Vitamin K uptake in hepatocytes and hepatoma cells

Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation.     

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes.

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.     

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identified in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal specific antiserum. The GP-N has an apparent molecular mass of 45-50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.     

Requirement of N-glycosylation for the secretion of recombinant extracellular domain of human Fas in HeLa cells

Apoptosis has been shown to be associated with altered glycosylation patterns and biosynthesis of glycoproteins. A major cell surface receptor involved in the induction of apoptosis is Fas that is activated by binding Fas ligand but can also be activated by binding anti-Fas antibody. In order to determine whether the Fas receptor is glycosylated, the extracellular domain of human Fas (shFas) was expressed as a cleavable fusion protein (shFas-Fc) in HeLa cells. These cells were shown to express activities of glycosyltransferases involved in N- and O-glycan biosynthesis. The secreted shFas-Fc was shown to be a glycoprotein with heterogeneous glycan chains. MALDI mass spectrometry revealed a disperse molecular weight of shFas with an average of 23.4kDa. Western blots of shFas-Fc secreted from tunicamycin treated transfected HeLa cells showed that only N-glycosylated glycoforms were secreted, while the unglycosylated shFas-Fc remained intracellular. The results suggest that both N-glycosylation sites of the extracellular domain of Fas are occupied with large N-glycans that play a role in the expression of the glycoprotein.     

Integral membrane antigens involved in cell-substratum adhesion of hepatocytes and hepatoma cells

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against purified plasma membranes of rat liver (anti-liver-antiserum) and Morris hepatoma 7777 (anti-hepatoma-antiserum). It is assumed that substances which block the adhesion-inhibiting activity of the antisera are involved in cell-substratum adhesion. Adhesion-involved molecules of rat liver monitored as 'blocking activity' were compared with those of Morris hepatoma 7777 and 9121. They were found to be integral membrane glycoproteins, which could be solubilized only by detergents. Fractionation of plasma membrane extracts by size exclusion HPLC revealed two blocking activity peaks representing molecules involved in the adhesion to plastic (P-AIM) and collagen (C-AIM). In rat liver both adhesion-involved molecules were found; yet P-AIM seemed to be the major type of adhesion-involved molecule. In the relatively well differentiated Morris hepatoma 9121 also both types were detected. In membrane extracts of the high malignant and poorly differentiated Morris hepatoma 7777, however, no P-AIM but only C-AIM were found. Estimation by size exclusion HPLC revealed molecular weights of 120 kD for C-AIM and approx. 105 kD for P-AIM. On SDS gel electrophoresis proteins in the region of 95 kD were found in C-AIM containing fractions, whereas proteins of 105 kD are likely candidates for P-AIM.     

Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The Kd values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.     

Interaction of laminin with the plasma membrane components of ascitic Zajdela hepatoma cells

The laminin affinity chromatography was used for isolating laminin-binding proteins from the plasma membrane of Zajdela hepatoma cells synthesizing laminin. These were components with mol. weights about 80, 67, 60, 55, 52, 48 and 43 kDa. The isolation of laminin integrin receptors from plasma membranes of Zajdela hepatoma cells in the presence of MnCl2 detected only a protein with mol. weight about 80 kDa in EDTA-elution conditions. This protein was identified by mass spectrometry method as the 78 kDa glucose-regulated protein precursor (GRP78). It belongs to the family of 70 kDa heat shock proteins, recently GRP78 was reported to be localized on the surface of different cell types, including hepatocytes.     

Biosynthesis and intracellular transport of alpha-2,6-sialyltransferase in rat hepatoma cells.

We investigated biosynthesis, intracellular transport and release of beta-galactoside alpha-2,6-sialyltransferase in a dexamethasone-inducible rat hepatoma cell line. Confluent cells were induced by 10 microM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S-labelled enzyme was synthesized as a 46-kDa precursor, converted to an intermediate 47-kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N-glycosylation or cleavage of N-glycans from isolated sialyltransferase using N-glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4-h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N-acetyllactosamine-type or one oligomannose-type and one N-acetyllactosamine-type N-linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4-h chase. The half-life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion-blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments. Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post-Golgi degradation pathway. This route contrasts with the post-Golgi trafficking of beta-1,4-galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623-7628].     

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.     

On the autocrine growth regulation of Morris hepatoma cells

Chemically induced, transplantable Morris hepatoma 7777 cells (MH) were examined for their anchorage-dependent and anchorage-independent growth properties. MH cells were found to grow on solid surface in a density-dependent manner, with respect to serum factors and gelatinization. The correlation between anchorage independent growth of MH cells and agar or serum concentrations in the culture medium was described. The ability of MH cells to stimulation of soft agar colony formation of NRK-49F indicator cells in coculture assay was presented. Autocrine control of proliferation of MH cells by TGFs-like factor(s) was suggested.     

