Incorporation of [3H]hypoxanthine by short-term cultured Theileria sergenti and its inhibition by drugs.

Red blood cells parasitized with Theileria sergenti were cultured in vitro for a short period in microplate wells. Addition of [3H]hypoxanthine to cultures enabled us to titrate the intraerythrocytic parasite growth because a significant amount of [3H]hypoxanthine was incorporated into the parasitized red blood cells (PRBC) in proportion to the number of PRBC. The incorporation of [3H]hypoxanthine was almost completed in an early phase of incubation. Bovine peripheral blood leukocytes incubated with lysate of PRBC did not incorporate [3H]hypoxanthine, indicating that contaminating leukocytes were not involved in the incorporation in the PRBC culture. The incorporation of [3H]hypoxanthine was decreased markedly by the addition of certain anti-parasitic drugs such as chloroquine, quinine, and pyrimethamine. This inhibition test was performed quantitatively with high sensitivity. Based on these results, this short-term culture seemed to be useful for drug screening or studying the mechanisms of theilerial infection. However, anti-T. sergenti antibodies failed to inhibit the [3H]hypoxanthine incorporation.     

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.     

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

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

Summary Novikoff rat hepatoma cells were propagated in suspension culture in the presence of 1m 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 m hypoxanthine and 30 m thymidine. The results illustrate a dependence of the growth rate of mammalian cells on the rate of transport of essential nutrients into the cell.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells

The ability of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells to drive their total purine requirements from inosine 5'-monophosphate, inosine, or hypoxanthine was compared. Inosine 5'-monophosphate first must be converted to inosine by the action of the enzyme ecto-5'-nucleotidase before it can be transported into the cell; inosine and hypoxanthine, however, can be transported directly. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and to make the cells dependent on an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 30 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA or [3H]leucine incorporation into protein at rates equal to that of untreated control cultures. Similar results were found when azaserine was used to inhibit purine synthesis de novo, and thus DNA synthesis. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells, and suggest that this enzyme may be important for purine salvage when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

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.     

Alternative modes of population growth inhibition in a human lymphoid cell line growing in suspension

Using a human lymphoid cell line grown under continuous culture conditions, two distinct plateau states were induced, either by lack of sufficient medium-supplied nutrient, or by other unknown mechanisms dependent on cell density. Flow microfluorometric measurements show that growth arrest due to nutritional insufficiency results in an accumulation of cells with G1 DNA content. In contrast, growth arrest due to high cell density is not associated with an altered distribution of cells with respect to DNA content as the population progresses from exponential to plateau state growth. Cell size decreases with progression of the plateau state induced by either type of growth arrest. Cells in a plateau state induced by high cell density utilize glucose and incorporate exogenous amino acid into protein at approximately the same rate as exponential cells. Proliferating, high cell density, plateau state cells have cell cycle phase durations similar to exponential cells. The stable, plateau state cell density is maintained by cell loss. No stable, unbound growth inhibitory factor was found in the medium of density-inhibited plateau state cultures.     

Inosine 5'-monophosphate vs inosine and hypoxanthine as substrates for purine salvage in human lymphoid cells

The ability of inosine 5'-monophosphate vs inosine or hypoxanthine to supply the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells was evaluated. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and make the cells dependent upon an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 25 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA at rates equal to that of untreated control cultures. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line, WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line No. 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, only inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells and suggest that this enzyme may have functional significance when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells.

