Inhibition of transport systems in cultured rat hepatoma cells by colcemid and ethanol

The transport of uridine, hypoxanthine, and choline in cultures of Novikoff rat hepatoma cells is competitively inhibited by colcemid with apparent K_i values of 135, 60, and 250 μM respectively, whereas the transport of 2-deoxy-D-glucose is not affected. Ethanol at high concentrations inhibits the transport of all four substrates in an apparent competitive manner.     

Zajdela hepatoma cells cultured in vitro

The goal of this work was to study cellular mechanisms of tumor progression and metastasizing. As a result of explantation of cells of rat Zajdela ascitic hepatoma, we obtained two transplantable cell cultures—monolayer (ZH-ad) and suspension (ZH-fl)—that differ in levels of cell differentiation and tumorigenicity. By using tumor-specific immune serum, we revealed tumor-associated antigens, synthesis of which is reduced or inhibited in ZH-ad cells, in outer membranes of the ZH-fl cells. Intraperitoneal injection into rat of 0.5–12 × 10⁶ ZH-fl cells leads to development of an ascitic tumor and death of 100% of animals, whereas, in the case of administration of ZH-ad cells, to achieve a tumorigenic effect, the minimal dose needs to be elevated to 20 × 10⁶ cells. Clonogenic analysis of the ZH-fl cells revealed three types of the formed clones—nonadhesive sphere colonies and two types of monolayer clones differing in proliferative potential, shape of colonies, and cell composition. Upon reaching a critical size, the spheres disintegrated, with separation of single cells and islands of different sizes, some of them being attached with monolayer formation. Three clonal cell lines were obtained: 1C as a result of expansion of a spherical clone and 4G and 10E from monolayer clones. We established that there is tumorigenicity of the 1C cell line, which, at a dose of 107 cells, led to the development of ascites and to the death of 50% of animals. The presented results indicate the existence in the ZH-fl cell population of tumor-initiating cells generating spherical clones—floating multicellular islets that, in culturing in the complete growth medium, are partly differentiated and are attached with monolayer formation.     

Inhibition of synthesis of alpha-fetoprotein by glucocorticoids in cultured hepatoma cells

alpha-Fetoprotein (AFP) synthesis was studied in the presence and absence of glucocorticoids in rat hepatoma Mc-A-RH-7777 cells. Radioimmunoassay of media from cell cultures grown in the presence of glucocorticoid (dexamethasone or cortisol) showed a reduction in AFP, an increase in albumin, and no significant change in transferrin accumulation, as compared to controls. Labeling experiments with L-[35S]methionine indicated that in both cells and media of dexamethasone-treated cultures there was a 50--80% reduction in polypeptide precipitated by anti-AFP serum, as compared with controls; no change was seen in polypeptide precipitated by anti-transferrin serum. Pulse and pulse-chase experiments demonstrated that dexamethasone inhibited the synthesis of AFP but not its secretion. The half-time for secretion of AFP in the presence and absence of dexamethasone was 43 min.     

Inhibition of uridine transport in cultured mammalian cells by theophylline

Theophylline (theobromine, caffeine) reversibly inhibits the incorporation of labeled RNA precursors both in confluent 37 RC and in exponentially growing HeLa cells. As measured in 37 RC after 2 h labeling, 20 mM theophylline reduces the incorporation of [3H]UTP and [14C]uridine into acid-precipitable material to 5% and 9% of the control, respectively. This reduction is paralleled by a comparably lowered incorporation of the same precursors into the acid-soluble pool. The initial rate of incorporation into total cell material is similarly affected by theophylline, the inhibition being of a simple competitive type. Theophylline does not alter the turnover rate of pulse labeled RNA during actinomycin D chase nor does it preclude the utilization of the endogenous pool of nucleoside phosphates. Upto a concentration of 10 mM, it does not inhibit uridine kinase neither in 37 RC nor in HeLa cells. The mentioned inhibitory effects of theophylline cannot be mimicked by exogenously added cyclic AMP. All the data support the conclusion that theophylline inhibits the transport of uridine into the cell.     

