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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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: <it>KLK7 </it>(<it>kallikrein 7</it>), <it>SOD2 </it>(<it>superoxide dismutase 2</it>), <it>100P </it>(<it>S100 calcium binding protein P</it>), <it>PI3 </it>(<it>protease inhibitor 3, skin-derived</it>), <it>CSTA </it>(<it>cystatin A</it>), <it>RARRES1 </it>(<it>retinoic acid receptor responder 1</it>), and <it>LXN </it>(<it>latexin</it>). The differential expression of the <it>KLK7 </it>and <it>SOD2 </it>transcripts was confirmed by Northern blot. Moreover, we observed that <it>SOD2 </it>expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells.</p> <p>Conclusion</p> <p>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.</p

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment-6

Inkage. After sample grouping the genes (p values <p><b>Copyright information:</b></p><p>Taken from "Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment"</p><p>http://www.biomedcentral.com/1755-8794/1/29</p><p>BMC Medical Genomics 2008;1():29-29.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2459201.</p><p></p

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment-4

Clear 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.<p><b>Copyright information:</b></p><p>Taken from "Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment"</p><p>http://www.biomedcentral.com/1755-8794/1/29</p><p>BMC Medical Genomics 2008;1():29-29.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2459201.</p><p></p

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment-2

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).<p><b>Copyright information:</b></p><p>Taken from "Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment"</p><p>http://www.biomedcentral.com/1755-8794/1/29</p><p>BMC Medical Genomics 2008;1():29-29.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2459201.</p><p></p

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment-1

Inkage. After sample grouping the genes (p values <p><b>Copyright information:</b></p><p>Taken from "Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment"</p><p>http://www.biomedcentral.com/1755-8794/1/29</p><p>BMC Medical Genomics 2008;1():29-29.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2459201.</p><p></p

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment-5

Inkage. After sample grouping the genes (p values <p><b>Copyright information:</b></p><p>Taken from "Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment"</p><p>http://www.biomedcentral.com/1755-8794/1/29</p><p>BMC Medical Genomics 2008;1():29-29.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2459201.</p><p></p

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment-0

Inkage. After sample grouping the genes (p values <p><b>Copyright information:</b></p><p>Taken from "Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment"</p><p>http://www.biomedcentral.com/1755-8794/1/29</p><p>BMC Medical Genomics 2008;1():29-29.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2459201.</p><p></p