L1210/B23.1 cells express equilibrative, inhibitor-sensitive nucleoside transport activity and lack two parental nucleoside transport activities

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 24, Iasue of August 25, pp. 16951-16956,1992 Printed in U.S.A.

L12 1O/B23.1 Cells Express Equilibrative, Inhibitor-sensitive Nucleoside Transport Activity and Lack Two Parental Nucleoside Transport Activities* (Received for publication, December 23, 1991)

Damaraju VijayalakshmiS, Lina DagninoSO, Judith A. Beltq, WendyP. GatiS, Carol E. Cassll **, and Alan R. P. PatersonS 11 ** $I From the Departments of $Pharmacology and 11 Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and the (IDepartment of Biochemical and Clinical Pharmacology, St. JudeChildren’s Research Hospital, Memphis, Tennessee 38101

Cultured mouse leukemia L1210 cells express the have been shown by these tactics: (i) NBMPR inhibition of nucleoside-specific membrane transport processes des- an equilibrative N T process (5-9), (ii) replacement of the Na+ ignated es, ei, and cif. The es and ei processes are content of incubation media with other cations to impair Na+equilibrative, but may be distinguished by the high coupled N T processes (10, l l ) , and (iii) use of mutation/ sensitivity of the former to 6-[(4-nitrobenzyl)thio1-98-0-ribofuranosylpurine (NBMPR); the cif process is selection techniques to generate clones with N T deficiencies mediated bya Na+/nucleosidecotransporter of lowsen- (4, 8, 9). This report describes the isolation by mutation and sitivity to NBMPR. Cellsof an ei-deficient clonal line, selection of the clone L1210/B23.1, cells of which express L12 10/MC5-1,were mutagenized, and clones were se- mutational deficiencies in two of the three NT systems reclected in soft agar medium that contained (i) NBMPR ognized in L1210 cells. (aninhibitor of es processes), (ii)erythro-9-(2-hyTransporter-independent permeation of nucleosides isa droxy-3-nony1)adenine (an inhibitor of adenosine de- minor process in cultured leukemia cells, as shown bylow aminase), and (iii) arabinofuranosyladenine (a cyto- inward fluxes of the 3H-labeled L-enantiomers of adenosine toxic substrate for the three nucleoside transporters). in L1210 cells (12) and of adenosine, uridine, and thymidine The selection medium did not allow es activity and selected against cells that expressed the Na+-linkedcif in S49 cells (13),relative to fluxes of the corresponding process. Cells of the L1210jB23.1 clonal isolate were physiological nucleosides, which are the D-enantiomers. This deficient in cif transport activity,and inward fluxes of report alsodemonstrates thepoor permeability to nucleosides formycin B, a poorly metabolized analog of inosine, of L1210 cells in which NT functions have been impaired by were virtually abolished by NBMPR in these cells. In chemical or genetic means or by Na+ deficiency. The expression of separate NBMPR-sensitive and the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of[‘HlforNBMPR-insensitive N T activities in L1210 cells was first mycin B, and inward fluxes of the latterwere compet- shown by Belt (6, 7) and Belt and Noel (8). The former itively inhibited by purine andpyrimidine nucleosides. activity is attributable to an equilibrative (facilitated diffuThe transportbehavior of L121OjB23.1 cells indicates sion) nucleoside transporter subtype that is distinctive bethat (i) the mutationjselection procedure impaired or deleted the Na+-linkedcif process and (ii)es nucleoside cause of its high NBMPR sensitivity; in the terminology of Belt and co-workers (4), thisis the es2 transporter. In erythtransport activity isexpressed in themutant cells. rocytes, NBMPR is bound tightly (KO< 1 nM) to an externally accessible site on the es transporter polypeptide (14,15), and site occupancy blocks transporter function (16). Transporter-mediatedprocesses of several types contribute NBMPR-insensitive NT processes of two types occur in to thepermeation of physiological nucleosides in animalcells wild-type L1210 cells as (i) equilibrative and (ii) Na+-linked (1-5). In recent studies with cultured mouse leukemia L1210 concentrative processes and are here designated as ei and cif cells, contributions to nucleoside fluxes by three NT1 systems transporters, respectively. Cells of the L1210/MC5-1 clonal * This work wassupported in partby the National Cancer Institute line isolated by Belt and Noel (8)are deficient in ei transport of Canada and theResearch Initiative Program of the Alberta Cancer activity and express es and cif NT activities (4). A clone of cells (L1210/MA-27.1) (9) that lack the es transporter and Board. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby express only the Na+-linked, formycin @-accepting transmarked “aduertisement” in accordance with 18 U.S.C. Section 1734 porter, cif, has been isolated from a mutagenized population solely to indicate this fact. of L1210/MC5-1 cells. § Recipient of a research studentship from the Alberta Heritage Dagnino et al. (17) reported that cultured L1210/C2 cells Foundation for Medical Research. Present address: Dept. of Cell N T activity, a finding since Biology, Vanderbilt University School of Medicine, Nashville, T N express concentrative, Na+-linked confirmed in other clonal lines (L1210/MC5-1 (4), L1210/ 37232. ** Senior Research Scientists of the National Cancer Institute of AM (10, l l ) , and L1210/MA-27.1 (9). Na+-coupled NT procCanada during this study. esses have also been identified in cell and vesicle preparations $$ To whom correspondence should be addressed. from intestinal and renal epithelia from several species, in

