Cultivation of hepatitis A virus in various cell lines and isolation of DNA sequences

Using the MBB/11/5 strain originally adapted to the PLC/PRF/5 cell line, reproduction of the virus in diploid cells of human embryo fibroblasts, continuous primate lines (RAMT, FRhK-4) and human urinary bladder tumor (T-24) lines was studied. We obtained HAV DNA sequences (about 99% from vRNA) for the MBB/11/5 strain which were used as a probe for demonstration of vRNA synthesis in the infected cells.     

Inhibition of human hepatocellular carcinoma and hepatoblastoma cell lines by deferoxamine.

Inhibition of human hepatocellular carcinoma (PLC/PRF/5 and Hep3B) or hepatoblastoma (Hep G2) cell lines by inclusion of deferoxamine mesylate (desferrioxamine) (DFX) in the culture medium was evaluated. When PLC/PRF/5 cells were maintained for 7 days in 30 or 60 microM DFX, the cell number was decreased by 30-60%, little or no alpha-fetoprotein (AFP) was produced, and supernatant endpoint dilution titers of hepatitis B surface antigen (HBsAg) were reduced 1-2 logs. PLC/PRF/5 cells maintained for 7 days without DFX (simultaneous controls) grew to confluence, produced AFP that reached 10-60 ng/ml in the supernate, and the HBsAg titer remained constant or increased 1 log. Similar effects were observed in Hep3B and Hep G2 cells maintained in DFX (except that Hep G2 cells do not produce HBsAg), compared to simultaneous control cells grown in the absence of DFX. The growth of a human embryonic lung fibroblast cell line (Wl 38) was not significantly inhibited by DFX, although it grew at a slower rate than simultaneous control cells grown without DFX. Subsequent growth in FeSO4 of PLC/PRF/5, Hep3B, and Hep G2 cells that previously had been maintained in DFX did not reverse the effects of DFX. PLC/PRF/5 cells were also inhibited when maintained in medium containing equimolar concentrations of DFX and FeCl3 and in medium containing equimolar concentrations of DFX and FeSO4. PLC/PRF/5 cells were not inhibited by maintenance in up to 60 microM of another chelating agent that has a similar affinity for iron, calcium disodium versenate (EDTA). These studies show that DFX inhibits the growth of human hepatocellular carcinoma and hepatoblastoma cell lines regardless of the presence (PLC/PRF/5, Hep3B) or absence (Hep G2) of integrated hepatitis B virus DNA. The findings also suggest that the inhibition may have been due to mechanisms other than iron chelation.     

Expression of oncogenes and tumor suppressor genes in human hepatocellular carcinoma and hepatoblastoma cell lines.

The expression of nine oncogenes (c-myc, N-myc, N-ras, H-ras, k-ras, abl, fos, src, and raf) and two tumor suppressor genes (p53 and RB) were studied by northern blot hybridization in six human hepatocellular carcinoma or hepatoblastoma cell lines (PLC/PRF/5, Hep3B, Hep G2, 2.2.15, HLE, and HLF) and in a human embryonic lung fibroblast cell line (WI-38) to look for differences that might be associated with the presence (PLC/PRF/5, Hep3B, and 2.2.15) or absence (Hep G2, HLE, and HLF) of integrated hepatitis B virus (HBV) DNA. The levels of expression of the oncogenes and tumor suppressor genes were unrelated to the presence or absence of integrated HBV-DNA. Furthermore, the intensity of expression of these oncogenes was no greater in the 2.2.15 cell line (consisting of Hep G2 cells transfected with hepatitis B virus) than in untransfected Hep G2 cells.     

Secretion of polyalbumin receptors in vitro

The nature of hepatitis B surface antigen (HBsAg)-associated receptors for polymerized human serum albumin (pHSA-R) and their relationship to hepatitis B e antigen (HBeAg) and human serum proteins have not been defined. We studied by radioimmunoassay and by electron microscopy HBsAg-associated pHSA-R secreted in vitro by a human hepatocellular carcinoma cell line (PLC/PRF/5) and by mouse 3T3 fibroblasts after transfection with cloned hepatitis B virus (HBV) DNA (4.10 cells). PLC/PRF/5 cells expressed only HBsAg, whereas 4.10 cells secreted also HBeAg. There was no significant difference in the production of HBsAg, HBeAg, and pHSA-R when the cells were cultured in the presence or absence of fetal calf serum. Secretion of pHSA-R by the two cell lines for a given amount of HBsAg was equal irrespective of the presence or absence of HBeAg. Supernatants from both cell lines grown in serum-free medium did not contain any Clq or albumin when tested by immunodiffusion. The ability of a transfected mouse cell line to produce HBsAg with pHSA-R activity strongly suggests that pHSA-R is coded by the HBV genome and does not depend on the presence of human serum proteins. In addition, our findings fail to demonstrate any correlation between HBeAg production and pHSA-R.     

