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PubMed2005

The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia.

Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=-0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=-0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=-0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.

Acute Disease↗

PubMed2006

Expression of human equilibrative nucleoside transporter 1 (hENT1) and its correlation with gemcitabine uptake and cytotoxicity in mantle cell lymphoma.

BACKGROUND AND OBJECTIVES: Nucleoside transporters might play a relevant role in the intracellular targeting of many nucleoside analogs used in anticancer therapy. Two gene families (SLC28 and SLC29) encode the two types of human nucleoside transporters, concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) proteins. Chronic lymphocytic leukemia (CLL) cells express both SLC28- and SLC29-related mRNA, although transport function seems to be mostly related to ENT-type transporters. Here we have analyzed the role of nucleoside transporters in nucleoside-derived drug bioavailability and action in mantle cell lymphoma (MCL) cells. DESIGN AND METHODS: The relative amounts of hENT1 and hENT2-related mRNA and protein were analyzed in five MCL cell lines and 20 primary MCL tumors by real-time quantitative reverse transcriptase polymerase chain reaction and western blots. Cell viability, measured by annexin V-FITC staining, and nucleoside-derived drug transport were also studied. RESULTS: MCL cells express higher levels of hENT1 protein than do CLL cells, and a good correlation was found between protein and mRNA levels of hENT1, thus indirectly suggesting that hENT1 might be transcriptionally regulated in MCL cells. More importantly, a significant correlation between these two parameters, drug uptake and sensitivity to gemcitabine, was also observed. INTERPRETATION AND CONCLUSIONS: These results further support the concept that nucleoside transporters are implicated in the therapeutic response to nucleoside analogs, and suggest a particular and novel role for hENT1 in the genotoxic response to selected nucleoside analogs, such as gemcitabine, in MCL cells.

Cell Line, Tumor↗

PubMed2002

Immunohistochemical variation of human equilibrative nucleoside transporter 1 protein in primary breast cancers.

Gemcitabine and capecitabine are nucleoside analogues used in chemotherapy strategies for the treatment of breast cancer. We previously demonstrated that deficiency in hENT1, the most abundant and widely distributed plasma membrane nucleoside transporter in human cells, confers high-level resistance to gemcitabine toxicity in vitro, whereas the relationship between hENT1 activity and capecitabine toxicity is unknown. To determine the relationship between capecitabine cytotoxicity and hENT1 abundance, cultured MDA-MB-435s human mammary carcinoma cells were exposed to graded concentrations of the capecitabine metabolites, 5'-deoxy-5-fluorouridine or 5-fluorouracil, in the presence and absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a tight-binding inhibitor of hENT1. The presence of NBMPR reduced the cytotoxic effects of 5'-deoxy-5-fluorouridine, indicating that hENT1 also enabled cellular uptake of this capecitabine metabolite by breast cancer cells. We report here the development of an immunohistochemical method to assess the hENT1 abundance of malignant cells in solid tumors. Frozen sections of 33 primary breast cancers were stained with monoclonal antibodies raised against a synthetic peptide derived from the large intracellular loop of hENT1, and staining intensity was scored on a 0-4+ scale. hENT1 staining intensity varied markedly among breast samples (4 with score 0, 5 with score 1+, 7 with score 2+, 14 with score 3+, 3 with score 4+), suggesting that at least 9 of the tumors were hENT1 deficient. We conclude that because hENT1 deficiency has previously been associated with nucleoside drug resistance, immunohistochemical staining of hENT1 warrants further study as a predictive tool for guiding the appropriate use of gemcitabine and capecitabine in the treatment of breast cancer.

Affinity Labels↗

PubMed2006

Localization of equilibrative nucleoside transporters in the rat brain.

