concanavalin-a and Carcinoma--Hepatocellular

Liver cancer is the predominant cause of cancer mortality in males of Southern China and Taiwan. The current therapy is not satisfactory, and more effective treatments are needed. In the search for new therapies for liver tumor, we found that Concanavalin A (Con A), a lectin from Jack bean seeds, can have a potent anti-hepatoma effect. Con A after binding to the mannose moiety on the cell membrane glycoprotein is internalized preferentially to the mitochondria. An autophagy is triggered which leads to cell death. Con A as a T cell mitogen subsequently activates the immune response in the liver and results in the eradication of the tumor in a murine in situ hepatoma model. The liver tumor nodule formation is inhibited by the CD8+ T cells, and a tumor antigen-specific immune memory is established during the hepatic inflammation. The dual properties (autophagic cytotoxicity and immunomodulation) via the specific carbohydrate binding let Con A exert a potent anti-hepatoma therapeutic effect. The novel mechanism of the Con A anti-hepatoma effect is discussed. The prototype of Con with an anti-hepatoma activity gives support to the search for other natural lectins as anti-cancer compounds.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Autophagy; Cancer Vaccines; Carcinoma, Hepatocellular; Concanavalin A; Glycosylation; Humans; Liver Neoplasms

Affinity electrophoresis is based on the reaction between interacting components during electro phoresis. In this review is given the general analytical technology. The main advantages of the analytical electrophoresis approach appear to be: 1. It can separate macromolecules that interact with a specific ligand from those that do not. 2. It can be used for studies of interacting macromolecules. 3. It is not necessary to purify interacting components. 4. A multitude of proteins reacting with the same ligand may be studied simultaneously. 5. It can be generalized to interactions other than those between lectins and glycoproteins.

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Female; Glycoproteins; Humans; Immunoelectrophoresis; Immunoelectrophoresis, Two-Dimensional; Liver Neoplasms; Mice; Pregnancy; Pregnancy Proteins

Concanavalin A (Con A) affinity electrophoresis of serum alpha-fetoprotein (AFP) can distinguish hepatocellular carcinoma (HCC) from other malignancies when the serum AFP concentration is elevated. However, Con A has not been able to distinguish HCC from benign chronic liver disease such as cirrhosis or chronic hepatitis.. The Con A affinity electrophoresis of serum AFP was analyzed in patients with a serum AFP concentration greater than 50 ng/mL by antibody affinity electrophoresis and Western blotting in an attempt to distinguish hepatocellular carcinoma from benign chronic liver disease. Before the assay, the serum AFP concentrations were adjusted between 100 ng/ml and 300 ng/ml by concentrating or diluting the samples.. Of 180 patients with HCC, 44 (24%) had a single band and 91 (51%), 35 (19%), and 10 (6%) had 2, 3, and 4 bands, respectively. All 35 patients with chronic hepatitis had a single band. All but 1 of 72 patients with cirrhosis had a single band. Multiple AFP bands on Con A affinity electrophoresis appear to be diagnostic of HCC. This method has a sensitivity of 76%, a specificity of 99%, a positive predictive value of 99%, and a negative predictive value of 71% for detecting HCC. The number of AFP bands correlated with serum AFP concentration and tumor size in patients with HCC.. This assay is useful for distinguishing HCC from benign chronic liver diseases.

Topics: Adult; Aged; alpha-Fetoproteins; Blotting, Western; Carcinoma, Hepatocellular; Chi-Square Distribution; Chronic Disease; Concanavalin A; Diagnosis, Differential; Electrophoresis; Female; Hepatitis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Predictive Value of Tests; Sensitivity and Specificity

Altered glycosylation associated with hepatocellular carcinoma (HCC) is well documented. However, few reports have investigated the association between dedifferentiation and glycosylation. Therefore, the aim of this study was to analyze glycosylation associated with dedifferentiation of HCC within the same nodule and to investigate glycosyltransferase related to the glycosylation.. We analyzed resected HCC specimens (n = 50) using lectin microarray to comprehensively and sensitively analyze glycan profiles, and identify changes to glycosylation between well- and moderately-differentiated components within the same nodule. Moreover, we performed immunohistochemical staining of mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1), which is an essential glycosyltransferase that converts high-mannose glycans to complex- or hybrid-type N-glycans.. Four lectins from Narcissus pseudonarcissus agglutinin (NPA), Concanavalin A, Galanthus nivalis agglutinin, and Calystegia sepium agglutinin were significantly elevated in moderately-differentiated components of HCC compared with well-differentiated components, and all lectins showed binding specificity to high-mannose glycans. Therefore, these structures were represented to a greater extent in moderately-differentiated components than in well-differentiated ones. Immunohistochemical staining revealed significantly increased NPA expression and decreased MGAT1 expression in moderately-differentiated components. Low MGAT1 expression in moderately-differentiated components of tumors was associated with intrahepatic metastasis and had tendency for poor prognosis.. Dedifferentiation of well-differentiated HCC is associated with an increase in high-mannose glycans. MGAT1 may play a role in the dedifferentiation of HCC.

Topics: Aged; Calystegia; Carcinoma, Hepatocellular; Concanavalin A; Disease-Free Survival; Female; Glycosylation; Humans; Immunohistochemistry; Liver Neoplasms; Male; Mannose-Binding Lectins; N-Acetylglucosaminyltransferases; Narcissus; Optical Imaging; Plant Lectins; Polysaccharides; Staining and Labeling

The previous studies have demonstrated that the polysaccharide isolated from Tarphochlamys affinis (PTA) exhibits anti-tumor effect on S180 tumor-bearing mice and protective effects against hepatic injury.. In this study, we investigated the anti-tumor activity and possible underlying mechanism of PTA on liver cancer using a murine H22 hepatocarcinoma model.. PTA was capable of repressing transplanted H22 solid hepatic tumor cell growth in vivo. The relative weight of immune organs (spleen and thymus) and lymphocyte proliferation induced by ConA or LPS were improved after PTA treatment. Furthermore, treatment with PTA promoted immune-stimulating serum cytokine secretion in H22 tumor-bearing mice. Additionally, the percentage of CD4+ T lymphocytes, CD8+ T lymphocytes and NK cells was increased in tumor-bearing mice following PTA administration. In tumor tissue, PTA significantly up-regulated the expression of Bax and p53 proteins and down-regulated the expression of Bcl-2 protein. In addition, at the therapeutic dose, PTA displayed very few toxic effects to major organs, such as the liver and kidney, in tumor-bearing mice.. In H22 tumor-bearing mice, PTA exhibited prominent anti-tumor activity in vivo. The possible mechanism of action might be related to enhanced host immune system function and induction of H22 tumor cell apoptosis.

Topics: Acanthaceae; Animals; Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Concanavalin A; Female; Gene Expression Regulation, Neoplastic; Injections, Subcutaneous; Killer Cells, Natural; Lipopolysaccharides; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Polysaccharides; Primary Cell Culture; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Spleen; Thymus Gland; Tumor Suppressor Protein p53

Concanavalin A (ConA) is a lectin and T-cell mitogen that can activate immune responses. In recent times, ConA-induced cell death of hepatoma cells through autophagy has been reported and its therapeutic effect was confirmed in a murine in situ hepatoma model. However, the molecular mechanism of ConA-induced autophagy is still unclear. As macrophage migration inhibitory factor (MIF), which is a proinflammatory cytokine, can trigger autophagy in human hepatoma cells, the possible involvement of MIF in ConA-induced autophagy was investigated in this study. We demonstrated that cell death is followed by an increment in MIF expression and secretion in the ConA-stimulated human hepatoma cell lines, HuH-7 and Hep G2. In addition, ConA-induced autophagy and cell death of hepatoma cells were blocked in the presence of an MIF inhibitor. Knockdown of endogenous MIF by small hairpin RNA confirmed that MIF is required for both ConA-induced autophagy and death of hepatoma cells. Furthermore, signal pathway studies demonstrated that ConA induces signal transducer and activator of transcription 3 (STAT3) phosphorylation to trigger MIF upregulation, which in turn promotes Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)-dependent autophagy. By using a murine in situ hepatoma model, we further demonstrated that MIF contributes to anti-hepatoma activity of ConA by regulating STAT3-MIF-BNIP3-dependent autophagy. In summary, our findings uncover a novel role of MIF in lectin-mediated anti-hepatoma activities by regulating autophagy.

Topics: Animals; Autophagy; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Concanavalin A; Hep G2 Cells; Humans; Intramolecular Oxidoreductases; Liver Neoplasms; Liver Neoplasms, Experimental; Macrophage Migration-Inhibitory Factors; Male; Mice; Mice, Inbred BALB C; Signal Transduction

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, "ConA-binding proCD" (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5-5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA-pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Blotting, Western; Carboxylic Ester Hydrolases; Carcinoma, Hepatocellular; Cathepsin D; Cell Line, Tumor; Concanavalin A; Enzyme Precursors; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Haptoglobins; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Proteomics; RNA Interference; RNA, Small Interfering

Malignant transformation of hepatocellular carcinoma (HCC) occurs through repetitive liver injury in a context of inflammation and oxidative DNA damage. A spectrum of natural sesquiterpenoids from curcuma oil has displayed antioxidant, anti-inflammatory and anti-carcinogenic properties. The aim of the study was to investigate the hepatoprotective and anti-HCC effects of curcuma oil in vivo and in vitro. Mice were pretreated with curcuma oil (100 mg/kg) for 3 days, then treated with Concanavalin A (30 mg/kg). The hepatic tissue was evaluated for histology, CD4+ cell, interferon-γ, apoptosis, lipid peroxidation, 8-hydroxy-deoxyguanosine and MnSOD. C57L/J mice were treated with curcuma oil and 107 Hepa1-6 cells directly inoculated into liver lobes. The effects of curcuma oil on cell growth and cell death were evaluated. In addition, MnSOD, HSP60, catalase, NF-κB and caspase-3 were also investigated in the Hepa1-6 cells treated with curcuma oil. Pretreatment with curcuma oil significantly attenuates inflammation and oxidative damage by Concanavalin A. Treatment with curcuma oil can decrease the incidence of HCC. Curcuma oil inhibits cell growth and induces cell death in Hepa1-6 cells. Curcuma protected mice with hepatic injury from inflammatory and oxidative stress. Curcuma oil can inhibit hepatoma cell growth in vivo and in vitro.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Chemical and Drug Induced Liver Injury; Concanavalin A; Curcuma; Fluorescent Antibody Technique; Gas Chromatography-Mass Spectrometry; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mitogens; Oxidative Stress; Plant Extracts; Tumor Cells, Cultured

Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4(-/-) ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1(-/-) ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background.. Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Chemical and Drug Induced Liver Injury; Concanavalin A; Galectin 1; Hepatitis, Chronic; Liver; Liver Neoplasms; Male; Methionine Adenosyltransferase; Mice; Mice, Inbred Strains; Mice, Knockout

Hepatocyte growth factor (HGF) is critical for triggering metastasis of hepatocellular carcinoma cell (HCC). Extracellular signal-regulated kinase (ERK) mediates HGF-induced cell migration via focal adhesion signaling. Protein kinase C (PKC) is a negative regulator of ERK activation, however, both PKC and ERK were required for HGF-induced cell migration. To address this intriguing issue, the signal mechanisms for HGF-induced HepG2 cell migration were investigated in a long-term fashion. HGF-induced phosphorylations of ERK, Src (at Tyr 416) and paxillin (at Ser178 and Tyr31) were up and down for 3 times within 24h. HGF also induced fluctuant PKC activation and Rac degradation. Consistently, HGF induced intermittent actin polarization within 24h, which can be blocked by the inhibitors of PKC (Bisindolymaleimide) and ERK. Inhibitor studies revealed that ERK was required for HGF-induced paxillin phosphorylation at Ser178, whereas PKC and Rac-1 may suppress HGF-induced phosphorylation of ERK and paxillin (at Ser178) and upregulate phosphorylation of paxillin at Tyr31. Based on shRNA technique, PKCα and δ were responsible for suppressing HGF-induced phosphorylation of ERK and paxillin (at Ser178), whereas PKC ε and ζ were required for phosphorylation of paxillin at Tyr31. The HGF-induced fluctuant signaling is reminiscent of c-Met endocytosis. Using Concanavalin A, an inhibitor of endocytosis, we found that c-Met endocytosis was required for PKC to suppress ERK phosphorylation. Moreover, HGF-induced c-Met degradation was also fluctuant, which can be prevented by Bisindolymaleimide. In conclusion, PKC is critical for mediating HGF-induced fluctuant ERK-paxillin signaling during cell migration, probably via triggering endosomal degradation of c-Met.