A permanent, clonal strain of rat pituitary tumor cells (GH3-cells) spontaneously synthesizes and secretes prolactin (rPRL) and growth hormone (rGH) into the culture medium. The rates of hormone production (microng extracellular hormone/mg cell protein/24 hours) and synthesis (vida infra) as well as the rate of [3H]thymidine incorporation into DNA (DNA synthesis) have been studied. During logarithmic growth rPRL and rGH production increased to 160 and 250% of the value at day 2 after plating, while during the plateau phase of cell growth hormone production decreased to initial values. The fluctuations in rPRL production could be fully explained by variations in the rate of rPRL synthesis: [3H]eucine incorporated into rPRL as measured with immunoprecipitation and polyacryl-amide gel electrophoresis. Also the rates of synthesis and production of rGH showed parallel changes during exponential and plateau phase of growth, but this hormone was probably degraded intracellularly. The relative reduction in the rate of synthesis of rPRL and rGH during the plateau of growth corresponded closely to the fall in the rate of DNA synthesis. The reduction in rPRL synthesis could not be explained through an inhibition by extra-cellular rPRL accumulation or by cell to cell interaction occurring in dense cultures. The intracellular concentrations of both hormones were unaltered during logarithmic growth, but rose to 500% for rPRL and 200% for rGH during the plateau phase. In spite of the marked variations in basal rPRL and rGH production the GH3 cultures of different ages were equally able to increase rPRL and decrease rGH production in response to thyrotropin releasing hormone (3 X 10(-7) M) and 17beta-estradiol (10(-8)M).     

Incorporation of [1-14C]-linoleic acid by LLC-WRC256 tumour cells

The incorporation of [(14)C]-linoleic acid (LA) into total lipid fractions was higher in LLC-WRC256 cells from the log phase of growth as compared to those of the plateau phase. LA was mainly incorporated into the phospholipid (PL) fraction of cells during the log phase, whereas in the plateau phase it was mostly taken into cholesterol ester. The proportion of radioactivity was higher in phosphatidylserine of cells from the log phase, whereas in the plateau phase it was higher in phosphatidylcholine. This feature of LA incorporation may be an important factor in determining the proliferative capacity of tumour cells.     

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.     

Adenine-induced hypoxanthine release from IMP-enriched human erythrocytes

Adenine uptake and hypoxanthine release by IMP-enriched human erythrocytes has been studied. The presence of IMP within the erythrocytes leads to an increase in the rate of adenine incorporation. Adenine is taken up by IMP-enriched erythrocytes as AMP, even when intracellular 5-phoshorobosyl-1-pyrophosphate concentration is undetectable and too low to allow IMP synthesis from hypoxanthine. During adenine uptake and AMP synthesis, hypoxanthine is released by the cells. The possibility that 5-phosphoribosyl-1-pyrophosphate, necessary for AMP synthesis, is formed through the hypoxanthine guanine phosphoribosyltransferese-catalyzed IMP pyrophosphorolysis is considered.     

Protein synthesis in 3T3 cells stimulated to proliferate at various rates

Reproducible conditions were defined for using rates of leucine incorporation as a valid measure of rates of de novo protein synthesis in mouse 3T3 cells. Upon stimulation of quiescent cultures, rates of de novo synthesis of proteins increased and pool levels of amino acids decreased in proportion to the concentration of serum in the stimulating medium. Rates of de novo protein synthesis (per cell) exhibited a biphasic pattern of increase. These rates approached a plateau value at the end of the lag phase and increased again as cells entered S phase. This pattern of behaviour helps to explain the observed relationships between cell growth (increase in mass) and cell proliferation (increase in cell number).     

Mechanism of cytotoxic action of azaguanine and thioguanine in wild-type V79 cell lines and their relative efficiency in selection of structural gene mutants

The cytotoxic effects of azaguanine and thioguanine have been compared in two wild-type V79 cells. To achieve equitoxic effects in both cell lines a 10–20-fold higher concentration of azaguanine than thioguanine was required. Affinity of HGPRT for azaguanine was 10-fold lower than for hypoxanthine in both cell lines and was similar to that for thioguanine in V79S cells. Affinity for thioguanine differed by a factor of 3 in the two cell lines. The rate of cell kill by azaguanine was markedly slower than by thioguanine in both cell lines. Reduction of whole cell uptake of [¹⁴C]hypoxanthine incorporation by unlabelled azaguanine was only demonstrable after prolonged incubation periods as was incorporation of [¹⁴C]azaguanine into acid-insoluble material. Experiments with cell-free extracts indicated that hypoxanthine acts as a non-competitive inhibitor of the enzyme. The slow rate of dissociation of the HGPRT—azaguanine complex is reflected in the slow rate of killing of wild-type cells. Clones resistant to the cytotoxic effects of these analogues have been selected from both cell lines and have been shown to possess HGPRT with altered kinetic properties. Our data suggest that azaguanine and thioguanine may select for mutations at different sites on the HGPRT molecule in V79 cells and provide possible explanations for the differences in effectiveness of these two agents reported in other cell lines.     

