Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions Keltie L. Dall, 1 Cinzia G. Scarpini, 1 Ian Roberts, 1 David M. Winder, 1 Margaret A. Stanley, 2 Balaji Muralidhar, 1 M. Trent Herdman, 1 Mark R. Pett, 1 and Nicholas Coleman 1,2 1 Medical Research Council-Cancer Cell Unit, Hutchison/Medical Research Council Research Center; 2 Department of Pathology, University of Cambridge, Cambridge, United Kingdom Abstract As the high-risk human papillomavirus (HPV) integrants seen in anogenital carcinomas represent the end-point of a clonal selection process, we used the W12 model to study the naturally occurring integration events that exist in HPV16- infected cervical keratinocytes before integrant selection. We performed limiting dilution cloning to identify integrants present in cells that also maintain episomes. Such integrants arise in a natural context and exist in a noncompetitive environment, as they are transcriptionally repressed by episome-derived E2. We found that integration can occur at any time during episome maintenance, providing biological support for epidemiologic observations that persistent HPV infection is a major risk factor in cervical carcinogenesis. Of 24 different integration sites isolated from a single nonclonal population of W12, 12 (50%) occurred within chromosome bands containing a common fragile site (CFS), similar to observations for selected integrants in vivo . This suggests that such regions represent relatively accessible sites for inser- tion of foreign DNA, rather than conferring a selective advantage when disrupted. Interestingly, however, integrants and CFSs did not accurately colocalize. We further observed that local DNA rearrangements occur frequently and rapidly after the integration event. The majority of integrants were in chromosome bands containing a cancer-associated coding gene or microRNA, indicating that integration occurs com- monly in these regions, regardless of selective pressure. The cancer-associated genes were generally a considerable dis- tance from the integration site, and there was no evidence for altered expression of nine strong candidate genes. These latter observations do not support an important role for HPV16 integration in causing insertional mutagenesis. [Cancer Res 2008;68(20):8249–59] Introduction Cervical carcinoma is caused by persistent infection with high- risk human papillomavirus (HR-HPV), most commonly HPV16 and HPV18(1). Squamous cell carcinoma (SCC) accounts for 80% to 85% of cases(2) and arises from noninvasive squamous intra- epithelial lesions (SIL). Integration of HR-HPV into the host genome is seen in most cervical SCC (3, 4). Cervical neoplasms are clonal, usually with a single integration site (5), suggesting that particular integration events are selected during carcinogenesis. Mechanisms of integration are not well understood, but the pro- cess most likely involves double-strand breaks (DSB) in viral and host genomes, followed by DNA ligation by host proteins (6). Previous studies of the sites of HPV integration into host chro- mosomes have used clinical samples of SCC and SIL, as well as cell lines derived from SCC (4, 5, 7). The integrants in such specimens reflect the end point of a clonal selection process. It is not known how typical they are of the range of integration events that occur in HR-HPV infected cells nor what factors confer a selective advantage to particular integrants. Over 200 selected HPV16 and HPV18 integration sites have been reported. These are widely distributed across the genome, although many are mapped at low resolution. There seems to be preferential integration near com- mon fragile sites (CFS), specific chromosomal loci that are parti- cularly prone to forming DSBs (8, 9), with around 50% of selected HPV16 and HPV18 integration sites being in the same chromo- somal band as a CFS (5, 7, 10, 11). Important unanswered questions in HR-HPV biology concern the mechanisms by which the site of integration contributes to selection of a particular integrant. One area of controversy is whether CFSs are frequently associated with selected integrants because integration at these sites confers a competitive advantage to the cell or simply because CFSs are relatively accessible sites for integration (4, 5, 11). In addition, it is not well understood whether integrated HPV affects transcription of adjacent host genes by a process of insertional mutagenesis and/or whether the site of integration affects viral transcription. There is some evidence to suggest that both scenarios may occur (10, 12, 13), although there is very little supporting functional data. Many previous studies have suffered from a variety of limitations, including a lack of suitably matched controls, use of cells in which chromosome breaks are induced or occur at a high spontaneous rate, and minimal investigation of host protein levels. In addi- tion, and of particular importance, analysis of HR-HPV integrants in clinical samples does not allow distinction between the process of integration itself and the factors associated with selection. In the present study, we have used the W12 cervical keratinocyte model to examine the range of HPV16 integration sites that occur before integrant selection during cervical neoplastic progression. W12 is a nonclonal cell culture, propagated from a cervical low- grade SIL (LSIL) that arose after natural cervical infection with HPV16, which represents a unique system for studying early events in HPV-associated carcinogenesis (14). At early passages, Southern blotting reveals f100 HPV16 episomes per cell with no detectable Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Nicholas Coleman, Medical Research Council-Cancer Cell Unit, Medical Research Council/Hutchison Research Center, Hills Road, Cambridge, CB2 0XZ, United Kingdom. Phone: 44-1223-763285; Fax: 44-1223-763284; E-mail: nc109@cam.ac.uk. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-1741 www.aacrjournals.org 8249 Cancer Res 2008; 68: (20). October 15, 2008 Research Article