From the Department of Molecular Pharmacology and
§ Center for Biotechnology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBankTM data base (human HNP36, 326 residues) that has been identified as a growth factor-induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5'-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter.
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INTRODUCTION |
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Nucleoside transporters play an important role in the salvage of exogenous physiological nucleosides such as thymidine and uridine and in the uptake of antitumor and antiviral nucleoside analogs. At least five distinct nucleoside transport activities have been identified that differ in their permeant selectivity, sensitivity to inhibitors, and distribution in normal tissues and tumors (reviewed in Refs. 1 and 2). Two of these are equilibrative mechanisms that mediate both the influx and efflux of nucleosides across the plasma membrane, whereas the other three are concentrative, sodium-dependent mechanisms that under physiological conditions mediate only the influx of nucleosides. The major equilibrative carrier in most cells, es (equilibrative, sensitive), is highly sensitive to the inhibitor nitrobenzylmercaptopurine riboside (NBMPR)1 with IC50 values of 0.1 to 1 nM. Many cells, however, have a second equilibrative transporter (ei, equilibrative, insensitive) that is insensitive to nanomolar concentrations of NBMPR but can be inhibited by higher (µM) concentrations. Both of the equilibrative transporters accept a broad range of physiological nucleosides and their cytotoxic and antiviral analogs as permeants, although there appear to be differences in the affinity of the two transporters for some nucleosides. In addition to the equilibrative transporters, there are at least three sodium nucleoside co-transporters that can mediate the concentrative uptake of nucleosides. These transporters are insensitive to NBMPR and differ from each other in their permeant specificity. Two of the concentrative transporters have been cloned from rat and human tissues (3-5) and found to belong to the nupC family of bacterial nucleoside transporters. Another potential nucleoside transporter belonging to the sodium/glucose co-transporter family has also been cloned (6). More recently the human NBMPR-sensitive equilibrative nucleoside transporter es has been cloned (7). The predicted protein has 10-11 potential membrane-spanning regions and appears to represent a new family of membrane transport proteins designated ENT for equilibrative nucleoside transporter.
We report here the cloning of a human cDNA that encodes the NBMPR-insensitive equilibrative nucleoside transport activity ei. This cDNA is highly homologous to ENT1, with 69% similarity of the deduced amino acid sequences, and has therefore been designated ENT2. A preliminary report of these studies has been presented (8).
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EXPERIMENTAL PROCEDURES |
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Cells and Growth Conditions-- HeLa S3 and COS-1 cells from the American Type Culture Collection were grown at 37 °C in a humidified air and 5% CO2 atmosphere in DMEM supplemented with 10% heat-inactivated fetal calf serum (HeLa S3) or with 5% heat-inactivated fetal calf serum plus 5% NuSerum IV (COS-1). A nucleoside transport-deficient subline (CEM/ARAC-8C) (9) of CEM human leukemia cells was provided by Dr. Buddy Ullman (Oregon Health Sciences University).This cell line and its derivatives were grown in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and the following additions: CEM/ARAC-8C: 0.25 µM tubercidin, 0.5 µM Ara-C; CEM/C19: 0.25 µM tubercidin, 0.5 µM Ara-C, 50 µg/ml G418; CEM/N1-7: 20 µM DUP-785, 100 µM uridine.
Isolation of the EBNA-1-positive, Nucleoside Transport-deficient
Cell Line CEM/C19--
To attain a high frequency of stable
transfection for the functional selection of cells expressing rare
nucleoside transporter cDNAs, the Epstein-Barr virus-based vector
pDR2 was utilized (10). This vector requires the use of a human cell
line expressing the EBNA-1 gene product in trans. Thus an
EBNA-1-positive, nucleoside transport-deficient human cell line was
constructed by co-transfection of the EBNA-1 expression cassette
pCMVEBNA with pRSVneo (20:1 molar ratio) into CEM/ARAC-8C cells. After
selection in G418 (200 µg/ml Geneticin; Life Technologies), surviving
cells were cloned in soft agarose (0.35%) as described previously
(11). Colonies were transferred to liquid culture and characterized for
transfection frequency with pDR2 (CLONTECH). A
clone designated CEM/C19 had a stable transfection frequency of
approximately 102, which was 4 orders of magnitude
greater than that of the parental EBNA-negative cell line (data not
shown).
