A hepatocellular carcinoma cell line producing mature hepatitis B viral particles

Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10II, exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10II cell genome. The presence of HBV in the FLC4A10II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10II, can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents.     

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.     

Structure and expression of integrated hepatitis B virus genes in an HBs antigen producing human cell line (huGK-14)

A human continuous cell line (huGK-14) within a lineage of passaged cultures was investigated in the mode of integration and expression of hepatitis B virus (HBV) genes. HBV DNA was integrated in eight different sites of the cellular DNA, in each of which HBV genome was rearranged, fragmented, and/or partly deleted. Complete HBV genome that may lead to production of infectious virus particles was not detected in the cells nor in the culture medium. Clones of cDNA containing a complete coding frame for small HBs antigen protein (type adr) were obtained from mRNA of the cells. The cells were stable over the period of six months of cultivation and more than 60 population doublings in the mode of HBV integration and HBs mRNA expression. These results provide substantial evidence for the absence of an ability for the integrated DNA to create an infectious product in the cell; for the stable production of HBs mRNA from the cells, and suggest the usefulness of this cell line as a substrate for HBV vaccine production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.     

Integration of hepatitis B virus: analysis of unoccupied sites.

Hepatitis B virus (HBV) sequences integrated in the PLC/PRF/5 cell line (Alexander cells), which was derived from a human primary liver carcinoma, were previously extensively studied. Here we describe the analysis of the unoccupied sites of two linearly integrated forms of HBV DNA, AL-14 and AL-26, that were characterized previously. No major cellular DNA rearrangements were seen at the integration sites except for small deletions of host sequences: 2 kilobases of DNA in AL-14 and 17 base pairs (bp) in AL-26. The unoccupied site of AL-26 was found to be missing 182 bp, which previously mapped next to the right end of the integration sites of several independent clones. These were believed to be of cellular origin, but we show here that these 182 bp are in fact from unusual HBV sequences. Surprisingly, a region of this newly detected HBV DNA sequence is more homologous to that of woodchuck HBV DNA. Our analysis shows that the normal counterparts of both AL-14 and AL-26 contain minisatellite-like repetitive sequences. Based on the data presented here and our previous finding of HBV DNA integration at satellite sequences, we propose that genomic simple repetitive sequences are hot spots for HBV DNA integration.     

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus.

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.     

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。     

The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

A human hepatocellular carcinoma cell line (FOCUS--Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular origin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, alpha 1-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectable in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and its contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous injection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Nucleotide sequences in human chromosomal DNA from nonhepatic tissues homologous to the hepatitis B virus genome

DNA extracted from human nonhepatic tissues (placenta and kidney) have been digested with restriction endonucleases and examined by the Southern procedure with cloned 32P-labelled DNA of hepatitis B virus (HBV). In placental DNAs of women with the history of a hepatitis B infection and in one out of four cases of patients with no known HBV exposure or manifestation, HBV-related chromosomal nucleotide sequences were detected. The integration of HBV-related sequences was observed also in human kidney DNA. Moreover, in the placenta of women who had hepatitis B infection prior to delivery, unusual unintegrated forms of HBV have been found. We conclude that HBV sequences can be found not only in hepatic tissue but also in placental and kidney DNA, both of HBV-exposed and in one case even of a nonexposed patient.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Late transient expression of human hepatitis B virus genes in monkey cells

The expression of human hepatitis B virus (HBV) surface (HBS) and e (HBe) antigens has been studied comparatively in monkey and mouse cell lines co-transfected with HBV DNA and the dominant selective marker aminoglycoside 3'-phosphotransferase gene. We have found that the kinetics and stability of expression of the HBS gene varies with the cell lines used. Only a late transient expression of both HBS and HBe is observed between 1 and 5 weeks after transfection in monkey kidney Vero cells transfected with the complete HBV genome, while a permanent expression of HBS and HBe is obtained in mouse cells. HBS and HBe are excreted into the cell culture medium. HBe is expressed in cells transfected with the complete HBV genome, but not with isolated HBS gene. In clones of Vero cells transformed with the HBS gene, HBV sequences were rearranged or lost.     

Rearrangement of the surface antigen gene of hepatitis B virus integrated in the human hepatoma cell lines.

The rearrangement of integrated HBV DNA sequences in three different hepatoma cell lines, huH-1, huH-2, KG-55-T from Japanese patients, were studied by blot hybridization using whole HBV genome or a HBsAg or HBcAg DNA as a probe. The characteristic existence of multiple integration sites of HBV DNA sequences in each HindIII-restricted hepatoma cell DNA was revealed by the HBV genome probe. Detection of the isolated HBsAg gene in the HindIII fragment indicates that the integration of HBV DNA was not always related to the maintenance of the whole viral genome, and that movement of the HBsAg gene to another location occurred by rearrangement. On the other hand, the presence of the HBV DNA sequence without the intact HBcAg gene was shown in some of the HindIII fragments, when the HBcAg gene, probe was used, but a HindIII fragment, containing only the HBcAg gene, was not detected so far. The absence of the intact HBcAg gene suggests that the viral genome may lose a part of the HBcAg gene in the process of integration. This is consistent with recent findings of Ogston et al. (1982) that in Woodchuck hepatocellular carcinoma viral sequences are extensively rearranged.