Cultured Novikoff Rat Hepatoma Cells Research Articles

Phosphatidylcholine as the choline donor in sphingomyelin synthesis.

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [methyl-14C]choline resulted in rapid accumulation of essentially all the label (approximately 95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 microM choline), loss of label from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 microM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 microM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9-12 hr until it reached the specific radioactivity of PC. In the presence of 300 microM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth rate of cultured Novikoff rat hepatoma cells as a function of the rate of thymidine and hypoxanthine transport

Novikoff rat hepatoma cells were propagated in suspension culture in the presence of 1 micron methotrexate and various concentrations of hypoxanthine (or adenosine plus guanosine) and thymidine and with or without the inhibitor of nucleoside and purine transport, Persantin (dipyridamole). Methotrexate-treated cells failed to replicate and died even if the medium was supplemented with either thymidine or a purine source, but normal replication occurred when both were present. The additional presence of Persantin reduced the rate of transport of thymidine or hypoxanthine and thus their incorporation into the nucleotide pool and decreased the rate of cell replication. The growth rate of the cells was directly proportional to the rate of incorporation of thymidine (in the presence of excess hypoxanthine) or of hypoxanthine (in the presence of excess thymidine) until the normal maximum growth rate was obtained. Normal cell replication in the presence of methotrexate and Persantin occurred only when the medium was supplemented with 500 micron hypoxanthine and 30 micron thymidine. The results illustrate a dependence of the growth rate of mammalian cells on the rate of transport of essential nutrients into the cell.

Effect of temperature and of cytochalasin B and persantin on the nonmediated permeation of non-electrolytes into cultured Novikoff rat hepatoma cells.

The nonmediated permeation of L-glucose, cytosine, and prednisolone into Novikoff rat hepatoma cells followed first order kinetics with rate constants of 0.00404, 0.173, and 2.4 min-1, respectively. The constants were estimated from a nonlinear least squares fit of the integrated first order rate equation. The rate constants were independent of substrate concentration and correlated with the partition coefficients of the substances in octanol-balanced salt solution (0.00158, 0.0352, and 17.8, respectively) and olive oil-balanced salt solution mixtures which were between 10- and 100-fold lower. Arrhenius plots for the permeation of L-glucose, cytosine, and prednisolone were linear and indicated activation energies of 24.2, 28.0, and 19.6 kcal/mol, respectively. The permeation of L-glucose and cytosine, but not of prednisolone, was impeded in a concentration-dependent manner by the presence of cytochalasin B and Persantin, heretofore thought of as specific inhibitors of facilitated diffusion processes. The relative degree of decrease of the permeation rates of L-glucose and cytosine, however, differed for cytochalasin B and Persantin.

Transport, Phosphorylation, and Toxicity of a Tricyclic Nucleoside in Cultured Novikoff Rat Hepatoma Cells and Other Cell Lines and Release of Its Monophosphate by the Cells 2

1,4,5,6,8-Pentaazaacenaphthylene-3-amino-1,5-dihydro-5-methyl-(5-14C)-1-beta-D-ribofuranysly (NSC-154020), a tricyclic 7-deazapurine nucleoside (TCN), was rapidly incorporated into the acid-soluble pool by cultured Novikoff rat hepatoma cells, mouse L-cells, HeLa cells, and HEp-2 cells, but little incorporation into nucleic acids occurred. More than 90% of the intracellular radioactivity was associated with the monophosphate (MP) of the substrate concentration followed normal Michaelis-Menten kinetics. Comparison of the kinetic constants for the uptake of adenosine and the TCN and of the inhibition of their uptake by each other suggests that both were transported by the same system (Michaelis constant approximately 6-10 muM) and with about the same efficiency. The TCN was also phosphorylated in a cellfree extract containing adenosine kinase activity at about the same rate as was adenosine, but not further phosphorylation of the analogue MP occurred. No significant deamination or degradation of the adenosine analogue to its base and ribose-1-phosphate was observed. TCN inhibited the replication of all four types of cells propagated in suspension culture; however, Novikoff cells were several times more sensitive than were the other three cell types, despite the finding that the TCN-MP, probably the main toxic principle, accumulated to about the same concentration in cells of all four lines. Complete inhibition of replication of Novikoff cells were several times more sensitive than were the other three cell types, despite the finding that the TCN-MP, probably the main toxic principle, accumulated to about the same concentration in cells of all four lines. Complete inhibition of replication of Novikoff cells and cell death occurred at concentration as low as 15 muM TCN. At these concentrations, TCN, within 2 hours of its addition, completely inhibited the incorporation of [14C]formate int0 nucleotides and nucleic acids of Novikoff cells. An inhibition of the denovo synthesis of purine and pyrimidines, however, was not the only toxic effect of the TCN since high concentrations of uridine, adenine, guanine, and hypoxanthine, either alone or combined, failed to prevent the inhibition of cell replication by TCN. Also, between 1 and 3 hours of treatment, 70-80% of the Novikoff cells fragmented into four to eight vesicles per cell. These fragments were impermeable to trypan blue, still exhibited some metabolic activity such as the phosphorylation of AMP and TCN, but failed to replicate when the drug was removed. No similar fragmentation was observed with the other cell lines. Novikoff and L-cells rapidly released TCN-MP into the culture fluid. After 4 hours of incubation, 70-100% of the total radioactivity in the medium was associated with the MP. Only a little TCN-MP was released from HeLa and HEp-2 cells. A TCN-resistant mutant of Novikoff cells failed to phosphorylate the analogue and was deficient in adenosine kinase.