Topics: Animals; Carcinoma, Hepatocellular; Cell Movement; Concanavalin A; Endocytosis; Extracellular Signal-Regulated MAP Kinases; Hep G2 Cells; Hepatocyte Growth Factor; Humans; Indoles; Isoenzymes; Liver Neoplasms; Maleimides; Mice; Mice, SCID; Mitogens; Paxillin; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-met; rac GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

Topics: Animals; Autophagy; Carcinoma, Hepatocellular; Cathepsin B; Cathepsin L; Cell Line, Tumor; Cell Membrane; Concanavalin A; Endocytosis; GTP-Binding Proteins; Hep G2 Cells; Hepatocytes; Humans; Interferon-gamma; Liver Neoplasms; Lysosomes; Mice; Mice, Inbred BALB C; Necrosis; Permeability

Protein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N-glycosylated protein investigation. In this study, we developed Con A-immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid-functionalized magnetic nanoparticles was performed by forming boronic acid-sugar-Con A bond in sandwich structure using methyl alpha-D-mannopyranoside as an intermedium. The selective capture ability of Con A-modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein-immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A-modified magnetic nanoparticles by the facile and low-cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large-scale glycoproteomic research in complicated biological samples.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Concanavalin A; Glycomics; Glycoproteins; Humans; Liver Neoplasms; Magnetics; Nanoparticles; Proteomics

A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media.. Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity.. Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

Topics: Adsorption; Antineoplastic Agents, Phytogenic; Carbohydrates; Carcinoma, Hepatocellular; Cell Proliferation; Chromatography; Concanavalin A; DEAE-Cellulose; Hep G2 Cells; HIV Reverse Transcriptase; Hot Temperature; Humans; Hydrogen-Ion Concentration; Mitogens; Molecular Structure; Phaseolus; Phytohemagglutinins; Reverse Transcriptase Inhibitors; Spleen

A dimeric 62-kDa lectin exhibiting a novel N-terminal amino acid sequence was purified from caper (Capparis spinosa) seeds. The purification protocol involved anion-exchange chromatography, cation-exchange chromatography and, finally, gel filtration by FPLC on Superdex 75. Approx. 100-fold purification was achieved. The haemagglutinating activity of the lectin, which was stable in the pH range 1-12 and up to 40 degrees C, could be inhibited by D(+) galactose, alpha-lactose, raffinose and rhamnose at 1 mM concentration, by 25 mM L(+)-arabinose and by 100 mM D(+)GlcN (glucosamine). The lectin potently inhibited HIV-1 reverse transcriptase with an IC50 of 0.28 microM and proliferation of both hepatoma HepG2 and breast cancer MCF-7 cells with an IC50 of approx. 2 microM. It induced apoptosis in HepG2 and MCF-7 cells. It manifested a weaker mitogenic activity on mouse splenocytes than ConA (concanavalin A). It inhibited mycelial growth in Valsa mali with an IC50 of 18 microM.

Topics: Amino Acid Sequence; Animals; Antifungal Agents; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Capparis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Concanavalin A; Dimerization; Female; Fungi; HIV Reverse Transcriptase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Liver Neoplasms; Mice; Mitosis; Molecular Sequence Data; Molecular Weight; Mycelium; Plant Lectins; Protein Stability; Seeds; Spleen; Temperature

Concanavalin A (ConA), a lectin with mannose specificity that can induce acute hepatic inflammation, was tested for its therapeutic effect against hepatoma. ConA is cytotoxic or inhibitory to hepatoma cells, which is mediated by the autophagic pathway through mitochondria. Once it was bound to cell membrane glycoproteins, the ConA was internalized and preferentially localized onto the mitochondria. The mitochondria membrane permeability changed, and an autophagic pathway including LC3-II generation, double-layer vesicle, BNIP3 induction, and acidic vesicular organelle formation was induced. Either 3-MA or siRNA for BNIP3 and LC3, but neither beclin-1 nor ATG 5, partially inhibited the ConA-induced cell death. In addition to the autophagy induction, ConA is known to be a T cell mitogen. Using an in situ hepatoma model, ConA can exert an anti-hepatoma therapeutic effect, inhibiting tumor nodule formation in the liver and prolonging survival.. ConA can be considered as an anti-hepatoma agent therapeutically because of its autophagic induction and immunomodulating activity. This dual function of ConA provides a novel mechanism for the biological effect of lectin.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Concanavalin A; Disease Models, Animal; Lectins; Liver Neoplasms; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, SCID; Microtubule-Associated Proteins; Mitochondrial Membranes; Mitochondrial Proteins; Mitogens; Proteins; RNA, Small Interfering

A recent study has suggested that the methylation silencing of the suppressor of cytokine signaling-3 (SOCS3), a negative regulator of interleukin-6-related cytokines, could be involved in hepatocellular carcinoma (HCC). However, the roles of SOCS3 in hepatocellular carcinogenesis and hepatitis have not been established. We investigated the effect of deleting the SOCS3 gene on the development of hepatitis and HCC in hepatitis C virus-infected patients and mouse models.. The expression of SOCS genes in HCC and non-HCC regions of patient samples was determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The conditional knockout approach in mice was used to determine the hepatocyte-specific roles of SOCS3. To generate a liver-specific deletion, floxed SOCS3 (SOCS3(fl/fl)) mice were crossed with albumin-Cre transgenic mice. Hepatitis and HCC were induced by administering concanavalin A and diethylnitrosamine, respectively.. SOCS3 expression was reduced in the HCC regions compared with the non-HCC regions. Carcinogen-induced hepatic tumor development was enhanced by deletion of the SOCS3 gene, which was associated with higher levels of the targets of signal transducers and activators of transcription (ie, B-cell lymphoma-XL, B-cell lymphoma-2, C-myelocytomatosis, cyclin D1, and vascular endothelial growth factor). In the concanavalin A-mediated hepatitis model, deletion of the SOCS3 gene in the hepatocytes protected against liver injury through suppression of interferon-gamma signaling and induction of the antiapoptotic protein Bcl-XL.. Deletion of the SOCS3 gene in hepatocytes promotes the activation of STAT3, resistance to apoptosis, and an acceleration of proliferation, resulting in enhanced hepatitis-induced hepatocarcinogenesis.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Concanavalin A; Female; Gene Deletion; Hepatocytes; Humans; Immunohistochemistry; In Vitro Techniques; Liver Neoplasms; Male; Mice; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

Quantitative changes in plasma protein concentrations during tissue injury or inflammation (acute phase response) are often accompanied by specific alterations in the carbohydrate moieties of these proteins. The glycosylation changes comprise alterations in the type of branching of the carbohydrate structures as revealed by modulated reactivity of acute phase glycoproteins with the lectin concanavalin A. Interestingly, inflammation-induced changes in the glycosylation of acute phase proteins have been shown to affect the functional properties of these proteins. In this study we demonstrate that synthesis of acute phase protein alpha(1)-PI, the controlling inhibitor of neutrophil elastase, is significantly up-regulated in hepatic and lung-derived epithelial cells by the inflammatory mediator oncostatin M. Although oncostatin M markedly altered the concanavalin A reactivity of hepatic alpha(1)-PI, lung-derived epithelial cells did not change the pattern of alpha(1)-PI glycan branching upon stimulation with oncostatin M. These results indicate that inflammation-induced changes in glycosylation of alpha(1)-PI may have different impacts on functional properties of liver and lung-synthesized alpha(1)-PI.

Topics: alpha 1-Antitrypsin; Carcinoma, Hepatocellular; Cell Line, Tumor; Concanavalin A; Epithelial Cells; Glycosylation; Humans; Lung; Oncostatin M; Peptides; Protein Binding

The central role of T cell activation in hepatocellular injury has been well documented. In this article, we provide evidence suggesting that T cells may also play a protective role in liver disease by releasing interleukin-22 (IL-22), a recently identified T cell-derived cytokine whose biological significance is unclear. IL-22 messenger RNA and protein expression are significantly elevated in T cell-mediated hepatitis induced by concanavalin A (ConA) but are less extensively elevated in the carbon tetrachloride-induced liver injury model. Activated CD3(+) T cells are likely responsible for the production of IL-22 in the liver after injection of ConA. The IL-22 receptor is normally expressed at high levels by hepatocytes and further induced after ConA injection. IL-22 blockade with a neutralizing antibody reduces signal transducer and activator of transcription factor 3 (STAT3) activation and worsens liver injury in T cell-mediated hepatitis, whereas injection of recombinant IL-22 attenuates such injury. In vitro treatment with recombinant IL-22 or overexpression of IL-22 promotes cell growth and survival in human hepatocellular carcinoma HepG2 cells. Stable overexpression of IL-22 in HepG2 cells constitutively activates STAT3 and induces expression of a variety of antiapoptotic (e.g., Bcl-2, Bcl-xL, Mcl-1) and mitogenic (e.g., c-myc, cyclin D1, Rb2, CDK4) proteins. Blocking STAT3 activation abolishes the antiapoptotic and mitogenic actions of IL-22 in hepatic cells. In conclusion, the T cell-derived cytokine IL-22 is a survival factor for hepatocytes; this suggests that T cell activation may also prevent and repair liver injury by releasing hepatoprotective cytokine IL-22 in addition to its previously documented central role in hepatocellular injury.

Topics: Agar; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Survival; Chemical and Drug Induced Liver Injury; Concanavalin A; DNA-Binding Proteins; Hepatitis; Hepatocytes; Humans; Interleukin-22; Interleukins; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, Interleukin; STAT3 Transcription Factor; T-Lymphocytes; Trans-Activators

Cardiotrophin-1 (CT-1) is a member of the interleukin 6 (IL-6) family of cytokines, which protect cardiac myocytes against thermal and ischemic insults. In this study, we investigated the expression of CT-1 by liver cells and its possible hepatoprotective properties.. We analyzed the production, signaling, and antiapoptotic properties of CT-1 in hepatocytes and the expression of this cytokine during liver regeneration. We also investigated whether CT-1 might exert protective effects in animal models of liver damage.. We found that CT-1 is up-regulated during liver regeneration and exerts potent antiapoptotic effects on hepatocytic cells. Hepatocytes cultured under serum starvation or stimulated with the pro-apoptotic cytokine transforming growth factor beta (TGF-beta) produce CT-1, which behaves as an autocrine/paracrine survival factor. Treatment with an adenovirus encoding CT-1 efficiently protects rats against fulminant liver failure after subtotal hepatectomy, an intervention that causes 91% mortality in control animals whereas 54% of those receiving CT-1 gene therapy were long-term survivors. This protective effect was associated with reduced caspase-3 activity and activation of the antiapoptotic signaling cascades signal transducer and activator of transcription (Stat-3), extracellular regulated kinases (Erk) 1/2, and Akt in the remnant liver. Gene transfer of CT-1 to the liver also abrogated Concanavalin A (Con-A) liver injury and activated antiapoptotic pathways in the hepatic tissue. Similar protection was obtained by treating the animals with 5 microg of recombinant CT-1 given intravenously before Con-A administration.. We show that CT-1 is a hepatocyte survival factor that efficiently reduces hepatocellular damage in animal models of acute liver injury. Our data point to CT-1 as a new promising hepatoprotective therapy.

Topics: Adenoviridae; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Survival; Concanavalin A; Cytokines; Genetic Therapy; Hepatectomy; Hepatocytes; Injections, Intravenous; Liver Failure; Liver Neoplasms; Liver Regeneration; Mice; Mice, Inbred BALB C; Rats; Rats, Wistar; Tumor Cells, Cultured; Up-Regulation

We have characterized the regulation of expressed human ecto-ATPase (E-NTPDase 2), a cell surface integral membrane glycoprotein. Ecto-ATPase activity is inhibited by parameters that decrease membrane protein interaction, i.e., detergents and high temperatures. These inhibitory effects are overcome when membranes are pretreated with concanavalin A or chemical cross-linking agents that increase the amounts of ecto-ATPase oligomers. Cross-linking agents also abrogate substrate inactivation of the ecto-ATPase, a unique characteristic of the enzyme. These effects indicate that the magnitude of negative substrate regulation is dependent on quaternary structures of the protein, which likely involves interaction of transmembrane domains. The importance of transmembrane domains of ecto-ATPase in activity modulation is demonstrated further by the stimulatory effect of digitonin, a steroid glycoside that preferentially interacts with cholesterol in the membranes but does not promote oligomer formation. These results indicate that ecto-ATPase activity is regulated by a multitude of mechanisms, some of which may have physiological significance. Ecto-ATPase is also susceptible to transcriptional regulation. Ecto-ATPase gene expression is increased in a human hepatoma whereas it is undetectable in the normal liver.

