Rearrangement of both alleles of human chromosome 8 in HeLa cells, one of them as a result of papillomavirus DNA integration

Integration of papillomavirus in the genome of the host cell has been found associated with malignant cases of cervical carcinoma. To determine what role viral integration plays as part of the pathogenic mechanism resulting in a cancer cell, the structure of integrated papillomavirus DNA (human papillomavirus DNA 18) segments and its cellular flanking sequences in HeLa cells as well as the corresponding normal human allele have been characterized. All integrated viral DNA segments have the same human DNA sequences in their 5' flank. The use of human sequence flanking the viral DNA as a probe detected the presence of four different forms of this human DNA region based on restriction fragment length polymorphism. Three of these forms can be linked to integrated viral DNA from human papillomavirus 18. The remaining form could not be linked to viral DNA and did not have a germline pattern in its 5'-end suggesting that it was also structurally altered. None of the forms of the human sequence present in HeLa cells has the complete structure of the germline normal allele characterized in DNA from placenta and human fibroblasts IMR-90. This observation suggests that HeLa cells carry a structural alteration in both alleles of the same locus, one of which was caused by integration of papillomavirus DNA. This locus is located on a chromosome fragile site. These rearrangements will result in a homozygous situation which is interpreted as affecting a recessive phenotype which might be involved in some aspect of tumorigenesis.     

Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene

Transgenic animals are extensively used to model human disease. Typically, the transgene copy number is estimated, but the exact integration site and configuration of the foreign DNA remains uncharacterized. When transgenes have been closely examined, some unexpected configurations have been found. Here, we describe a method to recover transgene insertion sites and assess structural rearrangements of host and transgene DNA using microarray hybridization and targeted sequence capture. We used information about the transgene insertion site to develop a polymerase chain reaction genotyping assay to distinguish heterozygous from homozygous transgenic animals. Although we worked with a bacterial artificial chromosome transgenic mouse line, this method can be used to analyse the integration site and configuration of any foreign DNA in a sequenced genome.     

DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer

Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160 232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy—in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.     

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.     

Recombination events during integration of transfected DNA into normal human cells.

The mechanisms of recombination responsible for random integration of transfected DNA into the genome of normal human cells have been investigated by analysis of plasmid-cell DNA junctions. Cell clones containing integrated plasmid sequences were selected by morphological transformation of primary human fibroblasts after transfection with a plasmid containing simian virus 40 sequences. Nucleotide sequence analysis of the plasmid-cell DNA junctions was performed on cloned DNA fragments containing the integration sites from two of these cell clones. Polymerase chain reaction was then performed with human cell DNA from primary fibroblasts to isolate the cell DNA from the same sites before plasmid integration. Comparison of the sequences at the plasmid-cell DNA junctions with those of both the original plasmid and the cell DNA demonstrated short sequence similarities and additional nucleotides, typical of nonhomologous recombination. Evidence of short deletions in the cell DNA at the plasmid integration sites suggests that integration occurred by a mechanism similar to that used for repair of spontaneous or gamma ray-induced strand breaks. Plasmid integration occurred within nonrepetitive cell DNA with no major rearrangements, although rearrangements of the cell DNA at the integration site occurred in one of the clones after integration.     

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.     

DNA-PK and ATM are required for radiation-enhanced integration

Ionizing radiation is known to improve transfection of exogenous DNA, a process we have termed radiation-enhanced integration. Previous observations have demonstrated that Ku proteins are critical for radiation-enhanced integration. Since Ku proteins form the DNA-binding domain of DNA-PK and since DNA-PK is important in nonhomologous DNA end joining, it was hypothesized that DNA-PK function might be important for radiation-enhanced integration. The ATM protein has been shown to be important in the recognition of a variety of types of DNA damage and to associate with DNA-PK under certain conditions. It was thus hypothesized that ATM might also play a role in radiation-enhanced integration. To test these hypotheses, radiation-enhanced integration was measured in hamster cells that are defective in the catalytic subunit of DNA-PK and in human cells containing mutant ATM. Radiation-enhanced integration was not detected in any of the cell lines with mutant PRKDC (also known as DNA-PKcs), but it was present in cells of the same lineage with wild-type PRKDC. Radiation-enhanced integration was defective in cells lacking kinase activation. ATM-deficient cell lines also showed defective radiation-enhanced integration. These data demonstrate that DNA-PK and ATM must both be active for radiation-enhanced integration to be observed.     

Fidelity of Target Site Duplication and Sequence Preference during Integration of Xenotropic Murine Leukemia Virus-Related Virus

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity.     

Sites of retroviral DNA integration: From basic research to clinical applications

One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency.     

