Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma

Hepatitis B virus (HBV) may be one of the agents involved in the aetiology of human primary liver cancer. This hypothesis is supported by (1) the similarity between the geographical distribution of chronic carriers of the viral surface antigen (HBsAg) and that of hepatocellular carcinoma (HCC); (2) the increase in the prevalence of HBV markers in serum of patients with primary liver cancer when compared with the general population; (3) the observation that HBV infection precedes the development of the tumour. Moreover, these epidemiological indications of an association between HBV infecton and hepatocellular carcinoma are supported by the detection of HBV markers such as HBsAg or viral DNA sequences, although in a non-integrated form in tumour tissue. To study the relationship between HBV and primary liver cancer further, we looked for the presence of free or integrated viral DNA in tumour tissue of human hepatocellular carcinomas and in a HBsAg-producing human hepatoma cell line. Using the blot-transfer hybridization technique and cloned HBV DNA as a probe, we have now demonstrated that the viral DNA is integrated in the cellular genome both in tumour tissue and in a hepatoma cell line.     

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.     

广西肝癌中HBV感染与N-ras基因突变的研究

应用ＰＣＲ－ＳＳＣＰ和免疫组化法检测２９例广西南部肝癌组织中的Ｎ－ｒａｓ基因突变和ＨＢＶ感染状况，结果，肝癌中Ｎ－ｒａｓ基因在第２￣３７密码子之间的突变率为７９．３％，其中２２例有２￣５个突变位点，该基因突变也见于癌旁组织，肝组织中ＨＢｓＡｇ和ＨＢｓＡｇ和ＨＢｘＡｇ检出率分别为８６．２％和７９．３％，两者具有相关性，并与Ｎ－ｒａｓ基因突变率呈相平行的趋势。因广西南部的肝癌与ＡＦＢ１污染有关，本研究     

Targeted integration of baboon endogenous virus in the BEVI locus on human chromosome 6

The infection of cultured human cells with baboon endogenous virus (BEV) frequently leads to an association of viral DNA with a specific genetic locus (termed BEVI, for baboon endogenous virus infection) on chromosome 6. Restriction endonuclease digestion of DNA from BEV-infected human cells and their derived somatic cell clones frequently revealed a common cellular DNA sequence in the proximity of one of the junctions between cellular DNA and the integrated virus. We propose that a short cellular DNA sequence, repeated on chromosome 6 and separated by unique DNA sequences, presents a high-affinity target for the integration of BEV in human cells.     

广西肝细胞癌与丙型肝炎的关系

用４种抗丙型肝炎病毒蛋白的单克隆抗体，免疫酶染色法直接检测肝细胞癌的病理组织中丙肝病毒蛋白。结果８／５２例（１５．４％），一至数种抗体阳性，阳性反应物主要定位于肝和癌细胞的胞浆中，值得注意的是本组几乎全部病例为乙肝，丙肝两种病毒混合感染，表明广西丙型肝炎的混合感染的比较高，乙型肝炎病毒（ＨＢＶ）加上黄曲霉毒素（ＡＦＢ１）或丙型肝炎病毒（ＨＣＶ）可能是广西肝细胞癌（ＨＣＣ）高发的原因。     

Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa.

Hepatocellular carcinoma (HCC) is a prevalent cancer in sub-Saharan Africa and eastern Asia. Hepatitis B virus and aflatoxins are risk factors for HCC, but the molecular mechanism of human hepatocellular carcinogenesis is largely unknown. Abnormalities in the structure and expression of the tumour-suppressor gene p53 are frequent in HCC cell lines, and allelic losses from chromosome 17p have been found in HCCs from China and Japan. Here we report on allelic deletions from chromosome 17p and mutations of the p53 gene found in 50% of primary HCCs from southern Africa. Four of five mutations detected were G----T substitutions, with clustering at codon 249. This mutation specificity could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1 (ref. 7), a food contaminant in Africa, which is both a mutagen that induces G to T substitution and a liver-specific carcinogen.     

原发性肝癌与乙型肝炎病毒感染的关系

目的　探讨HBV感染模式与肝病的关系.方法　选择经检测HBV5项血清学标志的肝癌患者120例为研究对象,并以非癌肝病180例作对照.结果　120例肝癌患者HBV感染以HbsAg75.1%(90/120),抗 Hbe70.2%(84/120),抗 Hbc80.5%(97/120)为主.与对照组58.9%,48.3%,66.1%比较,差异非常显著(P<0.01).肝癌组HBV总感染率高达98.3%.感染模式以HbsAg,抗 Hbe,抗 Hbc;HbsAg,抗 Hbc;抗 Hbe,抗 Hbc;HbsAg,HbeAg,抗 Hbc四种为主,占83.3%(100/120).肝炎、肝硬化、肝癌的HBV感染主要模式一致.结论　HBV是肝癌的主要原因之一,尤其4种感染模式与肝癌发生密切相关,定期随访复查是发现早期肝癌的最佳途径.     

