F. J. Casado

Nucleoside transporters in chronic lymphocytic leukaemia

Nucleoside derivatives have important therapeutic activity in chronic lymphocytic leukaemia (CLL). Experimental evidence indicates that in CLL cells most of these drugs induce apoptosis ex vivo, suggesting that programmed cell death is the mechanism of their therapeutic action, relying upon previous uptake and metabolic activation. Although defective apoptosis and poor metabolism often cause resistance to treatment, differential uptake and/or export of nucleosides and nucleotides may significantly modulate intracellular drug bioavailability and, consequently, responsiveness to therapy. Two gene families, SLC28 and SLC29, encode transporter proteins responsible for concentrative and equilibrative nucleoside uptake (CNT and ENT, respectively). Furthermore, selected members of the expanding ATP-binding cassette (ABC) protein family have recently been identified as putative efflux pumps for the phosphorylated forms of these nucleoside-derived drugs, ABCC11 (MRP8) being a good candidate to modulate cell sensitivity to fluoropyrimidines. Sensitivity of CLL cells to fludarabine has also been recently correlated with ENT-type transport function, suggesting that, besides the integrity of apoptotic pathways and appropriate intracellular metabolism, transport across the plasma membrane is also a relevant event during CLL treatment. As long as nucleoside transporter expression in leukaemia cells is not constitutive, the possibility of regulating nucleoside transporter function by pharmacological means may also contribute to improve therapy.

Regulation of nucleoside transport by lipopolysaccharide, phorbol esters, and tumor necrosis factor-alpha in human B-lymphocytes.

Nucleoside transport systems and their regulation in human B-lymphocytes have been characterized using the cell lines Raji and Bare lymphoma syndrome-1 (BLS-1) as experimental models. These cells express at least three different nucleoside transport systems as follows: a nitrobenzylthioinosine-sensitive equilibrative transport system of the es-type, which appears to be associated with hENT1 expression, and two Na+-dependent transport systems that may correspond to N1 and to the recently characterized N5-type, which is nitrobenzylthioinosine-sensitive and guanosine-preferring. B cell activators such as phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS) up-regulate both concentrative transport systems but down-regulate the equilibrativees-type transporter, which correlates with lower hENT1 mRNA levels. These effects are dependent on protein kinase C activity. Phorbol 12-myristate 13-acetate and LPS also induce an increase in tumor necrosis factor-α (TNF–α) mRNA levels, which suggest that this cytokine may mediate some of the effects triggered by these agents, since addition of TNF-α alone can increase N1 and N5 transport activities by a mechanism that also depends on protein kinase C activation. Interestingly, TNF-α down-regulates esactivity, but this effect cannot be abolished by inhibiting protein kinase C. This study reveals differential regulation of nucleoside transport systems following activation of human B-lymphocyte cell lines by agents of physiological relevance such as TNF-α and LPS. Moreover, it indicates that the recently characterized N5 transport system can also be regulated following B cell activation, which may be relevant to lymphocyte physiology and to the treatment of lymphocyte malignancies.

SLC28 genes and concentrative nucleoside transporter (CNT) proteins

The human concentrative nucleoside transporter (hCNT) protein family has three members, hCNT1, 2, and 3, encoded by SLC28A1, A2, and A3 genes, respectively. hCNT1 and hCNT2 translocate pyrimidine- and purine-nucleosides, respectively, by a sodium-dependent mechanism, whereas hCNT3 shows broad substrate selectivity and the unique ability of translocating nucleosides both in a sodium- and a proton-coupled manner. hCNT proteins are also responsible for the uptake of most nucleoside-derived antiviral and anticancer drugs. Thus, hCNTs are key pharmacological targets. This review focuses on several crucial aspects of hCNT biology and pharmacology: protein structure–function, structural determinants for transportability, pharmacogenetics of hCNT-encoding genes, role of hCNT proteins in nucleoside-based therapeutics, and finally hCNT physiology.

Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells

Fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). We have analyzed the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in CLL cells. Among the known plasma membrane transporters, we have previously observed a significant correlation between fludarabine uptake via ENT carriers and ex vivo sensitivity of CLL cells to fludarabine, although mRNA amounts of the equilibrative nucleoside transporters hENT1 and hENT2 do not show any predictive response to treatment. In this study, using polyclonal monospecific antibodies we have observed a significant correlation between the expression of hENT2 by Western blot and fludarabine uptake via hENT carriers and also with ex vivo sensitivity of CLL cells to fludarabine. These results suggest that the equilibrative nucleoside transporter hENT2 plays a role in fludarabine responsiveness in CLL patients.