The abbreviations and trivial names used are: NT, nucleoside transport; formycin B, 3-[P-~-ribofuranosyl]pyrazolo-7-[4,3-d]pyrimidone; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; NBMPR, The terminology of Belt and co-workers (4) refers to functional 6-[(4-nitrobenzyl)thio]-S-~-~-ribofuranosylpurine (nitrobenzylthioi- properties of the nucleoside transporters: es and ei refer to equilibranosine); NBTGR, 2-amino-6-[(4-nitrobenzyl)thio]-9-~-~-ribofurative processes that are respectively sensitive or insensitive to 1 p~ nosylpurine; NMG’, N-methyl-D-ghcammoniumion; araA, arabi- NBMPR, and cif refers to the concentrative transport process that is nofuranosyladenine. insensitive to NBMPR andaccepts formycin B as a substrate.

16951

16952

L121OIB23.1 Cells Nucleoside Lack Two Transport Activities

nucleoside fluxes at 22 ‘C were determined as initial rates of cellular uptake of 3H-nucleosides from uptake time courses. In measuring the latter, replicate assay mixtures were assembled over 100-pl portions of “transport oil” (27) in 1.5-ml microcentrifuge tubes. Measurement of the cellular content of 3H-permeant in the replicate mixtures after graded intervals of permeant uptake yielded uptake time courses. Flux assay mixtures contained 1-2 X lo6 cells/tube in Dulbecco’s phosphate-buffered saline (31) with 5 mM glucose (termed Na+ medium) or in variations of this medium in which Na+ was replaced by NMG+, K+, or choline+ in the volumes specified in individual experiments. In some experiments,intervals of permeant uptake were initiated by rapid manual addition of 3H-permeant in response to a metronome and were ended by centrifugal pelleting of cells under the oil (16,000 X g, 30 s), in which case, the cellular uptake of 3Hpermeant during the cell pelleting operation was considered equivalent to thatduring a 2-s incubation interval (30). In several experiments,a Digiflex-CX automaticpipettor(ICN Biomedicals, Costa Mesa, CA) was used to deliver 3H-permeant uptake at successive 1.5-sintervals tosets of six replicate assay mixtures waiting in the 12-place rotor of an Eppendorf 5412 microcentrifuge. Pipettor delivery volumes were 188 pl at this delivery interval. As soon as possible after the last delivery of 3H-permeant, cells were pelleted under oil (16,000 X g, 30 s) to end permeant uptake in all assay mixtures. The interval between the start of pipettor delivery and centrifuge switch-on was recorded electronically. The 3H-permeant content of cells in the six assay mixtures of each set comprised a time course of permeant uptake. In other experiments, time courses were obtained by methods that employed automated pipettors to both start and end intervals of cellular uptake of 3H-permeant in replicate assay mixtures. Uptake intervals were started by completing assay mixtures with permeant addition by means of a Digiflex-TPautomated pipettor (ICNBiomedicals) programed to deliver 188 pl at 1.5-s intervals. Those deliveries EXPERIMENTALPROCEDURES began at switch-on. A Digiflex-CX automated pipettor, linked to the Cells and Cell Culture-Stock cultures of the L1210 clonal lines Digiflex-TP pipettor by a common switch, was used to deliver, at 1.5used in this work were maintained at 37 “C in unstirred suspension s intervals, 188-pl portions of the flux stopper, 300 p~ dilazep in cultures in Fischer’s medium or in RPMI 1640 medium containing Dulbecco’s phosphate-buffered saline to achieve a final stopper con10% horse serum in a humidified atmosphere of 5% C 0 2 in air. Cell centration of 100 p ~ Because . of pipettor design, those deliveries concentrations were kept between 0.5 and 5 X lo5 cells/ml to ensure began 0.75 s after switch-on. By discarding 0-4 dilazep deliveries, exponential proliferation by dilution to 0.5 X lo6 cells/ml with fresh dilazep could be delivered to assay mixtures at 0.75-, 2.25-, 3.75-, medium every 2-3 days. For transport andbinding experiments, cells 5.25-, and 6.75-s intervals after 3H-permeant delivery started, making were propagated at 37 “C as 600-ml cultures in 2-liter roller bottles possible those intervals of permeant uptake. This assay presumes that thedilazep blockade of nucleoside uptake under these conditions gassed with 10% COZ in air and turned at 1.5 rpm. Cell population is almost instantaneous, as we have shown in earlier quenched-flow doubling times were 12-14 