Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line

1998 ◽  
Vol 76 (5) ◽  
pp. 843-851 ◽  
Author(s):  
Charles R Crawford ◽  
Carol E Cass ◽  
James D Young ◽  
Judith A Belt
Keyword(s):  
Cell Line ◽  
Nucleoside Transporters ◽  
Stable Expression ◽  
Nucleoside Transport ◽  
Leukemia Cell Line ◽  
Nucleoside Transporter ◽  
Mouse Leukemia ◽  
Concentrative Nucleoside Transporter ◽  
Sodium Dependent ◽  
Cloned Gene

Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17 757 - 17 760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2prime-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2prime-deoxyadenosine, 2-chloro-2prime-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.Key words: nucleoside transporter, CNT1, rat, sodium-dependent, recombinant, cloned gene, transfection, stable transfectant.

Allosteric and transport modulation of human concentrative nucleoside transporter 3 at the atomic scale

2021 ◽  
Author(s):  
Huaichuan Duan ◽  
Zhou Yanxia ◽  
XiaoDong Shi ◽  
Qing Luo ◽  
Gao Jiaxing ◽  
...  
Keyword(s):  
Nucleoside Transporters ◽  
Atomic Scale ◽  
Nucleoside Transport ◽  
Transmembrane Transport ◽  
Nucleoside Transporter ◽  
Nucleotide Synthesis ◽  
Physiological Processes ◽  
Concentrative Nucleoside Transporter ◽  
Transport Modulation ◽  
In Cells

Nucleosides are important precursors of nucleotide synthesis in cells, and nucleoside transporters play an important role in many physiological processes by mediating its transmembrane transport and absorption. During nucleoside transport,...

H-2 antigen variants in a cultured mouse leukemia cell line

1977 ◽  
Vol 5 (1) ◽  
pp. 295-308 ◽  
Author(s):  
C. Flores ◽  
T. V. Rajan
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Mouse Leukemia

scholarly journals Ophiobolin O and 6-Epi-Ophiobolin O, Two New Cytotoxic Sesterterpenes from the Marine Derived Fungus Aspergillus Sp

2012 ◽  
Vol 7 (11) ◽  
pp. 1934578X1200701 ◽  
Author(s):  
Dahai Zhang ◽  
Seketsu Fukuzawa ◽  
Masayuki Satake ◽  
Xianguo Li ◽  
Takafumi Kuranaga ◽  
...  
Keyword(s):  
Cell Line ◽  
Catalytic Reaction ◽  
Nmr Spectra ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Mouse Leukemia ◽  
Ir And Nmr Spectra ◽  
Aspergillus Sp

Two new sesterterpenes, ophiobolin O (1) and 6-epi-ophiobolin O (2), together with the known ophiobolins G (3), H (4), and K (5), and 6-epi-ophiobolin K (6) were isolated from the marine derived fungus Aspergillus sp. The structures of these compounds were elucidated based on chemical and physicochemical evidence, including MS, UV, IR and NMR spectra. The stereochemistry of 1 was further confirmed by catalytic reaction of 5 with p-TsOH as a catalyst. Compounds 1 to 6 showed cytotoxicity against mouse leukemia cell line P388, with IC50 values of 4.7, 9.3, 24.6, 105.7, 13.3 and 24.9 μM, respectively.

scholarly journals Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

1996 ◽  
Vol 317 (2) ◽  
pp. 457-465 ◽  
Author(s):  
Xiao FANG ◽  
Fiona E. PARKINSON ◽  
Delores A. MOWLES ◽  
James D. YOUNG ◽  
Carol E. CASS
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Functional Characterization ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Single Type ◽  
Nucleoside Transporter ◽  
Pyrimidine Nucleosides ◽  
Transporter Protein ◽  
Sodium Dependent

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Selective low level of protein kinase C isozyme in a tumor promoter-dependent mouse leukemia cell line

1989 ◽  
Vol 164 (3) ◽  
pp. 1397-1401 ◽  
Author(s):  
Toshio Tanaka ◽  
Masatoshi Hagiwara ◽  
Hiroyoshi Hidaka ◽  
Kazuo Nunoki ◽  
Hisataka Ohta ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Leukemia Cell ◽  
Tumor Promoter ◽  
Leukemia Cell Line ◽  
Mouse Leukemia ◽  
Low Level ◽  
Kinase C

scholarly journals Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2038-2042 ◽  
Author(s):  
CL Smith ◽  
LM Pilarski ◽  
ML Egerton ◽  
JS Wiley
Keyword(s):  
Cytosine Arabinoside ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
S Phase ◽  
Nucleoside Transporters ◽  
Labeling Index ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

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

scholarly journals Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line

2001 ◽  
Vol 280 (5) ◽  
pp. F879-F885 ◽  
Author(s):  
Lara M. Mangravite ◽  
Joshua H. Lipschutz ◽  
Keith E. Mostov ◽  
Kathleen M. Giacomini
Keyword(s):  
Mdck Cells ◽  
Green Fluorescence Protein ◽  
Nucleoside Transporters ◽  
Epithelial Cell Line ◽  
Nucleoside Transporter ◽  
Functional Studies ◽  
Kinetic Activity ◽  
Concentrative Nucleoside Transporter ◽  
Fluorescence Protein ◽  
Darby Canine Kidney

Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK1 cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway.

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

2005 ◽  
Vol 386 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Meaghan STOLK ◽  
Elizabeth COOPER ◽  
Greg VILK ◽  
David W. LITCHFIELD ◽  
James R. HAMMOND
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Interacting Protein ◽  
Human Osteosarcoma Cells ◽  
Specific Regulation ◽  
Membrane Lifetime ◽  
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 α′ and α subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2α′ (decreased CK2α′ activity) increased the number of binding sites (∼1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (∼1.8-fold) the Vmax for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the Vmax of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2α′ activity had no effect, however, on either the KD of [3H]NBMPR binding or the Km 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 CK2α′ subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2α′ 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 CK2α′-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.

scholarly journals Interferon-γ regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways

2003 ◽  
Vol 375 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Concepció SOLER ◽  
Antonio FELIPE ◽  
José GARCÍA-MANTEIGA ◽  
Maria SERRA ◽  
Elena GUILLÉN-GÓMEZ ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Interferon Γ ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Nucleoside Transporter ◽  
Protein Levels ◽  
Ifn Γ

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-γ (interferon-γ). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-γ was studied using macrophages from STAT1 knockout mice. IFN-γ triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-γ is required for DNA synthesis. Interestingly, IFN-γ led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-γ up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-γ is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-γ in macrophages.

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