Topics: Adenosine Triphosphatases; Carcinoma, Hepatocellular; Carcinoma, Small Cell; Cell Membrane; Concanavalin A; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Liver Neoplasms; Lung Neoplasms; Protein Conformation; Protein Structure, Quaternary; Recombinant Proteins; Transcription, Genetic; Tumor Cells, Cultured

Intravenous administration of tumor necrosis factor-alpha (TNF-alpha) (0.5 microg/mouse) caused hepatocyte apoptosis in BALB/c mice when they were sensitized with D-galactosamine (GalN, 20 mg/mouse). Activation of nuclear factor kappa B (NF-kappa B) and expression of apoptotic Bcl-2 family members were not significantly different between livers of mice treated with TNF-alpha alone and GalN + TNF-alpha, indicating that neither activation of NF-kappa B nor expression of Bcl-2 family is involved in the sensitization by GalN against TNF-alpha-induced hepatocyte apoptosis. To identify differentially expressed genes implicated in GalN-induced hepatocyte sensitization, we adopted mRNA fingerprinting using an arbitrarily primed polymerase chain reaction. The present analysis revealed that mRNA expression of extracellular antioxidant, selenoprotein P, was up-regulated in the livers after GalN administration. GalN-induced increase in its protein level was confirmed by Western blotting. Increased expression of this gene was also observed in the liver of mice treated with concanavalin A, but not anti-Fas antibody. mRNA of another antioxidant, glutathione peroxidase-1, was also up-regulated, and lipid peroxides were produced in the liver after GalN administration. Selenoprotein P mRNA level also increased in Huh-7 human hepatoma cells incubated with GalN (5 or 10 mM). Accordingly, formation of reactive oxygen species (ROS) was observed in GalN-treated Huh-7 cells. H(2)O(2) induced up-regulation of selenoprotein P mRNA and sensitized Huh-7 cells to TNF-alpha-induced apoptosis. These results suggest that ROS produced by GalN may play a pivotal role in hepatocyte sensitization toward TNF-alpha-induced apoptosis.

Topics: Animals; Antibodies; Apoptosis; Carcinoma, Hepatocellular; Cell Line; Concanavalin A; Drug Synergism; fas Receptor; Galactosamine; Gene Expression Profiling; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Hepatocytes; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; NF-kappa B; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; Selenoprotein P; Selenoproteins; Specific Pathogen-Free Organisms; Tumor Necrosis Factor-alpha

N-Acetylglucosaminyltransferase III (GnT III) catalyses the addition of N-acetylglucosamine through a beta 1-4 linkage to the mannose of the trimannosyl core, resulting in conversion of the concanavalin A (Con A)-reactive glycan into a non-reactive state. In this study, we measured GnT III activity to evaluate its diagnostic efficacy and its therapeutic effect on hepatocellular carcinoma (HCC). Concanavalin A-non-reactive fraction of serum transferrin (Tf) was also determined since the sugar chains of Tf are one of the possible candidates for the product of GnT III. Serum samples (159) were used from patients with HCC (89), liver cirrhosis (30), chronic hepatitis (19), alpha-fetoprotein (AFP) producing gastric carcinoma metastatic to the liver (five) and healthy controls (16). N-Acetylglucosaminyltransferase III activity was determined by high performance liquid chromatography. The reactivity of serum Tf to Con A was also analysed in 21 paired HCC samples before and after treatment by crossed immuno-affinoelectrophoresis. N-Acetylglucosaminyltransferase III activity from the HCC group (153 +/- 72pmol/mL/h) was significantly higher than that from liver cirrhosis (99 +/- 67 pmol/mL per h), chronic hepatitis (84 +/- 39 pmol/mL per h) and the normal controls (62 +/- 16 pmol/mL per h). N-Acetylglucosaminyltransferase III activity of 21 patients with HCC was significantly reduced after treatment such as transcatheter arterial chemoembolization and/or percutaneous ethanol infection therapy, (123 +/- 77 to 100 +/- 60 pmol/mL per h). Commensurate decreases of AFP and des-gamma-carboxy prothrombin with GnT III activity were also observed after treatment. The Con A-non-reactive fraction (n = 21; 6.4 +/- 2.3%) in patients with HCC after treatment was significantly lower than before (8.2 +/- 2.4%). The present study suggests that GnT III activity is a possible aid in the diagnosis and evaluation of HCC, especially when other tumour markers are negative.

Topics: Adult; Aged; Aged, 80 and over; alpha-Fetoproteins; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Clinical Enzyme Tests; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Middle Aged; N-Acetylglucosaminyltransferases; Protein Precursors; Prothrombin; Transferrin

Heterogeneous reactivity of human serum transferrin (Tf) with lectins was analysed using patient sera to determine whether it can be used to distinguish patients with hepatocellular carcinoma (HCC) from those with liver cirrhosis (LC). Microheterogeneity of Tf was analysed by crossed immunoaffinity electrophoresis (CIAE) with concanavalin A (Con A) and Lens culinaris agglutinin (LCA). Sample sera from 58 patients with HCC, 43 patients with LC and 10 normal controls were used in this study and the results were evaluated statistically. The increments of Con A-non-reactive (C1) and -weakly reactive (C2) species of Tf were observed in HCC compared with those of LC and Norm. Significant increase in the combined percentage of Con A- C1 + C2 species was also revealed in HCC (35.5 +/- 8.5%, mean+/-s.d.) compared with those of LC (29.1 +/- 6.8%; P < 0.001) and normal controls (17.1 +/- 2.3%; P < 0.001). The elevation of LCA-reactive (L2) species of Tf was recognized in HCC (8.2 +/- 3.8%) in comparison with those of LC (4.8 +/- 3.1%; P < 0.001) and normal controls (1.3 +/- 1.7%; P < 0.001). The increment of C1 + C2 species and/or L2 species of Tf was observed in 78% (sensitivity) of patients with HCC. The specificity, the positive predictive value and the overall accuracy were 81, 88 and 72%, respectively. Positive ratio of C1 + C2 and/or L2 species was 77 and 70% in alpha-fetoprotein low and -high producing HCC patients, respectively. These results indicate that the microheterogeneity analysis of human serum Tf is useful for distinguishing patients with HCC from those with LC and normal controls.

Topics: Aged; alpha-Fetoproteins; C-Reactive Protein; Carcinoma, Hepatocellular; Concanavalin A; Electrophoresis; Female; Humans; Lectins; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Plant Lectins; Reference Values; Transferrin

Topics: Adult; Aged; Arthritis, Rheumatoid; Carcinoma, Hepatocellular; Cell Line; Chromatography, Affinity; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Orosomucoid; Protein Binding; Sepharose; Tumor Cells, Cultured

Serum or ascites alpha-fetoprotein (AFP) from patients with hepatocellular carcinoma and from a cord blood were analyzed by affinity electrophoresis with two lectins mixed in agarose gel in a combination of concanavalin A (Con A) and Lens culinaris agglutinin A (LCA-A) or of erythroagglutinating phytohemagglutinin (E-PHA) and Allomyrina dichotoma lectin (allo A). Con A- and LCA-A-reactive AFP-C2-L3 was not further retarded by mixing with either of the other lectin. It showed a mobility identical with that of AFP-C2 or AFP-L3. E-PHA- and allo A-reactive AFP-P4-A3 showed similar results. It migrated with intermediate mobilities of AFP-P4 and AFP-A3 depending on the concentrations of the two lectins mixed in the gel. The results indicate that the two mixed lectins compete with each other for the topologically different lectin-binding sites on the oligosaccharide of AFP molecule.

Topics: alpha-Fetoproteins; Carbohydrate Metabolism; Carbohydrate Sequence; Carcinoma, Hepatocellular; Concanavalin A; Electrophoresis, Agar Gel; Electrophoresis, Gel, Two-Dimensional; Fetal Blood; Humans; Lectins; Ligands; Molecular Sequence Data; Phytohemagglutinins; Plant Lectins

We analyzed the Con A affinity of serum AFP in patients with a serum AFP concentration greater than 50ng/ml by antibody affinity electrophoresis and Western blotting to distinguish hepatocellular carcinoma (HCC) from benign chronic liver diseases (CLD). Of 164 patients with HCC, 48 (29.3%) had a single band, while 116 (70.7%) had multiple bands. All but three of 65 patients with cirrhosis had a single band. All but one of 32 patients with chronic hepatitis had a single band. We concluded that multiple AFP bands are diagnosis of HCC. This method is a useful assay for distinguishing HCC from CLD.

Topics: Adult; Aged; alpha-Fetoproteins; Blotting, Western; Carcinoma, Hepatocellular; Chronic Disease; Concanavalin A; Diagnosis, Differential; Electrophoresis, Agar Gel; Female; Hepatitis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity

The importance of the oncofetal glycoprotein antigen alphafetoprotein (AFP) as a tumor marker is well documented. Structural heterogeneity of AFP molecules due to its associated carbohydrate moieties has also been demonstrated. In the present study, molecular variants of AFP, Concanavalin A reactive (R Con A) and Concanavalin A nonreactive (NR Con A) were quantified in five cases of hepatocellular carcinoma (HCC), three cases of hepatoblastoma, five gonadal and two extragonadal germ cell tumors, and two suspected liver secondaries by employing crossed immunoaffino electrophoresis (CIAE). AFP peaks were localized using anti-AFP antibodies conjugated to alkaline phosphatase. Characteristic patterns of AFP R Con A and NR Con A fractions were obtained in different AFP-secreting malignancies. Serum samples of HCC and hepatoblastoma were predominantly composed of R Con A AFP, while gonadal and extragonadal germ cell tumors showed significant reduction of R Con A AFP and elevation of NR Con A AFP. Analysis of AFP variants in sera from two patients of suspected liver metastasis with elevated AFP confirmed liver secondaries arising from germ cell tumor in one patient and HCC in the other patient. The present study highlights the importance of AFP microheterogeneity analysis not only as diagnostic aid for the differential diagnosis of AFP-secreting tumors, but also in providing better management and prognosis.

Topics: Adolescent; Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Child, Preschool; Concanavalin A; Diagnosis, Differential; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Female; Germinoma; Hepatoblastoma; Humans; Infant; Liver Neoplasms; Male; Middle Aged

In heparinized human platelet-rich plasma (PRP), J-7 human hepatoma cells initially induced platelet aggregation; then a clot formed. ADP-scavenger systems, apyrase and creatine phosphate/creatine phosphokinase did not inhibit this tumor-cell-induced platelet aggregation (TCIPA), whereas hirudin and concanavalin A completely blocked it. J-7 cells also shortened the recalcification time of normal and of Factor-VIII- and IX-deficient human plasma, although it was inactive in shortening the recalcification time of Factor-VII-deficient plasma. After treatment with phorbol 12,13-dibutyrate (PDBu) for 5 to 90 min, the aggregation and coagulation abilities of J-7 cells were unaffected. Prolonged treatment of J-7 cells with PDBu but not with alpha-PDBu for 24 and 72 hr resulted in gradual loss of aggregation and coagulation. Staurosporine antagonized the effect of PDBu and restored aggregation and coagulation in J-7 cells. Protracted treatment with PDBu (24 or 72 hr) did not affect adherence of J-7 cells to the extracellular-matrix proteins (i.e., fibrinogen, fibronectin, laminin, vitronectin and collagen types I and IV) or to the surface of plastic culture dishes. The treatment also did not affect J-7 cell detachment from plastic culture dishes. These in vitro results demonstrate that protracted phorbol ester treatment diminishes TCIPA and blood coagulation of tumor cells.

Topics: Alkaloids; Blood Coagulation; Blood Platelets; Carcinoma, Hepatocellular; Cell Adhesion; Concanavalin A; Extracellular Matrix Proteins; Factor IX; Factor VII; Heparin; Hirudins; Humans; Liver Neoplasms; Phorbol 12,13-Dibutyrate; Platelet Aggregation; Staurosporine; Tumor Cells, Cultured

Elevated synthesis of 23 K protein by human hepatoma PLC/PRF/5 cells was observed after their treatment with conditioned medium from concanavalin A stimulated peripheral-blood monocytes. Increased amount of this protein was first determined 4 hr after the treatment and its maximal level was reached 48 hr later. The role of the 23 K protein remains so far unknown.

