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Cancer Research 68, 8249, October 15, 2008. doi: 10.1158/0008-5472.CAN-08-1741
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions

Keltie L. Dall1, Cinzia G. Scarpini1, Ian Roberts1, David M. Winder1, Margaret A. Stanley2, Balaji Muralidhar1, M. Trent Herdman1, Mark R. Pett1 and Nicholas Coleman1,2

1 Medical Research Council-Cancer Cell Unit, Hutchison/Medical Research Council Research Center; 2 Department of Pathology, University of Cambridge, Cambridge, United Kingdom

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{at}cam.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
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 insertion 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 commonly in these regions, regardless of selective pressure. The cancer-associated genes were generally a considerable distance 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
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 intraepithelial 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 process 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 chromosomes 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 common fragile sites (CFS), specific chromosomal loci that are particularly prone to forming DSBs (8, 9), with around 50% of selected HPV16 and HPV18 integration sites being in the same chromosomal 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 addition, 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 ~100 HPV16 episomes per cell with no detectable integrants, and the cells recapitulate an LSIL in organotypic tissue culture. Long-term in vitro culture series are characterized by spontaneous clearance of episomes and the emergence of cells containing integrated HPV16, with different integrants being selected in different culture series. These changes are associated with the development of high-level genomic instability and phenotypic progression through high-grade SIL to SCC (15).

We recently showed that early passage W12 cells also contain latent integrants that are repressed by episomally derived E2 protein (16). After episome clearance by mechanisms such as induction of IFN-stimulated genes, cells containing only episomes undergo apoptosis whereas those containing latent integration events emerge by virtue of retention of E6 and E7 expression (17). This creates a competitive environment in which the integration event(s) conferring the strongest growth advantage comes to dominate the population. We reasoned that within a noncompetitive environment, wherein E2 is retained, a range of integrants would be present, regardless of whether or not they confer a selective growth advantage after episome loss. Such integrants would represent those that occur naturally after HPV16 infection, as they arise in a background of episomally expressed E6 and E7, rather than in a setting where chromosome breaks are chemically induced or occur at a high spontaneous rate (7).

To characterize the range of naturally occurring HPV16 integration sites that exist in a noncompetitive environment, we isolated single-cell clones from an early point of a nonclonal W12 culture series (W12 Series 2; ref. 16) and observed 24 different integration sites across the genome. We undertook high-resolution mapping of the integration sites, which we performed directly after clone isolation to minimize any confounding effects of integrant-associated genomic instability. Our data provide detailed information concerning the relationships between naturally occurring HPV16 integrants and the host genome.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture and single-cell cloning. The starting population from which clones were isolated was nonclonal W12 Series 2 at passage 12 or 13 (W12Ser2p12 or W12Ser2p13), which is diploid and harbors ~100 HPV16 episomes per cell, with no integrated HPV16 DNA detectable by Southern blot (16, 18). During previous long-term passage of this nonclonal population, cells with HPV16 integrated at 8q24.21 emerged and were selected by passage 24 (this integrant is, henceforth, called the "selected 8q24.21 integrant").

W12 cells were propagated in monolayer culture, as described in detail elsewhere(14, 19). The cloning strategy is summarized in Fig. 1A . For single-cell cloning, cells were seeded on 96-well plates at a density of 0.2 cells per well on a layer of lethally irradiated mouse 3T3J2 fibroblast feeder cells (density 1 x 105 cells/cm2). Colonies that arose were expanded for ~30 population doublings in total from the initial single cell of origin and DNA then extracted for analysis of HPV16 physical state. In all but one experiment, feeder cells were maintained for all ~30 population doublings. The exception was an initial training round of cloning (see Results), in which feeder cells were allowed to diminish over the first ~15 population doublings (to test the hypothesis that this would encourage episome loss), after which they were reintroduced for ~15 population doublings to encourage growth (Fig. 1A). The clones isolated from this round were named A(f)-Q(f) after isolation, with the suffix "f" to indicate that their expansion had included feeder cell support and the parentheses to denote that feeders had not been present throughout. In the four subsequent cloning rounds, we used the suffix f without parentheses, as feeders were present for the whole expansion phase. Clones from round 2 were named Xf-Zf and A2f-U2F, whereas clones from rounds 3, 4, and 5 were named A3f-J3f, A4f-F4f, and A5f-C5f, respectively.