h under these conditions. Stock cultures were reinitiated after 30-35 subculture generationsfrom mycoplasma- studies (32). Flux assays by this method were conducted in quadrufree stocks cryopreserved in growth medium that contained10% plicate; all microcentrifuge tubes were capped, and centrifugation to dimethyl sulfoxide and were stored under liquid nitrogen. The clonal pellet cells under oil was begun within 10 s of dilazep addition. Cells lines L1210/MC5-1 and L1210/C2 have been described previously (8, were pelleted under oil without washing. To measure thecontent of 3H-permeants in cell pellets,tube 28). L121O/AM cells were kindly provided by Dr. L. L. Bennett, Jr. (Southern Research Institute, Birmingham, AL). These cells, origi- contents above the oil layers were removed by aspiration; tube intenally designated L1210/AraC/MeMPR, are deficient in adenosine, riors above the oil were rinsed twice with water and, afterremoval of deoxyadenosine, and deoxycytidine kinase activities (29). the oil layers, cell pellets were lysed in 250 pl of 5% Triton X-100. L1210/B23.1Cells-In the isolation of the double transport mutant Assay tubes with their contents were placed in counting vials conL1210/B23.1, a population of L1210/MC5-1 cells (4.4 X lo8 cells in taining 8 ml of scintillation fluid (33); and, after thorough mixing to 270ml of growth medium) was incubated (1 h, 37 “ C ) in growth disperse the tube contents, 3H activity was measured by liquid scinmedium that contained 3 pg/ml N-methyl-N-nitro-N’-nitrosoguani-tillation counting. The cellular water space in replicate cell pellets dine, a mutagen that induces point mutations. The cells were washed was determined from the difference between the total3H20space and once, resuspended in fresh, mutagen-free growth medium, and cul- the extracellular space in cell pellets measured with [3H]polyethyltured for -10 population doublings under nonselective conditions. eneglycol. Permeant present in extracellular medium trapped in cell For selection of mutant clones, cell suspensions were incubated for 1 pellets was subtracted from the total pellet content of permeant. When adenosine fluxes were measured, cell cultures were treated h at 37 ‘C in growth medium containing 1.5 p~ EHNA and then plated at lo6 cells/ml in soft agarcloning medium using 5-ml portions with 1 p~ 2’-deoxycoformycin at 37 “ C for 1 h prior to flux assays, in 60-mm culture dishes. The cloning medium (which consisted of and assay medium contained 1PM 2’-deoxycoformycin (final concenFischer’s medium with 0.13% agar, 10% horse serum, and 10% con- tration). Equilibrium Binding of [3H]NBMPR-Assay mixtures in 1.5-ml ditioned medium (filtered growth medium from 24-h cultures of L1210/MC5-1 cells)) contained 50 pg/ml gentamicin, 1.5 p~ EHNA, microcentrifuge tubes contained 2 x lo6cells in 1.0 ml of Na+ medium 7 pM araA, and 10 P M NBMPR. Thecultures were incubated at 37 “C with graded concentrations (0.04-40 nM) of [3H]NBMPR (34) layered in a humidified atmosphere of 7% COz in air for 14 days, and the 34 over 0.15 ml of transport oil. Assay mixtures with and without 20 pM NBTGR were incubated at 22 “C for 30 min before recovery of cells surviving colonies were transferred todrug-free RPMI 1640 medium to establish clonal cultures. After 4-6 weeks of passage in drug-free by oil layer pelleting. The 3H content of cell pellets and concentrations medium, the ability of the clonal cultures to proliferate in a secondary of [3H]NBMPR in the medium fraction of assaymixtures were selection medium (growth medium containing 7 p~ araA, 1.5 p~ 2‘- determined as in theflux assays. Site-specific binding of NBMPR was determined by subtracting deoxycoformycin, and 5 p~ dipyridamole) was confirmed. As well, absence of Na’-dependent N T activity in the clonal cultures was the cell content of [3H]NBMPR measured in the presence of NBTGR (“nonspecific binding”) from that measured in the absence of NBTGR demonstrated by the equivalence of formycin B uptake ratesin clonal cells suspended in Na+ or NMG’ medium. Clone B23, selected in this (“total binding”). Chemicals-NBMPR was synthesized in this laboratory (35). Lmanner, was recloned by limiting dilution and designated clone B23.1 Nucleoside Fluxes-As in earlier reports from this laboratory (30), Adenosine and 2’-deoxycoformycin were gifts from the Division of