Incorporation and turnover of phospholipid precursors in normal and tumoral glial cells in culture

The incorporation and turnover of phospholipid precursors in cultured normal and tumoral glial cells was investigated during the plateau phase of growth. Glycerol was incorporated similarly by all cell types, and was renewed with a half-life of 19-37 hr. Acetate had a much longer half-life in primary cultures (50-75 hr) than in proliferative tumor cells (20-40 hr). Phosphate had a more rapid turnover rate in primary cultures (25 hr) than in proliferative tumor cells (50 hr). For all precursors, inositol- and choline phosphoglycerides had a faster turnover rate than other phospholipids.     

Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth. (c) 1993 John Wiley & Sons, Inc.     

Rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after cessation of dihydrofolate reductase activity in stationary versus log phase L1210 leukemia cells.

Previous studies from this laboratory established that the rapid but partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure of L1210 leukemia cells to antifolates cannot be due to direct feedback inhibition of thymidylate synthase by dihydrofolate or any other endogenous folylpolyglutamates when dihydrofolate reductase activity is abolished by antifolates. Rather, the data suggested this preservation of tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to dihydrofolate. This paper explores the role of cell cycle phase in L1210 leukemia cells in logarithmic versus stationary phase growth as a factor in the rate and extent of tetrahydrofolate cofactor interconversion to dihydrofolate after exposure of cells to the dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures. 5-Bromo-2'-deoxyuridine incorporation into DNA decreased 80 and 96%, while [3H]dUrd incorporation into DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of tetrahydrofolate cofactor interconversion to dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total DNA content. This suggests that tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and 5-bromo-2'-deoxyuridine incorporation into DNA in comparison with the decline in the S phase fraction measured by DNA content along with the reduced rate of tetrahydrofolate cofactor interconversion to dihydrofolate indicates that the rate of DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparison of purine metabolism and nucleotide pools in normal and hypoxanthine-guanine phosphoribosyltransferase-deficient neuroblastoma cells

Purine nucleotide synthesis and interconversion were examined over a range of purine base and nucleoside concentrations in intact N4 and N4TG (hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient) neuroblastoma cells. Adenosine was a better nucleotide precursor than adenine, hypoxanthine or guanine at concentrations greater than 100 μM. With hypoxanthine or guanine, N4TG cells had less than 2% the rate of nucleotide synthesis of N4 cells. At substrate concentrations greater than 100 μM the rates for deamination of adenosine and phosphorolysis of guanosine exceeded those for any reaction of nucleotide synthesis. Labelled inosine and guanosine accumulated from hypoxanthine and guanine, respectively, in HGPRT-deficient cells and the nucleosides accumulated to a greater extent in N4 cells indicating dephosphorylation of newly synthesized IMP and GMP to be quantitatively significant. A deficiency of xanthine oxidase, guanine deaminase and guanosine kinase activities was found in neuroblastoma cells. Hypoxanthine was a source for both adenine and guanine nucleotides, whereas adenine or guanine were principally sources for adenine (>85%) or guanine (>90%) nucleotides, respectively. The rate of [¹⁴C]formate incorporation into ATP, GTP and nucleic acid purines was essentially equivalent for both N4 and N4TG cells. Purine nucleotide pools were also comparable in both cell lines, but the concentration of UDP-sugars was 1.5 times greater in N4TG than N4 cells.