Nucleoside Uptake Research Articles

Conserved surface accessible nucleoside ABC transporter component SP0845 is essential for pneumococcal virulence and confers protection in vivo.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.

Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets.

Nucleoside and nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases such as Crohn. Two gene families are implicated in the uptake of nucleosides and nucleoside analogs into cells, SCL28 and SLC29. The former encodes hCNT1, hCNT2, and hCNT3 proteins. They translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNT1 and hCNT2 pyrimidine- and purine-preferring, respectively, and hCNT3 a broad selectivity transporter. SLC29 genes encode four members, being hENT1 and hENT2 the only two which are unequivocally implicated in the translocation of nucleosides and nucleobases (the latter mostly via hENT2) at the cell plasma membrane. Some nucleoside-derived drugs can also interact with and be translocated by members of the SLC22 gene family, particularly hOCT and hOAT proteins. Inter-individual differences in transporter function and perhaps, more importantly, altered expression associated with the disease itself might modulate the transporter profile of target cells, thereby determining drug bioavailability and action. Drug transporter pharmacology has been periodically reviewed. Thus, with this contribution we aim at providing a state-of-the-art overview of the clinical evidence generated so far supporting the concept that these membrane proteins can indeed be biomarkers suitable for diagnosis and/or prognosis. Last but not least, some of these transporter proteins can also be envisaged as drug targets, as long as they can show “transceptor” functions, in some cases related to their role as modulators of extracellular adenosine levels, thereby providing a functional link between P1 receptors and transporters.

Two nucleoside receptors from Streptomyces coelicolor: Expression of the genes and characterization of the recombinant proteins

Streptomyces coelicolor is a soil-dwelling bacterium that undergoes an intricate, saprophytic lifecycle. The bacterium takes up exogenous nucleosides for nucleic acid synthesis or use as carbon and energy sources. However, nucleosides must pass through the membrane with the help of transporters. In the present work, the SCO4884 and SCO4885 genes were cloned into pCOLADuet-1 and overexpressed in Escherichia coli BL21. Each protein was monomeric. Using isothermal titration calorimetry, we determined that SCO4884 and SCO4885 are likely nucleoside receptors with affinity for adenosine and pyrimidine nucleosides. On the basis of bioinformatics analysis and the transporter classification system, we speculate that SCO4884–SCO4888 is an ABC-like transporter responsible for the uptake of adenosine and pyrimidine nucleosides.

EF0176 and EF0177 from Enterococcus faecalis V583 are substrate-binding lipoproteins involved in ABC transporter mediated ribonucleoside uptake.

One of the ABC transporter systems in Enterococcus faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knockout mutants, we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI 1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad and overlapping, but not identical, substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.

Highlights of This Issue

Although SMAD4 has been suggested to inhibit the activity of the WNT/β-catenin signaling cascade in cancer, the mechanism of this inhibition remains largely unknown. Jia and colleagues have discovered a novel interaction between SMAD4 and Aurora A kinase (AURKA) in cancer cells that facilitates AURKA degradation and suppresses WNT/β-catenin signaling. Furthermore, manipulation of TGFβ did not alter the ability of SMAD4 to repress AURKA and β-catenin signaling. Elucidation of this unprecedented molecular mechanism demonstrates how the balance between suppression and activation of the WNT/β-catenin signaling pathway in dysplastic precancerous cells could be broken to promote carcinogenesis in SMAD4-deleted advanced cancerous cells.The ETV6–RUNX1 genomic fusion is a frequent initiating event in childhood leukemia; however, its role in leukemogenesis is only partly understood. Here, Palmi and colleagues show defective adhesion and migration properties in ETV6–RUNX1-positive B-cell progenitors (BCP). In particular, alterations in the expression of adhesion molecules impaired chemotactic response to the chemokine SDF1 (CXCL12) and a block in the GTPase CDC42 signaling pathway are described. The molecular details revealed in this study posit an altered interaction of ETV6–RUNX1-positive BCP with the microenvironment and represent initiating events in the evolution of the disease, from its preleukemic phase to clinical onset, with potential implications to develop strategies for effective leukemia eradication.Most breast cancers are estrogen receptor (ER) positive and many exhibit intrinsic or acquired resistance to commonly used endocrine therapies. Wright and colleagues demonstrate that the proto-oncogene AGR2 is required for the viability of tamoxifen-sensitive and -resistant ERα-positive breast cancer cells and highlight the therapeutic utility, in vitro and in vivo, of interfering with the production and/or activity of this secreted protein. Further, a new regulatory pathway is defined wherein ligand activated ERα facilitates AGR2 expression, which is required for the maintenance of ERα expression. Surprisingly, AGR2 expression was shown to occur in an ERα-independent manner in tamoxifen-resistant breast cancer cells, an activity that was attributed to dysregulated signaling by the transcription factor FOXA1.Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, tumor-promoting, and other signaling effects through activation of G-protein–coupled adenosine receptors. Along with “classical” receptor-mediated pathways, adenosine also exerts cytotoxic and other biologic effects through intrinsic mechanisms. Virtanen and colleagues establish that pretreatment of either human prostate or breast cancer cells with low concentrations of adenosine markedly inhibits cell invasion and migration capacity. Molecularly, these inhibitory effects involve cellular uptake of nucleosides, their intracellular interconversion into ADP/ATP, and eventually, the inhibition of phospho-AMPK1α and other signaling pathways. This study provides novel insight into the ability of adenosine to prevent tumor invasion via two different adenosine receptor-dependent and -independent mechanisms.

