Hepatoma-derived Cell Line HepG2 Research Articles

Influence of Selected Carbon Nanostructures on the CYP2C9 Enzyme of the P450 Cytochrome.

Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.

The antitumor effect of epigallocatechin-3-gallate on gastroenterological cancer.

335 Background: Green tea is one of the most popular beverages it contains many catechins such as epigallocatechin-3-gallate (EGCG) inhibits cancer growth. The aim of this study was to elucidate the anti-tumor effect and the effect for spheres of EGCG. Methods: Human hepatoma-derived cell line (HepG2) and human colorectal carcinoma cell line (HCT-116) were used. The anticancer effects of EGCG was studied by MTT assay. We made cancer sphere from HepG2 cells, and we added EGCG to cancer sphere. And we compared the gene expressions of RT-PCR between normal HepG2 and cancer sphere groups. Results: EGCG had the anti-tumor effect in dose-dependent. Furthermore, EGCG cancelled this spheroid formation, EGCG down-regulated the Nanog and CD133, related stemnesss genes. And then, EGCG down-regulated the ABCG1and Nek2, related ABC transporter genes, expression in sphere. Conclusions: EGCG had the anti-tumor effects on gastroenterological cancer, and EGCG had the effect to sphere, same as cancer stem cell thorough the down-regulation of ABC transporter related genes. Therefore, EGCG might be used as the safe and novel antitumor agents.

The evaluation of in vitro toxicity of the protein (LL 4218) isolated from leaves of Argemone mexicana in human hepatoma-derived cell line (HepG2) using multiplexing approach

The evaluation of in vitro toxicity of the protein (LL 4218) isolated from leaves of Argemone mexicana in human hepatoma-derived cell line (HepG2) using multiplexing approach

Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue® and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose–response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

The inducibility of human cytochrome P450 1A by environmental-relevant xenobiotics in the human hepatoma derived cell line HepG2

Overexpression of the CYP1 family, independent of gender, is focal to the evaluation of the risk of human cancer. We have analysed the ability of 17 anthropogenic environmental xenobiotics widely used in Europe within households and agriculture to induce the human cytochrome P450 1A (CYP1A) in the human hepatoma derived cell line HepG2. The xenobiotics were potent to concomitantly induce both CYP1A mRNA and CYP1A activity in a dose-response relationship. Exceptions were shown by the organophosphate insecticide chlorpyrifos and the imidazole fungicide prochloraz in high concentrations which were capable of both inhibiting the basal or abolishing the initially induced CYP1A activity, respectively. A CYP1A induction has been shown for the first time by the aromatic xenobiotics irgasan, permethrin and azoxystrobin, the nonaromatic tributyltinoxide and for humans by the piperonylbutoxide. The xenobiotics additionally differed by their induced CYP1A isoenzyme pattern. A pronounced CYP1A1 and CYP1A2 mRNA induction was given by the phenyl urea herbicide diuron and benzodiazole insecticide piperonylbutoxide, respectively. In conclusion, out of the environmental xenobiotics, we described new members of human CYP1A inducers which extend chemical structures of biotransformation activators.

Construction and Evaluation of Drug-Metabolizing Cell Line for Bioartificial Liver Support System

Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.

Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg- siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2.

Time‐ and concentration‐dependent effects of resveratrol in HL‐60 and HepG2 cells

Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 microm in both HL-60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 microm for HL-60 cells and 60 microm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.

Abnormal activation of the synuclein-gamma gene in hepatocellular carcinomas by epigenetic alteration

Liver cancer is the fifth most common neoplastic disease and the fourth leading cause of cancer-related death. Identification of the key molecular targets involved in hepatocarcinogenesis has significant therapeutic implications. In this study, by conducting immunohistochemistry, we show that the neuronal protein, synuclein-gamma (SNCG), is abnormally expressed in a high percentage of liver cancer (46/70, 65.7%). The expression of SNCG in liver cancer exhibits a clear stage-specific pattern of low expression in stage I (1/19, 5.3%) and high expression in stages III to IV (44/50, 88%). Importantly, all patients with metastatic diseases expressed SNCG in their primary tumors (15/15, 100%). Consistent with the IHC results, RT-PCR assays demonstrate that SNCG mRNA is highly expressed in the tumor tissue of advanced hepatocellular carcinomas. Analyses of the methylation status of the CpG island of the SNCG gene by methylation-specific PCR confirmed that all tumor samples contained the demethylated gene. To determine whether demethylation of SNCG is an early event of genetic abnormality in the process of hepatocarcinogenesis, we examined the methylation status of SNCG in 70 non-malignant cirrhotic liver samples and showed that 64.3% cirrhotic liver samples contained the partially or completely demethylated gene. We further show that SNCG expression in liver cancer is not restricted to HBV- and HCV-infected tumors, implying the involvement of other hepatocarcinogenic risk factors in SNCG gene reactivation. Utilizing human hepatoma-derived cell line HepG2 as an in vitro model, we demonstrate that hepatic carcinogens aflatoxin B1 and N-nitrosodimethylamine (DMN) are strong inducers of SNCG expression. Collectively, these new findings suggest that SNCG protein expression in primary tumors is a strong indicator of distant metastasis and demethylation of SNCG CpG island is an early sign of genetic abnormality in liver cirrhosis preceding hepatocarcinogenesis. Our studies also suggest that inducing demethylation of SNCG by hepatocarcinogens may represent one underlying mechanism for the aberrant expression of SNCG in HCC.