Synthesis, transport and processing of cathepsin C in Morris hepatoma 7777 cells and rat hepatocytes

The synthesis, transport and processing of cathepsin C was studied in Morris hepatoma 7777 cells by metabolic labelling, immunoprecipitation and characterization of labelled polypeptides by gel electrophoresis and fluorography. The largest detectable precursor of cathepsin C was a polypeptide of Mr = 92 500. Even 3 min after synthesis this precursor was accompanied by four polypeptides with Mr values ranging from 63 000 to 54 000, indicating cleavage of the precursors within the endoplasmic reticulum. The early forms of cathepsin C were associated with low-buoyant-density organelles containing the markers of endoplasmic reticulum and Golgi complex. About 30% of these early forms were secreted within 3 h after synthesis. The remaining 70% were transferred into dense lysosomes and processed between 2 and 3 h after synthesis to a mixture of the least five major and nine minor polypeptides with Mr values ranging from 73 000 to 12 000. These forms remained stable for at least 3 days. In freshly isolated hepatocytes cathepsin C was processed to forms closely related to those found in the hepatoma cells. Cathepsin C was synthesized in Morris hepatoma 7777 cells as a glycoprotein with mannose-6-phosphate residues that mediated mannose-6-phosphate-specific receptor-dependent uptake in human skin fibroblasts. In contrast to hepatocytes, synthesis of mannose-6-phosphate receptors in Morris hepatoma 7777 cells was below the limit of detection. The hepatoma cells did not express at the cell surface these or other receptors mediating endocytosis of lysosomal enzymes. Further, processing and transport of newly synthesized cathepsin C was largely resistant to NH4Cl. Apparently, cathepsin C is transferred in Morris hepatoma 7777 cells by a mechanism independent of mannose-6-phosphate-specific receptors.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Differential regulation of insulin-like growth factor-II receptors in rat hepatocytes and hepatoma cells

This study compares the regulation of IGF-II receptors in three rat hepatoma lines, HTC, H-35 and 5123tc, and primary rat hepatocytes. In all cell types [125I]IGF-II bound solely to a species of approximately 250 kDa. Cell surface IGF-II receptors in hepatoma cells had slightly lower affinities (1-2 liters/nmol) than in hepatocytes (4 liters/nmol), but slightly higher IGF-I cross-reactivity (2-4% compared to 1% in hepatocytes). In confluent cultures, the three hepatoma lines expressed 5- to 15-fold more cell-surface receptors per cell than hepatocytes. However, while hepatocyte receptors showed marked inverse density-dependence, increasing over 6-fold between dense (3 x 10(5) cells/3.8 cm2) and sparse (0.16 x 10(5) cells/3.8 cm2) cultures, receptors in all hepatoma lines remained at a constant high level regardless of culture density. These distinct regulatory patterns resemble those described for growth-related functions in hepatocytes and hepatoma cells, and are thus consistent with a role for IGF-II receptors in liver cell proliferation.     

Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense. Protection against proteolytic attack

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied. Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species. In contrast to the glycosylated molecule only marginal amounts of high-molecular-mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin-treated cells. Most of the material removed by proteases from the cell surface of tunicamycin-treated cells has a molecular mass lower than 2 kDa. Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same preferential protease-sensitive site exists as is found in the glycosylated species. These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.     

DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.     

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.     

Role of oligomannosidic N-glycans in the proliferation,adhesion and signalling of C6 glioblastoma cells

The potential role of glycoprotein N-glycans in the proliferation and adhesion of C6 glioblastoma cells was investigated using a set of N-glycosylation inhibitors (tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, swainsonine), and traffic (monensin). It was observed that both the proliferative and adhesive properties of C6 cells were dependent upon the expression at the cell surface of glycoproteins with oligomannosidic and hybrid type N-glycans, whereas the absence of N-glycans (tunicamycin) or the presence of glucosyl-oligomannosides (deoxynojirimycin and castanospermine) and the absence of glycoproteins at the cell surface (monensin) reduced both the proliferative and adhesive properties of C6 cells. Studies of the classical elements of signalling pathways indicated that the different inhibitors have a low impact on tyrosine phosphorylations and oncogene product expression (except the ras oncogene product), except on phosphorylations on other residues. An endogenous soluble lectin (CSL; J. Neurochem. 49 (1987) 1250), specific for oligomannosidic and hybrid type N-glycans, was present and externalised by the cells through a pinching-off of large intracellular vesicles, a mechanism that was not blocked by monensine; in contrast with the externalisation of its glycoprotein ligands. The inhibitory effect of anti-CSL Fab fragments on adhesion indicates that the polyvalent CSL acts as a bridging molecule for a family of surface glycoproteins expressed at the surface of C6 cells. The inhibitory effect of the same Fab fragments on the proliferation indicates that CSL is a mitogen for these cells, possibly involved in clustering its surface glycoprotein ligands. A mechanism for the loss of contact inhibition is discussed based on the over-expression of CSL ligands in C6 glioblastoma cells relative to normal cells.     

Effects of dexamethasone and insulin on the acute phase response of Morris hepatoma cells and of rat hepatocytes in culture

Dexamethasone and insulin stimulate production of several plasma proteins in primary cultures of adult rat hepatocytes but inhibit their production in primary cultures of Morris hepatoma cell line 7777W. The acute phase response elicited in cultured cells by crude cytokines from activated rat peritoneal macrophages is considerably higher in hepatocytes in the presence of hormones, and especially of dexamethasone. In hepatoma cells the hormones enhance the cytokine-induced formation of fibrinogen and cysteine proteinase inhibitor but are without significant effect on suppression of albumin and alpha-fetoprotein synthesis by macrophage supernatants.