Adenosine is a recognized inhibitory neuromodulator and neuroprotective agent in the central nervous system. It is produced both intra- and extracellularly and transported across the cell membrane. Nucleoside transporters thus have a major impact on the extracellular adenosine levels, and consequently adenosine signalling. We have raised and characterized polyclonal antibodies against both the equilibrative nucleoside transporters 1 and 2, and report for the first time their distribution in rat brain at the cellular level. Double staining studies were performed to assess the localization of the transporters in neural and glial cells. Both transporters were present in practically all neurons. Some astrocytes showed equilibrative nucleoside transporter 1 staining, while equilibrative nucleoside transporter 2 staining on astrocytes was observed only sporadically.

Animals↗

PubMed2006

Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier.

The purpose of the present study was to characterize the adenosine transport system(s) at the inner blood-retinal barrier (inner BRB). A conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), used as an in vitro model of the inner BRB, expresses equilibrative nucleoside transporter 1 (ENT1), ENT2, concentrative nucleoside transporter 2 (CNT2), and CNT3 mRNAs. TR-iBRB2 cells exhibited an Na+-independent and concentration-dependent [3H]adenosine uptake with a Michaelis-Menten constant of 28.5 microM and a maximum uptake rate of 814 pmol/(min mg protein). [3H]Adenosine uptake by TR-iBRB2 cells was strongly inhibited by 2 mM adenosine, inosine, uridine, and thymidine. On the other hand, this process was not inhibited by 100 nM nitrobenzylmercaptopurine riboside and dipyridamole. These uptake studies suggest that ENT2 is involved in [3H]adenosine uptake by TR-iBRB2 cells. Quantitative real-time PCR revealed that the expression of ENT2 mRNA is 5.5-fold greater than that of ENT1 mRNA. An in vivo study suggested that [3H]adenosine is transported from the blood to the retina and significantly inhibited by adenosine and thymidine. The results of this study show that ENT2 most likely mediates adenosine transport at the inner BRB and is expected to play an important role in regulating the adenosine concentration in the retina.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Quantitative analysis of nucleoside transporter and metabolism gene expression in chronic lymphocytic leukemia (CLL): identification of fludarabine-sensitive and -insensitive populations.

Resistance to fludarabine is observed in the clinic, and molecular predictive assays for benefit from chemotherapy are required. Our objective was to determine if expression of nucleoside transport and metabolism genes was associated with response to fludarabine therapy in patients with chronic lymphocytic leukemia (CLL). CLL cells from 56 patients were collected prior to treatment with fludarabine. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on sample RNA to determine the relative levels of mRNA of 3 nucleoside transporters that mediate fludarabine uptake (human equilibrative nucleoside transporter 1 [hENT1], human equilibrative nucleoside transporter 2 [hENT2], and human concentrative nucleoside transporter 3 [hCNT3]), deoxycytidine kinase (dCK), and 3 5'-nucleotidases (ecto-5'nucleotidase [CD73], deoxynucleotidase-1 [dNT-1], and cytoplasmic high-Km 5-nucleotidase [CN-II]). Two-dimensional hierarchical cluster analysis of gene expression identified 2 distinct populations of CLL. Cluster 2 patients experienced a 3.4-fold higher risk of disease progression than cluster 1 patients (P = .0058, log-rank analysis). Furthermore, independent analysis of the individual genes of interest revealed statistically significant differences for risk of disease progression (adjusted hazard ratios [HRs]) with underexpression of dNT-1 (HR = 0.45; P = .042), CD73 (HR = 0.40; P = .022), and dCK (HR = 0.0.48; P = .035), and overexpression of hCNT3 (HR = 4.7; P = .0007) genes. Subjects with elevated hCNT3 expression experienced a lower complete response rate to fludarabine therapy (11% vs 69%; P = .002). No hCNT3-mediated plasma membrane nucleoside transport was detected in CLL samples expressing hCNT3 message, and hCNT3 protein was localized to the cytoplasm with immunohistochemical and confocal microscopy.

Adult↗

PubMed2006

Ribavirin uptake by cultured human choriocarcinoma (BeWo) cells and Xenopus laevis oocytes expressing recombinant plasma membrane human nucleoside transporters.