Isolation and propagation of hepatitis A virus in hepatoma cell cultures

Hepatitis A virus (HAV) was isolated directly from human faeces in PLC/PRF/5 cells. In the first passage cell-bound and supernatant viruses were found by immune electron microscopy and by enzymeimmunoassay. Serial passaging of HAV in PLC/PRF/5 cells resulted in its adaptation to the cell line and in reduction of the incubation time. HAV was still detectable after 10 cell passages. Cell-bound as well as supernatant HAV were employed as antigens in anti HAV IgM-enzymeimmunoassay.     

Adenovirus infection induces amplification of persistent viral DNA sequences (simian virus 40, hepatitis B virus, bovine papillomavirus) in human and rodent cells

Adenoviruses, types 2 and 12 induce amplification of SV40 DNA sequences in cells of the SV40-transformed human newborn kidney cell line, NB-E. Similarly, integrated hepatitis B virus DNA sequences in the human hepatoma cell line, PLC/*PRF/5, and bovine papillomavirus (BPV) DNA sequences in BPV-transformed mouse cells (ID13) are amplified by adenovirus infection. Thus, similar to herpes group or vaccinia viruses or DNA damaging agents, adenoviruses are able to mediate selective DNA amplification in addition to their reported mutagenic and chromosome damaging effects. The role of amplification of integrated viral DNA sequences in development and progression of specific tumors (e.g. hepatocellular carcinoma) remains to be determined.     

Detection of integrated hepatitis B virus DNA in hepatocellular carcinoma cell lines by nonradioactive in situ hybridization

A sensitive and specific nonradioactive in situ hybridization method capable of detecting single-copy integrated hepatitis B virus (HBV) DNA sequences in hepatocellular carcinoma (HCC) cell lines was developed. In situ hybridization of biotinylated HBV (adr, adw) DNA probes with nine different human HCC cell lines were carried out in 96-well microtiter plates. Integration was detected in HCC cell lines HCCM, Hep3B, huH-1, huH-4, and PLC/PRF/5. Detection of single-copy HBV DNA sequences was also achieved in Hep3B and huH-4. HCC cell lines HepG2, HUH-6, HUH-7, Mahlavu, and the non-HCC control MCF-7, gave clear negative results. These results show a 100% correlation with those obtained by Southern blot hybridization assay. The results demonstrate that nonradioactive detection of single-copy integrated HBV DNA sequences in HCC cell lines is possible by the method described, which has potential application for viral genome analysis requiring in situ hybridization.     

Discovery of the sequences of herpes simplex virus type 1 in the genomes of normal and transformed mouse and hamster cell lines

Sequences capable of hybridization with cloned fragments of herpes simplex virus type I (HSVI) DNA were found in genome DNA of Syrian hamster fibroblast lines transformed either by intact HSVI genome (cell line 14.012.81) or this virus DNA fragments (cell line EH/A44). The method of pinpoint hybridization demonstrated the presence in DNA of the cell lines under study of sequences capable of hybridization both with unique areas and with HSVI genome replicas. A correlation was established between the tumorigenic properties of the transformed lines and the number of HSVI sequences present in genomes of these lines. The genome of mouse cell line BaLB/3T3 was found to contain sequences homologous to HSVI replica areas, their number exceeding that of virus sequences present in the genome of nontransformed hamster cells (BHK/21 line).     

