Uridine transport and phosphorylation in mouse cells in culture: effect of growth-promoting factors, cell cycle transit and oncogenic transformation

The rapid increase in uridine uptake produced by the addition of serum to quiescent cultures of fibroblasts is primarily caused by an enhanced rate of nucleoside phosphorylation. While quiescent and serum-stimulated cells display identical initial rates of transport, they show a considerable change in the composition of the acid-soluble pools labelled with [3H] uridine for five seconds. The radioactivity recovered in the phosphorylated pools increases 2-, 3-, 4- and 6-fold after addition of serum to cultures of Swiss 3T3 cells, tertiary mouse embryo fibroblasts, Swiss 3T6 and Balb 3T3, cells respectively. Furthermore, insulin, a growth factor isolated from medium conditioned by SV40 BHK cells (FDGF) and epidermal growth factor (EGF) also stimulate uridine phosphorylation within minutes. The initial rate of uridine uptake is 2- to 3-fold faster in rapidly growing normal and Simian virus 40 or polyoma virus transformed 3T3 cells as compared to untransformed 3T3 cells in the quiescent state. When quiescent cultures of 3T3 or mouse embryo cells are stimulated to leave G1 and enter into DNA synthesis, transport increases several hours after addition of serum and apparently coincides with the S phase of the cell cycle. The results demonstrate that an increase in uridine phosphorylation is a rapid metabolic response elicited by growth-promoting agents in a variety of cell types and that uridine transport and phosphorylation are independently regulated.     

Selective high metabolic lability of uridine triphosphate in response to glucosamine feeding of untransformed and polyoma virus-transformed hamster fibroblasts

Derepression of hexose transport in a line of Syrian hamster fibroblasts (Nil) and polyoma-transformed (PyNil) hamster fibroblasts is obtained when cells are either starved for glucose or fed with fructose as the only hexose source. D-glucosamine feeding of these cells does not alter the repressed state with regard to hexose transport. High, derepressed rates of galactose transport were changed to low, repressed rates, within 18 hours of refeeding glucose-starved cells with D-glucosamine as the only hexose source. Nil and PyNil cells, when cultured in the presence of D-glucosamine, undergo rapid reductions in total cellular uridine 5′-triphosphate (UTP) pool sizes. By contrast, the total cellular pools of adenosine 5′-triphosphate, guanosine 5′-triphosphate, and cytosine 5′-triphosphate (ATP, GTP, and CTP) were only moderately affected by the treatment of the cells with glucosamine. The metabolic drain of the UTP pools in PyNil cells was much more pronounced than in the untransformed cells. The larger and more rapid metabolic lability of UTP pools in the transformed cells may be the primary reason for the selective toxicity of glucosamine on tumor cells. A comparison of the effects of glucosamine on hexose-starved Nil and PyNil cells demonstrated that only the untransformed cells were able to utilize glucosamine to increase the hexose starvation-depleted pools of all nucleoside triphosphates. Accumulation of UDP-glucosamine and UDP-N-acetylglucosamine followed the reduction in the UTP pools. Inhibition of protein synthesis by cycloheximide during glucosamine feeding led to higher levels of UDP-glucosamine and UDP-N-acetylglucosamine accumulation. It is suggested that the drain of UTP pools during glucosamine treatment proceeds through the formation of the UDP-aminosugars which turn over due to the action of intracellular UDP-aminosugar pyrophosphatase activities.     

Is glucose transport enhanced in virus-transformed mammalian cells? A dissenting view

Much of the literature on the uptake of glucose by untransformed and transformed animal cells is based on experiments carried out with 2-deoxy-D-glucose (2-DOG). Results obtained with this analog can be ambiguous, since 2-DOG can be phosphorylated by hexokinases of animal cells. An intracellular trapping mechanism is thus provided. Therefore, the total flux of 2-DOG into the cell is a resultant of both transport and hexokinase action, and the measurement of total 2-DOG incorporation is a valid measurement of transport only if 2-DOG is phosphorylated as rapidly as it enters the cell. Evidence is presented here that this is not necessarily the case, significant levels of free intracellular 2-DOG approaching external concentrations were found in untransformed and transformed mouse 3T3 cells even at early times during uptake. Differences in total intracellular 2-DOG between untransformed and transformed cells were accounted for entirely by 2-deoxyglucose phosphate. Thus, it appears the apparent increase of 2-DOG uptake accompanying transformation in these cell lines is not due to an effect on the transport process, but on enhanced phosphorylation, which is a reflection of an alteration in the regulation of glycolysis. The ambiguity introduced by phosphorylation can be oviated by the use of an analog that cannot be phosphorylated, such as 3-O-methyl-D-glucose. The rate of transport and efflux of this sugar was not found to be different in untransformed versus transformed 3T3 cells. Moreover, deficiencies of this analog as a substrate for the glucose transport system are pointed out.     

