Genetic and epigenetic alterations in hepatitis B virus-associated hepatocellular carcinoma

Hepatitis B virus(HBV) is a major cause of hepatocellular carcinoma(HCC). Its chronic infection can lead to chronic liver inflammation and the accumulation of genetic alterations to result in the oncogenic transformation of hepatocytes. HBV can also sensitize hepatocytes to oncogenic transformation by causing genetic and epigenetic changes of the host chromosomes. HBV DNA can insert into host chromosomes and recent large-scale whole-genome sequencing studies revealed recurrent HBV DNA integrations sites that may play important roles in the initiation of hepatocellular carcinogenesis. HBV can also cause epigenetic changes by altering the methylation status of cellular DNA, the post-translational modification of histones, and the expression of micro RNAs. These changes can also lead to the eventual hepatocellular transformation. These recent findings on the genetic and epigenetic alterations of the host chromosomes induced by HBV opened a new avenue for the development of novel diagnosis and treatments for HBV-induced HCC.     

Advances in research on hepatitis B virus DNA integration

Since HBV DNA integration was discovered for the first time in 1980, various methods have been used to detect and study it, such as Southern Blot, in situ hybridization, polymerase chain reaction and so on. HBV DNA integration is thought to be random on the whole although some hot spots of integration were described by some researchers, one of which might be the repetitive sequences of the genomic DNA. Besides, DNA damage, especially double-strand breaks could promote HBV DNA integration into host genome. HBV DNA integration into cells may damage the stability of the genome, cause DNA rearrangement, promote DNA deletion and induce the formation of HCC.     

Advances in Research on Hepatitis B Virus DNA Integration

Since HBV DNA integration was discovered for the first time in 1980, various methods have been used to detect and study it, such as Southern Blot, in situ hybridization, polymerase chain reaction and so on. HBV DNA integration is thought to be random on the whole although some hot spots of integration were described by some researchers, one of which might be the repetitive sequences of the genomic DNA. Besides, DNA damage, especially double-strand breaks could promote HBV DNA integration into host genome. HBV DNA integration into cells may damage the stability of the genome, cause DNA rearrangement, promote DNA deletion and induce the formation of HCC.     

Identification and extensive analysis of inverted-duplicated HBV integration in a human hepatocellular carcinoma cell line

Hepatitis B virus (HBV) DNA is often integrated into hepatocellular carcinoma (HCC). Although the relationship between HBV integration and HCC development has been widely studied, the role of HBV integration in HCC development is still not completely understood. In the present study, we constructed a pooled BAC library of 9 established cell lines derived from HCC patients with HBV infections. By amplifying viral genes and superpooling of BAC clones, we identified 2 clones harboring integrated HBV DNA. Screening of host-virus junctions by repeated sequencing revealed an HBV DNA integration site on chromosome 11q13 in the SNU-886 cell line. The structure and rearrangement of integrated HBV DNA were extensively analyzed. An inverted duplicated structure, with fusion of at least 2 HBV DNA molecules in opposite orientations, was identified in the region. The gene expression of cancer-related genes increased near the viral integration site in HCC cell line SNU-886.     

Tight clustering of human hepatitis B virus integration sites in hepatomas near a triple-stranded region.

The open circular genome of human hepatitis B virus (HBV) is known to contain a partially double-stranded DNA with a single-stranded gap region of variable length. This circular structure of the genome is maintained by base-pairing of the 5' ends of the two DNA stands, the long or L(-) strand and the short or S(+) strand. By cloning, mapping, and sequencing studies, we have localized three recombinational junctions of the integrated HBV in two hepatoma samples, HT14 and FOCUS. Breakpoints of recombination derived from these results and those of others appear to be clustered and coincidental with the identified 5' or the deduced 3' end of the long-strand DNA, respectively. Statistical analysis of these results supports the hypothesis that integration preferentially occurs in an extremely narrow region on the HBV genome. This site-specific recombinational mechanism appears to be conserved among different HBV subtypes. No extensive sequence homology was found between each pair of the recombining parental molecules; however, at the site of crossover, 2- to 3-base-pair junctional homology was consistently observed. Examination of the patterns of the integrated HBV DNAs allowed us to categorize these various patterns into four different groups according to their end specificity and strand polarity. The molecular form of relaxed circle is proposed to be one major substrate for HBV integration. The effect of free strand in the integration of HBV is emphasized in this model. Unlike any other known DNA animal viruses, the site specificity of HBV integration appears to be similar to that of the retroviruses.     

Detection and expression of hepatitis B virus X gene in one and two-cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying HBV DNA

The main objectives of this study were to introduce motile human sperm carrying Hepatitis B virus (HBV) DNA to golden hamster oocytes in a co-culture environment and to detect the replication and expression of the HBx gene in early embryonic cells. Zona-free hamster oocytes were inseminated with human sperm carrying pBR322-HBV DNA plasmid using the in vitro fertilization (IVF) technique. Both the one- and two-cell stages of early embryonic development were studied. PCR, RT-PCR, and Dot hybridization were performed to observe the HBx gene and its expression in these stages. "Fluorescence in situ hybridization" (FISH) was carried out to confirm the integration of HBV into the pronucleus, nucleus, and the chromosomes of embryos. The results showed that we have the ability to obtain a fertilization rate of 80%. RT-PCR showed that the HBx gene could be expressed in both one- and two-cell stages of embryonic development. The data suggested the possibility of sperm as a vector for the vertical transmission of HBV DNA to the next generation.     

Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children.

Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences.     