Temperature-dependent changes in activation energies of the transport systems for nucleosides, choline and deoxyglucose of cultured Novikoff rat hepatoma cells and effects of cytochalasin B and lipid solvents.

The initial rates of transport of uridine, thymidien, purines, choline and 2-deoxy-D-glucose by cultured Novikoff rat hepatoma cells were determined as a function of temperature between 5 and 41 degrees C. Arrhenius plots of all transport systems exhibited sharp breaks in slope; between 17 and 23 degrees for uridine, thymidine and hypoxanthine-guanine transport and between 29 and 32 degrees for choline and 2-deoxy-D-glucose transport. The activation energies for the transport systems changed from 15-26 kcal/mole below the transition temperatures to 4-9 kcal/mole above the transition temperatures. Propagation of the cells in the presence of cis-6-octadecenoic acid which results in marked changes in the lipid composition of cell membrane, had little effect on the temperature characteristics of the various transport systems. Similarly, propagation of the cells for 24 hr in media containing Tween 40 or nystatin had no effect on the capacity of the cells to transport the various substrates or on the temperature dependence of the transport systems. The presence of ethanol, phenethyl alcohol or Persantin at concentrations that inhibited thymidine and 2-deoxy-D-glucose transport between 40 and 70% also did not alter the transition temperatures or activation energies for the transport of these substrates. Cytochalasin B, on the other hand, shifted the transition temperature for 2-deoxy-D-glucose transport to higher temperatures in a concentration-dependent manner, whereas it had no effect on the temperature dependence of thymidien transport.

Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells.

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.

Intracellular conversions of deoxyribonucleosides by Novikoff rat hepatoma cells and effects of hydroxyurea

AbstractThe incorporation of 3H‐labeled deoxyadenosine and deoxyguanosine into nucleic acids by cultured Novikoff rat hepatoma cells is about 80% into RNA and 20% into DNA. The pathways of incorporation have been elucidated in studies with whole cells and cell‐free extracts. Deoxyadenosine is very rapidly deaminated to deoxyinosine. Most of the deoxyinosine formed by whole cells is transported out of the cells and accumulates in the medium. A portion of the deoxyinosine, and deoxyguanosine are phosphorolyzed by purine nucleoside phosphorylase to hypoxanthine and guanine, respectively. The latter are subsequently converted by hypoxanthine‐guanine phosphoribosyl transferase to IMP and GMP, respectively. Incorporation of the purine deoxyribonucleosides into DNA is mainly via this pathway and the subsequent reduction of ADP and GDP by ribonucleoside reductase, although a small proportion of the deoxyadenosine and deoxyguanosine taken up by the cells seems to be directly phosphorylated to dAMP and dGMP, respectively. Deoxyguanosine is incorporated only into guanine residues of RNA and DNA. Deoxyadenosine is also mainly incorporated into guanine residues of RNA and DNA, although the radioactivity of deoxyadenosine in the acid‐soluble pool is almost exclusively associated with ATP. A similar labeling pattern is observed with labeled deoxyinosine, inosine or hypoxanthine. The pyrimidine deoxyribonucleosides, on the other hand, are specific precursors for their respective bases in DNA.Hydroxyurea inhibits the incorporation of all deoxyribonucleosides into DNA. Results from pulse‐chase experiments indicate that the inhibition of DNA synthesis is prevented by the presence of high concentrations of deoxyadenosine plus deoxyguanosine in the medium. Either purine deoxyribonucleoside alone or deoxycytidine, hypoxanthine or inosine alone or in combination with deoxyadenosine or deoxyguanosine are ineffective. The results are consistent with the conclusion that the inhibition of DNA synthesis is due to a depletion of the dATP and dGTP pools as a result of the hydroxyurea treatment. On the other hand, hydroxyurea causes an increased incorporation of thymidine and deoxycytidine into the dTTP and dCTP pools, respectively. Evidence is presented to indicate that this effect of hydroxyurea is due to an increased synthesis of dTTP and dCTP rather than to an inhibition of their turnover.

