Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

  • Lara Termini1, 2Email author,

    Affiliated with

    • Enrique Boccardo1,

      Affiliated with

      • Gustavo H Esteves3,

        Affiliated with

        • Roberto HirataJr3,

          Affiliated with

          • Waleska K Martins1, 2,

            Affiliated with

            • Anna Estela L Colo1, 2,

              Affiliated with

              • E Jordão Neves3,

                Affiliated with

                • Lina Luisa Villa1 and

                  Affiliated with

                  • Luiz FL Reis1, 2

                    Affiliated with

                    BMC Medical Genomics20081:29

                    DOI: 10.1186/1755-8794-1-29

                    Received: 01 February 2008

                    Accepted: 27 June 2008

                    Published: 27 June 2008

                    Abstract

                    Background

                    Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours.

                    Methods

                    In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts.

                    Results

                    We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells.

                    Conclusion

                    This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

                    Background

                    Human papillomaviruses (HPVs) are double-stranded DNA tumor viruses that infect keratinocytes of the anogenital tract epithelium [1]. Persistent infection by high risk HPV types (e.g., HPV-16, -18, -31, and -45) is the main risk factor for the development of cervical intraepithelial neoplasia and cervical cancer [2, 3]. High-risk HPV DNA is detected in more than 90% of cervical carcinomas worldwide [4] and it has been shown that HPV types 16 and 18 can immortalize normal cells in culture, a function that is attributed to E6 and E7 oncogenes [5]. These are the only HPV genes consistently retained and expressed in cervical carcinomas. Besides, their continued expression is required to maintain the malignant phenotype [68]. The proteins encoded by these genes disturb cell proliferation and differentiation by physical and functional interaction with several cellular factors involved in cell cycle regulation [9]. E6 is best known for its ability to bind to p53 and induce its ubiquitin-dependent degradation [10, 11], whereas E7 was initially recognized by its ability to interact with members of the retinoblastoma protein family, namely pRb, p107 and p130 [12] and its capacity of enhancing their degradation [13].

                    Persistence of HPV infections and development of neoplasia is influenced by local cell-mediated immune response [14]. Tumor necrosis factor-alpha (TNF) is one of the main mediators of skin and mucosa inflammation and has a potent antiproliferative effect on normal primary human keratinocytes (PHKs). This cytokine is a key regulator of diverse inflammatory and immune processes in human epithelia and its expression by keratinocytes is enhanced in response to tissue injury, inflammation, viral infection, and UV radiation [1517]. Furthermore, TNF has been identified as a key mediator for the regression of HPV-induced lesions [1821]. Previous studies from our group had shown that TNF exerts a potent cytostatic effect on normal and HPV16 immortalized keratinocytes. On the other hand, keratinocytes immortalized by HPV18 or SV40, as well as HPV16 or HPV18-positive cervical tumor-derived cell lines continue to proliferate normally in the presence of this cytokine [22, 23]. In addition, it has been observed that continuous HPV18-gene expression in malignant HeLa-fibroblasts hybrids, as well as increased tumorigenicity of HPV16-transformed human keratinocytes is associated with TNF resistance [24, 25]. These observations underscore the importance of TNF-resistance acquisition in HPV mediated pathogenesis and suggest that this event could be an important factor in HPV-associated neoplasia outcome. However, the molecular basis of HPV-mediated TNF resistance has not been elucidated.

                    The aim of the present study was to characterize and compare the global transcription profile of normal and HPV-immortalized keratinocytes. Furthermore, we sought to analyze their response to TNF in order to identify differences that contribute to explain their divergent response to this cytokine. For this purpose, we used microarray analysis to determine transcriptional changes upon 3 and 60 hours after TNF exposure. The 3 hours treatment would favor the identification of immediate early TNF regulated genes. On the other hand, the 60 hours treatment was used because the cytostatic effect exerted by this cytokine on normal and HPV16-immortalized keratinocytes reaches its maximum at this time-point [22, 23]. Our experimental setting allowed us to: 1) identify genes that are differentially expressed between TNF-sensitive and TNF-resistant cells; 2) identify genes that are differentially modulated by TNF at two-time points (3 and 60 hours); 3) analyze the effect of HPV-induced immortalization on TNF-regulated genes and, 4) find genes that are differentially expressed between cells immortalized by two different high-risk HPV types. Using this approach, we identified differentially expressed genes that are involved in different cell processes such as immune and inflammatory responses, cell differentiation, cell death, proliferation, extracellular matrix remodeling and DNA repair. The implications of these results are discussed.

                    Methods

                    Cell Culture and TNF treatment

                    Cultures of primary human keratinocytes (PHK), recovered from newborn foreskin (Cambrex, Walkersville, MD, USA), were maintained in keratinocyte serum-free medium – KSFM (Life Technology, Inc., Gaithersburg, MD, USA) for 3 to seven passages [26]. HF698 and HF18Nco are cell lines obtained from human keratinocytes immortalized by HPV16 and HPV18 whole genome, respectively. These cell lines (from now on referred as HPV16 and HPV18, respectively) were kindly provided by R. Schlegel, Georgetown University Medical Center, Washington, DC [27], were grown in 3+1 medium, consisting of a mixture of 3 parts KSFM and 1 part DMEM, supplemented with 10% fetal calf serum. Cells were grown in 100-mm tissue culture dishes to 30% confluence and treated with 2 nM of human recombinant TNF (Boehringer Mannheim, Germany), for 3 or 60 hours. Cells were then trypsinized, washed 3 times with PBS and frozen until RNA extraction. For all time points, RNA was obtained from two independent experiments, including the control plates.

                    RNA extraction, amplification, labeling, and hybridizations

                    For each sample, total RNA was extracted using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY, USA) following the procedure recommended by the manufacturer. Three micrograms of target and reference (a pool of RNA from all control conditions) total RNA were linearly amplified using T7-based protocol, converted to cyanine-modified cDNA, and labeled as described previously [28].

                    Hybridizations were performed in duplicate, using dye-swap, on a cDNA platform of ORESTES representing 4,600 unique genes with known full-length sequence selected from the clone collection derived from the Human Cancer Genome Project [29]. cDNA amplification, purification, identity verification and printing were performed as previously described [28]. A detailed description of the cDNA microarray platform used and the raw data of this study are available at the GEO website under the accession numbers GPL1930 and GSE4524, respectively [30]. Slides were scanned on a confocal laser scanner (Arrayexpress; Packard Bioscience, USA) and, for each spot, signal and background intensities were measured using histogram method of Quantarray software (version 3.0, Packard BioScience, BioChip Technologies LLC, USA).

                    Statistical Analysis

                    Data analysis was performed with R project for statistical computing [31] and tools of the associated project, Bioconductor [32]. Prior to analysis, signal intensity was corrected by background subtraction, and data normalized by loess method, using span = 0.4 and degree = 2. For the identification of differentially expressed genes, we used ANOVA model when just one variable was considered. For the identification of differentially expressed genes in a pair-wise manner, we used t-test and determined the nominal p-value for each individual gene. Those nominal p-values can be conservatively adjusted for multiple testing with the Bonferroni correction by multiplying them by the number of genes in our chip. For clustering samples on the basis of their expression profile, we applied hierarchical clustering based on correlation distance and complete linkage.

                    Northern Blotting

                    For Northern blot analysis, 15 μg of total RNA was fractionated through a 1% denaturing agarose gel and transferred by capillarity onto Hybond N filters (GE Healthcare BioSciences, NJ, USA). Prehybridization, hybridization, and washes were performed as described by Church and Gilbert [33]. The KLK7 and SOD2 cDNA probes were the same used for immobilization in the array. The human GAPDH cDNA probe was used as control for ensuring equal RNA loading. Probes were labeled by random priming, using Ready-To-Go Labeling Beads (GE Healthcare Bio-Sciences, NJ, USA) and [α-32P]dCTP (3000 Ci/mmol). Nylon filters were exposed to Kodak Hyperfilm (GE Healthcare BioSciences, NJ, USA) with intensifying screen.