Expression Cloning-- A CLONTECH HeLa S3 cDNA library in the pDR2 vector was transfected into CEM/C19 cells (2 × 108) by electroporation (190 V at a capacitance of 960 microfarads, Bio-Rad Gene Pulser). After 48 h of recovery, the electroporated cells were sequentially selected in bulk culture in medium containing 200 µg/ml hygromycin (14 days), 20 µM DUP-785, 100 µM uridine (27 days), and finally 20 µM DUP-785, 100 µM uridine, 1 µM NBMPR (14 days). Plasmids were extracted (QIA-prep spin plasmid miniprep kit; Qiagen) from the surviving cell population and rescued back into electrocompetent WM 1100 Escherichia coli (Bio-Rad). Plasmids from individual E. coli colonies were analyzed for inserts and individually reintroduced into CEM/C19 cells, which were then selected as before.
Sequencing of the pDR2/N1-71 Insert-- Both strands of the insert were sequenced to a level of 3-7-fold redundancy by Taq DyeDeoxy terminator cycle sequencing on an automated Model 373A DNA Sequencer (Applied Biosystems).
Transient Expression in COS-1 Cells--
The full N1-71 open
reading frame plus the 3'-untranslated region was excised from
pDR2/N1-71 in a BglI/XbaI fragment (bp 233-2610) and directionally cloned into the multiple cloning site of
pcDNA3 (Invitrogen) to give pcDNA3/ENT2. Likewise, a
BamHI/XbaI fragment (bp 393-2610) lacking the
first start codon was ligated into pcDNA3 to give
pcDNA3/ENT2N. These constructs were transfected into COS-1 cells
by the DEAE-dextran method, and uptake of uridine was determined as
described by Fang et al. (12).
Northern Analysis-- Poly(A)+ RNA was isolated from the indicated cell lines using the FastTrack 2.0 kit from Invitrogen. RNA (5 µg/lane) was separated on formaldehyde-reducing 1% agarose gels and transferred to charged nylon membranes (Hybond-N, Amersham Corp.). A BamHI/NheI fragment of pDR2/N1-71 (1.8 kbp, bp 393-2183) was gel-purified and labeled with [32P]dCTP using the PrimeIt kit from Strategene. Hybridization was for 16 h at 42 °C in 50% formamide containing 10% dextran sulfate. The blot was washed at high stringency (0.2 × saline/sodium phosphate/EDTA at 65 °C) and analyzed using a PhosphorImager and ImageQuant software. Two human multiple tissue blots (CLONTECH) with 2 µg of poly(A)+ RNA/lane were also probed under identical conditions.
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RESULTS |
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Cloning Strategy-- Since the NBMPR-insensitive equilibrative nucleoside transport protein had not been identified, and there were no antibodies or probes available, a cloning strategy based on the functional expression of ei transport activity in a nucleoside transport-deficient cell line was devised. The primary selecting agent was DUP-785, an inhibitor of de novo uridylate synthesis (13), in combination with uridine. The toxicity of DUP-785 can be circumvented by the salvage of exogenous uridine in transport-competent cells (13) but not in the transport-deficient CEM/C19 cell line (data not shown). Thus, it was expected that the combination of DUP-785 with uridine would provide a strong positive selection for transfected cells expressing a functional nucleoside transporter. The choice of a cDNA library from HeLa cells was based on previous observations that these cells have high levels of both ei and es transport activity (14) but do not have any detectable sodium-dependent nucleoside transport activity.2 After transfection, batch cultures of cells were subjected to sequential selection as follows: 1) hygromycin to select for transfected cells, 2) DUP-785 plus uridine to select for cells expressing any nucleoside transporter, and 3) DUP-785 plus uridine and NBMPR to select for cells expressing NBMPR-insensitive uridine transport activity. Surviving cell cultures were found to be positive for NBMPR-insensitive uridine uptake (data not shown). By rescue of plasmids from positive cultures back into E. coli followed by reintroduction into the transport deficient CEM/C19 cells, a plasmid (pDR2/N1-7) containing a 2.6-kbp insert was identified that permitted survival of CEM/C19 cells in DUP-785/uridine/NBMPR selection medium. A stable cell line expressing the 2.6-kbp N1-7 insert was established and designated CEM/N1-7. This cell line was used for further functional characterization of the plasmid.
Uridine Transport in CEM/N1-7 Cells-- To confirm the presence of transport activity in the transfected cells, uridine influx was compared in CEM/N1-7 and CEM/C19 cells (Fig. 1A). CEM/N1-7 cells displayed a large component of uridine influx that was at least 10-fold greater than that of the recipient CEM/C19 cells. The addition of a large excess of unlabeled uridine blocked uridine transport in the transfected cells, indicating the presence of a saturable, carrier-mediated process. In contrast, excess uridine had no effect on nucleoside uptake in CEM/C19 cells, suggesting that the slow rate of uptake in these cells was due to simple diffusion. Neither removal of sodium (Fig. 1A) or the addition of low concentrations of NBMPR (0.1 µM) (Fig. 1B) had a significant effect on transport in CEM/N1-7. These data strongly suggested that the cDNA insert encodes the NBMPR-insensitive equilibrative nucleoside transporter ei. Partial inhibition of transport at higher concentrations of NBMPR (Fig. 1B) was also consistent with the properties of the human ei transporter in HeLa cells where the IC50 values for inhibition of uridine transport via es and ei are 1 nM and 6 µM, respectively (14). Also consistent with the ei transporter of HeLa cells (14), transport in CEM/N1-7 cells was completely blocked by 10 µM dipyridamole (Fig. 1B). As expected from the permeant selectivity of ei (1), uridine influx in CEM/N1-7 cells was inhibited by both purine and pyrimidine nucleosides (Fig. 1C) but not by the corresponding nucleotides (data not shown). Interestingly, uridine transport in CEM/N1-7 cells was inhibited by the nucleobase hypoxanthine (Fig. 1C) but not by adenine or uracil. Although ei is generally considered a nucleoside transporter, previous studies have suggested that it may also transport hypoxanthine (1). This was recently confirmed by Jarvis and co-workers (15) using direct measurements of hypoxanthine transport in human vascular endothelial cells. Thus, the uridine transport activity in CEM/N1-7 cells was Na+-independent, inhibited by physiological nucleosides, hypoxanthine, and dipyridamole but relatively insensitive to inhibition by NBMPR. All these features are consistent with those of the ei transporter.
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Rescue and Analysis of the Plasmid-- Plasmids were extracted from CEM/N1-7 cells and rescued back into E. coli. Restriction analysis of the plasmids from individual E. coli colonies demonstrated the presence of a single plasmid containing a 2.6-kbp insert that was identical to the plasmid initially introduced into CEM/C19 cells to produce the CEM/N1-7 cell line. The insert from one of the recovered plasmid clones (designated N1-71) was sequenced. The N1-71 cDNA was 2610 bp and contained a 1368-bp open reading frame flanked by approximately 200 bp of 5'-untranslated region and 1 kbp of 3'-untranslated region including a poly(A) tail. The open reading frame was 61% identical to the recently cloned human NBMPR-sensitive equilibrative nucleoside transporter ENT1 (7), and the deduced proteins exhibited 69% similarity (Fig. 2). There was no significant homology to the sodium-dependent nucleoside transporters (3, 4, 6). Based on its similarity to ENT1, we have designated the N1-71 cDNA as ENT2 to reflect that it is a member of the equilibrative nucleoside transporter family of membrane proteins. Similar to ENT1, ENT2 has 11 potential membrane-spanning regions (Fig. 2). As seen with other membrane transporter families (16), the most highly conserved regions of the ENT proteins fall in the transmembrane domains. ENT2 has three potential N-glycosylation sites, two of which lie in a predicted (17) extracellular loop between transmembrane domains 1 and 2.
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Expression of ENT2 in Human Cell Lines and Tissues--
Northern
blots of poly(A)+ RNA were probed at high stringency with a
BamHI/NheI fragment of pDR2/N1-71. In the HeLa
cell line from which the cDNA library was derived, a single
transcript of approximately 2.6 kbp was observed (Fig.
4A). A slightly larger transcript (approximately 3.0 kbp) was found in the stable transfectant CEM/N1-7. The larger size in CEM/N1-7 cells could be accounted for by
the fact that the message is derived from transcriptional start and
termination sites of the pDR2 vector, which add 460 nucleotides to
the message. These data suggest that N1-71 is a full-length, or nearly
full-length, cDNA for the ei transporter. As shown in
Fig. 4A, no message was detected in the transport-deficient recipient cell line CEM/C19.
