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Vol. 60, Issue 5, 1143-1152, November 2001
Canadian Institute of Health Research Membrane Protein Research Group (T.T.L., M.S., J.D.Y., C.E.C.), Departments of Oncology (T.T.L., M.S., C.E.C.) and Physiology (J.D.Y.), University of Alberta; and the Cross Cancer Institute (T.T.L., M.S., C.E.C.), Edmonton, Alberta, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results and Discussion
References
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CEM-ARAC leukemia cells with resistance to cytarabine were shown to lack equilibrative transporter (hENT1) expression and activity. Stable transfer of hCNT2 cDNA into CEM-ARAC enabled Na+-dependent transport of purine and pyrimidine nucleoside analogs and provided a unique in vitro model for studying hCNT2. Analysis of [3H]uridine inhibitory activity by test substances in hCNT2 transfectant ARAC/D2 revealed structural requirements for interaction with hCNT2: 1) ribosyl and 2'-deoxyribosyl nucleosides were better inhibitors than 3'-deoxyribosyl, 2',3'-dideoxyribosyl or arabinosyl nucleosides; 2) uridine analogs with halogens at position 5 were better inhibitors than 5-methyluridine or thymidine; 3) 2-chloroadenosine was a better inhibitor than 2-chloro-2'-deoxyadenosine (cladribine); and 4) cytosine-containing nucleosides, 7-deazaadenosine and nucleobases were not inhibitors. Quantification of inhibitory capacity yielded Ki values of 34-50 µM (5-halogenated uridine analogs, 2'-deoxyuridine), 82 µM (5-fluoro-2'-deoxyuridine), 197-246 µM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 µM (5-fluoro-5'-deoxyuridine, capecitabine metabolite). Comparisons of hCNT2-mediated transport rates indicated halogenated uridine analogs were transported more rapidly than halogenated adenosine analogs, even though hCNT2 exhibited preference for physiologic purine nucleosides over uridine. Kinetics of hCNT2-mediated transport of 5-fluorouridine and uridine were similar (Km values, 43-46 µM). The impact of hCNT2-mediated transport on chemosensitivity was assessed by comparing antiproliferative activity of nucleoside analogs against hCNT2-containing cells with transport-defective, drug-resistant cells. Chemosensitivity was restored partially for cladribine, completely for 5-fluorouridine and 5-fluoro-2'-deoxyuridine, whereas there was little effect on chemosensitivity for fludarabine, 7-deazaadenosine, or cytarabine. These studies, which demonstrated hCNT2 uptake of halogenated uridine analogs, suggested that hCNT2 is an important determinant of cytotoxicity of this class of compounds in vivo.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Fluoropyrimidine
nucleosides have been used in the
treatment of disseminated human cancers, especially of the
gastrointestinal tract, breast, and ovary (Focan et al., 1999
; Kim et
al., 2001). The differences in metabolism, mechanism of action, and
favorable pharmacokinetics of 5-fluorouridine compared with other
fluoropyrimidine nucleosides have contibuted to its efficacy in the
treatment of superficial bladder cancers, which represent about 80% of
bladder cancers (Song et al., 1997). 5-Fluorouridine and
5-fluoro-2'-deoxyuridine are also useful radiopharmaceuticals in tumor
imaging using positron emission tomography and in spectroscopic
analysis with 19F NMR to follow their
intracellular metabolism in vitro and in vivo (Chen et al., 1999).
Since serum levels of diagnostic radiopharmaceuticals are
typically very low, active transport will influence their cellular and
tissue distribution.
Human cells possess multiple nucleoside transporters that are
either equilibrative or concentrative (Cass et al., 1998; Baldwin et
al., 1999), of which five have been identified by molecular cloning.1 The equilibrative nucleoside transporters (ENTs),
which include the nitrobenzylthioinosine (NBMPR)-sensitive and
-insensitive transporters (hENT1, hENT2), exhibit broad permeant
selectivities and appear to be widely distributed among cells and
tissues (Griffiths et al., 1997a; Crawford et al.,
1998b). The concentrative nucleoside transporters (CNTs) have
been identified in specialized mammalian cells (e.g., intestinal and
renal epithelia) and in several neoplastic cell types (Gutierrez et
al., 1992; Belt et al., 1993; Roovers and Meckling-Gill 1996;
Chandrasena et al., 1997; Flanagan and Meckling-Gill 1997; Patil and
Unadkat, 1997). The cognate proteins (hCNT1, hCNT2, hCNT3) responsible
for three (cit, cif, cib, respectively) of the
human concentrative processes (Ritzel et al., 1997, 1998, 2000; Wang et
al., 1997) have been recently identified by molecular cloning.
The multiplicity of nucleoside transporters and their overlapping
substrate selectivities in human cells (Crawford et al., 1990b;
Roovers and Meckling-Gill 1996; Boleti et al., 1997) have made the
functional analysis of CNTs difficult. There are only a few examples of
human cell types (e.g., erythrocytes and cultured CCRF-CEM leukemia
cells) that naturally exhibit a single nucleoside-transport process
(the prototypic equilibrative NBMPR-sensitive (es) process); these cell types have been used to study the es transport
process in the absence of other processes, and much is known about its functionality (Cass 1995; Griffith and Jarvis 1996). A clonal derivative of the CEM cell line, CEM-ARAC, which was selected for
resistance to cytarabine, exhibits deficiencies in transporting cytidine analogs and binding NBMPR and is cross-resistant to
gemcitabine (Ullman et al., 1988; Mackey et al., 1998).
