A comparison of the ability of normal liver,a premalignant liver,a solid hepatoma and the zajdela ascitic hepatoma,to take up amino acidsin vitro

Summary The net total uptake of several amino acids at low (0.8–3.1 moles/liter) as well as high (800–1200 moles/liter) extracellular concentrations, by normal rat liver, a premalignant liver, a solid hepatoma, and the Zajdela ascitic hepatoma cells, has been compared under conditions in which protein synthesis continues. At low amino acid concentrations, the initial (3 min) total uptake of the various amino acids in the Zajdela cells, was 3–10 (average 7) times more, and the intracellular concentration of the labeled amino acids taken up 14–45 (average 31) times more, than in normal liver. At the high amino acid concentrations, the total uptake in the Zajdela cells, at 60–120 min was 2–5 (average 3.5) times more, and the intracellular concentration of the amino acids taken up 8–19 (average 13) times more, than in normal liver; the corresponding values for the premalignant liver and the solid hepatoma were in between those for normal liver and the Zajdela cells. Further, the rate of the total uptake of amino acids, their intracellular concentration, the proportion of the amino acid taken up utilized for protein synthesis, the rate of incorporation of the amino acid taken up into protein, and the cellular growth rate, seemed to be correlated in the four cell/tissue preparations studied. In most cases, the rate of the net uptake fell drastically with time, the uptake virtually stopping after 90–180 min, probably due to lack of serum in the incubation medium.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

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.     

The uptake of homologous ribonucleic acid by rat-liver parenchymal cells in suspension

1. Native or partially degraded RNA derived from intact rat liver, or from the parenchymal-cell or the non-parenchymatous fraction of liver, has been shown to be transported into rat parenchymal cells in suspension, without prior degradation to acid-soluble components, when the cell suspension is incubated with the RNA at 37 degrees . The amount of RNA of exogenous origin present in the parenchymal cells in an acid-precipitable form increased rapidly up to 30-60min., after which it gradually decreased, indicating intracellular degradation to acid-soluble components of the RNA taken up by the cells. 2. The RNA taken up by the parenchymal cells from the medium, and the acid-soluble products of its degradation within the cells, could be released back into the medium. 3. The RNA of exogenous origin present in acid-precipitable form in the parenchymal cells represented up to 5% of the RNA of the cells after 60min. of incubation. 4. When the concentration of RNA in the medium was less than 200mug./ml., over 10% of the RNA was transported in an acid-precipitable form in 60min. into the parenchymal cells incubated at a concentration of 2.3x10(6)/ml. 5. Ribonuclease inhibited the uptake of exogenous RNA by the parenchymal cells, whereas 2,4-dinitrophenol, sodium azide, protamine sulphate and polyvinyl sulphate had no significant effect. 6. The uptake of exogenous RNA by liver slices proceeded at a rate which was 4-20% of that obtained in the parenchymal-cell suspensions; the RNA taken up did not appear to become degraded, unlike that taken up by the cell suspensions. 7. It is concluded that dispersion of liver tissue to a suspension of single cells increases the permeability of the parenchymal cells to macromolecular RNA and creates conditions that lead to a rapid degradation of the RNA taken up.     

The nuclear matrix of slowly and rapidly proliferating liver cells.

The nuclear matrix of slowly proliferating rat liver is compared with rapidly proliferating regenerating liver and Zajdela ascites hepatoma cells. While no differences are detected in overall ultrastructure, composition or polypeptide profiles of normal liver versus regenerating liver matrices, significant alterations are observed in the polypeptides of Zajdela hepatoma nuclear matrices.     

Effect of cell concentration on the uptake of amino acids by rat liver parenchymal cells in suspension.

The accumulation of several amino acids in the acid-soluble fraction and their incorporation into protein in rat liver parenchymal cell suspensions, has been shown to depend on the concentration of cells in the incubation medium; the uptake, both in the acid-soluble and the acid-insoluble fractions, decreased as the cell concentration increased from 0.03 X 10(6) cells/ml upwards, reaching a plateau at high cell concentrations (3-5 X 10(6) cells/ml). The uptake values at high cell concentrations were the same as those obtained in liver slices in which a similar effect was not observed. Evidence is presented which suggests that this phenomenon is mediated by a material released from the cells in suspension, which is inhibitory to enhancement of the uptake of amino acids by these cells over and above the value obtained in normal, adult liver slices.     

