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.     

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.     

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 phosphatidylcholine, 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 spectrofluorimetric 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.     

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 cells 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 200000 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.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Immunoelectron microscopic localization of ubiquitin in hepatoma cells.

Ubiquitin, a 76 amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Free (unconjugated) ubiquitin was localized in hepatoma cells using affinity purified anti-ubiquitin antibodies and colloidal gold immunoelectron microscopy. The anti-ubiquitin antibodies recognize only unconjugated ubiquitin. Ubiquitin is found within the cytoplasm, nucleus, the microvilli, autophagic vacuoles and lysosomes.     

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.     

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.     

Cytochemical localization of peroxidase activity in root cells

Summary The distribution of peroxidase in the apical 3 mm of pea roots has been investigated using the histochemical method employing 3,3-diaminobenzidine as a substrate. At the tissue level the enzyme is localized predominately in the root cap, epidermis, inner cortical cells, endodermis, phloem and maturing xylem. At the subcellular level peroxidase is found mainly in the intercellular regions of the cortex cell walls and in the cytoplasm and vacuoles of the steler cells. Root microbodies, unlike those of leaves, do not appear to be able to oxidize this substrate. The significance of these observations is discussed in relation to the validity of the technique and the proposed roles of the enzyme in cellular metabolism.     

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.     

A Polydeoxythymidylic acid-specific deoxyribonuclease from rat ascites hepatoma cells.

A deoxyribonuclease has been purified 950-fold from rat ascites hepatoma cells and has been separated from another deoxyribonuclease that appears to have DNase III type activity. The enzyme preferentially degrades single stranded poly(dT), requires Mg²⁺ for maximum activity and has a pH optimum at 8.5 in Tris-HCl buffer. Poly(dA), poly(dC), poly(rA), and poly(rU) are not effective substrates. The hydrolysis of poly(dT) is strongly inhibited when poly(dA) or poly(rA) is annealed with poly(dT). Poly(dT) is degraded ultimately into 5′-deoxythymidylic acid via the formation of oligodeoxythymidylate intermediates.     

Interrelationships between Golgi, GERL and synaptic vesicles in the nerve cells of insect and gastropod ganglia.

In addition to demonstrating synaptic vesicles, staining with the zinc-iodide-osmium tetroxide (ZIO) method reveals the presence of positively reacting GERL membranes in association with the Golgi complex and lysosomes in the nerve cell bodies within ganglia from the locust Schistocerca gregaria and the gastropod molluscs, Limnaea stagnalis and Helix aspersa. A positive response to ZIO occurs in certain Golgi vesicles and saccules, in GERL (Golgi-endoplasmic-reticulum-lysosomes), in multivesicular bodies as well as residual bodies and in small vesicles and cisternae of axonal smooth endoplasmic reticllum (ER). The interrelationships between these organelles are considered in view of the similarity of the ZIO localization to phosphatase-rich sites in the neuronal perikarya and with respect to the possibility that components of the synaptic vesicles are formed in the Golgi region of the cell and migrate via the axonal smooth ER to the synaptic regions.     

Cytochemical localization and biochemical analysis of the enzyme markers in human hepatoma cell lines

Three enzyme makers, glucose-6-phosphatase, ATPase and gamma-glutamyl transpeptidase, have been used in studying carcinogenesis of hepatocellular carcinoma. They have been investigated in animal models and human hepatocellular carcinoma in vivo and in vitro. But the inconsistent levels of these three enzymes associated with this type of carcinoma raised the possibility that the carcinoma cells might have derived from the cells originating from different stages of differentiation. To evaluate this possibility, three human cell lines, Hep G2, Hep 3B, and HA 22T, all thought to be arrested in different stages of differentiation based on their biochemical and morphological characteristics, were used as models. The three enzyme markers glucose-6-phosphatase, ATPase and gamma-glutamyl transpeptidase were examined cytochemically and biochemically. Our results showed that there was no correlation between the ATPase levels and the stages of the cell line's differentiation. But both glucose-6-phosphatase and gamma-glutamyl-transpeptidase were higher in cells that were more differentiated.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

Cytochemical localization of NADH-ferricyanide oxido-reductase in hypocotyl segments and isolated membrane vesicles of soybean

Summary NADH-ferricyanide oxido-reductase activity was demonstrated at the inner (cytoplasmic) aspect of plasma membranes and plasma membrane vesicles from hypocotyls of etiolated soybean (Glycine max L.) seedlings by cytochemical procedures. The plasma membrane-associated activity, observed in both tissue and vesicle preparations, resisted fixation in 0.1 % glutaraldehyde, required the presence of exogenous pyridine nucleotide and was inhibited by adriamycin. With tissue, the activity could be demonstrated only with broken cells where reactants could penetrate freely. With vesicles of plasma membrane origin, activity was seen only with cytoplasmic side out vesicles (fraction E) prepared by free-flow electrophoresis. Activity was observed also on the cytoplasmic surface of the tonoplast and on putative tonoplast vesicles oriented cytoplasmic side out.Recipient of a NSF/CNRS post doctoral fellowship.     

Cytochemical localization of phosphatase in differentiating secondary vascular cells

Summary Phosphatase activity was studied in the cambium and differentiating vascular cells of beech by using a modified Washstein and Meisel method. After fixation in glutaraldehyde or crotonaldehyde and incubation in a medium containing ATP and lead nitrate at pH 7.2, a deposit of electron-opaque granules was found in the nucleoli, nucleoplasm, nuclear envelope, endoplasmic reticulum, plastids, mitochondria, and at the plasmalemma. Although located at these different sites, the distribution varied both inter- and intra-cellularly. This is thought to be a true reflection of the variation in activity between closely adjacent cells in this part of the stem.Some reaction was obtained when ADP replaced ATP in the reaction mixture, but there was no reaction at all when both ATP and ADP were omitted. Fixation in hydroxyadipaldehyde, or incubation at pH 6.4 both produced very little reaction.