Hepatitis B Virus Integration Sites Research Articles

The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients

Hepatitis B virus (HBV) infection is a leading risk factor for hepatocellular carcinoma (HCC). HBV integration into the host genome has been reported, but its scale, impact and contribution to HCC development is not clear. Here, we sequenced the tumor and nontumor genomes (>80× coverage) and transcriptomes of four HCC patients and identified 255 HBV integration sites. Increased sequencing to 240× coverage revealed a proportionally higher number of integration sites. Clonal expansion of HBV-integrated hepatocytes was found specifically in tumor samples. We observe a diverse collection of genomic perturbations near viral integration sites, including direct gene disruption, viral promoter-driven human transcription, viral-human transcript fusion, and DNA copy number alteration. Thus, we report the most comprehensive characterization of HBV integration in hepatocellular carcinoma patients. Such widespread random viral integration will likely increase carcinogenic opportunities in HBV-infected individuals.

Pathogenesis of hepatitis B virus‐associated hepatocellular carcinoma

Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) remains the most common form of HCC in large areas of Asia and Africa. It remains common even in some countries where hepatitis C virus (HCV)-associated HCC has become the predominant form, such as Japan. Integration of HBV in HCC DNA is found at random sites in the host genome in nearly all patients with HBV-associated HCC. It is not clear how often this integration results in insertional mutagenesis, but previously unknown growth regulating genes and cancer-associated genes have been found frequently near HBV integration sites in HCC in recent studies. In addition, HBV encodes a transactivating protein, the X protein, which could enable the randomly integrated HBV to alter the function of host genes that are not near the integration site. Mutations at two adjacent codons in HBV (1762(T)/1764(A) mutations) within the X gene are frequently found in HCC patients, and may play a role in the mutagenic or transactivational role of HBV in HCC. The presence of cirrhosis in most patients with HBV-associated HCC, and the presence of mutations in tumor suppressor genes in many, suggests that these are also factors in hepatocarcinogenesis. Few studies have examined the mutations of more than one gene in the same HCC patients. Fewstudies have evaluated the interactions between HBV mutations, host gene mutations, cirrhosis, and other potentially mutagenic stresses at the cellular level, with progression to HCC, and few studies have been conducted to determine whether these changes must accumulate in succession to lead to HCC. The recent availability of rapid sequencing methods and DNA microarray technologies has permitted expression profiling and permutation analysis of an array of genes to explore the pattern and succession of molecular changes leading to HBV-associated HCC. To date, these methods have been used to show patterns of molecular changes that differ in HBV-associated HCC (compared to HCV-associated HCC or to HCC in patients lacking either virus) and patterns that can predict survival (and hence may directly indicate different mechanisms of disease), and may soon make possible a universally accepted clinical classification scheme for HCC.

Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B

Hepatitis B virus (HBV) integration into the host genome is frequently detected in HBV positive hepatocellular carcinoma (HCC) in China. The aim of this study is to carry out a large-scale screening for the HBV integrations sites in HCC samples from Chinese patients. Cellular DNA was extracted from 40 HBV-related HCC by proteinase K digestion/phenol extraction method. One primer specific to HBV sequence and another primer directed to human Alu repeat were used to amplify the virus/cellular DNA junction. To avoid undesirable amplifications between Alu sequences, primers were constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 15 initial cycles of amplification. Only desirable fragments were then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-Specific primer. The PCR product was purified and subject to direct sequencing by ABI 3700 Auto sequencer. NCBI (national center for biotechnology information) BLAST and MapViewer search were used for identification of HBV location on human genomes. In 40 HBsAg positive HCC samples, 34 (85%) were showed to have at least one copy of HBV fragment in host genome, indicating HBV-Alu-PCR is a rapid way for identification of new cellular DNA sequences adjacent to HBV. Analysis from the 68 isolated viral-cellular junctions, X gene was found to be interrupted at any length, not specifically at DR1 and DR2 regions. Three-prime-deleted X gene was observed in 65 (96%) cases. HBV preferred to integrate into the intron and the up-stream regulatory region of the cellular genes. In no case HBV inserted into the exon. Our results also demonstrated that the cellular genes targeted by HBV are usually key regulators of cell proliferation and cell death. Three genes, myeloid/lymphoid or mixed-lineage leukemia 4, G protein alpha transducing activity polypeptide 1 and fibronectin, were found to be recurrently targeted by HBV. HBV-Alu-PCR is a powerful tool for the study of HBV integration sites. Truncated X is a major form existed in the HBV integrants. HBV integration is not distributed evenly throughout the host genome and viral insertional mutagenesis may play an important role in the development of HCC.

