A monoclonal antibody disrupting cell-cell adhesion of rat ascites hepatoma cells

A cell surface-associated adhesive factor (AF) separated from differentiated rat ascites hepatoma AH136B cells (forming cell islands in vivo) has been highly purified by chromatography. AF is assumed to mediate the cell-cell adhesion essential to island formation of the hepatoma cells. A substance, immunologically crossreactive with AF, is present in the ascites fluid or culture medium of the AH136B cells. Because the substance is almost identical to AF in molecular weight and aggregation-promoting activity, it has been concluded that AF is released into the ascites fluid where it is concentrated. Monoclonal antibodies have been raised against AF purified from ascites fluid of AH136B cells. We have obtained a monoclonal antibody, coded MoAF-6D6, that strongly abolishes the aggregation-promoting activity of AF. When AH136B cell islands are incubated in the presence of Fab fragments of MoAF-6D6, cell detachment from the islands is evident within 24 h. Cell islands following 36-h culture show a distinct dissociation and islands completely lose their organization 48 h after culture. The dissociating effect of MoAF-6D6 is neutralized by the addition of AF. These results suggest that AF plays a significant role in the maintenance of cell islands.     

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.     

A new type of blood group B active glycosphingolipid in rat bone marrow cells. Occurrence of the glycolipid in rat immunocytes and ascites hepatoma

A major glycosphingolipid in rat bone marrow cells was purified, and its structure was studied. The glycolipid was found to exhibit blood group B activity by the hemagglutination inhibition test. The structure was determined to be (formula; see text) by studies of nuclear magnetic resonance, sequential hydrolysis by exoglycosidases, linkage analysis of methylated sugars by gas chromatography-mass spectrometry, and immunological tests. The blood group B active glycolipid was detected not only in the bone marrow cells but also in spleen, thymus, and rat ascites hepatoma AH 7974F cells. Besides the glycolipid, gangliotriaosylceramide, gangliotetraosylceramide, and fucogangliotetraosylceramide were commonly detected in these cells. The similarity between the glycolipid species on the cell surfaces of the immunocytes and the tumor cells is discussed with the respect to an escape mechanism of the tumor cells from the immunosurveillance system.     

Potentiation of invasive activity of hepatoma cells by reactive oxygen species is mediated by autocrine/paracrine loop of hepatocyte growth factor

We have already reported that reactive oxygen species (ROS) promote rat ascites hepatoma cell invasion beneath mesentery-derived mesothelial cell monolayer. To investigate the mechanism for this, we examined the involvement of motility factors, particularly hepatocyte growth factor (HGF). Rat ascites hepatoma cell line of AH109A expressed HGF and c-Met mRNAs. Treatment with ROS augmented amounts of HGF mRNA in AH109A and HGF concentration in the medium. ROS also induced HGF gene expression in mesothelial cells. Exogenously added HGF enhanced invasive activity of AH109A cells, but exerted no effect on proliferation. AH109A cells pretreated with ROS showed an increased invasive activity, which was cancelled by simultaneous pretreatment with anti-HGF antibody. These results suggest that the invasive activity of AH109A is mediated by the autocrine and paracrine pathways of HGF, and ROS potentiate invasive activity by inducing gene expression of HGF in AH109A and mesothelial cells.     

Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.     

Degradative removal of heparan sulfate from the surface of an ascites hepatoma, AH 66, by heparitinase

Heparitinase [EC 4.2.2.8, heparitin sulfate lyase] was prepared from an extract of cultured cells of Flavobacterium heparinum. Purification of the enzyme was achieved by repeating the hydroxyapatite column chromatography. The enzyme was used to degrade heparan sulfate occurring on the surfaces of ascites hepatoma cells, AH 66. From the supernatant of the enzyme-treated cells, breakdown products from heparan sulfate could be detected by paper chromatography. The heparitinase was found to be more effective than trypsin in removing heparan sulfate from the cells. Furthermore, on analyzing glycosaminoglycans and glycopeptides from the enzyme-treated cells and control cells, it was concluded that heparan sulfate was exclusively present on the cell surface and accessible to the heparitinase whereas other cell surface complex carbohydrates remained intact.     