Characterization of the human C6 promoter: requirement of the CCAAT enhancer binding protein binding site for C6 gene promoter activity.

The sixth complement component (C6) is a late-acting complement protein that participates in the assembly of the membrane attack complex. C6 and most of the complement proteins are mainly synthesized in the liver. However, the human hepatoma-derived cell line Hep-G2, which produces the majority of complement proteins, synthesizes traces of C6. Here, we have isolated and characterized the human C6 promoter. Approximately 1 kb of C6 upstream sequence is shown to be sufficient to achieve tissue-specific expression of a luciferase reporter gene in two hepatic (Hep-G2 and Hep-3B) and two extrahepatic cell lines (fibroblast M1 and HeLa) in a manner similar to endogenous C6. There are wide differences in C6 mRNA expression among the four cell lines, whereas Hep-3B expresses high levels of C6, Hep-G2 and M1 poorly synthesize C6, and HeLa completely lacks C6 expression. Deletional and mutational analysis demonstrates that a C/EBP (CCAAT/enhancer binding protein) site located at -67 is required for C6 expression in Hep-3B cells, but it has little effect in M1 and Hep-G2 cells. Electrophoretic mobility shift assays show that this sequence binds to a C/EBP alpha using Hep-3B nuclear extract, but a negligible activity is detected using a Hep-G2 extract. To further investigate whether C/EBP alpha is the limiting factor for C6 expression, we have transfected a C/EBP alpha expression vector into Hep-G2 and Ml cells. C/EBP alpha expression vector dramatically trans-activates the luciferase reporter gene controlled by the C6 promoter, and it partially restores C6 mRNA expression in Hep-G2 cells.

Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung.

The small-complement C5 activation fragment, C5a, is a potent phlogistic molecule that, on binding to the C5a Receptor (C5aR), mediates contraction of smooth muscle, enhances vascular permeability, and promotes leukocyte functions such as directed chemotaxis, degranulation, mediator release, and production of superoxide anions. Although C5aR expression has traditionally been thought to be limited primarily to myeloid blood cells, including neutrophils, monocytes, macrophages, and eosinophils, we report here that C5aR is expressed by liver and lung cells as well as by cells in several other tissues. By Northern blot analysis, it was determined that mouse liver, baboon liver, human liver, and the human hepatoma-derived cell line HepG2 express a normal size (2.3 kb) C5aR mRNA; in HepG2 cells, the quantity of C5aR mRNA was comparable to that contained in dbcAMP-differentiated U937 cells. HepG2 cells were demonstrated to express the C5aR on their cell surface by flow cytometric and immunofluorescence analyses as well as by 125I-C5a binding assays. The binding data indicated that HepG2 cells express a single class of C5aR with a Kd of 1.18 nM and approximately 28,000 receptors per cell. In vivo expression of C5aR in human liver cells was demonstrated by in situ hybridization and immunohistochemistry analyses. Northern blot analysis of murine and baboon organs shows that, in addition to the liver, other tissues express C5aR mRNA in significant quantities, including the spleen, lung, heart, kidney, and intestine. Moreover, mice treated with LPS show a large increase in C5aR mRNA in all these tissues except the intestine. Immunostaining of human lung tissue demonstrated that bronchial and alveolar epithelial cells, as well as vascular smooth muscle and endothelial cells, also express the C5aR. Collectively, these data indicate that the C5aR is expressed in several different types of cells in liver and lung, and in yet undetermined cell types in spleen, heart, intestine, and kidney. Furthermore, these data suggest that the C5a anaphylatoxin mediates previously unrecognized functions by binding to tissue cells that express the C5aR.

