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PubMed1990

Integration of hepatitis B virus DNA and its implications for hepatocarcinogenesis.

Hepatitis B virus (HBV) DNA integrates into human hepatocyte DNA. We have gathered the available data on the structure of the integrants from human hepatocellular carcinomas, and classified them into those that seem to represent primary integrants and those that are the products of secondary rearrangements. By means of structural analyses of the possible primary integrants, we deduced that the replication intermediates of the viral genome are the preferred substrates for integration. The integrated HBV DNA and the target cellular DNA are invariably associated with deletions, possibly reflecting the substrate for, and the mechanism of, the integration reaction. The target cell DNA sequence, as well as the target site of integration in chromosomes, seems to be selected randomly, suggesting that HBV DNA integration should bring about random mutagenic effects. Several samples recovered from hepatocellular carcinomas show that the integrated HBV DNA can mediate secondary rearrangements of chromosomes, such as translocations, inversions, deletions and (possibly) amplifications. Thus, HBV DNA integration causes multiple mutagenic effects. We argue that during hepatitis infection, the tendency of rearrangement of hepatocyte chromosomes is combined with the forcible turnover of cells. This is a constantly operating system for the selection of cells that grow better than average cells, possibly involving important steps in multistaged hepatocarcinogeneses.

Carcinoma, Hepatocellular↗

PubMed1984

Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.

Base Sequence↗

PubMed1986

Hepatitis B virus DNA contains a glucocorticoid-responsive element.

It has recently been shown that hepatitis B virus (HBV) contains a transcriptional enhancer element. In order to determine whether this enhancer responds to glucocorticoids, a series of derivatives of plasmid pA10CAT2 was constructed containing the HBV enhancer and variable lengths of further upstream sequences. Transient expression of chloramphenicol acetyltransferase (CAT) was determined after introduction of these plasmids into PLC/PRF/5, Hep 3B, Hep G2, HeLa, and mouse L cells. Highest CAT activity was noted in the human hepatocellular carcinoma line PLC/PRF/5, which contains integrated HBV DNA sequences. Dexamethasone augmented CAT expression in all cell lines tested with 40% of maximal induction at 10 nM and maximum stimulation (3- to 8-fold) at 1 microM dexamethasone. Dexamethasone augmentation of CAT expression was observed only when constructs contained HBV DNA sequences residing upstream to map position 735 from the EcoRI site. This indicates that the glucocorticoid-responsive region is distinct from the previously defined HBV enhancer sequence located at map position 1080-1234. These studies suggest that HBV DNA contains a glucocorticoid-responsive element, which may mediate expression of HBV genes in infected mammalian cells.

Acetyltransferases↗

PubMed1999

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization.

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.

Adult↗

PubMed1986

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.

Animals↗

PubMed1997

Natural history of chronic hepatitis B virus infection: an immunopathological study.

Three clinicopathological phases of chronic hepatitis B virus (HBV) infection are identified. First, is immune tolerance of HBV. High levels of viraemia are associated with normal alanine aminotransferase (ALT) levels and minimal histological lesions. More than 30-40% of hepatocytes have the hepatitis B core antigen (HBcAg), predominantly in their nuclei. Maternally derived hepatitis B e antigen (HBeAg) crossing the placenta may result in the elimination of T helper cells responsive to HBeAg/HBcAg. This phase can last for periods ranging from a few weeks to 10 or more years until the immune tolerance is lost. Second, is the immune clearance of HBV. Intermediate levels of viraemia are associated with fluctuating ALT levels and active and ongoing hepatitis. Approximately 20-30% of hepatocytes have HBcAg, predominantly in their cytoplasm. Expression of pre-core defective HBV mutants during chronic HBV infection may lead to a reduction in the secretion of HBeAg and may trigger the beginning of the immuno-elimination phase. The mechanism of intrahepatic shift of HBcAg from the nucleus to the cytoplasm and the decreased levels of viraemia in this phase may be, at least in part, secondary to liver damage and regeneration. Third, is latent infection with residual integrated HBV. Undetectable viraemia is associated with normal ALT levels and no virus-induced liver damage. With regard to hepatocyte expression of HBsAg in chronic HBV infection, membrane staining of HBsAg on hepatocytes has been shown to correlate well with the presence of viraemia. The degree of cytoplasmic hepatitis B surface antigen (HBsAg) expression inversely correlates with the level of viraemia. Therefore, HBsAg carriers with high levels of viraemia have low levels of cytoplasmic hepatitis B surface antigen (HBsAg) expression, while those with low levels of viraemia have high levels of cytoplasmic HBsAg expression. However, several exceptions have been identified. High levels of viraemia associated with high levels of cytoplasmic HBsAg expression were recognized in patients with fibrosing cholestatic hepatitis. In contrast, low levels of viraemia associated with low levels of cytoplasmic HBsAg expression were recognized in patients with hepatitis C virus but not hepatitis D virus superinfection.

