Stimulation of hepatic fatty acid synthetase activity in chick embryo by cycloheximide

The regulation of hypoxanthine transport activity by Chinese hamster lung fibroblasts grown in culture was examined in wild-type clones and 8-azaguanine-resistant mutant clones which lack hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine transport activity increases with increased rates of cellular growth expressed as viable cell number, total cell protein, and DNA synthesis. The transport activity for hypoxanthine declines when the fibroblasts approach confluence or after exposure to cycloheximide or actinomycin D. In vivo incubation of either fibroblast subline with 100 μm dibutyryl cyclic AMP decreases transport activity over 50%, whereas exposure to 10 μm dibutyryl cyclic GMP increases hypoxanthine uptake by 40%. A synergistic effect is observed when fibroblasts are incubated with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine or theophylline) plus glucagon, an adenylate cyclase stimulator. Such additions result in a 70% decrease in the cellular transport capacity. Stimulation of hypoxanthine transport by 40% is observed following incubation with insulin. Addition of all agents produces maximum changes in the rate of hypoxanthine transport only after a 6-h in vivo incubation with the fibroblasts. These findings suggest that hypoxanthine transport is regulated by the intracellular concentration of cyclic nucleotides. This control may occur at the level of gene expression for a hypoxanthine transport protein.     

Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation.

The zero-trans uptake of purines and pyrimidines was measured in suspensions of Novikoff rat hepatoma, mouse L, P388 mouse leukemia, and Chinese hamster ovary cells by a rapid kinetic technique which allows the determination of uptake time points in intervals as short as 1.5 s. Kinetic parameters for purine/pyrimidine transport were determined by measuring substrate influx into cells in which substrate conversion to nucleotides was negligible either due to lack of the appropriate enzymes or to depletion of the cells of ATP (5'-phosphoribosylpyrophosphate), and by computer fitting exact, integrated rate equations derived for various carrier-mediated transport models directly to zero-trans influx data. The results indicate that different carriers function in the transport of hypoxanthine/guanine, adenine, and uracil with substrate:carrier association constants (K) at 24 degrees C of 300 to 400 muM, 2 to 3 mM, and about 14 mM, respectively, for Novikoff cells. K and Vmax for hypoxanthine transport by L and P388 cells are similar to those for Novikoff cells, but the transport capacity of Chinese hamster ovary cells is much lower and K = 1500 muM. All transport systems are completely symmetrical. Hypoxanthine transport is so rapid that an intracellular concentration of free hypoxanthine (90%) close to that in the medium is attained within 20 to 50 s of incubation at 24 degrees C, at least at extracellular concentrations below K. In cells in which conversion to nucleotides is not blocked free hypoxanthine accumulates intracellularly to steady state levels with equal rapidity and thereafter the rate of hypoxanthine uptake into total cell material is strictly a function of the rate of phosphoribosylation. The low Km systems for hypoxanthine (1 to 9 muM) and adenine (0.2 to 40 muM) uptake detected previously in many types of cells reflect the substrate saturation of the respective phosphoribosyltransferases rather than of the transport system.     

Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [³H]hypoxanthine for 5 or 10 min, followed by storage of ³H-labelled cells at ?70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [³H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K_m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.     

Hexose transport derepressed and refractory to purine regulation in NAD(H)-depleted Nil cells

Nil hamster fibroblasts depleted of NAD(H) by growth in medium devoid of nicotinamide (NAm^?MEM) exhibit up to 2-3-fold higher rates of glucose transport. Derepression of glucose transport is observed only when Nil cells have become severely depleted of both intracellular NAD(H) and ATP, despite the continued presence of 5.5 mM D-glucose in the growth medium. Neither the initial rate of transport, approximated from 3-O-methylglucose uptake, nor accumulation of D-glucose itself is repressed upon restoring nicotinamide to the medium. Exposure of the cells to NAD⁺ (10^?5 M), however, leads to a sharp curtailment of transport within 2 to 3 hours. The purines, hypoxanthine and guanine, that sharply reduce glucose transport capacity of normal cells, have no significant effect upon transport activity of NAD(H)-depleted cells.     

Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase.

