The effect of two isomeric octadecenoic acids on the lipid metabolism and growth of Novikoff hepatoma cells.

The origin and metabolism of octadecenoic acid (18 : 1) was examined in intact Novikoff rat hepatoma cells by using labeled precursors and two isomeric octadecenoic acids which differed in their abilities to stimulate cell growth in a serum-free medium. The isomers (ci-6-18 : 1 and cis-9-18 : 1) were measured in the cellular lipid by ozonolysis and reduction of the ozonides. The results indicate that the 18 : 1 fatty acid accumulated in the cell lipid by uptake of the preformed acid from the medium. The cis-9-18 : 1 to 16 : 1 and 20 : 1 fatty acids by chain shortening and chain elongation. Both isomers inhibited de novo fatty acid synthesis from acetate by cells suspended in a serum-free medium. The isomers did not exert coordinate control of both fatty acid and cholesterol biosynthesis in the Novikoff cells.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

L-Lactate or pyruvate stimulated growth of Novikoff rat hepatoma cells

Novikoff rat hepatoma cells (subline N1S1-67) grew when 30 mM L-lactate or pyruvate was substituted for D-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lacking L-lactate, pyruvate or D-glucose, cell growth of 0.42 generation was obtained. Growth with L-lactate was dependent on the L-lactate concentration up to 30 mM at which the greatest increase in cell number occurred. Significant growth did not occur when D-lactate, glycerol, acetate, alpha-ketoglutarate, succinate or malate, each at 30 mM, was substituted for D-glucose. Growth in the medium containing L-lactate was not due to the utilization of D-glucose or some other substrate carried into the culture with the inoculum. Medium contamination by D-glucose was insufficient to explain the growth obtained in the medium containing L-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration of L-lactate in the medium remained unchanged. The increase in cell number cannot be explained by L-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoprotein in the medium. L-Lactate does not serve as a significant carbon or energy source in the growth of these cells.     

Osmotic permeability of Novikoff hepatoma cells

The osmotic permeability coefficient (P_f) for water movement across Novikoff hepatoma cells was found to be 82 ± 3 (S.E.) · 10^?5 cm · s^?1 at 20°C. The corresponding diffusional permeability coefficient for ³HHO (P_d) was 97 ± 10 (S.E.) · 10^?5 cm · s^?1, therefore the ratio $\text{P}{}_{\mathrm{<mtext>f</mtext>}}\text{P}{}_{\mathrm{<mtext>d</mtext>}}$ is close to unity. The apparent activation energy for water filtration was 10.4 ± 0.4 (S.E.) kcal · mol^?1. This value is significantly greater than the activation energy for the self diffusion of water. The product of the hydraulic permeability coefficient and the viscosity coefficient for water was temperature-dependent. However, the product of the hydraulic permeability coefficient and the viscosity coefficient for membrane lipid did not vary with temperature. These data are interpreted as evidence for water movement across a lipid membrane barrier rather than through aqueous channels.     

The biochemical determinants of hypoxanthine uptake in Novikoff rat hepatoma cells

The rate with which Novikoff rat hepatoma cells took up exogenous hypoxanthine increased sharply towards the end of the logarithmic growth phase, remained high for several hours into the stationary phase, and then decreased again. In an effort to account for these phenomena, several biochemical parameters were monitored during culture growth: the activities of the hypoxanthine transporter, of hypoxanthine phosphoribosyltransferase, and of P-Rib-PP synthetase; and the intracellular concentrations of ATP and P-Rib-PP. All of these parameters remained virtually constant during growth of the culture, except for P-Rib-PP, which increased greater than 10-fold in a pattern similar to that for hypoxanthine uptake. The activities of the transporter, synthetase, and phosphoribosyltransferase remained stable over 7 h of treatment with cycloheximide.     

The deoxyribonucleoside transport systems of cultured Novikoff rat hepatoma cells

The transport of various deoxyribonucleosides by cultured Novikoff rat hepatoma cells (subline N1S1-67) follows normal Michaelis-Menten kinetics. The transport reactions are competitively inhibited by most heterologous deoxy- and ribonucleosides and by Persantin and Cytochalasin B. Comparisons of the transport kinetics of the various deoxyribonucleosides (K_m and V_max ) and of the K_m/K_i ratios for the inhibitions indicate that deoxythymidine, deoxyuridine and 5-fluordeoxyuridine are transported by a single system, whereas deoxycytidine and the purine deoxyribonucleosides are transported by other systems. The data suggest that deoxyadenosine, deoxyguanosine and deoxyinosine, are not transported by a single system, but the number of transport systems involved could not be established unequivocally. Similar comparisons also suggest that the deoxyribonucleosides are transported by different systems than the ribonucleosides. All deoxyribonucleoside transport systems are inhibited to about the same extent by Persantin (K_i = 1–2 μM) and Cytochalasin B (K_i = 4–12 μM). The inhibitions of deoxynucleoside transport resulted in corresponding apparent competitive inhibitions of their incorporation into nucleic acids.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells.

N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.     

Pentose utilizing variants of Novikoff hepatoma cells: Modification of growth and morphological properties

A series of variant lines that utilize multiple pentoses for growth in place of glucose have been isolated from an 8-azaguanine resistant line of Novikoff hepatoma cells (N1S167). These variants utilize for growth ribose, xylose, arabinose, and/or deoxyribose. The variants growing on pentose containing medium (a) exhibit a density dependent cessation of growth, (b) have a morphology change to a more flattened cell type, (c) become binucleated in the presence of cytochalasin B, and (d) show an altered sensitivity to trypsin treatment.