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Schaner, Marci E.; Wang, Juan; Zevin, Shoshana; Gerstin, Karin M.; Giacomini, Kathleen M.

doi:10.1023/a:1012148016794

Cited by 30 publications

(7 citation statements)

References 24 publications

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“…2-Chlorodeoxyadenosine (cladribine) and fluoroarabinosyladenosine (fludarabine) are putative substrates for the CNT2 gene product (N1 transport system) (15,48). Little is known about the pharmacological properties of this new N5-type nucleoside transport system.…”

Section: Tnf-␣ Modulation and Role Of Pkc In The Regulation Of Nucleomentioning

confidence: 99%

Regulation of Nucleoside Transport by Lipopolysaccharide, Phorbol Esters, and Tumor Necrosis Factor-α in Human B-lymphocytes

Soler¹,

Felipe²,

Mata³

et al. 1998

Journal of Biological Chemistry

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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 equilibrative es-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-␣ (TNF-␣) mRNA levels, which suggest that this cytokine may mediate some of the effects triggered by these agents, since addition of TNF-␣ alone can increase N1 and N5 transport activities by a mechanism that also depends on protein kinase C activation. Interestingly, TNF-␣ down-regulates es activity, 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-␣ 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.Nucleosides and some of their metabolites trigger a variety of regulatory effects in biological systems. Indeed, guanosine derivatives exert immunostimulatory responses (1) and may trigger mitogenic effects in mature B-lymphocytes and, to a lesser extent, in immature B cells (2). These actions are independent of cGMP, a second messenger in B cell activation (3). Moreover, nucleosides can mimic, both in vitro (4) and in vivo (5), a T cell-like signal for B cells that enables them to elicit antigenspecific responses to T cell-dependent antigens in the absence of T cells (6). These regulatory properties of nucleosides may be dependent on their uptake into the cell (1). Thus, the characterization of nucleoside transport systems and their regulation in these cell types may contribute to a better understanding of the role of nucleosides in lymphocyte physiology. Moreover, evidence that most antiviral and antiproliferative drugs used in lymphocyte malignancies can be substrates of these transport systems (7) provides additional stimulus in the attempt to identify the major routes for nucleoside uptake into lymphocytes and how these transport systems are regulated during B cell activation.Several nucleoside transport systems have been described in mammalian cells (8). Two of them, es and ei, are equil...

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Section: Tnf-␣ Modulation and Role Of Pkc In The Regulation Of Nucleomentioning

confidence: 99%

Regulation of Nucleoside Transport by Lipopolysaccharide, Phorbol Esters, and Tumor Necrosis Factor-α in Human B-lymphocytes

Soler¹,

Felipe²,

Mata³

et al. 1998

Journal of Biological Chemistry

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“…Similarly, two CNT isoforms have been cloned, CNT1 which is pyrimidine‐preferring and CNT2 which is purine‐preferring [1–4]. Heterologous expression of these membrane proteins in mammalian cells and Xenopus laevis oocytes has recently been used to determine the pharmacological profile of the CNT and ENT transporters, by measuring either the influx of radiolabeled substrates or the interaction with the carrier by inhibition of the uptake of the natural substrate [2–9]. The first approach is direct but requires a labeled drug and is time‐consuming, which may not be suitable for pharmacological screenings.…”

Section: Introductionmentioning

confidence: 99%

Electrogenic uptake of nucleosides and nucleoside‐derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes

et al. 2000

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The concentrative pyrimidine-preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two-electrode voltage-clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from 3 350 to 3 3150 mV) did not affect the K 0X5 for uridine, although it increased the transport current approximately 3-fold. Gemcitabine (a pyrimidine nucleoside-derived drug) but not fludarabine (a purine nucleosidederived drug) induced currents in oocytes expressing the hCNT1 transporter. The K 0X5 value for gemcitabine at 3 350 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1-mediated transport of nucleoside-derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside-derived drugs. ß

