Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

In vitro interaction of Zajdela ascites hepatoma cells with lipid vesicles.

We studied the in vitro interaction between Zajdela ascites hepatoma cells and small unilamellar vesicles, consisting of 14C-labeled phosphatidylacholine, cholesterol, and phosphatidylserine (molar ratio 5 : 4 : 1), containing high intravesicular concentrations of carboxyfluorescein or fluorescein isothiocyanate tagged dextran. The entrapped markers were found to be associated with the cells to a lesser degree than the vesicle membrane marker. This discrepancy, which is slightly less pronounced for fluorescein isothiocyanate tagged dextran than for carboxyfluorescein, increases with incubation time and decreases with increasing vesicle lipid concentration in the incubation mixture. Vesicle-plasma membrane exchange of the vesicle lipid marker could not entirely explain the observed discrepancy. It is tentatively concluded that the gap mainly arises from a selective loss of entrapped dyes from vesicles actually interacting with the cell surface. Both spectrofluorimetry and fluorescence microscopic observations, as well as the relative insensitivity of vesicle uptake towards the presence of metabolic inhibitors, exclude a major contribution of endocytosis as a vesicle uptake route. We therefore conclude that vesicles are primarily internalized by a vesicle-cell fusion-like process. The observed discrepancy in uptake between entrapped materials and vesicle lipid is discussed in terms of a two-site vesicle-cell surface interaction model.     

Morphological and functional heterogeneity of Zajdela rat ascites hepatoma cells

This work studies the mechanisms of dysdifferentiation at cell neoplastic transformation based on the example of heterogeneity of the cell populations that form malignant tumors. Two natural fractions of Zajdela rat hepatoma cells are revealed that differ in the type of growth in the primary culture. Cells of one fraction are attached to substrate and are growing in monolayer (S-fraction), whereas cells of the other fraction are floating in the culture medium (F-fraction). Using the method of supravital observation of the primary culture cells (of 1–2 passages) at the limit of resolution of DIC microscopy, it has been established that both fractions contain cells of several types. Some of these cells are specific to one of the fractions and others are present in both fractions, but with different frequencies. Using the same method, it has been shown that, at the long-term separate cultivation of the fractions in vitro (more than 50 passages), both the cell composition and the initial ratio of cells of different types are changed in both of them. According to the data of flow DNA cytometry, cells of both fractions are hypotetraploid and have insignificant differences in the amount of DNA. After adaptation to conditions of cultivation in vitro, S-fraction cells have been found to have elevated proliferative activity compared to the cells of F-fractions; after long cultivation, the fractions already differ significantly (2.3 times) by this criterion. The content of the cell surface laminin, a marker of hepatocellular carcinomas, is higher on cells of the F-fraction than on those of the S-fraction. The interfraction differences are confirmed by immunologic estimations of the resistance of hepatoma cells to lyses of natural killer cells; cells of the S-fraction of the primary culture are 2.4 times more sensitive than cells of the F-fraction, while, after long-term cultivation, cells of the F-fraction become almost resistant to the cytotoxic action of natural killer cells. Based on the obtained data, the most probable pathways of the dysdifferentiation of rat hepatocytes upon the establishment of Zajdela hepatoma and at the long-term cultivation of cells of this tumor in vitro are discussed.     

Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells

The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane.     

Altered metabolic states do not change the intracellular distribution of hexokinase in Zajdela hepatoma ascites cells.

The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.     

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

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

Impairment of enzyme induction by glucocorticoids in Zajdela hepatoma cells.

Whereas glucocorticoids induce TAT, TRP, GPT in liver and only TAT in HTC cells, no hormonal effect on the synthesis of these enzymes was found in Zajdela hepatoma cells grown in vivo as an ascitic tumor, or in vitro as layer cultures. Although these cells remain uninducible, the hormone penetrates normally, but a strong decrease of the specific binding of cytosol and nuclear proteins with the hormone was observed. The impairment at the level of the hormone receptors could account for the non-inducibility of enzyme synthesis in ZHC cells.     