The emergence of drug-resistant cells following drug exposure mitigates their therapeutic potential and poses a major problem in chemotherapy. In the present study, CEM-ARAC cells were shown to be cross-resistant to purine and pyrimidine nucleoside drugs due to the lack of detectable hENT1 mRNA and the associated deficiency in nucleoside transport capability. CEM-ARAC cells were transfected with hCNT2 cDNA and a stable transfectant (ARAC/D2) was identified by screening cultures for acquisition of Na+-dependent uptake of [3H]uridine, thereby providing the first example of a cultured cell line with hCNT2 activity. The structural determinants for interaction with hCNT2 were assessed by comparing the ability of test compounds to inhibit transport of [3H]uridine. The transportability and pharmacological properties of adenosine and uridine analogs were compared in cells containing hCNT2, hENT1, or no nucleoside transporters. CEM cells producing the broadly selective hENT1 were sensitive to both purine and pyrimidine nucleoside drugs, whereas ARAC/D2 cells producing the purine-nucleoside-selective hCNT2 were more sensitive to 5-fluorouridine and 5-fluoro-2'-deoxyuridine than either cladribine, fludarabine, or tubercidin. Chemosensitivity was correlated with the relative affinity of the nucleoside drugs for hCNT2. These results demonstrated that nucleoside drug resistance could be overcome by introduction of hCNT2 into nucleoside drug-resistant cells, and suggested the importance of hCNT2 in the transport of several fluoropyrimidine nucleoside drugs, but not that of 5-fluoro-5'-deoxyuridine.
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Experimental Procedures |
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Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Plasmid Construction.
The open reading frame of the cDNA
encoding hCNT2 (Ritzel et al., 1998) was excised from the original
cloning vector, pBluescript II KS(+) (Stratagene, La Jolla, CA) using
EcoRI and XbaI restriction enzymes and was
subcloned into the polylinker region downstream of the
enhancer/promoter sequences of the immediate early gene of human
cytomegalovirus of the mammalian expression vector pcDNA3 (Invitrogen,
Carlsbad, CA). The pcDNA3/hCNT2 construct was transformed into
electrocompetent JM109 Escherichia coli (Invitrogen) using a
CELL-PORATOR (Invitrogen). Plasmid DNA for use as template in DNA
sequencing and transfections was prepared using the Qiagen plasmid
purification kit (Qiagen Corp., Mississauga, ON) according to the
manufacturer's instructions. The structure of pcDNA3/hCNT2 was
verified by restriction endonuclease mapping and DNA sequencing. DNA
sequences were determined by Taq DyeDeoxy terminator cycle sequencing with an automated model 310 DNA sequencer (Applied Biosystems Inc., Foster City, CA). Sequence analysis used MacVector DNA
analysis software (Oxford Molecular Ltd., Bethesda, MD).
RT-PCR Analysis.
Poly(A)+ RNA was
isolated from actively proliferating cells using the FastTrack 2.0 isolation kit (Invitrogen) according to the manufacturer's
instructions. mRNA concentrations were determined spectrophotometrically. For cDNA synthesis reactions, Superscript RT
kits (Invitrogen) were used following the manufacturer's instructions. Oligonucleotide primers specific for hENT1 were ES2 and ES4, which corresponded, respectively, to hENT1 cDNA residues
4-21 (sense,
5'-CACCATGACAACCAGTCACCAGCCT-3', start codon
underlined) and +414-+437
(antisense, 5'-GACCTGATATACTCCATTCTCC-3') derived from the 3'-untranslated regions. Primers specific for hCNT2 were NT2D and JM23,
which corresponded, respectively, to residues 1528-1549 (sense,
5'-GGAATGGAGGAGTGGATGAGG-3') and 1949-1971 (antisense, 5'-GCTGTGGATTCTACAACAATACC-3'). Nested PCR was performed on
"first-round" hCNT2 products using NT2KG25 and NT2KG26 internal
primer sets, which corresponded, respectively, to residues 1562-1584
(sense, 5'-GGATTTCTGTGAGAGCTGAAATC-3') and 1920-1939 (antisense,
5'-GAATTTCAATGCTATGGCCC-3'). Amplifications were performed on a
Robocycler temperature cycler (Stratagene) as follows: 5 min at 94°C,
25 cycles (94°C for 1 min, 57°C for 2 min, 72°C for 2 min), and
20 min at 72°C. PCR products were subjected to electrophoresis on a
1% agarose gel with size markers (1-kb DNA ladder, Invitrogen) and
bands that migrated with the expected mobilities of hENT1 and hCNT2
were excised, gel-purified, and sequenced in one direction for
confirmation of identity.
Southern Blot Analysis.