Rapid kinetic techniques were employed to measure the transport of adenine in adenine phosphoribosyltransferase-deficient L929 and Chinese hamster ovary (CHO) cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled adenine. Adenine transport conformed to the simple carrier model with directional symmetry and equal mobility of loaded and empty carrier. The Michaelis-Menten constants and maximum velocities for various strains of L929 cells fell between 2.3 and 3.5 mM and 90 and 150 pmol/microliters of cell water per s, respectively, values similar to those previously reported for CHO and Novikoff hepatoma cells. The corresponding values for hypoxanthine transport in L929 cells were 413 microM and 16 pmol/microliters of cell water per s. Adenine transport velocities were directly proportional to adenine concentrations between 0.03 and 50 microM in both CHO and Novikoff cells. The results indicate that adenine is transported in these cells by a single, low-affinity, high-capacity transporter. Adenine transport was inhibited by hypoxanthine in some cell strains, but not in others. Adenine also rapidly bound to L929 cells in a saturable manner (KD = 18 microM), presumably to the cell surface (about 3 X 10(7) sites per cell).     

Transport of hypoxanthine by human diploid skin fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase

Transport of purine bases and nucleosides by a variety of mammalian cell lines is generally accomplished by facilitated diffusion, a non-concentrative, saturable process. However, previous investigators have been unable to detect a saturable component for the transport of hypoxanthine by human fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase, implying that in normal cells the enzyme actively participates in transport. In the present study we have used the phenomenon of countertransport to demonstrate the existence of a saturable transport mechanism in hypoxanthine-guanine phosphoribosyltransferase-deficient human diploid skin fibroblasts.     

The transfer of purine metabolites via the medium rather than through cell contacts

When the medium in which mouse B82 cells had been grown for 24 h with [³H]hypoxanthine was given to HGPRT⁻ Chinese hamster cells (CHW-1102), the acid-insoluble fraction of these cells became radioactive. When the medium in which mouse B82 cells had been grown for 24 h without [³H]hypoxanthine was given to CHW-1102 cells at the same time as [³H]hypoxanthine was added, the acid-insoluble fraction of the cells did not become radioactive. This indicates that CHW-1102 cells acquire from the B82 medium ³H material and not the ability to utilize hypoxanthine. Very little radioactivity was found in the acid-insoluble fraction of the B82 medium and when the medium was given to CHW-1102 cytoplasms, they did not become labelled. These results suggest that ³H purine metabolites (and not ³H nucleic acids) are responsible for the radioactivity in the CHW-1102 cells. Such ³H metabolites were also present in the medium of mouse L929 cells, but were absent in the medium of Chinese hamster (V79), mouse (A9), Syrian baby hamster kidney (BHK) and human fibroblasts. The cells were judged to be free of mycoplasma by three different criteria. This exchange of metabolites through the medium is referred to as contact-independent metabolite transfer (CIMT).     

Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT+ and HGPRT- cells

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT- Chinese hamster and human fibroblasts were able to incorporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT- cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT- mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT- cells became labeled.     

Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.

A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage.     

Use of latex particles for analysis of heterokaryon formation and cell fusion.

A simple method for the quick and accurate detection of cell fusion utilizing latex particles as cytoplasmic markers was developed and is reported here. The method is particularly useful for demonstrating human skin fibroblast heterokaryons. Ingestion of latex particles did not affect the growth of primary human and established BHK(21)/C(13) hamster fibroblasts. In addition, somatic cell hybridization between hypoxanthine phosphoribosyltransferase-deficient (HPRT-) and thymidine kinase-deficient (TK-) mutants of BHK(21)/C(13) was also unaffected by lates particle ingestion.     

Role of adenine phosphoribosyltransferase in adenine uptake in wild-type and APRT− mutants of CHO

Adenine uptake in cultured Chinese hamster fibroblasts showed biphasic saturation kinetics. The transport system was highly specific for adenine and was competitively inhibited by adenosine. Utilizing mutant clones of Chinese hamster fibroblasts that have either reduced or negligible adenine phosphoribosyltransferase (APRT) activity, we found that (1) adenine was not accumulated against a concentration gradient in the absence of APRT activity and (2) after rapid initial uptake equal to that of the parent the rates of adenine accumulation found for the mutants correlated strongly with their residual APRT activities. Furthermore, using either artificially depressed phosphoribosylpyrophosphate pool size and APRT activities or the mutants with decreased APRT activity, we found that adenine transport was independent of phosphorylation by APRT. These studies suggest that adenine is transported as the free base by facilitated diffusion and is subsequently phosphorylated by APRT.     

Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.

HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.     

Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.