Interaction between the dexamethasone-receptor complex and Zajdela ascites hepatoma DNA]

The activated dexamethasone--receptor complex from rat liver cell cytoplasm and from Zajdela ascite hepatoma cell cytoplasm is bound to Sepharose-immobilized native DNA containing different numbers of recurrent sequences. The DNA-bound hormonereceptor complex is eluted as three peaks by varying concentrations of NaCl. The binding in independent of the number of DNA recurrent sequences and of the source of the cytoplasmic complex used.     

Cytochemical localization of lectin labeled vesicles in GERL region of hepatoma ascites cells.

The fate of lectin labeled internalized plasma membrane in the ascites tumor form of the Chang rat hepatoma growing under in vivo and in vitro conditions was investigated cytochemically. Ascites cells were incubated in Convanavalin A (Con A) and horseradish peroxidase (PO), either with or without prior glutaraldehyde fixation and subsequently treated with 3',3-diaminobenzidine. In cells fixed before Con-A-PO labeling the reaction product was localized as a continuous and even layer upon the external surface of the plasma membrane. If unfixed cells were treated with Con A, coupled with PO at 4 degrees C and reincubated in phosphate buffered saline at 37 degrees C for varying periods of time, the Con-A-PO layer was of irregular thickness. In as little as 15 min of reincubation endocytotic vesicles containing PO positive material were closely associated with GERL components of the Golgi Apparatus. Localization of acid phosphatase (ACPase) within GERL vesicles, similar in size and location to those containing Con-A-PO reaction product, indicates that the Con-A-PO labeled vesicles may be a component of the Golgi apparatus in hepatoma cells.     

Cell membrane polarity in rat ascites hepatoma cells

Summary As previously described, a cell surface-associated adhesive factor (AF) was separated from differentiated rat ascites hepatoma AH136B cells (forming cell islands in vivo) and highly purified by chromatography. AF induces not only aggregation of dissociated AH136B cells or undifferentiated rat ascites hepatoma AH109A cells (present as free cells in vivo), but also adhesiveness characterized by the development of junctional complexes. The localization of AF on the surfaces of AH136B cell islands was investigated using anti-AF IgG (Fab fragment) coupled to peroxidase. AF was detected in the contact region of the lateral surfaces of the AH136B cells and in the intercellular spaces. In contrast, no AF was detectable on the apical non-contacted free cell surfaces of AH136B cells. Fluorescence studies revealed that biotin-labeled AF did not bind to the apical surface of AH136B cell islands. These results indicate a distinct difference between apical and lateral surfaces of AH136B cells; neither AF nor binding site for AF were localized on the apical surface of AH136B cells, whereas both were localized on the lateral surface. On the other hand, AH136B cells detached from the cell islands, or during the process of partial dissociation from them, showed the loss of the AF localization and binding site of AF on the surfaces. The results suggest that AH136B cell surfaces may be polarized in terms of the AF localization, and this polarization may be lost after cell dissociation.     

Analysis of substances released from Zajdela ascites hepatoma cells exposed to UV radiation of different wavelengths. III. Release of nucleoproteins and carbohydrates]

Irradiation of the Zaidela ascite hepatoma cells with physiological doses of shortwave length (254 nm) and longwave length (300-380 nm) UV light (far and near UV radiation) is accompanied by the release of ribonucleoproteins (RNP) from the cells, whose amounts increase with dose. Irradiation with far and near UV light leads to the release of high-molecular and low-molecular RNP, respectively. No deoxyribonucleoprotein were found among the released substances. Non-protein fractions, released from irradiated cells, contain carbohydrate-like substances. At maximum far and near UV doses the amounts of these substances constitute 180-190% of the control and 6% of their amount in intact cells. After irradiation with far UV light, relatively high-molecular carbohydrates are released, while near UV light treatment induces the release of low-molecular carbohydrates. The criteria tested show that the efficiency of far UV light exceeds that of near UV light by one order.     