Genomic DNA was prepared from cells
by the proteinase K-RNase method (Ausubel et al., 1997), cleaved with
EcoRI (Invitrogen), subjected to electrophoresis on an 0.8%
agarose gel with size markers (1-kb DNA ladder, Invitrogen) and
transferred to Hybond-N+ membranes (Amersham
Pharmacia Biotech, Piscataway, NJ). The hybridization probes were
prepared by 1) PCR amplification of the full-length hCNT2 cDNA from
pcDNA3/hCNT2; 2) separation on agarose gels; 3) purification on a G10
Sepharose column; 4) 32P-radiolabeling using a
Pharmacia random primer oligolabeling kit (5 × 108-109 dpm/µg of DNA);
and 5) purification by gel filtration chromatography on a Nick column
(Amersham Pharmacia Biotech). Hybridization was conducted under high
stringency (65°C for 1 h) using Express Hybridization solution
(Amersham Pharmacia Biotech) according to the manufacturer's instructions. Autoradiography was carried out by exposure to Kodak X-ray films at 
80°C.
Growth and Maintenance of Cell Lines.
The CCRF-CEM (American
Type Culture Collection, Manassas, VA; CCL-119) cell line (hereafter
referred to as CEM) is a human T-lymphoblast line that was originally
derived from a patient with acute lymphocytic leukemia and exhibits
es nucleoside transport activity (Cass et al., 1992).
CEM-ARAC-8C (hereafter referred to as CEM-ARAC) is a nucleoside
transport-defective subline that was selected for resistance to
cytarabine by Dr. B. Ullman (Oregon Health Sciences University,
Portland, OR) (Ullman et al., 1988). CEM and its derivatives were
maintained in RPMI 1640 (Invitrogen) medium supplemented with 10%
fetal bovine serum (v/v) and, for CEM-ARAC cells, 0.25 µM tubercidin
and 0.5 µM cytarabine or, for hCNT2 transfectants, 0.25 µM
cytarabine and 0.2 µg/µl G418; cells were subcultured every 3 to 4 days. HeLa S3 cells (American Type Culture Collection; CCL-2.2) were
maintained as adherent cultures in RPMI 1640 medium supplemented with
10% calf serum and subcultured at weekly intervals. Cells were
incubated at 37°C in a humidified (95%) atmosphere of 5%
CO2 in air, and cell numbers were determined using a Coulter Z2 electronic particle counter equipped with a size
analyzer (Coulter Electronics, Burlington, ON, Canada). The cell lines
used in this study were periodically shown to be free of
Mycoplasma by direct culture in agar/cell-free medium
(Medical Microbiology Laboratory, Edmonton, AB, Canada).
Transfection and Selection of Transfectants.
For
transfections, pcDNA3 or pcDNA3/hCNT2 plasmids were 1) purified on a
Qiagen Midi column from JM109 cultures that had been grown in the
presence of 50 µg/ml ampicillin, 2) linearized with BlgII
restriction enzyme, and 3) diluted to 0.25 µg/µl with RPMI 1640 that contained no glutamine (hereafter referred to as Gln-free RPMI).
For production of transient transfectants, hCNT2/pcDNA3 was introduced
into HeLa cells as described previously (Graham et al., 2000). Actively
proliferating cultures (2 × 106 HeLa cells
per 100-mm dish) were transfected with plasmid DNA (4 µg/dish) using
DEAE-dextran (Amersham Pharmacia Biotech, Quebec, Canada) and uptake
assays were performed 72 h later.
-galactosidase staining method. Parallel cultures were transfected with pcDNA3 or pcDNA3 containing the Escherichia coli lacZ
gene (pcDNA3/lacZ) using either the DEAE-dextran (HeLa) or
the electroporation procedure (CEM). Cultures were grown for 72 h;
after which they were harvested as described above, fixed (0.5%
glutaraldehyde in PBS) for 15 min at room temperature, and washed twice
(PBS adjusted to pH 7.4) by centrifugation (800g, 5 min).
The resulting cell pellets were incubated in 1-ml staining solution
that consisted of 1 mg/ml
5-bromo-4-chloro-3-indoyl--D-galactopyranoside,
20 mM potassium ferricyanide/ferrocyanide, and 2 mM
MgCl2 in PBS for 24 h at 37°C.
Transfection efficiencies were estimated by determination of the
proportion of blue-stained cells using a hemacytometer and were about
30% for HeLa cells and 0.1 to 1% for CEM cells.
Cloning of Stable Transfectants in Semisolid Medium.
G418-resistant cells were selected by growth in soft agarose cloning
medium that consisted of equal volumes of fresh RPMI 1640 supplemented
with 0.2 µg/µl G418, 20% HIHS, 1 mM 
-ketoglutarate (Sigma,
Oakville, ON, Canada), and 6 mM glutamine, and the same medium that had
been "conditioned" by exposure to actively proliferating CEM-ARAC
cells for 24 h. G418 selection cultures were suspended in cloning
medium with 1% Seaplaque agarose (Mandel, Guelph, ON, Canada) to yield
5 × 103 to 1 × 104 cells/100-mm plate (Fisher Scientific,
Ottawa, ON, Canada) and incubated for 3 weeks at 37°C in a humidified
(95%) atmosphere of 5% CO2 in air. Surviving
colonies were picked, expanded, and screened for nucleoside transport
activity as described below.