Activation and inactivation of thyroxine by cultured rat hepatoma cells

The metabolism of thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with ¹²⁵I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography.When thyroxine was incubated with the cells at 37°C, its glucuronide was the major product and a little increase in ¹²⁵I^? was detected. Although 3,3′,5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation.This cell line also metabolized labeled 3,3′,5-triiodothyronine added to culture medium. After 24 h incubation, 3,3′,5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3′,5-triiodothyronine and 3,3′-diiodothyronine sulfates and an unknown component.In the metabolism of 3,3′,5′-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3′,5′-triiodothyronine and 3,3′-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3′-diiodothyronine sulfate.These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.     

Studies of inhibition of rat spermidine synthase and spermine synthase

1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5′-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70–98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5′-Methylthioadenosine, 5′-ethylthioadenosine and 5′-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10–15μm and 50% inhibition of spermidine synthase at 30–45μm. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20μm respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5′-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Inhibition of sodium-independent amino acid transport by dexamethasone in rat hepatoma cells

We studied the uptake of leucine, phenylalanine, and the amino acid analog, 2-aminonorborane-2-carboxylic acid, by rat hepatoma cells in tissue culture. The uptake of these amino acids was partially mediated by a plasma membrane transport system similar to the L agency described in other cell types in that it does not require extracellular sodium and is subject to trans-stimulation. Initial rates of sodium-independent transport of these amino acids were calculated using mathematical transformations of the uptake time course curves. The glucocorticoid dexamethasone inhibits the activity of this transport system; the initial rates of sodium-independent uptake of leucine, phenylalanine, and 2-aminonorborane-2-carboxylic acid are decreased by approximately one-third (average = 30%, n = 19) after incubation of HTC cells with 0.1 microM dexamethasone. This inhibition requires at least 15 h, reaching a maximum at 24 h of exposure of the cells to the hormone. Dexamethasone has an asymmetrical effect on sodium-independent amino acid transport in that exposure of the cells to the hormone does not inhibit the rates of outflow of leucine or phenylalanine from preloaded cells into medium without sodium. Inhibition of uptake is blocked by 0.1 mM cycloheximide and 4 microM actinomycin D, indicating the need for continuous protein synthesis for dexamethasone action. Insulin, which is known to partially reverse the inhibitory effect of dexamethasone on the A amino acid transport system in HTC cells, does not alter the action of dexamethasone on the L system. Previous investigations have demonstrated inhibition by dexamethasone of at least two distinct sodium-dependent amino acid transport activities in HTC cells. The data presented here, showing inhibition by the glucocorticoid of a sodium-independent transport activity, indicate that the effect of the hormone is independent of the energy source of the amino acid transport systems affected.     

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate

Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis.     

Metabolism of testosterone and dihydrotestosterone in cultured rat hepatoma cells.

Steroid metabolism in hepatoma tissue culture (HTC) cells derived from a male rat was investigated. Steroids in ethanol were incubated with the cells for various lengths of time. Volume of ethanol never exceeded 1% of incubation volume. Thin-layer and paper chromatography were used. Incubation was with tritiated steroids. It was demonstrated that testosterone as well as dihydrotestosterone is transformed. The main enzyme activities detected were 5alpha-reduction and 3alpha-, 3beta, and 17beta-hydroxysteroid dehydrogenation. The pattern of metabolism was reproducible and varied with time, substrate concentration, and number of cells incubated. Some steroids interfered with androgen metabolism. 17beta-estradiol, 17-epitestosterone, and progesterone competed for the 17beta-hydroxyprogesterone dehydrogenase. it is concluded that 3beta and 17beta reduction in the HTC cells may be catalyzed by the same enzyme which might differ considerably from the 3beta-hydroxysteroid dehydrogenase assayed in intact liver cells. A hepatoma derived from a female rat also produced considerable amounts of 3beta-derivatives of testosterone.     

