Case selections and microarray construction
Following approval from our institutional review board, a tissue microarray (TMA) was constructed with 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. The cases were retrieved from the archived Tissue Bank at Liège University (Liège Biothèque: BTULg) and represented biopsies diagnosed between the years 1998 and 2006.
These cases were selected on the basis of availability of at least one evaluable tissue representative hematoxylin and eosin-stained slide and a paraffin block. For each case, slides were reviewed to select a representative area. The corresponding spot on the associated paraffin block was then cored and placed on a tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA). The TMA blocks were constructed in doublets (2 spots for each diagnostic entity) using 1 mm tissue cores (Alphelys, Plaisir, France).
The whole series supplied a total number of 194 spots including duplicate of 11 L-SIL, 18 H-SIL, 12 SCC, 12 primary cervical adenocarcinoma (ADC) and 9 adenocarcinoma in situ (AIS). From the cases without cervical lesions, duplicate of 16 ectocervix and 19 endocervix tisues were arrayed. The finalized arrays were then cut into 5 μm-thick sections and mounted on glass slides.
In addition to the formalin-paraffin embedded tissues that were used for the TMA preparation, additional frozen cervical biopsy specimens were retrieved from the Tumor Bank of Liege University (BTULg). These biopsies included 10 high-grade squamous intraepithelial lesions (HSIL), 10 invasive squamous cell carcinoma (SCC) and 10 paired normal exocervical tissues from the same patients. These biopsies were used to perform immunofluorescence studies under confocal microscopy.
Immunohistochemistry and immunofluorescence
The tissue microarray slides were stained with antibodies against VHR (Clone 237020 dilution 1:2500, R&D Systems, Minneapolis, MN) using a standard avidin-biotin complex method. Tissue microarray slides were deparaffinized with xylene, graded alcohol then rehydrated with distilled water. Endogenous peroxidase activity was blocked by placing the slides in 0.5% hydrogen peroxidase/methanol for 10 minutes followed by a tap water rinse. Background staining was reduced by incubating slides in 0.3% bovine serum albumin/Tris-buffered saline. Antigen retrieval entailed placing the slides in a pressure cooker with an antigen unmasking solution (0.01 M citrate buffer, pH 6.0) for 1 minute. Slides were subsequently incubated with the primary (4°C overnight), then biotinylated secondary antibodies and streptavidin-biotin peroxidase. 3'3' diaminobenzidine (DAB) was used as chromogen and sections were counterstained with hematoxylin.
Immunofluorescence staining on the cervix biopsies was performed with a monoclonal antibody directed against VHR (Clone 24 dilution 1:2500, BD, San Diego, CA). The primary antibodies were revealed with Alexa-488 conjugated secondary antibody together with TOTO-3 to stain nuclei. The sections were mounted and viewed under a confocal laser scanning microscopy TCS SP2 (Leica TCS SP2, Van Hopplynus, Belgium).
For immunofluorescence and immunohistochemistry on cells in culture, the cells were grown on poly-L-lysine coated coverslips and fixed with 4% formaldehyde. The fixed cells were permeabilized with 0.3% of Triton X-100/PBS buffer then stained either with anti-VHR mAb (4 μg/ml) (BD-transduction laboratories) or with p16 antibody (NeoMarkers). After 3 washes, the primary Ab was revealed with an Alexa-488 conjugated secondary Ab together with TOTO-3 (Invitrogen) to stain nuclei and visualized under confocal microscopy. For immunohistochemistry, the anti-VHR antibody was revealed using an HRP secondary detection kit (Universal LSABTM 2 KIT/HRP, Rabbit/Mouse, DakoCytomation). The stained cells were mounted and visualized on light microscopy.
Scoring of immunohistochemical staining
The VHR immunostaining was scored semi-quantitatively. For staining intensity, 0 represented samples in which VHR nuclear and/or cytoplasmic staining was undetectable, whereas 1+, 2+, and 3+ denoted samples with low, moderate, and strong staining, respectively. For staining extent, in normal ectocervix and the various grades of SIL, 1+ represented samples in which VHR expression was detectable in the lower 1/3 of the epithelium whereas 2+ denoted samples in which the lower 2/3 of the epithelium showed detectable VHR expression and 3+ represented those in which the immunoreactive cells reached the upper epithelial 1/3. For the extent of staining in SCC, normal endocervix, AIS and ADC, 1+ represented samples in which VHR expression was detectable in up to 33% of the epithelium whereas 2+ denoted samples in which 33–66% of the epithelium showed detectable VHR expression and 3+ represented those in which more than 66% of the cells were stained. In order to provide a global score for each case, the results obtained with the two scales were multiplied, yielding a single scale with steps of 0 to 9. The microarrays were scored by 2 independent observers and discrepancies were resolved during a consensus session. To externally validate the staining patterns observed in the TMA, full representative tissue sections of 10 SCC were randomly selected, stained with VHR and scored using the same system as used with the microarray.
Cell Lines and primary keratinocytes
Five different cell lines derived from cervix cancer were used in this study: HeLa, SiHa and CaSki (all positive for HPV) and C33 and HT3 (HPV negative). Immortalized human foreskin keratinocytes stably transfected with E6 and E7 (E6/E7) were previously described  and kindly provided by F. Rosl (Heidelberg, Germany). The cells were grown in DMEM medium (Dulbecco's modified Eagle's medium; ICN; Flow Laboratories) complemented with 10% heated inactivated fetal calf serum (FCS), 30 units/ml of penicillin, 30 μg/ml of streptomycin and 2 mM of L-glutamine.
Primary keratinocytes (KN) were prepared from hysterectomies. Fragments were plunged in a solution containing gentamycin, fungizon and anti-mycoplasm. These fragments were cut in smaller pieces, and then incubated in trypsin-EDTA (Invitrogen) at 37°C under agitation for 1–2 hours. The epithelium was scraped and cells were recovered in FCS. After centrifugation, the cells were resuspended in K-SFM medium (Serum Free Media; Invitrogen) complemented with EGF (0.1 ng/ml), pituitary hormone (20–30 μg/ml) and gentamycin (5 μg/ml).
Cell lysates and Immunoblotting
Cells were lysed in 20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA containing 1% NP-40, 1 mM Na3VO4, 10 μg/ml aproptinin and leupeptin, 100 μg/ml soybean trypsin inhibitor and 1 mM phenylmethylsulfhonyl fluoride, incubated on ice for 30 min then centrifuged at 20,000 g for 20 min. The proteins were then resolved by SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were immunoblotted with optimal dilutions of monoclonal primary antibodies, followed by an HRP conjugated anti-mouse secondary Ab. The blots were developed by enhanced chemiluminescence (ECL kit, Amersham) according to the manufacturer's instructions.
RNA preparation and RT-PCR
Total RNA was extracted from primary keratinocytes and the different cell lines using the High Pure RNA Isolation Kit (Roche Diagnostics, Germany) according to the procedures supplied by the manufacturer. Reverse transcription was performed using the RT-PCR kit (Applied Biosystem, Foster City, CA). The PCR reaction was performed using VHR specific primers (5'-ATGTCGGGCTCGTTCGAGCTC-3' and 5'-CTAGGGTTTCAACTTCCCCTC-3') and normalized with HPRT (5'-GTTGGATACAGGCCAGACTTTGTTG-3' and 5'-GATTCAACTTGCGCTCATCTTAGGC-3').