Selective high metabolic lability of uridine, guanosine and cytosine triphosphates in response to glucose deprivation and refeeding of untransformed and polyoma virus-transformed hamster fibroblasts.

Sugar deprivation of hamster fibroblasts (NIL) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the folloing fashion: the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma-transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar-supplemented medium.Refeeding glucose to sugar-depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.     

Changes in the uptake of 2-deoxy-D-glucose in BALB-3T3 cells chemically transformed in culture

Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50–100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.     

The Use of Glucosamine and Actinomycin D in Radioactive Labeling of RNA in Cells in Culture

Pretreatment of confluent cultures of mouse L cells or of well-differentiated nervous system cells in primary cultures with 20–120 mM glucosamine resulted in a stimulation of the uptake of tritiated uridine, but not of adenosine. A marked stimulation of the incorporation of radioactive uridine into acid-precipitable macromolecules was also obtained, while adenosine incorporation was unchanged. Cultures of L cells in log phase of growth were similarly affected by glucosamine pretreatment. Uridine and cytidine uptakes were stimulated by 50%. Tritiated uridine incorporation was stimulated in a biphasic manner, with maximal stimulation (115%) after 15–60 min of labeling and at later times an inhibition of incorporation. The stimulation of cytidine incorporation paralleled the stimulation of its uptake. The data indicate that there is: a) a glucosamine-induced stimulation of pyrimidine nucleoside uptake, b) a marked stimulation of tritiated uridine incorporation into RNA due to depletion of the cellular pools of unlabeled uridine nucleotides during glucosamine pre-treatment, and c) a decrease in the rate of RNA synthesis after several hours of glucosamine treatment, probably related to diminished intracellular supplies of uridine nucleotides. In the presence of glucosamine, high concentrations of actinomycin D could be used to increase nuclear retention of pulse-labeled nascent RNA. Cordycepin treatment did not result in similar retention of RNA. These techniques will be useful in autoradiographic and biochemical studies of nuclear RNA synthesis.     

The high rate of aerobic glycolysis of transformed 3T3 cells persists after cell homogenization.

The high rate of lactic acid production by 3T3 cells which have been transformed by simian virus 40 or by polyoma virus as compared to confluent untransformed 3T3 cells persists after cell homogenization. This difference is also reflected by increased phosphofructokinase activity in the viral transformed cells. The findings imply that the increased aerobic glycolysis in the transformed cells results from changes in the glycolytic pathway rather than changes in sugar transport.     

Antagonistic effects of insulin and cortisol on coordinate control of metabolism and growth in cultured fibroblasts

A variety of metabolic and biosynthetic pathways in chick embryo fibroblasts are stimulated coordinately by many unrelated exogenous agents. Three of the best characterized components of this coordinate response are the uptake of 2-deoxy-D-glucose (2-dGlc) and of uridine and the incorporation of thymidine into DNA. Insulin stimulates and cortisol inhibits the coordinate response. In cortisol-treated cultures, as little as 10^?3 units/ml of insulin may stimulate thymidine incorporation 4-fold and 10^?1 units/ml may stimulate as much as 40-fold. The higher concentrations of insulin completely override the inhibitory effect of cortisol. They also cause about a 5-fold stimulation of the uptake of 2-dGlc and of uridine and a 2-fold stimulation of proline incorporation into protein. The uptake rates of 2-dGlc and uridine double within 30 minutes after addition of insulin to cortisol-inhibited cultures, but the incorporation of thymidine only begins to increase markedly after a 4-hour delay. When cortisol is added to cultures in the absence of insulin, the rates of uptake of 2-dGlc and uridine begin to decrease within two hours, but the incorporation of thymidine remains constant for two hours before beginning to decrease. Deprivation of Mg²⁺ inhibits the accelerated coordinate response maintained by insulin, but does not further the inhibition induced by cortisol. Results with metabolic inhibitors indicate that the stimulation of 2-dGlc and uridine uptake by insulin do not require RNA synthesis, and also suggest that they do not require protein synthesis. These and other findings can be explained by a model for coordinate control in which insulin increases and cortisol decreases the availability of Mg²⁺ for a wide spectrum of regulatory reactions in different metabolic pathways. In this model both hormones affect only the rates of ongoing reactions and do not instruct the cell to carry out specific new reactions unless the cell was predetermined to do so.     

Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.     