Competitive inhibition of the transport of nucleosides, hypoxanthine, choline and deoxyglucose by theophylline, papaverine and prostaglandins

Summary Theophylline, papaverine and prostaglandins E 1 and F 2∝ competitively inhibit the transport of uridine, thymidine, hypoxanthine, choline and deoxyglucose by cultured Novikoff rat hepatoma cells. The transport processes are maximally inhibited immediately upon addition of the drugs and the effects are readily reversed by their removal.

Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells.

Cytochalasin B competitively inhibits the transport of uridine and thymidine by Novikoff rat hepatoma cells growing in suspension culture with apparent K(i)'s of 2 and 6 microM, respectively, but has no effect on the intracellular phosphorylation of the nucleosides. Choline transport is not affected by cytochalasin B. Results from pulse-chase experiments indicate that cytochalasin B has no direct effect on the synthesis of RNA, DNA, or uridine diphosphate-sugars. The inhibition of uridine and thymidine incorporation into nucleic acids by cytochalasin B is solely the consequence of the inhibition of nucleoside transport.

THYMIDINE TRANSPORT BY CULTURED NOVIKOFF HEPATOMA CELLS AND UPTAKE BY SIMPLE DIFFUSION AND RELATIONSHIP TO INCORPORATION INTO DEOXYRIBONUCLEIC ACID

The initial rate of thymidine-(3)H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 microM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22 degrees , 27 degrees , 32 degrees , and 37 degrees C with an apparent K(m) of 0.5 microM, and the V(max) values increased with an average Q(10) of 1.8 with an increase in temperature. The intracellular acid-soluble (3)H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 microM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 microMp-chloromercuribenzoate for 15 min or heat-shock (49.5 degrees C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 microM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K(m) and K(i) values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 microM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.

Permeation of Glucose by Simple and Facilitated Diffusion by Novikoff Rat Hepatoma Cells in Suspension Culture and Its Relationship to Glucose Metabolism

Abstract The incorporation of 2-deoxy-d-[14C]glucose by cultured Novikoff rat hepatoma cells was investigated as a function of the deoxyglucose concentration in the medium. The intracellular radioactivity was mainly associated with deoxyglucose 6-phosphate, to a small extent with 6-phosphodeoxygluconate, and the remainder with free deoxyglucose. Below a concentration of 1 to 2 mm in the medium, the initial rate of deoxyglucose incorporation followed simple Michaelis-Menten kinetics at 22, 27, 32, and 37° with an apparent Km of about 2 mm. The Vmax values increased with a Q10 of about 2.5 with an increase in temperature. Between 2 and 10 mm, on the other hand, the initial rate of incorporation increased linearly with an increase in deoxyglucose concentration and the relative increase in incorporation rate with increase in deoxyglucose concentration was about the same at the temperatures tested. Treatment of the cells with 0.5 mm p-chloromercuribenzoate for 15 min or heat shock (48.5°, 5 min) abolished the saturable uptake component without affecting the nonsaturable component. When combined, the results indicate that at low concentrations, deoxyglucose is taken up mainly by carrier-mediated transport (probably facilitated diffusion), whereas at concentrations above 2 mm, simple diffusion becomes the principal mode of entry of deoxyglucose into the cells. Results from studies on d-glucose and 3-O-methyl-d-glucose incorporation support this conclusion and suggest that all three substrates are transported by a single system. Persantine competitively inhibited deoxyglucose uptake by whole cells and decreased the metabolism of glucose without affecting the phosphorylation of the substrates. Prior treatment with p-chloromercuribenzoate or heat shock also had no effect on the hexokinase activity of the cells. Little if any hexokinase activity was associated with the plasma membrane and the kinetic properties of the in vitro hexokinase reaction differed from that for glucose or deoxyglucose uptake by whole cells. These results indicate that transport of glucose into the cell is a reaction distinct from phosphorylation and is the rate-limiting step in the metabolism of glucose by the cells. Since the cells possess an excess of hexokinase, glucose is phosphorylated as rapidly as it enters the cells and thereby trapped. At low concentrations of glucose in the medium (5 to 50 µm), the glucose 6-phosphate formed was exclusively used by the cells for macromolecular synthesis and oxidative processes without net production of lactate. The rate of CO2 production and incorporation of glucose in macromolecules approached a maximum at about 1 mm glucose in the medium and the excess glucose 6-phosphate formed, particularly at higher concentrations of glucose in the medium, was converted to lactate.

Glucocorticoids: Competitive inhibition of glucose transport

Summary Prednisolone competitively inhibits the transport of 2-deoxy-D-glucose by cultured Novikoff rat hepatoma cells without affecting deoxyglucose phosphorylation. The Ki for the inhibition (2 mM) is about the same as the Km for deoxyglucose transport. Transport is maximally inhibited immediately upon addition of the drug. Glucose metabolism by the cells is inhibited in direct proportion to the inhibition of glucose uptake.