title: Integration of Human Papillomavirus Type 16 DNA in Cervical Carcinogenesis : Design of a novel strategy for HPV16 integration site determination in cervical scrapes and analysis of HPV16-induced c-myc insertional mutagenesis
creator: Xu, Bo
subject: ddc-570
subject: 570 Life sciences
description: Persistent infection with high-risk human papillomavirus (HPV) types, most frequently HPV16, is a necessary cause of cervical cancer. Correlated with tumor progression, the circular HPV DNA usually becomes integrated into the host cell genome with diverse target sites. HPV DNA integration may induce alterations of flanking cellular genes by insertional mutagenesis that contribute to the multistep process of carcinogenesis. The primary goal of this study was the design of a novel strategy to determine HPV16 DNA integration sites at the sequence level in selected pre-cancerous and cancerous lesions obtained from cervical scrapes.  While testing restriction-site PCR for HPV16 DNA integration analysis, cell line MRI-H186 was identified as an interesting case of HPV16-induced insertional mutagenesis of the c-myc proto-oncogene. Associated with HPV16 DNA integration into the c-myc locus, over-expression of c-myc was found at both mRNA and protein levels. In addition, several HPV16 integration variants were characterized at the sequence level. One of these variants is strongly expressed as myc-HPV16 hybrid transcript and MycHPV16E2 fusion protein both unique for MRI-H186. In MycHPV16E2, the amino-terminal part of the c-MYC trans-activation domain is fused to the linker and DNA-binding domain of HPV16 E2. These data strengthen the assumption that insertional mutagenesis, in this case c-myc activation/mutation, contributes to cervical carcinogenesis.  For HPV16 DNA integration analysis in clinical samples from which genomic DNA is available only in nanogramme amounts and often degraded, a novel strategy enabling the simultaneous sequencing of multiple DNA samples was developed and examined in the present study. This strategy was named ASP16 because it combines whole genome amplification (A), HPV16 DNA selection (S) and high-throughput pyrosequencing (P) followed by bioinformatics data analysis. Two ASP16 experiments were performed, which have proven the feasibility of the novel strategy. The known 3’ viral-cellular junction sequence of integrated HPV16 DNA in MRI-H186 was identified in both experiments. HPV16 DNA integration sites were determined in clinical samples harbouring high percentages of integrated HPV16 DNA. Cancer-related cellular genes near or at the integration sites, namely Gli1 and Grem2, hold great potentials as candidates for HPV16-induced insertional mutagenesis. Future optimizations of the ASP16 method will focus primarily on increasing the average sequence length for a complete coverage of all possible viral DNA breakpoints in the HPV16 E1/E2 region.  Taken together, the ASP16 strategy offers for the first time the opportunity to systematically explore HPV16 DNA integration sites in a multiplex manner in clinical samples obtained from cervical scrapes, with particular respect to low quantity and poor quality of the template DNA. This unique feature is of great interest for the incorporation of HPV16 DNA integration analysis into routine cervical screening programs. Application of APS16 will contribute unequivocally to identify lesions of progressive or highly advanced disease and to obtain further insights into the underlying molecular mechanisms of cervical carcinogenesis.
date: 2010
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/10679/1/Bo_Xu_Doktorarbeit_2010.pdf
identifier: DOI:10.11588/heidok.00010679
identifier: urn:nbn:de:bsz:16-opus-106792
identifier:   Xu, Bo  (2010) Integration of Human Papillomavirus Type 16 DNA in Cervical Carcinogenesis : Design of a novel strategy for HPV16 integration site determination in cervical scrapes and analysis of HPV16-induced c-myc insertional mutagenesis.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/10679/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng