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.     

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.     

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.     

Cyclic AMP,membrane transport and cell division. I. Effects of various chemicals on cyclic amp levels and rate of transport of nucleosides,hypoxanthine and deoxyglucose in several lines of cultured cells

Nutrient transport rates and cyclic AMP levels have been implicated in the regulation of cell proliferation. In the present study, however, changes in intracellular cyclic AMP level induced in several lines of cultured cells (normal 3T3 and SV₄₀ and polyomavirus-transformed 3T3 cells; 3T6, C6 glioma, mouse L, and Novikoff rat hepatoma cells) by treatment with papaverine, prostagladine E, or isoproterenol did not correlate with the inhibition of the uridine, hypoxanthine or deoxyglucose transport rates by these chemicals. Transport inhibitions by above chemicals or Persantin or Cytochalasin B occurred in most cell lines in the absence of any measurable change in intracellular cyclic AMP concentration. Furthermore, treatment of several cell lines with 1 mM dibutyryl cyclic AMP had no immediate effect on the transport of uridine, thymidine or deoxyglucose, although the transport capacity of the cells for uridine and thymidine, but not that for deoxyglucose, decreased progressively with time of treatment. We also observed that the uridine transport system of all cell lines derived from 3T3 cells and the hypoxanthine transport system of L cells exhibited high degrees of resistance to inhibition by the various chemicals. On the other hand, deoxyglucose transport was inhibited to about the same extent by these chemicals in all the cell lines investigated.     

A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis

5,10-Dideazatetrahydrofolate (DDATHF) is a new antimetabolite designed as an inhibitor of folate metabolism at sites other than dihydrofolate reductase. DDATHF was found to inhibit the growth of L1210 and CCRF-CEM cells in culture at concentrations in the range of 10-30 nM. The inhibitory effect of DDATHF on the growth of L1210 and CCRF-CEM cells was reversed by either hypoxanthine or aminoimidazole carboxamide. Growth inhibition by DDATHF was prevented by addition of both thymidine and hypoxanthine, but not by thymidine alone. 5-Formyltetrahydrofolate reversed the effects of DDATHF in a dose-dependent manner. DDATHF had no appreciable inhibitory activity against either dihydrofolate reductase or thymidylate synthase in vitro, but was found to be an excellent substrate for folylpolyglutamate synthetase. DDATHF had little or no effect on incorporation of either deoxyuridine or thymidine into DNA, in distinct contrast to the effects of the classical dihydrofolate reductase inhibitor, methotrexate. DDATHF was found to deplete cellular ATP and GTP over the same concentrations as those inhibitory to leukemic cell growth, suggesting that the locus of DDATHF action was in the de novo purine biosynthesis pathway. The synthesis of formylglycinamide ribonucleotide in intact L1210 cells was inhibited by DDATHF with the same concentration dependence as inhibition of growth. This suggested that DDATHF inhibited glycinamide ribonucleotide transformylase, the first folate-dependent enzyme of de novo purine synthesis. DDATHF is a potent folate analog which suppresses purine synthesis through direct or indirect inhibition of glycinamide ribonucleotide transformylase.     

DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate

Previous results from this laboratory have shown that thymidylate deprivation results in dramatic elevation of intracellular dUTP and incorporation of dUMP into DNA. The goal of the present studies was to determine whether the latter changes may play a part in the associated cytotoxicity ("thymineless death"), which is ordinarily assumed to be a direct result of reduced intracellular dTTP. The approach used here was to increase intracellular dUTP without allowing dTTP to diminish and observe the effects on cell viability. dUMP pools were expanded by exposure of cells to deoxyuridine [in cell growth medium containing hypoxanthine, methotrexate, and thymidine (HAT medium)], resulting in accumulation of dUTP to levels that approached those of dTTP, which were at, or higher than, the levels in untreated cells. In conjunction with this the cells became nonviable, and newly synthesized DNA was fragmented, both of which occur with thymidylate depletion and, we assume, result from the active process of excision repair at the many uracil-containing sites in DNA. The results indicate that, although the relative importance of low dTTP remains unknown, elevated dUTP can account for the cytotoxicity caused by thymidine starvation. Most of the "dTTP" measured by the DNA polymerase assay in cells treated with methotrexate (MTX) (plus purine supplement) was, in fact, dUTP, which may explain some previous observations of only modest depression of dTTP in cells treated with MTX or similarly acting drugs.     

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.     

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.     

Control of cytolysis of BALB/c-3T3 cells by platelet-derived growth factor: a model system for analyzing cell death

In culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c-3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with ³H-thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c-3T3 cells were transferred from serum-supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet-poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime. The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of-PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c-3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40-transformed cells occurred at a constitutively low rate and was insensitive to PDGF.     

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.     

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.     

Population-dependent requirements of vitamin B12 and metabolically related substances of several mouse cell types in serum-free, albumin-fortified medium

Mouse FM3A cells propagated well in serum-free medium containing 0.5% serum albumin and 1 microgram of insulin/ml. The vitamin B12 (B12) requirement of the cells depended on the population density. This requirement disappeared when a sufficiently large cell population was present. A combination of 1-100 ng of B12/ml and 4 micrograms of hypoxanthine/ml resulted in a synergistic increase in cell growth at low cell densities. A similar growth response was obtained when the B12 plus hypoxanthine was replaced by 4 micrograms of hypoxanthine/ml in combination with 100 ng of thymidine/ml, 1 microgram of folic acid/ml or 1 microgram of folinic acid/ml, even though 1 microgram of folic acid/ml already was present in the medium. Experiments on single cell inoculation showed that colony size and the yield of cells grown in B12-supplemented medium were much larger than those for cells grown in B12-free medium. A more critical population-dependent B12 requirement was demonstrated in mouse Ehrlich and L cells and their hybrids. At less than 100 cells there was no propagation in serum-free medium lacking B12, folinic acid and thymidine; whereas, a satisfactory growth response was obtained in medium supplemented with these substances.     

The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells

When cultured human choriocarcinoma (BeWo) cells are exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast. Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines. This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype. When methotrexate exposures (1 microM, 48 h) were conducted in a purine-free basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur. Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis. In contrast, morphologically differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.     

Genetic demonstration that the mutationally expressed nucleobase transporter of mouse S49 cells is nonconcentrative

Somatic cell genetic analysis of purine base transporters in mouse S49 cells has demonstrated the existence of a unique high-affinity purine base transporter, which is mutationally expressed and is not found in wild-type S49 cells or any other cells of the animal kingdom (B. Aronow, et al. (1986) Mol. Cell. Biol. 6, 2957). In order to determine whether this nucleobase transport system is active and concentrative, a secondary mutation in hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) was inserted into the cell line expressing this novel base transporter. The HGPRTase-deficient cells were capable of transporting hypoxanthine at increased rates but did not accumulate the base to concentrations in excess of that in the culture medium. Moreover, neither sodium azide nor ouabain had significant effects on hypoxanthine transport rates, indicating that energy metabolism and the maintenance of a sodium gradient were not required for transport function. These studies suggest that the novel mutationally expressed base transporter is independent of subsequent metabolism and does not require energy or a functioning Na+-K+-dependent ATPase activity.     

Genetic demonstration of bidirectionality in the high affinity purine base transporter of mutant mouse S49 cells

A mutant cell line was selected from wild type S49 lymphoblasts that expressed a novel high affinity purine base transport system not found in parental cells or any other mammalian cell line (Aronow, B., Toll, D., Patrick, J., Hollingsworth, P., McCartan, K., and Ullman, B. (1986) Mol. Cell. Biol. 6, 2957-2962). In order to determine whether this nucleobase transport system was bidirectional, mutant cell lines possessing this high affinity base transport capability were derived from a nucleoside transport-deficient derivative of an adenylosuccinate synthetase-deficient S49 cell line. The resulting progeny excreted significantly greater amounts of purine into the cell culture medium than parental cells. This purine was identified as hypoxanthine. These results demonstrate genetically that the high affinity purine base transport system can mediate both the influx and efflux of hypoxanthine.     

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.     

The relative contribution of somatomedin to the serum-stimulated growth of human fibroblasts

Culture conditions were defined allowing to demonstrate a stimulatory effect of both serum-contained and purified Somatomedin activity on incorporation of [3H]thymidine and replication of cultured normal human fibroblasts. The use of dialyzed human serum in MEM medium supplemented by 0.2 mM serine offered the necessary and sufficient culture conditions. A significant difference between normal and hypopituitary patients sera was found in their effect on the rate of [3H]thymidine incorporation (p < 0.0001) and on cell replication (p < 0.01). Purified Somatomedin-C, in MEM without serum, is a poor mitogen. Its activity was strongly enhanced by the addition of 0.1 % dialyzed serum and 0.2 mM serine without, however, exceeding the stimulatory level of 1 % whole normal serum. The requirement of concomitant presence, for optimal $\text{in}\text{vitro}$ cell growth, of different low and high MW serum components is discussed.     

Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [¹⁴C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.     

Enhancement of proliferation in cultures of Chinook salmon embryo cells by interactions between inosine and bovine sera

The influence of inosine on DNA synthesis by Chinook salmon embryo cells (CHSE-214) was investigated because previously cell number was shown to increase from six- to thirtyfold if inosine was added to the basal medium (L-15) supplemented with either dialyzed fetal bovine serum (dFBS), calf serum (CS), or dCS. Relative to L-15, ³H-thymidine incorporation was inhibited by these sera alone but elevated in nondialyzed (intact) FBS. Inosine at 10 μM stimulated ³H-thymidine incorporation from ten- to seventyfold in dFBS, CS, and dCS but was only slightly stimulatory in FBS and in L-15 alone. As well as inosine, hypoxanthine, cIMP, IMP, IDP, and ITP were just as stimulatory, but the nonsalvageable purines (xanthine, xanthosine, and XMP) were not. The stimulatory action of inosine was highest in low density cultures. Dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibitors of facilitated nonconcentrative nucleoside transport, did not completely block the enhancement of cell number by inosine and by themselves increased proliferation in CS and dCS. Overall, these results suggest that exogenous inosine promoted CHSE-214 proliferation by overcoming factors in the nondialyzable fraction of sera that led to purine loss and by raising intracelular purine nucleotides to levels necessary for cells to respond to growth factors in the nondialyzable fraction of sera. © 1994 Wiley-Liss, Inc.     

A critical intracellular concentration of fully reduced non-methylated folate polyglutamates prevents macrocytosis and diminished growth rate of human cell line K562 in culture.

Growth rate of human leukaemic cell line K562 was independent of intracellular folate concentration when this was greater than 1.5 microM. When intracellular folate concentration was less than 1.5 microM, the rate of growth was proportional to the logarithm of intracellular concentration of non-methylated fully reduced folates, but not to the logarithm of the intracellular concentration of N5-methyltetrahydropteroylglutamate. Intracellular folate concentration sufficient to support an optimal growth rate was maintained by either DL-N5-formyltetrahydropteroylglutamate or DL-N5-methyltetrahydropteroylglutamate at a 100-fold lower concentration than pteroylglutamate. Addition of hypoxanthine to culture medium partially restored growth of folate-depleted cells: thymidine had no effect on growth rate either alone or in combination with thymidine. Folate-depleted cells with diminished growth rate were larger than replete cells, but did not have megaloblastic morphology. The mitotic index was not decreased in cultures with diminished growth rate. The rate of growth and cell size of K562 cells is thus dependent on a critical intracellular concentration of non-methylated tetrahydrofolates, which may be maintained by different concentrations of either reduced folates or pteroylglutamate.