spleen cells and peritoneal macrophages (18-25), in choroid plexus (26), and in cultured cells of several types (11, 23, 27). It has been a general finding that theNa+-coupledNT systems are insensitive to NBMPR. In vesicle systems, macrophages, and L121O/AM cells, the Na’/nucleoside stoichiometry has been shown to be 1:l (20,23,25). Substrate specificity studies of Na+-coupled nucleoside transport in fresh mouse enterocytes identified Na+-coupled transport activities of two types, cif and cit, with preferences, for purine and pyrimidine nucleosides, respectively; both transporters accept uridine, adenosine, anddeoxyadenosine as substrates(18). In theenterocyte system, formycin B and thymidine appeared to be exclusive substrates for the cif and cit transporters, respectively. Two Na+-coupled transporters with distinguishing substrate specificities have also been recognized in bovine kidney (21). Wild-type L1210 cells exhibit NT activities of two equilibrative types (es and ei) and a single type of concentrative, Na+-coupled NT activity ( c i f ) . In the study here described, L1210/MC5-1 cells (which express es and cif NT activities) were mutagenized, and clones were selected in growth medium that was cytotoxic to cells expressing cif activity. Cells of the isolated clone (L1210/B23.1) exhibit equilibrative, Na+-independent NT activity that is of high NBMPR sensitivity, suggesting that mutational loss of the cif system occurred during the mutation/selection process. This study supports other evidence showing that wild-type L1210 cells express three distinct types of NT activity, es, ei, and cif (4-6, 8-11).

L121OlB23.1 Lack Cells

Two Nucleoside Transport Activities

Cancer Treatment, National Cancer Institute (Bethesda, MD), and other nucleosides were obtained from Sigma. 3H-Labeled nucleosides were purchased from Moravek Biochemicals (Brea, CA) and, after storage, were purified by high performance liquid chromatography on a Whatman Partisil-10 ODs-3 M9 column (25cm)elutedwith methanol/water solutions to achieve radiochemical purities of 98% or greater. [1,2-3H]Polyethyleneglycol (2 mCi/g) and 3Hz0 (100 mCi/ ml) were purchased from Du Pont-New England Nuclear and ICN Radiochemicals (Irvine, CA), respectively. EHNA was provided by Dr. H. J. Schaeffer (Wellcome Research Laboratories, Research Triangle Park, NC), and dilazep was a gift from Hoffmann-La Roche. Paraffin oil (Saybolt viscosity 125-135) and silicone 550 oil, components of transport oil, were obtained from Fisher and Dow Corning Corp., respectively. Cell culturematerials were purchased from GIBCO. RESULTS AND DISCUSSION

16953

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201

o t ,~, 0

9

8

7

6

0 5

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,~ % ,

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-Log [NBMPR] (M)

FIG. 1. Concentration-effect relationships in NBMPR inhibition of formycin B transport in L1210m23.1 and L1210/MC5-1 cells. Cells were incubated for 20 min at 22'C in Na' (0) or NMG' (0)medium containing NBMPR prior to assays of cellular uptake of [3H]formycin B (FB) by a procedure that employed oil-stop methodology to end brief intervals (2-20 s) of permeant uptake ( 5 ) . The assay methodyielded time courses of permeant uptake from which initial rates (fluxes) were derived. Those rates are expressed as percentages of control rates (100%) in cells in Na' medium without exposure to NBMPR, which were as follows: L1210/ MC5-1 cells, 1.7 pmol/d of cell water/s; and L1210/B23.1 cells, 6.9 pmollpl of cell water/s.