Cell Cycle↗

PubMed1997

Frequent rearrangements at minisatellite loci D1S7 (1p33-35), D7S22 (7q36-ter) and D12S11 (12q24.3-ter) in hepatitis B virus-positive hepatocellular carcinomas from Thai patients.

Primary hepatocellular carcinoma (HCC) is one of the most common cancers in Thailand; chronic infection with hepatitis B virus (HBV) is endemic and represents a major risk factor for the development of this cancer. Several mechanisms for HBV-related hepatocarcinogenesis have been proposed, among them a direct role of HBV in the promotion of genetic recombination leading to chromosomal alterations. Minisatellite DNA sequences are hypervariable regions dispersed throughout the genome which are susceptible to genetic recombination events. In the present study, somatic rearrangements affecting minisatellite sequences were examined in a total of 26 HCC from Thai patients. Multilocus DNA fingerprinting using probes 33.15 and 33.6 detected rearrangements in 11 and 12 HCC, respectively, all of them carrying integrated HBV DNA. The frequency of rearranged bands was calculated for each probe based on the total number of rearrangements observed in the 26 tumours and the total number of bands revealed by DNA fingerprinting in the non-tumour DNA. With each probe a total of 23 rearrangements was observed, yielding rearrangement frequencies of 3.7% and 4.2% for the 33.15 and 33.6 minisatellite families, respectively. To test for possible clustering of these rearrangements at specific loci, we used minisatellite locus-specific probes previously cloned from 33.15 and 33.6. Minisatellites located at 1p33-35, 7q36-ter and 12q24.3-ter were shown to be frequently affected by rearrangement events in this series of HBV-positive HCC. Frequent rearrangements at minisatellite locus D7S22 (7q36-ter) in HBV-positive human HCC have not been reported so far.

Adolescent↗

PubMed2002

[Antiviral activity of a hammerhead ribozyme against HBV in HepG2.2.15 cells].

The carboxy terminal arginine rich domain of core protein is highly conserved among HBV subtypes, and plays important roles in the packaging of pregenomic RNA and viral replication. Therefore, the 3' highly conserved region of HBV C gene is an attractive target for antiviral therapy mediated by ribozymes. A hammerhead ribozyme RzC, targeting the above site, was designed; meanwhile, as controls, the disabled form of RzC, dRzC, was also designed. In order to investigate its antiviral effects in cultured cells, in vitro-transcribed ribozymeRzC, dRzC and ribozyme expression vectors pCRzC, pCdRzC were delivered, respectively, into HepG2.2.15 cells ( clonal cells derived from HepG2 cells that contain integrated HBV ayw DNA). The preliminary results demonstrated that in vitro-transcribed ribozyme RzC had weak inhibition on HBV replication, possibly due to its quick degradation by RNases in cells, while the endogenously expressed ribozyme RzC showed significant inhibitory effect on HBV replication. In conclusion, the results suggested the possibility of the hammerhead ribozyme RzC to be used for the gene therapy of HBV infection.

Antiviral Agents↗

PubMed2006

Differentially expressed genes associated with hepatitis B virus HBx and MHBs protein function in hepatocellular carcinoma.