Abstract 4478: Biopharmaceutic studies of novel nucleoside transporter inhibitor prodrugs that can protect against nucleoside drug-induced mitochondrial toxicity

Abstract Nucleoside analog antimetabolites are used as chemotherapy agents to treat cancer by interfering with DNA biosynthesis and therefore cell division and the growth of tumors. However, nucleoside drugs, such as the nucleoside reverse transcriptase inhibitor antiviral agents and gemcitabine, a drug approved for use against various solid tumors, have been found to cause severe mitochondrial toxicities that can limit their use. Nucleoside analogs cause toxicity by inhibiting mitochondrial DNA (mtDNA) synthesis. Nucleoside drugs can be transported from cytoplasm into mitochondrial by mitochondrial membrane nucleoside transporters (NTs) where they form the active triphosphate analogs and incorporate into mtDNA and inhibit mtDNA polymerase, Pol γ. Currently, there is no proven general treatment or prevention strategy for nucleoside drug-induced mitochondrial toxicity. Thus, the development of innovative approaches to preempt or treat mitochondrial toxicity will be an important advance in the field of nucleoside drug therapy. In our research to overcome the mitochondrial toxicity, we developed a set of novel nucleoside transporter inhibitor (NTI) prodrugs that lack of NT inhibition but can release active drugs intracellularly to inhibit mitochondrial nucleoside uptake. In vitro biopharmaceutics studies of cell entry, in-cell hydrolysis profiles, and metabolic stability in human liver S9 faction and human plasma were carried out on a set of novel prodrugs that have shown protection against nucleoside drug-induced mitochondrial toxicity. Financial support by NIH grant RO1GM10405 is acknowledged. Citation Format: Shan Sun, Hilaire C. Playa, Hemantkumar S. Deokar, Wenwei Lin, John K. Buolamwini. Biopharmaceutic studies of novel nucleoside transporter inhibitor prodrugs that can protect against nucleoside drug-induced mitochondrial toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2014-4478

FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.

Potential for developing purinergic drugs for gastrointestinal diseases.

Treatments for inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), functional dyspepsia, or motility disorders are not adequate, and purinergic drugs offer exciting new possibilities. Gastrointestinal symptoms that could be targeted for therapy include visceral pain, inflammatory pain, dysmotility, constipation, and diarrhea. The focus of this review is on the potential for developing purinergic drugs for clinical trials to treat gastrointestinal symptoms. Purinergic receptors are divided into adenosine P1 (A(1), A(2A), A(2B), A(3)), ionotropic ATP-gated P2X ion channel (P2X(1-7)), or metabotropic P2Y(1,2,4,6,11-14) receptors. There is good experimental evidence for targeting A(2A), A(2B), A(3), P2X(7), and P2X(3) receptors or increasing endogenous adenosine levels to treat IBD, inflammatory pain, IBS/visceral pain, inflammatory diarrhea, and motility disorders. Purine genes are also potential biomarkers of disease. Advances in medicinal chemistry have an accelerated pace toward clinical trials: Methotrexate and sulfasalazine, used to treat IBD, act by stimulating CD73-dependent adenosine production. ATP protects against NSAID-induced enteropathy and has pain-relieving properties in humans. A P2X(7)R antagonist AZD9056 is in clinical trials for Crohn's disease. A(3) adenosine receptor drugs target inflammatory diseases (e.g., CF101, CF102). Dipyridamole, a nucleoside uptake inhibitor, is in trials for endotoxemia. Drugs for pain in clinical trials include P2X(3)/P2X(2/3) (AF-219) and P2X(7) (GSK1482160) antagonists and A(1) (GW493838) or A(2A) (BVT.115959) agonists. Iberogast is a phytopharmacon targeting purine mechanisms with efficacy in IBS and functional dyspepsia. Purinergic drugs have excellent safety/efficacy profile for prospective clinical trials in IBD, IBS, functional dyspepsia, and inflammatory diarrhea. Genetic polymorphisms and caffeine consumption may affect susceptibility to treatment. Further studies in animals can clarify mechanisms and test new generation drugs. Finally, there is still a huge gap in our knowledge of human pathophysiology of purinergic signaling.

Oxidative stress modulates nucleobase transport in microvascular endothelial cells

Purine nucleosides and nucleobases play key roles in the physiological response to vascular ischemia/reperfusion events. The intra- and extracellular concentrations of these compounds are controlled, in part, by equilibrative nucleoside transporter subtype 1 (ENT1; SLC29A1) and by equilibrative nucleobase transporter subtype 1 (ENBT1). These transporters are expressed at the membranes of numerous cell types including microvascular endothelial cells. We studied the impact of reactive oxygen species on the function of ENT1 and ENBT1 in primary (CMVEC) and immortalized (HMEC-1) human microvascular endothelial cells. Both cell types displayed similar transporter expression profiles, with the majority (>90%) of 2-chloro[3H]adenosine (nucleoside) uptake mediated by ENT1 and [3H]hypoxanthine (nucleobase) uptake mediated by ENBT1. An in vitro mineral oil-overlay model of ischemia/reperfusion had no effect on ENT1 function, but significantly reduced ENBT1 Vmax in both cell types. This decrease in transport function was mimicked by the intracellular superoxide generator menadione and could be reversed by the superoxide dismutase mimetic MnTMPyP. In contrast, neither the extracellular peroxide donor TBHP nor the extracellular peroxynitrite donor 3-morpholinosydnonimine (SIN-1) affected ENBT1-mediated [3H]hypoxanthine uptake. SIN-1 did, however, enhance ENT1-mediated 2-chloro[3H]adenosine uptake. Our data establish HMEC-1 as an appropriate model for study of purine transport in CMVEC. Additionally, these data suggest that the generation of intracellular superoxide in ischemia/reperfusion leads to the down-regulation of ENBT1 function. Modification of purine transport by oxidant stress may contribute to ischemia/reperfusion induced vascular damage and should be considered in the development of therapeutic strategies.

ProGem1: A phase I/II study of a first-in-class nucleotide, Acelarin, in patients with advanced solid tumors.

2531 Background: Acelarin (NUC-1031) is a first-in-class nucleotide designed to overcome key cancer cell resistance by having nucleoside transporter-independent cellular uptake, activity independen...

Steroid hormones are novel nucleoside transport inhibitors by competition with nucleosides for their transporters

Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

FLT3 Is Involved In Ara-C Transport By Human Equilibrative Nucleoside Transporter (hENT1) In Pediatric Acute Leukemia