                    Electrophoretic mobility shift assay (EMSA)

                    Nuclear extracts were obtained from monolayer cultures of PHK, and from cell lines HPV16 and HPV18 treated with 2 nM of human recombinant TNF for 1 h. Briefly, cell plates were washed with ice-cold PBS and cells were scraped in 5 ml of PBS. Cells were transferred to a 15 ml Falcon tube and centrifuged at 3000 rpm for 3 min. Cell pellets were ressuspended in 4 ml of lysis buffer (10 mM HEPES pH 7,9, 10 mM KCl, 0,2 mM EDTA, 1 mM DTT), incubated on ice for 5 min, centrifuged and ressuspended in 4 ml of lysis buffer. Nuclei obtained were centrifuged at 2000 rpm for 2 min, ressuspended in 100 μl of extraction buffer (20 mM HEPES pH7,9, 0,42 M NaCl, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 2 μM pepstatin, 0,6 μM leupeptin, 25 mU/ml aprotinin) and incubated on ice for 30 min. Finally, the samples were centrifuged at 12000 rpm for 15 min at 4°C. The supernatants were stored at -80°C. The protein concentration was determined by the Bradford method (Bio-Rad, CA, USA).

                    For gel retardation the following double-stranded oligonucleotide, corresponding to the NF-κB binding sequence, was used: forward-5'-GCCTGGGAAAGTCCCCTCAACT-3' (Invitrogen, CA, USA) was used. The annealed oligonucleotide was labeled with [γ-32]ATP (Amersham, Buckinghamshire, UK; 3,000 Ci/mmol) using TK polynucleotide kinase according to the manufacturer instructions (Biolabs, MA, USA) and purified using Sephadex G50 columns followed by phenol:chloroform extraction and precipitation using 10 μg of salmon sperm DNA as a carrier (Invitrogen, CA, USA). DNA pellets were ressuspended in binding buffer (20 mM HEPES pH 7,9, 20% glycerol, 0,1 M KCl, 2 mM EDTA, 1 mM PMSF, 2 μM pepstatin, 0,6 μM leupeptin, 25 mU/ml aprotinin) to a final concentration of 2,5 fmol/μl. The incorporated radioactivity was quantitated using a LS6500 scintillation counter (Beckman Coulter, CA, USA).

                    The binding of NF-κB was performed in a reaction containing 5 μg of protein extract, 5 μg of BSA, 5 μg of salmon sperm DNA and binding buffer to a final volume of 32 μl on ice. After 10 min, 8 μl of the [γ-32]ATP 5'-end-labeled double-stranded oligonucleotide probe was added, and the incubation was continued for an additional 15 min at 30°C. The DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide gels (29:1 cross-linking ratio), dried, and exposed overnight to X-ray films (Amersham, Buckinghamshire, UK).

                    Results

                    Glass arrays containing 4.800 cDNA sequences were used in order to determine the effects of HPV infection in human keratinocytes as well as the impact of TNF treatment on global gene expression, in HPV negative or positive cells.

                    In order to identify differentially expressed genes as a function of a unique variable (cell type or TNF-treatment) our dataset was first analyzed by one way ANOVA. The comparisons performed allowed us to determine 1) genes that are differentially expressed between TNF-sensitive and TNF-resistant cells; 2) identify genes that are differentially modulated by TNF at two-time points (3 and 60 hours); 3) analyze the effect of HPV-induced immortalization on TNF-regulated genes and, 4) find genes that are differentially expressed between cells immortalized by two different high-risk HPV types (Figure 1).
                    http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-1-29/MediaObjects/12920_2008_Article_29_Fig1_HTML.jpg
                    Figure 1

                    Experimental setup for the analysis of HPV and TNF effects on keratinocytes gene expression. In order to characterize and compare the global transcription profile of normal and HPV-immortalized keratinocytes and to analyze their response to TNF we used an experimental setting that allowed us to: 1) identify differential expressed genes between normal PHK, HPV16 and HPV18-immortalized keratinocytes (comparisons represented by dashed arrows); 2) identify genes modulated by TNF upon treatment for three and sixty hours (comparisons represented by solid arrows) and; 3) compare the effect of TNF between normal PHK and cells immortalized by two different high-risk HPV types (comparisons represented by round dot arrows).

                    Differentially expressed genes as a function of cell type or TNF treatment

                    The identification of differentially expressed genes, with statistical significance, as a function of cell type was performed by ANOVA. Samples and differentially expressed genes (cutoff p-value <10-10) were grouped hierarchically, using correlation distance and complete linkage (Figure 2). As it can be observed, normal (PHK) and HPV16-immortalized keratinocytes (HPV16), which are sensitive to TNF cytostatic effect, grouped together while TNF-resistant HPV18-immortalized cell line (HPV18) formed an independent branch. This indicates that TNF-sensitive cell lines share a group of genes which are regulated in a way that clearly differentiate them from the TNF-resistant one. Samples were further clusterized by the time in culture after the last medium change (3 or 60 hs) and finally separated as a function of TNF treatment. This clusterization pattern may reflect differences in cell density and other cultures variables such as nutrients availability or medium conditioning. Initially, all treatments were performed using 30% cell density cultures. As expected, due to TNF cytostatic effect on normal and HPV16-immortalized keratinocytes, cell density at the end of the 60 hours period was different between treated (40–50%) and control cells (70–80%) for these cell lines. On the other hand, both cytokine treated and control HPV18-immortalized cells reached 80–90% cell density by the end of the 60 hours period. Flow-cytometry analysis revealed that the TNF effect on sensitive cells was characterized by the accumulation of cells in the G1-phase of the cell cycle. Conversely, TNF-induced G1-arrest was not observed in HPV18-immortalized keratinocytes [[22, 23] and data not shown]. Finally, no differences in cell density were observed for cultures corresponding to 3 hours-treatment group.
                    http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-1-29/MediaObjects/12920_2008_Article_29_Fig2_HTML.jpg
                    Figure 2

                    Hierarchical grouping based on differentially expressed genes as a function of cell type. These genes where identified by the ANOVA method and the samples where grouped considering the correlation distance and complete linkage. After sample grouping the genes (p values <10-10) were hierarchically grouped by their correlation distances. High gene expression is shown in red, low gene expression is shown in green and black indicates non-differential gene expression. Samples: Primary human keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively (PHK_3H, PHK_60H, PHK_3H.TNF, PHK_60H.TNF); HPV16-immortalized keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively (HPV16_3H, HPV16_60H, HPV16_3H.TNF, HPV16_60H.TNF); HPV18-immortalized keratinocytes: controls and treated with 3 or 60 hours for TNF, respectively (HPV18_3H, HPV18_60H, HPV18_3H.TNF, HPV18_60H.TNF).

                    Among the differentially regulated genes we found some related with inflammatory response (SOD2, TGFB1, CD44, INHBA, OAS1, SIMP), epidermal development, differentiation and proliferation (ADAMST1, RARRES1, CREG, HBP17, MCM2, PRSS1, S100P, CREG1), proteolysis regulation (KLK7, PI3, LXN), and cell adhesion (CD44, PARVA, PROS1). The name and function of the genes described are listed in Table 1. We next determined the global changes in gene expression as a function of TNF treatment. The name and annotated function of the identified genes that best distinguish samples based on TNF treatment (cutoff p-value <10-2,9) are listed in Table 2. As expected, many of these genes are involved in the inflammatory response and/or are direct targets of TNF e.g. CCL20, CD44, HLA-F, IL1F9, NFKBIA, INHBA, SOD2, MARCKS, RFX5. Samples and genes were hierarchically clusterized on the basis of their correlation distance using complete linkage (Figure 3). Samples from TNF-treated normal keratinocytes (PHK) grouped together and apart from the others. HPV-positive samples exhibited a complex clusterization pattern suggesting that the presence of either HPV16 or 18 has an impact on TNF-regulated gene expression. Furthermore, the grouping of treated PHKs apart from the other samples could reflect the fact that PHKs are the only normal cells used in this study and, as such, the only cell type expected to have an unaltered TNF-signaling network. This could contribute to explain the differences in gene expression upon TNF treatment observed between normal and HPV-immortalized keratinocytes.
                    Table 1