Proteoglycan synthesis by rat lung cells cultured in vitro

Cells having a fibroblast-like morphology were cultured from explants of adult rat lung tissue. (35S)Sulfate was incorporated into sulfated proteoglycans in the medium at a linear rate for up to 96 h, while the rate of incorporation into the cell layer increased gradually until reaching a plateau at 40 h. The culture medium contained proteoglycans which migrated as a single peak with Kav = 0.10 on Bio-Gel A-15. Their glycosaminoglycan components (Kav = 0.70 on Bio-Gel A-15) contained predominantly chondroitin sulfate (33 to 44% of the total galactosaminoglycans) or dermatan sulfate chains. Based on the results of chondroitinase AC-II and periodate degradation, disaccharide repeating units of the dermatan sulfate were composed of 36% iduronic acid, 50% 2-sulfoiduronate, and 14% glucuronic acid. A similar composition was found for the dermatan sulfate in the cell fraction. Almost one-half of the sulfate label in the cell fraction was in a heparan sulfate proteoglycan which migrated on Bio-Gel A-15 with Kav = 0.30. The heparan sulfate chains (Kav = 0.81 on Bio-Gel A-15) had few, if any, sulfated N-acetylglucosamine residues and did not contain 2-sulfoiduronic acid in neighboring disaccharide repeat sequences. These results indicate that fibroblast-like lung cells synthesize several types of multichain sulfated proteoglycans which have properties in common with those found in lung tissues.     

Infection of cultured rat hepatoma cells by mouse mammary tumor virus.

A continuous line of buffalo rat hepatoma (HTC) cells has been successfully infected with mouse mammary tumor virus (MMTV) produced by the GR mammary tumor cell line. Uniform infection required initial exposure of the HTC cells to greater than 10(5) MMTV particles per cell. The resultant chronically infected cell population was found to have stably acquired 20-30 copies of MMTV DNA. The infected cells contain viral RNA and express viral antigens; however, very few MMTV particles are released into the culture medium. In spite of the biochemical evidence for infection, we have not detected any alterations in the morphology or growth properties of the infected HTC cells. As is the case in mammary tumor cells, the intracellular concentration of viral RNA is strongly stimulated (50-150 fold) by the synthetic glucorcorticoid, dexamethasone. Thus it appears that the mechanisms by which glucorticoids regulate MMTV gene expression in mouse cells are maintained when this virus infects nonmurine cells.     

Glycogen synthase turnover and phosphorylation in rat H4IIE hepatoma cells

Glycogen synthase was isolated from rat H4IIE hepatoma cells by the use of specific antibodies. Immunoprecipitates from cells grown in the presence of [35S]methionine contained two 35S-labeled polypeptides, designated GS1 and GS2, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of both species was half-maximal after 3 h and remained constant up to 48 h. When cells were incubated with [32P]-phosphate, 32P was incorporated into both species with similar kinetics, half-maximal labeling occurring after 2-3 h. The steady-state ratio 32P/35S was significantly higher for the lower mobility GS2 polypeptide. Pulse-chase experiments showed that the two subunits followed similar kinetics with respect to 35S-labeling. However, the turnover of 32P on the GS2 subunit was significantly faster (t1/2 approximately 30 min) than that on the GS1 subunit (t1/2 approximately 2 h). We suggest that the two polypeptides represent different phosphorylation states of the glycogen synthase subunit and are rapidly interconverted.     

Inhibition of mammalian aspartate transcarbamylase by quinazolinone derivatives

Quinazolinone derivatives have been studied as both in vitro and in vivo inhibitors of aspartate transcarbamylase (ATCase). In vitro treatment of mammalian ATCase with four compounds revealed that they inhibited enzyme activity and that 2-phenyl-1,3-4(H)benzothiazin-4-thione was the most potent one. This compound acts as a noncompetitive inhibitor towards both aspartate and carbamoyl phosphate. The values of the inhibition constant (K_i) indicate that this compound exerts a potent inhibitory effect upon ATCase activity. Moreover, in vivo treatment with different doses of these derivatives showed also an inhibitory effect upon ATCase, the relative activity being decreased by 40%–58% with a 1 mg dose. These data support the inhibition of ATCase by quinazolinone derivatives as a new type of inhibitor for the enzyme.