Figure 1
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  		Figure 1. Summary of W12 cell cloning strategy and HPV16 integration sites identified. A, summarizes the cloning strategy used in the initial training experiment (left stem) and subsequent cloning rounds (right stem). B, summarizes the routes by which the 24 different HPV16 integration sites were identified. The preliminary cloning experiments referred to on the right of this panel were separate from those shown in A but used the method of single-cell cloning from W12Ser2p12 shown in the right stem of A.

 
In all cases, cells were next transferred to feeder-free conditions to encourage episomal loss or further loss in the case of the initial training experiment (Fig. 1A). After four passages (~25 population doublings) without feeder support, genomic DNA (gDNA) was extracted for analysis of HPV16 physical state and integration site. From this point, the cells were called clones A-Q, etc., without a suffix. Finally, total RNA and protein were extracted for use in viral physical state assessment and quantification of host gene expression. To establish culture conditions more representative of the in vivo environment before RNA/protein isolation, cells were returned to growth with feeder support for a single passage (about six population doublings; Fig. 1A).

Normal ectocervical keratinocytes were obtained after hysterectomy for disease unrelated to the cervix and cultured as described in detail elsewhere (19). Other cells used were the cervical carcinoma lines SiHa (HPV16-positive), C33A (HPV-negative), and HeLa (HPV18-positive), the Burkitt's lymphoma line BJAB, and the alveolar rhabdomyosarcoma line SJRH30. All were obtained from American Type Culture Collection and grown as recommended.

Southern analysis of HPV16. Southern blotting was performed as described in detail elsewhere (18). A total of 5 to 10 µg of gDNA was restriction enzyme digested, electrophoresed through a 1% agarose gel, and transferred to a Hybond-N+ nylon membrane for subsequent hybridization. The restriction enzymes used were HindIII, BamHI, EcoRI, and PstI. Probe was prepared by excision of full-length HPV16 DNA from the pspHPV16 plasmid (14), followed by labeling with [{alpha}-32P]dCTP by random priming.

Restriction site PCR. Restriction site PCR (RS-PCR) enables amplification of unknown nucleotide sequences adjacent to known nucleotide sequences (20). The use of RS-PCR to generate PCR products spanning HR-HPV host junction fragments in cervical carcinomas has been described previously (11). A total of 100 ng gDNA from each clone was amplified with various combinations of eight HPV16-specific primers and six restriction site oligonucleotide primers, using low-stringency cycling conditions followed by high-stringency nested PCR. PCR products were then sequenced using the appropriate HPV16-specific sequencing primers. All primers and PCR conditions were as described previously (11).

Amplification of papillomavirus oncogene transcripts. Cells were lysed in situ by application of Bio-RNA Xcell2 solution (BioGene), and total RNA extracts were prepared according to the manufacturer's instructions. Contaminating DNA was removed using Turbo DNA-free (Ambion). Amplification of papillomavirus oncogene transcripts (APOT) was performed as described previously using the same primers and PCR cycling conditions (21). Briefly, 1 µg total RNA was reverse transcribed and then used as a template for low-stringency nested PCR. Products were analyzed by gel separation, gel excision, and purification, followed by direct sequencing (Medical Research Council Geneservice Ltd.).

PCR screening for integration sites and quantification of host RNA and protein levels. PCR screening for integration sites detected by RS-PCR was applied to individual clones, as well as to the starting W12Ser2p12 nonclonal population. Levels of host mRNA and protein were determined by quantitative PCR and quantitative Western blotting, respectively. For full details of these techniques, refer to Supplementary Methods.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Isolation of HPV Integrants in Noncompetitive Conditions
To assess the range of naturally occurring HPV16 integration events in low-passage W12Ser2, we performed limiting dilution cloning to sample single cells within the population. We reasoned that the integrants present would coexist with episomes expressing inhibitory E2.