Hepatitis B Virus DNA Integration in Hepatocellular Carcinoma After Interferon-Induced Disappearance of Hepatitis C Virus

Hepatocellular carcinoma (HCC) has been reported in patients in whom hepatitis C virus (HCV) was eliminated by interferon (IFN) therapy. We examined the pathogenesis of HCC in patients with sustained viral response. Operable HCC developed in 7 of 342 patients cured of HCV infection by IFN monotherapy. No patient abused alcohol or had diabetes mellitus or obesity. Resected specimens of HCC were histologically evaluated. DNA extracted from HCC was examined by polymerase chain reaction (PCR) to locate hepatitis B virus (HBV) DNA. HBV integration sites in human genome were identified by cassette-ligation-mediated PCR. HBV DNA was not amplified in serum samples from any of the seven patients with HCC and was found in liver in four patients. In the latter four patients, HBV DNA was integrated into the human genome of HCC. In two of these patients, covalently closed circular HBV (cccHBV) was also detected. The patients with HBV DNA integration were free of HCV for more than 3 yr. In two of the three patients without HBV DNA integration, the surrounding liver showed cirrhosis. The liver of HCC with HBV DNA integration had not progressed to cirrhosis. Three of the four tumors with HBV integration had one integration site each, located at chromosomes 11q12, 11q22-23, and 22q11, respectively. The other tumor had two integration sites, situated at chromosomes 11q13 and 14q32. At chromosome 11q12, HBV DNA was integrated into protein-coding genome, the function of which remains unclear. Integrated HBV DNA may play a role in hepatocarcinogenesis after the clearance of HCV by IFN treatment.

Hepatitis B virus-transfected Hep G2 cells demonstrate genetic alterations and de novo viral integration in cells replicating HBV

Hepatitis B virus (HBV) is a major etiological factor associated with hepatocarcinogenesis, but its role in the transformation process remains unclear. We previously documented the accumulation of genetic alterations in a HBV-transfected cell line. In the present study, we addressed the effect of HBV and its replication on the genome and phenotype of the host cell. Parental HBV-free Hep G2 cells and two HBV-transfected variant lines Hep G2215 and Hep G2T14.1, which do and do not replicate HBV, respectively, were used to monitor genetic alterations in conjunction with HBV profile in vitro and in vivo. Comparison of in vitro growth rates showed that Hep G2T14.1 cells grew more rapidly, while Hep G2215 cells, replicating HBV, grew slower than parental Hep G2 cells. Molecular analysis confirmed an HBV integration site (s) in both variants, and reverse trancriptase-polymerase chain reaction (RT-PCR) amplification documented expression of transcript for the HBX protein, which has recently been implicated in the compromised efficiency of cellular DNA repair. Tumorigenisity testing indicate a comparable rate of tumor formation in nude mice of both HBV-transfected variants, giving rise to tumors in 3 weeks; parental Hep G2 cells did not form tumors in nude mice. Tumor tissue from nude mice injected with Hep G2T14.1 cells showed no change in HBV status. However, a new HBV integration site was detected in tumor tissue from Hep G2215-injected mice. Two cell lines derived from the respective tumor tissue grew in vitro at rates compatible to those observe before passage in nude mice. The Hep G2215 tumor-derived line continued to replicate HBV, while HBV status remained unchanged in the Hep G2T14.1 tumor-derived line. Unique genetic alterations were detected in both transfected cell lines, and Hep G2215 cells particularly showed cellular mosaicism and clonal selection when analyzed after the passage in nude mice. Further genetic alterations were detected in tumor-derived cell lines. Interestingly, the de novo genetic alterations in the Hep G2215 cells, which maintain the ability to replicate HBV, included a new HBV integration site, several chromosome rearrangements and loss of heterozygosity (LOH) of one p53 allele. Western analyses of p21/Waf1 protein indicate an upregulation of the protein in cells that replicate HBV. Based on the combined data, we hypothesize that the genetic alterations in the cellular genome could also be generated as a function in the presence of HBV and HBV replication. Possible mechanisms that could be implicated in cumulative mutagenetic events are discussed.