Noncoordinate control of RNA synthesis in eucaryotic cells

Inhibition of protein synthesis in confluent monolayers of chick fibroblasts stimulates selectively the synthesis of 4S RNA, resulting in a net accumulation of 4S RNA in the inhibited cells. Under these conditions, inhibition of ribosomal RNA synthesis and processing occurs, as does a decrease in soluble uridine phosphate concentrations; increased pools of certain amino acids are also apparent. Recovery of cells from inhibition is accompanied by a rapidly increasing rate of protein synthesis that lasts for several hours. The small molecular weight RNA synthesized during inhibition of protein synthesis appears properly methylated, and in the presence of cycloheximide and actinomycin D shows a precursor-product conversion. Radiolabeled RNA synthesized during inhibition of protein synthesis is stable following the recovery of cells from inhibition. Stimulation of uridine incorporation into 4S RNA during arrest of protein synthesis is also demonstrated in high-density cultures of L- and Hep-2 cells, suggesting that this non-coordinate stimulation of 4S RNA may be a general property of eucaryotic cells.     

Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: a good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose transport in a galactokinase null-allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D-galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose transport of 8.6 +/- 2.6 mM and 26.1 +/- 7.2 nmol/mg p/min, 4.1 +/- 1.2 mM and 40.3 +/- 9.5 nmol/mg p/min, and 7.01 +/- .85 mM and 11.6 +/- 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 +/- 3.7% of galactose uptake was cytochalasin B inhibitable) and L-glucose uptake (7.5% of the galactose uptake). D-Galactose was not metabolized and effluxed rapidly from preloaded cells. The Kls for the inhibition of D-galactose transport were 4.5 +/- 2.5 mM for D-glucose, 7.0 +/- 2.0 mM for 2-deoxy-D-glucose, 6 mM for 2-deoxy-D-galactose and 6.0 +/- 0.6 mM for 3-O-methyl-D-glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50-60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum-deprived cells. Hexose transport rates increased substantially in glucose-deprived, D-fructose- or D-galactose-fed cells as compared to glucose-fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKl. The kinetic and regulatory characteristics of D-galactose transport in the GalKl cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.     

Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

Transformed BHK cells exhibiting normal or subnormal sugar uptake

The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior at 38.5° but not at 32°. At 32°, DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5°, the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32° and 38.5°. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed at 38.5° rather than 32°. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32°) can be normalized by growth at 38.5°, a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se.     

Action of delta 9-tetrahydrocannabinol on the pool of acid soluble nucleotides

The effects of delta 9-tetrahydrocannabinol (THC) treatment on acid soluble pools of uridine nucleoside and nucleotides were investigated in Tetrahymena pyriformis and in isolated mouse lymphocytes and spermatogenic cells. In THC treated Tetrahymena and mouse lymphocytes the uptake of labelled precursor into acid soluble pools of uridine nucleoside and nucleotides fluctuated, whereas in pachytene spermatocytes and round spermatid cells the labelled pool was reduced. The reduction in the labelled pool measured in mouse spermatogenic cells was attributed primarily to a reduction in radioactively labelled uridine nucleoside. Treatment of Tetrahymena in high concentrations of THC (960 and 3,200 microM) resulted in an increase of labelled uridine nucleoside and a reduction in the amount of labelled uridine nucleotides. Expansion of the acid soluble pool with radioactive uridine resulted in small differences in labelled nucleoside and nucleotides in control and THC treated Tetrahymena and mouse lymphocytes. The results are discussed in terms of the effects of THC on macromolecular synthesis in various cellular systems.     

Myxospore coat synthesis in Myxococcus xanthus: in vivo incorporation of acetate and glycine.

Myxospore coat synthesis in Myxococcus xanthus was studied by incorporation of [(14)C]acetate into intermediates in the biosynthesis of coat polysaccharide and into acid-insoluble material during vegetative growth and after glycerol induction of myxospores. During short labeling periods at 27 degrees C, the radioactivity was shown to be located primarily in N-acetyl groups rather than sugar moieties. Two hours after glycerol induction, the pools of N-acetylglucosamine 6-phosphate and uridine 5'-diphosphate-N-acetylgalactosamine (UDPGalNAc) plus uridine 5'-diphosphate-N-glucosamine increased about twofold and were labeled at twice the rate measured for vegetative cells. The increased rate of synthesis of UDPGalNAc and its precursors could be correlated with increased enzyme activities measured in vitro. Controlled acid hydrolysis revealed that the galactosamine portion of the myxospore coat was N-acetylated. After glycerol induction, the incorporation of acetate into acid-insoluble material increased threefold. This enhanced incorporation was sensitive to neither penicillin nor d-cycloserine. In contrast, bacitracin inhibited the incorporation of [(14)C]acetate into acid-insoluble material more effectively 2 h after myxospore induction than during vegetative growth. Chloramphenicol added to cells 90 min after induction blocked further increase in the rate of [(14)C]acetate incorporation. Since the myxospore coat contains glycine, polymer synthesis was also measured by chloramphenicol-insensitive [(14)C]glycine incorporation into acid-insoluble material. Although protein synthesis decreased after glycerol induction, glycine incorporation increased. Two hours after induction, glycine incorporation was only 75% inhibited by chloramphenicol and rifampin. The chloramphenicol-insensitive rate of incorporation of [(14)C]glycine increased during the first hour after myxospore induction and reached a peak rate after 2 to 3 h. The chloramphenicol-resistant incorporation of [(14)C]glycine was resistant to penicillin but sensitive to bacitracin.     