Initial cloning round. In the initial training experiment (Fig. 1A) after cloning on feeder cells, we deliberately allowed the feeder layer to diminish to test the hypothesis that this would encourage loss of episomes through a stress response (17). We predicted that episome clearance would induce apoptosis of clones that contained episomes only and allow emergence of clones containing integrants (17) whether or not the integrants would confer a selective advantage in a mixed population. Colonies that emerged using this strategy (after ~15 population doublings from the originating cell) were expanded for a further ~15 population doublings (using feeder cells to encourage growth; Fig. 1A) and then screened by Southern blot to assess HPV16 physical state. Consistent with our predictions, episomes were maintained in only 5 of the 17 clones (29%) isolated in this round [named A(f)-Q(f)]. Moreover, 11 of the 12 clones with episome loss showed the same banding pattern as the selected 8q24.21 integrant (i.e., the integrant that emerged during long-term passage of nonclonal W12Ser2).

All clones were further expanded in feeder-free conditions for ~25 population doublings, after which they were renamed clones A-Q (Fig. 1A). This step induced episomal loss in the five clones that had retained episomes. All 17 clones then underwent Southern blotting and/or sequence analysis using RS-PCR. The 8q24.21 integrant was seen in 14 clones overall, two of which contained a second integrant; at 4q13.3 in clone F and 12q14.3 in clone Q. The remaining three clones each contained an integrant at a different site, namely 3q24, 4q21.23, and 4q35.2. Subcloning of clones F and Q showed that the two detected integrants coexisted in the same cells rather than being present in different subpopulations (data not shown).

Based on RS-PCR data, we designed PCR primers to amplify the host-virus junctions identified. Whereas the selected 8q24.21 integrant and the 4q35.2 integrant were detectable in the starting W12 Series 2 population (and were therefore latent integrants), the other integrants were not (Supplementary Fig. S1). Although we could not exclude that these other integrants were present in an extremely small number of cells in the starting population, we considered it more likely that the relevant integration events occurred de novo after cell cloning in a background of E2-expressing episomes, as has been observed previously in W12 (6).

Subsequent cloning rounds. Based on these observations, further revised cloning rounds were designed to encourage the clonal expansion of cells containing episomes. We predicted that this would allow further de novo integration events to occur in a noncompetitive environment, as well as enable isolation of any other latent integration sites present in the starting population. In our revised protocol, the feeder cell layer was maintained throughout the cloning and expansion phase (Fig. 1A). In four cloning rounds performed (using 2,208 wells, with ~442 cells seeded), W12 grown in feeders showed an overall colony forming efficiency of 10.5%, a value similar to that of nonclonal early passage W12 when seeded at standard density (15), supporting the notion that the feeder cell layer did not exert significant cell stress. Isolation of integration sites was then achieved by feeder-free culture to encourage episome loss, as before.

In these cloning rounds, 33 of the 42 isolated clones (79%) retained episomes before growth in feeder-free conditions (Fig. 2A ). After removal of feeders, only 11 of 42 clones (26%) contained the 8q24.21 integrant, whereas the majority of the other clones contained a single, different integration site. Given that cells containing these different sites were able to grow in feeder-free conditions, we concluded that they had the potential to be isolated during the initial cloning round had they been present in the starting population. The increase in the number of different isolated integration sites (relative to the 8q24.21 integrant), therefore, suggested that the subsequent cloning rounds allowed the development and isolation of naturally occurring integration events.


Figure 2
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  		Figure 2. Southern analysis of HPV16 physical state in W12 clones. The panels show representative blots after HindIII digests. Red arrows, sizes of the fragments expected after digestion of HPV16 episomes; blue arrows, fragments seen after digestion of clones with HPV16 integrated at the 8q24.21 common integration site. Molecular weight markers (kb) are shown on the left of each blot. The control samples were W12Ser2p11, in which only HPV16 episomes were detectable; W12Ser1p25, which contained HPV16 integrated at 5p15 and had cleared all episomes (15); and the cervical SCC cell line SiHa, which contained HPV16 integrated at 13q21 and no episomes. A, clones after expansion on a well-maintained feeder layer before transfer to feeder-free conditions. Most retain episomes, exceptions being clones P2f and K2f. The 8-kb band (third red arrow from top) is due to the presence of nicked (linearized) episomes, which vary in their representation in different clones. B and C, clones with unique integrant bands identified after transfer to feeder-free conditions. Note that clone O2f is included in C to show the bands produced by episomal HPV16. D, clones in which the same host-viral junction (at 128,476,710 bp of 8q24.21 adjoined to 1,757 bp in HPV16) was isolated by RS-PCR. The different banding patterns indicate local rearrangement at the site of integration.