                    Name and function of the differentially expressed genes that best distinguish samples by cell type variable

                    GENE

                    UniGene ID

                    GENE NAME

                    FUNCTION

                    ADAMTS1

                    Hs.534115

                    disintegrin-like and metalloprotease (reprolysin type)

                    negative regulation of cell proliferation

                    ARHGEF10

                    Hs.443460

                    Rho guanine nucleotide exchange factor (GEF) 10

                    GTPase activator activity

                    CD44

                    Hs.502328

                    CD44 antigen

                    cell adhesion

                    CITED1

                    Hs.40403

                    Cbp/p300-interacting transactivator

                    transcription regulator activity

                    CREG

                    Hs.5710

                    cellular repressor of E1A-stimulated genes 1

                    cell proliferation

                    CSTA

                    Hs.518198

                    cystatin A (stefin A)

                    cysteine protease inhibitor activity

                    D4S234E

                    Hs.518595

                    DNA segment on chromosome 4 (unique) 234 expressed sequence

                    dopamine receptor signaling pathway

                    DHRS3

                    Hs.289347

                    dehydrogenase/reductase (SDR family) member 3

                    fatty acid metabolism

                    EPB41L1

                    Hs.437422

                    erythrocyte membrane protein band 4.1-like 1

                    structural molecule activity

                    FLJ20105

                    Hs.47558

                    FLJ20105

                    regulation of transcription

                    FLJ21511

                    Hs.479703

                    FLJ21511

                    unknown function

                    FLJ21616

                    Hs.591836

                    FLJ21616

                    regulation of transcription

                    FLJ30525

                    Hs.7962

                    FLJ30525

                    unknown function

                    GALNT11

                    Hs.647109

                    UDP-N-acetyl-alpha-D-galactosamine

                    transferase activity, transferring glycosyl groups

                    GC20

                    Hs.315230

                    translation factor sui1 homolog

                    regulation of translational initiation

                    GLDC

                    Hs.584238

                    glycine dehydrogenase

                    glycine metabolism

                    GSR

                    Hs.271510

                    glutathione reductase

                    glutathion metabolism

                    HBP17

                    Hs.1690

                    fibroblast growth factor binding protein 1

                    regulation of cell proliferation

                    INHBA

                    Hs.28792

                    inhibin, beta A

                    cell cycle arrest, negative regulation of immune cell differentiation

                    JPH3

                    Hs.592068

                    junctophilin 3

                    unknown function

                    KIAA0368

                    Hs.368255

                    KIAA0368

                    ER-associated protein catabolism

                    KLK7

                    Hs.151254

                    kallikrein 7 (chymotryptic, stratum corneum)

                    epidermis development, proteolysis and peptidolysis, chymotrypsin activity

                    LCN2

                    Hs.204238

                    lipocalin 2 (oncogene 24p3)

                    transporter activity

                    LXN

                    Hs.478067

                    latexin

                    enzyme inhibitor activity

                    MAL2

                    Hs.201083

                    T-cell differentiation protein 2

                    Unknown function

                    MCM2

                    Hs.477481

                    minichromosome maintenance deficient 2

                    cell cycle

                    MGC45400

                    Hs.389734

                    transcription elongation factor A (SII)-like 8

                    translation elongation factor activity

                    MGEA5

                    Hs.500842

                    meningioma expressed antigen 5 (hyaluronidase)

                    glycoprotein catabolism

                    MYO5B

                    Hs.200136

                    acetyl-Coenzyme A acyltransferase 2

                    fatty acid metabolism

                    NMES1

                    Hs.112242

                    normal mucosa of esophagus specific 1

                    unknown function

                    NMU

                    Hs.418367

                    neuromedin U

                    neuropeptide signaling pathway, digestion

                    NT5E

                    Hs.153952

                    5'-nucleotidase, ecto (CD73)

                    DNA metabolism

                    OAS1

                    Hs.524760

                    2',5'-oligoadenylate synthetase 1

                    immune response to viral infections

                    ODC1

                    Hs.467701

                    ornithine decarboxylase 1

                    polyamine biosynthesis

                    PARVA

                    Hs.607144

                    parvin, alpha

                    cell adhesion, actin binding

                    PEX3

                    Hs.7277

                    peroxisomal biogenesis factor 3

                    peroxisome organization

                    PI3

                    Hs.112341

                    protease inhibitor 3, skin-derived (SKALP)

                    elastase-specific inhibitor

                    PLAU

                    Hs.77274

                    plasminogen activator

                    chemotaxis

                    PPGB

                    Hs.517076

                    protective protein for beta-galactosidase

                    intracellular protein transport

                    PROS1

                    Hs.64016

                    protein S (alpha)

                    cell adhesion, endopeptidase inhibitor activity

                    PRSS11

                    Hs.501280

                    protease, serine, 11 (IGF binding)

                    insulin-like growth facto binding, regulation of cell growth

                    RARRES1

                    Hs.131269

                    retinoic acid receptor responder

                    negative regulation of cell proliferation

                    RDH-E2

                    Hs.170673

                    epidermal retinal dehydrogenase 2

                    oxidoreductase activity

                    RPL15

                    Hs.381219

                    ribosomal protein L15

                    protein biosynthesis

                    RUTBC3

                    Hs.474914

                    RUN and TBC1 domain containing 3

                    unknown function

                    S100P

                    Hs.440880

                    S100 calcium binding protein P

                    cell cycle progression and differentiation

                    SEPT10

                    Hs.469615

                    septin 10

                    cell cycle

                    SF3B4

                    Hs.516160

                    myotubularin related protein 11

                    inositol or phosphatidylinositol phosphatase activity

                    SIMP

                    Hs.475812

                    immunodominant MHC-associated peptides

                    protein amino acid glycosylation

                    SMOC2

                    Hs.487200

                    SPARC related modular calcium binding

                    calcium ion binding

                    SOD2

                    Hs.487046

                    superoxide dismutase 2

                    age-dependent response to reactive oxygen species, cellular defense response

                    STAF65 (gamma)

                    Hs.6232

                    SPTF-associated factor 65 gamma

                    regulation of transcription, DNA- dependent

                    SYTL3

                    Hs.436977

                    synaptotagmin-like 3

                    intracellular protein transport

                    TFRC

                    Hs.529618

                    transferrin receptor (p90, CD71)

                    endocytosis

                    TGFB1

                    Hs.645227

                    transforming growth factor, beta 1

                    cell proloferation

                    TPD52

                    Hs.368433

                    tumor protein D52

                    morphogenesis

                    TPX2

                    Hs.244580

                    microtubule-associated protein homolog

                    cell proliferation

                    TRIM31

                    Hs.493275

                    tripartite motif-containing 31

                    protein ubiquitination, ubiquitin ligase activity

                    YME1L1

                    Hs.499145

                    YME1-like 1 (S. cerevisiae)

                    protein catabolism

                    ZNF198

                    Hs.644041

                    zinc finger protein 198

                    regulation of transcription, DNA- dependent

                    *Genes are listed in alphabetical order. The cutoff p-value was set as <10-10.