We investigated the mechanism of the transport of ribavirin (1-beta-D-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na(+)-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 microM) of ribavirin was used, Na(+)-independent uptake was observed, but there was only a slight Na(+)-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K(m) values were 18.0 microM, 14.2 microM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na(+)-dependent transporter hCNT3 and the low-affinity Na(+)-independent transporters hENT1 and hENT2.

Animals↗

PubMed2004

Xenobiotic and endobiotic transporter mRNA expression in the blood-testis barrier.

A major function of xenobiotic and endobiotic transporters is to move a wide range of organic substances across cell membranes. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. The aim of this study was to quantitatively determine the constitutive expression of various transporters in isolated Sertoli cells from adult Sprague-Dawley rats. The following mRNA levels were measured in isolated Sertoli cells by the branched DNA signal amplification method, multidrug resistance (Mdr) protein 1a, 1b, and 2; multiple drug resistance protein (Mrp) 1, 2, 3, 4, 5, 6, 7, and 8; sodium taurocholate cotransporting polypeptide; bile salt excretory protein; ileal bile acid transporter; AbcG5 and AbcG8; organic anion transporting polypeptide (Oatp) 1, 2, 3, 4, 5, 9, and 12; prostaglandin transporter (Pgt); testis-specific transporter (Tst) 1 and Tst2; organic anion transporter (Oat) 1, 2, 3, and K; organic cation transporter (Oct) 1, 2, 3, N1, and N2; divalent metal transporter (Dmt) 1, Menke's, and Wilson's; zinc transporter (Znt) 1; equilibrative nucleoside transporter (Ent) 1 and 2; concentrative nucleoside transporter (Cnt) 1 and 2; and peptide transporter (Pept) 1 and 2. Levels were also determined in whole testis, liver, kidney, and ileum to provide a reference for determining relative expression levels. Mrp8, Tst1 and 2, and Ent1 and 2 were expressed in Sertoli cells at higher levels than in liver, kidney, or ileum, whereas Mrp1, 5, and 7, Mdr2, Oatp3, Oat2, OctN2, Dmt1, Menke's, Wilson's, and Znt1 were all significantly expressed in Sertoli cells, but Sertoli cell expression was not the tissue of highest expression. The remaining transporters were expressed at low levels in isolated Sertoli cells. Additionally, expression levels of Mrp1, Mrp7, Mrp8, Tst1, Tst2, OctN2, Wilson's, Znt1, Ent1, and Ent2 were greater in isolated Sertoli cells than in whole testis. Constitutive expression of transporters in Sertoli cells may provide an insight into the range of xenobiotics that can potentially be transported by Sertoli cells and thereby provide a mechanistic under standing of blood-testis barrier function.

Animals↗

PubMed2001

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system.

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell-surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A(1) receptor abundance. The regional co-localization of hENT1 and A(1) receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A(1) receptors in human brain.

Adenosine↗

PubMed1998

Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells.

Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na+-dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na+-independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purine-preferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na+-dependent nucleoside uptake and occurs before the peak of 3H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene.

Animals↗

PubMed1997

Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta.

Mammalian equilibrative nucleoside transporters are typically divided into two classes, es and ei, based on their sensitivity or resistance respectively to inhibition by nitrobenzylthioinosine (NBMPR). Previously, we have reported the isolation of a cDNA clone encoding a prototypic es-type transporter, hENT1 (human equilibrative nucleoside transporter 1), from human placenta. We now report the molecular cloning and functional expression in Xenopus oocytes of a cDNA from the same tissue encoding a homologous ei-type transporter, which we designate hENT2. This 456-residue protein is 46% identical in amino acid sequence with hENT1 and corresponds to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in a form bearing a sequence deletion. In addition to placenta, hENT2 is found in brain, heart and ovarian tissue. Like hENT1, hENT2 mediates saturable transport of the pyrimidine nucleoside uridine (Km 0.2+/-0.03 mM) and also transports the purine nucleoside adenosine. However, in contrast with hENT1, which is potently inhibited by NBMPR (Ki 2 nM), hENT2 is NBMPR-insensitive (IC50<1 microM). It is also much less sensitive to inhibition by the coronary vasoactive drugs dipyridamole and dilazep and to the lidoflazine analogue draflazine, properties that closely resemble those reported for classical ei-type transport in studies with intact cells.