Nucleoside Transport Assays. Initial rates of nucleoside uptake were measured under zero-trans conditions at room temperature with cells harvested from actively proliferating cultures (4 × 105 cells/ml). Briefly, cells were harvested by centrifugation (800g, 10 min), washed twice in either Na+-containing transport buffer (5 mM D-glucose, 20 mM Tris-HCl, 3 mM K2HPO4, 1 mM MgCl2·6H2O, 2 mM CaCl2, and 130 mM NaCl, pH 7.4) or Na+-free transport buffer (NaCl was substituted with N-methyl-D-glucammonium chloride), and then resuspended in the appropriate transport buffer. Time courses of uptake of 3H-labeled nucleosides were determined using rapid sampling procedures in which the transport process was initiated by addition of cells to the 3H-labeled nucleoside solution (1:1) and terminated by rapid addition of excess cold nucleoside solution followed by immediate centrifugation (16,000g, 30 s) through transport oil [mixture of paraffin oil (Fisher Scientific, Ottawa, ON, Canada) and silicone 550 oil (Dow Corning, Mississauga, ON, Canada) with final specific gravity of 1.03 g/ml] to separate the cells from the permeant solution. Replicate assay mixtures were exposed to either [14C]polyethyleneglycol or 3H2O to determine trapped extracellular and intracellular water volumes. The cell pellets were solubilized in 0.5 ml of 5% Triton X-100, and cell-associated radioactivity was determined by liquid scintillation counting. Transport rates were derived from regression analysis of the linear component of uptake and kinetic parameters (apparent Km and Vmax values) were calculated using Prism (GraphPad Software Inc., San Diego, CA).
Materials. Radioisotopes were purchased from Moravek Biochemicals Inc. (Brea, CA) and 3H-labeled nucleosides were purified by high-performance liquid chromatography using water-methanol gradients on a C18 reverse-phase column. Natural nucleosides, nucleoside analogs, and doxorubicin were purchased from either Sigma Chemical Corp. or Cross Cancer Institute Pharmacy (Edmonton, AB, Canada). Dilazep was a gift from F. Hoffman La Roche and Co. (Basel, Switzerland). All other chemicals were of analytical grade and obtained from commercial sources. Cell culture supplies were from Invitrogen.
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Results and Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Demonstration of the Absence of hENT1 mRNA in CEM-ARAC Cells.
CEM-ARAC cells lack NBMPR-sensitive nucleoside transport activity and
the capacity for high-affinity binding of NBMPR (Ullman et al., 1988).
These characteristics suggested an absence of functional es
transporters in the mutant cells. RT-PCR analysis with primers designed
to detect full-length hENT1 transcripts was undertaken with RNA
isolated from CEM and CEM-ARAC cells to determine whether the hENT1
gene was expressed in the mutant cells (Fig.
1A). A PCR product of the expected
mobility was observed in reactions conducted with RNA from CEM cells
whereas there was no such product in reactions with RNA from CEM-ARAC
cells. Sequencing of the CEM-derived PCR product showed correspondence
to the hENT1 coding sequence that was recently cloned from human
placenta (Griffiths et al., 1997). A comparison of initial rates
of uptake of 10 µM [3H]uridine by CEM and
CEM-ARAC cells is shown in Fig. 1B. The results shown in Fig. 1, A and
B, established an association between the absence of hENT1 mRNA and the
inability to transport uridine and other nucleosides in CEM-ARAC cells,
indicating that the hENT1 protein was responsible for mediating
nucleoside uptake in CEM cells as predicted from its transport
characteristics.
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Production and Isolation of hCNT2-Producing Transfectants of CEM-ARAC Cells. Gene-transfer techniques were used to introduce the hCNT2 coding sequence into transport-deficient CEM-ARAC cells to assess the role of hCNT2 in the delivery of cytotoxic nucleosides. Isolation of hCNT2 stable transfectants with the introduced pcDNA3/hCNT2 construct was based on selection for neomycin resistance by growth in G418. Surviving G418-resistant clones were individually expanded and their nucleoside-transport activities were compared with those of CEM-ARAC cells by quantitating cellular uptake of radioactivity during 5-min exposures to 10 µM [3H]uridine. Of the greater than 4000 clones that were produced and tested, most did not exhibit nucleoside transport activity, despite their resistance to G418 (results not shown). Several of the G418-resistant transfectants exhibited uridine-uptake activity. The clone (D2) with the highest activity was selected and recloned by limiting dilution. Subsequent uridine uptake measurements with the CEM-ARAC/hCNT2-D2 reclone (hereafter referred to as ARAC/D2) yielded no further enrichment in uridine transport activity. Values ± S.D. ranged from 0.11 ± 0.01 to 0.14 ± 0.008 pmol/µl cell water/s, indicating that the transport-competent phenotype was stable.
Genomic Integration and Expression of hCNT2 cDNA in ARAC/D2 Cells. Genomic DNA from ARAC/D2 cells was examined to determine whether the pcDNA3/hCNT2 construct was integrated into the host cell genome. High-stringency hybridization with full-length hCNT2 cDNA of EcoRI-digested genomic DNA revealed a single unique band of 3.4 kb in ARAC/D2 but not in CEM-ARAC preparations (data not shown), indicating integration of hCNT2 cDNA into host genomic DNA.