HBx and MHBst products from hepatitis B virus-DNA (HBV-DNA), which become transcriptional transactivators of cellular and viral genes, are known to play causative roles in the development of hepatocellular carcinoma (HCC). However, the biomolecular mechanism(s) for their roles in hepatocarcinogenesis in vivo remain poorly understood. To identify authentic cellular genes involved in HBx and MHBst-transactivated carcinogenesis,we used mRNA differential display polymerase chain reaction (DD-PCR). We examined HBx and MHBs-positive or -negative HCC, which had chromosomally integrated HBV DNA, vs nontumor tissues, respectively, and differentially expressed genes in either type of HCC were identified and compared with each other. Using 240 different combinations of three one-base anchored oligo-dT primers and 80 arbitrary 13mers, 16 genes were differentially expressed in the HBx and MHBs-positive HCC including RoRNA hY1, glutamine synthetase, factor H homologue 3' end, voltage-dependent anionc hannel 3 (VDAC3), three ribosomal proteins, four mitochondrial genes, and four novel genes. Unexpectedly, upregulated genes in association with functional HBV proteins were different from those reportedly transactivated by HBV viral proteins in vitro. Ten genes were downregulated, including three novel genes. In contrast, 15 genes in HCC tissue negative for HBx and MHBs-expression were preferentially expressed including pancreatic secretory trypsin inhibitor (PSTI), H19, guanidine nucleotide-binding protein alpha-1 subunit (GNAZ), carbamyl phosphate synthetase I (CPS I), insulin-like growth factor (IGF)-II, and 10 ribosomal proteins genes. Eighteen genes were downregulated including acute phase genes, a novel gene, and particularly the retinoblastoma susceptibility gene. Only two genes (ribosomal protein P0 and L37a) were commonly upregulated in both types of HCC tissues. These results suggest that cellular genes involved in the viral protein-transactivation may generally differ from those not associated with transactivation in established HCC, and that the specific oncogenic coordination through the transactivation by viral proteins which works in experiments in vitro, may play only a potential role in hepatocarcinogenesis in vivo. In addition, the functional analyses of the eight novel genes identified in this study might be valuable to further understand the mechanism(s) of hepatocarcinogenesis.

Blotting, Northern↗

PubMed2005

High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus.

The serious result of hepatitis B (HBV) virus infection is development of hepatocellular carcinoma (HCC). However, the reason of development of HCC in HBV infected patients is still unclear. Recently, the suppression of cell apoptosis is found to relate with the development of cell carcinogenesis, therefore, the expression of apoptosis inhibitor in the virus related cancer line such as hepatoma cell line HepG2.215 was investigated. There are at least six Human apoptosis inhibitors (IAP) have been identified now. They are cIAP1, cIAP2, XIAP, NAPI, survivin and pIAP. Using gene-assay technology, we have recently compared the expression of IAPs in the HepG2.215 cells that persistently expresses Hepatitis B virus by integrated HBV genome with its parent cell line HepG2. The results suggest that there was obviously increase of cIAP2 and cIAP1 in the HepG2.215 cells versus HepG2 cells. Those observations imply a possibility of long time HBV infection could induce the over-expressing apoptosis inhibitors, furthermore, causing the liver cancer. The high expression of cIAP1 and cIAP2 in HBV expressing cells was confirmed by RT-PCR and Northern blot analysis. However, we did not find the change of NIAP and suvivin in HepG2.215 cells. In contrast, the expression of XIAP was down in the HepG2.215 cells comparing with HepG2 cells. How HBV triggers the over-expression of apoptosis inhibitor is unclear. Transient transfection of HepG2 cells with the plasmids expressing different HBV proteins such as S, M, L, X and core proteins did not give a decisive conclusion. Further study is going on now.

Journal Article↗

PubMed1986

Interactive effects of chemical carcinogens and hepatitis B virus in the pathogenesis of hepatocellular carcinoma.

Liver cancer is one of the most prevalent forms of cancer in the world. Hepatitis B virus (HBV) is considered to be a major aetiological factor. Evidence from epidemiological studies has also indicated that environmental contaminants such as mycotoxins may, either in combination with HBV or independently, be important aetiological factors in the pathogenesis of primary hepatocellular carcinoma (PHC). Laboratory data also suggest an interplay between viral and chemical factors in the multifactorial aetiology of PHC. Aflatoxin B1, the chemical carcinogen most frequently implicated in the aetiology of hepatocellular carcinoma is a procarcinogen that must be activated by mixed-function oxidases to an electrophilic metabolite before it can exert its carcinogenic effects. Interindividual differences (greater than 10-fold) in the metabolic activation of aflatoxin B1 are observed. These differences may play a part in an individual's oncogenic susceptibility to aflatoxin B1. Chemical carcinogens and integrated HBV may activate cellular oncogenes, eg N-ras, and inactivate tumour suppressor genes. Recently developed methods that allow monitoring of aflatoxin B1 and HBV exposures and also genetic damage caused by these agents in individuals should help in biochemical and molecular epidemiological studies concerning the aetiology of hepatocellular carcinoma. We identify areas of uncertainties and of future experimentation and propose a hypothesis of liver carcinogenesis.