                    Table 2

                    Name and function of the differentially expressed genes that best distinguish samples by TNF treatment variable

                    GENE

                    UniGene ID

                    GENE NAME

                    FUNCTION

                    ADORA2b

                    Hs.167046

                    adenosine A2b receptor

                    activation of MAPK

                    AKAP1

                    Hs.463506

                    A kinase (PRKA) anchor protein 1

                    RNA binding

                    BTG2

                    Hs.519162

                    BTG family, member 2

                    negative regulation of cell proliferation

                    C3

                    Hs.529053

                    complement component 3

                    inflammatory response

                    CCL20

                    Hs.75498

                    chemokine (C-C motif) ligand 20

                    inflammatory response

                    CD44

                    Hs.502328

                    CD44 antigen

                    cell adhesion

                    cig5

                    Hs.17518

                    radical S-adenosyl methionine domain containing 2

                    Catalytic activity

                    CLCA4

                    Hs.546343

                    chloride channel, calcium activated, family member 4

                    chloride transport

                    DC-UbP

                    Hs.179852

                    dendritic cell-derived ubiquitin-like protein

                    Protein modification

                    FAD104

                    Hs.159430

                    fibronectin type III domain containing 3B

                    cell differentiation

                    FLJ21511

                    Hs.479703

                    FLJ21511

                    Unknown function

                    FMNL3

                    Hs.179838

                    formin-like 3

                    cell organization and biogenesis

                    GFPT2

                    Hs.30332

                    glutamine-fructose-6-phosphate transaminase 2

                    carbohydrate biosynthesis

                    HLA-F

                    Hs.519972

                    major histocompatibility complex, class I, F

                    antigen presentation, endogenous antigen

                    IL1F9

                    Hs.211238

                    interleukin 1 family, member 9

                    inflammatory response

                    INHBA

                    Hs.28792

                    inhibin, beta A

                    cell cycle arrest, negative regulation of immune cell differentiation

                    KIAA0303

                    Hs.133539

                    microtubule associated serine/threonine kinase family member 4

                    protein kinase activity

                    KIAA1279

                    Hs.279580

                    KIAA1279

                    Unknown function

                    LAP3

                    Hs.479264

                    leucine aminopeptidase 3

                    Protein metabolism

                    MARCKS

                    Hs.75061

                    MARCKS-like 1

                    calmodulin binding, macrophage activation

                    MGAT4B

                    Hs.437277

                    mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isoenzyme B

                    cytokine activity

                    MGC45400

                    Hs.389734

                    transcription elongation factor A (SII)-like 8

                    translation elongation factor activity

                    MMP9

                    Hs.297413

                    matrix metalloproteinase 9

                    proteolysis and peptidolysis

                    NFKBIA

                    Hs.81328

                    nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

                    cytoplasmic sequestering of NF-kappaB

                    NMES1

                    Hs.112242

                    normal mucosa of esophagus specific 1

                    Unknown function

                    OAS1

                    Hs.524760

                    2',5'-oligoadenylate synthetase 1

                    immune response to viral infections

                    PLAU

                    Hs.77274

                    plasminogen activator

                    chemotaxis

                    RDH-E2

                    Hs.170673

                    epidermal retinal dehydrogenase 2

                    oxidoreductase activity

                    RFX5

                    Hs.166891

                    regulatory factor X, 5

                    inflammatory response, HLA class II expression

                    RIG-1

                    Hs.17466

                    retinoic acid receptor responder (tazarotene induced) 3

                    negative regulation of cell proliferation

                    RIPK2

                    Hs.103755

                    receptor-interacting serine-threonine kinase 2

                    inflammatory response

                    SASH1

                    Hs.193133

                    SAM and SH3 domain containing 1

                    Negative regulation of cell cycle

                    SDCBP

                    Hs.200804

                    syndecan binding protein (syntenin)

                    intracellular signaling cascade, interleukin-5 receptor binding

                    SEC24A

                    Hs.211612

                    SEC24 related gene family, member A

                    intracellular protein transport

                    SERPINB2

                    Hs.514913

                    encoding serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2

                    anti-apoptosis

                    SF3B4

                    Hs.412818

                    myotubularin related protein 11

                    RNA splicing

                    SOD2

                    Hs.487046

                    superoxide dismutase 2

                    age-dependent response to reactive oxygen species, cellular defense response

                    TMSB4

                    Hs.522584

                    thymosin, beta 4, X-linked

                    cytoskeleton organization and biogenesis

                    VMP1

                    Hs.444569

                    transmembrane protein 49

                    Unknown function

                    *Genes are listed in alphabetical order. The cutoff p-value was set as <10-2,9.

                    http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-1-29/MediaObjects/12920_2008_Article_29_Fig3_HTML.jpg
                    Figure 3

                    Hierarchical grouping based on differentially expressed genes as a function of TNF treatment. These genes where identified by the ANOVA method and the samples where grouped considering the correlation distance and complete linkage. After sample grouping the genes (p values <10-2,9) were hierarchically grouped by their correlation distances. High gene expression is shown in red, low gene expression is shown in green and black indicates non-differential gene expression. Samples: Primary human keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively (PHK_3H, PHK_60H, PHK_3H.TNF, PHK_60H.TNF); HPV16-immortalized keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively (HPV16_3H, HPV16_60H, HPV16_3H.TNF, HPV16_60H.TNF); HPV18-immortalized keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively (HPV18_3H, HPV18_60H, HPV18_3H.TNF, HPV18_60H.TNF).

                    Since our experimental setting included the comparative analysis of global gene expression at two time points (3 or 60 hours), we searched for genes that best differentiate our samples as a function of time. We found 48 genes that clearly differentiate samples from the analyzed time points. The name and annotated function of the identified genes (cutoff p-value <10-9) are listed in additional file 1. Hierarchical clusterization divided samples in two main branches (additional file 2). Each branch was exclusively composed of samples from the same time point, namely, 3 or 60 hours. Samples from the 3 hours-time point formed a secondary branch that divided normal from HPV-immortalized keratinocytes. On the other hand, samples from the 60 hours-time point formed a secondary branch that divided normal and HPV16-immortalized keratinocytes (TNF-sensitive samples) from HPV18-immortalized keratinocytes (TNF-resistant samples).

                    Differentially expressed genes between TNF-sensitive and TNF-resistant cells

                    In order to identify differentially expressed genes between specific samples we performed a series of pair-wise comparisons. For each pair-wise comparison, we generated a list of differentially expressed genes with p-value lower than 0,01. The complete list of all pair-wise comparisons performed is presented in additional file 3. We next aimed to characterize genes that were differentially expressed between TNF-sensitive (PHK and HPV16) and TNF-resistant cells (HPV18). To achieve this goal we selected the thirty genes with the lowest p-values that best distinguish both PHK and HPV16 from HPV18 and the thirty genes with lowest p-values that best distinguish both PHK+TNF and HPV16+TNF from HPV18+TNF (considering treatment with TNF for 3 h). Twelve genes were common to both lists giving a total of 48 different genes identified (Table 3). Using the expression profile of these 48 genes, samples were grouped hierarchically, based on their correlation distance and complete linkage (Figure 4).
                    Table 3

                    List of differentially expressed genes that best distinguish TNF-resistant cells (HPV18) from TNF-sensitive cells (PHK and HPV16), in normal culture conditions or upon treatment with TNF for 3 hours

                      

                    (PHK and HPV16) vs HPV18

                    (PHK_TNF and HPV16_TNF) vs HPV18_TNF

                    GENE

                    GENE NAME

                    FOLD

                    p VALUE

                    FOLD

                    p VALUE

                    ABCE1

                    ATP-binding cassette, sub-family E (OABP), member 1

                    0.592

                    0.001384

                    ----

                    ----

                    ACBD5

                    acyl-Coenzyme A binding domain containing 5

                    ----

                    ----

                    0.378

                    0.00086

                    ALDH3A2

                    encoding aldehyde dehydrogenase 3 family, member A2

                    1.623

                    0.00122

                    ----

                    ----

                    APG12L

                    APG12 autophagy 12-like (S. cerevisiae)