Poly(A)+ RNA from actively proliferating cultures of ARAC/D2, CEM, and CEM-ARAC cells was analyzed by RT-PCR (Fig. 2), followed by nested PCR (data not shown). When pcDNA3/hCNT2 was used as the template, fragments with gel mobilities expected for the 458- and 425-base pair products of the external and nested hCNT2 primers were produced. Although PCR products that migrated with the same mobilities as those obtained with the hCNT2-plasmid preparations were detected in both rounds of PCR with the ARAC/D2 preparations, PCR products were not detected after either round (of 25 cycles each) conducted with the CEM and CEM/ARAC preparations.2 Sequencing of the nested PCR product from the ARAC/D2 preparations demonstrated correspondence with hCNT2 DNA (data not shown). These results established that 1) CEM and CEM/ARAC cells lacked hCNT2 mRNA, indicating that the absence of endogenous hCNT2 activity was due to low (or no) expression of the hCNT2 gene; and 2) ARAC/D2 cells contained hCNT2 mRNA, evidently produced by expression of the introduced hCNT2 cDNA.
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Transport Characteristics of ARAC/D2 Cells: Na+
Dependence and cif-Type Activity.
The nature of the
acquired nucleoside-transport capability of ARAC/D2 cells was assessed
by comparing time courses of uptake of 10 µM
[3H]uridine in the presence or absence of
Na+ by ARAC/D2 cells with those of nucleoside
transport-deficient CEM-ARAC cells and hENT1-containing CEM cells (Fig.
3A). In the presence of
Na+, ARAC/D2 cells exhibited initial uptake rates
(i.e., transport) that were 25-fold greater than those of CEM-ARAC
cells, whereas uptake rates were negligible in the absence of
Na+, indicating that the observed stimulation of
uridine transport activity required the presence of an inwardly
directed Na+ gradient. Uridine uptake by CEM
cells, which possessed only hENT1 activity, reached equilibrium at
around 1 min and was not dependent on the Na+
gradient. No measurable uridine uptake was observed in CEM-ARAC cells
in either the presence or absence of Na+. In the
experiments shown in Fig. 3B, uptake of 35 µM
[3H]uridine by ARAC/D2 cells was completely
inhibited by the presence of either 0.5 or 1 mM nonradioactive uridine.
These results demonstrated that ARAC/D2 cells possessed functional
recombinant hCNT2.
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; Patil and
Unadkat, 1997).
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Structure-Activity Relationships of hCNT2: Ability of Purine and
Pyrimidine Nucleosides and Nucleobases to Inhibit Uridine
Transport.
The experiments shown in Table 1 were undertaken to
identify structural determinants for interaction with hCNT2. Rates of uptake of 10 µM [3H]uridine were measured in
the absence or presence of 1 mM concentrations of 1) physiologic purine
and pyrimidine nucleosides, nucleobases, and ribose; 2) purine and
pyrimidine arabinonucleosides and arabinose; and 3) analogs of
adenosine and uridine. Studies involving adenosine and
deaminase-sensitive adenosine analogs were conducted in the presence of
2 µM 2'-deoxycoformycin to prevent deamination (Barros et al., 1991).
Deoxycoformycin did not inhibit hCNT2-mediated [3H]uridine influx when tested at
concentrations of 1 mM (data not shown).
[3H]Uridine influx by hCNT2 was inhibited
completely by the physiologically occurring ribosyl and 2'-deoxyribosyl
purine nucleosides, uridine, 2'-deoxyuridine, partially by thymidine
and uracil, and not at all by ribose, cytidine, 2'-deoxycytidine or the
other nucleobases that were tested. Although 2'-deoxyuridine and
2'-deoxyribosyl purine nucleosides inhibited uridine transport
completely, 3'-deoxyuridine and 2',3'-dideoxyribosyl derivatives of
uridine and adenosine had no effect, suggesting a requirement of the
hydroxyl group at position 3' of the ribose moiety for interaction with hCNT2.
-D-arabinofuranosyladenine, 9--D-arabinofuranosylhypoxanthine), whereas it was not
inhibited or only partially inhibited by pyrimidine arabinonucleosides
(1--D-arabinofuranosyluracil, 1--D-arabinofuranosylcytosine),
2-fluoro-9--D-arabinofuranosyladenine (fludarabine), and
arabinose. Complete inhibition of hCNT2-mediated [3H]uridine influx was observed for
2'-deoxyadenosine and 85% inhibition for 2-chloroadenosine,
whereas 2-chloro-2'-deoxyadenosine (cladribine) exhibited 28%
inhibition. The poor ability of 2-chloro-2'-deoxyadenosine and
2-fluoro-9--D-arabinofuranosyladenine, compared with
that of 2'-deoxyadenosine and
9--D-arabinofuranosyladenine, to inhibit hCNT2-mediated
uridine transport suggested a low tolerance for the presence of a
halogen at position 2 of the purine base. Modifications of the purine
ring to create 7-deazaadenosine (tubercidin) had no effect on
hCNT2-mediated [3H]uridine transport, which was
consistent with previous reports demonstrating that 7-deazaadenosine
was not a permeant of the murine cif-type transport process
(Crawford et al., 1990).
