos SRE, the –600 region of the c-fos transcriptional start (c-fos –600 bp), and the coding region of the GAPDH gene. PCR products were analyzed in 1.5% agarose gels. B, schematic overview of the c-fos promoter, showing the location of the primers (indicated by arrows).
      Exogenous Net Suppresses c-fos in Tumorigenic Cells Whereas Net siRNA Induces c-fos Transcription in the Non-malignant Counterparts—To prove a causal relationship between the lack of repressor function at the SRE and increased c-fos transcription, we first examined the effect of ectopically expressed Net in transient transfection assays. As shown for CGL3 cells, Net expression inhibited SRE and c-fos promoter activity, but had no effect on the CRE (Fig. 8A). To exclude potential squelching of general transcription factors because of the high copy number of the effector plasmid under transient transfection conditions, we established stable cell lines of CGL3 expressing a net cDNA under a heterologous promoter. In comparison with parental cells, transfected clones showed distinct net expression levels that inversely correlated with an endogenous amount of c-fos mRNA and protein (Fig. 8B). In addition, supershift EMSA demonstrated that the amount of c-Fos associated with AP-1 was reduced (Fig. 8C). To further corroborate the repressor function, non-malignant cells were transfected with siRNA directed against Net. As shown in Fig. 8D, in the same way as endogenous Net, mRNA and protein were diminished, c-fos transcription was increased. Delivery of a nonspecific siRNA showed no effect on both genes. These results provide strong evidence that constitutive expression of c-fos transcription in tumorigenic cells is because of the loss of Net repressor activity.
      Figure thumbnail gr8
      Fig. 8Effects of modulation of Net expression on c-fos promoter activity and expression. A, effect of Net overexpression on SRE-, CRE-, and c-fos-directed transcription (see for details). Co-transfected SV40 driven Renilla luciferase was used for normalization. Ordinate, relative luciferase activity of transfections with the Net expression vector compared with the empty vector. Abscissa, activity of SRE-, CRE-, and c-fos-reporter plasmids. B, effects of ectopic Net expression on endogenous c-fos transcription. Semiquantitative RT-PCR was used to monitor the levels of net,c-fos, and GAPDH mRNA in 444, CGL3, and the stable clones (CGL3-Net clone 1–3). c-Fos and actin were detected by Western blotting as indicated above. C, EMSA showing the disappearance of c-Fos from the AP-1 complex in “clone 1,” expressing the highest amount of Net. 2 μg of an anti-c-Fos antibody was used for the supershifts. Arrows mark the AP-1 and the retarded complexes. D, down-regulation of endogenous Net by siRNA increases c-fos expression in 444 cells. Control, non-transfected cells; siRNA luc, luciferase siRNA; siRNA net 01 and 02, siRNA targeting two distinct region of net mRNA. Total RNA was purified 48 h post-transfection and semiquantitative RT-PCR was used to measure the amounts of net, c-fos, and the internal control 28 S rRNA. Net and actin were detected by Western blot analysis of 50 μg of total lysates/lane.