Endocytosis of N-acetylglucosamine-containing glycoproteins by rat fibroblasts expressing a single species of chicken liver glycoprotein receptor

A cDNA clone for the chicken liver receptor which mediates endocytosis of glycoproteins containing terminal N-acetylglucosamine has been isolated and sequenced, confirming the previously obtained amino acid sequence of this protein (which is also known as the chicken hepatic lectin). This cDNA was introduced into Rat-1 fibroblasts and expressed using the promotor in the long terminal repeat of Moloney murine leukemia virus. Cells expressing chicken receptor were identified by screening with antireceptor antibodies followed by fluorescein-conjugated second antibodies. Receptor expressed in these cells was indistinguishable on gel electrophoresis from receptor isolated from liver. Three clonally isolated lines were examined for their ability to bind agalacto-alpha 1-acid glycoproteins at 0 degrees C and to take up and degrade this ligand at 37 degrees C. The receptor number (50,000/cell), affinity for ligand (35 nM), and uptake rate (5 molecules ligand/surface receptor/h) are similar to those previously observed for chicken hepatocytes, and for the uptake of asialoglycoproteins by rat hepatocytes and hepatoma cells. These findings indicate that the chicken receptor correctly traverses the endocytic pathway in a rat cell even though the cytoplasmic domain of this protein shows no primary structural homology with the corresponding portion of the rat liver receptor or with receptors found in fibroblasts.     

The effects of dietary conditions and glycerol concentration on glycerol uptake by rat liver and kidney-cortex slices

1. Glycerol utilization by rat liver and kidney-cortex slices was studied in an attempt to define factors that might be important in the regulation of glycerol utilization by these tissues in vivo; the formation of glucose from glycerol by kidney-cortex slices was also studied. 2. The rate of glycerol uptake by liver slices was not changed (in comparison with the normal fed control) by starvation (48hr.), feeding with a low-carbohydrate diet (4-8 days) or feeding with a diet containing 25% glycerol (up to 18 days). Similarly, starvation or a low-carbohydrate diet had no effect on uptake by kidney-cortex slices; however, feeding with the glycerol diet increased glycerol uptake by kidney-cortex slices. 3. The rates of glycerol uptake by slices from both tissues were increased on raising the glycerol concentration from 0.2mm to 2.5 or 5.0mm. 4. Starvation increased the conversion of glycerol into glucose by kidney-cortex slices, but there was no effect of the low-carbohydrate diet; the rate of glucose formation was increased by feeding with the 25%-glycerol diet and was proportional to the increase in glycerol uptake. The rate of glucose production by these slices was increased by raising the glycerol concentration in the incubation medium from 0.2mm to 1.0mm, but, except for the slices from animals receiving the 25%-glycerol diet, there was no effect above 1.0mm-glycerol. 5. The significance of plasma glycerol concentration in regulating glycerol uptake by these tissues is discussed.     

Receptor mediated endocytosis of formaldehyde treated albumin, yeast invertase and chondroitin sulfate in suspensions of rat liver endothelial cells

Isolated rat liver endothelial cells take up and degrade formaldehyde serum albumin (FSA), invertase and chondroitin sulfate (CS) efficiently. Degradation products start to appear in the medium after 5-30 min. Calcium was necessary for binding of invertase to the cells, but not for the two other ligands. Ammonia and monensin inhibited uptake as well as degradation of all three ligands, whereas leupeptin only inhibited the degradation of FSA and invertase. Uptake of CS was strongly inhibited in the presence of 1 microM FSA. The possibility that these two ligands bind to a common receptor is discussed.     

Uptake of amino acids and nucleic acid precursors by regenerating rat liver

1. At 0.5-1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [(3)H]orotic acid, [(3)H]uridine and [(3)H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [(3)H]Lysine entry into regenerating liver at this time was increased by 60%; [(3)H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.     

Localization of antigens in the chromatin of normal rat liver cells, not detectable in hepatoma chromatin

Antibodies against rat liver chromatin interact with homologous chromatin as well as with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, but not with the nuclear matrix isolated from these hepatomas. Rat liver chromatin regions hypersensitive to DNAase I and endogenous Mg2+-dependent nuclease are enriched with immunogenic nonhistone proteins. Using antiliver IgG pretreated with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, it was shown that liver chromatin antigens that are not detectable in hepatoma cells are localized in hypersensitive to nucleases chromatin regions buy not in actively transcribed ones.     

Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

The effect of actinomycin D on the synthesis of ribonucleic acid and protein in rat liver parenchymal cells in suspension and liver slices

1. Rat liver parenchymal cells in suspension are shown to require a higher concentration of actinomycin D than liver slices for equivalent inhibition of the incorporation of [(14)C]adenine, [(14)C]uracil and [(32)P]phosphate into RNA, and of (14)C-labelled amino acids into protein; protein synthesis is much less susceptible to actinomycin D inhibition than RNA synthesis in both the tissue preparations. Possible causes for these differences are discussed. 2. The uptake of [(3)H]actinomycin D in the first few minutes was much greater in the cell suspensions than in the tissue slices; that in the next 1-4hr. was about the same in both the cases. The uptake by both the tissue preparations was at all times proportional to the concentration of the drug within the range 0.5-2.0mug./ml. 3. In the slices actinomycin D taken up initially was concentrated almost exclusively in the nuclei; with time the concentration of the drug in the mitochondria and the supernatant increased more rapidly than in the nuclei though at no stage did it exceed that in the nuclei. In the cell suspension the largest concentration of the drug taken up initially was found in the supernatant; most of the drug taken up subsequently also stayed in the supernatant. 4. When the drug concentration in the incubation medium was 1mug./ml., its concentration within the parenchymal cells in suspension and the parenchymal cells in the slices reached 2.2 and 1.6mug./cm.(3) of cellular volume respectively. On average, 7% of the drug was removed from the medium by the cells in suspension and 23% by the cells in the slices; the average ratio of intracellular to extracellular concentration was 2.4 in the former and 2.1 in the latter case.     

Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.     

A tumor-cell-agglutinating lectin in snail mucus from Arion lusitanicus (MAB) and Arion empiricorum (Fér.)

A lectin which agglutinates Zajdela hepatoma cells; rat red cells and lymphocytes, but no normal rat liver cells, was detected in the mucus, yielded by simple saline extraction, of the two snail species Arion empiricorum (Fér.) and Arion lusitanicus (MAB). The agglutination spectrum involves also human erythrocytes and red cells of several animal species.     

Uptake studies in adipocytes isolated from rainbow trout (Salmo gairdnerii). A comparison with adipocytes from rat and cat

Adipocytes were isolated from mesenteric adipose tissue of rainbow trout (Salmo gairdnerii) by incubation of tissue slices at 20 degrees C in a buffer containing 3 mg collagenase per ml. These cells were compared to adipocytes from the cat and the rat, isolated by conventional technique (1 mg collagenase per ml buffer, incubation temperature 37 degrees C). Uptake studies of some metabolites were performed with fish, rat and in some cases cat adipocytes. At a glucose concentration of 0.33 mM, the glucose uptake into rat cells was more than twice as fast as in cells from the cat, and more than five times as fast as in trout cells. 2-Amino butyrate resembled glucose in relative uptake rates between species. Metabolite uptake into rat cells was specific, with different uptake rates for different metabolites. The uptake into trout adipocytes proceeded at similar rates for all metabolites tested, provided the concentrations were the same. The uptake rate of glucose into rat cells was stimulated by insulin. Insulin had no effect on glucose uptake into adipocytes from trout.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel

Brain tissue has become a challenging therapeutic target, in part because of failure of conventional treatments of brain tumors and a gradually increasing number of neurodegenerative diseases. Because antisense oligonucleotides are readily internalized by neuronal cells in culture, these compounds could possibly serve as novel therapeutic agents to meet such a challenge. In previous in vitro work using cell culture systems, we have demonstrated that intracellular delivery requires a vector such as cationic liposomes since free oligonucleotides remain largely trapped in the endocytic pathway following cellular uptake. Here we studied the cellular uptake properties of oligonucleotides by explants of rat brain (brain slices), and by in vivo brain tissue after administration of oligonucleotides by bolus injection. In contrast to in vitro uptake, we show that in brain slices oligonucleotides were taken up by neuronal and nonneuronal cells, irrespective of their assembly with cationic liposomes. In either case, a diffuse distribution of oligonucleotides was seen in the cytosol and/or nucleus. Uptake of oligonucleotides by brain slices as a result of membrane damage, potentially arising from the isolation procedure, could be excluded. Interestingly, internalization was inhibited following treatment of the tissue with antibody GN-2640, directed against a nucleic acid channel, present in rat kidney cells. Our data support the view that an analogous channel is present in brain tissue, allowing entry of free oligonucleotides but not plasmids. Indeed, for delivery of the latter and accomplishment of effective transfection, cationic lipids were needed for gene translocation into both brain slices and brain tissue in vivo. These data imply that for antisense therapy to become effective in brain, cationic lipid-mediated delivery will only be needed for specific cell targeting but not necessarily for delivery per se to accomplish nuclear deposition of oligonucleotides into brain cells and subsequent down-regulation of disease-related targets.     

Activation of a ribonuclease on dispersion of rat liver to a single cell suspension and incubation of the cells at 37 degrees

Incubation of rat liver parenchymal cell suspensions at 37° results in degradation, to acid-soluble material, of 15% of cellular RNA at 30 minutes and 40% at three hours, beyond which there is little, if any, further degradation. The RNA which remains in the acid-insoluble form in the cells up to 30 minutes appears to exist largely in the native state. However, after 30 minutes, the acidinsoluble RNA of the cells is found to be partially depolym-erised. These observations suggest the activation of an intracellular nuclease on dispersion of the liver tissue to a single cell suspension and incubation at 37°. This nuclease appears to be responsible also for the degradation, reported earlier, of exogenous RNA taken up by the cells. Activation of the nuclease is not due to depletion of pool of ATP or of other ribonucleotides from the cells, during either dispersion of the tissue or incubation at 37°. Incubation of the cells at 28°, or of liver slices at 37°, does not lead to any significant degradation to acid-soluble material, or to partial depolymerisation, of RNA. Analysis of RNA obtained from cells incubated at 37° for various periods showed that chromatography on methylated albumin-kiesulguhr (MAK) and Sephadex columns is not suitable for detecting partial depolymerisation of cellular RNA; RNA shown to be partially depolymerised by analysis on sucrose density gradient, in an analytical ultracentrifuge, and on a cellulose column, gave the normal pattern in MAK or Sephadex runs.