Substitutions with a series of halogens with decreasing
electronegativities at position 5 of uridine (5-fluorouridine,
5-bromouridine, 5-iodouridine) resulted in complete inhibition of
hCNT2-mediated uridine transport. However, 5-methyluridine and
thymidine were less potent inhibitors of
[3H]uridine transport (62 and 26% inhibition,
respectively). The different 5-halogenated 2'-deoxyribosyl uridine
analogs showed different magnitudes of inhibition of
[3H]uridine transport, with
5-fluoro-2'-deoxyuridine being the most effective (82% inhibition),
followed by 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine (44%
and 31% inhibition, respectively). 5-Fluoro-5'-deoxyuridine, an
intermediate metabolite of the oral fluoropyrimidine capecitabine (Di
Costanzo et al., 2000), showed only 20% inhibition of hCNT2-mediated
[3H]uridine transport. The combined results
shown in Table 1 demonstrated that hCNT2 exhibited 1) tolerance for
substitution of the hydroxyl group for a hydrogen in the 2' but not 3'
position of the ribosyl moiety, 2) preference for the 2'-hydroxyl
substituent in the - rather than -configuration, 3) low tolerance
for modifications of position 2 on the purine base of adenosine analogs
with halogen, and 4) higher tolerances for modifications of position 5 on the pyrimidine base of uridine analogs with halogen than with methyl substituents.
Interaction of hCNT2 with Halogenated Uridine Analogs.
Since
hCNT2 exhibited remarkably high tolerance for halogens at position 5 in
several uridine analogs, inhibition experiments were performed with
graded concentrations of the halogenated analogs to quantify the
relative effects of these modifications in ribosyl and deoxyribosyl
derivatives of uridine. As shown in the representative dose-response
curve of Fig. 4 comparing
5-fluorouridine, 5-fluoro-2'-deoxyuridine, and
5-fluoro-5'-deoxyuridine, hCNT2-mediated
[3H]uridine transport was inhibited by
5-halogenated uridine analogs to varying degrees. The calculated
Ki values (Table
2) for 5-fluorouridine, 5-bromouridine, and 5-iodouridine (34, 46, and 50 µM,
respectively) were close to the Km
value of 46 ± 4 µM observed for zero-trans influx of
uridine. A much higher Ki value (197 µM)
was obtained for 5-methyluridine, which was consistent with its poor
ability to inhibit hCNT2-mediated [3H]uridine
transport in the inhibition studies of Table 1. Both thymidine and
1--D-arabinofuranosyluracil exhibited very
high Ki values, indicating that they were
likely not permeants of hCNT2. The halogen substituents in the
pyrimidine ring of ribosyl analogs did not alter the apparent
Ki values, whereas there was a progressive increase in Ki values that paralleled the
increase in size and/or decrease in electronegativities of the halogen
atoms in the 2'-deoxyribosyl analogs (5-fluoro-2'-deoxyuridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine). A comparison
of 5-fluoro-2'-deoxyuridine and 5-fluoro-5'-deoxyuridine demonstrated
the importance of the hydroxyl group at the 5'-position for
inhibitor-transporter interaction. Removal of the hydroxyl group at
position 5' in the ribosyl moiety of 5-fluorouridine increased the
Ki value from 34 to 411 µM, whereas removal of the hydroxyl group at position 2' increased the
Ki value to 82 µM. These results
indicated that 5-halogenated uridine analogs, 2'-deoxyuridine and
5-fluoro-2'-deoxyuridine bound well to hCNT2. Although inhibition of
nucleoside influx indicated interaction between the inhibiting
substance and hCNT2, it did not demonstrate that the inhibiting
substance utilized the transporter to move through plasma membrane.
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Transport Characteristics of ARAC/D2 Cells: hCNT2-Mediated
Transport of Physiological and Modified Nucleosides.
Uptake of
several of the adenosine and uridine analogs used in this study was
examined to determine whether these compounds were transported by hCNT2
and, if so, to compare their rates with those of the universal permeant
uridine (Fig. 5). ARAC/D2 cells were
included as a control to show the absence of mediated transport for
several of the physiological and modified nucleosides. The results
showed that the Na+-dependent transport rates,
which represented the hCNT2-mediated component, were generally higher
for purine nucleosides than for pyrimidine nucleosides. Both adenosine
and 2'-deoxyadenosine, which completely inhibited hCNT2-mediated
[3H]uridine transport (Table 1), exhibited
approximately 2-fold higher transport rates than uridine
(adenosine > 2'-deoxyadenosine). Guanosine exhibited slightly
higher rates of transport than 2'-deoxyguanosine.
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-D-arabinofuranosyl-2-fluoroadenine) and cladribine
(2-chloro-2'-deoxyadenosine), exhibited low transport rates
(Fig. 5), which was consistent with their poor ability to inhibit
hCNT2-mediated [3H]uridine transport in the
inhibition experiments of Table 1. These results are in contrast to
previous reports showing that cladribine was a permeant of the rat
homolog of hCNT2 (also termed SPNT) in transiently transfected cervical
cancer cell lines (Schaner et al., 1997). This difference in cladribine
transportability suggested species differences in substrate
selectivities between the rat and human CNT2 proteins.
Since hCNT2-mediated uridine transport was inhibited by several
modified uridine analogs, a series of pyrimidine nucleosides and
nucleobases were also examined for transportability in the experiments
of Fig. 5. The Na+-dependent component of the
transport rate for 5-fluoruridine (0.132 ± 0.003 pmol/µl cell
water/s) was rapid and higher than that observed for uridine
(0.097 ± 0.009 pmol/µl cell water/s). Although uracil and
thymidine partially inhibited hCNT2-mediated [3H]uridine transport (Table 1), direct
measurement of [3H]uracil and
[3H]thymidine influx into ARAC/D2 cells (Fig.