Comparison of manganese superoxide dismutase precursor induction ability in human hepatoma cells with or without hepatitis B virus DNA insertion

In a previous study (Hajnická et al., Acta virol. 38, 55-57 (1994)), we described synthesis of a 23 K protein in high amounts in the PLC/PRF/5 human hepatoma cell line after stimulation with sera of patients suffering from liver cirrhosis. In this study we identified this protein as manganense superoxide dismutase (Mn-SOD). When PLC/PRF/5 cells stimulated by various cytokines (interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, IL-6, tumor growth factor-alpha (TGF-alpha), TGF-beta, and interferon-gamma (IFN-gamma) were compared, the most effective was IL-1, followed by TNF-alpha and IL-6. Other cytokines had no effect on the stimulation of Mn-SOD. IL-1 alpha was selected for stimulation of Mn-SOD production in four human hepatoma cell lines (PLC/PRF/5, Hep-3B, Hep-G2 and Sk-Hep 1). Maximum Mn-SOD production occurred in PLC/PRF/5 cells. In other cell lines, Mn-SOD production was lower, reaching 35.7% and 31.5% in Hep-3B and Sk-Hep-1 cells, respectively, while it was only 4.3% in Hep-G2 cells.     

Human astrovirus isolation and propagation in multiple cell lines

Summary.  Laboratory adapted human astrovirus serotypes 1 through 7 were tested for growth in 15 human, 7 simian, and 10 other non-primate mammalian cell lines. Propagation of all seven serotypes was successful in the human cell lines Caco-2, T84, HT-29, and in the African green monkey kidney cell line MA-104. Both primary and secondary African green monkey kidney cells were more effective than Rhesus monkey kidney cells for cultivation of astrovirus. Except for human foreskin cells, all of the other human and simian cell lines supported growth of at least one astrovirus serotype. The only non-primate cell line that permitted sustained passage of astroviruses was the BHK-21 (C13) cell line for astrovirus serotype 2. Seventeen human stool specimens that had previously been shown to be astrovirus positive by ELISA were cultured in Caco-2, T84, HT-29, SK-CO-1, PLC/PRF/5, MA-104, and VERO cells. Caco-2 cells (13 isolates), T84 cells (12 isolates) and PLC/PRF/5 cells (12 isolates) were the cell lines most effective for isolation of human astroviruses from clinical stool specimens. By immunofluorescent staining of infected cells, culturing of the same 17 specimens in shell vials for 18 h was positive for astroviruses in all 17 specimens in Caco-2 cells, 12 in T84 cells, and 7 in PLC/PRF/5 cells. Shell vial assay is suitable as a rapid and sensitive culture technique for detection of astroviruses in clinical specimens. Received December 12, 1999/Accepted March 16, 2000     

Insulin reduces HBsAg production by PLC/PRF/5 human hepatoma cell line

Summary Although its action at the molecular level is not completely understood, insulin, as well as its antagonist glucagon, certaily plays an important role in the modulation of protein synthesis.In order to observe whether insulin is involved in virus gene expression, we studied its effect on PLC/PRF/5 human hepatoma cell line, which posses HBV DNA sequences integrated at several sites.While human insulin had no effect on cell growth and increased the production of two plasma proteins, a selective inhibitory effect on HBsAg production could be detected.This observation might be useful for further studies both on virus gene expression and insulin action at the molecular level.With 3 Figures     

A virus antigen (HBsAg) indicates metastases of human carcinoma cells in the nude mouse

The human hepatoma cell line PLC/PRF/5 produces tumours and lung metastases in nude athymic mice. Here we describe how detection of hepatitis B virus surface antigen, a marker of these cells, has led to identification of pancreatic and muscle metastases. The antigen was shown in the tissues, and antigen-producing PLC/PRF/5 cells were isolated from them. The number of cells detectable by this method is between 5 and 10 million. The principle used here may be adaptable be useful in studying micrometastasis formation in other systems.     

The PLC/PRF/5 human hepatoma cell line. II. Chromosomal assignment of hepatitis B virus integration sites

The chromosomal sites at which hepatitis B virus (HBV) DNA is integrated into the genome of the hepatocellular carcinoma (HCC) cell line, PLC/PRF/5 were investigated in an attempt to understand the mechanisms by which hepatitis B virus may induce malignant transformation. In situ hybridization of an HBV DNA probe to metaphase chromosomes of the PLC/PRF/5 cell line, followed by statistical analysis, identified three integration sites; these were 15q22-q23, 11q22, and 18q12. In particular, hybridization to chromosome #15, which is present in four copies in complete metaphases of this cell line, was highly significant (p much less than 0.0005).     

