Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma

To investigate whether integration of HPV DNA in cervical carcinoma is responsible for structural alterations of the host genome at the insertion site, a series of 34 primary cervical carcinomas and eight cervical cancer‐derived cell lines were analysed. DNA copy number profiles were assessed using the Affymetrix GeneChip Human Mapping 250K Sty array. HPV 16, 18 or 45 integration sites were determined using the DIPS‐PCR technique. The genome status at integration sites was classified as follows: no change, amplification, transition normal/gain, normal/loss or gain/LOH. A single HPV integration site was found in 34 cases; two sites were found in seven cases; and three sites in one case (51 sites). Comparison between integration sites and DNA copy number profiles showed that the genome status was altered at 17/51 (33%) integration sites, corresponding to 16/42 cases (38%). Alterations detected were amplification in nine cases, transition normal/loss in four cases, normal/gain in three cases, and gain/LOH in one case. A highly significant association was found between genomic rearrangement and integration of HPV DNA (p < 10^?10). Activation of the replication origin located in viral integrated sequences in a cell line derived from one of the primary cervical carcinomas induced an increase of the amplification level of both viral and cellular DNA sequences flanking the integration locus. This mechanism may be implicated in the triggering of genome amplification at the HPV integration site in cervical carcinoma. Structural alterations of the host genome are frequently observed at the integration site of HPV DNA in cervical cancer and may act in oncogenesis. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Preferential sites for the integration and disruption of human papillomavirus 16 in cervical lesions

BackgroundPersistent infection with high-risk human papillomavirus (HPV) is necessary to cause cervical cancer, and integrating viral DNA into the host genome may contribute to the process of carcinogenesis. The underlying mechanisms are still unclear.ObjectiveIn this study, we aimed to investigate the distribution of HPV 16 integration in the host genome and disrupted sites in the viral genome.Study designThe physical status of HPV 16 genomes in 46 cervical precancerous and cancerous lesions was determined via ligation-mediated chain reaction (DIPS) using 15 previously published primer sets and 12 newly designed primer sets.ResultsA total of 60 viral-cellular junctions were identified in 31 of 46 specimens, and over 80% of the integration sites in the human genome were located in regions of repetitive elements. The proportion of LSIL-, HSIL-, and SCC-containing integration sites near cancer-relevant genes was 10%, 18.8%, and 33.3%, respectively. The frequency of viral gene disruption was significantly higher (P < 0.05) in the L2 gene than in other regions of the viral genome.ConclusionThere are sites of preferential HPV 16 integration. The integration sites tend to be located in repetitive regions of the host genome, and some sites are found near cancer-relevant genes. In addition, the HPV 16 genome is more likely to be disrupted in the L2 gene locus.     

Integration of human papillomavirus type 16 into cellular DNA of cervical carcinoma: preferential deletion of the E2 gene and invariable retention of the long control region and the E6/E7 open reading frames

The integration patterns of human papillomavirus (HPV) type 16 in the cellular DNA of six cervical carcinoma samples were analyzed by the Southern blot procedure. None of the HPV integrants retained the entire viral genome. Double HPV integration was found in one case while all other cases were single integrants. In some samples, internal deletion and selective amplification of the viral sequences were observed. On integration, the E2 open reading frame (ORF) was invariably lost but the E6/E7 ORFs and the long control region of the HPV-16 genome were retained in all seven integrations analyzed and may play a role in cellular transformation and/or maintenance of the transformed phenotype.     

Chromosomal location of endogenous geminivirus-related DNA sequences inNicotiana tabacum L.

TheN. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivrus infection, duringNicotiana evolution. Each GRD repeat carries sequences similar to the geminiviralAL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus thecis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescencein situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA fromN. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool.     