5) indicated that these inhibitors were not permeants of hCNT2. The
relatively high rates of uptake observed for
[3H]uracil in the presence and absence of
extracellular sodium gradient may have been due to uptake by a
nucleobase transporter.
Transport of 5-Fluorouridine by hCNT2.
The kinetics of
transport of 5-fluorouridine by hCNT2- and hENT1-containing cells were
compared with those of the naturally occurring nucleoside uridine
(Table 3). Initial rates of uptake by
hCNT2-containing ARAC/D2 cells were determined as a function of
concentration and the relationship so obtained conformed to Michaelis-Menten kinetics, with apparent Km
and Vmax values (mean ± S.D.) of
43 ± 7 µM and 0.38 ± 0.07 pmol/µl cell water/s,
respectively. The apparent Km value for
5-fluorouridine was comparable to the Ki
value determined from the inhibition experiments of Table 2. The
Km and Vmax
values for 5-fluorouridine were almost identical to those obtained for
uridine, indicating that hCNT2 indiscriminately transports either
substance with equal efficiency. Higher apparent Km values were observed for the transport
mechanism mediated by hENT1, with the 5-fluorouridine values being
slightly higher than those for uridine.
|
Enhanced Sensitivity to Purine and Pyrimidine Nucleoside Drugs in
hCNT2-Producing Cells.
To assess relationships between efficiency
of transport and cellular toxicity, several cytotoxic nucleoside drugs
were compared for their ability to inhibit cell proliferation in cancer
cell lines that possessed either hCNT2, hENT1, or no nucleoside
transporters (Table 4). Doxorubicin was
included to demonstrate that the resistance of CEM-ARAC cells was not
associated with membrane transport-associated multidrug-resistant (MDR)
proteins (van den Heuvel-Eibrink et al., 2000), since all the cell
lines tested were equally sensitive. CEM cells were sensitive, whereas
CEM-ARAC cells were highly resistant to all of the nucleoside drugs
tested, indicating a requirement for mediated permeation across the
plasma membrane to achieve cytotoxicity. Cladribine and fludarabine
were less effective against ARAC/D2 cells than CEM cells, which was
consistent with the apparent low-affinity interaction with hCNT2 and
the very low transport rates observed in isotopic flux analyses. Their
relatively high cytotoxicities against CEM cells were probably
influenced by the greater capacity for uptake via hENT1.
|
; Johansson and Karlsson, 1995).
The unique ability of cladribine compared with fludarabine to affect
mitochondrial function upon cell entry may also be a factor in the
greater cytotoxic effects observed against ARAC/D2 cells (Genini et
al., 2000).
Tubercidin (7-deazaadenosine), a known permeant of es
processes (Cass et al., 1992), exhibited markedly different
cytotoxicities against hENT1-containing CEM and hCNT2-containing
ARAC/D2 cells, with IC50 values of 0.08 and 16.7 µM, respectively, a 200-fold difference. A qualitatively similar
result was obtained with cytarabine, which is a known es
permeant (Wiley et al., 1982); IC50 values of
0.10 and > 100 µM, respectively, were obtained with CEM and ARAC/D2 cells. The relative insensitivities of hCNT2-containing cells
to tubercidin and cytarabine were consistent with the conclusion that
these drugs are not permeants of hCNT2. Cytarabine and tubercidin are
also poor permeants of the human and rat pyrimidine-nucleoside selective (CNT1) transporters (Crawford et al., 1998a; Graham et
al., 2000).
5-Fluorouridine, which was shown in the present study to be a
high-affinity hCNT2 permeant, was considerably more toxic to CEM and
ARAC/D2 cells (IC50 values of 0.06 and 0.05 µM,
respectively) than to CEM-ARAC cells (IC50 > 50 µM). For ARAC/D2 cells, the acquired sensitivity to 5-fluorouridine
was associated with, and most likely due to, the acquisition of hCNT2
activity, whereas for CEM cells, the intrinsic sensitivity to
5-fluorouridine was attributed to hENT1-mediated uptake.
5-Fluorouridine was equally cytotoxic to ARAC/D2 and CEM cells,
although the
Vmax/Km ratio was greater for CEM cells as shown in Table 3. The similar
chemosensitivities, despite the different kinetic characteristics, were
attributed to differences in the intrinsic properties of the
transporters in the two cell lines. ARAC/D2 cells possess hCNT2, which
is unidirectional and capable of concentrating nucleoside drugs inside
cells, whereas CEM cells possess hENT1, which is bidirectional and
transports nucleoside drugs in either direction, depending on the
concentration gradient. Thus, hCNT2-containing ARAC/D2 cells could not
lose drug by outward transport whereas hENT1-containing CEM cells
could, if intracellular trapping of drug via phosphorylation was slower than inward transport. Studies with murine leukemia L1210 cells (which
possess two equilibrative and one concentrative nucleoside transporter)
demonstrated that treatment of cells with inhibitors of equilibrative
transport reduced initial rates of nucleoside drug uptake but enhanced
net accumulation of nucleoside drug and therefore toxicity by blocking
drug efflux (Dagnino and Paterson 1990).