We have isolated clones of an established cell line which express defects in intracellular cholesterol metabolism. Chinese hamster ovary cells were mutagenized, and clones unable to mobilize low density lipoprotein (LDL)-derived cholesterol to the plasma membrane were selected. Biochemical analysis of two mutant clones revealed a phenotype characteristic of the lysosomal storage disease, Niemann-Pick type C. The mutant cell lines were found to be defective in the regulatory responses elicited by LDL-derived cholesterol. LDL-mediated stimulation of cholesterol esterification was grossly defective, and LDL suppression of 3-hydroxy-3-methylglutaryl-CoA reductase was impaired. However, the mutants modulated these activities normally in response to 25-hydroxycholesterol or mevalonate. The LDL-specific defects were predicated by the inability of these mutants to mobilize LDL-derived cholesterol from lysosomes. Cell fractionation studies showed that LDL-derived, unesterified cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, commensurate with defective movement of cholesterol to other cellular membranes. Characterization of cell lines defective in intracellular cholesterol transport will facilitate identification of the gene(s) required for intracellular cholesterol movement and regulation.     

Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.     

Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes.

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10-30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.     

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.     

The influx of uric acid and other purines into everted jejunal sacs of the rat and hamster.

The in vitro transport of [2-14-C]uric acid, [8-14-C]hypoxanthine, and [8-14-C]xanthine, each dissolved in Krebs--Ringer bicarbonate buffer, was studied with everted jejunal sacs from rat and hamster. No evidence could be obtained for the development of a concentration gradient between the intracellular fluid and the incubation medium or between the sac contents and the incubation medium, for any of the three oxypurines. Inhibitiors of active transport, such as anaerobiosis for dinitrophenol, had no significant effect on the rate of transport. A large percentage of hypoxanthine and xanthine was oxidized to urine acid in the sac-wall homogenate, sac contents, and incubation medium during the course of the incubation. This oxidation could be prevented by addition of allopurinol (3 mM) to the incubation medium, but concentration gradients were still not obtained. No active transport mechanism could be demonstrated for uric acid, hypoxanthine, or xanthine in rat or hamster jejunum.     

Gene linkage analysis in the mouse by somatic cell hybridization: assignment of adenine phosphoribosyltransferase to chromosome 8 and alpha-galactosidase to the X chromosome.

Somatic cell hybridization techniques were applied to gene linkage analysis in the laboratory mouse. Cells of an established line of Chinese hamster lung fibroblasts were fused with mouse embryo fibroblasts and with mouse peritoneal macrophages obtained from different inbred strains. From 3 hybridization experiments, 123 primary and secondary clones were isolated in HAT selective medium and 24 were back-selected in 8-azaguanine. Hybrid clones were characterized for the expression of 16 murine isozymes by starch, acrylamide, and Cellogel electrophoresis, and on the basis of segregation data, 3 syntenic associations could be made. Malate oxidoreductase decarboxylating (MOD) and mannose phosphate isomerase (MPI) segregated concordantly, confirming an established linkage relationship; adenine phosphoribosyltransferase (APRT) segregated concordantly with glutathione reductase (GR) which is known to be on chromosome 8; alpha-galactosidase was observed to be syntenic with hypoxanthine phosphoribosyltransferase (HPRT), and X-linked enzyme. All other isozymes examined segregated independently of one another.     

Mammalian cells with defective mitochondrial functions: a Chinese hamster mutant cell line lacking succinate dehydrogenase activity

A mutant cell line derived from Chinese hamster fibroblasts is described which is defective in oxidative energy metabolism. Glucose is continuously required in the medium. As a result of a block in the Krebs cycle, these cells are auxotrophs for carbon dioxide and asparagine. Several experiments support our conclusion that the mutant cells lack appreciable levels of succinate dehydorgenase activity. Other components of the electron transport chain appear to be fully functional, although there is the possibility that electron transport and oxidative phosphorylation are uncoupled.     

Hypoxanthine and thymidine compete for transport in Chinese hamster fibroblasts

The transport of thymidine and hypoxanthine was investigated in mutant Chinese hamster lung fibroblasts deficient in both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase. Kinetic data from rapid uptake experiments (0.5–4.5 s) indicate that thymidine is transported by a monophasic saturable system (K_m = 0.29 mM, V = 6.7 nmol/min · mg) which is competitively inhibited by hypoxanthine (K_i = 3.3 mM). The cells displayed a single transport system for hypoxanthine (K_m = 2.0 mM, V = 8.9 nmol/min · mg) that is inhibited competitively by thymidine (K_i = 0.43 mM). Both hypoxanthine and thymidine entry were noncompetively inhibited by nitrobenzylthioinosine, but thymidine transport was more sensitive. A kinetic model in which hypoxanthine and thymidine share a common transporter can account for the competitive inhibition and the observation that the inhibition constants are similar to the Michaelis constants.