Topics: Acute-Phase Proteins; Carcinoma, Hepatocellular; Concanavalin A; Culture Media, Conditioned; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Humans; Liver Neoplasms; Lymphocyte Activation; Lymphokines; Neoplasm Proteins; T-Lymphocytes; Tumor Cells, Cultured

Concanavalin A (Con A) and lentil lectin (LCA) analysis of alpha-fetoprotein (AFP) glycosylation heterogeneity is used in a variety of clinical situations. We studied the influence of analytical conditions on the separation of AFP glycoforms by using lectin-crossed affinoimmunoelectrophoresis, regardless of the AFP concentration, which can vary over a wide range in biological fluids. We defined the optimal concentration of Con A (2 g/L) and LCA (0.35 g/L) in the first-dimension gel, together with the optimum antigen (AFP)/antibody ratio in the second-dimension gel. The presence of protein in the diluent used for AFP samples was found to change the shape of crossed affinoimmunoelectrophoresis patterns without changing the percentage composition of AFP fractions. The within-run CV was less than 4% for both lectins, and the between-run CV was less than 6.3%. The minimal quantity of AFP that provided a visible pattern with both lectins was 4 ng, corresponding to 50 microL of an 80 micrograms/L AFP sample. These technical conditions allow the cellular origin of AFP to be determined, regardless of the concentration in the sample. Typical AFP lectin patterns of secreting tumors are compared with fetal and cord serum AFP.

Topics: alpha-Fetoproteins; Amniotic Fluid; Carcinoma, Hepatocellular; Concanavalin A; Fetal Blood; Humans; Immune Sera; Immunoelectrophoresis, Two-Dimensional; Lectins; Liver Neoplasms; Plant Lectins; Teratoma

The induction of proteolytic enzymes is an important mechanism in the migration of monocytes into tissues and body fluids. The monocytic cell line THP-1 was used as a model system to study the production of a particular gelatinase. Upon stimulation with phorbol myristate acetate (PMA) the cells differentiated to the adherent phenotype and produced significant amounts of a 96-kD gelatinase in a dose-dependent way. The secretion rate was maximal between 12 and 24 h after induction. Study of gelatinase mRNA steady state levels showed that the synthesis of THP-1 gelatinase is regulated by PMA at transcriptional or posttranscriptional levels. Stimulation of signal transduction pathways with other substances, including calcium ionophore A 23187, dibutyryl cyclic AMP, and dexamethasone, were ineffective in inducing gelatinase mRNA or enzyme activity. However, THP-1 cells were responsive to the cytokine interleukin (IL)-1 beta, to bacterial lipopolysaccharide (LPS), and the lectin concanavalin A (Con A), the kinetics of gelatinase induction being similar to those of induction by PMA. The THP-1 cells did not synthesize and/or secrete detectable levels of IL-6 after stimulation with PMA, Con A, LPS, or IL-1 beta. The 96-kD monocytic THP-1 gelatinase was shown to be a neutral metalloproteinase that cross-reacted with hepatoma-derived and neutrophil gelatinases in immunoprecipitation experiments. The active enzyme produced by THP-1 cells consistently showed, however, a molecular mass different from that of normal granulocyte-, monocyte-, and tumor cell-derived gelatinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line; Concanavalin A; Gelatinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Kinetics; Leukemia, Monocytic, Acute; Lipopolysaccharides; Liver Neoplasms; Molecular Sequence Data; Molecular Weight; Pepsin A; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription, Genetic

In rat hepatocytes; the tumorigenic rat liver cell line ARL-16; and the human hepatoma line, Hep G2, 50% of the total gamma-glutamyl transpeptidase (GGT) activity was bound by a Concanavalin-A Sepharose 4B column, calling for alpha-methylmannoside elution (Peak I). Non-binding GGT was distributed between a rapidly eluting Peak II and a slightly retained Peak III. The Km for gamma-glutamyl-p-nitroanalide for either hydrolysis or transpeptidation, or glutathione (GSH) transpeptidation did not vary with peak number or cell type. The GSH hydrolysis Km was essentially constant in Peak I and II GGT. Peak III GGT exhibited a lower Km for GSH hydrolysis with Hep G2 Peak III GGT being the lowest. Peak III GGT increased to 50% of the GGT activity in Hep G2 cells cultured with GSH as the sole cysteine source.

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cells, Cultured; Concanavalin A; Cysteine; gamma-Glutamyltransferase; Glutathione; Hydrolysis; Kinetics; Liver; Liver Neoplasms; Rats; Tumor Cells, Cultured

1. The concentrations of four serum glycoproteins, thyroxine-binding globulin, alpha 2-macroglobulin, alpha 1-antitrypsin and transferrin, as well as their reactivities with concanavalin A and lentil-lectin, were measured in patients with hepatocellular carcinoma or fulminant hepatic failure and in normal subjects. 2. Serum concentrations of thyroxine-binding globulin and alpha 1-antitrypsin were significantly greater in patients with hepatocellular carcinoma than in normal subjects, and the percentage lentil-lectin reactivity of these two proteins was markedly increased. 3. With the exception of transferrin, which did not bind to lentil-lectin, an enhancement of lentil-lectin reactivity was observed for the glycoproteins in serum from patients with fulminant hepatic failure. No difference in concanavalin A binding was found between the groups for any of the glycoproteins. 4. Altered fucosylation, as indicated by increased lentil-lectin binding, occurs in several glycoproteins arising in malignant and non-malignant conditions associated with abnormal hepatic regeneration.

Topics: Adult; Aged; alpha 1-Antitrypsin; alpha-Macroglobulins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Glycoproteins; Humans; Lectins; Liver Diseases; Liver Neoplasms; Liver Regeneration; Middle Aged; Plant Lectins; Thyroxine-Binding Proteins; Transferrin

Serum alpha-fetoprotein levels were determined in patients (268) with liver disease. Markedly elevated concentrations (greater than 100 micrograms/l) were found in twelve patients with malignant tumours and two with cirrhosis. Molecular variants of alpha-fetoprotein were distinguished by lectin affinity chromatography of these sera. Reversible binding to concanavalin A (86 +/- 5%) and to lentil agglutinin (61 +/- 19%) conformed to expected values for primary hepatocellular carcinoma except in one patient with a metastatic carcinoma whose alpha-fetoprotein binding to concanavalin A was similar to non-liver alpha-fetoprotein (44 +/- 13%), and the two patients with cirrhosis in whom binding to lentil agglutinin was typical for benign liver disorders (less than 20%). Since low levels of serum alpha-fetoprotein and non-characteristic alpha-fetoprotein binding patterns assisted in the regrouping of eleven out of 24 patients initially thought to have primary hepatocellular carcinoma, it was concluded that alpha-fetoprotein determination and lectin affinity chromatography are helpful in distinguishing primary hepatocellular carcinoma from metastatic and benign liver diseases. Slight increases in the alpha-fetoprotein level in the presence of serum hepatitis B surface antigen indicated seven patients at risk for primary hepatocellular carcinoma who should be monitored frequently.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Female; Genetic Variation; Humans; Lectins; Liver Diseases; Liver Neoplasms; Male; Middle Aged

To establish whether alpha-fetoprotein (AFP) produced in the early post-treatment phase of a patient with a germ cell tumour of the testis or the ovary originates from the tumour or is due to an underlying disturbance in liver function, the binding of AFP to concanavalin A (Con A) was investigated as a discriminative variable. A two-step assay is described that can distinguish the type of AFP produced at levels as low as 10 ng/ml. A Con A-binding ratio of 12-43% was found in the patients with disseminated germ cell tumours and in patients with AFP-positive gastrointestinal carcinomas. AFP from the liver gives ratios below 10%.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Concanavalin A; Female; Gastrointestinal Neoplasms; Humans; Liver Neoplasms; Male; Methods; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Protein Binding; Testicular Neoplasms

We have previously shown that changes in acute-phase protein glycosylation result from alterations occurring within hepatocytes as a result of regulation by cytokines, that the glycosylation patterns of proteins secreted by Hep 3B and Hep G2 cells respond differently to the crude mixtures of cytokines found in conditioned medium from LPS-stimulated monocytes, and that interleukin-6 (IL-6) causes increased concanavalin A (Con A) binding of alpha 1 protease inhibitor in Hep 3B cells and decreased Con A binding of this protein in Hep G2 cells. In the present study we found that transforming growth factor beta 1 (TGF-beta), like IL-6, led to secretion of forms of alpha 1-protease inhibitor with increased Con A binding in Hep 3B cells, and that IL-6 and TGF-beta in combination were additive. In contrast, in Hep G2 cells, TGF-beta had an effect opposite to that produced by IL-6, leading to secretion of forms of alpha 1-protease inhibitor with increased Con A binding. When employed in combination with IL-6. TGF-beta abolished the effect of that cytokine. These studies indicate that TGF-beta influences glycosylation of alpha 1-protease inhibitor in two human hepatoma cell lines in a manner that can be differentiated from that of IL-6. The identification of TGF-beta as a second defined cytokine capable of influencing glycoprotein glycosylation and the demonstration that the effect of one cytokine can be modulated by another cytokine support the view that changes in glycosylation of plasma proteins are mediated by combinations of cytokines.

Topics: alpha 1-Antitrypsin; Carcinoma, Hepatocellular; Concanavalin A; Drug Interactions; Glycosylation; Humans; Interleukin-6; Liver Neoplasms; Neoplasm Proteins; Protein Binding; Protein Processing, Post-Translational; Transforming Growth Factor beta; Tumor Cells, Cultured

Serum samples from 20 patients with acute exacerbation of chronic hepatitis due to hepatitis B virus and 20 patients with hepatocellular carcinoma arising from B viral cirrhosis with elevated levels of alpha-fetoprotein (AFP) were analyzed by affinity column chromatography for concanavalin A binding. Serum AFP was tested at regular intervals in all of these patients. Acute exacerbation was defined as elevation of serum transaminase greater than 300 IU/L in patients with chronic hepatitis B. In hepatocellular carcinoma, serum AFP levels fluctuated but remained higher than 92 ng/ml, whereas, in acute exacerbation of chronic hepatitis B, serum AFP levels returned to normal within 3-12 months of follow-up. The results of concanavalin A-binding assay revealed that AFP from both these groups had a high affinity for concanavalin A, and this assay could not be used to discriminate between the two conditions.

Topics: Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Chronic Disease; Concanavalin A; Diagnosis, Differential; Female; Follow-Up Studies; Hepatitis B; Humans; Liver Neoplasms; Male; Middle Aged

The effects of various cytokines on synthesis and microheterogeneity of carbohydrate structure of alpha 1-proteinase inhibitor (PI) and alpha-fetoprotein (AFP) in the human hepatoma cell lines Hep 3B and Hep G2 were studied. In both lines, crude cytokine preparations from LPS-activated human monocytes (CM) and several cell lines led to increased PI and decreased AFP synthesis, while recombinant interleukin 1 (IL-1), recombinant tumor necrosis factor (TNF) and hepatocyte stimulating factor preparations (HSF) affected AFP but not PI production. Several of the crude cytokine preparations, but not IL-1, TNF, or HSF, caused Hep 3B cells to secrete forms of PI and AFP showing increased reactivity with Con A upon testing by affinity electrophoresis, while decreased reactivity with Con A was seen in these proteins secreted by Hep G2 cells. Determination of molecular size of PI inducing activity in CM showed a sharp peak at about 17 kD while AFP inhibiting activity was present in a very broad range of molecular size fractions maximal at 17-30 kD. Changes in patterns of glycosylation of these proteins were attributable to cytokines of about 30 kD in Hep 3B and 44 kD in Hep G2 cells. These findings demonstrate the existence of a family of glycosylation regulating cytokines, and suggest that distinct mechanisms within hepatocytes, responsive to different cytokines, may lead to increased or decreased Con A binding of glycoproteins and to altered gene expression.

Topics: Acute-Phase Proteins; alpha 1-Antitrypsin; alpha-Fetoproteins; Blood Proteins; Carcinoma, Hepatocellular; Concanavalin A; Glycosylation; Interleukin-6; Interleukins; Liver Neoplasms; Molecular Weight; Monocytes; Proteins; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In order to differentiate yolk sac-type alpha-fetoprotein (AFP) from hepatic-type AFP, 5 yolk sac tumors (YSTs) and 6 hepatocellular carcinomas (HCCs) were examined immunohistochemically by the peroxidase-antiperoxidase (PAP) method for AFP, and paradoxical concanavalin A (P-Con A) staining, which has been reported to detect glycoprotein including AFP. In all 5 YSTs, AFP was negative for P-Con A staining. On the other hand, AFP was strongly positive for the same staining in all 6 HCCs. A similar staining pattern for AFP was observed in human yolk sac endodermal cells and embryonal hepatocytes. Thus, it was clarified that yolk sac-type AFP was unable to bind with Con A, in contrast with hepatic-type AFP, on tissue sections. It was concluded that the PAP method for AFP and P-Con A staining might facilitate the immunohistochemical differentiation of these two types of AFP, and that it would be useful for clarifying the histogenesis of various AFP-secreting tumors.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Female; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Mesonephroma; Ovarian Neoplasms; Staining and Labeling; Yolk Sac

A sensitive new technique for lectin-affinity immunoelectrophoresis was applied to samples from 28 infants and children in order to distinguish the origin of elevated alpha-fetoprotein (AFP) in sera. This new immunoelectrophoresis was successfully performed within 24 hours in sera with AFP as small as 910 ng/mL. With combined use of concanavalin A (Con A) and lentil agglutinin (LCH) binding tests, AFPs were classified into three subtypes: benign hepatic condition type (six patients), hepatocellular carcinoma type (nine patients) and yolk sac type (12 patients). AFP was of hepatocellular carcinoma type in all seven patients with hepatoblastoma, and of benign hepatic condition type in six of seven patients with elevated AFP due to conditions such as hepatitis, biliary atresia, and normal newborn. The question as to whether AFP produced in "hepatoblastoma" is of benign hepatic condition type or hepatocellular carcinoma type was first answered by the information in this present report. The differentiation between yolk sac and general hepatic AFPs was completed with the Con A binding test.