Assay systems for screening food components that have anti-proliferative and anti-invasive activity to rat ascites hepatoma cells: In vitro and ex vivo effects of green tea extract

We have developed in vitro and ex vivo assay systems for screening food components and natural substances that suppress the proliferation and/or invasion of a rat ascites hepatoma cell line of AH109A and have used them to study the effect of green tea extract. AH109A cells were found to penetrate underneath the monolayer of primary cultured mesothelial cells isolated from Donryu rat mesentery in the presence of 10% newborn bovine serum. Green tea extract inhibited this AH109A penetration in a dose dependent manner and also inhibited AH109A proliferation in vitro dose-dependently. Green tea tannin, the major polyphenolic substances in green tea extract, also inhibited the proliferation and invasion of AH109A in vitro in a dose dependent manner. When rat serum obtained 0.5 h after oral intubation of green tea extract was added to the culture media instead of newborn bovine serum at a concentration of 10%, the invasion of AH109A was significantly inhibited as compared to control rat serum (before green tea extract intubation); the inhibitory effect lasted for 1 h and disappeared 3 h after oral intubation of green tea extract, but those rat sera showed no inhibition of AH109A proliferation. These results suggest that green tea extract has an inhibitory effect on the invasion of AH109A both in vitro and ex vivo, but on the proliferation of AH109A only in vitro, and that these assay systems are effective for the screening of food components which inhibit tumor cell proliferation and invasion.     

Anti-invasive activity of niacin and trigonelline against cancer cells

The effects of niacin, namely, nicotinic acid and nicotinamide, and trigonelline on the proliferation and invasion of cancer cells were studied using a rat ascites hepatoma cell line of AH109A in culture. Niacin and trigonelline inhibited the invasion of hepatoma cells at concentrations of 2.5-40 microM without affecting proliferation. Hepatoma cells previously cultured with a reactive oxygen species (ROS)-generating system showed increased invasive activity. Niacin and trigonelline suppressed this ROS-potentiated invasive capacity through simultaneous treatment of AH109A cells with the ROS-generating system. The present study indicates for the first time the anti-invasive activities of niacin and trigonelline against cancer cells.     

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.     

Differential expression of fucosyl GM1 and a disialoganglioside with a NeuAc alpha 2-6GalNAc linkage (GD1e) in various rat ascites hepatoma cells

A distinct difference in ganglioside composition among various rat ascites hepatomas and Yoshida sarcoma was observed on TLC-immunostaining with anti-fucosyl GM1 antibody, and chemical and enzymatic analyses. Yoshida sarcoma and ascites hepatomas, AH13, AH66F and AH66, but not the other 9 tumor cell lines investigated, specifically contained a disialoganglioside, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1ceramide (GD1e), whereas the 9 ascites hepatoma cells without GD1e contained fucosyl GM1. The differential expression of fucosyl GM1 and GD1e in various tumor cell lines indicates that different cell lineages express distinct metabolic pathways for gangliosides, and that the gangliosides are useful markers for distinguishing tumor cell lines.     

Lectin binding sites related with rat ascites hepatoma cell adhesion

In order to elucidate the correlation between cell surface lectin binding sites and the degree of cell adhesiveness, quantitative lectin binding assays were performed using three types of rat ascites hepatoma cell lines (free cell, mixed cell, and island-forming cell types). The lectin binding site patterns showed no remarkable differences among the intact tumor cell lines, but treatment of the cells with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or neuraminidase induced remarkable differences in the modulation of the number of lectin binding sites. TPCK-trypsin treatment caused a marked decrease in the number of peanut agglutinin binding sites on the island-forming and mixed cell types, concomitant with disaggregation of the cells, showing that trypsin sensitive binding sites are involved in the cell-cell adhesion. Neuraminidase treatment caused a decrease in wheat germ agglutinin binding sites and an increase in castor bean agglutinin binding sites, and these effects were greater for the free cell type. These results indicated that alpha-sialyl-beta-D-galactosyl residues are more abundant on the cell surface of the free cell type than the other cell types. Therefore, it was suggested that electrostatic repulsion due to negative charges of the cell surface sialic acid contributes to the low cell adhesiveness of the free cell type.     