ARAC/D2 cells were sensitive to 5-fluoro-2'-deoxyuridine, albeit
moderate compared with 5-fluorouridine, whereas CEM-ARAC cells were
resistant. These results suggested that 5-fluoro-2'-deoxyuridine utilized hCNT2 to cross the cell membrane, since transport-deficient cells were far less sensitive to this analog than hCNT2-containing cells. In contrast, 5-fluoro-5'-deoxyuridine was equally ineffective against ARAC/D2 and CEM-ARAC cells as compared with CEM cells, indicating that its entry was not mediated by hCNT2.
Conclusion.
For many cell types, the presence of heterogeneous
nucleoside transporters and complex overlapping substrate selectivities poses difficulty in the interpretation of flux measurements and determination of mechanism of drug sensitivity. The studies presented here provide the first demonstration of the transport properties of
hCNT2 in structure-activity and structure-cytotoxicity relationships in
a human cancer cell line that possessed only hCNT2-mediated transport.
The results demonstrated that the resistance of CEM-ARAC cells to
purine and pyrimidine nucleoside drugs was due to the absence of
expression of the hENT1 gene leading to a loss of nucleoside transport
activity. Introduction of hCNT2 by gene transfer conferred the capacity
for nucleoside transport and drug sensitivity to the resistant cells.
The 3'- and 5'-hydroxyl groups of the ribosyl moiety were important for
interaction of uridine analogs with hCNT2, whereas removal of the
hydroxyl at position 2' did not significantly affect interaction with
hCNT2. The transporter exhibited a preference for the 2'-hydroxyl group
in the - rather than -configuration and a tolerance for
modifications of the 5 but not the 3 position of the base with halogen
substituents. The transport activity of some adenosine analogs such as
cladribine, which is a known substrate for CNT2 from rat or mouse
species (Crawford et al., 1990a; Schaner et al., 1997), and
fludarabine were low compared with several halogenated uridine analogs.
5-Fluorouridine, 5-fluoro-2'-deoxyuridine, and 5-fluoro-5'-deoxyuridine
exhibited progressively decreasing affinities for hCNT2, and this was
reflected in the degree of their cytotoxicity against hCNT2-containing
cells. The high transportability of 5-fluorouridine and
5-fluoro-2'-deoxyuridine by hCNT2 suggests a role for this transporter
for fluoropyrimidine nucleoside chemotherapy and radiopharmaceutical imaging.
| |
Acknowledgments |
|---|
We thank Dr. Kathryn Graham and Pat Carpenter (Cross Cancer Institute) for help in sequencing and Dr. Charles Crawford (St. Jude Children's Research Hospital) for helpful advice in production of stable transfectants. We are grateful to Dr. Buddy Ullman (Oregon Health Sciences University) for generously providing the CEM-ARAC-8C cell line.
| |
Footnotes |
|---|
Received June 11, 2001; Accepted August 3, 2001
Supported by the National Cancer Institute of Canada and the Alberta Cancer Board. J.D.Y. is a Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research and C.E.C. is Canada Research Chair in Oncology.
2 Northern blot and RT-PCR analysis of mRNA from 10 other cultured human cancer cell lines of either hematopoietic or solid-tumor origin also gave negative results for hCNT2 transcripts (K. Graham, J. R. Mackey, D. Mowles, and C. E. Cass, unpublished results).
1 The correspondence between human nucleoside transporter proteins (GenBank accession numbers given in parentheses) and their activities is: hENT1 (U81375), es; hENT2 (AF029358), ei; hCNT1 (U62966), cit; hCNT2 (AF036109), cif; hCNT3 (AF305210), cib.
Carol E. Cass, Department of Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, T6G 1Z2. E-mail: carol.cass{at}cancerboard.ab.ca
| |
Abbreviations |
|---|
ENT, equilibrative nucleoside transporter;
CNT, concentrative nucleoside transporter;
h, human;
r, rat;
cytarabine, 1--D-arabinofuranosylcytosine (araC);
NBMPR, nitrobenzylmercaptopurine ribonucleoside
(6-[(4-nitrobenzyl)thio]-9--D-ribofuranosylpurine);
PBS, phosphate-buffered saline;
kb, kilobase(s);
MDR, multidrug-resistant;
HIHS, heat-inactivated horse serum;
RPMI, Roswell
Park Memorial Institute;
RT-PCR, reverse transcriptase polymerase chain
reaction;
abbreviations used in transporter acronyms, c,
concentrative;
e, equilibrative;
s and i, sensitive and insensitive to inhibition by NBMPR,
respectively;
f, formycin B (nonmetabolized purine nucleoside);
t, thymidine.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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) and CEM-ARAC (
) cells was measured at the
time intervals shown in Na+-containing transport buffer as
described under Experimental Procedures. Each value
represents the mean ± S.D. of three determinations, and error
bars are not shown where S.D. values were smaller than that represented
by the symbols.
)
(0.1-1000 µM), or 5-fluoro-5'-deoxyuridine (
) (5 µM-10 mM).
Na+-dependent transport rates were calculated
from the uptake measurements performed in both
Na+-containing and Na+-free
transport buffer. Results are presented as the fraction of uridine
transport remaining as a function of the logarithm of the concentration
of inhibitor and are the means ±S.D. of data obtained from triplicate
determinations in two experiments. The IC50
values for 5-fluorouridine, 5-fluoro-2'-deoxyuridine, and
5-fluoro-5'-deoxyuridine inhibition of uridine transport were 41, 100, and 500 µM, respectively.