Topics: Adolescent; alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Female; Hepatitis; Humans; Immunoelectrophoresis; Infant; Lectins; Liver Neoplasms; Mesonephroma; Neoplasms; Ovarian Neoplasms; Plant Lectins

A model system for studies of mechanisms governing the alterations of glycosylation of plasma glycoproteins was developed. The system employs two human hepatoma cell lines, Hep 3B and Hep G2, as target cells and agarose affinity electrophoresis with lectins for studies of microheterogeneity of alpha 1-protease inhibitor (PI), a model glycoprotein synthesized by hepatocytes. As an example for the application of the system, the effect of cytokines on major microheterogeneity of plasma proteins is demonstrated. The results indicate that interleukin 6, transforming growth factor beta 1 and, to some extent, tumor necrosis factor alpha are directly involved in regulating the pattern of glycosylation of plasma proteins in vitro, but the major effect is obtained by using combinations of interleukin 6, transforming growth factor beta 1, tumor necrosis factor alpha and interleukin 1. In addition, the results underline the dissociation between alteration of gene expression and the changes in the pattern of plasma protein glycosylation.

Topics: alpha-Fetoproteins; Blood Proteins; Carcinoma, Hepatocellular; Ceruloplasmin; Concanavalin A; Electrophoresis; Electrophoresis, Agar Gel; Glycoproteins; Glycosylation; Humans; Liver Neoplasms; Protease Inhibitors; Tumor Cells, Cultured

Hepatoma-associated abnormal (des-gamma-carboxy)prothrombin (HAPT) is a newly described tumor marker for hepatocellular carcinoma. HAPT has been measured in the blood of patients with hepatoma by immunoassay but has not been isolated or characterized. This paper describes the quantitative isolation and structural characterization of HAPT. Purified HAPT has the same molecular weight, amino-terminal sequence, and amino acid analysis (exclusive of gamma-carboxyglutamic acid) as native prothrombin and abnormal prothrombin isolated from the blood of patients taking sodium warfarin. HAPT is heterogeneous in gamma-carboxyglutamic acid (Gla) content with an average of 5 Gla residues/molecule compared to 10 Gla residues for native prothrombin and 2 Gla residues for abnormal prothrombin. HAPT is glycosylated in a manner equivalent to that for native prothrombin when evaluated by a concanavalin A-binding assay. These studies find structural identity between HAPT and abnormal prothrombin. Therefore the findings support the hypothesis that HAPT results from an acquired defect in the posttranslational vitamin K-dependent carboxylation of the prothrombin precursor and not an intrinsic defect in the prothrombin precursor molecule.

Topics: Ascites; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Liver Neoplasms; Protein Precursors; Prothrombin

Topics: Carcinoma, Hepatocellular; Chronic Disease; Concanavalin A; Diagnosis, Differential; Electrophoresis; Electrophoresis, Cellulose Acetate; gamma-Glutamyltransferase; Hepatitis; Humans; Isoenzymes; Liver Cirrhosis; Liver Neoplasms; Octoxynol; Polyethylene Glycols; Sensitivity and Specificity

Hyperestrogenemia in humans increases both the concentration of serum T4-binding globulin (TBG) by 2- to 3-fold and the proportion having anodal mobility on isoelectric focusing (IEF). As TBG is synthesized in the liver, we studied the effect of estrogen on TBG synthesis, secretion, and degradation by cultured human hepatocarcinoma cells (Hep G2). beta-Estradiol in concentrations in the range found in pregnancy (10(-7) M) had no effect on the accumulation of immunoreactive TBG in medium over 4 days. The absence of fetal calf serum or phenol red did not alter these findings. The amount of [35S]TBG accumulated 6 h after addition of [35S]methionine was not influenced by exposure to estrogen or to serum obtained from pregnant women. However, 10(-5) M beta-estradiol suppressed TBG more severely than albumin synthesis (34% vs. 9%). The lack of an estrogen effect on TBG synthesis and secretion was supported by experiments showing no effect of estrogen on the disappearance of TBG added to the medium or the accumulation of cytoplasmic TBG mRNA. The same cultures responded to estrogen by a 10-fold increase in nuclear estrogen receptor binding sites and a 2-fold increase in apolipoprotein CII. As TBG in serum, the rate of heat denaturation was not altered in TBG synthesized by Hep G2 cells in the presence of estrogen. In contrast to the effect on TBG in serum, in Hep G2 cells estrogen did not produce an anodal shift on IEF, or increased its proportion not bound to Concanavalin A, nor reduced its clearance rate when injected into rats. However, even untreated Hep G2 cells synthesized TBG with a larger number of anodal IEF bands and proportion of Concanavalin A excluded material than TBG in pregnancy serum. Results support our hypothesis, based on analysis of TBG in pregnancy, that estrogen-induced serum TBG elevation may not be mediated through an increase in synthesis. The failure to observe estrogen induced changes in oligosaccharide structure does not exclude estrogen responsivity of Hep G2 cells. Such effect could be masked by the marked constitutive increase in number of oligosaccharide chain antennae typical in this and other neoplastic tissues.

Topics: Animals; Blood; Carcinoma, Hepatocellular; Cell Nucleus; Concanavalin A; Estradiol; Female; Hot Temperature; Humans; Liver Neoplasms; Male; Phenolsulfonphthalein; Pregnancy; Protein Denaturation; Rats; Rats, Inbred Strains; Receptors, Estrogen; RNA, Messenger; Testosterone; Thyroxine-Binding Proteins; Tumor Cells, Cultured

Using affinity chromatography on concanavalin A Sepharose, the authors studied the molecular heterogeneity of the serum alpha-fetoprotein of 53 patients with hepatocellular carcinoma, 16 patients with metastatic tumors to the liver, and 16 patients with germ cell tumors. Mean concanavalin binding of alpha-fetoprotein in the sera of patients with hepatocellular carcinoma was 79%, whereas the mean binding in metastatic tumors was 52% and that of germ cell tumors was 45%. This striking molecular variation of the alpha-fetoprotein produced by these different tumors is helpful in the clinical distinction of these tumors.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Diagnosis, Differential; Humans; Liver Neoplasms; Male; Testicular Neoplasms

The activity and the affinity for concanavalin A-Sepharose (Con A) of liver gamma-glutamyltransferase (gamma GT) were investigated in man, under various clinical conditions and in rats during experimental hepatocarcinogenesis. In man, gamma GT activity was higher than normal in hepatomas and (except for 1 case of hemochromatosis) also higher in the surrounding cirrhotic liver. The proportion of gamma GT which did not bind to Con A (Con A- form) was also increased in the tumors and in the surrounding liver, yet (with the same exception as above) to a greater extent in the hepatomas. In rat, gamma GT activity was higher in fetal liver (15-fold) and in hepatocarcinomas (10-fold) than in normal adult liver; total liver gamma GT activity gradually increased during progression from foci of altered cells to neoplastic nodules and tumors. The proportion of the Con A- form of gamma GT in the early or late stage of the carcinogenic process did not significantly differ from that in normal adult or regenerating rat liver, i.e. about 20% of the total activity. By contrast, nearly all the gamma GT from fetal rat liver bound to Con A. This suggests that gamma GT expression in rat liver carcinoma does not correspond to so-called retrodifferentiation process.

Topics: Adolescent; Adult; Animals; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Female; gamma-Glutamyltransferase; Humans; Isoenzymes; Liver; Liver Neoplasms; Liver Neoplasms, Experimental; Liver Regeneration; Male; Middle Aged; Rats; Rats, Inbred Strains

Using a modified method of concanavalin A (Con A), lentil lectin (LCH) or phytohemagglutinin-E (PHA-E) affinity crossed-line immunoelectrophoresis (ACIE), we studied alpha-fetoprotein (AFP) subfractions in 69 sera, including 58 from patients with primary liver cancer and 11 from patients with hepatic metastasis of gastric cancer. We found that Con A non-reactive subfraction (type b) or LCH weakly-reactive subfraction (type B) was more frequently detected in metastatic liver cancer, as compared with liver cancer hepatoma. The amount of Con A non-reactive subfraction (type b) or of PHA-E reactive subfraction (type X) was significantly higher in case of metastatic liver cancer than in primary liver cancer. Since different affinities between AFP and lectins are due to the microheterogeneity in AFP sugar chain, our findings suggest that AFP in primary liver cancer and metastatic liver cancer is glycosylated in a different manner. It is also indicated that different patterns of AFP subfractions identified by the combination of Con A, LCH or PHA-E ACIE facilitate a differential diagnosis of these hepatic malignancies.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Humans; Immunoelectrophoresis, Two-Dimensional; Lectins; Liver Neoplasms; Phytohemagglutinins; Plant Lectins; Stomach Neoplasms

Serum alpha fetoprotein (AFP) is heterogeneous, one form binding to the lectin concanavalin A (conA) and the other not. The relative amounts, of the two forms in the serum of patients has diagnostic applications in differentiating between primary hepato-cellular carcinoma and metastatic liver disease. In 36 patients with primary hepatocellular carcinoma, the conA-nonreactive form of AFP comprised less than 20% of the total (range 1.6%-19.2%; median 8.7%), whereas in 13 patients with metastatic liver disease the conA-nonreactive form comprised more than 20% of the total (range 26.6%-91.7%; median 57.6%). Four patients with primary hepatocellular carcinoma were treated with CB3717, and serial changes in the serum AFP characteristics were examined. In two patients in whom the total serum AFP concentration fell, the percentage of the conA-nonreactive fraction, initially less than 20% rose steadily. In two other patients the total serum AFP did not fall significantly and the proportion of the conA-nonreactive fraction remained below 20%.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Folic Acid; Humans; Liver Neoplasms; Quinazolines

Topics: Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Diagnosis, Differential; Female; Humans; Immunoelectrophoresis, Two-Dimensional; Lectins; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Plant Lectins

Techniques have been studied which distinguish two variants of human alpha-fetoprotein (AFP) on the basis of characteristics of the carbohydrate moiety of this glycoprotein. AFP in serum samples from six children with tumors of yolk sac origin showed little concanavalin-A (Con A) binding. In contrast, Con A binding of AFP was almost complete in serum samples from 14 other subjects with elevated AFP, including two with liver-cell tumors, eight with neonatal cholestasis, and four normal newborn infants. Differences were confirmed by immunoelectrophoretic studies. Thus, AFP from cells of yolk sac origin can be distinguished from AFP from liver cells or from tumors of hepatic cell origin.

Topics: alpha-Fetoproteins; Bile Ducts; Carcinoma, Hepatocellular; Child; Child, Preschool; Cholestasis; Chromatography, Affinity; Concanavalin A; Female; Hepatitis; Humans; Immunoelectrophoresis, Two-Dimensional; Infant; Infant, Newborn; Jaundice, Neonatal; Liver Neoplasms; Male; Mesonephroma; Pancreatic Neoplasms; Teratoma

Using a modified method of Con A, LCH or PHA-E affinity crossed-line immunoelectrophoresis, we studied AFP subfractions in 78 sera including 58 from patients with primary hepatoma, 11 from patients with hepatic metastasis of gastric cancer and 9 from patients with germ cell tumors of the gonads (yolk sac tumor, immature solid teratoma or mature solid teratoma). It was found that AFP in primary hepatoma, metastatic hepatoma or germ cell tumors of the gonads were differently glycosylated, and different patterns of AFP subfractions identified by Con A, LCH or PHA-E affinity crossed-line immunoelectrophoresis facilitated a differential diagnosis of such AFP related malignancies.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Female; Humans; Immunoelectrophoresis; Immunoelectrophoresis, Two-Dimensional; Lectins; Liver Neoplasms; Male; Mesonephroma; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Teratoma; Testicular Neoplasms

Major molecular species of human alpha-fetoprotein(AFP), which were separated as single components by serial affinity chromatography with concanavalin A(Con-A) and Lens culinaris agglutinin, were further resolved into several bands by affinity electrophoresis with erythroagglutinating phytohemagglutinin of Phaseolus vulgaris lectin(E-PHA). Among the newly separated main molecular species, both Con-A- and E-PHA-reactive AFP(AFP-1X1) was demonstrated, contrary to the known sugar specificity of Con-A and E-PHA, in addition to molecular species of AFP reacting with Con-A but not with E-PHA(AFP-1X0) and of AFP reacting with E-PHA but not with Con-A(AFP-0X1). AFP-0X1 was formed from AFP-0X0, and AFP-1X1 from AFP-1X0 by neuraminidase treatment; thus, AFP-0X1 and AFP-1X1 represent asialylated and AFP-0X0 and AFP-1X0 sialylated molecular species. AFP-1X1' and AFP-0X0' were present as minor components. AFP-0X0' had no affinity for E-PHA, and the affinity increased in the order of AFP's-0X0(or 0X1), -1X1', -1X1 and -0X1. Proportions of those components varied depending on the pathophysiological conditions of AFP production.