                    1.739

                    0.000924

                    ----

                    ----

                    APPBP1

                    amyloid beta precursor protein binding protein 1

                    0.540

                    0.000283

                    ----

                    ----

                    ARF4L

                    ADP-ribosylation factor 4-like

                    ----

                    ----

                    0.578

                    5.70E-05

                    BCLAF1

                    BCL2-associated transcription factor 1

                    0.611

                    0.000876

                    ----

                    ----

                    BOC

                    brother of CDO

                    ----

                    ----

                    2.337

                    6.00E-06

                    CCNA2

                    cyclin A2

                    0.577

                    0.000604

                    ----

                    ----

                    CDCA2

                    cell division cycle associated 2

                    ----

                    ----

                    0.539

                    7.20E-05

                    CDK2AP1

                    CDK2-associated protein 1

                    ----

                    ----

                    1.523

                    0.000148

                    CPSF3

                    cleavage and polyadenylation specific factor 3

                    0.586

                    0.000466

                    ----

                    ----

                    CYP1B1

                    cytochrome P450, family 1, subfamily B, polypeptide 1

                    0.499

                    0.001386

                    ----

                    ----

                    DEK

                    DEK oncogene

                    0.480

                    0.000278

                    ----

                    ----

                    FAM31C

                    family with sequence similarity 31, member C

                    ----

                    ----

                    2.548

                    0.000663

                    FLJ20105

                    hypothetical protein LOC54821

                    0.026

                    5.00E-06

                    0.029

                    2.00E-06

                    GALNAC4S-6ST

                    B cell RAG associated protein

                    1.827

                    0.000145

                    1.673

                    0.000215

                    H105E3

                    encoding NAD(P) dependent steroid dehydrogenase-like

                    ----

                    ----

                    0.569

                    6.80E-05

                    HLCS

                    holocarboxylase synthetase

                    ----

                    ----

                    1.551

                    0.00011

                    JPH3

                    junctophilin 3

                    0.154

                    0.000192

                    ----

                    ----

                    KIAA0795

                    kelch-like 18 (Drosophila)

                    0.857

                    0.001325

                    ----

                    ----

                    KIAA1023

                    IQ motif containing E

                    1.575

                    0.000723

                    ----

                    ----

                    KIF1B

                    kinesin family member 1B

                    ----

                    ----

                    0.570

                    0.000632

                    KLK7

                    encoding kallikrein 7 (chymotryptic, stratum corneum)

                    0.421

                    0.000416

                    0.374

                    2.30E-05

                    LCN2

                    lipocalin 2 (oncogene 24p3)

                    ----

                    ----

                    0.216

                    0.000686

                    LOC151242

                    protein phosphatase 1, regulatory (inhibitor)

                    1.928

                    0.000268

                    ----

                    ----

                    Lrp2bp

                    low density lipoprotein receptor-related protein binding protein

                    0.629

                    0.000574

                    ----

                    ----

                    MAPRE1

                    encoding microtubule-associated protein, RP/EB family, member 1

                    0.410

                    0.001068

                    0.503

                    4.20E-05

                    MBD2

                    methyl-CpG binding domain protein 2

                    0.680

                    0.001247

                    ----

                    ----

                    MGC35048

                    hypothetical protein MGC35048

                    ----

                    ----

                    0.499

                    0.000211

                    MRPS6

                    mitochondrial ribosomal protein S6

                    ----

                    ----

                    1.460

                    0.000161

                    MYO5B

                    acetyl-Coenzyme A acyltransferase 2

                    0.353

                    0.000104

                    0.292

                    4.10E-05

                    NMES1

                    normal mucosa of esophagus specific 1

                    0.324

                    0.000294

                    0.244

                    7.00E-06

                    NPR2

                    encoding natriuretic peptide receptor B/guanylate cyclase B

                    ----

                    ----

                    0.435

                    0.000234

                    ODC1

                    ornithine decarboxylase 1

                    1.660

                    0.000886

                    ----

                    ----

                    PI3

                    protease inhibitor 3, skin-derived (SKALP)

                    0.213

                    0.000274

                    0.207

                    1.80E-05

                    PROS1

                    protein S (alpha)

                    1.807

                    0.000633

                    ----

                    ----

                    PTP4A1

                    protein tyrosine phosphatase type IVA, member 1

                    0.478

                    0.000385

                    0.497

                    1.80E-05

                    RRAGA

                    Ras-related GTP binding A

                    ----

                    ----

                    1.510

                    0.000301

                    RUTBC3

                    RUN and TBC1 domain 3

                    0.518

                    0.000704

                    0.450

                    0.000131

                    S100P

                    S100 calcium binding protein P

                    0.101

                    0

                    0.102

                    0

                    SDCBP

                    syndecan binding protein (syntenin)

                    0.456

                    0.000395

                    ----

                    ----

                    SFRP1

                    secreted frizzled-related protein 1

                    ----

                    ----

                    0.502

                    5.10E-05

                    SLC35B3

                    solute carrier family 35, member B3

                    ----

                    ----

                    1.990

                    0.000304

                    STAF65 (gamma)

                    SPTF-associated factor 65 gamma

                    0.021

                    1.00E-06

                    0.028

                    0

                    THBS1

                    thrombospondin 1

                    ----

                    ----

                    2.712

                    0.000225

                    VMP1

                    likely ortholog of rat vacuole membrane protein 1

                    ----

                    ----

                    1.547

                    0.000899

                    YME1L1

                    YME1-like 1 (S. cerevisiae)

                    0.373

                    2.60E-05

                    0.333

                    5.00E-06

                    *Genes are listed in alphabetical order. Underlined genes were identified as differentially expressed between TNF sensitive and TNF resistant cells in both culture conditions.

                    http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-1-29/MediaObjects/12920_2008_Article_29_Fig4_HTML.jpg
                    Figure 4

                    Supervised hierarchical grouping based on differentially expressed genes between normal/HPV16-immortalized keratinocytes and HPV 18-immortalized ones after treatment with TNF for 3 hours. High gene expression is shown in red, low gene expression is shown in green and black indicates non-differential gene expression. Samples: Primary human keratinocytes: controls and treated for 3 hours with TNF, respectively (PHK_3H, PHK_3H.TNF); HPV16-immortalized keratinocytes: controls and treated for 3 hours with TNF, respectively (HPV16_3H, HPV16_3H.TNF); HPV18-immortalized keratinocytes: controls and treated for 3 hours with TNF, respectively (HPV18_3H, HPV18_3H.TNF).

                    Using this approach we observed that genes involved with cell cycle control (CCNA2, CDCA2, CDK2AP1), epidermis development, differentiation and proliferation (KLK7, ALDH3A2, PI3, APG12L, BCLAF1, DEK, MAPRE1, S100P, RRAGA, SFRP1), protein ubiquitination (APPBP1) and cell adhesion (BOC, PROS1, SDCBP, THBS1, JPH3), among others, were differentially expressed between TNF-sensitive and TNF-resistant cells (Table 3). These analyses were also performed considering TNF treatment for 60h (available as additional files 4 and 5).

                    Validation on KLK7 and SOD2 as differentially expressed genes

                    We identified a group of genes whose differential expression could be associated with the differential response to TNF of the cell lines studied, namely: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), S100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). Based on the reported function as well as the expression profile observed, KLK7 and SOD2 genes were selected for further analysis. The expression pattern of these genes observed by microarray was confirmed by Northern Blot in control and TNF-treated (60 hours) samples from all cell lines used (Figures 5A and 5B). As it can be observed, KLK7 is equally expressed in TNF-treated or untreated HPV18-immortalized cells but is not detected in PHK or HPV16-immortalized cells, even after cytokine treatment. On the other hand, we observed that SOD2 expression is up-regulated by TNF in both PHK and HPV16-immortalized cells but not in HPV18-immortalized cells, confirming the data obtained by microarray (Figures 5A and 5B).
                    http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-1-29/MediaObjects/12920_2008_Article_29_Fig5_HTML.jpg
                    Figure 5

                    Differential expression of KLK7 and SOD2 transcripts. A. Detail of the supervised hierarchical grouping based on differentially expressed genes between normal/HPV16-immortalized keratinocytes and HPV 18-immortalized ones, after treatment with TNF for 60 hours. B. Northern blot analysis of KLK7 and SOD2 transcription levels. Arrows indicate the two alternative splicing products of KLK7 in HPV18-immortalized keratinocytes (GenBank # NM_005046); the SOD2 transcript is induced by TNF in both PHK and HPV16-immortalized cells but not in HPV18-immortalized cells (GenBank # NM_00636). A probe against GAPDH was used to monitor comparable loading between samples.