Electrophysiological Characterization of the Human Na+/Nucleoside Cotransporter 1 (hCNT1) and Role of Adenosine on hCNT1 Function

We previously reported that the human Na+/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5′-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na+-leak currents, and the presteady-state currents, with a Ki of 6.5 μm. The kinetic parameters for uridine, gemcitabine, and 5′-deoxy-5-fluorouridine were studied as a function of membrane potential; at -50 mV, K0.5 was 37, 18, and 245 μm, respectively, and remained voltage-independent. Imax for gemcitabine was voltage-independent and accounts for ∼40% that for uridine at -50 mV. Maximal current for 5′-DFUR was voltage-dependent and was ∼150% that for uridine at all membrane potentials. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{0.5}^{\mathrm{Na}^{+}}\) \end{document} for Na+ was voltage-independent at hyperpolarized membrane potentials (1.2 mm at -50 mV), whereas \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(I_{\mathrm{max}}^{\mathrm{Na}^{+}}\) \end{document} was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of 3H-nucleoside or 22Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na+ per positive inward charge, suggesting a stoichiometry of two Na+/nucleoside.

Concentrative nucleoside transporters (CNTs) in epithelia: from absorption to cell signaling.

Concentrative and Equilibrative Nucleoside Transporter proteins (CNT and ENT, respectively) are encoded by gene families SLC28 and SLC29. They mediate the uptake of natural nucleosides and a variety of nucleoside-derived drugs, mostly used in anticancer therapy. CNT and ENT proteins are mostly localized in the apical and basolateral sides, respectively, in (re)absorptive epithelia. This anatomic distribution determines nucleoside and nucleoside-derived vectorial flux. CNT expression (particularly CNT2) is associated with differentiation and is also nutritionally regulated in intestinal epithelia, whereas ENT protein amounts (mostly ENT1) are increased when cells are exposed to proliferative stimuli such as EGF, TGF-α or wounding. Although all these features suggest a role for NT proteins in nucleoside salvage and (re)absorption, recent data demonstrate that CNT2 might be under purinergic control, in a manner that is dependent on energy metabolism. A physiological link between CNT2 function and intracellular metabolism is also supported by the evidence that extracellular adenosine can activate the AMP-dependent kinase (AMPK), by a mechanism which relies upon adenosine transport and phosphorylation. Thus the complex pattern of NT isoform expression in mammalian cells can fulfill physiological roles other than salvage.

Nitric oxide regulates nucleoside transport in activated B lymphocytes.

Activation of human B lymphocytes by lipopolysaccharide (LPS) or phorbol 12‐myristate 13‐acetate (PMA) results in the differential regulation of nucleoside uptake [Soler, C., Felipe, A., Mata, J. F., Casado, F. J., Celada, A., Pastor‐Anglada, M. (1998) J. Biol. Chem. 273, 26939–26945]. Because nitric oxide (NO) is involved in the modulation of the apoptotic response of B cells, the effects of NO on the regulatory responses of these transport systems to phorbol esters has been studied in Raji cells by a combination of approaches that involve arginine depletion, inhibition of nitric oxide synthase, and non‐enzymatic production of NO using a donor. Human B lymphocytes express three transport systems involved in nucleoside uptake: N1 and N5, which are concentrative and Na+‐dependent, and the nitrobenzylthioinosine‐sensitive equilibrative system es. Raji cells do not express significant amounts of iNOS mRNA or protein; thus, NO production is presumably constitutive. The data are consistent with a role of NO in maintaining the basal transport activities of the three systems: N1, N5, and es. However, the up‐regulatory effect of PMA on N1 and N5 does not require NO, whereas the inhibition of es transport activity does. In summary, NO differentially modulates nucleoside transport systems in activated human B lymphocytes and thus, NO may also be involved in the regulation of nucleoside (i.e., adenosine) disposal by activated B cells. J. Leukoc. Biol. 67: 345–349; 2000.