Effects of 2-deoxy-D-glucose on the synthesis of RNA and protein in Saccharomyces carlsbergensis G-517

When Saccharomyces carlsbergensis G-517 was grown in 10 mM galactose as the carbon source, the addition of 2-deoxy-D-glucose restricted the uptake of galactose, [3H]uridine and [3H]leucine, and restricted invertase synthesis (beta-D-fructofuranoside fructohydrolase; EC 3.2.1.26) for a period of 60-90 min. During this time, the radioactive antimetabolite was taken up by the cells; afterwards, invertase synthesis was enhanced, and the utilizaton rate of galactose, [3H]uridine and [3H]leucine increased until it reached that of the control culture. When glucose was used as a carbon source, sugar utilization and uptake of radioactive precursors were unaffected by addition of the deoxysugar.     

Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates.

Dense cell cultures of the hamster lines, NIL, and polyoma transformed NIL were exposed to culture media containing various sugars (or no sugar). Various responses to these culture conditions were observed as changes in the uptake of galactose and its subsequent metabolism. Cells deprived of sugar have higher uptake rates for galactose and markedly different accumulation products from identical cells treated with sugar. A persistent increase in the transport of the amino acid, cycloleucine, was also observed as a response to culture conditions devoid of sugar     

Culture kinetics of glycolytic enzyme induction, glucose utilization, and thymidine incorporation of extended-exposure phytohemagglutinin-stimulated human lymphocytes

Glycolytic enzyme activity is significantly (P less than 0.05) induced between 24 and 48 hours of incubation in phytohemagglutinin-stimulated human lymphocytes. Nonstimulated cultured cells do not show this induction although these cells have an approximate daily doubling of thymidine incorporation. Maximal glycolytic enzyme activity is reached between 96 and 120 hours of culture in stimulated cells (3.5-fold increase) and maintained until at least 168 hours. There is no significant induction of the hexosemonophosphate shunt or the TCA cycle during seven-day transformation. Induction of glucose utilization becomes significantly (P less than 0.05) greater in stimulated as compared to nonstimulated cultures between 48 and 72 hours of culture and is significantly elevated for at least an additional 96 hours. There is a 17% increase in total protein in the stimulated cells after 24 hours of culture and higher levels of protein content are then maintained over the control. Thymidine incorporation is significantly greater in stimulated cells from 24-144 hours of culture but is not significantly different from the nonstimulated cells at 168 hours (P = 0.98) although glycolytic enzyme activity remains elevated in the stimulated cells. There is a greater enzyme induction of the latter phase of glycolysis during transformation and this phenomenon continues in extended cultures. Increases in glycolytic enzyme activity during mitogenesis appear to be an intrinsic phenomenon independent of cell proliferation and glucose transport. The mitogen-induced increase in the activity of the glycolytic enzymes accompanies blastogenesis and the sustained elevated activity of these enzymes to be related to the high metabolic rate of transformed cells.     

Hormonal studies of uridine utilization in an insect cell line CP-1268 derived from the codling moth Laspeyresia pomonella.

Ecdysterone decreased cellular growth and the incorporation of uridine into RNA following 4 days of hormone exposure. This hormone did not affect uridine incorporation following short-term exposure up to 25 hours. Juvenile hormone and farnesol both significantly decreased uridine uptake and incorporation into RNA; however, uridine uptake was inhibited to a greater extent than uridine incorporation. Cyclic AMP increased the incorporation of uridine into RNA but had no demonstrable effect on the uptake process. This stimulation was not the result of cAMP degradation products. Cyclic AMP and ecdysterone together produced a significant increase in uridine incorporation into RNA. These studies demonstrate the potential utilization of insect cell lines for studying the mode of action of insect developmental hormones.     

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.