                    NF-κB is differentially activated in HPV-16- and HPV-18-infected cells

                    It has been reported that NF-κB activation plays an important role in SOD2 induction by TNF. So we hypothesized that the differential expression of SOD2 could be due to the presence of different levels of activated NF-κB after TNF treatment between TNF-sensitive and TNF resistant cells. In order to address this hypothesis NF-κB activation was determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Interestingly, we observed that normal as well as HPV16-immortalized keratinocytes exhibited a clear activation of NF-κB as shown by the increase of this factor levels in nuclear protein extracts after TNF treatment (Figure 6). On the other hand, NF-κB activation in TNF-resistant HPV18-immortalized cells was below the level of detection (Figure 6, lanes 9 and 10). This prompted us to analyze if NF-κB activation was also altered in other HPV-positive cell lines previously reported to be resistant to TNF cytostatic effect [[22, 23], and data not shown]. To address this issue we performed EMSA using nuclear protein extracts obtained from HPV16-positive (SiHa) or HPV18-positive (HeLa and SW756) cervical cancer derived cell lines cultures. We observed that TNF-resistant cells exhibited reduced NF-κB activation when compared to normal PHK (additional file 6). Altogether, these observations suggest that alteration of TNF-signaling pathway leading to NF-κB activation is a common event in HPV-positive cell lines resistant to this cytokine.
                    http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-1-29/MediaObjects/12920_2008_Article_29_Fig6_HTML.jpg
                    Figure 6

                    Analysis of TNF-induced NF-κB activation in normal and HPV16 or 18-immortalized keratinocytes. Subconfluent cultures of normal and HPV16 or 18-immortalized keratinocytes treated with 2 nM of TNF for 1 h were used to obtain nuclear protein extracts. For each EMSA reaction, 5 μg of nuclear protein were incubated with 50 fmol of [γ-32P]ATP-labeled double-stranded oligonucleotide and a 50X excess of unlabeled oligonucleotide (lanes 3 and 7). Specificity of binding was further demonstrated by incubation of 1 μg of nuclear protein with the described amount of labeled consensus oligonucleotide and a 50X excess of a labeled oligonucleotide carrying a single-base mutation at the NF-κB binding site (lane 4), and incubation of nuclear extract in the absence of any labeled probe (lane 8). NF-κB DNA binding reactions were carried out as described under "Material and Methods". DNA binding complexes are indicated.

                    Discussion

                    Production and secretion of inflammatory cytokines are among the main events that take place upon viral infection. These molecules coordinate host cell-mediated immune response by recruiting cellular elements from the immune system and by regulating gene expression on target cells [34, 35]. The pleiotropic cytokine TNF is a key regulator of inflammation of the epithelia with a well-documented capacity to induce growth arrest in normal or HPV16-immortalized keratinocytes, mainly in the G0/G1 phase of the cell cycle [36]. Conversely, we have previously reported that HPV18-immortalized and both HPV16 or HPV18-transformed cell lines are resistant to TNF-induced growth arrest [22, 23].

                    In order to address the yet unknown molecular bases of this difference we applied cDNA microarray technology to compare the global gene expression profiles of TNF-sensitive normal and HPV16-immortalized keratinocytes with that of TNF-resistant HPV18-immortalized ones. Some limitations of this study are the use of a reduced number of samples and the existence of differences in cell culture conditions which are inherent to our experimental setting, i.e. the differences in cell density at the different time-points described above. However, using this approach we identified a group of genes that clearly distinguish both cells groups (Figure 2 and Table 1). This indicates that TNF-sensitive cell lines share a group of genes which are regulated in a way that clearly differentiate them from the TNF-resistant one.

                    On the other hand, when we analyzed changes in global gene expression as a function of TNF treatment we observed that HPV16 and HPV18 samples could not be distinguished from each other while normal keratinocytes could be readily discriminated (Figure 3). This observation suggests that the presence of either HPV16 or 18 has an impact on TNF-regulated gene expression. In line with these observations, several studies have shown that HPV positive cells exhibit impaired TNF pathways [37, 38]. Moreover, it has been reported that the effects of TNF on HPV-harboring cells depends on variables as cell type studied, the virus type present and culture conditions (i.e., growth factors availability). This cytokine is capable of inducing the proliferation of HPV16-immortalized human cervical epithelial cells cultures in the absence of growth factors through an autocrine, EGF receptor-dependent, pathway [39]. Besides, TNF can upregulate E6/E7 RNA expression and cyclin-dependent kinase activity in these cells [40]. Conversely, it has been reported that TNF exerts a potent cytostatic effect on HPV16-immortalized keratinocytes while HPV18-immortalized as well as cervical carcinoma-derived HPV-positive cell lines remain unaffected [22, 23]. Furthermore, it has been observed that increased tumorigenicity of human keratinocytes transformed by HPV16 is associated with resistance to TNF cytostatic effect [24]. Finally, it was demonstrated that TNF downregulates HPV18 transcription in non-malignant HeLa-fibroblasts hybrids, while viral expression in tumorigenic hybrids segregants as well as in parental HeLa cells remained undisturbed [25]. On the other hand, it has been consistently observed that TNF negatively regulates normal keratinocytes proliferation in monolayer [22, 23, 36] as well as in organotypic cell cultures [41, 42]. Altogether, these data support the notion that acquisition of resistance to TNF by HPV-infected cells may represent an important step towards malignancy.

                    Despite the existence of similarities between the two high-risk HPV types used to generate the cell lines studied, the fact that HPV16 and HPV18 are different viruses that exhibit clear differences in their biological activities must be highlighted. For instance, epidemiological studies have shown that HPV18 is more associated to cervical adenocarcinomas while HPV16 is more prevalent in squamous cell carcinomas [4345]. Furthermore, compared to other HPV types HPV18 has been associated with increased transforming potential in cell culture systems and with poorer cancer prognosis at the clinical level [26, 27, 46, 47]. On the other hand, HPV16 exhibits a greater potential to establish persistent infections that can progress to high-grade lesions [48, 49]. Although we cannot explain the molecular bases of the differences in gene expression between these cell lines, we believe that this may reflect the divergences that exist between these HPV types.

                    We next searched for genes that best distinguish between TNF-sensitive and TNF-resistant cells by pair-wise comparison both before and after cytokine treatment for 3 or 60 hours. By this means we identified 48 and 52 different genes, respectively, that set apart TNF-sensitive from TNF resistant cells (Figure 4, Table 3, additional files 4 and 5). The functional characterization of these genes shows that they are involved in critical cellular processes such as regulation of proliferation, differentiation and cell adhesion. Altogether, the differential expression of these genes may contribute to the differential response to the cytostatic effect of TNF observed in these cells.

                    Two genes, namely KLK7 and SOD2, were selected for further analysis based on their reported function and expression profile (Figure 5A). KLK7 expression pattern was validated by Northern blot and showed that it is equally expressed in TNF-treated or untreated HPV18-immortalized cells but is not detected in PHK or HPV16-immortalized cells (Figure 5B). Kallikreins are a sub-group of serine proteases with different physiological functions. In humans, kallikreins are encoded by 15 structurally similar, steroid hormone-regulated genes that co-localize to chromosome 19q13.4, representing the largest cluster of contiguous protease genes in the entire genome [5052]. These proteins mediate the proteolytic degradation of cohesive intracellular structures associated to epithelial differentiation. Recent data also suggest that kallikreins may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion, and, thus, may represent attractive drug targets to consider for therapeutic intervention [50]. Consistent with our findings, it has been observed that KLK7 expression is up-regulated in cervical tumors as well as in cells lines derived from them. On the other hand, normal keratinocytes express low levels of this protein [53, 54]. Furthermore, KLK7 expression has been found up-regulated in breast [55] and ovary tumors [56] and is being considered a new tumor progression marker.

                    The superoxide dismutase 2 (SOD2) expression pattern was also validated by Northern blot (Figure 5B). This gene is up-regulated in TNF-sensitive but not in TNF-resistant cells. The superoxide dismutase 2 (SOD2) belongs to a family of enzymes involved in the conversion of superoxide radicals in molecular oxygen. Reactive oxygen metabolites have multifactorial effects on the regulation of cell growth and malignant invasion. Furthermore, numerous in vivo studies have shown that the superoxide dismutases can be highly expressed in aggressive human solid tumors [5759].