Investigation of the Expression of Bin1, a Putative Suppressor, in Human Hepatoma Cells

Recent data suggest that Bin1, a novel C-MYC interacting protein, is a suppressor gene whose loss of expression is a frequent aberration associated with several malignancies. The mechanism responsible for loss of BIN1 expression is not understood. The purpose of this study is to investigate DNA profile of the BIN1 gene in human hepatoma Hep G2 cells, previously documented with lack of BIN1 expression. Chromosome and molecular analyses of Hep G2 cells were initiated to exclude the possibility of genetic alterations as a factor affecting BIN1 gene expression in these cells. We used Hep G2 cell line and its hepatitis B virus (HBV) transfected variants—Hep G2T14.1 and Hep G2215 cell lines. The cytogenetic localization of BIN1 was identified in the 2q14 region. Fluorescence in situ hybridization (FISH) with the chromosome 2 whole chromosome painting probe (WCP) demonstrated three or four intact copies of chromosome 2 in all three hepatoma cell lines studied. FISH analyses with the BIN1-specific probe of the Hep G2, Hep G2T14.1, and Hep G2215 metaphase chromosomes document no rearrangement of the BIN1 gene on any of the multiple copies of chromosome 2. FISH with the specific HBV probe did not identify the HBV integration site in Hep G2T14.1 and Hep G2215 cells within the BIN1 locus. Southern blot analyses revealed no genetic rearrangements in the BIN1 gene in any of the cell lines studied. Our RNA analyses (northern blot and RT-PCR) document lack of BIN1 message in Hep G2 cells in contrast to the presence of BIN1 in Hep G2T14.1 and Hep G2215 cells. No difference was identified in other transcripts analyzed, including c-myc. Analyses of BIN1 expression of Hep G2 cells at different passages were initiated and document low levels of BIN1 transcript in Hep G2 cells of passage <85. Furthermore, BIN1 transcript was identified in additional seven HCC cell lines analyzed. Our data indicate that lack of Bin1 expression in HepG2 cells previously documented is a characteristic of cells of passage >85 and is not due to genetic loss, or rearrangement within the BIN1 DNA sequence. Loss of the BIN1 transcript is not a characteristic of HCCs analyzed.

Accumulation of genetic alterations in a human hepatoma cell line transfected with hepatitis B virus

Chromosome and molecular analyses of the hepatitis B virus (HBV)-transfected HepG2T14.1 variant of the HepG2 cell line was conducted. In HepG2T14.1 cells several genetic alterations such as de novo aberrations of chromosomes 9, 14, 15, and 20 were identified that are not present in the parental HepG2 cell line. Furthermore, HepG2T14.1 cells showed loss of heterozygosity (LOH) in the q region of chromosome 14. The single HBV integration site in HepG2T14.1 cells mapped to the 2q35–36 region of one copy of chromosome 2 by fluorescence in situ hybridization (FISH). No genetic changes were identified at or near the HBV integration site at the level of these analyses. In addition, growth rates in vivo and in vitro were dramatically accelerated in HepG2T14.1 cells. These results document that a HBV-transfected hepatoma cell line has de novo genetic mutations at several sites of the host genome, one HBV integration site in an non-rearranged chromosome and an altered phenotype. These findings support our hypothesis that HBV might play a role in cellular transformation by interfering with cellular processes responsible for the stability of the genome.