Glucocorticoid-mediated degradation of DNA in thymocytes of rats with transplantable Zajdela ascites hepatoma

Tumour growth was shown to be associated with DNA breakdown in thymocytes of rats bearing Zajdela ascites hepatomas. The tumour action on the thymus is mediated through adrenal glands since bilateral adrenalectomy completely prevents DNA breakdown in thymocytes. Using Southern hybridization of DNA genome with probes for histone, ribosomal and heat shock gene (hsp 70), it was shown that the degradation products of specific DNA sequences are as heterogenous as those of total DNA, although marked differences in appearance of nucleosomal ladder were seen. These data were interpreted to indicate different patterns of DNA breakdown in dying thymocytes. DNA breakdown in thymocytes in vivo and in isolated rat liver nuclei in vitro seems to proceed by similar mechanisms.     

Cytochemical localization of lectin labeled vesicles in GERL region of hepatoma ascites cells

Summary The fate of lectin labeled internalized plasma membrane in the ascites tumor form of the Chang rat hepatoma growing under in vivo and in vitro conditions was investigated cytochemically. Ascites cells were incubated in Concanavalin A (Con A) and horseradish peroxidase (PO), either with or without prior glutaraldehyde fixation and subsequently treated with 3,3-diaminobenzidine. In cells fixed before Con-A-PO labeling the reaction product was localized as a continuous and even layer upon the external surface of the plasma membrane. If unfixed cells were treated with Con A, coupled with PO at 4°C and reinbated in phosphate buffered saline at 37°C for varying periods of time, the Con-A-PO layer was of irregular thickness. In as little as 15 min of reincubation endocytotic vesicles containing PO positive material were closely associated with GERL components of the Golgi Apparatus. Localization of acid phosphatase (ACPase) within GERL vesicles, similar in size and location to those containing Con-A-PO reaction product, indicates that the Con-A-PO labeled vesicles may be a component of the Golgi apparatus in hepatoma cells.Supported by NIH Grant CA 16663.     

Revelation and identification of laminin in the structure of plasma membrane of ascitic Zajdela hepatoma cells

Cell dysdifferentiation during neoplastic transformation is a crucial problem of cell biology and oncology. Antigenic diversion of cancer cells is a typical characteristic of dysdifferentiation. It involves the appearance of antigens which are unusual for normal tissue of this type. Components organospecific for membrane proteins of normal kidney were previously found among plasma membrane proteins of hepatocellular rat tumors, rat hepatocytes after carcinogen treatment, and regenerating liver, respectively. In the present work we showed that a protein with mol. weight about 200 kDa reacting with laminin-1 immunoserum is the basic component of plasma membranes of the rat Zajdela hepatoma cells, which is responsive for organospecific anti-kidney immunoserum in Western blot. A mass-spectrometer analysis of trypsin proteolysis fragments was carried out in SDS-PAGE slices containing the investigated component. The analysis showed the presence of beta1, beta2 and alpha4 laminin chains peptides. The component with mol. weight about 180 kDa, found in the Western blot with laminin-1 immunoserum, was also subjected to the mass spectrometer analysis. As a result, a gamma1 laminin chain was found. An increased amount of laminin was revealed in the ascitic liquid and sera of rat with developed Zajdela hepatoma, in comparison with sera of normal rats. In addition, we found the appearance of laminin on the hepatocyte surface on the 4th day after hepatocarcinogen injection (N-diethylnitrosamine, DENA). Thus, for the first time tumor associated antigens were revealed and identified in the structure of plasma membranes of Zajdela hepatoma cells, being specific to rat kidneys. Our results allow to conclude that in the process of carcinogenesis in rat liver laminin synthesis occurs, which is also characteristic of the rat hepatoma Zajdela cells.