Polyethylene membranes were cut to size and mounted on non-charged Superfrost Color glass slides. Slides for immunohistochemistry staining were treated with 8% 3-aminopropyltriethoxysilane solution in acetone. Serial 5-mm paraffin sections were cut with a fresh knife, mounted on prepared slides, and incubated at 60uC for 2 hours to achieve better tissue adhesion to the membrane. Deparaffinization was carried out by incubation in xylene, followed by washing and rehydration in ethanol. Immunhistochemical staining for the expression of p16INK4a was performed using the CINtec p16INK4a Histology kit for manual staining according to manufacturer’s instructions. Cytospin preparations of the HPV18 positive cervical cancer cell line HeLa were used as positive controls. Immunostaining for L1 was perfomed using the Cytoactive kit for HPV L1 as prescribed by the manufacturer. Counterstaining was performed with Mayer’s Hematoxylin. Selected p16INK4a-positive clusters, as well as p16INK4a-negative non-tumour tissue cells, were collected separately in the cap of a microfuge tube by laser pressure catapulting using the PALMH Robot-Micro Beam for microdissection. DNA was isolated using QIAamp DNA FFPE Tissue Kit. The methylation status of the HPV16 URR was determined by bisulfite treatment. Genomic DNA from microdissected specimen or cell line was bisulfite-modified using the EZ DNA methylation kit according to the manufacturer’s instructions. One microgram of DNA from the Caski and SiHa cell lines was used as control and treated concurrently with the samples to ensure complete bisulfite treatment. After treatment, the resulting bisulfite-modified DNA was eluted in 30 ml of the kit elution buffer and stored at 220uC. Five microliter of the bisulfite-modified DNA was used for each PCR reaction. A nested PCR system was developed using primers that span the URR of HPV 16. PCR reaction mixtures were performed in a total of 50 ml containing 106PCR buffer, 1.5 ml 50 mM MgCl2, 1 ml 10 mM deoxynucleotide triphosphates, 0.5 ml of each PCR primer, 2.0 U Platinum Taq and 5 ml of the bisulfite modified DNA. Amplification conditions were as follows: initial denaturation at 94uC for 2 min followed by 40 cycles and 30 cycles for the nested PCR of 94uC for 40 s, annealing at 50uC for 30 s, extension at 72uC for 1 min and finally 72uC for 4 min. PCR products were electrophoresed and isolated from 1.2% agarose gels stained with ethidium bromide. Isolated PCR products were then purified by QIAquick Gel Extraction Kit according to the manufacturer’s instructions. Purified PCR fragments were cloned the TA Cloning System and 12 individual clones were sequenced to identify the presence of methylated CpGs within the HPV 16 LCR. Sequencing of bisulfite modified sample DNA was performed using the BigDye terminator sequencing kit according to the manufacturer’s recommendations. The sequencing PCR products were analyzed on the ABI Prism 3100 Genetic Analyzer. The degree of methylation of the 12 independent clones each isolated from basal, intermediate and superficial cell layers from the three latent, three permissive and 5 transforming epithelial regions was analyzed by using the Systat statistical data evaluation software. In vitro DNA methylation was accomplished with CpGmethylase, by following the procedure recommended by New England Biolabs, the commercial provider of SssI. Completion of DNA methylation was assessed by digestion with the Hpa II restriction Publications Using Abomle Temozolomide enzyme, which cleaves at its recognition sequence only if the DNA is not methylated at the cytosine residue within it. For the generation of methylated and unmethylated LCR HPV16 containing DNA fragments, the double-stranded LCR 16 DNA fragment, which was or was not subjected to in vitro methylation with the SssI CpGmethylase, was cloned into the HindIII and BamHI-linearized reporter plasmid pGluc-promoter. Ligated products were purified using the PCR purification kit. Two micrograms of the ligated products generated with methylated or unmethylated LCR was transfected into C33A cells.