Adenosine↗

PubMed2002

Transport characteristics of HL-1 cells: a new model for the study of adenosine physiology in cardiomyocytes.

Adenosine is a physiologically important nucleoside in the cardiovascular system where it can act as a cardioprotectant and modulator of energy usage. Adenosine transporters (ATs) modulate cellular adenosine levels, which, in turn, can affect a number of processes such as receptor activation and glucose uptake, but their role in cardiac physiology is poorly understood. Therefore, we have developed a new cell model by determining various adenosine-related characteristics of HL-1, an immortalized atrial cardiomyocyte murine cell line. Adenosine uptake in HL-1 cells is sodium independent, saturable, and inhibitable by nucleoside transport inhibitors (nitrobenzylthioinosine (NBTI), dipyridamole, dilazep). Reverse transcription--polymerase chain reaction analysis confirmed that HL-1 cells possess mouse equilibrative nucleoside transporters 1 and 2 (mENT1, mENT2) and kinetic analyses indicate moderate-affinity (Km = 51.3 +/- 12.9 microM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (B(max) = 520 +/- 10 fmol/mg protein, Kd = 0.11 +/- 0.04 nM, 1.6 x 10(5) NBTI-binding sites/cell). HL-1 cells possess adenosine receptor, metabolic enzyme, protein kinase C isoform, and insulin-stimulated glucose transport profiles that match normal mouse heart. Therefore, HL-1 is an excellent model to study ATs within cardiomyocytes and the first model for evaluating in detail the role of the ATs in modulating effects of adenosine.

Adenosine↗

PubMed2006

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium.

L-Arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D-glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D-glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D-glucose. Insulin increased hENT2 protein abundance in normal or high D-glucose, but reduced hENT1 protein abundance in normal D-glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D-glucose. Insulin effect on hENT1 mRNA expression in normal D-glucose was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development.

Adenosine↗

PubMed2000

Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine.

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.

Biological Transport↗

PubMed2005

Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2.

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

2-Chloroadenosine↗

PubMed2005

Residue 33 of human equilibrative nucleoside transporter 2 is a functionally important component of both the dipyridamole and nucleoside binding sites.

Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) differ functionally in that hENT2 generally displays lower affinity for its nucleoside permeants and is less sensitive to inhibition by the coronary vasodilators dilazep and dipyridamole. In previous work, we demonstrated that mutation of residues 33 (Met versus Ile) of hENT1 and hENT2 altered sensitivity to dilazep and dipyridamole and that the hENT2 mutant (I33M) displayed a K(m) value for uridine that was lower than that of hENT2 and similar to that of hENT1 (J Biol Chem 277:395-401, 2002). In this study, we report results of an in-depth investigation of the role of residue 33 in hENT2. We found that hENT2-I33M displayed decreased K(m) values for both pyrimidine and purine nucleosides and increased V(max) values for purine nucleosides. Cys or Ser at position 33 had similar effects on the kinetic parameters of hENT2 as Met, indicating that hydrophobic (Met and Cys) or hydrogen-bonding energy (Ser) contributed to permeant binding by these residues. hENT2-I33M and I33C displayed increased sensitivities to dipyridamole compared with wild-type hENT2, hENT2-I33A, and hENT2-I33S, suggesting interaction of the sulfur atom of Met and Cys with aromatic moieties on dipyridamole. hENT2-I33C was inhibited by the membrane-impermeant sulfhydryl reactive reagent p-chloromercuribenzyl sulfonate, and uridine, adenosine, and dipyridamole protected against inhibition. Our results indicated that residue 33 resides in an extracellular domain as predicted by the current hENT2 topology model and suggested that it is a functionally important component of both the permeant and dipyridamole binding sites.