Selection of Clone LI210/B23.1-Cells of the L1210/MC5-1 clonal line, isolated from a mutagen-treated population of wild-type L1210 cells (8), express the following: (i) NBMPRsensitive NT activity, (ii) sites at which NBMPR is bound tightly (site abundanceand KOfor bound NBMPR aresimilar t o those parameters in parental cells), and (iii)concentrative, Na'-linked, NBMPR-insensitive N T activity (8). Inthis study, the selection strategy was intended to eliminate cells expressing cif activity. Accordingly, L1210/MC5-1 cells were L121O/MC5-1 200 mutagenized and plated in a soft agar selection medium that 20 LIM FB contained 7 p~ araA, 1.5 p~ EHNA, and 10 p~ NBMPR. 10 pM NBMPR araA was chosen as the toxic agent in the selection medium because (i) it is a substrate for the cif NT system in L1210 cells (36), and (ii) asa substrate of both adenosine kinase (37) and deoxycytidine kinase (38), the probability of isolating kinase-deficient mutants was reduced. NBMPR was included NMG' in the selection medium because of the possibility that the I N T inhibitor might cause araA retention in the cells by 0 1.0 3.0 5.0 0 1.0 3.0 5.0 blocking efflux of the analog via the es system, as in an earlier study (39). The inclusion of NBMPR in the selection medium L1210iB23.1 L1210iB23.1 20 IIM FB 20 LIM FB also served to protect cif mutants against araA cytotoxicity. .~ , N'G* 50 - 10LIM NBMPR The selection procedure yielded clone B23, which was recloned and designated clone B23.1. L1210/B23.1 cells were Na' 2 5 similar to L1210/MC5-1 cells with respect to cell volume (mean values (S.D., n = 12) were 0.63 k 0.08 and 0.64 4 0.3 .-: 0I p1/106 cells, respectively) and with respect to proliferation rates (population doubling times were 11 and 12 h, respec0 1.0 3.0 5.0 0 1.0 3.0 5.0 tively). Time (min) Cation Dependence of Formycin B Fluxes in Mutant and FIG. 2. Formycin B retention in presence ofNBMPR in Parental Cells-Formycin B, a C-nucleoside analog of inosine, L1210 cells that express Na+-linked nucleoside transport acis poorly metabolized in L1210 cells3 and is a substrate for tivity and in mutant clone. Cells in Na' (0) or NMG' (A) medium the three NTsystems expressed in wild-type L1210 cells (4). with (0,A) or without (0,A) 10 pM NBMPR were exposed to 20 F M The NBMPRsensitivity of formycin B fluxes in L1210/MC5- [3H]formycin B ( F B ) at 22 "C for the time intervals specified, after 1 and L1210/B23.1 cells was explored in the experiment of which the cellular content of labeled permeant was measured. The Fig. 1, which shows that the NBMPR concentration-effect means of triplicate assays are shown. Assay mixtures contained 2 X lo6 cells in 200 pl of medium of the specified content layered over relationship in L1210/B23.1 cells was monophasic and was transport oil. not affected by replacement of Na' with NMG'. Formycin B fluxes in L1210/B23.1 cells were virtuallyeliminated by NBMPR at concentrations in excess of 10 nM. In contrast, suspended in either Na' or NMG' medium. When L1210/ L1210/MC5-1 cells exhibitedacomponent of formycin B MC5-1 cells were assayed in Na' medium that contained transport that was insensitive to inhibition by NBMPR a t NBMPR, a cellular concentration of formycin B of 180 PM concentrations as high as 10 p~ and dependent on the pres- was achieved in 5 min, whereas in L1210/MC5-1 cells susence of Na' in the assay medium. These characteristics indi- pendedinNMG+ medium with NBMPR, formycin Bupcated that cif activity was present in the parentalclone, but take was negligible. The remarkable enhancement in formycin B uptake by L1210/MC5-1 cells that resulted from the was not expressed in the mutant clone. The relative abilities of the parental and mutant cells to presence of NBMPR in Na' medium is attributedto (i) concentrate and retain formycin B were compared in the NBMPR blockade of es-mediated efflux of formycin B and (ii) unimpaired influx of formycin B via the cif system in the experiments of Fig.2, which assessed the effects of 10 p~ presence of NBMPR. The insensitivity to NBMPR of the cif NBMPR o n cellular uptake of20 PM formycin B by cells system in L1210 cells was established clearly in the studies of '' In earlier work, conversion of the cell content of [3H]formycin B Crawford et al. (9) with the clonal line L1210/MA-27.1, which t o "metabolites" in L1210/C2 cells in 5 min at 22 "C was -5% (36). is deficient in the es and ei NT systems. As shown in Fig. 2,

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16954 L1210lB23.1

20

Cells Lack Two Nucleoside Transport Activities

1

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Formycin B

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Loaded wlih non-isotopic FB

Not loaded

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Uptake interval (rnin) FIG. 4. Formycin B countertransport in L1210B23.1 cells.