Integration of hepatitis B virus and alteration of the 1p36 region found in cancerous tissue of primary hepatocellular carcinoma with viral replication evidenced only in noncancerous, cirrhotic tissue

We have studied the genetic profile of the host genome and hepatitis B virus (HBV) in HBV-associated primary hepatocellular carcinoma (HCC). Comparative analyses of HCC cell line Hep 40 and the original biopsy specimens showed the episomal and replicating form of HBV only in the biopsy specimen from nontumor (NT) cirrhotic liver tissue, where a molecular change in the 1p36 region was detected (NT tissue showed a normal 46XY karyotype). In contrast, only integrated HBV was detected in HCC tumor (T) tissue and Hep 40 cells. Two HBV integration sites were identical in HCC tissue and the hyperdiploid Hep 40 cell line, where genetic alteration in the 1p36 region was identified. These data indicate that viral replication is ongoing only in NT cirrhotic-hyperplastic, chromosomally normal tissue with evidence for genetic instability. Only the tumor cell with altered genotype has virus integrated

Integration of hepatitis B virus and alteration of the 1p36 Region found in cancerous tissue of primary hepatocellular carcinoma with viral replication evidenced only in noncancerous, cirrhotic tissue

We have studied the genetic profile of the host genome and hepatitis B virus (HBV) in HBV-associated primary hepatocellular carcinoma (HCC). Comparative analyses of HCC cell line Hep 40 and the original biopsy specimens showed the episomal and replicating form of HBV only in the biopsy specimen from nontumor (NT) cirrhotic liver tissue, where a molecular change in the 1p36 region was detected (NT tissue showed a normal 46XY karyotype). In contrast, only integrated HBV was detected in HCC tumor (T) tissue and Hep 40 cells. Two HBV integration sites were identical in HCC tissue and the hyperdiploid Hep 40 cell line, where genetic alteration in the 1p36 region was identified. These data indicate that viral replication is ongoing only in NT cirrhotic-hyperplastic, chromosomally normal tissue with evidence for genetic instability. Only the tumor cell with altered genotype has virus integrated.

Deletion of integrated hepatitis B virus genome and cellular flanking sequences in hepatocellular carcinoma cells in BALB/c Mice

We have previously reported the establishment of well-differentiated BALB/c mouse liver (ML) cell lines. Transfection of these cell lines with hepatitis B virus (HBV) DNA led to the expression of HBV-specific antigens and integration of HBV sequences in the cellular genome. Two cloned HBV-transfected ML cell lines, ML-2(HBV) and ML-3(HBV), expressed viral antigens and were highly tumorigenic in nude mice. However, the tumorigenicity of the two cell lines was significantly reduced in BALB/c mice. Southern blot analyses showed that the integrated HBV sequences were retained in tumors growing in nude mice but deleted in tumors growing in BALB/c mice. Furthermore, the deletion of HBV DNA was accompanied by deletion of chromosomal sequences flanking the HBV integration sites. In ML-2(HBV) cells, a significant reduction in chromosomal number was also observed. These results suggest that the immune response of BALB/c mice selected against hepatocellular carcinoma (HCC) cells expressing viral antigens and led to the proliferation of cells with deleted HBV sequences and concomitant chromosome aberrations. By using this mechanism, HCC cells escape the immune surveillance and gain the advantage of cell growth.