In situ Detection of Specific DNA Double Strand Breaks using Rolling Circle Amplification

We have developed a method to localize DNA double strand breaks (DSBs) insitu in cultured mammalian cells. Adenoviruses encoding Saccharomyces cerevisiae HOendonuclease and its cleavage site were used to induce site-specific DSBs. Rolling circleamplification (RCA), a sensitive method that allows the detection of single molecularevent by rapid isothermal amplification, was used to localize the broken ends in situ.Punctate RCA signals were only seen in the cells that had been infected with bothadenoviruses encoding HO endonuclease and HO cleavage site, but not in the cells mockinfectedor infected with the site or endonuclease virus only. With use of a chemicalcrosslinker, in situ RCA and immunofluorescence (IF) can be performed simultaneouslyon the same sample. This methodology provides a novel approach for investigation ofDNA recombination, DNA repair, and checkpoint controls in mammalian cells.     

The transient expression of mRNA coding for Rep protein from AAV facilitates targeted plasmid integration

BACKGROUND: There is a risk of insertional mutagenesis when techniques that facilitate random integration of exogenous DNA into the human genome are used for gene therapy. Wild-type adeno-associated virus (AAV) integrates preferentially into a specific site on human chromosome 19 (AAVS1). This is mediated by the interaction of the viral Rep68/78 proteins with Rep-binding elements in the AAV genome and AAVS1. This specificity is often lost when AAV is used as a gene therapy vector due to removal of the sequences coding for Rep. METHODS: Messenger RNA coding for the Rep68/78 proteins was prepared in vitro and co-transfected with a 21 kb DNA plasmid containing the P5 integration efficiency element (P5IEE) from AAV. Single cells were seeded in plates to establish clonal cell lines that were subsequently analysed by dual colour fluorescent in situ hybridisation (FISH) to determine whether site-specific plasmid integration had occurred on chromosome 19. RESULTS: The co-transfection of plasmid DNA with Rep68/78 mRNA gave a 2.5-fold increase in DNA integration when compared to transfection of cells with plasmid DNA alone. Rep68/78 mRNA expression facilitated site-specific plasmid integration to chromosome 19 in 30% (14/44) of all analysed integration sites, while no targeted integration events were observed following transfection of cells with plasmid DNA alone. CONCLUSIONS: These results demonstrate that transient expression of Rep protein using transfected mRNA facilitates site-specific integration of plasmid DNA. This approach allows expression of Rep for only a short time, and may circumvent the toxicity and chromosome instability associated with long-term expression of Rep.     

Factors predicting successful DNA recovery from archival cervical smear samples

Polymerase chain reaction (PCR)-based DNA testing of archival cervical smear slides is a useful method of retrospectively establishing the presence of the human papillomavirus (HPV) in cervical cells. A cellular DNA recovery test is performed in parallel to HPV DNA testing to ensure that sufficient cells are present and purification of sample DNA has been successfully performed. Previous studies have not comprehensively assessed DNA recovery rates in slides older than 13 years. We undertook a study to determine the factors impacting DNA recovery in 436 UK slides dating from 11 to 33 years prior to testing. Overall, a low cellular DNA recovery success rate of 29% was obtained but a strong trend was observed with increasing recovery rates the older the slides (P < 0.001). Recovery rates increased from 22% in the most recent slides collected from 1988 to 1992, to 61% in the oldest slides, collected in 1970-72. It is likely that fixation compounds incorporating acetic acid, introduced in the UK through the 1980s, have compromised subsequent attempts at PCR amplification. These findings emphasize the importance of the original fixation method in the success of DNA recovery from archival smear samples.     

Human immunodeficiency virus type 1 DNA nuclear import and integration are mitosis independent in cycling cells

An essential step in human immunodeficiency virus type 1 (HIV-1) replication is the movement of the viral preintegration complex from the cytoplasm into the nucleus. The pathway(s) and timing for HIV-1 DNA nuclear entry in cycling cells have not been established. Here, we show that if cycling cells are infected before S phase, viral DNA can be integrated prior to passage of the host DNA replication fork through the integration site, as indicated by stable inheritance in both daughter cells. We conclude that efficient nuclear entry can occur independently of mitotic nuclear disassembly in cycling cells.     

Radiation-induced DNA single-strand breaks in freshly isolated human leukocytes.

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.     

Human immunodeficiency virus integration in a cell-free system.

Integration of the viral genome into the nuclear DNA of a host cell plays a pivotal role in the replication of retroviruses. We have developed an in vitro method for studying the biochemistry of human immunodeficiency virus (HIV) integration by using extracts from HIV-infected cells. Analysis of the reaction products showed that HIV integration in vitro accurately reproduces the in vivo process. Integration occurred without apparent specificity for the target sequence, and the integrated provirus was directly flanked by a 5-base-pair duplication of DNA from the target site. HIV integration did not require a high-energy cofactor, and the enzymatic activities required for integration were recovered with the viral DNA when cell extracts were fractionated by gel exclusion chromatography.     

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors.

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. We have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possibly related to HBV integration.