 
Summary of Clones Derived
In total, 59 clones were isolated from the five limiting dilution cloning rounds combined (Fig. 1B). Of these, 55 cleared episomes and showed virus-host junction fragments on Southern analysis. Interestingly, four clones retained episomes, suggesting that they were resistant to stress induced by feeder-free conditions. Using a variety of techniques (see Materials and Methods), we identified HPV16 integration sites in 50 of the 55 clones that cleared episomes, with some sites being confirmed by more than one method (Fig. 1B). The integration site of five clones could not be found by any of the techniques applied. Two clones (F and Q) contained two integration sites on different chromosomes, giving a total of 52 integration sites found. The integration sites were initially identified by RS-PCR (n = 23), APOT (n = 6), PCR screening for integration sites identified by the other approaches (n = 6), and Southern blotting using a panel of four restriction enzymes (n = 17). In the latter technique, the integration site was identified based on identical banding patterns to other clones where host-virus junction sequence information was available.

An additional three clones that had been isolated in preliminary experiments (separate to those described here but using the same method of single-cell cloning from W12Ser2p12) were added to the analysis at this stage (Fig. 1B). These clones were labeled clone 1, clone 3, and clone 5. The integration site was identified in each clone using RS-PCR. Finally, an integration site was identified by RS-PCR screening in one of the four clones that failed to clear episomes, clone B5, and this was also added to the analysis, giving a total of 56 naturally occurring HPV16 integration sites, isolated in a noncompetitive environment (Fig. 1B).

From this final panel, we identified 24 different integration sites (Table 1 and Fig. 2B and C, for example). Details of the junctional sequence used to identify each site by BLAST searching are given as supplementary data. Two of the 24 sites were seen in more than two clones (i.e., were "common" integration sites). Both of these were shown by RS-PCR to have been latent integrants in the starting W12Ser2p12 population (see above), suggesting that they were isolated at high frequency by virtue of being available for clonal expansion in feeder-free conditions rather than representing discrete de novo integration events. The first of these sites was at 8q24.21, the same as the selected 8q24.21 integrant that emerged in long-term nonclonal culture of the starting population; 25 clones had this integration site (including clones B, F, and Q in Table 1). These represented 18 clones that gave an identical pattern of bands on Southern blot, as well as seven further clones that gave unique bands, indicating that rearrangement had occurred at the site of integration (Fig. 2D). The second common integration site was at 4q35.2. All eight clones with this integration site gave unique bands on Southern blot, indicating a different pattern of rearrangement in each clone. The representative clone in Table 1 with this integration site is clone J.


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  		Table 1. Summary of the 24 different HPV16 integrants isolated from W12

 
In several clones, we identified two host-virus junctions within very close proximity. The available evidence in these cases was most consistent with a single integration event with local rearrangement. Firstly, clone J contained two sites of integration at 4q35.2, one of which showed a complex pattern of host and viral sequences in different orientations (Fig. 3 ). Importantly, identical breakpoints in host and viral DNA were seen at both integration sites. Secondly, clone B contained the 8q24.21 common integration site and a second junction 418 bp further upstream in 8q24.21. The banding pattern of clone B on Southern blotting was identical to that of other clones containing the 8q24.21 common integrant, suggesting that both host-virus junctions were related to a single integration event. Thirdly, clone H2 contained two host-viral junctions at 17q12 that were separated by 1,170 kb. We cannot exclude the possibility that two separate integration events occurred in this case, although the close proximity of the two junctions in a single clone is more likely to represent local rearrangement similar to that in clones J and B. In our further analysis, clone H2, as well as clones J and B, was regarded as containing a single integration event with local rearrangement.


