Plasma carriers influence the uptake of cholecalciferol by human hepatoma-derived cells.

The uptake of [3H]cholecalciferol by the human hepatoma-derived cell lines Hep G2 and Hep 3B was examined as a function of the sterol's presentation on various plasma proteins at their native concentrations. Control cultures utilized devitalized cells cross-linked with glutaraldehyde and estimated nonspecific sterol adherence to cells. With both cell lines, neither albumin nor plasma vitamin D binding protein permitted cholecalciferol uptake above control values. With Hep G2 cells, only low-density lipoprotein presentation of the sterol resulted in significant cellular uptake that had features resembling a receptor-mediated process. With Hep 3B, only high-density lipoprotein presentation of the sterol resulted in a significant uptake that was cell, carrier, and time dependent. These results support the hypothesis that lipoprotein carriers could account for the efficient hepatic accumulation of cholecalciferol in vivo.

Characterization of thyroxine-binding globulin secreted by a human hepatoma cell line.

T4-binding globulin (TBG) is a glycoprotein synthesized by the liver and is the principal carrier of T4 and T3 in serum. In this report, we demonstrate that the Hep G2 cell line, derived from a human hepatoblastoma, synthesizes and secretes TBG, the properties of which were characterized. Hep G2 cells secreted TBG into the medium after more than 100 transfers in tissue culture conditions. At confluency and after changing to serum-free culture conditions, TBG accumulation into the medium was linear for 3 days and constituted approximately 0.16% of the proteins synthesized over 24 h. Its abundance relative to albumin is 10-fold greater than that found in normal human serum. TBG secreted by the Hep G2 cells was indistinguishable from native normal human serum TBG, as determined immunologically, by electrophoresis on polyacrylamide gel in denaturing and nondenaturing conditions, and by isoelectric focusing. It also specifically bound T4 and T3, albeit with slightly reduced affinity, and had increased heat lability. Although slightly different from normal serum TBG in caucasians, the physical and biological properties of the Hep G2-derived TBG are similar to those of the variant TBG found in the serum of some healthy Australian Aborigines.

Histone complements of human tissues, carcinomas, and carcinoma-derived cell lines.

The pattern of subtypes of the nucleosomal histones and of histone H1 was investigated in human cells from adult and fetal lung and liver, from carcinoma tissues and from carcinoma-derived cell lines, with the object of comparing these patterns, and their relationship to cell growth rate, with those in cells of other species. The subtype pattern of the nucleosomal histones H2A and H3 shows a correlation with replication rate. In adult tissues, subtype H3-3 predominates over H3-2 and H3-1, and the subtype H2A-1 and H2A-2 are approximately equally abundant. In fetal tissues, lung carcinoma and cultured carcinoma-derived cell lines, the subtype H3-1 is predominant and H2A-1 is more abundant than H2A-2. The subtype pattern of H1 also differs between normal and carcinoma cells, among different tissues, and in different cell lines derived from the same type of carcinoma. In particular, the relative level of H1 degrees differs in several cell lines showing relatively high rates of replication, and in some cases represents more than 25% of the total H1, similar to the level in slowly replicating normal adult liver and lung tissues. The relative level of H1 degrees does not therefore appear to be correlated in a simple manner with cell growth rate in these human cells.

Hormonal influences on the secretion of steroid-binding proteins by a human hepatoma-derived cell line.

We examined the influence of numerous substances on the secretion of corticosteroid-binding globulin (CBG) and testosterone-estradiol binding globulin (TeBG) by a human hepatoma-derived cell line. Thyroxine, at physiologic concentrations, resulted in an increased secretion of TeBG but not CBG. Estrogens, antiestrogens, and androgens were without effect on either of these binding proteins.

Characterization of the human plasma binding protein for vitamin D and its metabolites synthesized by the human hepatoma-derived cell line, Hep 3B.