Characteristics and regulation of bile salt synthesis and secretion by human hepatoma HepG2 cells.

Bile salt uptake, synthesis and secretion by the human hepatoma-derived cell line HepG2 were studied. The cells transported and secreted bile salts largely by means of passive mechanisms. The cells synthesized and secreted the normal human primary bile salts. The ratio of cholate to chenodeoxycholate was 1.5:1. The degree of conjugation, about 35%, was lower than normal, and the glycine-to-taurine ratio was abnormal (4.5:1). This was not due to amino acid deficiency in the medium. Contrary to the report of others, little 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid was secreted. This was confirmed by gas chromatography-mass spectrometry. The total rate of synthesis was about 33% that of normal liver. The specific activity of bile salts synthesized from [3H]mevalonate was about 20 times higher than that of the cellular cholesterol derived from the same precursor. The regulation of bile salt synthesis by two compounds that could alter the precursor pool of cholesterol was studied. After a 24-hr incubation in serum-free medium, the compound 25(OH)cholesterol inhibited the rate of bile salt synthesis compared with control values, possibly by depleting the intracellular free cholesterol pool. Surprisingly, however, progesterone, which inhibits cholesterol esterification and should have expanded this pool, also inhibited bile salt synthesis under those conditions. The effect of these compounds on the level of mRNA for cholesterol 7 alpha-hydroxylase was also determined by Northern-blot analysis. The cholesterol 7 alpha-hydroxylase mRNA was 3.7 kb, similar to that in the rat. The incubation of cells in 25(OH)cholesterol or progesterone, as above, resulted in a decreased level of mRNA. The reduction was proportional to the reduction in bile salt synthesis, suggesting that these compounds act at a pretranslational level. Taken together, these results suggest that our particular subclone of HepG2 cells will be useful for studies of the regulation of bile salt synthesis, but not of transport, by human liver-derived tissue.

Novel apoprotein A-I-containing lipoprotein produced by a human hepatoma-derived cell line HuH-7

The apoprotein A-I (apo A-I)-containing lipoprotein (LP HuH.7apoA-I) was isolated from the concentrated conditioned medium of human hepatoma-derived cell line HuH-7 by immunoaffinity chromatography. Lp HuH-7apoA-I consists of two kinds of lipoproteins. One is a lipoprotein of large particle size (Lp L) with broad electrophoretic mobility on agarose gel ranging from the origin to the position of prebeta-lipoprotein. Lp L is protein-rich in composition (protein, 75.4% by weight) and is heterogeneous in size (34-17 nm in diameter) electron microscopically. However, the most intriguing properties of Lp L are its partial electrophoretic mobility towards the cathode on agar gel. The other lipoprotein is of small particle size (Lp s). It demonstrates prebeta-electrophoretic mobility on agarose gel. Lp s is also protein-rich in composition (protein, 95.4% by weight) and is heterogeneous in size (16.5–8.4 nm in diameter) electron microscopically. Lp L is obviously different from LP-X and LP-Y in property, although LP-X, LP-Y and a part of Lp L migrate towards the cathode on agar gel elecrrophoresis. Lp s is also different from human apo A-I-containing lipoprotein without apo A-II in property, although these two lipoproteins possess the same mobility on agarose gel elecrrophoresis. These results indicate that both Lp L and Lp s are novel lipoproteins which have not yet been reported. The major isoproteins of the apo A-I of Lp HuH-7apoA-I are aPo A-I isoprotein 2 (apo A-I 2), apo A-I isoprotein 4 (apo A-I 4) and apo A-I isoprotein 5 (apo A-I 5), and are different from those of apo A-I in human plasma and in the conditioned medium of hepatoma-derived cell line HepG2. This result suggests the presence of a proteinase which converts proapoprotein A-I (apo A-I 2) to apoprotein A-I (apo A-I 4) in the conditioned medium of HuH-7.

Hormonal regulation of complement biosynthesis in human cell lines—I. Androgens and gamma-interferon stimulate the biosynthesis and gene expression of C1 inhibitor in human cell lines U937 and HepG2

Cl inhibitor (Clinh), a member of the serine protease inhibitor gene superfamily, is a glycosylated plasma protein inhibiting the proteolytic activities of Clr and Cls and involved in the regulation of coagulation, fibrinolysis and kinin-releasing systems. In this study, the in vitro effect of androgen hormones, dehydroepiandrosterone (DHEA), testosterone (TEST) and recombinant human γ-interferon (γ-IFN), has been determined on the production of Clinh in human cell lines. In both human monocytoid/histiocytoid cell line U937 and in hepatoma derived cell line HepG2, DHEA and TEST upregulated the gene expression and secretion of Clinh. The most pronounced effect was detected in the concn range 10 −7−10 −9 M of the hormones. Under the same conditions DHEA and TEST had no detectable effect on the biosynthesis of C3, C2 and factor B by these cells, but DHEA at higher concn (10 −4M) slightly increased that of C4 in HepG2 cells. Both in U937 and in HepG2 cells recombinant γ-IFN markedly increased the gene expression and secretion of Clinh. This effect of γ-IFN was abolished by histamine.