The role of viral integration in the development of cervical cancer

The development of invasive cervical cancer is associated with human papillomavirus (HPV) infection and subsequent integration into the host epithelium. More than 99% of cervical cancers contain HPV sequences, and many of these contain a truncated HPV genome integrated into a single position within the host genome. Studies examining the role of viral integration in cervical cancer development have found that the sites of integration appear randomly distributed throughout the genome. This, and the observation that it frequently takes years after HPV infection for cervical cancer to develop, has led to the current paradigm that the site of HPV integrations is unimportant to the invasive cervical cancer that eventually develops. In our previous studies of HPV16 and HPV18 integration in cervical cancers, we also found integrations throughout the genome, but observed as well that more than half of the integrations occurred within common fragile site regions. To determine if HPV integration might play an important role in cervical cancer, we conducted two complementary studies. We first localized 40 new HPV16 integration sites from cervical tumors from women in Hong Kong; this, together with previous integration studies, provided a better picture of the distribution of integration sites throughout the genome. We then analyzed the sites of viral integration in an in vitro model of HPV integration. By comparing the sites of HPV integration in vivo (in multiple primary cervical tumors) to those obtained in vitro, the data can help to determine if HPV integrations observed in vivo are the result of random and nonselected integrations.     

FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites

The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes for the formation of gaps and breaks when DNA replication is perturbed by aphidicolin or folate stress. Although rare fragile sites are known to arise through the expansion of CCG repeats, the mechanism responsible for common fragile sites is unknown. Beyond being a basic component of chromosome structure, no biological effects of common fragile sites have been convincingly shown, although suggestions have been made that breakage and recombination at these sites may sometimes be mechanistically involved in deletions observed in many tumors and in constitutional deletions. In an observation related to the high rate of recombination at fragile sites, a number of studies have shown a statistical association between the integration of transforming DNA viruses and chromosomal fragile sites. Using FISH analysis we recently identified a 1.3 Mb YAC spanning both FRA3B and the t(3;8) translocation associated with hereditary RCC. Here we report the further localization of FRA3B within this YAC. Using lambda subclones of the YAC as FISH probes, gaps and breaks were found to occur over a broad region of at least 50 kb. Neither CCG nor CAG repeats were found in this region suggesting a different mechanism for fragility than seen with rare fragile sites. We further show that an area of frequent gaps and breaks within FRA3B, defined by a lambda contig, coincides with a previously characterized site of HPV16 integration in a primary cervical carcinoma. The HPV16 integration event gave rise to a short chromosomal deletion limited to the local FRA3B region within 3p14.2. Interestingly, 3p14.2 lies within the smallest commonly deleted region of 3p in cervical cancers, which are often HPV16 associated. To our knowledge this is the first molecular characterization of an in vivo viral integration event within a confirmed fragile site region, supporting previous cytogenetic observations linking viral integration sites and fragile sites.      

Human papillomavirus genomes in squamous cell carcinomas of the uterine cervix

The association between invasive cervical carcinoma and human papillomavirus (HPV) has now been established beyond doubt, but this is not necessarily a direct-and-effect association. To assess the causality of HPV, we analyzed HPV genomes in squamous cell carcinomas (SCCs) [corrected] of the uterine cervix by both blot hybridization and PCR. Genital HPV sequences were found in 231 (79%) of 294 SCCs by blot hybridization with more than five copies of entire HPV genomes identified in some cases including HPV 16 (92 cases), HPV 58 (32 cases), and HPV 52 (24 cases). By PCR-direct sequence analysis in 250 of 294 SCCs, genital HPV sequences were found in 240 samples (96%). The partial L1 sequences of HPV 16 were identified in 123 cases, and those of HPVs 18 and 31 were found in 24 and 20 cases, respectively. In addition, multiple HPV types were identified in 29 (12%) of 250 SCCs, and the HPV copy number, detected by PCR only, was less than 0.05. Marked discrepancies were therefore evident between the two analytical techniques. In this report, we discuss the causality of HPV for SCC with regard to the length of the viral genome, the amount of viral DNA, and multiple HPVs in single SCCs.     

Genomic deletions and precise removal of transposable elements mediated by short identical DNA segments in primates

Insertion of transposable elements is a major cause of genomic expansion in eukaryotes. Less is understood, however, about mechanisms underlying contraction of genomes. In this study, we show that retroelements can, in rare cases, be precisely deleted from primate genomes, most likely via recombination between 10- to 20-bp target site duplications (TSDs) flanking the retroelement. The deleted loci are indistinguishable from pre-integration sites, effectively reversing the insertion. Through human-chimpanzee-Rhesus monkey genomic comparisons, we estimate that 0.5%-1% of apparent retroelement "insertions" distinguishing humans and chimpanzees actually represent deletions. Furthermore, we demonstrate that 19% of genomic deletions of 200-500 bp that have occurred since the human-chimpanzee divergence are associated with flanking identical repeats of at least 10 bp. A large number of deletions internal to Alu elements were also found flanked by homologies. These results suggest that illegitimate recombination between short direct repeats has played a significant role in human genome evolution. Moreover, this study lends perspective to the view that insertions of retroelements represent unidirectional genetic events.     