IntroductionDespite the remarkable improvement in survival of children with acute leukemia in the last decades, some patients still have a poor outcome. FMS-like tyrosine Kinase 3 (FLT3) is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Infant leukemias (those diagnosed at age<1 year) are a special subgroup characterized by distinctive clinico-biological features, including frequent MLL (Mixed Lineage Leukemia) gene rearrangements, high FLT3 expression and high sensitivity to cytarabine (Ara-C), but a dismal prognosis. Ara-C is a deoxycytidine analog known to be transported by the human equilibrative nucleoside transporter 1 (hENT1) across the cell membrane. Once inside the cell Ara-C is activated by deoxycitidine kinase (DCK) to finally exert its cytotoxic action. High hENT1 expression levels have been involved in Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are scarcely known. AimTo study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric leukemia. Patients, Materials, and MethodsBone marrow samples of 56 pediatric patients diagnosed with acute leukemia in Hospital Sant Joan de Déu and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used for screening of Ara-C transporters and ME expression. In all cases the patients or their parents signed an informed consent. We performed a positive selection of cases with acute lymphoblastic leukemia (ALL) with hyperdiploidy (>50 chromosomes) and of MLL-rearranged cases presenting either as ALL or acute myeloblastic leukemia (AML), as these are the subgroups with highest expression of FLT3 reported in the literature, although other leukemia subtypes were also represented. The mRNA expression levels of FLT3, nucleoside transporters (NT) (hENT1, hENT2, hCNT1, hCNT3) and Ara-C ME, dCK and CNII, were quantified using real-time PCR and analyzed with the 2-ΔΔCt method, with non-neoplastic samples used as controls for the relative quantification. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters in these cell lines was addressed by repressing FLT3 function with its specific inhibitor PKC412 (sold only for research purposes). ResultsWe included 56 patients (68% males) diagnosed with acute leukemia. The median age was 5.3 years (range 0-16.2), with 3 cases of infant leukemia. Fifty cases (89%) were precursor B-ALL (24 hyperdiploid cases, 5 MLL rearranged, 3 BCR-ABL+, 4 E2A-PBX1+, 5 TEL-AML1+, 9 other subtypes), 5 cases were AML (4 MLL positive cases and one case with mutated FLT3) and one case was a T-ALL harboring FLT3 mutation. As expected, the FLT3 expression was higher in cases with ALL and MLL rearrangement and, to a less extent, in ALL with hyperdiploidy. Interestingly, a significant positive correlation was found between FLT3 and hENT1 expression (mRNA levels) with all patient samples (figure 1). hENT1 expression and cytarabine-mediated uptake was significantly repressed when MV4-11 and SEM cell lines were exposed to the FLT3 inhibitor PKC412 (figures 2 & 3) without affecting hENT2, CNT1 and CNT3 expression and activity. [Display omitted] [Display omitted] [Display omitted] ConclusionsOur results show a strong correlation between FLT3 and the Ara-C transporter hENT1 in pediatric leukemia patients. This observation was consistent with the in vitro evidence that FLT3 inhibition resulted in hENT1 repression and down-regulation of Ara-C uptake in leukemic derived cell lines. Taken together, our data suggest that FLT3 regulates hENT1, thereby modulating the ability of cancer cells to incorporate Ara-C and promote its cytotoxic action. As FLT3 inhibitors are currently being tested as mono or combined therapy with Ara-C in several clinical trials, based upon our observations we suggest that a better schedule design might eventually be needed when dealing with treatments involving FLT3 inhibitors and Ara-C, thereby improving the outcome of this subset of patients. Disclosures:Off Label Use: The presentation include the results of in vitro studies with the FLT3 inhibitor PKC412. This drug was only used for in vitro studies, exclusively for research purposes.

Abstract C46: Implication of FLT3 in human equilibrative nucleoside transporter 1 (hENT1)-mediated uptake of Ara-C in pediatric acute leukemia.