Aflatoxin B1↗

PubMed1996

Hepatitis B virus transcriptional activators: mechanisms and possible role in oncogenesis.

The hepatitis B virus (HBV) genome encodes a 154 amino acid protein termed X (HBx, hepatitis B x protein), which is a promiscuous transcriptional activator of polymerase II and III promoters. HBx upregulates a wide range of cellular and viral genes and is thought to facilitate viral pregenome and mRNA transcription; however, its precise role in the viral replication cycle remains to be elucidated. The functional mechanisms of HBx appear very complex. It was shown to activate transcription factors AP-1 and NF-kappa B vis cytoplasmic pathways including ras-MAP kinase. In contrast, nuclear HBx is thought to activate the transcriptional machinery directly. A second transcriptional activator protein (Mst, middle s transactivator) is encoded by 3'-truncated preS2/S sequences of integrated HBV DNA, but not by the intact viral gene. HBx and Mst may contribute to the pathogenicity of chronic hepatitis B and are suggested to promote hepatocyte transformation via upregulation of cellular proto-oncogenes. Further, HBx may enhance HBV related carcinogenesis by inactivation of the tumour suppressor gene product p53.

Animals↗

PubMed1997

Identification of hepatitis B virus integration in hepatitis C virus-infected hepatocellular carcinoma tissues.

BACKGROUND/AIMS: The integration of HBV DNA is thought to be involved in the initial stage of hepatocarcinogenesis, and it has been reported that transactivating factors encoded by the X and preS2/S genes stimulate transcription of multiple viral and cellular genes. We assessed the possible contributions of hepatitis B virus integration to the occurrence of hepatocellular carcinoma in hepatitis C virus-infected as well as in hepatitis B virus-infected patients by identifying the integrated HBV DNA sequence, and the X and preS2/S regions were further investigated in HBV DNA-integrated cases. METHODS: Southern blot hybridization for detecting HBV DNA in tumor tissues from 28 hepatocellular carcinoma patients was carried out with full-length HBV DNA, and then with X and preS2/S regions as probes. We also carried out reverse transcription-polymerase chain reaction for detecting HCV RNA to confirm hepatitis C virus-infection in liver tissues. RESULTS: Clonally integrated HBV DNA sequences were demonstrated in 16 of 28 patients (57.1%), including five HBsAg seropositive and 11 HBsAg seronegative patients. Of these 11 HBsAg seronegative patients, 10 were also positive for anti-HCV in their sera, and all nine examined cases had HCV RNA in liver. Furthermore, the X region was identified in 14 of 16 HBV DNA integrated cases (87.5%), and the preS2/S region in 6/16 (37.5%). CONCLUSIONS: The present Southern blot analysis demonstrates that clonally integrated HBV DNA sequences were identified even in hepatitis C virus-infected hepatocellular carcinoma patients at a high rate (10/18, 55.6%), and suggests that integrated hepatitis B virus, whose major component is the X gene, may play an important role in hepatocarcinogenesis in hepatitis B virus-integrated cases with and without hepatitis C virus infection.

Adult↗

PubMed1996

Mutation of p53 gene in hepatocellular carcinoma cell lines with HBX DNA.

It has been known that the incidence of p53 mutation is very rare in HBX-positive primary human hepatocellular carcinoma (HCC) tissues. The frequency of p53 mutation, however, in established cell lines with integrated HBV DNA and/or HBX has not been well studied. To know p53 mutational frequency, and to investigate whether the presence of HBX DNA sequence correlates with the absence of p53 mutation in the established HCC cell lines, we studied the p53 mutation and the presence of HBX sequence in 12 recently characterized HCC cell lines. As a result, all 12 (100%) lines showed mutation in the p53 gene. Three (25%) cell lines had transversion of codon 215 while no mutation of codon 249 was found. In contrast with previous reports, although HBX DNA was present in 11 cell lines, p53 mutation had occurred, indicating that the presence of HBX viral DNA does not correlate with a lack of p53 mutation in established HCC cell lines. Our results suggest that the frequency of p53 mutation is extremely high even in HBX DNA positive HCC cell lines.