Figure 3
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  		Figure 3. Schematics showing host-viral junctions at the different integration sites. In all schematics, host chromosomal DNA is shown in blue, orientated with the forward strand running from left to right. Integrated HPV16 DNA is shown in yellow, with the viral oncogenes and URR highlighted in orange, and the direction of transcription from the viral promoter shown by an arrow from the URR. The location of the viral breakpoint in base pairs is given above the junction, whereas the cellular DNA breakpoint in base pairs is given below the junction. Sequence overlap or unrecognized sequence at the host-viral junction is indicated where appropriate. Where two junctions were identified in a clone, the second is referred to using the suffix ".2". Note that for each integration site, only one host-virus junction was determined by RS-PCR, and although other viral genes not involved in the junction are shown in the schematics, they may have been partially or completely deleted. The last four schematics show viral-host fusion transcripts that were isolated by APOT and used to identify the integration sites in clones Z, E3, O2, and G2. Each virus-host fusion transcript is depicted in the direction of transcription from the viral promoter, i.e., 5' to 3' left to right, as depicted by the arrow. The viral and host portions of each transcript are shown by a solid black line and striped line, respectively. Note that the size of the fusion transcripts was not determined, and all are depicted at the same arbitrary length.

 
The 24 different integration sites were spread relatively evenly throughout the genome (Supplementary Fig. S2), although two clusters were apparent. The first represented a 600-bp region of 8q24.21, which encompassed each single integration site detected in clones I2, F3, and I3 (which carried the same integration site as F3), as well as the 8q24.21 common integration site (for which we observed evidence of local rearrangement in clone B; Fig. 4A ). As clones I2 and F3/I3 contained discrete single integration sites, with distinct virus and host breakpoints, we considered it unlikely that such sites were derivatives of the 8q24.21 common integrant. The second integrant cluster was at 12q14.3, where there were two integration events 3 kb apart. As for the 8q24.21 cluster, each 12q14.3 integrant was seen in isolation in an individual clone (clones Q and 5). Moreover, the viral genome was integrated in opposite orientations in the two clones with different viral breakpoints. These observations again suggested that the 12q14.3 junctions most likely represented different integration events within the same genomic region. We conclude that 8q24.21, and perhaps 12q14.3, may represent particularly susceptible sites for HR-HPV integration.


Figure 4
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  		Figure 4. Detailed mapping of the 8q24.21 integrants and HPV16 breakpoints. A, the integration site at 8q24.21 is flanked by two CFSs (FRA8C and FRA8D; top). The Ensembl view of the region is shown in the middle panel, whereas the schematic in the bottom panel depicts the 600-bp region containing the cluster of integration sites. In this, the blue line represents cellular DNA and the red lines HPV16 DNA (not to scale), with the orientation of the viral promoter shown by the black arrows. The red numbers indicate viral breakpoints. Note that POU5F1P1, which maps to 8q24.21, is not shown by Ensembl. Clone B contained the common integrant, as well as the specific junction also shown. B, top, schematic representation of the linearized HPV16 genome, with the viral base pairs indicated below. Locations of the viral breakpoints in the 20 integrants mapped at the DNA level are indicated by the vertical blue arrows. Bottom, a detailed view of the E2/E4 region, with the base pair location for each viral breakpoint shown above the blue arrow.

 
Clones F and Q had two integration sites in different chromosomes, as has been reported in a minority of cervical carcinoma samples (22–24). Several chromosome bands or sub-bands containing integration sites corresponded to those reported in clinical carcinoma samples (2p24.1, 3q28, 4q13.3, 4q31.21, 8q24.21, 10q22, and 17q12; refs. 10, 22, 25), which is of interest, as there are relatively few examples of recurrent sites of HPV16 integration in the literature.