Topics: alpha-Fetoproteins; Asialoglycoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Fetuins; Humans; Liver Neoplasms; Mesonephroma; Neoplasms; Neoplasms, Germ Cell and Embryonal; Phytohemagglutinins; Stomach Neoplasms; Substrate Specificity

A major 110-kDa phosphoprotein in rat liver and hepatoma (P 110) was identified in nuclear 0.2 M HCl extracts from rat lymph node cells. Stimulation with concanavalin A altered both the amount of P110 and, more strikingly, its in vivo phosphorylation in a typical biphasic manner with an initial maximum after 10-14 h. RNA synthesis showed a similar biphasic increase. Inhibition of hnRNA synthesis (5,6 dichlorobenzimidazoleriboside), but not of DNA synthesis (hydroxyurea), depressed both the amount of P110 and its phosphorylation markedly.

Topics: Animals; Carcinoma, Hepatocellular; Cell Nucleus; Cells, Cultured; Chemical Phenomena; Chemistry; Concanavalin A; DNA; Hydrolysis; Liver; Liver Neoplasms; Lymph Nodes; Lymphocyte Activation; Molecular Conformation; Peptide Hydrolases; Phosphoproteins; Phosphorylation; Rats; RNA; Time Factors

gamma-Glutamyltransferase from human hepatoma and the surrounding non-neoplastic liver tissue was purified by immunoaffinity column chromatography and characterized with regard to molecular weight, isoelectric point (pI), amino acid composition, hexosamine content and affinity for various lectins. Both enzymes showed the same molecular weight (the heavy subunit 64 000; the light subunit 26 000) and pIs (3.7-3.9) on SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, respectively. After neuraminidase treatment, the pIs for both enzymes shifted to a more alkaline pH (pI 5.7). Both enzyme preparations exhibited similar amino acid compositions; however, the glucosamine content of the hepatoma enzyme was 362 nmol/mg protein, about 3-fold higher than that of the enzyme isolated protein from the non-neoplastic tissue. Binding of the two enzymes to lectins revealed that less of the hepatoma enzyme bound to Sepharose-conjugated wheat germ agglutinin, erythroagglutinating phytohemagglutinin and Ricinus communis agglutinin. These results suggest that the two enzymes possess similar peptide moieties and degree of sialylation, but differ with respect to other aspects of their heterosaccharide moieties.

Topics: Acetylglucosamine; Amino Acids; Carbohydrates; Carcinoma, Hepatocellular; Concanavalin A; Electrophoresis, Polyacrylamide Gel; gamma-Glutamyltransferase; Hexosamines; Humans; Isoelectric Focusing; Lectins; Liver; Liver Neoplasms; Methylmannosides; Molecular Weight; Peptides; Phytohemagglutinins; Plant Lectins; Wheat Germ Agglutinins

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Humans; Immunoelectrophoresis; Liver Diseases; Liver Neoplasms

We used affinity chromatography on concanavalin A Sepharose to study the serum alpha-fetoprotein of 10 patients with histologically proven germ-cell tumors and 12 patients with primary liver cancer. Less than 50% of the fetoprotein from germ-cell tumors bound to concanavalin A, as compared with more than 80% of the alpha-fetoprotein from primary liver cancers.

Topics: Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Mesonephroma; Middle Aged; Ovarian Neoplasms; Sepharose; Testicular Neoplasms

Topics: Adenocarcinoma; alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Concanavalin A; Diagnosis, Differential; Humans; Isoenzymes; L-Lactate Dehydrogenase; Liver Neoplasms; Lung Neoplasms; Mediastinal Neoplasms; Mesonephroma; Mice; Protein Binding

The concanavalin A binding characteristics of serum alpha-fetoprotein (AFP) were investigated in patients with primary hepatocellular carcinoma and hepatic secondaries using affinity column chromatography and radioimmunoassay. The primary hepatocellular carcinoma (n = 21) was associated with a median concanavalin A non-reactive AFP fraction of 7.4% (range 1.6 - 18.8) while the hepatic secondaries (n = 8) had a median concanavalin A non-reactive AFP fraction of 50.7% (range 26.6 - 91.7). A simple diagnostic test for differentiating between the two groups of patients is proposed.

Topics: Adolescent; Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Diagnosis, Differential; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis

The reactivity of alpha-fetoprotein (AFP) with concanavalin A (Con A) and Lens culinaris agglutinin (LCA) was studied by crossed immuno-affinoelectrophoresis of the serum samples of 146 patients from three groups (groups I, II and III). Fifty-one patients with benign liver diseases were included in group I, 83 patients with hepatocellular carcinoma in group II, and 12 patients with carcinoma metastatic to the liver from digestive organs in group III. In group I, the percentage of Con A-reactive species of AFP was high (97 +/- 5%, mean +/- SD), but that of LCA-reactive species was very low (3 +/- 5%). The percentage of LCA-reactive species of AFP in group II (45 +/- 33%) was higher than that in group I (P less than 0.001), while the Con A binding pattern of AFP in this group, as demonstrated by immuno-affinoelectrophoresis, was similar to that of group I. The percentage of Con A-reactive species of AFP in group III (55 +/- 24%) was much lower than that in group II (97 +/- 5%) (P less than 0.001). The above results indicate that measurement of the reactivity of AFP in serum samples with Con A and LCA is useful for the differentiation of AFP species which are found in association with benign liver diseases, hepatocellular carcinoma and carcinoma metastatic to the liver.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Concanavalin A; Humans; Immunoelectrophoresis, Two-Dimensional; Lectins; Liver Neoplasms; Plant Lectins

We determined by affinity chromatography on concanavalin A-Sepharose the carbohydrate variant patterns of alphafetoprotein in the sera of 15 infants and children with endodermal sinus tumors (five cases), a neonatal mature teratoma (one case), hepatoblastomas (two cases), pancreatic carcinoma (one case), biliary atresia (four cases), neonatal hepatitis (one case) and neonatal hyperbilirubinemia (one case), in the sera from four normal neonates, and in the sera from two kinds of nude mice bearing human endodermal sinus tumors. Sera from patients with endodermal sinus tumors and pancreatic carcinoma were found to contain a relatively high proportion (48.4 +/- 4.5 and 52.6%) of alphafetoprotein which did not bind to concanavalin A. Sera from nude mice with human endodermal sinus tumors contained AFP, 96.2% of which did not bind to concanavalin A. Sera from patients with other lesions (nine cases) and from normal neonates, whose AFPs are all presumed to be of hepatic origin, contained much less (5.9 +/- 3.6%) of the concanavalin A non-binding AFP variant. These results indicate that human AFP has three distinct patterns of reactivity with concanavalin A and that studies in xenograft models may give important information relating to the glycosylation and secretion process of AFP.

Topics: Adolescent; Adult; alpha-Fetoproteins; Animals; Biliary Tract Diseases; Carcinoma, Hepatocellular; Child; Child, Preschool; Chromatography, Affinity; Concanavalin A; Female; Humans; Infant; Infant, Newborn; Liver Neoplasms; Male; Mesonephroma; Mice; Mice, Nude; Neoplasm Transplantation; Teratoma

Resolution of human alpha-fetoprotein (AFP) into four distinct molecular species was demonstrated by a combination of two affinity chromatographies with crossed-immuno-affino-electrophoresis (CIAE) using concanavalin A (Con A) and Lens culinaris hemagglutinin (LcH)-A and LcH-B as affinity media. Of the four AFPs, AFP1 had no affinity for Con A, LcH-A, or LcH-B; AFP2 showed a high affinity for Con A, a low affinity for LcH-A, and an intermediate affinity for LcH-B (or a low affinity, depending on the lot of LcH-B preparations used); AFP3 revealed strong affinities for all of the three lectins; and AFP4, a trace component of hepatoma AFP in the present study, showed no interaction with Con A, but a definite interaction with LcH-A or LcH-B. These results were based on the determination of dissociation constants (Kd) of AFP-lectin complex by CIAE on isolated preparations of the three major hepatoma AFPs. These AFPs had identical electrophoretic mobilities of 0.86-0.87 (relative to human albumin) in the absence of lectins. The calculated mobilities of AFP2 and AFP3 were both reduced to 0.50-0.58 by saturation with lectins, but these two AFPs were clearly separated by 1 mg/ml LcH-A or LcH-B because of their large differences in Kd.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Humans; Immunoelectrophoresis, Two-Dimensional; Isomerism; Kinetics; Lectins; Liver Neoplasms

Glycosylation of alpha-fetoprotein (AFP) by human primary hepatocellular carcinoma (PHC) is abnormal. Concanavalin A (Con A)-affinity molecular variant patterns of serum AFP from patients with PHC are different from those of cord-serum AFP. Different patients with PHC exhibit different Con-A-affinity AFP molecular variant patterns, and the pattern remains constant over time in a given individual. The degree of deviation of the AFP molecular variant pattern from the molecular pattern of AFP secreted by neonatal liver cells is independent of the total serum AFP concentration. We propose that analysis of the AFP lectin-affinity molecular heterogeneity will improve the discrimination between malignant and nonmalignant liver disease in cases when the degree of elevation of the serum AFP concentration is nondiagnostic.

Topics: Adult; Aged; alpha-Fetoproteins; Carbohydrates; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Female; Fetal Blood; Humans; Liver Neoplasms; Male; Middle Aged; Phenotype; Pregnancy

The effect of concanavalin A on insulin binding was studied in cells cultured in a monolayer. Preliminary incubation of concanavalin A with Reuber rat hepatoma cells ( R117 - 21B ) showed an inhibitory effect on insulin binding at 25 degrees C and 4 degrees C; a "blocking" effect. But, even at high concentrations (greater than 50 micrograms/ml), concanavalin A could not completely inhibit the specific binding of insulin to these cells. Under these conditions, the number of high affinity insulin-binding sites decreased when compared to the number of sites in the control. In contrast, when cells first were incubated with insulin then with insulin plus concanavalin A, the binding of insulin increased, even at high concentrations of the lectin. This is a "trapping" effect. This effect increased the number of insulin-binding sites as compared to the number in the control. The apparent equilibrium constant of insulin for the cells was changed slightly by the concanavalin A in R117 - 21B cells. The binding of concanavalin A to these cells also was investigated. Insulin did not affect its binding to the cells. This study shows that insulin-binding sites can be classified into two groups in which insulin binding is affected by concanavalin A or is independent of the lectin. The "blocking" effect of concanavalin A on insulin binding suggests that the binding sites of insulin and concanavalin A on the insulin receptors are very close.

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Concanavalin A; Insulin; Liver Neoplasms; Rats; Receptor, Insulin; Receptors, Concanavalin A

The human hepatoma cell line SK-HEP-1 has been shown by radioimmunoelectrophoresis to synthesize and secrete a protein which coprecipitates with human alpha 1-antitrypsin. This protein was indistinguishable from serum alpha 1-antitrypsin in terms of electrophoretic mobility, apparent subunit molecular weight (47,000), and binding to concanavalin-A. The protein identified as alpha 1-antitrypsin (alpha 1 AT) was secreted by seven clones derived from SK-HEP-1 and by twelve out of eighteen hybrid clones derived from the fusion of SK-HEP-1 with mouse RAG cells. There was no correlation between the expression of alpha 1 AT and that of human enzymes assigned to sixteen different autosomes. There was an imperfect correlation between the expression of alpha 1 AT and of the two chromosome 9 marker enzymes AK1 and AK3 (two discordant clones).