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DISCUSSION |
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Cloning of a human cDNA encoding the NBMPR-insensitive equilibrative nucleoside transporter ei was accomplished by functional complementation of the nucleoside transport defect in a mutant cell line derived from the human CEM T-cell leukemia (9). The conclusion that the ENT2 cDNA encodes the ei transport protein is supported by direct measurements of uridine transport in the stable transfectant CEM/N1-7 (Fig. 1) and in COS-1 cells transiently expressing ENT2 (Fig. 3) and by inhibition of transport by nucleosides, hypoxanthine, and dipyridamole but not by low concentrations of NBMPR (Fig. 1). The deduced amino acid sequence of the ENT2 protein is 69% similar to the human NBMPR-sensitive equilibrative transporter ENT1 (7) but lacks homology to the concentrative nucleoside transporters (3, 4, 6) or other known membrane transport proteins. Although these studies demonstrate that ENT1 and ENT2 belong to the same gene family, they also confirm our previous hypothesis that the es and ei nucleoside transport activities are mediated by separate and distinct proteins rather than isoforms of a single gene product produced by alternate splicing or post-translational modification (1, 2, 23, 24).
The CEM/N1-7 cell line established in these studies, which stably expresses ENT2 in a nucleoside transport-deficient background, will provide a valuable tool for further characterization of this transporter. This is the first cell line available that has ei as its sole nucleoside transport activity. Whereas several cell lines have been identified that have endogenous ei activity, they all have additional nucleoside transport activities that have complicated the characterization of the ei transporter. Thus, the functional characterization of ei has lagged behind that of es, where several good model systems are available. It is also anticipated that the CEM/N1-7 cell line and additional derivatives of CEM/C19 expressing single recombinant human nucleoside transporters will provide valuable tools to screen for transportability in the development of antitumor and antiviral nucleoside analogs.
Although our studies have demonstrated that the delayed early response gene HNP36 encodes a truncated form of the nucleoside transporter ENT2, the functional relationship between these two proteins is still unclear. The full-length human ENT2 cDNA conferred uridine transport activity to COS-1 cells, whereas a 5'-truncated construct encoding the 36-kDa protein did not (Fig. 3). The lack of activity of the truncated construct could reflect an integral role for the amino terminus of the protein in the transport process or in the proper insertion of the protein into the membrane. On the other hand, the 36-kDa protein might play another role in the cell that is not detected in the uridine uptake assays used here, perhaps the transport of nucleosides or related compounds between intracellular compartments. Further studies are required to determine if a 36-kDa protein is produced and whether it has a role in nucleoside transport. It is also unclear whether ENT2 is a delayed early response gene as reported for mouse HNP36 (18, 19). In preliminary studies using serum stimulation of the human fibroblast cell line IMR-90, we have been unable to detect regulation of ENT2, although regulation of cyclin D1 was clearly evident.2 We have also been unable to detect regulation of ENT2 in phytohemagglutinin/interleukin-2-stimulated peripheral blood lymphocytes. Furthermore, previous studies of nucleoside transport in a bone marrow-derived murine macrophage cell line have demonstrated colony stimulating factor 1 stimulation of es transport activity (ENT1) with no change in ei transport activity (ENT2) (25). Thus, a role for the ENT2 nucleoside transporter in the proliferative response is unclear and will require further study. During the revision of this manuscript, Young and co-workers (26) cloned a rat ENT2 cDNA. They concluded that ENT2 is the full-length form of HNP36 and that the previously described delayed early response gene (18, 19) is therefore the ei nucleoside transporter. As discussed above, this may not be the case, and further experimental evidence is needed to determine whether ei is a delayed early response gene and to clarify the functional relationship between ENT2 and HNP36.
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ACKNOWLEDGEMENTS |
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We thank John Morris, Jr., Doris Phelps, and Karen Rakestraw for excellent technical assistance and Dr. Buddy Ullman at Oregon Health Sciences University for providing the CEM/ARAC-8C cell line.
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FOOTNOTES |
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* This work was supported by NCI, National Institutes of Health Research Grant R01-CA55056 and Cancer Center (CORE) Support Grant P30-CA21765 and by the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF034102.
¶ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale Ave., Memphis, TN 38105. Tel.: 901-495-3461; Fax: 901-521-1668; E-mail: judi.belt{at}stjude.org.
1
The abbreviations used are: NBMPR,
nitrobenzylmercaptopurine riboside
(6-[(-4-nitrobenzyl)thio]-9--D-ribofuranosyl purine); tubercidin,
4-amino-7-
-D-ribofuranosylpyrolo[2,3-D]
pyrimidine; DUP-785,
6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt; dipyridamole,
2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-D]-pyrimidine; Ara-C, 1-
-D-arabinofuranosylcytosine; ENT, equilibrative
nucleoside transporter; EBNA, Epstein-Barr nuclear antigen; CMV,
cytomegalovirus; bp, base pair(s); kbp, kilobase pair(s).
2 J. A. Belt and C. R. Crawford, unpublished data.
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REFERENCES |
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