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PubMed2003

Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents.

l-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of l-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of l-nucleoside analogs (but not d-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of l-nucleotides in the cells, thereby playing a key role in the activity of l-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of l- and d-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.

Antineoplastic Agents↗

PubMedDate not supplied

Purine nucleoside and nucleobase cell membrane transport in Giardia lamblia.

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines. This study investigated purine nucleoside and nucleobase transport utilizing rapid uptake determinations. Nucleoside substrate/velocity curves exhibited the hyperbolic kinetics of a saturable carrier-mediated system. Deoxynucleosides exhibited a much lower affinity for the transporter. Inhibition studies confirmed the relative carrier affinities of these ribonucleosides and deoxyribonucleosides. The nucleobase adenine did not exhibit saturation kinetics at a comparable substrate range, and did not inhibit nucleoside transport. Dipyridamole markedly inhibited nucleoside but not nucleobase transport, confirming the separate entry pathways. When cells were depleted of ATP, the velocity of nucleoside and nucleobase transport was unchanged, indicating that it is a non-energy-dependent process. Three nucleoside analogs, formycin A, adenine arabinoside and 7-deazaadenosine, were studied. Transport kinetics ranged widely among this group and could not completely account for their cytotoxic effect. When the apparent Km and Vmax of the nucleosides were compared, an approximately linear relationship (r2 = 0.95) was noted. This suggests that a high affinity of the nucleoside permease for the substrate retards disassociation of the substrate-carrier complex, slowing net influx.

Adenosine↗

PubMed1992

Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells.

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.

Adenosine Diphosphate↗

PubMed1975

Preparative enzymic synthesis of nucleoside-5'-phosphates.

1. Wheat shoot phosphotransferase has been employed, with p-nitrophenylphosphate as a phosphate donor, to specifically phosphorylate the 5'-position of a variety of nucleosides and nucleoside analogues. The specificity of the enzyme towards the 5'-position of pentose nucleosides is testified to by the complete resistance to phosphorylation of 5'-O-methylcytidine. 2. With the use of ion-exchange chromatography, the foregoing procedure has been applied to the large-scale preparation of nucleoside-5'-phosphates with overall yields of the order of 80-90%. Quantitative recovery of unreacted nucleoside makes it possible to use this method without risk of losses either on a small or large scale with rare nucleosides. It is also applicable to acid- and alkali-labile nucleosides which cannot readily be phosphorylated by chemical procedures. 3. The wheat shoot phosphotransferase also phosphorylated a galactopyranosyl nucleoside, as well as such derivatives as 1-(beta-hydroxyethyl)cytosine and 5-(beta-hydroxyethyl)uracil, showing that the enzyme does not have an absolute requirement for a 5-membered sugar ring, but rather for the presence of a primary hydroxyl group. 4. The phosphorylated derivatives of galactopyranosyluracil, and of both hydroxyethyl pyrimidines, were resistant to 5'-nucleotidase. E. coli alkaline phosphatase converted all three nucleotides quantitatively to the starting compounds. 5. A synthesis of 1-(beta-hydroxyethyl)cytosine is described.

Alkaline Phosphatase↗

PubMed1991

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.

Animals↗

PubMed1987

3'-azido-3'-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion.

