A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells

A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10⁻⁷ M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K_m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

Reversible arrest of Chinese hamster V79 cells in G2 by dibutytyl AMP.

Mouse L cells 929 were cloned in supplemented Eagle's minimal medium enriched with lactalbumin and yeast extract and buffered with HEPES. Multiplication was followed photographically in single clones from the 8-cell stage through 6–7 days. Addition of the folic acid analogue methotrexate (amethopterin) in 5 × 10^?6 M concentration slowed growth only after two cell generations; 10^?4 M uridine had no effect on growth except when combined with methotrexate. The two agents together blocked cell division quickly and symptoms of thymine-less death developed in few days. The cells could be rescued before 48 h by removal of the inhibitors, or by addition of folic acid or thymidine. The combination of methotrexate with uridine blocks DNA synthesis in Tetrahymena by inhibition of thymidylate synthesis and of thymidine uptake from the complex medium. Apparently the same mechanisms operate in L cells grown in a complex medium containing thymidine.     

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.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

Thymidine transport by Novikoff rat hepatoma cells synchronized by double hydroxyurea treatment

Suspension cultures of Novikoff rat hepatoma cells were synchronized by a double hydroxyurea block. About 80% of the cells of the population doubled 5 to 8 h after the reversal of the second hydroxyurea block. At all stages of the cell cycle, thymidine was rapidly incorporated into the acid-soluble pool of the cells (mainly dTTP) and the rate of incorporation was limited by the rate of thymidine transport. The rate of thymidine transport per cell roughly doubled during the S or late S phase and decreased again to the base level during cell division. This was reflected by corresponding changes in V_max for thymidine transport, whereas the apparent K_m remained constant throughout the cell cycle.     

Thymidine transport by cultured Novikoff hepatoma cells and uptake by simple diffusion and relationship to incorporation into deoxyribonucleic acid

The initial rate of thymidine-³H 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 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent K_m of 0.5 µM, and the V_max values increased with an average Q₁₀ of 1.8 with an increase in temperature. The intracellular acid-soluble ³H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, 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 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°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 µM, 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 µM 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.     

Inhibition of macromolecular synthesis in tumors by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

L-1-tosylamido-2-phenylethyl chloromethyl ketone was observed to inhibit the incorporation of [³H] amino acids into protein and [³H] thymidine incorporation into DNA in Novikoff hepatoma ascites cells $\text{in vitro}$ Similar effects were seen with several Morris hepatomas and a transplanted colon tumor in rats, and were accompanied by decreased uptake of isotope into acid soluble tissue fractions. Under the same conditions, there was no significant inhibition in regenerating liver and there was an increased uptake of [³H] amino acids in the livers of normal and tumor bearing rats.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

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 µM, 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.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-³H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-³H]thymidine studies of aquatic bacterial production.     

AUTORADIOGRAPHIC EVIDENCE FOR MANY SEGREGATING DNA MOLECULES IN THE CHLOROPLAST OF OCHROMONAS DANICA

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-³H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.     

Adrenal cells in tissue culture. The effect of papaverine and amytal on steroidogenesis, respiration and replication

The smooth-muscle relaxant papaverine has been shown to be a potent inhibitor of cyclic AMP phosphodiesterase activity (Kukovetz, W. R., and Poch, G. (1970) Naunyn Schmiedebergs Arch. Pharmakol.267, 189). Because of this, papaverine was tested in monolayer cultures of functional mouse adrenal cortex tumor cells for possible stimulatory effects on Steroidogenesis. At 10^?5m, papaverine was found to inhibit ACTH-stimulated steroidogenesis 50% and at 10^?4m, > 95%. This was associated with a > 10-fold increase in [¹⁴C]lactate production from [¹⁴C]glucose and a 50% reduction in ³²P_i, incorporation into macromolecules. These findings were similar to those observed with the barbiturate amytal, an inhibitor of the mitochondrial electron-transport chain at the level of oxidation of NADH (Site I). Papaverine was 100 times more effective than amytal in inhibiting steroidogenesis and 1000 times more effective in initiating an increase in glycolysis. In intact tumor cells and mitochondria isolated from normal rat adrenals, papaverine (10^?4m) completely inhibits oxygen uptake supported by malate or α-ketoglutarate. Oxygen uptake is restored by the addition of succinate, suggesting that, like amytal, papaverine inhibits respiration at Site I.Papaverine does not inhibit NADPH-supported cholesterol side-chain cleavage in bovine adrenal acetone powders or 11β-hydroxylation in normal rat adrenal cortex mitochondria. By contrast, amytal inhibits both these activities at concentrations comparable to that effective in intact adrenal cells, suggesting a direct interaction of amytal with cytochrome P-450. Both papaverine and amytal inhibit incorporation of thymidine into nuclear DNA to an extent far greater than that observed with either maximally stimulating levels of cyclic AMP or high concentrations of ACTH. Succinate does not reverse the inhibitory effects of either papaverine or amytal on thymidine incorporation into DNA. Papaverine increases intracellular cyclic AMP in both resting and ACTH-treated cells. However, the effects of papaverine on steroidogenesis, glycolysis, ATP-P_i exchange, and DNA synthesis in adrenocortical cells are not directly attributable to this action.     

Human C-apolipoproteins promote hydrolysis of dimyristoyl phosphatidylcholine by snake venom phospholipase A2

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10^?5 M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [³H]thymidine incorporation. Proliferation is completely restored by 5 × 10^?5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 10^?5 M each, fail to re-establish proliferation but produce an isotropic dilution of [³H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (<0.001 nmol.min^?1.10^?6 cells).     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

A radioimmunoassay for measurement of serum thymidine

A simple and sensitive radioimmunoassay for the measurement of serum thymidine has been developed based on the competition between thymidine and an analog (¹²⁵IUdR) for sites on anti-thymidine antibody. Antibody-bound ¹²⁵IUdR was measured in ammonium sulfate precipitates. None of the structurally related compounds that are normal body metabolites showed thymidine's ability to displace the label except at 200-fold higher concentration. A thymidine concentration of slightly greater than 10^?6m has been measured in mouse blood serum. The rate of disappcarance of a thymidine load from the bloodstream has been followed after intravenous administration. The sensitivity and specificity of the technique have been discussed, and applications in radiotherapy and chemotherapy depending on altered DNA synthesis or cell death have been suggested.     

Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-³H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-³H]thymidine or [6-³H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [³H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [³H]thymidine labeling of protein and RNA, but caused some inhibition of [³H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [³H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [³H]thymidine incorporation.     

DNA-cellulose column chromatography of phosphorylated nucleolar nonhistone proteins

Summary A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [³²P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA.     

Cloned mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones

We have cloned circular unintegrated mouse mammary tumor virus (MMTV) DNA from infected rat hepatoma cells in bacteriophage lambda. Seven independent clones containing MMTV DNA of homogeneous length of 9 kb (five) or 10 kb (two) were identified. The five 9 kb clones had identical restriction maps consistent with that of 9 kb unintegrated DNA; the other two were aberrant. MMTV DNA inserts were purified, ligated and used for cotransfection of Ltk^? cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. All Tk⁺ cell clones acquired new MMTV sequences and those transfected with the 9 kb MMTV DNA synthesized normal viral RNA and proteins. Viral gene expression was increased by the addition of dexamethasone.