Invasion ability, MMP expression and activity of C33A cells stably expressing different HPV16 oncoproteins.

<p>(A) <i>In vitro</i> invasion and (B) wound healing assays for C33A cells expressing different HPV16 oncoproteins. (C) A representative gelatin zymographic gel showing MMP-2 activity normalized with equal amounts of loading protein (30 µg). Bands corresponding to the gelatinolytic activity of MMP-2 were quantified by densitometric analysis and compared with those obtained from control pcDNA3 expressing cells. (D) Western blot analysis of MT1-MMP using anti-MT1-MMP antibody (ab78738) at 1∶1,500 dilution with GAPDH as loading control. *<i>p</i>-value<0.05, **<i>p</i>-value<0.01.</p

<i>In vitro</i> invasion and <i>MMP</i> expression of HPV positive, HPV negative and HPV oncoprotein expressing C33A.

<p>(A) Quantitation of invasion using migration through filters coated with Matrigel for HPV positive (SiHa, Caski and HeLa) and negative (C33A and 293T) cells presenting as numbers of invading cells. (B) Relative expression of <i>MMP-1</i>, <i>-2</i>, <i>-7</i>, <i>-9</i>, <i>-10</i>, <i>-11</i> and <i>MT1-MMP</i> measured in cell lines using qPCR. Data was normalized to C33A. *:<i>p</i>-value<0.05 relative to C33A. (C) Relative expression of <i>MMP-2, -7</i> and <i>MT1-MMP</i> in C33A cells stably expressing different HPV16 (<i>16E6, 16E6F, 16E6*I, 16E7, 16E6E7</i>, <i>16E6*IE7</i>), HPV18 <i>(18E6E7)</i> and low-risk HPV6 <i>(6E6)</i> oncoproteins as measured by qPCR and normalized to pcDNA3. *<i>p</i>-value<0.05 compared to pcDNA3 expressing cells. (D) The level of HPV transcripts compared to the control, HPRT, in each cell line as shown by RT-PCR. (E) P53 degradation activity of 16E6, 16E7 and 16E6E7 was compared by western blot analysis. GAPDH was used as loading control.</p

Fluorescent immunohistochemistry of MMP-2 and MT1-MMP in invasive cervical cancers and their HPV typing.

<p>CIN 1, 2, 3 are various stages of control carcinoma <i>in situ</i>. Adenomyosis represents normal uterine control.</p

Inhibition of cell invasion by specific inhibitors for MMP-2 and MT1-MMP and assay of TIMP-2.

<p>(A) Cell invasion as determined using <i>in vitro</i> invasion and wound healing of HPV16E6E7 expressing cells and (B) SiHa cells treated with various concentrations of MMP-2 inhibitor, OA-Hy. (C) The same cell lines were also treated with anti-MT1-MMP antibody (20 µg/ml). Relative fold of cell invasion with pcDNA3 vehicle control or untreated cells set as 1 at φ<i>p</i><0.05 compare to pcDNA3 and *<i>p</i>-value<0.05 compare to untreated cells is shown. (D) TIMP-2 protein (normalized with equal loading protein of 5 µg) was detected using western blot analysis with an anti-TIMP-2 antibody (abcam, 1∶1,000 dilution) and TIMP-2 transcripts were measured by qPCR (normalized to HPRT) for HPV16 oncoprotein expressing cells.</p

Representative phase contrast and fluorescent images of MMP-2 and MT1-MMP expression in cervical cancer tissues.

<p>Presence of MMP-2 and MT1-MMP are shown in red (Alexa Fluor 647) and cell nuclei in blue (Hoechst 33258). (A, B) no primary antibodies; (C, D) and (G, H) negative staining of MMP-2 and MT1-MMP respectively; (E, F) and (I, J) positive staining of MMP-2 and MT1-MMP respectively.</p

HPV oncogenes, <i>MMP</i> transcripts and MMP protein levels in SiHa cells transfected with shRNA directed at E6 oncogene.

<p>(A) RT-PCR of <i>16E6F, 16E6*I</i>, and <i>16E7</i> transcripts normalized to the control, <i>HPRT</i>. SiHa cells transfected with non-targeting shCtrl and vehicle control pSUPER were used as negative controls. shE6A and shE6B are two different shRNAs used in knock-down experiments. (B) qPCR of <i>MMP-2</i>, <i>-7</i> and <i>MT1-MMP</i> transcripts and (C) MMP-2 activity as assayed by gelatin zymography, with equal amounts of protein (20 µg), in shRNA transfected cells. (D) MT1-MMP protein in knocked down cells as determine by western blot analysis with GAPDH as loading control. (E) Invasion ability of SiHa cells transfected with different shRNAs as determined by <i>in vitro</i> invasion and wound healing assays. Relative fold of cell invasion with untreated cells were set as 1 is reported. *<i>p</i>-value<0.05 compared to shCtrl cells.</p

Caspase-1 activity did not account for decreased IL-1β in E6-positive cells.