Association of Integration Sites with CFSs and Other Regions of DSBs
Of the 24 different unselected integrants identified, 12 (50%) occurred within a chromosomal band containing a CFS, based on the 87 CFS listed by National Center for Biotechnology Information.3 However, integration was not seen in bands containing FRA3B and FRA16D, the two most commonly expressed CFSs (26). Moreover, 9 of the 12 integrants were actually in different chromosomal sub-bands to the respective CFS, at a distance of 335 kb to 5.7 Mb from the sub-band reported to contain the CFS (Table 1). Only one integration event, in clone R2, occurred in the same sub-band as a CFS; at 10q22.1, although it should be noted that this is a large sub-band of 5.8 Mb. In another two clones, clones O2 and Q2, the relevant CFS was only mapped at the chromosomal band level, so there was insufficient information to determine the proximity of colocalization of CFS and integration site. In clone O2, the integrant was at 19q13.31, although the CFS is mapped only to 19q13, which covers 24.7 Mb. In clone Q2, HPV16 was integrated in 1q44, which is 5.5 Mb in length and not divided into sub-bands. FRA1I is also mapped to 1q44, but no further location information was available, and FRA1I may not cover the full 5.5 Mb.

We found no association between unselected HPV16 integration sites and other regions prone to forming DSBs, namely AT-rich regions and matrix attachment regions (see Supplementary Information and Supplementary Fig. S3).

Viral Breakpoints and Host-Viral Junctional Sequence
The HPV16 breakpoints determined from the RS-PCR and APOT sequences were all outside the E6 and E7 open reading frames (ORF) and the upstream regulatory region (URR; Fig. 4B). Of the 20 different integration sites where viral breakpoints were resolved at the DNA level (i.e., rather than by APOT alone), eight occurred within the E2/E4 ORFs, as is commonly seen in carcinomas in vivo (23), whereas a further eight occurred within E1, consistent with deletion of the downstream E2 gene. In the remaining four integrants, the breakpoints identified were within the L1/L2 region, most likely representing the upstream host-viral junctions, and it was not possible to determine whether or not E2 was deleted.

The 24 different integrants could be divided into three groups of approximately equal size, according to the sequence at the host-virus junction. In the first group (n = 8), the host sequence directly adjoined the viral sequence with no homology, suggesting a role for nonhomologous end joining (NHEJ) in the integration process (Table 2 ). In the second group (n = 7), regions of microhomology (1–10 bp) were seen between the host and viral sequences, suggesting microhomology-mediated NHEJ (6, 27, 28), whereas in the third group (n = 9), stretches of unrecognized "orphan" DNA (1–36 bp) were present between the host and viral sequences.


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  		Table 2. Host-viral junctional sequences in the HPV16 integrants isolated

 
Significance of Host Genes at or near Integration Sites
Using the Ensembl genome browser, we determined the genomic distance between each integration site and the nearest known cancer-associated host gene4 and whether or not the host gene was orientated in the direction of transcription from the viral promoter (Table 1). We also determined the nearest host gene [coding gene, pseudogene, or microRNAs (miR)] to each integration site in the direction of transcription using the Ensembl BIOMART function5 and the miR-base website.6 Twenty of the 24 different integration events (83%) occurred within the same chromosomal band as a cancer-associated gene, which included C-MYC, TP73L(TP63), MAPK10, MYCN, RASSF6, and MDM2 (Table 1). However, the cancer-associated genes were generally a considerable distance from the integration site (median, 2,379 kb; range, 0–12,343 kb) and often in the opposite orientation to that of transcription from the viral promoter. For example, whereas integrants were seen in chromosome bands containing C-MYC and MYCN (genes previously implicated in HPV-related insertional mutagenesis; refs. 12, 29), the integration cluster at 8q24.21 was 330 kb from C-MYC and the integrant in clone 3 was 4,814 kb from MYCN. On the other hand, some integrants were present within intronic sequences—in clone H (MAPK10), clone N (SLC9A9), clone F (RASSF6), clone C5 (TP73L), clone S2 (TTC28), and clone H2 (PLXDC1).

Of the 24 different integration events, 14 were in the same chromosomal band as an miR gene. Five integrants were within 2.5 Mb of the miR gene (30), with a median separation for these five integrants of 1,205 kb (range, 11.9–2,347 kb; Table 1 and Supplementary Table S2).