Screening of three human hepatoma-derived cell lines revealed the presence of an immunologically similar plasma binding protein for vitamin D and its metabolites in media from Hep 3B cells. Approximately 3% of protein synthesized and secreted by these cells was immunoprecipitated by specific antiserum to the D-binding protein. Medium content of the protein increased over 11 days following cell seeding, and negligible amounts of 125I-labeled binding protein added to cultures were degraded over 48 h. The hepatoma-derived binding protein was indistinguishable from plasma binding protein or reference pure protein in gel filtration, sucrose gradient ultracentrifugation, and polyacrylamide gel electrophoresis systems. The Hep 3B cell product was found to bind mole/mol with monomeric actin, and bind vitamin D sterols with an affinity and specificity characteristic of the human plasma binding protein. The findings argue strongly for the identity of the Hep 3B cell product and the human plasma protein. The continuous availability of the Hep 3B cell line provides a reasonable model for investigations of biosynthesis and release of this important plasma protein.

Demonstration that a human hepatoma cell line produces a specific insulin-like growth factor carrier protein.

Insulin-like growth factors (IGF's) circulate in blood complexed to specific carrier proteins. The BRL 3A and BRL 3A2 rat liver cell lines secrete a 30,000-50,000 mol wt IGF carrier protein. Since the liver parenchymal cell is a likely source of IGF carrier protein synthesis, we have evaluated medium conditioned by three human hepatocellular carcinoma- or hepatoblastoma-derived cell lines for the presence of IGF carrier protein. The HEP G2 and the HEP 3B, but not the PLC/PRF/5, cell lines secrete a specific IGF carrier protein(s) into serum-free medium. This carrier protein is specific for IGF molecules. Multiplication-stimulating activity, IGF I, and IGF II were equipotent in competing for the binding of [125I]multiplication-stimulating activity to HEP G2 medium. The HEP G2 IGF carrier protein is trypsin sensitive, acid stabile, and does not contain a glycoprotein moiety. It has a molecular size of 30,000-50,000, as assessed by Sephadex G-200 gel filtration. These studies suggest that the HEP G2 cell line is a useful model to study the synthesis and secretion of human IGF carrier protein.

Identification of human hepatoma-defined cell surface molecules.

BALB/c mouse splenocytes from mice immunized with cells of the human hepatoma line Hep G2 were fused with SP2/0-Ag 14 mouse myeloma cells. Two monoclonal antibodies recognizing antigenic determinants (Hag-1, Hag-2) of hepatoma cell surface molecules were investigated. Analysis of immunoprecipitates by sodium dodecyl sulfate (SDS) gel electrophoresis revealed that the Hag-1 antigenic determinant is born ona 115, kD MW glycoprotein, and that the second antibody immunoprecipitates a group of surface proteins with MW of 230 kD, 79 kD, 23 kD, and 20 kD from human hepatoma cells. These antigenic determinants are present on cell lines derived from other human tumors, thus neither of the antibodies is hepatoma-specific; cross-reactivity with human colorectal carcinoma and some mammary carcinoma cell lines is notable. Using indirect immunofluorescence on frozen sections Hag-1 was detected in one of three liver biopsies tested whereas Hag-2 was demonstrated in all three. Both antigens were detected in sections of human kidney with Hag-2 localized to the proximal tubules.

Complement biosynthesis by the human hepatoma-derived cell line HepG2.

The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.

Synthesis and secretion of alpha 2-plasmin inhibitor by established human liver cell lines.