<p>A) RT-PCR analysis of caspase-1 mRNA in comparison to the GAPDH steady state levels in PK and HPV-positive immortalized keratinocytes. B) Fluorometric measurement of caspase-1 activity was performed incubating the cells for 4 h at 37°C with 20 µM of the specific caspase-1 substrate R110-YVAD. RFU: Relative fluorescence units. C) Quantification of intracellular IL-1β levels by ELISA after 5 h of caspase-1 inhibition using 250 nM of caspase-1 inhibitor. The graphs in B and C represent the mean values (± SEM) of three independent experiments. ANOVA **p<0.01, ***p<0.001.</p

Proteasome inhibition or knock-down of E6-AP increases the levels of pro-IL-1β in immortalized E6-positive cells.

<p>A) ELISA of intracellular IL-1β from untreated immortalized HPV-positive cells and cells incubated with 10 µM of the proteasome inhibitor MG132 for 6 h. B) Western blot analysis of pro-IL1β and p53 in immortalized keratinocytes after inhibition of deubiquitinases using PR-619 (10 µM) for 6 h prior to protein extraction. C) Detection of poly-ubiquitinated pro-IL-1β in immortalized keratinocytes by Western blotting. Cells were treated with MG132 for 6 h prior to protein extraction and pull-down of ubiquitinated proteins was performed by the tandem ubiquitin-binding entities technique (TUBE-PD, right panel). Left panel: input, representing 2.5% of the total protein extract. D) Confocal microscopy analysis of IL-1β (green) in immE6 cells after knock-down of E6-AP by siRNA or scrambled siRNA delivery used as a control. Nuclei (blue) were stained using Hoechst dye solution; scale bars represent 10 µm. E) Western blot analysis of pro-IL-1β after the knock-down of E6-AP. F) ELISA of intracellular IL-1β from immortalized HPV-positive cells after the knock-down of E6-AP by siRNA (+) or control knock-down.(−) G) ELISA of IL-1β secretion from immortalized HPV-positive cells after E6-AP knock-down and/or after NALP3 inflammasome activation using 50 µM of nigericine for 6 h. H) Knock-down of p53 and/or E6-AP in immortalized cells. Cells were transfected with 30 pmoles of the respective siRNA against E6-AP or p53 and incubated for 72 h prior to protein extraction and Western blot analysis. The bar graphs shown in A (ANOVA ***p<0.05), D (ANOVA ***p<0.001, **p<0.01) F and G (ANOVA ***p<0.001) represent the mean levels (± SEM) of three independent experiments.</p

Expression and secretion of IL-1β in human primary keratinocytes and HPV-positive cell lines.

<p>A) Quantification of secreted IL-1β (expressed as pg/ml) by ELISA in human primary keratinocytes (PK), keratinocytes immortalized by individual oncogenes (E6, E7, E6/E7) and HPV16/18-positive cervical carcinoma cell lines (SiHa, CaSki, HeLa, respectively) 24 h after infection with a recombinant GFP-expressing adenovirus 5 (Ad) in comparison to uninfected cells. B) Quantification of basal intracellular IL-1β levels by ELISA. C) Western blot analysis of pro-IL-1β in human primary keratinocytes (PK) and HPV-positive cells. Actin was used as a loading control. D) qPCR analysis of IL-1β cDNA obtained from PK and HPV-positive cells (Ordinate: expressed as fold changes using the average IL-1β steady state level of 3 different primary human keratinocyte preparations PK cells as a reference which was arbitrarily set as 1. The graphs in A, B and D represent the mean values (± SEM) of three independent experiments. ANOVA ***p<0.05.</p

Analysis of IL-1β expression in normal and HPV16-positive cervical tissues.

<p>A) Immunohistochemical analysis of IL-1β expression in normal epithelium and HPV-positive lesions differing in their progression grades CIN I to CIN III and cervical tumors; scale bars represent 25 µm. B) qPCR analysis of IL-1β cDNA derived from samples negative for intraepithelial lesion and malignancy (NT), different HPV-positive lesions (CIN I, II, III) and cervical tumors (CxCa); ordinate: expression as fold changes using SiHa cells as reference which was arbitrarily set as 1. The pictures in A are a representative example of 25 biopsies analyzed from normal tissue (n = 5) and HPV-positive lesions of different donors (n = 25). The box-and-whisker blot in B represents the mean values from three to five samples for each group depicted in the graph (± SEM). ANOVA *p<0.01.</p