Illegitimate recombination in plants: a model for T-DNA integration

Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA (the T-DNA) to the genome of higher plants, where it is then stably integrated. Six T-DNA inserts and their corresponding preinsertion sites were cloned from Arabidopsis thaliana and analyzed. Two T-DNA integration events from Nicotiana tabacum were included in the analysis. Nucleotide sequence comparison of plant target sites before and after T-DNA integration showed that the T-DNA usually causes only a small (13-28 bp) deletion in the plant DNA, but larger target rearrangements can occur. Short homologies between the T-DNA ends and the target sites, as well as the presence of filler sequences at the junctions, indicate that T-DNA integration is mediated by illegitimate recombination and that these processes in plants are very analogous to events in mammalian cells. We propose a model for T-DNA integration on the basis of limited base-pairing for initial synapsis, followed by DNA repair at the junctions. Variations of the model can explain the formation of filler DNA at the junctions by polymerase slipping and template switching during DNA repair synthesis and the presence of larger plant target DNA rearrangements.     

Chromosomal DNA loops may constitute basic units of the eukaryotic genome organization and evolution

In eukaryotic cell nuclei the genome is organized into large loops attached to the nuclear matrix. Rearrangements of the genome frequently occur via an illegitimate recombination between loop anchorage sites resulting in deletion or repositioning of DNA loops. The illegitimate recombination between loop anchorage sites is possibly mediated by topoisomerase II. Treatments stabilizing intermediate covalent complexes of topoisomerase II with DNA seem to increase the possibility of illegitimate recombination between loop anchorage regions. On the basis of these and some other observations we suggest that chromosomal DNA loops constitute basic units of the genome evolution, or, in other words, structural blocks of the eukaryotic genome.     

Analysis of six distinct integrated hepatitis B virus sequences cloned from the cellular DNA of a human hepatocellular carcinoma

Six distinct hepatitis B virus (HBV) integrations and the flanking cellular sequences were cloned from a hepatoma DNA preparation. None of the cloned fragments retains the entire HBV sequences but the surface antigen (HBsAg) gene and the HBV enhancer are retained in three of the six clones. The other three clones carry only short and possibly highly rearranged HBV genomic sequences and seem to contain some GC-rich clusters. Members of the repetitive Alu family are also found in the vicinity of five of the six integration regions which may have contributed to genome instability. In these six clones, the preferred integration sites are shown to lie within the single-strand region of the HBV genome. None of the clones carries in the flanking cellular sequences any of the 17 oncogenes tested, although the possibility still exists that an oncogene may be found on the side of the genome which has not been cloned. This work thus paves the way for detailed sequence analysis of virus-host junctions, for transfection studies of the HBV integration events, and for a search of genes in the flanking cellular sequences which may have been activated by the retained HBV enhancer using the clones described.     

Homologous junctions formed between a vector and human genomic repetitive line-1 elements as a result of one-sided invasion

Studies on homologous recombination in mammalian cells between an exogenous DNA molecule containing a double-strand break and a homologous genomic sequence have indicated that there were at least two distinct types of homologous recombination processes, one that involved the formation of two homologous junctions and another that involved the formation of one homologous junction and one illegitimate junction. Both types of events are produced in gene targeting experiments. We have proposed a model to account for the later process called one-sided invasion. One-sided invasion has now been reported in numerous species belonging to different phyla and appears to be a universal mechanism. It has also been observed in normal human germ cells. The role of one-side invasion is still unknown. Using a recombination assay between LINE-1 elements from the human genome and exogenous LINE-1 sequences, we have characterized the process of homologous junction formation in one-sided invasion. We found that at each of the homologous junctions, variable lengths of the vector L1 sequences had been replaced by genomic L1 sequences. We also found a homologous junction that involved three partners, suggesting that the homologous end could be released and become available for a second round of interaction.     