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“…The cloned N1 (659 amino acids) and N2 (648 amino acids) transporters share 64% identity and are both predicted to possess 14 putative transmembrane domains. These two cloned transporters are shown to be involved in the cellular uptake of nucleoside drugs including adenosine, cladribine, azidothymidine, and 2Ј,3Ј-dideoxycytidine (9,(15)(16)(17). More recently, the human N1 and N2 homologs (hSPNT1 and hCNT1) have been cloned (18,19).…”

mentioning

confidence: 99%

Molecular Determinants of Substrate Selectivity in Na+-dependent Nucleoside Transporters

Wang

Giacomini

1997

Journal of Biological Chemistry

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In mammalian cells, the salvage of purine and pyrimidine nucleosides is mediated by both facilitated and Na ؉ -dependent nucleoside transporters. These transporters also play important roles in the transmembrane flux of therapeutic nucleoside analogs, which are widely used in the treatment of cancer and viral infections. The N1, N2, and N3 Na ؉ -dependent nucleoside transporters differ in terms of their transport selectivity for purine and pyrimidine nucleosides. N1 is purine-selective, N2 is pyrimidine-selective, and N3 is broadly selective. To identify structural domains involved in substrate binding and molecular determinants responsible for distinct transport selectivity, chimeric transporters were made from the cloned rat N1 and N2 transporters. Of the 14 transmembrane domains (TM) of N1 and N2, transplanting TM8 -9 of N1 into N2 converted N2 from a pyrimidine-to a purine-selective transporter. Transplanting only TM8 generated a chimera with characteristics similar to the N3 transporter that has yet to be cloned. These data suggest that TM8 -9 confer substrate selectivity and may form at least part of a substrate-binding site in Na ؉ -dependent nucleoside transporters.Nucleosides and nucleoside analogs are increasingly being developed and used in the treatment of cancer, viral infections, and cardiac arrhythmias (1-3). Notable examples of therapeutic nucleoside analogs include cytosine arabinoside, cladribine, azidothymidine, and 2Ј,3Ј-dideoxyinosine used in the treatment of cancer and human immunodeficiency virus infection. The endogenous nucleoside, adenosine, exerts profound cardiac effects and is used in the treatment of cardiac arrhythmias (3). In mammalian cells, transmembrane flux of nucleosides and nucleoside analogs is mediated by both equilibrative and Na ϩ -dependent nucleoside transporters (4 -7). Consequently the distribution and functional characteristics of these transporters play important roles in determining the absorption, disposition, and elimination of nucleoside drugs (5, 6). Nucleoside transporters present in tumor cells and in the vicinity of purinergic receptors may also represent important gene targets for drug therapy (3, 7).The equilibrative nucleoside transporters mediate passive downhill transport of nucleosides and function bidirectionally in accordance with the concentration gradient of the substrate. Equilibrative nucleoside transporters exhibit a broad substrate selectivity for both purine and pyrimidine nucleosides and appear to be ubiquitous in mammalian cells. They have been further classified into two subtypes (es and ei) according to their sensitivity to inhibition by nitrobenzylthioinosine (5-7). The human es type transporter, hENT1, is recently cloned and has been implicated in the cellular uptake of some chemotherapeutic drugs (8). Na ϩ -dependent nucleoside transporters mediate active uphill transport of nucleosides into cells by coupling to the inwardly directed Na ϩ gradient across the plasma membrane. These transporters have been demonstrated in a variety of tis...

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Abstract: HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.

Cited by 30 publications

References 24 publications

Regulation of Nucleoside Transport by Lipopolysaccharide, Phorbol Esters, and Tumor Necrosis Factor-α in Human B-lymphocytes

Regulation of Nucleoside Transport by Lipopolysaccharide, Phorbol Esters, and Tumor Necrosis Factor-α in Human B-lymphocytes

Electrogenic uptake of nucleosides and nucleoside‐derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes

Molecular Determinants of Substrate Selectivity in Na+-dependent Nucleoside Transporters

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