Topics: Adenocarcinoma; alpha 1-Antitrypsin; Animals; Carcinoma, Hepatocellular; Cell Line; Clone Cells; Concanavalin A; Humans; Hybrid Cells; Karyotyping; Liver Neoplasms; Mice; Molecular Weight

The effects of several detergents commonly used to solubilize membrane glycoproteins have been investigated on the binding of hepatoma cell surface [3H]-galactoglycoproteins to, and their elution from, concanavalin A or Ricinus communis lectins conjugated to Sepharose 4B. The optimum conditions (pH, ionic strength) in the presence of ionic [sodium deoxycholate (DOC) and sodium dodecyl sulphate (SDS)] and non-ionic detergents (Triton X-100) at a constant concentration were determined in order to ascertain which would yield the better efficiency. The effects of different detergent concentrations on binding and elution were then studied. The range of concentrations for each detergent to be used without modifying efficiency was determined. Triton X-100 and DOC (0.1-1%) did not change the efficiency on Ricinus lectin-Sepharose, whereas SDS, at a concentration greater than 0.05%, caused a dramatic decrease in efficiency. On concanavalin A-Sepharose, by contrast, the non-ionic detergent had no effect on the efficiency at all the concentrations tested (0.1-1%), while concentrations of more than 0.5% DOC and 0.1% SDS significantly decreased both binding and elution.

Topics: Animals; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Detergents; Glycoproteins; Hydrogen-Ion Concentration; Lectins; Liver Neoplasms; Membrane Proteins; Protein Binding

Topics: Animals; Antibodies, Neoplasm; Carcinoma, Hepatocellular; Complement System Proteins; Concanavalin A; Cytotoxicity, Immunologic; Guinea Pigs; Immunoglobulin G; Immunoglobulin M; Liver Neoplasms; Neoplasms, Experimental; Rabbits; Sepharose; Staphylococcus aureus

Concanavalin A added to intact cells at 37 degrees caused rapid and reversible inactivation of a soluble enzyme, tyrosine aminotransferase, in two lines of rat hepatoma tissue culture cells grown in monolayer culture. This temperature-dependent process was independent of de novo protein and RNA synthesis and independent of increased uptake of Ca2+ and Mg2+ or glucose. The inactivation could be reversed by adding alpha-methyl-D-mannopyranoside a competing sugar for concanavalin A binding. Other lectins known to bind to different sugars did not bring about the inactivation of tyrosine aminotransferase. Addition of concanavalin A did not result in the inactivation of another soluble enzyme, lactic dehydrogenase. The maintenance of tyrosine aminotransferase in an inactive form after the binding of concanavalin A to the cells required the continued presence of concanavalin A. This effect of concanavalin A could not be mimicked either by dibutyryl cyclic adenosine or guanosine monophosphoric acid. Incubation of cell extracts with concanavalin A did not result in inactivation nor did mixing of extracts from concanavalin A-treated cells with extracts from untreated cells. On the basis of these results we conclude that the following are the essential requirements for concanavalin A to bring about the inactivation of tyrosine aminotransferase: (a) the binding of native concanavalin A to the cells; (b) integrity of certain structural elements of the cells.

Topics: Alanine Transaminase; Animals; Carcinoma, Hepatocellular; Cell Line; Concanavalin A; Cyclic AMP; Cyclic GMP; Enzyme Induction; Fluorouracil; Kinetics; L-Lactate Dehydrogenase; Liver Neoplasms; Rats; Theophylline; Tyrosine Transaminase

Topics: alpha-Fetoproteins; Animals; Animals, Newborn; Carcinoma, Hepatocellular; Concanavalin A; Female; Isomerism; Liver Neoplasms; Male; Neoplasm Proteins; Neoplasms, Experimental; Protein Binding; Rats; Rats, Inbred F344

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Clone Cells; Concanavalin A; Immunoelectrophoresis; Liver Neoplasms; Molecular Weight; Neoplasms, Experimental; Rats

Topics: Agglutination; Animals; Carcinoma, Hepatocellular; Cell Line; Cell Separation; Cell Survival; Concanavalin A; Glycopeptides; Lectins; Liver; Liver Neoplasms; Membrane Proteins; Papain; Rats; Receptors, Concanavalin A; Receptors, Drug; Surface Properties

Evidence for augmentation of antigenic recognition was investigated by measuring antigen-specific lymphocyte stimulation in animals which were minimally immunized with Con A-modified tumor cells. Comparison of lymphocyte stimulation response was made to animals immunized with unmodified tumor cells or sham immunization. Markedly heightened, tritiated Thymidine incorporation was observed in the lymph node cells from animals immunized with Concanavalin A-modified tumor cells, while minimal response was observed in control hosts. These preliminary findings imply that Con A-modified tumor cell immunotherapy may work by augmenting the host's capacity to recognized minimal differences in immunogenicity between tumor cells and the normal cells or origin. This modulation of the immune response may be a key factor in the success of active-specific immunotherapy.

Topics: Animals; Antibodies, Neoplasm; Antibody Formation; Antibody Specificity; Antigens, Neoplasm; Carcinoma, Hepatocellular; Concanavalin A; Female; Immunity; Immunotherapy; Liver Neoplasms; Lymphocyte Activation; Neoplasms, Experimental; Rats

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.

Topics: Animals; Aspartic Acid; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Liver; Liver Neoplasms; Neoplasms, Experimental; Rabbits; Rats; RNA, Transfer; Sepharose; Tyrosine

Concanavalin A (Con A) was found to inhibit the killing of antibody-sensitized line-1 tumor cells (TA) by guinea pig complement (GPC) but not by human complement (HuC). Other plant lectins (wheat germ, leucoagglutinin, and pokeweed mitogen) were also tested but Con A was the only lectin found to inhibit antibody-GPC-mediated killing. The inhibitory effect of Con A was observed when the GPC was mixed with Con A or when the antibody-sensitized cells were pretreated with Con A (TA-Con A) before the addition of GPC. The effect could be reversed by treatment of such cells with alpha-D-methylglucopyranoside or by incubation at 37 degrees C for approximately 2 hr. Con A appeared to act by preventing the binding of the first component of GPC (GPC1) to antibody-sensitized tumor cells. Differences in the binding of the first component of HuC (HuC1) and GPC1 to TA-Con A suggested that a difference in the binding site for HuC1 and GPC1 might exist. There was no difference in the number of GPC1 molecules fixed to antibody-sensitized sheep erythrocytes (EA) or EA treated with Con A in experiments using the same antibody as used with the tumor cells and the same Con A preparation. It would consequently appear that the inhibitory effect of Con A on the binding of GPC1 to TA is not due solely to an interaction of Con A with the antibody.

Topics: Animals; Antibodies; Binding Sites; Carcinoma, Hepatocellular; Cell Membrane; Complement C1; Complement Fixation Tests; Complement System Proteins; Concanavalin A; Cytotoxicity Tests, Immunologic; Forssman Antigen; Guinea Pigs; Liver Neoplasms; Neoplasms, Experimental; Species Specificity

Topics: Animals; Carcinoma, Hepatocellular; Cell Membrane; Concanavalin A; Female; Iodine Radioisotopes; Kinetics; Ligands; Liver Neoplasms; Methods; Neoplasms, Experimental; Protein Binding; Rats; Receptors, Concanavalin A; Receptors, Drug

Topics: Animals; Carbohydrates; Carcinoma, Hepatocellular; Cell Nucleus; Concanavalin A; Lectins; Liver; Liver Neoplasms; Membranes; Neoplasms, Experimental; Rats; Sarcoma, Experimental; Spectrometry, Fluorescence

Topics: Animals; Carcinoma, Hepatocellular; Chondroitinases and Chondroitin Lyases; Concanavalin A; Female; Kinetics; Lectins; Liver Neoplasms; Neoplasms, Experimental; Rats

Cell membranes from mouse L-cells (L-B82), rat hepatoma (HTC-H1), and three clones of their somatic cell hybrids (07, V4a, and V5) showing different degrees of density-dependent inhibition of growth were analyzed by polyacrylamide gel electrophoresis. The membrane polypeptides of the hybrid clones were all similar and all showed higher proportions of polypeptides with molecular weights of 56,000 and 45,000 than their parents of their normal counterparts. The major glycoprotein form cell hybrids appeared to be identical with that of rat liver or rat hepatoma cells and different from that of L-cells. One hybrid showed density-dependent inhibition growth; the other two, like both parents, did not. All produced tumors in nude mice, although tumor production by the hybrids was delayed. A large external protein (M.W. 240,000) iodinated by lactoperoxidase-catalyzed reaction was virtually missing in the parents but was present at high levels in all their hybrid clones. Thus, there was a lack of correlation between the presence of this protein, growth control in vitro, and tumorigenicity. Furthermore, no correlation was seen between agglutination of these cells by concanavalin A and tumorigenicity. The factors controlling these membrane properties thus are independent of density-dependent inhibition of growth and of those controlling the expression of cancer.

Topics: Agglutination; Animals; Carcinoma, Hepatocellular; Cell Fusion; Cell Membrane; Cell Transformation, Neoplastic; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Hybrid Cells; L Cells; Lactoperoxidase; Liver Neoplasms; Mice; Molecular Weight; Neoplasms, Experimental; Peptides; Rats

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Centrifugation; Concanavalin A; Culture Media; Dextrans; Lectins; Liver Neoplasms; Lymphocyte Activation; Neoplasm Transplantation; Rats; Time Factors

Topics: Animals; Blood; Carcinoma, Hepatocellular; Concanavalin A; Dextrans; Dose-Response Relationship, Drug; Lectins; Liver Neoplasms; Lymphocyte Activation; Neoplasms, Experimental; Rats; Time Factors

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.

Topics: Animals; Antigens, Bacterial; Antigens, Neoplasm; BCG Vaccine; Carcinoma, Hepatocellular; Concanavalin A; Culture Techniques; Female; Immunization; Lectins; Liver Neoplasms; Lymphocytes; Macrophages; Male; Mice; Mycobacterium bovis; Mycobacterium tuberculosis; Neoplasms, Experimental; Tuberculin

Various reagents used in the chemical modification of amino- and carboxy-groups of proteins, and of carbohydrates of glycoproteins and glycolipids, inhibit respiration in ascites tumor cells concomitant with release of potassium ion from those cells. The respiratory activity of washed ascites tumor cells is increased by exogenous addition of potassium ion. The lowered respiratory control index as well as oxidative phosphorylation of aged mitochondria are restored upon increasing the potassium concentration of the incubation mixture in the presence of respiratory substrates. The data suggest that the potassium ion level of cells is changed by modifying physicochemical properties of membrane components and that cellular energy metabolism is regulated by intracellular potassium ion concentration.

Topics: 4-Aminobenzoic Acid; Acrolein; Animals; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Membrane; Cells, Cultured; Cobalt; Concanavalin A; Dansyl Compounds; Dicyclohexylcarbodiimide; In Vitro Techniques; Lanthanum; Liver Neoplasms; Mitochondria, Liver; Ouabain; Oxidative Phosphorylation; Oxygen Consumption; Potassium; Rats; Ruthenium Red; Trinitrobenzenesulfonic Acid

An alpha-feto-protein (AFP) is present in many mammals, in birds, and in sharks during development. The AFP present in different species have similar physicochemical properties and often have common antigenic determinants. Their study, both in health and disease, has provided a useful model for the understanding of other phase-specific antigens and the activation of the genes which control their synthesis. In the human fetus, the level of AFP falls with increasing maturity. The more sensitive methods of detection have disclosed that this fetal protein persists in trace amounts throughout life and its level increases in maternal blood during pregnancy. The principal sites of synthesis are the fetal liver and in some mammals, the yolk sac splanchnopleur. In humans as well as in mice and cows, it is notable that the synthesis of AFP is increased in liver cancer cells and that high levels of this protein are present in serum. Elevated values of AFP have also been detected in human subjects with undifferentiated tumours of the testis and ovary. A fall to normal levels has been noted in cases of complete remission after surgery and a return to high levels in patients who develop metastases. In some patients with hepatitis a temporary rise in the level of AFP has also been observed. In recent years, the detection of high levels of AFP in amniotic fluid has proved to be of great value for the prenatal diagnosis of neural-tube defects. Abnormal levels have also been found in the amniotic fluid or in maternal serum in cases of spontaneous abortion. Such measurements are now being assessed as a methodof monitoring abnormal pregnancy.

