%0 Generic
%A Chotewutmontri, Sasithorn
%D 2011
%F heidok:11630
%K HPV16 , HPV68 , DNA Integration , Cervical cancer
%R 10.11588/heidok.00011630
%T Genome integration structures and genotype variants of oncogenic human papillomavirus types HPV16 and HPV68 in cervical carcinoma-derived cell lines, cervical precursor lesions and carcinomas
%U https://archiv.ub.uni-heidelberg.de/volltextserver/11630/
%X Persistent infection with high-risk human papillomavirus (hr-HPV) is essential for cervical carcinogenesis, and is frequently followed by integration of the viral DNA into the host genome. Upon integration, the viral E2 gene is usually disrupted or deleted leading to deregulated transcription of the E6/E7 oncogenes from the upstream regulatory region (URR). Integrated HPV DNA may also affect critical cellular genes through insertional mutagenesis, which can contribute to the multi-step process of cervical carcinogenesis. HPV16 is the most frequent and HPV68 is a rare hr-HPV type, present in about 55% and less than 1% of cervical carcinomas worldwide, respectively. In this work, HPV68 DNA structures in cervical carcinoma cell lines and clinical samples were analyzed. HPV16 integration and E1-E2 sequences were studied using the novel “amplification selection pyrosequencing of HPV16” (ASP16) strategy. HPV68 is divided into two subtypes, a and b. A hallmark of HPV68b is its presence as integrated DNA in the cervical carcinoma cell line ME180. In the mutant cell line ME180R, selected for resistance to growth inhibition by tumor-necrosis-factor alpha (TNFalpha), partial deletions in the integrated HPV68b DNA had been detected. In this study, the complete structures of the integrated HPV68b in ME180 and ME180R have been determined. ME180 cells contain two disrupted HPV68b copies, integrated in a unique head-to-head arrangement into chromosome 18q21. By selection of new TNFalpha-resistant ME180 sub-lines, it was found that the rearrangements and partial deletions of HPV68b in ME180R are unnecessary for the TNFalpha-resistance phenotype. In addition, a full-length and a mutant HPV68b genome were isolated from a cervical intraepithelial neoplasia grade 2 (CIN2) precursor lesion, cloned and completely sequenced. The mutant genome carrying a 1.2-kb deletion in the E1 gene is probably integrated. Based on partial URR sequences, ten HPV68b variants, nine of them new, and one HPV68a variant have been identified in eleven clinical samples, suggesting that HPV68b is more widely distributed than HPV68a and is present in a multitude of molecular variants. ASP16 was developed for simultaneous determination of HPV16 integration junctions in multiple clinical DNA samples. It consists of four main steps: GenomePlex whole genome amplification, HPV16 E1-E2 sequence enrichment, Roche/454 GS-FLX pyrosequencing, and data analysis. In this work, computer programs for ASP16 data analysis were developed and applied. The ASP16 strategy was further optimized and used for the analysis of 25 HPV16-positive samples. The optimized ASP16 delivered longer sequence read lengths and 89% average sequence coverage. HPV16 integration junctions were identified in 3 out of 4 cell lines, and 6 out of 21 clinical samples. The HPV16 integration sites identified in the clinical samples are all located near cellular proto-oncogenes or tumor suppressor genes, supporting the assumption that HPV integration contributes to cervical carcinogenesis by altering cancer-relevant cellular genes. The high E1-E2 sequence coverage also allowed HPV16 variant assignments. Altogether, the ASP16 strategy, which is the first method combining next generation sequencing technologies with HPV integration analysis in a multiplex format, shows the potential to identify HPV16 integration junctions in series of clinical samples in parallel and at the same time provides E1-E2 sequences suitable for mutation/variant analysis.