The demonstrated in vitro and in vivo activity of 3'-azido-3'-deoxythymidine (N3dThd) against the infectivity and the cytopathic effect of human immunodeficiency virus has prompted an investigation of the mechanism by which this nucleoside analogue permeates the cell membrane. As with the transport of thymidine, the influx of N3dThd into human erythrocytes and lymphocytes was nonconcentrative during short incubation times (less than 5 min) which did not allow significant metabolism of this nucleoside. However, in contrast with thymidine transport, the initial velocity of N3dThd influx was strictly a linear function of nucleoside concentration (0.5-10 mM), without evidence of saturability; insensitive to micromolar concentrations of potent inhibitors of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep); insensitive to a 1000-fold excess of other nucleosides (thymidine, uridine, 2-chloroadenosine); and relatively insensitive to temperature, with Q10 values (37-27 degrees C) of 1.4 and 2.7 for N3dThd and thymidine, respectively, determined in erythrocytes. Although the above results indicate that N3dThd permeates the cell membrane chiefly by nonfacilitated diffusion and not via the nucleoside transporter, millimolar concentrations of this nucleoside analogue were observed to inhibit both zero-trans influx of thymidine and efflux of thymidine from [3H]thymidine-loaded erythrocytes. The partition coefficients (1-octanol:0.1 M sodium phosphate, pH 7.0) of N3dThd and thymidine were determined to be 1.26 and 0.064, respectively. The unusual ability of N3dThd to diffuse across cell membranes independently of the nucleoside transport system may be attributed to the considerable lipophilicity imparted to this molecule by the replacement of the 3'-hydroxyl group of thymidine with an azido moiety.

Antiviral Agents↗

PubMed1994

Guanosine-inosine-preferring nucleoside N-glycohydrolase from Crithidia fasciculata.

Protozoan parasites are incapable of de novo purine biosynthesis and obtain purines by salvage pathways. A nucleoside hydrolase which prefers inosine and uridine as substrates (IU-nucleoside hydrolase) has been characterized and implicated in purine salvage in Crithidia fasciculata (Parkin, D. W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., and Schramm, V. L. (1991) J. Biol. Chem. 31, 20658-20665). Treatment of C. fasciculata with inhibitors of the IU-nucleoside hydrolase did not prevent cell growth, suggesting alternative enzymes. A guanosine-inosine-preferring enzyme (GI-nucleoside hydrolase) has been purified from extracts of C. fasciculata and characterized. The enzyme is an oligomer of M(r) 38,500 subunits. The Vmax/Km for guanosine, inosine, and adenosine are 3.2 x 10(6), 6.2 x 10(6), and 9.8 M-1 S-1, respectively. Deoxynucleosides, nucleotides, and pyrimidine nucleosides are poor substrates. The pH profile for Km is independent of pH, whereas both Vmax and Vmax/Km demonstrate that a single protonated base, pKa 7.7 is required for activity. The transition state inhibitors of IU-nucleoside hydrolase, 1,4-dideoxy-1,4-imino-1-(S)-phenyl-D-ribitol (Horenstein, B. A., and Schramm, V. L. (1993) Biochemistry 32, 9917-9925) and p-nitrophenylriboamidrazone (Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V.L., and Ganem, B. (1994) Biochemistry 33, 3994-4000), are unexceptional inhibitors of the GI-nucleoside hydrolase. The enzyme is inhibited by 3-deazaadenosine and 2-iodoadenosine with Km/Ki values of 145 and 61, respectively. The results demonstrate that this previously uncharacterized enzyme has distinct structure, kinetic, and chemical mechanisms relative to IU-nucleoside hydrolase. Metabolic studies with labeled inosine as the sole purine source indicated that the GI-enzyme is efficient for purine salvage in vivo.

Animals↗

PubMed1997

Interaction of dipyridamole, a nucleoside transport inhibitor, with the renal transport of organic cations by LLCPK1 cells.