      DISCUSSION

      The dimeric transcription factor AP-1 acts as a junction point for many regulatory mechanisms associated with proliferation, apoptosis, and tumorigenicity (
      • Eferl R.
      • Wagner E.F.
      ). AP-1 becomes activated upon a whole variety of extra/intracellular stimuli including growth factors, cytokines, tumor promoters, and oncogenes (
      • Karin M.
      • Liu Z.
      • Zandi E.
      ). The decision, however, which target genes finally turns out to be induced, is apparently depending on AP-1 composition (
      • van Dam H.
      • Castellazzi M.
      ). For HPV-positive human cells, AP-1 is part of an intracellular surveillance network determining not only the in vivo phenotype but also the sensitivity/response against growth inhibitory cytokines and chemokines (
      • zur Hausen H.
      ,
      • Soto U.
      • Das B.C.
      • Lengert M.
      • zur Finzer P.
      • Hausen H.
      • Rösl F.
      ,
      • Soto U.
      • Denk C.
      • Finzer P.
      • zur Hutter K.J.
      • Hausen H.
      • Rösl F.
      ).
      Raising the question how c-fos is deregulated in HPV18-positive cervical carcinoma cells, we took advantage of a model system where fusion of malignant and normal cells resulted in somatic hybrids that are non-tumorigenic in nude mice (
      • Stanbridge E.
      ). We found that only malignant cells transcribed high levels of c-fos under asynchronized growth conditions, whereas there was almost no detectable signal when non-tumorigenic cells were examined (Fig. 1). Although the intracellular availability of c-Fos can also be regulated in a post-translational manner (
      • Acquaviva C.
      • Ferrara P.
      • Bossis G.
      • Brockly F.
      • Salvat C.
      • Jariel-Encontre I.
      • Piechaczyk M.
      ), the amount of protein in Western blot closely matched with the steady state level of the corresponding mRNA (Fig. 1). Moreover, because actinomycin D treatment (
      • Loflin P.T.
      • Chen C.Y.
      • Xu N.
      • Shyu A.B.
      ) also did not reveal any differences of the mRNA half-life (data not shown), c-fos expression was evidently controlled at the level of initiation of transcription. Although both viral oncoproteins can stimulate the c-fos promoter in rodent cells (
      • Morosov A.
      • Phelps W.C.
      • Raychaudhuri P.
      ), transcriptional regulation of c-fos in the human system was actually independent of E6/E7 expression. This supports previous data demonstrating that the induction of particular cytokines/chemokines was determined by the in vivo phenotype, rather than by the expression of the viral oncoproteins per se (
      • Bachmann A.
      • Hanke B.
      • Zawatzky R.
      • Soto U.
      • zur van Riggelen J.
      • Hausen H.
      • Rosl F.
      ,
      • Rösl F.
      • Lengert M.
      • Albrecht J.
      • Kleine K.
      • Zawatzky R.
      • zur Schraven B.
      • Hausen H.
      ).
      We found that non-tumorigenic hybrids behave similarly to normal cycling cells (IMR-90), where both the extent and the temporal range of c-fos expression were tightly controlled (
      • Angel P.
      • Szabowski A.
      • Schorpp-Kistner M.
      ,
      • Treisman R.
      ). Such a negative regulatory loop is obviously missing in tumorigenic cells, where c-fos remained expressed both under asynchronous growth conditions and during serum starvation (Fig. 2). Remarkably, although not followed up in molecular terms at the time, an earlier study has already provided initial clues for a constitutive re-expression of higher c-fos levels after transition of non-tumorigenic HPV-positive somatic hybrids toward malignancy (
      • O'Hara B.M.
      • Klinger H.P.
      • Curran T.
      • Zhang Y.D.
      • Blair D.G.
      ). Accordingly, ectopic expression of c-fos in 444 hybrids provoked rapid tumor formation by shifting the AP-1 composition from Jun/Fra-1 to Jun/c-Fos. Hence, a potential linkage between c-fos deregulation and E6/E7 expression during human tumor progression has been predicted (
      • Soto U.
      • Das B.C.
      • Lengert M.
      • zur Finzer P.
      • Hausen H.
      • Rösl F.
      ).
      In fact, inappropriate transcriptional control of c-fos in malignant cells could be further substantiated in transient transfections (Fig. 3). Using either the entire c-fos promoter or individual cis-regulatory elements, luciferase reporter activity was consistently higher in malignant cells than in their non-malignant counterparts. This property was apparently mediated by the SRE, known to be the major target sequence responsive to extracellular signal transduction (
      • Shaulian E.
      • Karin M.
      ).
      Signal transduction is controlled both by the strength and duration of MAPK activation (
      • Yang S.H.
      • Bumpass D.C.
      • Perkins N.D.
      • Sharrocks A.D.
      ). Because increased MAPK activity is normally considered as a hallmark for cancer cells (
      • English J.M.
      • Cobb M.H.
      ), we reasoned that sustained activation of this pathway might account for enhanced SRE activity. However, neither JNK nor p38 MAPK were affected in its basal activity (Fig. 4). Only ERK1/2 showed constitutive phosphorylation in non-tumorigenic hybrids (444), arguing for a stronger functionality of the upstream kinases MEK1 and -2 (
      • Whitmarsh A.J.
      • Davis R.J.
      ). Elevated ERK1/2 activity, however, could not be responsible for constitutive c-fos expression, because treatment with the MEK1/2 inhibitor U0126 (
      • English J.M.
      • Cobb M.H.
      ) abrogates ERK1/2 phosphorylation without any consequences on the steady state level of the c-fos specific mRNA (Fig. 4B). Notably, even though enhanced ERK1/2 activity has been described for many other human malignancies as the result of Ha-ras proto-oncogene activation (
      • Hoshino R.
      • Chatani Y.
      • Yamori T.
      • Tsuruo T.
      • Oka H.
      • Yoshida O.
      • Shimada Y.
      • Ari-i S.
      • Wada H.
      • Fujimoto J.
      • Kohno M.