Nucleoside transporters in absorptive epithelia

There are two families of nucleoside transporters, concentrative (termed CNTs) and equilibrative (called ENTs). The members of both families mediate the transmembrane transport of natural nucleosides and some drugs whose structure is based on nucleosides. CNT transporters show a high affinity for their natural substrates (with Km values in the low micromolar range) and are substrate selective. In contrast, ENT transporters show lower affinity and are more permissive regarding the substrates they accept. Both types of transporters are tightly regulated in all cell types studied so far, both by endocrine and growth factors and by substrate availability. The degree of cell differentiation and the proliferation status of a cell also affect the pattern of expressed transporters. Although the presence of both types of transporters in the cells of absortive epithelia suggested the possibility of a transepithelial flux of nucleosides, their exact localization in the different plasma membrane domains of epithelial cells had not been demonstrated until recently. Concentrative transporters are found in the apical membrane while equlibrative transporters are located in the basolateral membrane, thus strengthening the hypothesis of a transepithelial flux of nucleosides.ResumenExisten dos familias de transportadores de nucleósidos, la de los concentrativos (denominada CNT) y la de equilibrativos (o ENT). Los miembros de ambas familias median el transporte transmembrana de los nucleósidos naturales y de algunos fármacos cuya estructura deriva de aquéllos. Los transportadores CNT presentan una alta afinidad por sus sustratos naturales (sus Km están en el rango micromolar bajo) y una cierta selectividad por sustrato. Los ENT, por el contrario, muestran menor afinidad y son más permisivos en cuanto a los sustratos que aceptan. Ambos tipos de transportadores están estrechamente regulados en los diversos tipos celulares estudiados, tanto por factores endocrinos o de crecimiento como por la disponibilidad de sus sustratos. También el grado de diferenciación y el estado de proliferación de una célula influyen sobre el patrón de transportadores expresado por esa célula. Aunque la presencia de ambos tipos de transportadores en las células de los epitelios absorbentes parecía sugerir la posibilidad de un flujo transepitelial de nucleósidos, hasta hace muy poco no se ha podido demostrar su localización en los diferentes dominios de la membrana plasmática de las células epiteliales; así, los transportadores concentrativos se encuentran en la membrana apical mientras que los equilibrativos se localizan en la membrana basolateral, lo que refuerza la tesis de un flujo transepitelial de nucleósidos.

The Human Concentrative Nucleoside Transporter-3 C602R Variant Shows Impaired Sorting to Lipid Rafts and Altered Specificity for Nucleoside-Derived Drugs

The human concentrative nucleoside transporter-3 C602R (hCNT3C602R), a recently identified human concentrative nucleoside transporter-3 (hCNT3) variant, has been shown to interact with natural nucleosides with apparent Km values similar to those of the wild-type transporter, although binding of one of the two sodium ions required for nucleoside translocation is impaired, resulting in decreased Vmax values (Mol Pharmacol 73:379–386, 2008). We have further analyzed the properties of this hCNT3 variant by determining its localization in plasma membrane lipid domains and its interaction with nucleoside-derived drugs used in anticancer and antiviral therapies. When expressed heterologously in HeLa cells, wild-type hCNT3 localized to both lipid raft and nonlipid raft domains. Treatment of cells with the cholesterol-depleting agent methyl-β-cyclodextrin resulted in a marked decrease in hCNT3-related transport activity that was associated with the loss of wild-type hCNT3 from lipid rafts. It is noteworthy that although exogenously expressed hCNT3C602R was present in nonlipid raft domains at a level similar to that of the wild-type transporter, the mutant transporter was present at much lower amounts in lipid rafts. A substrate profile analysis showed that interactions with a variety of nucleoside-derived drugs were altered in the hCNT3C602R variant and revealed that sugar hydroxyl residues are key structural determinants for substrate recognition by the hCNT3C602R variant.

Long-term osmotic regulation of amino acid transport systems in mammalian cells.

SummaryMammalian cells accumulate organic osmolytes, either to adapt to permanent osmotic changes or to mediate cell volume increase in cell cycle progression. Amino acids may serve as osmolytes in a great variety of cells. System A, a transport system for neutral amino acids, is induced after hypertonic shock by a mechanism which requires protein synthesis and gene transcription. Indirect evidence supports the view that system A activity increases due to the interaction of pre-existing A carriers with putative activating proteins. The intracellular accumulation of most neutral amino acids after hypertonic shock depends, exclusively, on the increase in system A activity. Long-term activation of system A is dependent on the integrity of cytoskeletal structures, but in a different way depending on whether cells are polarized or not.