Topics: alpha-Fetoproteins; Amniotic Fluid; Anencephaly; Animals; Antigen-Antibody Reactions; Carcinoma, Hepatocellular; Concanavalin A; Cystic Fibrosis; Down Syndrome; Female; Fetal Proteins; Gastrointestinal Neoplasms; Gestational Age; Hepatitis; Humans; Immunologic Techniques; Infant, Newborn; Liver; Liver Neoplasms; Metabolism, Inborn Errors; Neoplasm Metastasis; Neoplasms, Experimental; Pregnancy; Spinal Dysraphism; Teratoma

Concanavalin A-mediated agglutination reactions of hepatoma cells [AH-130F(N)] and isolated liver cells, as well as of plasma membranes prepared from the liver and hepatoma, were investigated kinetically together with the effect of colchicine upon them. Concanavalin A-mediated agglutination of hepatoma cells at 25 degrees proceeded with biphasic kinetics (first and second stages of agglutination), while no appreciable agglutination of liver cells was observed in the presence of concanavalin A. The plasma membranes from the liver and hepatoma cells were similarly agglutinated with concanavalin A at 25 degrees but not at 0 degrees. The concanavalin A-mediated membrane agglutination proceeded with monophasic kinetics and was incomplete. Colchicine inhibited preferentially the second stage of concanavalin A-mediated agglutination of hepatoma cells, but it did not affect the concanavalin A-mediated agglutination of plasma membranes of both types of cells.

Topics: Agglutination; Animals; Carcinoma, Hepatocellular; Cell Membrane; Colchicine; Concanavalin A; Kinetics; Liver; Liver Neoplasms; Male; Rats; Temperature

A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregatesmas predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates. The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [3-H]concanavalin A revealed that the binding to approx;0% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.

Topics: Agglutination Tests; Animals; Binding Sites, Antibody; Carcinoma, Hepatocellular; Concanavalin A; Kinetics; Lectins; Liver Neoplasms; Mathematics; Methods; Neoplasms, Experimental; Plant Lectins; Plants, Toxic; Protein Binding; Rats; Ricinus; Time Factors

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Chromatography, Affinity; Colonic Neoplasms; Concanavalin A; Freeze Drying; Glutaral; Humans; Liver Neoplasms; Neoplasm Metastasis

A previous study demonstrated that cells of transplantable hepatocellular carcinomas were agglutinated by the plant lectin concanavalin A, while normal hepatocytes were not. In the present experiments, 95% or more of cells obtained from primary hepatocellular carcinomas which resulted from exposure of rats to N-2-fluorenylacetamide were agglutinated by this lectin. Exposure to this carcinogen also produces grossly visible foci of morphologically and biochemically altered hepatocytes which have been termed hepatic (hyperplastic; premalignant, neoplastic) nodules. Although these hepatocyte aggregates are generally accepted as precursors of the hepatocellular carcinomas, no agglutination was detected when their cells were exposed to concanavalin A. These results indicate that concanavalin A agglutinability is not acquired as a result of tumor transplantation. Furthermore, they suggest that significant alterations must occur in the cells of hepatic nodules prior to the manifestation of malignant behavior.

Topics: 2-Acetylaminofluorene; Agglutination; Animals; Carcinoma, Hepatocellular; Concanavalin A; Liver Neoplasms; Male; Neoplasms, Experimental; Precancerous Conditions; Rats; Rats, Inbred ACI

The observation of BOREK et al. (1973) on nonagglutinability of transformed rat liver cells by Lens culinaris lectin and our ultrastructural findings of a greater mobility of the Lens culinaris lectin receptors on transformed rat liver cells as compared to normal rat liver cells (ROTH 1975) initiated the present agglutination experiments on liver cells with lectins. For agglutination assay the microhemadsorption technique after FURMANSKI et al. (1973) was used with exception of several tests on EDTA-detached cells. The transformed rat liver cells exhibited, in contrast to the findings of BOREK et al. (1973), a positive microhemadsorption with Lens culinaris lectin as well as with Concanavalin A, Ricinus communis lectin and wheat germ agglutinin whereas the normal rat liver cells became positive only after a brief trypsin treatment. The significance of the difference in agglutinability of rat liver cells with Lens culinaris lectin and the other lectins used is discussed with regard to the cell-cell interaction mediated by lectins.

Topics: Agglutination Tests; Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cell Wall; Concanavalin A; Hemadsorption; Lectins; Liver; Liver Neoplasms; Plant Lectins; Plants; Plants, Toxic; Rats; Ricinus; Surface Properties; Triticum

Topics: Animals; Antibodies, Neoplasm; Antibody Formation; Antibody Specificity; Antineoplastic Agents; Carcinoma, Hepatocellular; Concanavalin A; Epitopes; Liver Neoplasms; Neoplasms, Experimental; Rats

The distribution of Concanavalin A receptors in cultures of normal rat liver cells and of Zajdela ascites hepatoma cells of the rat was investigated by the Con A-po technique (Bernhard and Avrameas, 1971). Con A-po labelling of the cell surface showed a patchy distribution of Concanavalin A receptors in ascites hepatoma cells, whereas continuous Con A-po labelling was seen at the surface of normal rat liver cells. The different distribution of Concanavalin A receptors was caused by rapid internalization of Con A-po labelled material and not by a simple rearrangement of the receptors. The significance of this process is discussed with respect to Concanavalin A receptor mobility and membrane stability after cell transformation.

Topics: Animals; Carcinoma, Hepatocellular; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Culture Media; Golgi Apparatus; Liver; Liver Neoplasms; Microscopy, Electron; Neoplasm Transplantation; Neoplasms, Experimental; p-Dimethylaminoazobenzene; Peroxidases; Rats; Receptors, Drug; Staining and Labeling

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Chromatography, Gas; Chromatography, Gel; Concanavalin A; Glycopeptides; Haptens; Lectins; Liver; Liver Neoplasms; Models, Biological; Neoplasms, Experimental; Rats; Receptors, Drug; Spectrophotometry; Spectrophotometry, Ultraviolet; Trypsin

Numerous studies have reported the capacity of the lectin, concanavalin A, to agglutinate selected cell-types. The finding that cells transformed in culture, embryonic cells, and malignant cells are all agglutinated by this substance, may contribute to our understanding of the oncogenic process. The present study compared the response to concanavalin A of rat hepatocytes derived from livers of differing developmental and mitotic-status as well as those derived from malignant liver tumors (hepatomas). Fetal hepatocytes and hepatoma cells were highly susceptible to agglutination while hepatocytes from post-natal livers, whether dividing or quiescent, were not. Treatment with protease(s) did not make the interphase hepatocyte agglutinable. These data emphasize the importance of examining a wide variety of cells in attempting to understand the interaction of lectins on cell surfaces, and further, demonstrate the value of obtaining cells directly from tissue(s) during differing physiologic and pathologic states.

Topics: Agglutination; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Separation; Cell Transformation, Neoplastic; Concanavalin A; Erythrocytes; Liver; Liver Neoplasms; Male; Microbial Collagenase; Rats; Rats, Inbred ACI; Trypsin

Topics: Amino Acids; Animals; Binding Sites; Carcinoma, Hepatocellular; Chromatography, Gel; Chromatography, Ion Exchange; Concanavalin A; Female; Galactosamine; Glucosamine; Glycopeptides; Hexoses; Kinetics; Lectins; Liver Neoplasms; Neoplasms, Experimental; Papain; Plant Lectins; Pronase; Protein Binding; Rats; Receptors, Drug; Sialic Acids; Triticum

A substance capable of promoting tumour cell aggregation was released from rat ascites hepatoma cell (possibly from the cell surface) kept in Hanks' balanced salt solution (free of calcium and magnesium) in the cold, and then partially purified by chromatography with DEAE-Sephadex and gel filtration with Bio-gel. The thermostable substance seemed to be a glycoprotein and its molecular weight was about 72,000 when measured by gel filtration on Sephadex G-200. It had no proteolytic activity. The material was clearly effective for rat ascites hepatoma cells as well as SV40 transformed cells, but less effective for Chang's cells and apparently ineffective for normal rat liver cells and red blood cells. The action of this material was more potent than that of Jack bean concanavalin A when assayed for aggregation of SV40 transformed cells. Its effect was not influenced by concanavalin A inhibitors such as alpha-methyl-D-glucopyranoside, N-acetyl-D-glucosamine and D-glucose.

Topics: Animals; Carcinoma, Hepatocellular; Cell Aggregation; Cell Separation; Cell Transformation, Neoplastic; Chromatography, Gel; Concanavalin A; Erythrocytes; Glucosamine; Glucose; Glycoproteins; Liver; Liver Neoplasms; Male; Methylglucosides; Molecular Weight; Rats; Simian virus 40; Tissue Extracts

Topics: Animals; Carcinoma, Hepatocellular; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Concanavalin A; Cycloheximide; Epithelial Cells; Epithelium; Fibroblasts; Lectins; Liver; Liver Neoplasms; Neoplasm Proteins; Peptide Hydrolases; Protein Biosynthesis; Proteins; Rats; Surface Properties

Topics: Agglutination; Animals; Azides; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Membrane; Cells; Concanavalin A; Cycloheximide; Cytochalasin B; Dimethyl Sulfoxide; Dinitrophenols; Glycosides; Lectins; Liver Neoplasms; Rats; Sarcoma, Yoshida; Temperature; Tritium

Topics: Agglutination; Agglutination Tests; Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Aggregation; Cells, Cultured; Chromatography, Gel; Chromatography, Ion Exchange; Concanavalin A; Dose-Response Relationship, Drug; Glycopeptides; Guinea Pigs; Hemagglutination Inhibition Tests; Lectins; Liver Neoplasms; Neoplasms, Experimental; Neuraminic Acids; Papain; Plant Lectins; Pronase; Rats; Spectrophotometry, Ultraviolet; Triticum

Topics: Agglutination Tests; Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Cell Count; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Fibroblasts; Hydrogen-Ion Concentration; Kinetics; Lectins; Liver; Liver Neoplasms; Magnesium; Mice; Mitosis; Peptide Hydrolases; Polyomavirus; Receptors, Drug; Simian virus 40; Temperature; Tritium

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Centrifugation; Concanavalin A; Lectins; Liver Neoplasms; Liver Regeneration; Membranes; Mitochondria, Liver; Neoplasms, Experimental; Proteins; Rats; Spectrophotometry; Surface-Active Agents; Time Factors; Tritium

Topics: Agglutination; Animals; Carcinoma, Hepatocellular; Cell Movement; Concanavalin A; Electrophoresis; Liver; Liver Neoplasms; Neoplasms, Experimental; Neuraminidase; Rats; Surface Properties; Temperature; Trypsin

Topics: Animals; Carcinoma, Hepatocellular; Cell Aggregation; Cells, Cultured; Concanavalin A; Liver Neoplasms; Neoplasms, Experimental; p-Dimethylaminoazobenzene; Rats; Rotation

Differentiated epithelial cells in contact-inhibited monolayers derived from adult rat liver have been transformed in vitro into epithelioid neoplastic cells under conditions of nutritional stress. The transformed cells maintain their differentiated quality and manufacture serum proteins. They differ from control cultures in the following properties: They are aneuploid, can be agglutinated by wheat-germ agglutinin and concanavalin A, can grow in suspension, and are able to form colonies in semisoft agar. There is no intercellular communication at permeable junctions between the cells; this is demonstrable by electrical measurement or by injection of fluorescein. The cells show invasiveness in culture, and are not inhibited by contact with normal cells. The characteristics of the hepatocytes after transformation in vitro resemble those of epithelioid cells derived from a transplantable hepatoma.

Topics: Agglutination; Aneuploidy; Animals; Blood Proteins; Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Concanavalin A; Contact Inhibition; Culture Media; Epithelial Cells; Epithelium; Immunoelectrophoresis; Karyotyping; Lectins; Liver; Liver Neoplasms; Nutritional Physiological Phenomena; Rats; Stress, Physiological

Topics: Animals; Carbohydrates; Carcinoma, Hepatocellular; Cell Nucleus; Cells, Cultured; Chromatography; Chromatography, Gel; Concanavalin A; Galactose; Lectins; Liver; Liver Neoplasms; Mannose; Neoplasms, Experimental; Rats; Receptors, Drug; Tritium

Topics: Agglutination; Animals; Antigens, Neoplasm; Binding Sites; Carcinoma, Hepatocellular; Cell Fractionation; Cell Membrane; Chemical Precipitation; Chromatography, DEAE-Cellulose; Chromatography, Gel; Concanavalin A; Glycopeptides; Lectins; Liver Neoplasms; Molecular Weight; Neuraminic Acids; Papain; Peptides; Plant Lectins; Polysaccharides; Pronase; Trichloroacetic Acid; Triticum

Topics: Adult; Binding Sites; Carcinoma, Hepatocellular; Cell Differentiation; Cell Membrane; Concanavalin A; Female; Fetus; Fluoresceins; Humans; In Vitro Techniques; Infant; Infant, Newborn; Lectins; Liver; Liver Neoplasms; Male; Middle Aged; Spleen; Thymus Gland; Trypsin