L 20 pM L-dThd

0

0

6

3

9

12

Uptake Interval (s)

FIG. 3. Effect of Na+ ion replacement on transport of formycin B and thymidine by L1210B23.1 cells. Time courses of cellular uptake of20 pM [3H]formycin B (upper) were measured in Na+ (0,O) or choline+ (A, A) medium, without (0,A) or with 10 p~ NBMPR (0,A).Time courses of cellular uptake of 20 p~ D-[m!thyl"Hlthymidine (lower) or of20 p~ ~-[rnethyl-~H]thymidine (lower) were measured in Na+ (O), choline+ (A),or K+ (V) medium. Intervals of permeant uptake were achieved by successively completing replicate assay mixtures (layered over oil in microcentrifuge tubes) by means of a Digiflex-CX automatic pipettor programed to deliver six 188-pl portions of 3H-permeant at 1.5-s intervals. Intervals of permeant uptake in each set of six assaymixtures were ended by centrifugation of the set assoon as possible after the lastaddition of "H-permeants (see "Experimental Procedures"). Assay mixtures contained 2 X lo6cells in 376 pl (final concentration); means of triplicate assays are plotted.

TABLEI Site-specific binding of NBMPR to cells of L1210 clonal lines Site-specific binding of [3H]NBMPR to intactcells was measured by a previously described method (34) using triplicate samples. Nonspecific binding of [3H]NBMPR was that which occurred in the presence of 20 pM NBMPR. Mass law analysis of specifically bound NBMPR by the Scatchard method yielded the constants listed.

L1210/AM" 2.7L1210/C2b L1210/MC5-1' L1210/B23.1 L1210/MA27.1

nM

fmolecukslcell) X 10'

0.4 0.4 0.2 0.1 NSd

2.5 2.0 1.0 NS

' A clone (29) with a wild-type NT phenotype (10, 11). A cloned wild-type line (28). See Ref. 8. NS, no sites (9).

the accumulative uptake of formycin B did not occur in L1210/B23.1 cells, and the steady-state intracellular concentration of formycin B was roughly that of the extracellular medium and was similar for cells incubated in either Na+or NMG+ medium. In either medium, formycin B uptake was almost completely blocked by NBMPR, a result consistent with loss of the cif system and retention of the es system in the mutantclone. The absence of Na+-dependent nucleoside transport in L1210/B23.1 cells was confirmed in the experiments of Fig. 3, which evaluated the effect of replacing the medium content

Cells were incubated with and without 2 mM nonisotopic formycin B ( F B ) in Na+ medium (10' cells/ml, 10 min at 22 "C) andwere rapidly washed twice at 4 "C in Na+ medium. The cells were then resuspended in Na+ medium at 22 "C containing 20 p~ [3H]formycin B (final concentration), and the mixtures were sampled (100 pl, lo6 cells) after the indicated incubation intervals. The samples were immediately added to 100 pl of 20 p~ NBMPR in Na+ medium layered over 150 pl of transport oil and centrifuged to pellet cells under oil prior to measuring the pellet content of [3H]formycin B. Pellet water space and extracellular medium space were determined in parallel incubation mixtures not exposed to [3H]formycin B.

of Na+with K+ or choline on fluxes of formycin B and thymidine. Mutational deletion of the cif system, which accepts formycin B as a permeant, is evident in the equivalence of formycin B fluxes in Na+-containing and Na+-free media (Fig. 3). Wild-type L1210 cells have previously been shown to lack the cit NT system (4),and the absence of this process was confirmed in an experiment similar to that of Fig. 3 in which time courses for the uptake of20 p M ~-[methyl-~H] thymidine (the physiological enantiomer)in L1210/B23.1 cells were found to be similar whether the cells were suspended in media prepared with Na+, K', or choline+ salts (data not shown). Nonmediated permeation of thymidine in L1210/ B23.1 cells was a minor process under these conditions, as judged by the low fluxes of ~-[methyl-~H]thymidine, the unphysiological enantiomer (Fig. 3). The NT characteristics of the L1210/B23.1 cells so far discussed indicate that thees system expressed in the parental L1210/MC5-1 cells was also expressed in the mutant cells. This inference was supported by the experiments summarized in Table I, which showed that in L1210/B23.1 cells, (i) NBMPR was specifically bound to high affinity sites previously associated with the es nucleoside transporter, and (ii) characteristics of NBMPR binding at those siteswere similar in cells of several L1210 clones. Countertransport of Formycin B in Mutant Cells-The Na+ independence and NBMPR sensitivity of nucleoside fluxes, together with the presence of NBMPR-bindingsitesin L1210/B23.1 cells and the NT phenotype of the parentalcells, argue that the mutant cells express only the es NT system. The achievement of steady-state levelsof formycin Bin L1210/B23.1 cells during an uptake interval of 5 min (Fig. 2) provides evidence for the operation of an equilibrative NT process in these cells. The experiment of Fig. 4 was intended to ascertain if the NT process(es) evident in L1210/B23.1 cells would exhibit countertransport, classic behavior of a facilitated diffusion (equilibrative) transport system (40). After incubation of L1210/B23.1 cells with 2 mM nonisotopic formycin B, the loaded cells were washed twice and resuspended in Na' medium containing 20 pM [3H]formycin B, and the influx of [3H]formycinB was followed over a 10-min