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.     

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.     

Inhibitory effect of gingerol on the proliferation and invasion of hepatoma cells in culture

Effect of [6]-gingerol, a major pungent component in ginger, on the proliferation of a rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cultured cells and that on the invasion by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells. [6]-Gingerol inhibited both the proliferation and invasion of hepatoma cells in a dose-dependent manner at concentrations of 6.25–200 μM (proliferation) and 50–200 μM (invasion). [6]-Gingerol accumulated cells in S phase and elongated doubling time of hepatoma cells, and increased the rate of apoptosis. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. [6]-Gingerol suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with [6]-gingerol, HX and XO or with [6]-gingerol and hydrogen peroxide. Furthermore, [6]-gingerol reduced the intracellular peroxide levels in AH109A cells. These results suggest that the suppression of hepatoma cell proliferation by [6]-gingerol may be due to cell cycle arrest and apoptosis induction. They also suggest that the anti-oxidative property of [6]-gingerol may be involved in its anti-invasive activity of hepatoma cells.     

Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.     

Hepatocyte cell surface polarity as demonstrated by lectin binding

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.     

An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane.

The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.     

Inhibitory effect of serum from rats administered with coffee on the proliferation and invasion of rat ascites hepatoma cells

The action of coffee on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was investigated using in vitro and ex vivo assay systems. When rats were given oral intubation of instant coffee powder solution, the sera of those rats had the potent inhibitory activity on both the proliferation and invasion of AH109A. The activity of rat serum was both time- and dose-dependent. The instant coffee powder also inhibited the proliferation and invasion of AH109A in vitro. These results indicate that coffee has anti-proliferative and anti-invasive activity both in vitro and ex vivo. They also suggest that some anti-proliferative and anti-invasive material(s), which may be the ingredient(s) of coffee or their metabolites, appear in rat serum when rats are given oral intubation of coffee, although a possibility that host defense systems may be activated by the oral intubation of coffee cannot be ruled out. This revised version was published online in June 2006 with corrections to the Cover Date.     

The growth and differentiation of transitional epithelium in vitro

The development of rat transitional epithelial cells grown on conventional non-permeable surfaces was compared with development on permeable collagen supports. On glass or plastic surfaces, cells grew as expanding nomolayer sheets. Once confluent, growth continued with a bilayer being formed in most areas and apical cells being continuously sloughed off. Although most cells were interconnected by desmosomes, and junctional complexes were formed, no other indications of differentiation were observed. After 2-3 wk of growth, division stopped and cel death ensued. In contrast, single-cell suspensions plated on collagen-coated nylon disks reassociated into multicellular islands and commenced growth. Mitoses were confined to the basal cells in contact with the permeable substrate. The islands developed into epithelial trilayers, tapering to monolayers along spreading edges. Once the islands were confluent, stratification was completed and appeared similar to that observed in vivo. Germinal cells formed a basal lamina, and the upper layer was composed of large, flattened cells with an unusually thick asymmetrical plasma membrane on the apical surface. Electron microscopic and radioactive tracers demonstrated "leaky" zonulae occludentes with a restricted permeability to small molecules. The movement of urea was retarded in comparison to water. Unlike the slow turnover of adult epithelium in vivo, maturation and sloughing of apical cells were measurable. Transfer of cells could be effected and growth maintained for up to 4 mo. These results may indicate the necessity of a nutrient-permeable growth surface for the polarized differentiation of adult transitional epithelium.