                    Previous reports have shown that activation of the transcription factor NF-κB is essential for the induction of SOD2 by TNF and IL-1β [60, 61]. Here we show that TNF-sensitive cells exhibit higher levels of activated NF-κB than TNF-resistant ones after cytokine treatment (Figure 6 and additional file 6). Several studies have shown that NF-κB is a negative regulator of keratinocytes proliferation in the epidermis, and that it plays an important role in cell differentiation and tissue homeostasis [6264]. In stratified epithelia NF-κB is found in the cytoplasm of proliferating cells from the basal layer while it is detected in the nuclei of non-proliferating cells from the upper layers. Furthermore, it has been observed that NF-κB superexpression is associated with epidermal hypoplasia while its down-regulation promotes hyperplasia [62]. Overall, these data suggest that alterations in TNF-mediated NF-κB activation pathways can play a role in the development and progression of HPV-associated epithelial and mucosal lesions.

                    Conclusion

                    Progression of HPV-associated lesions depends on the many alterations caused by this virus in the infected cells. We have identified multiple genes differentially regulated by TNF in HPV16 and HPV18 immortalized keratinocytes. Among them we found KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), S100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was further confirmed at the RNA level. Moreover, we present evidence that differential SOD2 expression correlates with the levels of NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells.

                    This is the first time that the effect of TNF on global gene expression of normal and HPV-immortalized keratinocytes is addressed at two time points. The thorough analysis of the expression pattern of the identified genes may contribute to the understanding of critical differences between transient and chronic events. Furthermore, it may provide insights of the molecular mechanisms of HPV-induced TNF resistance, contribute to the identification of key functions and pathways associated to specific HPV types and, finally, lead to the identification of new cervical tumor progression markers.

                    Declarations

                    Acknowledgements

                    This work was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo grant 98/14335-2. L. Termini had a PhD fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant 01/01006-5).

                    We would like to thank Dr. Alex Fiorini, Chamberlein Neto, Mariana Santos and Aline Pacífico for technical assistance, Ana Carolina Quirino Simões and Lucas Fahham for the review of the statistical analysis, and Dr. Ana Paula Lepique and members of the Laboratory of Functional Genomics and the Laboratory of Virology of Ludwig Institute for Cancer research for helpful discussions.

                    Authors’ Affiliations

                    (1)
                    Ludwig Institute for Cancer Research
                    (2)
                    Hospital do Câncer A. C. Camargo
                    (3)
                    Instituto de Matemática e Estatística da Universidade de São Paulo