Innate immune recognition and regulation in liver injury: A brief report from a series of studies

The discovery of innate immune receptors and the emergence of liver immunology （high content of NK and NKT cells in liver） led to the second research summit in innate immunity since the finding of NK cells in the middle 1970s. Liver disease is one of the most dangerous threats to humans, and the progress in innate immunology and liver immunology made it possible to re-explain the cellular and mo- lecular immune mechanisms of liver disease. In the past ten years, we have found that innate recognition of hepatic NK and NKT subsets were involved in murine liver injury. We established a novel NK cell-dependent acute murine hepatitis model by activating Toll-like receptor-3 （TLR-3） with an injection of poly I：C, which may mimic mild viral hepatitis （such as Chronic Hepatitis B）. We observed that a network of innate immune cells including NK, NKT and Kupffer cells is involved in liver immune injury in our established NK cell-dependent murine,model. We noted that TLR-3 on Kupffer cells activated by pretreatment with poly I： C might protect against bacterial toxin （LPS）-induced fulminant hepatitis by down-regulating TLR-4 function, while TLR-3 pre-activation of NK cells might reduce Con A-induced NKT cell-mediated fulminant hepatitis by blocking NKT cell recruitment to the liver. We also found that the oversensitivity to injury by immune stimulation in HBV （hepatitis B virus） transgenic mice （full HBV gene-tg or HBs-tg） correlated to the over-expression of Real, an NKG2D （natural killer cell group 2D） ligand of NK cells or CDld, a ligand of TCR-V14 of NKT cells, on HBV＋ hepatocytes, which leads to an innate immune response against hepatocytes and is critical in liver immune injury and regeneration.     

Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours

The recent finding of c-myc activation by insertion of woodchuck hepatitis virus DNA in two independent hepatocellular carcinoma has given support to the hypothesis that integration of hepatitis B viruses into the host genome, observed in most human and woodchuck liver tumours, might contribute to oncogenesis. We report here high frequency of woodchuck hepatitis virus DNA integrations in two newly identified N-myc genes: N-myc1, the homologue of known mammalian N-myc genes, and N-myc2, an intronless 'complementary DNA gene' or 'retroposon' that has retained extensive coding and transforming homology with N-myc. N-myc2 is totally silent in normal liver, but is overexpressed without genetic rearrangements in most liver tumours. Moreover, viral integrations occur within either N-myc1 or N-myc2 in about 20% of the tumours, giving rise to chimaeric messenger RNAs in which the 3' untranslated region of N-myc was replaced by woodchuck hepatitis virus sequences encompassing the viral enhancer. Insertion sites were clustered in a short sequence of the third exon that coincides with a retroviral integration hotspot within the murine N-myc gene, recently described in T-cell lymphomas induced by murine leukaemia virus. Thus, comparable mechanisms, leading to deregulated expression of N-myc genes, may operate in the development of tumours induced either by hepatitis virus or by nonacute retroviruses in rodents. Activation of myc genes by insertion of hepadnavirus DNA now emerges as a common event in the genesis of woodchuck hepatocellular carcinoma.     

Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells

Moloney murine leukaemia virus (M-MuLV) infection of embryonal carcinoma (EC) cells results in the integration of proviral DNA into the host cell genome, but not in virus production. One suggested explanation for the lack of viral gene expression in EC cells has been methylation of the integrated viral DNA. However, subsequent reports indicated that integration of the M-MuLV DNA occurs soon after infection, but that viral DNA methylation occurs considerably later. Nevertheless, viral gene expression is not observed even at early times. One possible explanation is that certain M-MuLV regulatory sequences do not function in EC cells. We now present evidence which supports this hypothesis.     

Identification and nucleotide sequence of a human locus homologous to the v-myc oncogene of avian myelocytomatosis virus MC29

Avian myelocytomatosis virus MC29 is a replication-defective acute leukaemia virus which induces a variety of tumours in chickens including sarcomas, renal and hepatic carcinomas, and myelocytomatosis. The oncogenic potential of the virus is mediated by the gene v-myc, acquired from sequences (c-myc) present in normal uninfected chicken DNA. Sequences closely related to chicken c-myc have been highly conserved throughout evolution, from Drosophila to vertebrates. The hypothesis that c-myc may be involved in neoplastic transformation has been strengthened by the finding that B-cell lymphomas induced in chickens by avian leukosis virus (ALV) are often associated with increased expression of c-myc resulting from integration of the ALV provirus adjacent to the c-myc gene. More recently, it has been demonstrated that the malignant human cell line HL-60, derived from the peripheral blood leukocytes of a patient with acute promyelocytic leukaemia, expresses elevated levels of myc-related mRNA associated with an amplification of the c-myc gene. To explore the relationship of the human cellular myc gene with the corresponding viral oncogene from MC29, and to provide a framework for the analysis of the mechanism and significance of c-myc amplification in human tumours, we have isolated and determined the nucleotide sequence of a genomic clone prepared from a normal human library which contains all domains sharing homology with v-myc.     