Stimulation of Histamine Receptors of Human Monocytoid and Hepatoma‐Derived Cell Lines and Mouse Hepatocytes Modulates the Production of the Complement Components C3, C4, Factor B, and C2

The influence of histamine (and the related agonists and antagonists) alone or in the presence of recombinant human interleukin 1 alpha (IL-1 alpha) and gamma interferon (IFN-gamma) was studied on the production of complement components C3, C2, factor B, and C4 in vitro with human monocytoid cell line U937, hepatoma-derived cell line HepG2, and mouse hepatocytes. Both U937 and HepG2 cells responded to histamine through H1 and H2 histamine receptors. The effect of histamine on the biosynthesis and gene expression of complement proteins was predominantly enhancing via the H1 histamine receptors and inhibitory through the H2 receptors. The actual predominance of the histamine receptor involved (and the outcome of the ligand interaction) seemed to be greatly affected by the simultaneous activation of the cells by IL-1 or IFN-gamma.

Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides.

Effects of acidotropic compounds on the secretory pathway: Inhibition of secretion and processing of the third and fourth components of complement

Acidotropic compounds (also termed lysosomotropic) such as chloroquine and amantadine interfered with processing of the single-chain precursors to the third and fourth components of complement (C3 and C4) by the human hepatoma-derived cell line HepG2. When these compounds were added to culture medium, the precursors of C3 and C4 became the major secretory forms in contrast to the normal secretion of C3 and C4 as their mature forms. In addition, secretion of C3, C4, and total protein was inhibited by these compounds. Our results indicate that lysosomotropic agents, in addition to their well recognized effects on lysosomes and endosomes, inhibit functions of the secretory pathway.

Identification of the site of sulfation of the fourth component of human complement.

The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.

Distinct primary translation products from human liver mRNA give rise to secreted and cell-associated forms of complement protein C2.

The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.

Identification and structural characterization of two incompletely processed forms of the fourth component of human complement.

Immunoprecipitates of human C4 from EDTA-plasma were incubated with [14C]methylamine and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. In addition to finding label in the alpha-chains of the secreted (C4s) and predominant plasma (C4p) forms of C4, two additional molecules with apparent molecular weights of approximately 168,000 (p168) and approximately 125,000 (p125) covalently incorporated methylamine, indicating the presence of an internal thioester bond. These two molecules were present at a concentration of approximately 5% of total plasma C4 and were not immunoprecipitated by antisera to C3 or alpha 2-macroglobulin. A human hepatoma-derived cell line (HepG2), in addition to synthesizing C4s and small quantities of the polypeptide precursor of C4 (pro-C4), was found to secrete p168 and p125 at concentrations of 14 +/- 4.8 and 21 +/- 9.2% (mean +/- SD), respectively, of total secreted C4. These molecules were not found intracellularly. Both molecules were present on reduced, but not nonreduced, SDS-polyacrylamide gels. Chido (C4B) and Rodgers' (C4A) alloantisera precipitated the C4A and C4B variants of pro-C4, p168, p125, and C4s. Both tryptic and Staphylococcus aureus V8 protease peptide analyses showed homology between p168 and the beta- and alpha-chains and between p125 and the alpha- and gamma-chains. Partial NH2-terminal sequencing revealed that the beta-chain was NH2-terminal in p168 and that the alpha-chain was NH2-terminal in p125. Taken together, these data indicate that p168 and p125 represent uncleaved beta-alpha- and alpha-gamma-fragments of pro-C4, respectively. Thus, in most individuals, plasma C4 consists of five structurally distinct molecules, the single polypeptide precursor (pro-C4), the three-subunit secreted (C4s) and predominant plasma (C4p) forms of C4, and two incompletely processed two-subunit molecules with uncleaved beta-alpha- (p168) or uncleaved alpha-gamma (p125)-subunits. In addition, all five molecules are observed for both C4A (Rodgers) and C4B (Chido) structural genes.