HLA expression in hepatocellular carcinoma cell lines

The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B. HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu, The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B tymphoid-derived cell line, was shown to express negligible amounts of human leucocyte anligens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte anligens as well as their respective invariant chains, β₂-niicroglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium bulyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class 1 and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.     

Propagation of adenovirus types 40 and 41 in the PLC/PRF/5 primary liver carcinoma cell line.

The sensitivity of cell cultures to adenovirus types 40 and 41 (Ad40/41) was compared by means of cell culture infectious dose (ID50) assays using monolayer cultures in microtitre plates. The PLC/PRF/5 cell line derived from a primary human hepatocellular carcinoma was 100 times more sensitive to a laboratory strain of Ad41, and 10 times more sensitive to a laboratory strain of Ad40 and two Ad41 stool isolates, than Graham 293 and Chang conjunctival cells commonly used for the propagation of these viruses. In microtitre plate titration assays PLC/PRF/5 cells retained an optimal condition for longer and displayed cytopathogenic effects earlier and more clearly than the other cell lines. In contrast to previously used cells, PLC/PRF/5 cells also proved successful for the quantitation of Ad41, but not Ad40, by conventional plaque assays. The reason for the exceptional susceptibility of PLC/PRF/5 cells has not been elucidated, but the findings open attractive new doors for research on Ad40/41.     

Propagation of hepatitis A virus in human embryo fibroblasts

human diploid fibroblasts and human primary liver cell carcinoma cells (PLC/PRF/5) were infected with hepatitis A virus (HAV) adapted to growth in cell culture or derived directly from human stool. Viral antigen was expressed in PLC/PRF/5 cells 28 days after infection with cell culture-adapted HAV, and 50 days after infection with virus from human stool. In human fibroblasts the periods until first expression of viral antigen were 90 and 210 days, respectively. During further passages of HAV in fibroblasts the time of first appearance of antigen decreased to about 28 days. Biophysical properties of HAV extracted from infected fibroblasts were comparable to those of HAV derived directly from human stool. Immunofluorescence studies showed that the antigen was located exclusively within the cytoplasm of the infected fibroblasts. Kinetics of antigen production indicated that an equilibrium between virus multiplication and cell metabolism was reached in persistently infected fibroblasts.     

Cell type specific expression of pre S 1 antigen and secretion of hepatitis B virus surface antigen

Summary Production of the three hepatitis B surface (HBs) proteins was studied in a hepatoma cell line (PLC/PRF/5) and two HBs antigen secreting cell lines (HeLa and mouse L-cells), which had been transfected by a viral genome isolated by molecular cloning from PLC/PRF/5 chromosomal DNA. The DNA used for transfection contains the HBs-specific promoters and the enhancer which regulate the expression of HBs genes in the transfected cell lines. All three cell lines expressed well the small and middle HBs protein, but the larger pre S 1 containing protein was barely detectable in the L-cell. In vivo growth of the transfected HeLa cell as nude mouse tumour increased pre S 1 expression and suppressed secretion of HBsAg.     

Characterization and demonstration of human liver-specific protein (LSP) and apo-LSP

Liver-specific protein (LSP) prepared by standard methods from five normal human livers showed significant variations in terms of quantitative yield, lipid/protein ratio and migration characteristics of different components on SDS-polyacrylamide gel electrophoresis. This heterogeneity is probably related to varying amounts of different molecular species in the LSP preparations. Delipidation and re-chromatography of the LSP preparation appeared to result in relative enrichment of apo-LSP which showed immunological identity with LSP. Rabbit antiserum to LSP gave a precipitin line of identity with standard antisera to human LSP (anti-LSP) from two other laboratories. After extensive absorption, anti-LSP showed selective reactivity with a surface membrane antigen on a human hepatocellular carcinoma cell line (PLC/PRF/5) that exhibits functional and morphological characteristics of differentiated hepatocytes. The antiserum did not react with cell lines derived from other organs as determined by the indirect fluorescent antibody technique. The surface staining of viable PLC/PRF/5 cells was eliminated by absorption with LSP and apo-LSP, but not with the equivalent kidney fractions. These findings support the concept of a liver-specific antigen and suggest that the PLC/PRF/5 cell line may serve as a source of homogeneous LSP.