Expression of Candidate Host Genes
To investigate further the possibility of insertional mutagenesis associated with HPV16, expression levels of eight candidate coding genes and one pseudogene were investigated in clones with HPV16 integrated in the same chromosomal band as the candidate gene, relative to clones with integration sites at other chromosomal loci and normal ectocervical keratinocytes. It was not possible to compare expression levels with those at the stage when the integrants coexisted with episomes (i.e., before growth in feeder-free conditions), as inadequate amounts of RNA were available from this point in the cloning process. No evidence for increased expression of c-Myc was found at the RNA or protein level in any of the clones with integration at 8q24.21 nor in the 8q24.21 selected integrant from nonclonal culture of W12 Series 2 (Supplementary Fig. S4). These clones also showed no evidence of overexpression of other candidate genes at 8q24: FAM84B, Argonaute 2, and the cancer-associated pseudogene POU5F1P1/Oct4 (ref. 31; Supplementary Fig. S5). In addition, there was no evidence of altered expression of candidate genes at other integration sites, namely MYCN, MDM2, MAPK10, SLC9A9, and RASSF6 (Table 1 and Supplementary Fig. S5), despite the last three showing HPV16 integration within introns.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The HPV16 integration sites investigated in this study can be considered to represent natural events, because they occurred in naturally infected cervical keratinocytes containing viral episomes at copy numbers seen in basal squamous cells in productive infections in vivo (32). The viral oncogenes E6 and E7 would therefore be expected to be present at physiologic levels in episome-containing cells. In this regard, the W12 system compares favorably with previous studies that used cells in which DSBs were induced or were present at a high spontaneous level (7). Furthermore, HR-HPV integration in cells containing episomes expressing the inhibitory E2 protein represents a noncompetitive environment, from which integration events that would be out-competed in a mixed population can be isolated and studied.

Our data suggest that many of the integrants identified represented de novo events, indicating that integration can occur at any time during the period of HR-HPV episome maintenance when there is no quantitative deregulation of viral oncogene expression. The identification of clones that contained both the 8q24.21 common integrant (which was present within the starting population), and another integrant showed that, providing episomes are maintained, integration events can occur more than once within a single cell. Persistent infections with HR-HPV episomes in vivo will therefore increase the probability of integration events, which would initially be latent and may or may not be selected if episome loss is subsequently induced. These data provide biological support to the epidemiologic observation that persistent infection with HR-HPV is a major risk factor for cervical neoplastic progression (33).

Our evidence that 50% of the naturally occurring HPV16 integration events isolated from W12 were in the same chromosomal band as an CFS is very similar to observations made for HR-HPV in integrants selected in vivo (5, 7). This suggests that HR-HPV integration occurs close to CFSs because these regions are especially permissive, rather than conferring a selective advantage on the host cell. However, further questions arise from our findings. Firstly, although the unselected integration sites in W12 are associated with CFSs, they do not accurately colocalize with them, based on available data. This may reflect the fact that CFSs are mapped at a low resolution. Of the few CFS that have been mapped closely, many are under 1 Mb in length (34–40), whereas chromosomal bands can cover 30 Mb and sub-bands over 5 Mb. To add further complexity, CFSs recently mapped at high resolution were found to be larger than previously thought and to extend beyond the chromosomal bands to which they were initially mapped (22, 26, 41, 42). Secondly, we saw no colocalization of HPV16 integrants with FRA3B and FRA16D, the most commonly expressed CFSs. Although individuals are thought to express only 7 to 20 CFSs after CFS induction in vitro, FRA3B and FRA16D seem to be expressed in all individuals (26) and would be expected to be available for integration in W12.