Dipyridamole is a well-known inhibitor of nucleoside transport by various cell membranes and is frequently used in in vitro studies that characterize nucleoside transport properties. Because interactions between the renal transport of organic cations and nucleosides have previously been suggested, we studied the effect of dipyridamole on the renal transport of the typical organic cations cimetidine and N1-methylnicotinamide by LLCPK1 monolayer cells grown on a permeable support. [14C]Mannitol was used to correct for extracellular flux. Basolateral to apical transcellular flux (transepithelial flux-extracellular flux) of [3H]cimetidine was significantly reduced by the monolayer cells (90%) in the presence of 50 microM dipyridamole. In addition, the effect of dipyridamole on cimetidine renal transport was dose dependent (IC50 = 7.7 microM). The dipyridamole inhibitory effect was nearly comparable with the effect of 1 mM quinine (a typical organic cation transport inhibitor), which led to 95% inhibition of cimetidine renal transport over time. The dipyridamole effect on N1-methylnicotinamide renal transport was less potent. The effect of 1 mM of typical probes of the nucleoside transporters (i.e., thymidine, adenosine, uridine) and the effect of 100 nM of another nucleoside transport inhibitor, dilazep, were also studied on cimetidine transport by LLCPK1 monolayer cells. These compounds did not exert any significant effect. These results suggest that dipyridamole, a widely used nucleoside transport inhibitor, is also an inhibitor of organic cation renal transport and they alert us to possible interactions between the renal transport of nucleosides and organic cations. This finding also has relevance to the interpretation of in vitro studies using this agent as a nucleoside membrane transport inhibitor.

Animals↗

PubMed1993

Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells.

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

Adenosine↗

PubMed1989

Hydration of nucleosides in dilute aqueous solutions. Ultrasonic velocity and density measurements.

The values of the concentration increments of the ultrasound velocity and their temperature slopes, apparent molar volumes, apparent molar expansibilities, apparent molar adiabatic compressibilities and their temperature gradients for 12 nucleosides and their analogs, as well as for ribose and deoxyribose, have been obtained using precision measurements of ultrasound velocity and density. The following hydration parameters for the atomic groups of the nucleosides, reflecting the state of water in the hydration shells of these groups, have been analyzed: (1) the contribution of ribose to the values of the concentration increment of ultrasound velocity A, the apparent molar volumes phi v and apparent molar adiabatic compressibilities phi ks of nucleosides; (2) contributions of the CH3, NH2 and O = ... -H groups of nucleic bases to the A, phi v and phi ks values of nucleosides and free nucleic bases; (3) contributions of the 2'-OH group of ribose to the values of A, phi v and phi ks nucleosides; (4) changes in the A values of nucleosides and free nucleic bases upon their protonation and deprotonation. Data have been obtained on the mutual influence of the atomic groups of nucleosides on their hydration. It is shown that the GC pairs of free deoxynucleosides undergo hydration more vigorously than the AT pairs, which contrasts with the relation of the degree of hydration of the GC and AT pairs of the double helix.

Base Composition↗

PubMed2006

Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

Adenine Nucleotides↗

PubMed2005

Cell entry and export of nucleoside analogues.

Some nucleoside analogues currently used as antiretroviral agents might promote mutagenesis besides their putative ability to interfere with endogenous nucleotide metabolism and/or inhibit viral transcription. The intracellular concentration of nucleosides and nucleobases is to some extent the result of the metabolic background of the specific cell line used for infection studies, its particular suit of enzymes and transporters. This review focuses on the transporter-mediated pathways implicated in either the uptake or the efflux of nucleoside- and nucleobase-derivatives. From a biochemical point of view, four different types of transport processes for nucleoside-related antiviral drugs have been described: (1) equilibrative uniport, (2) substrate exchange, (3) concentrative Na+- or H+-dependent uptake and finally, (4) substrate export through primary ATP-dependent active efflux pumps. These mechanisms are mainly related to the following set of transporter families: Concentrative Nucleoside Transporter (CNT), Equilibrative Nucleoside Transporter (ENT), Organic Anion Transporter (OAT) and Organic Cation Transporter (OCT), Peptide Transporter (PEPT) and Multidrug Resistance Protein (MRP). The basic properties of these carrier proteins and their respective role in the transport across the plasma membrane of nucleoside-derived antiviral drugs are reviewed.

Animals↗

PubMed2004

Crystal structure of the bacterial nucleoside transporter Tsx.