L1210lB23.1 Cells Lack Two Nucleoside Transport Activities

16955 TABLE I1

Nucleoside inhibitors of inward transport of adenosine in L1210fB23.1 cells ) assayed in the presence Inward fluxes of [3H]adenosine (1p ~were and absence of graded concentrations of test nucleosides by the automated procedure described for Fig. 5. Concentration-effect plots for the inhibition of adenosine fluxes by test nucleosides (as described for Fig. 5) yielded the ICs0 values listed. ICbo forinhibition of Test nucleoside 0

2

inward flux of adenosine

4

Time (s)

Control

Of

IC50 flM __ 0 FB

160 A I N 0 40 0 Guo 40

>loo0

I

0

200

600

1000

[Inhibitor], p M

FIG. 5. Inhibition of inward fluxes of adenosine in L1210B23.1 cells by nucleosides. Adenosine fluxes at 22 "C were measured by a method that employed two electrically linked automatic pipettors to startintervals of adenosine uptake by adding [3H] adenosine to replicate assay mixtures and to end such intervals by adding the N T quencher dilazep. Cells were recovered by oil layer centrifugation for assay of 3H content. Pipettor programs enabled simultaneous addition of 3H-permeant and quencher (0)to starttime courses and allowed their addition in sequence at graded intervals. The data comprising the time course (inset) are means & S.D. ( n = 4); the cellular uptake of [3H]adenosine during the interval of 2.25 s is seen to measure the initial rate of adenosine uptake under these conditions and was therefore a measureof adenosine flux. To evaluate the inhibition of adenosine fluxes in L1210/B23.1 cells under these conditions (22 "C, Naf medium, 1p~ [3H]adenosine (finalconcentration)), replicate assay mixtures (total volume of 376 pl) were completed by the simultaneous additionof 94-pl portions of [3H]adenosine and test nucleoside by means of a Digiflex-TP automatic pipettor to start intervals of permeant uptake. After a programed interval of 2.25 s, cellular uptake of [3H]adenosine in the assay mixture was ended by the automated addition of 188 pl of quencher (300 ~ L Mdilazep) by a Digiflex-CX pipettor (100 pM dilazep final concentration).The assays were conducted in quadruplicate. Shown are the effects of graded concentrations of formycin B ( F B ) , inosine, and guanosine on adenosine flux in L1210/B23.1 cells as well as ICs0 values for the three nucleoside competitors. The ICs0 values for nucleoside inhibition of adenosine uptake shown in Table I1 were obtained by this procedure.

interval. Initially, [3H]formycin B influx proceeded against the concentration gradient of nonisotopic formycin B, indicatingthat nonisotopic molecules of formycin B initially dominated the competition between intracellular 3H-labeled and nonisotopic molecules for transporter-mediated movement of formycin B in the outward direction. After 5 min, the labeled and nonisotopic species were approaching equilibrium. Substrate Specificity of the L1210 es Transporter-In expressing only es NT activity, the L1210/B23.1 clonal line allowed exploration of the substrate specificity of the L1210 es system in the absence of the ei or cif transporters. Substrate specificity was examined in competition experiments (Fig. 5 ) in which graded concentrations of test nucleosides were evaluated for inhibition of inward fluxes of [3H]adenosine. This competitive approach yielded the ICsovalues for inhibition of adenosine flux at 22 "C listed in Table 11. It is apparent that the es system expressed by L1210/B23.1 cells is of broad substrate specificity and, in accepting both purine and pyrim-