Abstract Introduction: Infant acute leukemias (those diagnosed at age &amp;lt;1 year) are characterized by high FMS-like tyrosine Kinase 3 (FLT3) expression and high sensitivity to the nucleoside analogue Ara-C. FLT3 is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Ara-C is known to be transported by hENT1 undergoing metabolic activation within target cells. In fact, high hENT1 expression levels have been reported to determine Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are poorly known. Aim: To study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric acute leukemia. Experimental Procedures: Bone marrow samples from 56 pediatric patients diagnosed with acute leukemia in Hospital San Joan de Deu of Barcelona and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used in this study. In all cases an informed consent was signed. The mRNA amounts of FLT3, the putative Ara-C nucleoside transporters (NT), hENT1, hENT2, hCNT1, hCNT3, and the main Ara-C metabolic enzymes, DCK and CNII were quantified by real-time PCR. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters was addressed by exposure of cells to the FLT3 inhibitor PKC412. Results: A significant positive correlation was found between FLT3 and hENT1 mRNA levels when analysing the whole cohort of patients. DCK and CNII also showed a positive correlation. As expected, FLT3 expression was higher in cases with acute lymphoblastic leukemia (ALL) and mixed lineage leukemia (MLL) rearranged genes and, to a lesser extent, in ALL with hyperdiploidy (&amp;gt;50 chromosomes), as these are the subgroups with highest expression of FLT3 reported in the literature. Ara-C uptake into cells was mediated mainly by hENT1, hENT2, and hCNT1. The hENT1-mediated uptake of Ara-C was transiently abolished by the FLT3 inhibitor PKC412 reaching its minimum activity after 16 hours with the inhibitor. This down-regulation of hENT1 activity seemed to be dependent on the ability of PKC412 to inhibit hENT1 gene (SLC29A1) expression. Conclusions: The positive correlation between the FLT3 and hENT1 expression levels in leukemic cells from patients, along with the in vitro evidence that FLT3 inhibition represses hENT1 expression and down-regulates Ara-C uptake in leukemic derived cell lines, supports the idea that FLT3 modulates hENT1. This evidence should be taken into account at the therapy schedule level when therapeutic combinations including both AraC and FLT3 inhibitors are designed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C46. Citation Format: Paula Fernández-Calotti, Albert Català, MarÇal Pastor-Anglada, Roberta Malatesta, Susana Rives, Mireia Camos. Implication of FLT3 in human equilibrative nucleoside transporter 1 (hENT1)-mediated uptake of Ara-C in pediatric acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C46.

Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

Basolateral Uptake of Nucleosides by Sertoli Cells Is Mediated Primarily by Equilibrative Nucleoside Transporter 1

The blood-testis barrier (BTB) prevents the entry of many xenobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S-[(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport.

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

Abstract 3534: Detection of intracellular ara-C/ara-CTP following treatment with the new anti-leukemic drug elacytarabine using a bioluminescent bacterial biosensor.

Abstract Cytarabine (ara-C) is an analogue of deoxycytidine with an established role in treating AML. Cytarabine depends on the human equilibrative nucleoside transporter-1 (hENT-1) for cellular entry (1). Loss of hENT-1 function in vitro has been shown to result in ara-C resistance (2), and reduced hENT-1 expression and activity has been associated with adverse therapeutic outcomes in patients treated with cytarabine (3). The lipophilic ara-C-5’ elaidic acid ester of cytarabine (elacytarabine) has shown nucleoside transport independent uptake and long retention of the active nucleotide ara-CTP in a leukemic cell line (4) and may have the ability to overcome resistance due to insufficient transport in the clinical setting. (1). The whole cell bioluminescent biosensor, Escherichia coli HA1 was engineered to produce a measurable bioluminescent output in response to the nucleoside analogue ara-C (5). This was achieved through reduced expression of cytidine deaminase and the insertion of the human deoxycytidine kinase gene and the full lux operon from the Photorhabdus luminescens. The bacterial biosensor is able to monitor both ara-C and ara-CTP. The aim of this study was to evaluate the feasibility of using the Escherichia coli HA1 biosensor to measure intracellular ara-C and ara-CTP following ex vivo exposure of human cells to elacytarabine and cytarabine. CCRF-CEM cells or blast cells from patients were treated with ara-C or elacytarabine for up to 4 hours in the presence or absence of the hENT inhibitor dipyridamole (DPM). Cells were washed, lysed and the lysate incubated with E. coli HA1 and monitored on a plate reader luminometer. A cell viability assay was also performed to confirm biosensor results. E. coli HA1 produced a measurable response to lysate from CCRF-CEM cells treated with either ara-C or elacytarabine. In the case of ara-C, this response was reduced when cells were incubated with DPM. No such reduction was observed for elacytarabine. When used to treat blast cells derived from leukemic patients, elacytarabine treatment produced a stronger signal from E. coli HA1 than an equivalent concentration of ara-C. Furthermore, the efficacy of elacytarabine was not susceptible to hENT inhibition in patient samples, whereas the efficacy of ara-C was. The findings of this project suggest that the E. coli HA1 biosensor is compatible with elacytarabine and that elacytarabine is able to bypass the effect of hENT inhibition on CCRF-CEM cells and patient derived blast cells. 1. Burke &amp; Giles. Expert Opin Inv Drug. 2011; 20(12):1707-15. 2. Cai et al. Cancer Res. 2008; 68(7):2349-57. 3. Galmarini et al. Leukemia Res. 2002;26(7):621-9. 4. Adema et al. Invest New Drug. 2012; 30(5):1908-16. 5. Alloush et al. Clin Chem. 2010; 56(12):1862-70. Citation Format: Gareth M. Robinson, Elizabeth Anderson, Vyvyan C. Salisbury, Priyanka Mehta, Marit L. Sandvold, Darren M. Reynolds. Detection of intracellular ara-C/ara-CTP following treatment with the new anti-leukemic drug elacytarabine using a bioluminescent bacterial biosensor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3534. doi:10.1158/1538-7445.AM2013-3534

Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus

The degradation of purine nucleoside is the first step of purine nucleoside uptake. This degradation is catalyzed by purine nucleoside phosphorylase, which is categorized into two classes: hexameric purine nucleoside phosphorylase (6PNP) and trimeric purine nucleoside phosphorylase (3PNP). Generally, 6PNP and 3PNP degrade adenosine and guanosine, respectively. However, the substrate specificity of 6PNP and 3PNP of Thermus thermophilus (tt6PNP and tt3PNP, respectively) is the reverse of that anticipated based on comparison to other phosphorylases. Specifically, in this paper we reveal by gene disruption that tt6PNP and tt3PNP are discrete enzymes responsible for the degradation of guanosine and adenosine, respectively, in T. thermophilus HB8 cells. Sequence comparison combined with structural information suggested that Asn204 in tt6PNP and Ala196/Asp238 in tt3PNP are key residues for defining their substrate specificity. Replacement of Asn204 in tt6PNP with Asp changed the substrate specificity of tt6PNP to that of a general 6PNP. Similarly, substitution of Ala196 by Glu and Asp238 by Asn changed the substrate specificity of tt3PNP to that of a general 3PNP. Our results indicate that the residues at these positions determine substrate specificity of PNPs in general. Sequence analysis further suggested most 6PNP and 3PNP enzymes in thermophilic species belonging to the Deinococcus-Thermus phylum share the same critical residues as tt6PNP and tt3PNP, respectively.

Sodium Coupling and Nucleoside Specificity of a Concentrative Nucleoside Transporter

Concentrative nucleoside transporters (CNTs) are ion-coupled membrane transporters that utilize ion gradients to transport nucleosides and nucleoside-derived anticancer and antiviral drugs into cells. Humans have three subtypes of CNTs with varying nucleoside and nucleoside-drug specificity. The crystal structure of a CNT from Vibrio cholerae (vcCNT) in complex with uridine revealed the overall architecture, the nucleoside-binding site, and the putative sodium-binding site of this class of transporter 1.In our follow-up studies, we have addressed two issues: the role of sodium in nucleoside transport and the substrate specificity of vcCNT. With regard to the role of sodium in nucleoside transport, the hypothesis that we gained from the structure is that sodium binding stabilizes the nucleoside-binding site thereby increasing its affinity for nucleosides. To test this structural hypothesis, we performed mutagenesis, isothermal titration calorimetry, and radioactive nucleoside uptake experiments. Our results are consistent with our structural hypothesis. To dissect the substrate specificity of vcCNT, we first observed that the nucleoside-binding site of vcCNT is very similar to hCNT3 (>90% sequence identity). As a result, our functional studies showed that vcCNT displays similar substrate specificity as hCNT3. To understand the structural basis of nucleoside and nucleoside-drug specificity, we have performed crystallographic and functional studies of vcCNT in complex with several different nucleosides and nucleoside-derived drugs. Our results provide a structural basis for the substrate specificity of vcCNT and hCNT3.Reference1. Zachary Johnson, Cheom-Gil Cheong, and Seok-Yong Lee, Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 A. Nature. 2012, Mar 11;483(7390):489-93.

Development and identification of a SCAR marker linked to female gametophyte of Gracilariopsis lemaneiformis (Rhodophyta) based on AFLP technique

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.