                    References

                    1. zur Hausen H: Human papillomaviruses in the pathogenesis of anogenital cancer. Virology 1991, 184:9–13.View ArticlePubMed
                    2. Schlecht NF, Platt RW, Negassa A, Duarte-Franco E, Rohan TE, Ferenczy A, Villa LL, Franco EL: Modeling the time dependence of the association between human papillomavirus infection and cervical cancer precursor lesions. Am J Epidemiol 2003, 158:878–86.View ArticlePubMed
                    3. Schlecht NF, Kulaga S, Robitaille J, Ferreira S, Santos M, Miyamura RA, Duarte-Franco E, Rohan TE, Ferenczy A, Villa LL, Franco EL: Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia. JAMA 2001, 24:3106–14.View Article
                    4. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999, 189:12–9.View ArticlePubMed
                    5. Münger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen CL, Owens M, Grace M, Huh K: Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004, 78:11451–60.View ArticlePubMed
                    6. Wu S, Meng L, Wang S, Wang W, Xi L, Tian X, Chen G, Wu Y, Zhou J, Xu G, Lu Y, Ma D: Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA. Clin Cancer Res 2006, 12:2032–7.View ArticlePubMed
                    7. Horner SM, DeFilippis RA, Manuelidis L, DiMaio D: Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells. J Virol 2004, 78:4063–73.View ArticlePubMed
                    8. Goodwin EC, DiMaio D: Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc Natl Acad Sci USA 2000, 97:12513–8.View ArticlePubMed
                    9. zur Hausen H: Papillomavirus causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst 2000, 92:690–8.View ArticlePubMed
                    10. Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990, 248:76–9.View ArticlePubMed
                    11. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM: The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 1993, 75:495–505.View ArticlePubMed
                    12. Dyson N, Howley PM, Munger K, Harlow E: The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 1989, 243:934–7.View ArticlePubMed
                    13. Boyer SN, Wazer DE, Band V: E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res 1996, 56:4620–4.PubMed
                    14. Stanley M: Immune responses to human papillomavirus. Vaccine 2006, 24:16–22.View Article
                    15. Griffiths CE, Barker JN, Kunkel S, Nickoloff BJ: Modulation of leucocyte adhesion molecules, a T-cell chemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) in allergic contact dermatitis (rhus dermatitis). Br J Dermatol 1991, 124:519–26.View ArticlePubMed
                    16. Tizard IR: Immunity at body surfaces. Vet Immunol 2000, 222–34.
                    17. Gröne A: Keratinocytes and cytokines. Vet Immunol Immunopathol 2002, 88:1–12.View ArticlePubMed
                    18. Lunger TA, Kock A, Danner M: Production of distinct cytokines by epidermal cells. Br J Dermatol 1985, 113:145–56.View Article
                    19. Jenson AB, Kurman RJ, Lancaster WD: Tissue effects and host response to human papillomavirus infection. Obstet Gynecol Clin North Am 1987, 14:397–406.PubMed
                    20. Tay SK, Jenkins D, Maddox Campion M, Singer A: Subpopulation of Langerhans' cells in cervical neoplasia. Br J Obstet Gynecol 1987, 94:10–5.
                    21. Beutler BA, Cerami A: Cathetin: more than a tumor necrosis factor. N Engl J Med 1987, 316:379–85.View ArticlePubMed
                    22. Vieira KBL, Goldstein DJ, Villa LL: Tumor necrosis factor α interferes with the cell cycle of normal and papillomavirus-immortalized human keratinocytes. Cancer Res 1996, 56:2452–7.PubMed
                    23. Villa LL, Vieira KBL, Pei X, Schlegel R: Differential effect of tumor necrosis factor on proliferation of primary human keratinocytes and cell lines containing human papillomavirus types 16 and 18. Mol Carcinog 1992, 6:5–9.View ArticlePubMed
                    24. Malejczyk J, Malejczyk M, Majewski S, Breitburd F, Luger TA, Jablonska S, Orth G: Increased tumorigenicity of human keratinocytes harboring human papillomavirus type 16 is associated with resistance to endogenous tumor necrosis factor-alpha-mediated growth limitation. Int J Cancer 1994, 56:593–8.View ArticlePubMed
                    25. Rösl F, Lengert M, Albrecht J, Kleine K, Zawatzky R, Schraven B, zur Hausen H: Differential regulation of the JE gene encoding the monocyte chemoattractant protein (MCP-1) in cervical carcinoma cells and derived hybrids. J Virol 1994, 68:2142–50.PubMed
                    26. Schlegel R, Phelps WC, Zhang YL, Barbosa MS: Quantitative keratinocyte assay detects two biological activities on human papillomavirus DNA and identifies viral types associated with cervical carcinomas. EMBO J 1988, 7:3181–7.PubMed
                    27. Barbosa MS, Schlegel R: The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes. Oncogene 1989, 4:1529–32.PubMed
                    28. Gomes LI, Silva RL, Stolf BS, Cristo EB, Hirata R, Soares FA, Reis LF, Neves EJ, Carvalho AF: Comparative analysis of amplified and non-amplified RNA for hybridization in cDNA microarray. Anal Biochem 2003, 321:244–51.View ArticlePubMed
                    29. Brentani RR, Carraro DM, Verjovski-Almeida S, Reis EM, Neves EJ, de Souza SJ, Carvalho AF, Brentani H, Reis LF: Gene expression arrays in cancer research: methods and applications. Crit Rev Oncol Hematol 2005, 54:95–105.View ArticlePubMed
                    30. Gene Expression Omnibus-NCBI[http://​www.​ncbi.​nlm.​nih.​gov/​geo]
                    31. The R Project for Statistical Computing[http://​www.​r-project.​org]
                    32. Bioconductor software for bioinformatics[http://​www.​bioconductor.​org]
                    33. Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad Sci USA 1984, 81:1991–5.View ArticlePubMed
                    34. Pichlmair A, Reis e Sousa C: Innate recognition of viruses. Immunity 2007, 27:370–83.View ArticlePubMed
                    35. Allen SJ, Crown SE, Handel TM: Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol 2007, 25:787–820.View ArticlePubMed
                    36. Basile JR, Zacny V, Munger K: The cytokines tumor necrosis factor-alpha (TNF-alpha) and TNF-related apoptosis-inducing ligand differentially modulate proliferation and apoptotic pathways in human keratinocytes expressing the human papillomavirus-16 E7 oncoprotein. J Biol Chem 2001, 276:22522–8.View ArticlePubMed
                    37. Filippova M, Song H, Connolly JL, Dermody TS, Duerksen-Hughes PJ: The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis. J Biol Chem 2002, 14:21730–9.View Article
                    38. Bachmann A, Hanke B, Zawatzky R, Soto U, van Riggelen J, zur Hausen H, Rösl F: Disturbance of tumor necrosis factor alpha-mediated beta interferon signaling in cervical carcinoma cells. J Virol 2002, 76:280–91.View ArticlePubMed
                    39. Woodworth CD, McMullin E, Iglesias M, Plowman GD: Interleukin 1 alpha and tumor necrosis factor alpha stimulate autocrine amphiregulin expression and proliferation of human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells. Proc Natl Acad Sci USA 1995, 92:2840–4.View ArticlePubMed
                    40. Gaiotti D, Chung J, Iglesias M, Nees M, Baker PD, Evans CH, Woodworth CD: Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway. Mol Carcinog 2000, 27:97–109.View ArticlePubMed
                    41. Boccardo E, Noya F, Broker TR, Chow LT, Villa LL: HPV-18 confers resistance to TNF-alpha in organotypic cultures of human keratinocytes. Virology 2004, 328:233–43.View ArticlePubMed
                    42. Delvenne P, al-Saleh W, Gilles C, Thiry A, Boniver J: Inhibition of growth of normal and human papillomavirus-transformed keratinocytes in monolayer and organotypic cultures by interferon-gamma and tumor necrosis factor-alpha. Am J Pathol 1995, 146:589–98.PubMed
                    43. Clifford GM, Smith JS, Plummer M, Muñoz N, Franceschi S: Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003, 88:63–73.View ArticlePubMed
                    44. Zielinski GD, Snijders PJ, Rozendaal L, Daalmeijer NF, Risse EK, Voorhorst FJ, Jiwa NM, Linden HC, de Schipper FA, Runsink AP, Meijer CJ: The presence of high-risk HPV combined with specific p53 and p16INK4a expression patterns points to high-risk HPV as the main causative agent for adenocarcinoma in situ and adenocarcinoma of the cervix. J Pathol 2003, 201:535–43.View ArticlePubMed
                    45. IARC – International Agency for Research on Cancer-World Health Organization: IARC Monographs on the evaluation of carcinogenic risks to humans: human papillomaviruses.[http://​www-dep.​iarc.​fr/​]Lyon: Human papillomavirus 2007., 90:
                    46. Romanczuk H, Villa LL, Schlegel R, Howley PM: The viral transcriptional regulatory region upstream of the E6 and E7 genes is a major determinant of the differential immortalization activities of human papillomavirus types 16 and 18. J Virol 1991, 65:2739–44.PubMed
                    47. Villa LL, Schlegel R: Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region. Virology 1991, 181:374–7.View ArticlePubMed
                    48. Sichero L, Ferreira S, Trottier H, Duarte-Franco E, Ferenczy A, Franco EL, Villa LL: High grade cervical lesions are caused preferentially by non-European variants of HPVs 16 and 18. Int J Cancer 2007, 120:1763–8.View ArticlePubMed
                    49. Clifford GM, Smith JS, Aguado T, Franceschi S: Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. Br J Cancer 2003, 89:101–5.View ArticlePubMed
                    50. Borgono CA, Michael IP, Diamandis EP: Human tissue kallikreins: physiologic roles and applications in cancer. Mol Cancer Res 2004, 2:257–80.PubMed
                    51. Diamandis EP, Yousef GM: Human tissue kallikreins: a family of new cancer biomarkers. Clin Chem 2002, 8:1198–205.
                    52. Yousef GM, Diamandis EP: The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr Rev 2001, 22:184–204.View ArticlePubMed
                    53. Tian X, Shigemasa K, Hirata E, Gu L, Uebaba Y, Nagai N, O'Brien TJ, Ohama K: Expression of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in uterine endocervical glands and in cervical adenocarcinomas. Oncol Rep 2004, 5:1001–6.
                    54. Santin AD, Cane' S, Bellone S, Bignotti E, Palmieri M, De Las Casas LE, Roman JJ, Anfossi S, O'Brien T, Pecorelli S: The serine protease stratum corneum chymotryptic enzyme (kallikrein 7) is highly overexpressed in squamous cervical cancer cells. Gynecol Oncol 2004, 94:283–8.View ArticlePubMed
                    55. Talieri M, Diamandis EP, Gourgiotis D, Mathioudaki K, Scorilas A: Expression analysis of the human kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma. Thromb Haemost 2004, 91:180–6.PubMed
                    56. Dong Y, Kaushal A, Brattsand M, Nicklin J, Clements JA: Differential splicing of KLK5 and KLK7 in epithelial ovarian cancer produces novel variants with potential as cancer biomarkers. Clin Cancer Res 2003, 9:1710–20.PubMed
                    57. Kinnula VL, Crapo JD: Superoxide dismutases in malignant cells and human tumors. Free Radic Biol Med 2004, 36:718–44.View ArticlePubMed
                    58. Djavaheri-Mergny M, Javelaud D, Wietzerbin J, Besancon F: NF-kappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFalpha-treated Ewing sarcoma cells. FEBS Lett 2004, 578:111–5.View ArticlePubMed
                    59. Delhalle S, Deregowski V, Benoit V, Merville MP, Bours V: NF-kappaB-dependent MnSOD expression protects adenocarcinoma cells from TNF-alpha-induced apoptosis. Oncogene 2002, 21:3917–24.View ArticlePubMed
                    60. Kiningham KK, Xu Y, Daosukho C, Popova B, St Clair DK: Nuclear factor kappaB-dependent mechanisms coordinate the synergistic effect of PMA and cytokines on the induction of superoxide dismutase 2. Biochem J 2001, 353:147–56.View ArticlePubMed
                    61. St Clair DK, Porntadavity S, Xu Y, Kiningham K: Transcription regulation of human manganese superoxide dismutase gene. Methods Enzymol 2002, 349:306–12.View ArticlePubMed
                    62. Seitz CS, Lin Q, Deng H, Khavari PA: Alterations in NF-kappaB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kappaB. Proc Natl Acad Sci USA 1998, 95:2307–12.View ArticlePubMed
                    63. Komine M, Rao LS, Kaneko T, Tomic-Canic M, Tamaki K, Freedberg IM, Blumenberg M: Inflammatory versus proliferative processes in epidermis. Tumor necrosis factor alpha induces K6b keratin synthesis through a transcriptional complex containing NFkappaB and C/EBPbeta. J Biol Chem 2000, 275:32077–88.View ArticlePubMed
                    64. Hu Y, Baud V, Oga T, Kim KI, Yoshida K, Karin M: IKKalpha controls formation of the epidermis independently of NF-kappaB. Nature 2001, 410:710–4.View ArticlePubMed
                    65. Pre-publication history

                      1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1755-8794/​1/​29/​prepub

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                    © Termini et al. 2008

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