Adenosine 2'-Deoxyadenosine Guanosine 2'-Deoxyguanosine Inosine Xanthosine Formycin B Tubercidin Uridine 2"Deoxyuridine Cytidine 2'-Deoxycytidine Thymidine

e10 e20 40 150 40

185 20 165 165 300 50 60

I

FIG. 6. Saturability of inward fluxes of adenosine in L1210B23.1 cells. Time courses for the uptake of [3H]adenosine at various concentrations by L1210/B23.1 at 22 'C were obtained by the dilazep-stop method outlined for Fig. 5. At adenosine concentra, uptake of adenosine during the first 2.25 s tions up to60 p ~cellular of exposure measured initial rates of the uptake process and therefore measured unidirectional inward fluxes of adenosine. The concentration-flux relationship for adenosine transport in L1210/B23.1 cells is presented in the form of an s/v versus s plot.

idine nucleosides as permeants, resembles the es system of human erythrocytes. The relationship between inward fluxes of adenosine and concentration that was explored in the experiment of Fig. 6 showed flux saturation and a K,,, of 16 pM for adenosine at 22 "C.K,,, values reported for adenosine influx into various types of mammalian cells at 22 "C range between 10 and 120 FM, and V,,, values between 5 and 49 pmol/pl of cell water/s have been reported (32, 41-43). In conclusion, we report the isolation and characterization of a doubly mutated clone of L1210 cells in which the es nucleoside transporter is the sole NT mechanism for transport of both purine and pyrimidine nucleosides. REFERENCES 1. Gati, W. P., and Paterson, A. R. P. (1989) in Red Blood Cell Membranes (Agre, P., and Parker, J. C., e&) pp. 635-661, Marcel Dekker, Inc., New

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14. 15. 16. 17. 18. 19. 20. 21. 22.

L121 OIB23.1 Cells Lack Two Nucleoside Transport Activities

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23. Plagemann, P.G. W., and Aran, J. M. (1990) Biochim. Biophys. Acta 1 0 2 6 , 32-42 24. Plagemann, P. G. W., and Aran,J. M. (1990) Biochim. Biophys. Acta 1 0 2 8 , 289-298 25. Baer, H. P., and Mooji, A. (1990) Biochim. Biophys. Acta 1026,241-247 26. Spector R., and Huntoon, S. (1984) J. Neurochem. 42,1048-1052 27. Jakobs,'E. S., Van Os-Corby, D. J., and Paterson, A. R. P. (1990) J. Biol. Chem. 266,22210-22216 28. Cass, C. E., and Au-Yeung, T. H. (1976) Cancer Res. 36,1486-1491 29. Bennett, L. L., Jr., Bowdon, B. J., Shaddix, S. C., Dubois, N. F., Hughes, J., and Brockman, R. W. (1982) Proc. Am. Assoc. Cancer Res. 2 3 , 6 30. Harley, E. R., Paterson, A. R. P., and Cass, C. E. (1982) Cancer Res. 42, 1289-1295 ~~~. - ~ . . 31. Dulbecco, R., and VoM. (1954) J. Exp. Med. 99,167-182 32. Paterson, A.R. P., arley, E. R., and Cass, C. E. (1984) Biochem. J. 2 2 4 , 1nnl -1nna 33. Pande, S. V. (1976) Anal. Biochem. 7 4 , 25-34 34. Cass C. E Kolassa NUehara Y. Dahlig-Hade E., Harley E., and Piterson: A. R. P. '(19g1) B i o c h k biophys. Acta &9, 769-773 35. Paul, B., Chen, M. F., and Paterson, A. R. P. (1975) J. Med. Chem. 1 8 , 968-973 36. Dagnino, L. (1988) Sodium-driuen Nucleoside Transport in Mouse Leukemia L1210 Cells. Ph.D. thesis Universit of Alberta 37. Bloch, A., Leonard, R. J., and Nichol, C. (1967) Biochim. Biophys. Acta 1 3 8 , 10-25 38. Momparler, R. L., and Fischer,G. A. (1968) J. Biol. Chem. 243,4298-4304 39. Jakobs E. S. andPaterson, A. R. P. (1986) Biochem. Biophys. Res. Comkun. lhO,lO28-1035 40. Oliver, J. H., and Paterson, A. R. P. (1971) Can. J. Biochem. 49,262-270 41. Plagemann, P. G. W., Wohlhueter, R. M., and Kraupp, M. (1985) J. Cell. Physiol. 125,330-336 42. Paterson, A. R. P., Kolassa, N., and Cass, C. E. (1981) Pharmacol. & Ther. 12,515-536 43. Pla emann, P. G. W., and Wohlhueter, R. M. (1980) Curr. Top. Membr. 8 a n s p . 14,225-330

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