The site of synthesis of alpha 2-plasmin inhibitor (alpha 2-PI), a physiologic inhibitor of plasmin, is not known with certainty. We have studied the production and secretion of alpha 2-PI by three established human liver cell lines derived from hepatocellular carcinoma and hepatoblastoma (Hep G2, Hep 3B, and PLC/PRF/5). As measured by a specific radioimmunoassay, the titer of alpha 2-PI increased in the medium of Hep G2 and Hep 3B cells with time, but no significant amount of alpha 2-PI was found in the medium of PLC/PRF/5. There was no evidence for a significant intracellular pool of this protein. On immunodiffusion against anti-alpha 2-PI serum, alpha 2-PI secreted by Hep G2 (G2 alpha 2-PI) formed a simple precipitin line of complete identity with the alpha 2-PI present in plasma (plasma alpha 2-PI). G2 alpha 2-PI behaved similarly to plasma alpha 2-PI in Sephadex G-150 gel filtration, sucrose density-gradient centrifugation, and crossed immunoelectrophoresis. G2 alpha 2-PI inhibited plasmin activity instantaneously in a functional assay and formed a complex with plasmin demonstrable by crossed immunoelectrophoresis. De novo synthesis of alpha 2-PI was shown by the presence of specific immunoprecipitable radioactivity in the medium after 5 hr of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by NaDodSO4/polyacrylamide gel electrophoresis showed a single peak of radioactivity corresponding to Mr 68,000. These results indicate that the liver is a site of alpha 2-PI production.

Chromosomes of human hepatoma cell lines.

The karyotypes of three human hepatoma cell lines Hep G2, Hep 3B and PLC/PRF/5 were investigated by G- and C-banding techniques. In addition to ploidy changes, typical for most carcinoma cell lines, certain markers were found that remained stable throughout passage of these cultures. Chromosome I is involved in multiple translocations, resulting in at least three copies of the chromosome I heterochromatin region in each cell line. Inversion in the 9qh region is also seen. In addition, each of the cell lines consistently contains trisomy of 17q. The rearrangements of chromosome I are most striking in the Hep 3B and PLC/PRF/5 cell lines, which are derived from human hepatocellular carcinomas and contain integrated copies of the hepatitis B viral genome. These two cell lines are characterized by the presence of at least five copies of the I (p13 leads to q21) region that result from multiple deletions and/or translocations; by consistent trisomy and polymorphism of the 9qh region; and by trisomy of chromosome 10 (also involved in rearrangements). The Hep G2 and Hep 3B cell lines behave functionally as highly differentiated liver parenchymal cells and are karyologically distinguishable from PLC/PRF/5 both by the presence of trisomy of 6 (pter leads to q14) and by the finding that one of the homologues of chromosome 15 is 15q+.

Biosynthesis and processing of a human precursor complement protein, pro-C3, in a hepatoma-derived cell line.

A 180,000-dalton single-chain molecule (human pro-C3) is the precursor of the third component of human complement (C3), a disulfide-linked two-chain protein. The pro-C3 is converted by limited proteolysis to C3. The relationship between pro-C3 and C3 was established with the use of Hep G2, a cell line derived from a human hepatocellular carcinoma, which synthesizes at least 17 plasma proteins.

Characterization of the major apolipoproteins secreted by two human hepatoma cell lines.

Two newly described human hepatoma derived cell lines, Hep G2 and Hep 3B [Knowles, B. B., Howe, C. C., & Aden, D. P. (1980) Science (Washington, D.C.) 209, 497-499], synthesize and secrete into the culture medium most of the major plasma apoproteins (apoA-I, apoA-II, apoB, apoC-II, apoC-III, and apoE). The synthesized apoproteins were identified by direct two-dimensional gel analysis of the culture medium or by two-dimensional analysis following purification of the apoproteins by ultracentrifugation or immunoprecipitation. We found that the apoA-I synthesized by both of the hepatoma cell lines consists of two isoproteins designated 2 and 3 which are more basic than the major plasma apoA-I isoproteins designated 4 and 5. The apoE synthesized by both cell lines is composed mainly of an array of isoproteins with increasingly higher molecular weights and lower isoelectric points as compared to those of the major apoE isoproteins found in plasma. These precursors of apoE are converted to the major apoE isoproteins upon treatment with Clostridium perfringens neuraminidase and represent sialo apoE isoproteins. ApoA-II, apoC-II, apoC-III-1, and apoC-III-2 correspond to the protein forms present in plasma. The human hepatoma cell lines (Hep G2 and Hep 3B) provide a unique model for studies of the regulation of human apoprotein and lipoprotein synthesis and catabolism.