The variability of the host-viral junctional sequences that we observed suggests that more than one mechanism of HPV integration may occur and that regions of microhomology or even orphan DNA sequences may be required to ligate the host and viral sequences in some cases. Interestingly, while a similar analysis performed on 16 selected integrants in HPV16-associated and HPV18-associated anogenital neoplasia found the same three types of junction (43), the relative frequencies differed, with 2 incidences of clean joins, 2 with orphan DNA (4–9 bp), and 12 with regions of microhomology (1–6 bp). Together, these data raise the possibility that microhomology-mediated integration events may confer a selective advantage in mixed populations. The viral breakpoints that we observed were all outside E6, E7, and the URR, supporting the view that retention of viral oncogene expression is a prerequisite for growth of HPV16-containing cells. Assuming that HPV16 episomal DNA is opened at random during the process of integration, the E6/E7/URR region would be predicted to be disrupted in some integration events. However, it is unlikely that such integrants would have been detected using our cloning strategy, as they would not have shown viral oncogene expression after episome clearance and would therefore have been incapable of sustained growth.

Our findings indicate that local rearrangements occur frequently after natural HPV16 integration. They also seem to occur early, as we analyzed integration sites after only ~25 population doublings in feeder-free conditions. Local rearrangement has been described in previous cell line studies (29, 44, 45), but it is likely to have been underreported, particularly in clinical samples, as PCR-based methods of integration site analysis would not identify it. Interestingly, it has recently been shown that latent integrants may be able to induce genomic instability in the locality of the integration site, despite no quantitative deregulation of E6 and E7, as expression of E1 and E2 proteins in cells containing integrated HR-HPV origins can induce genomic rearrangements of flanking cellular sequences (45). The presence of a CFS near an integrant may also contribute to local rearrangement, although this seems not to be essential, as 4q35.2 is not associated with a CFS.

We found no evidence to suggest that HPV16-associated insertional mutagenesis occurs frequently. Expression of strong candidate genes (including C-MYC and MYCN) was not affected by integration in the same chromosomal band, including integration within introns (MAPK10, SLC9A9, RASSF6) or a relatively modest 20.1 kb upstream in the same orientation (POU5F1P1). Our conclusions are supported by a paucity of published data showing altered levels of host gene expression related to HR-HPV integration. Despite evidence for preferential integration of HPV18 within 8q24 in selected cervical carcinoma cells (46), there is no convincing functional evidence that C-MYC transcription is increased in this setting (4) nor in cervical carcinoma samples compared with normal cervix (47). An alternative explanation may be required to explain the observation that HR-HPV integration occurs close to a range of cancer-associated genes. One possibility is that these are simply transcriptionally active and therefore open regions, which are more prone to DSBs. This would be consistent with the overall notion that HR-HPV integration occurs within relatively accessible regions of the genome.

It may nevertheless be the case that HPV16 integration affects transcription of alternative genes to the candidates chosen in the present study. The genes closest to the integrated HPV16 genomes in the direction of viral transcription included pseudogenes and genes of unknown function (Table 1). Furthermore, it is feasible that insertional mutagenesis could involve looping of DNA, affecting transcription of genes that are not close to the integration site in base-pair distance but are brought into close proximity by DNA folding. However, in the absence of a detailed understanding of nuclear DNA organization and adequate functional investigations of HR-HPV–associated insertional mutagenesis, it is not possible to predict the likelihood or extent of these effects.

We conclude that the profile of naturally occurring integrants that can be isolated under noncompetitive conditions from cervical keratinocytes infected with HPV16 bears close similarity to the integrants that are selected in cervical SCC in vivo. The integration sites observed seem to be particularly suitable for the process of integration, potentially through an increased density and/or likelihood of DSBs. We observed a frequent association between integrants and genomic regions containing CFSs, although detailed mapping did not indicate colocalization. The panel of clones that we have generated will provide a valuable isogenic system for future experiments examining virus and host factors that confer a selective advantage in a mixed population.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Medical Research Council and Cancer Research UK program grants.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank David I. Smith for helpful discussions and for advice on the RS-PCR technique and Elizabeth Gray and Nicolas Wentzensen for advice and assistance with the APOT technique.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

3 http://www.ncbi.nlm.nih.gov/ Back

4 www.sanger.ac.uk/genetics/CGP/Census/ Back

5 www.ensembl.org/ Back

6 www.microrna.sanger.ac.uk/sequences/ Back

Received 5/ 8/08. Revised 7/11/08. Accepted 7/14/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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