Tsx is a nucleoside-specific outer membrane (OM) transporter of Gram-negative bacteria. We present crystal structures of Escherichia coli Tsx in the absence and presence of nucleosides. These structures provide a mechanism for nucleoside transport across the bacterial OM. Tsx forms a monomeric, 12-stranded beta-barrel with a long and narrow channel spanning the outer membrane. The channel, which is shaped like a keyhole, contains several distinct nucleoside-binding sites, two of which are well defined. The base moiety of the nucleoside is located in the narrow part of the keyhole, while the sugar occupies the wider opening. Pairs of aromatic residues and flanking ionizable residues are involved in nucleoside binding. Nucleoside transport presumably occurs by diffusion from one binding site to the next.

Amino Acid Sequence↗

PubMed2004

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells.

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.

Adaptation, Physiological↗

PubMed1994

Multiple myeloma: expression of nucleoside transporters on malignant plasma cells and their relationship to cellular proliferation.

The expression of nucleoside transporters is a limiting factor in the pharmacology of the nucleoside analogue, cytosine arabinoside (AraC) and is associated with cellular proliferation. We investigated the expression of nucleoside transporters on plasma cells from the bone marrow of 51 patients with multiple myeloma by 2-colour immunofluorescence flow cytometry, utilising 5-(SAENTA-x8)-fluorescein, a fluorescent ligand for the nucleoside transporter and anti-CD38 conjugated to phycoerythrin, as CD38 expression has unique characteristics on plasma cells. Mean nucleoside transporter expression on bone marrow plasma cells from patients with myeloma (1777 +/- 2181 transporters/plasma cell) was not significantly different from expression on plasma cells from normal bone marrow (997 +/- 1096 transporters/plasma cell). However, analysis of disease subgroups revealed a significant trend towards increased transporter expression in patients with progressive disease compared to those with stable disease (chi 2 = 4.0, p < 0.05). Nucleoside transporter expression correlated significantly with the plasma cell labeling index (LI) (r = 0.45, p < 0.01) and serum thymidine kinase levels (r = 0.66, p < 0.01), both markers of cellular proliferation but not with c-myc oncoprotein expression. These findings suggest that flow cytometric measurement of nucleoside transporter expression on plasma cells provides a rapid and convenient measurement of disease activity or quiescence in myeloma.

ADP-ribosyl Cyclase↗

PubMed1989

Nucleoside transport and proliferative rate in human thymocytes and lymphocytes.

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1994

Assessment of proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF) in acute myeloid leukaemia using a fluorescent ligand for the nucleoside transporter.

Nucleoside transporter expression has been linked to proliferation in a variety of haemopoietic cell types. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given for 72 h before commencing chemotherapy in 15 patients with relapsed or refractory acute myeloid leukaemia (AML) and in 11 patients serial bone marrows were taken for measurement of [3H]thymidine labelling index, Ki-67 positivity and maximal binding of 5-(SAENTA-x8)-fluorescein, a flow cytometry ligand which enumerates nucleoside transporter sites. GM-CSF caused proliferation of marrow myeloblasts in eight of 11 patients, while in three patients there was no change in proliferative indices. The expression of nucleoside transporters increased up to 4-fold in the myeloblasts from the patients showing a proliferative response to GM-CSF but there was no increase in transporters on the myeloblasts from the three non-responding patients. A close correlation was found between the fold increase in nucleoside transporter expression and the fold increase in labelling index of marrow myeloblasts (r = 0.86, n = 9, p < 0.01). In one patient with acute megakaryoblastic leukemia, GM-CSF caused parallel increases in labelling index, Ki-67 positivity and numbers of nucleoside transporters on peripheral blood blast cells. Thus induction of proliferation by cytokine increases the expression of nucleoside transporters on leukaemic myeloblasts studied in serial samples from the same source (bone marrow or blood). The suitability of 5-(SAENTA-x8)-fluorescein for two colour flow cytometric analysis allows the rapid enumeration of nucleoside transporters in the myeloblast compartment of heterogeneous marrow samples.

Acute Disease↗

PubMed2004

A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection.

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with single-parameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively.

Chromatography, High Pressure Liquid↗