Characterization of genomic breakpoints in MLL and CBP in leukemia patients with t(11;16)

The recurring chromosome translocation t(11;16)(q23;p13) is detected in leukemia patients, virtually all of whom have received previous chemotherapy with topoisomerase (topo) II inhibitors. In the t(11;16), 3' CBP, on 16p13, is fused to 5' MLL, on 11q23, resulting in an MLL-CBP fusion gene that plays an important role in leukemogenesis. In this study, we cloned genomic breakpoints of the MLL and CBP genes in the t(11;16) in the SN-1 cell line and in five patients with therapy-related leukemia, all of whom had received topo II inhibitors for previous tumors. In all patients except one, both the genomic MLL-CBP and the reciprocal fusions were cloned. Genomic breakpoints in MLL occurred in the 8.3-kb breakpoint cluster region in all patients, whereas the breakpoints in CBP clustered in an 8.2-kb region of intron 3 in four patients. Genomic breakpoints in MLL occurred in intron 11 near the topo II cleavage site in the SN-1 cell line and in one patient, and they were close to LINE repetitive sequences in two other patients. In the remaining two patients, genomic breakpoints were in intron 9 in Alu repeats. Genomic breakpoints in CBP occurred in and around Alu repeats in one and two patients, respectively. In two patients, the breaks were near LINE repetitive sequences, suggesting that repetitive DNA sequences may play a role. No specific recombination motifs were identified at or near the breakpoint junctions. No topo II cleavage sites were detected in introns 2 and 3 of CBP. However, there were deletions and duplications at the breakpoints in both MLL and CBP and microhomologies or nontemplated nucleotides at most of the genomic fusion junctions, suggesting that a nonhomologous end-joining repair mechanism was involved in the t(11;16).     

Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas.

Using a procedure based on restriction enzyme cleavage, self-ligation, and inverse polymerase chain reaction (rliPCR), the authors investigated 18 cervical intraepithelial neoplasia III (CIN III) cases and 37 invasive squamous carcinomas for integration of human papillomavirus type 16 (HPV16). All eighteen CIN III cases (severe dysplasia or high-grade squamous intraepithelial lesion) were found to harbor episomal HPV, but one of the samples contained mixed episomal and integrated forms. Seventeen of 37 invasive cervical carcinoma samples were identified previously as containing the completely integrated HPV16 genome by using PCR covering the entire E1/E2 gene, and this was confirmed by rliPCR in 16 cases. One case, however, showed a low level of episomal deoxyribonucleic acid in addition to the predominant integrated form. Of the remaining 20 carcinoma samples showing episomal forms in the previous analysis, 14 were found to contain integrated forms using rliPCR, and four contained multimeric episomal forms. Thus, in total, 31 of 37 of the carcinomas (84%) showed the integrated HPV16 genome. The rliPCR product from five carcinoma cases was cloned into a plasmid vector and used as a template for "primer walking" deoxyribonucleic acid sequencing to deduce human sequences flanking the integrated HPV genome. Based on this information, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were obtained and used as probes in fluorescent in situ hybridization experiments on human metaphase chromosomes. The results of the fluorescent in situ hybridization experiments showed evidence for HPV16 integration in chromosome regions 1q25, 3q28, 6p25, 11p13, and 18q22. Sixteen carcinoma samples, containing episomal HPV16, were sequenced in the long control region. Evidence for changes in E2 binding or silencer YY1 sequences was found in only two samples.     

Mechanism and application of genetic recombination in herpesviruses

Herpes simplex virus type 1 (HSV‐1) is a ubiquitous human pathogen that latently infects sensory ganglia and encodes over 80 genes in a 152 kbp DNA genome. This well characterised virus provides a model for analysing genetic recombination in herpesviruses, a fundamental biological process by which new combinations of genetic materials are generated. The frequency of homologous recombination was estimated to be 0·0048–0·007 (0·48%–0·7%)/kb of the HSV‐1 genome, determined using physical markers. The double‐strand break repair model, the current model of homologous recombination, adequately explains L‐S inversion of herpesvirus genomes and the recombinogenicity of the a sequence. Several herpesvirus genomes, including HSV‐1 consist of a unique sequence bracketed by a pair of inverted repeat sequences. This arrangement is attributed to illegitimate recombination between molecules arranged in an inverse orientation. Junctions of unique and repeated sequences that correspond to the crossover site of illegitimate recombination are recombinogenic. Recombination is important for virus evolution, construction of mutated virus, gene therapy and vaccination in which the potential for recombination between engineered input virus and wild type virus has to be considered. Copyright © 1999 John Wiley & Sons, Ltd.