Adenosine↗

PubMed2005

Cytosine arabinoside affects multiple cellular factors and induces drug resistance in human lymphoid cells.

Continuous in vitro cultivation of human lymphoid H9 cells in the presence of 0.5microM arabinosyl-cytosine (araC) resulted in cell variant, H9-araC cells, that was >600-fold resistant to the drug and cross resistant to its analogs and other unrelated nucleosides, e.g. dideoxycytidine (5-fold), thiacytidine (2-fold), 2-fluoro-adenine arabinoside (8.3-fold), and 2-chloro-deoxyadenosine (2.1-fold). Compared to the parental cell line, the resistant cells accumulated <1% araCTP, and had reduced deoxycytidine kinase (dCK) activity (31.4%) and equilibrative nucleoside transporter 1 (ENT1) protein. The expression of the dCK gene in araC resistant cells was reduced to 60% of H9 cells, which correlated with lower dCK protein and activity. Whereas, there was no difference in the expression of ENT1 mRNA between the cell lines, ENT1 protein content was much lower in the resistant cells than in H9 cells. The concentrative nucleoside transporter (CNT3) was slightly increased in H9-araC cells, but CNT2, and MDR1 remained unaffected. Although a definitive correlation remains to be established, the amount of Sp1 protein, a transcription factor, that regulates the expressions of dCK, nucleoside transporters and other cellular proteins, was found reduced in H9-araC cells. Like ENT1, the Sp1 mRNA levels remained unaffected in H9-araC whereas protein contents were reduced. These observations are indicative of differences in the production and/or turnover of ENT1 and Sp1 proteins in H9-araC cells. Since nucleoside transporters and dCK play an important role in the activity of potential antiviral and anticancer deoxynucleoside analogs, understanding of their regulation is important. These studies show that the exposure of cells to araC, in vitro, is capable of simultaneously affecting more than one target site to confer resistance. The importance of this observation in the clinical use of araC remains to be determined.

Cell Line↗

PubMed2003

Nucleoside transporter profiles in human pancreatic cancer cells: role of hCNT1 in 2',2'-difluorodeoxycytidine- induced cytotoxicity.

PURPOSE: Concentrative nucleoside transporter (CNT) 1, CNT3, equilibrative nucleoside transporter (ENT) 1, and, to a lesser extent, ENT2, appear to be the transporters responsible for 2',2'-difluorodeoxycytidine (gemcitabine; Gemzar) uptake into cells. Gemcitabine is used currently in the treatment of pancreatic cancer, but the role of specific nucleoside carrier proteins in gemcitabine cytotoxicity has not been elucidated. Indeed, it is not known which nucleoside transporters are expressed in human pancreas. EXPERIMENTAL DESIGN: In this study we have used four cell lines [pancreatic neoplasia (NP)9, NP18, NP29, and NP31] derived from human pancreatic adenocarcinomas to monitor the pattern of nucleoside transporter expression, and we have heterologously expressed the high-affinity gemcitabine transporter human orthologue (h) CNT1 to monitor its role in drug responsiveness. RESULTS: All of the cell lines take up gemcitabine mostly via the hENT1 transporter, which is expressed at high levels. Reverse transcription-PCR analysis of the other four cloned plasma membrane transporter mRNAs revealed very different expression patterns among NP cell lines, with apparent selective loss or decrease of hCNT mRNAs. NP cells transiently express hCNT1-type Na(+)-dependent nucleoside transport activity at low/medium cell density but not in confluent cultures. Cells expressing hCNT1 in a more constitutive manner were cloned after stable transfection of hCNT1. Despite high constitutive hENT1 activity, this increased sensitivity to gemcitabine. CONCLUSION: In summary, human pancreatic adenocarcinoma cells overexpress hENT1, although they retain the ability to express a functional hCNT1 transporter, an isoform that confers sensitivity to gemcitabine.

Antimetabolites, Antineoplastic↗