人肝癌细胞基因组中HBV DNA的整合与c-fos、p53基因表达之间的关系

为了探讨HBVDNA、c-fos和p53在肝癌发生中的作用及其关系。利用PCR技术和免疫组化ABC法,检测了肝癌基因组中HBVDNA的整合、c-fos和突变p53的表达。HBVDNA整合率为67%,C-FOS蛋白阳性率为67%,突变P53蛋白阳性率为58%。HBVDNA整合与c-fos激活、p53突变有显著的一致性(P＞0.05;P＞0.05),c-fos激活与p53突变之间呈负相关,但无显著意义(r=-0.2816,P＞0.05)。HBVDNA的整合可能引起c-fos的激活和/或p53的突变,从而导致肝癌的发生。     

Structural rearrangement of integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically infected hepatic tissues.

Cellular DNAs from human livers chronically infected with hepatitis B virus (HBV) were analyzed by Southern blot hybridization for the presence of integrated HBV DNA. In 15 of 16 chronically infected hepatic tissues, random HBV DNA integration was evident. By molecular cloning and structural analyses of 19 integrants from three chronically infected hepatic tissues, deletion of cellular flanking DNA in all cases and rearrangement of HBV DNA with inverted duplication or translocation of cellular flanking DNA at the virus-cell junction in some cases were noted. Thus, the rearrangement of HBV DNA or cellular flanking DNA is not a specific incident of hepatocellular carcinoma formation. Detailed analyses of various integrants bearing rearranged viral DNA failed to indicate any gross structural alteration in cellular DNA, except for a small deletion at the integration site, indicating that viral DNA rearrangement with inverted duplication possibly occurs before integration of HBV DNA. Based on nucleotide sequencing analyses of virus-virus junctions, a one- to three-nucleotide identity was found. A mechanism for this inverted duplication of HBV DNA is proposed in which illegitimate recombination between two complementary viral strands may take place by means of a nucleotide identity at the junction site in a weakly homologous region (patchy homology) on one side of adjoining viral sequences. For virus-cell junctions, the mechanism may be basically similar to that for virus-virus junctions.     

利用基因捕获技术建立稳定表达HBV的细胞模型

pGEM-HBV1.3质粒经HindIII限制性内切酶消化,将HBV1.3全长DNA切下,与同样经HindIII限制性内切酶降解过的PU21连接,得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中,G418筛选阳性克隆并以X-gal染色,RT-PCR、Southern blot等方法验证HBV DNA的插入和表达。 PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接,该重组质粒转染HepG2细胞后经G418筛选,得到一系列阳性克隆, Southern blot证实HepG2细胞基因组中含HBV DNA,RT-PCR结果表明HBV DNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立,为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。     

Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.     

Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity

When transgenic mice are created by microinjection of DNA into the pronucleus, the sites of DNA integration into the mouse genome cannot be predicted. Most methods based on polymerase chain reaction (PCR) that have been used for determining the integration site of foreign DNA into a genome require specific reagents and/or complicated manipulations making routine use tedious. In this report we demonstrate the use of a PCR-based method-TAIL-PCR (Thermal Asymmetric Interlaced PCR) which relies on a series of PCR amplifications with gene specific and degenerate primers to reliably amplify the integration sites. By way of example, using this approach, three separate integration sites were found (on chromosomes 8, 15 and 17) in one transgenic founder. As the sites on chromosomes 8 and 15 failed to segregate in any subsequent progeny, whole chromosome paints were done to determine if translocations involving chromosomes 8 and 15 occurred at the time of transgene integration. Whole chromosome painting could not detect translocations, suggesting that the rearrangements likely involve only small stretches of chromosomes. Site-specific primers were used to identify the progeny carrying only one integration site; these mice were then used as sub-founders for subsequent breedings. Integration site specific primers were used to distinguish homozygous progeny from heterozygotes. TAIL-PCR thus provides an easy and reliable way to (1) identify multiple integration sites in transgenic founders, (2) select breeders with one integration site, and (3) determine zygosity in subsequent progeny. Use of this strategy may also be considered to map integration sites in situations of unexpected phenotype or embryonic lethality while creating new transgenic mice.     

乙型肝炎病毒DNA在患者血清及肝组织中存在状态的研究

对29例肝炎,1例尸检肝组织和血清中乙型肝炎病毒的DNA(HBV DNA)进行了研究,发现HBsAg( )/HBeAg( )患者中,有9/17(52.94％)血清HBV DNA阳性;HBsAg( )/抗-HBe( )患者中,2/6(33.33％)也为阳性。从30例肝组织中提取DNA经琼脂糖电泳,Southern吸印转移及分子杂交试验结果表明,27例HBV DNA阳性,全部有游离型HBV DNA。27例中有5例经用标记pBR322探针杂交排除非特异杂交带后,在高分子量区有HBV DNA特异的杂交带,提示有HBV DNA整合。     

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.     

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors

Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At about 30 nM IN (20 min at 37 degrees C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of about 70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (about 125 microg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.     

广西HBsAg阳性母亲的胎儿肝、肾细胞中乙肝病毒DNA存在状态的研究

采用核酸分子杂交Ｓｏｕｔｈｅｒｎ印迹法,以３２Ｐ标记的ＨＢＶＤＮＡ为探针,检测ＨＢｓＡｇ阳性母亲引产的４０例胎儿的肝、肾组织。结果有２例胎肝和１例胎肾细胞ＤＮＡ出现大于３．２ｋｂ的杂交带,表明ＨＢＶＤＮＡ已处于整合状态。胎肾细胞基因组中查出ＨＢＶＤＮＡ整合为首次报道。     

Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.     

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.