      ), HeLa cells as well as primary tumor specimens directly obtained from cervical cancer patients apparently lack mutated Ha-ras allele expression (
      • Mammas I.N.
      • Zafiropoulos A.
      • Koumantakis E.
      • Sifakis S.
      • Spandidos D.A.
      ).
      Although c-fos expression was maintained at an elevated level through a mechanism independent of increased MAPK activity, constitutive SRE activity could be either because of deficient antagonistic phosphatase function (
      • Sugimoto T.
      • Stewart S.
      • Guan K.L.
      ,
      • Tian J.
      • Karin M.
      ) or by stimuli-independent phosphorylation by other kinases (
      • Galetic I.
      • Maira S.M.
      • Andjelkovic M.
      • Hemmings B.A.
      ). Alternatively, SRE activity could be also regulated by a pathway involving the Rho family GTPases (
      • Gineitis D.
      • Treisman R.
      ). These options, however, could be excluded, because both the steady state and phosphorylation levels of potential target proteins such as Elk1 and SRF (
      • Yang S.H.
      • Bumpass D.C.
      • Perkins N.D.
      • Sharrocks A.D.
      ) were the same (Fig. 5).
      In the absence of active MAPK signaling, TCF family members Net and to a lesser extent Elk-1 have been implicated in repression of transcription (reviewed in Buchwalter et al. (
      • Buchwalter G.
      • Gross C.
      • Wasylyk B.
      )). Moreover, there is also experimental evidence that silencing of Net in NIH3T3 cells was followed by an increase of basal c-fos expression (
      • Giovane A.
      • Pintzas A.
      • Maira S.M.
      • Sobieszczuk P.
      • Wasylyk B.
      ,
      • Maira S.M.
      • Wurtz J.M.
      • Wasylyk B.
      ). By using RT-PCR and Western blot analyses, we found considerable differences in Net expression in malignant and non-malignant HPV-positive cells. Whereas net was constitutively expressed in fibroblasts and in non-tumorigenic 444 cells, the corresponding mRNA level was significantly diminished in three of four cervical carcinoma cells (HeLa, SiHa, and SW756) and almost undetectable in tumorigenic CGL3 hybrids (Fig. 6). Notably, similar to non-malignant cells, HPV16-positive CaSki cells expressed high/low levels of Net/c-Fos. This was interesting in the context of our previous observation that in contrast to SiHa or SW756, CaSki cells were able to complement highly tumorigenic HeLa cells to a non-malignant phenotype after somatic cell hybridization (
      • Soto U.
      • Denk C.
      • Finzer P.
      • zur Hutter K.J.
      • Hausen H.
      • Rösl F.
      ). Moreover, a potential link between Net and the nucleosomal organization of the c-fos promoter was provided by the fact that its binding to native chromatin could be exclusively detected in non-malignant cells when formaldehyde cross-linked lysates were analyzed (Fig. 7). Intriguingly, Net harbors a repression domain, which is not conserved in other TCFs, but is capable of suppressing transcription by interacting with C-terminal-binding protein as co-repressor. This connection may provide a possible mechanism for inhibition, because C-terminal-binding protein is involved in the recruitment of histone deacetylase and in turn in silencing of chromatin (
      • Criqui-Filipe P.
      • Ducret C.
      • Maira S.M.
      • Wasylyk B.
      ). Interestingly, when asynchronized 444 cells were incubated in the presence of the histone deacetylases inhibitor trichostatin A, c-fos transcription could be induced (data not shown). This reinforces the notion that Net and probably also C-terminal-binding protein mediate both transcriptional repression and chromatin remodeling.
      In any case, ectopically expressed net cDNA resulted almost in a complete suppression of SRE- and c-fos promoter-directed reporter constructs under transient transfection conditions (Fig. 8A). Moreover, stable reconstitution of net expression in tumorigenic cells significantly reduced endogenous c-fos transcription (Fig. 8B), whereby c-Fos was diminished from the AP-1 transcription complex (Fig. 8C). However, the amount of ectopically expressed net was apparently not sufficient to completely suppress c-fos in CGL3 cells (Fig. 8B). Hence, we currently cannot answer the question, whether Net is acting as tumor suppressor, because its simple overexpression in malignant cells did not block cellular growth in nude mice (data not shown). However, transient delivery of siRNA against net resulted in increased c-fos transcription in asynchronized 444 hybrids, unequivocally demonstrating a causal relationship between Net and c-fos expression in non-tumorigenic cells (Fig. 8D). It will be worthwhile in future experiments whether a stably introduced Net siRNA transcription cassette leads to similar phenotypic changes (e.g. tumorigenicity) in 444 cells as previously reported after ectopical expression of c-fos (
      • Soto U.
      • Das B.C.
      • Lengert M.
      • zur Finzer P.
      • Hausen H.
      • Rösl F.
      ).
      In conclusion, our data show a direct functional link between the loss of Net, its absence at the SRE in a repressive chromatin constellation, and the constitutive c-fos transcription in tumorigenic HPV-positive cells. This has important implications for the role of Net function and regulation during HPV-induced carcinogenesis. Interestingly, loss of Net (Elk3) expression has recently been observed in malignant mesothelioma compared with normal mesothelial cells (
      • Mohr S.
      • Bottin M.C.
      • Lannes B.
      • Neuville A.
      • Bellocq J.P.
      • Keith G.
      • Henri Rihn B.
      ), suggesting that Net as a repressor may be important in other tumors.

      Acknowledgments

      We thank G. T. Bowden (Arizona Cancer Center, Tucson, AZ) for providing the c-fos luciferase construct, E. J. Stanbridge (University of California, Irvine, CA) for the in vitro cell model system, A. Bachmann (Deutsches Krebsforschungszentrum, Heidelberg) for helpful discussion, and E. Göckel-Krzikalla for technical support.

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