Secretion of testosterone-estradiol-binding globulin by a human hepatoma-derived cell line.

Using a sensitive radioimmunoassay for testosterone-estradiol-binding globulin (TeBG) we demonstrate that a human hepatoma-derived cell line secretes a protein which: (a) is immunologically indistinguishable from TeBG; (b) has the same Stokes radius as TeBG; (c) is absorbed by concanavalin A in the same way as TeBG in normal plasma.

A murine stage-specific embryonic antigen (SSEA-2) is expressed on some murine SV40-transformed cells.

A stage-specific embryonic antigen-2 (SSEA-2) found on murine preimplantation embryos is maximally expressed on 4- to 8-cell stage embryos and is present in decreasing amounts of morulae and blastocysts. This antigenic determinant is also expressed on murine teratocarcinoma cells, sperm, and some, but not all, SV40-transformed mouse cell lines. Analysis of solubilized immunoprecipitates by SDS-gel electrophoresis indicates that this cell surface molecule is not the 54,000 m.w. protein shared by teratocarcinoma and SV40-transformed cell lines.

Integration pattern of hepatitis B virus DNA sequences in human hepatoma cell lines.

Four human hepatoma cell lines established from primary hepatocellular carcinomas were examined for the presence of hepatitis B virus DNA sequences. Reassociation kinetic analysis indicated that the cell lines HEp-3B 217, HEp-3B 14, HEp-3B F1, and PLC/PRF/5 contained two, one, one, and four genome equivalents per cell, respectively. Southern blot hybridization analysis demonstrated that hepatitis B virus DNA was integrated into the cellular DNAs of these cell lines. Further liquid hybridization studies with 32P-labeled HincII restriction fragments of hepatitis B virus DNA established that DNA sequences from all regions of the HBV genome were represented in the integrated viral sequences. Although the three HEp-3B cell lines were derived from the same tumor, they differed significantly in their patterns of integration of hepatitis B virus DNA, the number of copies of viral DNA per cell, and their ability to produce the virus-coded surface antigen.

Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen.

Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B in injected into athymic mice, metastatic hepatocellular carcinomas appear. These cell lines provide experimental models for investigation of plasma protein biosynthesis and the relation of the hepatitis B viru genome to tumorigenicity.

Metabolic activation of benzo[a]pyrene by a human hepatoma cell line.

The liver-derived human cell line, Hep G2, has high benzo[a]pyrene-metabolizing activity and converts benzo[a]pyrene to intermediates that are mutagenic and that bind to DNA. This cell line will be useful for studying metabolic activation of polycyclic aromatic hydrocarbons and other xenobiotics by human tissue and as an activation system in short-term screening assays for identifying compounds with carcinogenic potential for humans.

Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line.

A significant aspect of primary hepatic carcinoma in man is the high positive correlation of hepatocellular carcinoma with infection with hepatitis B virus (HBV)1. Analysis of the relationship between HBV infection and oncogenesis is difficult because natural infection with HBV is limited to man and experimental infection has been achieved only in chimpanzees and gibbons. Furthermore, because HBV has not been successfully propagated in cell culture, basic study of virus-cell interaction of the aetiological agent of one of the most widespread infections of man has been impossible. Recently, however, a cell line (PLC/PRF/5) derived from a human hepatoma biopsy was described which produces the HRV surface antigen (HBsAg) and so provides a tool for the experimental investigation of HBV in viro. We now report the derivation and characterisation of two additional cell lines primary liver carcinomas. In contrast to the PLC/PRF/5 cell line, these cell lines retain the capacity to synthesise many human plasma proteins, including both albumin and alpha-fetoprotein (AFP). One of these lines also produces BHsAg. We also present evidence that HBsAg synthesis and secretion in this cell line are correlated with the growth state of the culture. This finding is in contrast to the continuous HBsAg production found in the PLC/PRF/5 cell line.