Carcinoma, Hepatocellular↗

PubMed2001

Hepatitis B virus X protein induced expression of the Nur77 gene.

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in development of HBV-associated hepatocellular carcinoma (HCC). Recently, we reported that HBx induces Fas Ligand (FasL) expression, which may help HCC cells to evade host-immune surveillance. The aim of this study was to investigate the role of HBx in expression of Nur77, an orphan nuclear receptor implicated in the upregulation of FasL. When Chang X-34 expressing HBx under the control of a doxycycline-inducible promoter was examined, induction of Nur77 was observed following HBx expression. Blocking of Nur77 function by introduction of an antisense or a dominant negative mutant Nur77 significantly inhibited the induction of FasL, indicating that Nur77 plays critical roles in FasL expression. Further, a high-level expression of transcripts and DNA binding of Nur77 were observed in the HBV-integrated cell lines established from HCC patients that express HBx. These results suggested that Nur77 may contribute to leading the HBx-induced Fas/FasL signaling pathway which eliminates invading Fas-expressing lymphocytes.

Carcinoma, Hepatocellular↗

PubMed1995

Hepatocarcinogenesis in rodents and humans.

In hepatocarcinogenesis in rodents, induction of foci and nodules comprising clonally proliferated initiated cells is considered to be essential for the future development of carcinomas. Nodules in human cirrhotic liver, though known to be associated with a high hepatocellular carcinoma risk, have generally been regarded as regenerative in nature, and not the result of clonal or neoplastic cell proliferation, on a morphological basis. However, when we analyzed 83 cirrhotic nodules from 11 HBV carrier patients, utilizing hepatitis B virus (HBV) integration as a marker for clonal proliferation, we found the existence of clonal populations of more than 10(5) hepatocytes in 26 (31.3%) of them. Although such clonal cell populations are morphologically not discernible from neighboring hepatocytes, they may have particular histogenetic significance in human hepatocarcinogenesis and clearly deserve further investigation. Allelotype analysis of mouse hepatocellular carcinomas (HCC), induced by a single dose of diethyl nitrosaminine in C3H/MSM F1 hybrids, revealed no remarkable alterations in the original tumors when microsatellite probes were used, but loss of heterozygosity of chromosome 4 at extremely high frequency (95%) in cultured cell lines derived from these HCC. The shortest common region was about 10 cM distal to the interferon alpha gene, in which the p16 gene is located. The results indicated that loss of gene function, most probably including that of the p16 gene, may be essential for immortalization of cultured hepatocytes but that it may not play any role in initiation or early events in mouse hepatocarcinogenesis in vivo. The mouse HCC used for analysis in this study may be comparable with human HCC at an early stage, for which only very limited genetic alterations have so far been identified.

Alleles↗

PubMed1994

An evaluation of the flow cytometric nuclear DNA analysis of intrahepatic multinodular hepatocellular carcinoma for a diagnosis of their multicentricity.

This study aims to evaluate the clinical significance of the nuclear DNA index (DI) for identification of multicentrically occurring (MC) hepatocellular carcinoma (HCC). In 14 multinodular HCC patients, the DI of 30 HCC specimens and 14 non-cancerous liver tissues were analyzed by flow cytometry. Histological studies of the 30 HCCs revealed MC in 6 cases and intrahepatic metastasis (IM) in 7 cases except for a histologically undetermined case who was found to be a hepatitis B virus (HBV) carrier, and the MC of this case was determined by a clonal study using the HBV integration pattern. In four of the seven specimens with MC HCC, the DI of all the intrahepatic tumors was 1.0 (diploid pattern), while the remaining three were different. On the other hand, five of the seven IM cases were identical (3 diploid and 2 aneuploid), one similar level (DI = 1.17-1.18) and one different (1.0 and 1.24). Moreover, in one IM case, the possibility of an alteration of the DI during the course of HCC development was investigated. Although the DI of the recurrent main tumor (DI = 1.17) of this case, which was identified as metastasis of the primary tumor by a clonal study, was also similar to that of subsequent metastatic lesions (DI = 1.18), the DI of the primary tumor was 1.0. These results indicate that DI analysis was not enough to make a differential diagnosis of the multicentric occurrence of HCC.

Adult↗

PubMed2000

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.

Adaptor Proteins, Signal Transducing↗