A new retinoic acid receptor identified from a hepatocellular carcinoma

Processes as diverse as growth, vision and reproduction depend on the presence of vitamin A and its metabolites (retinoids), but the molecular mechanisms which govern these diverse actions remain unclear (for reviews see refs 1,2). A crucial advance recently was the isolation of a specific nuclear receptor for retinoic acid, one of the physiologically active vitamin A derivatives. This nuclear receptor is a member of the steroid/thyroid hormone receptor family. Our analysis of an uncharacterized member of this class of intracellular receptors, encoded by a complementary DNA clone from a human placental library, has led us to discover a second retinoic acid receptor. This new receptor is expressed at high levels in a number of epithelial-type tissues. The gene for the receptor was first identified in a hepatocellular carcinoma where it surrounds a site of integration of hepatitis B virus. Activation by this virus may play a role in tumour development in liver cells, where it is normally not expressed.     

RNA interference-mediated inhibition of Hepatitis B Virus replication

Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.     

Biosynthesis of hepatitis B virus surface antigen in Escherichia coli

Hepatitis B is a widespread viral disease. In the absence of cell cultures capable of propagating the virus (HBV) an efficient vaccine has been prepared from viral envelopes isolated from the plasma of chronic carriers. The major polypeptide of the envelope is one of molecular weight 25,000 which carries the surface antigen (HBsAg). Therefore, the biosynthesis of this polypeptide in Escherichia coli may offer an alternative procedure to produce HbsAg free from human proteins. Recently, the HBV genome has been cloned in E.coli. Determination of its primary structure allowed the localization of the gene (called gene S) coding for HBsAg and the synthesis of the core antigen in E.coli has been reported. We have constructed a derivative of bacteriophage lambda carrying a fusion between the beta-galactosidase gene (lacZ) and the HBsAg coding sequence (lambdalacHBs-1). Infection of E.coli with lambdalacHBs-1 leads to the biosynthesis of a polypeptide of molecular weitht 138,000 carrying antigenic determinants of HBV surface antigen.     

树对人乙型肝炎病毒易感性的验证

为验证树对人乙型肝炎病毒 (HBV)的易感性 ,用含 HBV的人血清接种给 10只成年树 (雌雄各半 )。然后每周抽血 1次 ,每只动物共抽血 11次。用不同公司生产的 EL ISA试剂检测接种后的动物血清感染指标。实验观察至 13周 ,结果分别有 9只和 7只动物出现 HBs Ag阳性 ,持续最短 1周 ,最长 7周。用 PCR检测 ,结果有 1只动物连续 4周在血清中检测出 HBV DNA。表明树能感染人 HBV,但实验有待改进以延长感染持续时间     

A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma

We have previously isolated from a human hepatocellular carcinoma a hepatitis B virus integration in a 147-base-pair cellular DNA fragment, similar to steroid- and c-erb-A/thyroid-hormone receptor genes. We have now cloned the corresponding complementary DNA from a human-liver cDNA library. Nucleotide sequence analysis revealed that the overall structure of the cellular gene, which we have named hap, is similar to that of the DNA-binding hormone receptors. That is, it displays two highly conserved regions identified as the putative DNA-binding and hormone-binding domains of the c-erb A/steroid receptors. Six out of seven hepatoma and hepatoma-derived cell-lines express a 2.5-kilobase (kb) hap messenger RNA species which is undetectable in normal adult and fetal livers but present in all non-hepatic tissues analysed. The data suggest that the hap gene product may be a novel ligand-responsive regulatory protein whose inappropriate expression in liver may relate to the hepatocellular carcinogenesis.     

治疗性乙型肝炎疫苗研究反思

 “治疗性乙肝疫苗”是以乙肝病毒(HBV)的表面抗原(s抗原)为基础的生物制剂,目的是激发抗s抗原免疫反应,终止HBV慢性感染.HBV的e抗原与s抗原无抗原性关联,对e抗原的反应也与病毒及感染细胞的清除无关,因此以II期临床试验者血清有关病毒e抗原的数据结果,尚不能判断“治疗性乙肝疫苗”是否有效.疫苗的应用是抵御病毒入侵,而治疗性乙肝疫苗是在病毒已经进入人体后应用,在患者肝细胞可能广泛受累的情况下,疫苗一旦打破耐受激发抗s抗原免疫反应,除能清除血清中游离的病毒和s抗原颗粒外,将直接攻击被感染的肝细胞.由于无法估计慢性HBV感染者肝细胞感染程度,所以无法推测免疫反应发生后,免疫病理所致的肝损程度以及相应的风险.在应用上隐含如此风险,是“治疗性乙肝疫苗”走不出实验室的重要因素之一.