Deletion of integrated hepatitis B virus genome and cellular flanking sequences in hepatocellular carcinoma cells in BALB/c mice

We have previously reported the establishment of well-differentiated BALB/c mouse liver (ML) cell lines. Transfection of these cell lines with hepatitis B virus (HBV) DNA led to the expression of HBV-specific antigens and integration of HBV sequences in the cellular genome. Two cloned HBV-transfected ML cell lines, ML2(HBV) and ML-3(HBV), expressed viral antigens and were highly tumorigenic in nude mice. However, the tumorigenicity of the two cell lines was significantly reduced in BALB/c mice. Southern blot analyses showed that the integrated HBV sequences were retained in tumors growing in nude mice but deleted in tumors growing in BALB/c mice. Furthermore, the deletion of HBV DNA was accompanied by deletion of chromosomal sequences flanking the HBV integration sites. In ML-2(HBV) cells, a significant reduction in chromosomal number was also observed. These results suggest that the immune response of BALB/c mice selected against hepatocellular carcinoma (HCC) cells expressing viral antigens and led to the proliferation of cells with deleted HBV sequences and concomitant chromosome aberrations. By using this mechanism, HCC cells escape the immune surveillance and gain the advantage of cell growth.

Hepatitis B virus DNA integration and expression of an erb B-like gene in human hepatocellular carcinoma

Southern blot studies on Hepatitis B Virus (HBV) DNA integration in 13 human hepatocellular carcinomas (HCCs) patients revealed the presence of several distinct HBV integration sites in different human liver disease patients. In one HCC patient the DNA fragment containing the HBV integration also hybridized to an erb B probe. The erb B/HBV co-migrating DNA fragment was cloned and sequenced, and showed that HBV DNA is integrated next to a cellular DNA fragment which is homologous to the tyrosine protein kinase domain of the human epidermal growth factor receptor gene and other cell surface receptor genes. The virus-integrated cellular DNA sequence is expressed in this HCC patient, suggesting a possible role for this gene in hepatocarcinogenesis.

Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma

Hepatitis B virus (HBV) DNA frequently integrates into the genome of human primary liver cancer cells, but the significance of this integration in liver carcinogenesis is still unclear. Here we report the cloning of a single HBV integration site in a human hepatocellular carcinoma at an early stage of development, and of its germline counterpart. The normal locus was found to be transcribed into two polyadenylated messenger RNA species of 1.8 and 2.7 kilobases. We have isolated a complementary DNA clone from a normal adult human liver cDNA library which has an open reading frame with a coding capacity for a protein of 432 amino acids and relative molecular mass 48,536. The strong homology of the C-terminal half of the protein to the A-type cyclins of clam and Drosophila identifies it as a human cyclin A. The cyclin A gene has several exons, and the HBV integration occurs within an intron. As cyclins are important in the control of cell division, the disruption of a cyclin A gene by viral insertion might contribute to tumorigenesis.

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.

Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17;18 translocation.

Integrated hepatitis B virus (HBV) DNA is almost invariably found in hepatocellular carcinomas (HCC) which develop in HBV carriers. Integrated HBV DNAs from two single-integration HCCs (C3 and C4) have been cloned, and the cellular integration sites have been analyzed. Integrated HBV DNA of C3 is present in chromosome 6 and contains a nearly complete linear HBV genome. The HBV DNA integration in tumor C3 was not associated with major rearrangements of cellular DNA. In contrast, the integrated HBV DNA in C4 contains a large inverted repeat of HBV DNA, in which each repeat consists of a linear HBV DNA segment similar to that present in C3. The C4 integration was also accompanied by a cellular DNA translocation at the HBV integration site. The translocation occurred between chromosomes 17 and 18, along with a deletion of at least 1.3 kilobases of chromosome 18 DNA at the translocation site. Our data support a model in which postintegration rearrangement of integrated HBV and cellular DNA results in the generation of chromosomal aberrations. These chromosomal aberrations may function in a multistage mechanism leading to fully malignant HCC.

Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma

Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation.