MOLECULAR AND CELLULAR BIOLOGY, Apr. 1986, p. 1296-1303 Vol. 6, No. 40270-7306/86/041296-08$02.00/0Copyright © 1986, American Society for Microbiology

Incomplete Nucleoside Transport Deficiency with IncreasedHypoxanthine Transport Capability in Mutant

T-Lymphoblastoid CellsBRUCE ARONOW,l PAULA HOLLINGSWORTH,' JOSEPH PATRICK,' AND BUDDY ULLMAN2*

Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536,1 and Department ofBiochemistry, Oregon Health Sciences University, Portland, Oregon 972012

Received September 1985/Accepted 14 January 1986

From a mutagenized population of wild-type mouse (S49) T-lymphoma cells, a clone, 80-5D2, was isolatedin a single step by virtue of its ability to survive in 80 nM 5-fluorouridine. Unlike previously isolated nucleosidetransport-deficient cell lines (A. Cohen, B. Ullman, and D. W. Martin, Jr., J. Biol. Chem. 254:112-116, 1979),80-5D2 cells were only slightly less sensitive to growth inhibition by a variety of cytotoxic nucleosides and werecapable of proliferating in hypoxanthine-amethopterin-thymidine-containing medium. The molecular basis forthe phenotype of 80-5D2 cells was incomplete deficiency in the ability of the mutant cells to translocatenucleosides across the plasma membrane. Interestingly, mutant cells were more capable than wild-type cells oftransporting the nucleobase hypoxanthine. Residual transport of adenosine into 80-5D2 cells was just assensitive to inhibition by nucleosides and more sensitive to -inhibition by hypoxanthine than that in wild-typecells, indicating that the phenomena of ligand binding and translocation can be uncoupled genetically. The80'5D2 cells lacked cell surface binding sites for the potent inhibitor of nucleoside transport p-nitrobenzylthioinosine (NBMPR) and, consequently, were largely resistant to the physiological effects ofNBMPR. However, the altered transporter retained its sensitivity ¢o dipyridamole, another inhibitor ofnucleoside transport. The biochemical phenotype of the 80-5D2 cell line supports the hypothesis that thedeterminants that comprise the nucleoside carrier site, the hypoxanthine carrier site, the NBMPR binding site,and the dipyridamole binding site of the nucleoside transport function of mouse S49 cells are geneticallydistinguishable.

Transport of nucleosides across the mammalian plasmamembrane occurs by a facilitated diffusion mechanism whichappears to be mediated by a single carrier of broad speci-ficity (4, 11, 18, 21, 28). Mutant murine T-lymphoma (S49)cells have been generated in which the nucleoside transportfunction has been genetically altered (1, 5, 9, 11, 30, 32). Theexistence of nucleoside transport-deficient S49 cell lines hasdemonstrated genetically that the translocation of virtuallyall nucleosides requires a common component, presumablythe membrane carrier (la, 5, 11, 30, 32). The genetic defi-ciency in nucleoside transport can be pharmacologicallysimulated by p-nitrobenzylthioinosine (NBMPR), a highlyspecific inhibitor of nucleoside transport (1, 4-6, 19-22).NBMPR abrogates the transport of most nucleosides, suchas adenosine and cytidine, by S49 cells (1, 5). Nucleosidetransport-deficient cells have lost their sensitivity to andbinding sites for NBMPR (1, 5). Characterization of thesenucleoside transport-deficient cell lines provides powerfulgenetic evidence that the transport of all nucleosides by S49cells requires a common component that is sensitive tocomplete inhibition by NBMPR.

In this report, we describe the isolation and characteriza-tion of a unique nucleoside transport-deficient S49 clone,which still partially retained some nucleoside transport ca-pacity, had lost all high-affinity binding sites for NBMPR,yet possessed increased capacity to transport hypoxanthine.The characterization of this mutant cell line has allowedconsiderable insight into the properties of nucleoside andnucleobase transport in S49 cells.

* Corresponding author.

MATERIALS AND METHODS

Chemicals and reagents. [3H]adenosine (20 Ci/mmol),[3H]cytidine (22 Ci/mmol), [3H]guanosine (11 Ci/mmol),[3H]deoxyguanosine (7 Ci/mmol), [3H]deoxyadenosine (28Ci/mmol), [3H]inosine (17 Ci/mmol), [3H]thymidine (75Ci/mmol), [3H]adenine (40 Ci/mmol), [3H]hypoxanthine (25Ci/mmol), and [3H]NBMPR (16 Ci/mmol) were purchasedfrom Moravek Biochemicals, Inc., 3H20 (1.0 mCi/ml) and[methoxy-3H]methoxyinulin (466.4 mCi/g) were obtainedfrom the New England Nuclear Corp. Methotrexate wasobtained from Lederle Laboratories in a solution at 25 mg/ml.N-Methyl-N-nitroso-N'-nitroguanidine was provided by K &K Laboratories, Inc., of ICN Pharmaceuticals, Inc.Erythro-9-(2-hydroxy-3-nonyl)-adenine was bought from theBurroughs Wellcome Co. NBMPR, dipyridamole, and allnucleobases, nucleosides, and nucleotides were obtainedfrom the Sigma Chemical Co. All other materials and reagentswere of the highest quality commercially available.

Cell culture. S49 cells were originally derived from aBALB/c mouse by Horibata and Harris (14). The S49 cellline is a T-cell lymphoma, and its growth characteristics andlymphocytic properties have been described previously indetail (14, 29). S49 cells grew in suspension culture inDulbecco modified Eagle medium containing 3.7 g of sodiumbicarbonate and 4.5 g of D-glucose per liter supplementedwith 10% horse serum which had been heat inactivated at56°C for 30 min. S49 cells were propagated continuously in ahumidified 10% CO2 atmosphere at 37°C.

Mutagenesis and selection of the 80-5D2 cell line. Wild-typeS49 cells were expanded to a density of 106 cells per ml in avolume of 200 ml and mutagenized by incubation with 3 ,ug

12%

ALTERED NUCLEOSIDE TRANSPORT IN ANIMAL CELLS

of N-methyl-N-nitroso-N'-nitroguanidine per ml for 3 h at37°C as previously described (11, 30, 32). The cells wereseparated from the mutagen by centrifugation and suspendedin fresh growth medium. Approximately 75% of the cellswere killed by this N-methyl-N-nitroso-N'-nitroguanidineexposure. Surviving cells were expanded in nonselectivegrowth medium for 7 to 10 generations to ensure expressionof the mutant phenotype. The mutagenized cells were clonedin semisolid agarose (SeaKem-ME) overlying mouse embryofibroblast feeder layers, as originally described by Coffino etal. (8). The 80-5D2 cell line was picked from the surface of aselective plate containing 80 nM 5-fluorouridine. The fre-quency of appearance of nucleoside transport-deficientclones in a mutagenized wild-type population is about 10-8.The origin of the KAB5 cell line used in one experiment hasbeen described previously (1).Growth rate determinations. The abilities of wild-type and

80-5D2 cells to proliferate in the presence of increasingconcentrations of cytotoxic nucleosides were ascertained inCostar 24-well tissue culture plates as previously described(31). Two wells of each cell line were maintained in theabsence of drugs as controls. Cells were enumerated on amodel ZB1 Coulter Counter after 3 days of growth. Initialcell densities were subtracted from final cell densities, andthe resulting differences were plotted as the percentage ofcontrol growth as a function of the concentration ofexogenous nucleoside or nucleoside analog.Measurements of NBMPR binding sites. Binding of

[3H]NBMPR to intact wild-type and 80-5D2 cells was mea-sured by the glass fiber filter disk method previously de-scribed by Aronow et al. (1). Cells (7 x 106) were suspendedin 1.0 ml of phosphate-buffered saline at ambient tempera-ture and incubated with 0.1 to 1.6 nM [3H]NBMPR (16Ci/mmol) for 20 min in the absence or presence of 20 puMunlabeled NBMPR. Cells were separated from unbound[3H]NBMPR by filtration on glass fiber filter disks andwashed four times with 5-ml portions of ice-cold phosphate-buffered saline, and cell-associated radioactivity wascounted by liquid scintillation counting. The amount ofspecifically bound NBMPR at each concentration was takento be the difference in binding of [3H]NBMPR to cells in theabsence or presence of 20 ,uM nonradioactive NBMPRpresent in the initial incubation mixture.

Nucleoside and hypoxanthine transport measurements. Theinflux of nucleoside into wild-type and 80-5D2 cells oververy short time intervals was measured by the rapid sam-pling technique described by Cass et al. (5) and Aronow etal. (1). Aronow and Ullman (lb) have adapted this rapidsampling procedure for measuring hypoxanthine transportover short time intervals. When inhibitors of nucleosidetransport were tested for their effects on transport, cellswere pretreated for 20 min with that inhibitor. All transportmeasurements were performed in 1.5-ml polypropylenetubes (Sarstedt) with caps removed at 37°C unless otherwiseindicated. Transport medium (100 ,ul) containing 10 ,uCi ofradiolabeled nucleoside or hypoxanthine per ml at variousspecific activities was layered over 100 Rl of an inert oilmixture consisting of silicone oil (Aldrich Chemical Co.,Inc.; catalog no. 17,563-3)-paraffin oil (Fischer ScientificCo.; catalog no. 0-119) in a ratio of 94 to 6. The density ofthis mixture was 1.03 g/ml, sufficient to allow rapid sedimen-tation of S49 cells at 10,000 x g in a Brinkmann Instruments,Inc., microcentrifuge. Transport assays were initiated byaddition of 100 RI of the cell suspensions to the aqueousphase containing radiolabeled ligand and terminated bycentrifugation. The aqueous phase was aspirated, the sur-

face of the oil was washed twice with 1.0 ml of phosphate-buffered saline, the oil was removed by aspiration, and thecell pellet was lysed in 100 ,ul of 2% Triton X-100. Afteraddition of 1.0 ml of 3a70B scintillation fluid (ResearchProducts International Corp.) to each Eppendorf tube, ra-dioactivity was counted by liquid scintillation. Intracellularvolumes of wild-type and 80-5D2 cells were assessed to beequal as determined by the differences in amounts of 3H20and [methoxy-3Hlmethoxyinulin associated with cell pelletscentrifuged through the same inert oil mixture used in rapidtransport determinations.

RESULTS

Growth rate determinations. The 80-5D2 cell line wasisolated in semisolid agarose by virtue of its resistance to 80nM 5-fluorouridine, a cytotoxic pyrimidine analog that hasbeen used previously in this laboratory to isolate nucleosidetransport-deficient clones (la, 32). In comparative growthrate experiments in suspension culture, 80-5D2 cells wereless sensitive to 5-fluorouridine toxicity (Fig. 1). The effec-tive concentration of 5-fluorouridine which inhibited growthof wild-type cells by 50% (EC50) was 7 nM, whereas the EC50of 80-5D2 cells for the selective agent was 33 nM. The highcell density of the cells plated during the selection procedure

*-WT) a-WT +10pM NBMPR

100 ~~~~~~WT+IO0pM DPA

80

060

4400

20-0

c I

I.-4I

(-9

100 1,05- FLUOROURIDINE (yM)

FIG. 1. Effects of 5-fluorouridine on the growth of wild-type(WT) and 80-5D2 cells in the absence and presence of inhibitors ofnucleoside transport. The abilities of WT (panel A) and 80-5D2(panel B) cells to grow in the presence of increasing concentrationsof 5-fluorouridine in the absence or presence of either 10 .LMNBMPR or 10 F.M dipyridamole (DPA) were ascertained as de-scribed in Materials and Methods. The results are those of a single,typical experiment which was repeated five times with identicalresults.

VOL. 6, 1986 1297

1298 ARONOW ET AL.

could account for the ability of the 80-5D2 cell line to surviveand grow in semisolid medium containing 80 nM 5-fluorouridine. The ability of 80-5D2 cells to grow in thepresence of increasing concentrations of a spectrum ofcytotoxic nucleosides against which they were not selectedwas also examined (Table 1). The 80-5D2 cells were slightlyless sensitive to adenosine, arabinosylcytosine, deoxyaden-osine, 6-thioguanosine, deoxyguanosine, and guanosine thanwere wild-type parental cells, but they were equally sensi-tive to thymidine. Erythro-9-(2-hydroxy-3-nonyl)-adeninewas added to growth experiments with adenosine and de-oxyadenosine to prevent deamination (25). The partial re-sistance of 80-5D2 cells to multiple nucleosides and nucleo-side analogs was consistent with a deficiency in nucleosidetransport. However, 80-5D2 cells were much more sensitiveto these agents than previously characterized nucleosidetransport-deficient S49 cells (11, 32).The transport of nucleosides by wild-type S49 cells is

profoundly inhibited by two compounds, dipyridamole (26)and NBMPR (6, 7, 19, 20, 33). NBMPR can protect wild-type S49 cells from the adverse effects of most toxic nucle-osides, whereas dipyridamole protects S49 cells from somenucleosides, and then only to a small degree (1). NBMPRincreased the EC50 of wild-type cells for 5-fluorouridine1,000-fold to 7 ,uM, while dipyridamole augmented the EC50

TABLE 1. Sensitivities of wild-type and 80-5D2 cells to cytotoxicnucleosides in the absence and presence of inhibitors of

nucleoside transport

Nucleoside and EC50 (,uM)addition Wild-type 80-5D2

cells cells

AdenosineNone 5 35Dipyridamole 120 500NBMPR 1,000 60

ArabinosylcytosineNone 0.025 0.175Dipyridamole 0.2 0.5NBMPR 1.5 0.2

6-ThioguanosineNone 0.3 0.5Dipyridamole 3 3NBMPR 5 0.5

DeoxyadenosineNone 4 7Dipyridamole 20 30NBMPR 100 15

DeoxyguanosineNone 30 120Dipyridamole 150 400NBMPR 300 175

GuanosineNone 200 600Dipyridamole 400 600NBMPR 600 600

ThymidineNone 40 40Dipyridamole 40 150NBMPR 400 40

I

r-

03.

z

0

0$r

HYPOXANTHINE (juM)FIG. 2. Comparison of the abilities of wild-type (WT) and 80-5D2

cells to salvage hypoxanthine to overcome azaserine cytotoxicity.WT (panel A) and 80-5D2 (panel B) cells were exposed to 2 ,ug ofazaserine per ml and various concentrations of hypoxanthine in theabsence or presence of either 10 ,uM NBMPR or 10 ,uMdipyridamole (DPA). Cell densities were measured after 72 h asdescribed in Materials and Methods. The results depicted are thoseof a single experiment which was repeated five times with similarresults.

75-fold to 0.53 ,M (Fig. 1). NBMPR had virtually noprotective effect on the sensitivity of 80-5D2 cells to 5-fluorouridine, whereas dipyridamole increased the EC50 over30-fold to approximately 1.1 ,uM. The lack of a protectiveeffect ofNBMPR toward nucleoside toxicity for 80-5D2 cellscould be demonstrated with other toxic nucleosides as well(Table 1). On the other hand, dipyridamole was effective atprotecting both wild-type parental and mutant 80-5D2 cellsfrom nucleoside toxicity. Curiously, dipyridamole did notprotect wild-type cells from thymidine toxicity, whereas theEC50 of mutant cells for thymidine was raised from 40 to 150,M by 10 ,uM dipyridamole (Table 1).Unlike previously characterized nucleoside transport-

deficient S49 cell lines (la, lb), 80-5D2 cells could salvagehypoxanthine in the presence of azaserine, a potent inhibitorof purine biosynthesis (13), as efficiently as did wild-typeparental cells (Fig. 2) and were capable of proliferating inhypoxanthine-amethopterin-thymidine (HAT) medium (Fig.3). NBMPR was ineffective in blocking the salvage ofhypoxanthine by 80-5D2 cells in hypoxanthine-azaserine-containing medium, whereas dipyridamole inhibited hypo-xanthine salvage in both parental and mutant cells (Fig. 2).Finally, growth of wild-type cells in HAT medium wasblocked only by NBMPR, whereas growth of 80-5D2 cells inHAT medium was inhibited by dipyridamole but not byNBMPR (Fig. 3).NBMPR binding measurements. Since the growth sensitiv-

MOL. CELL. BIOL.

ALTERED NUCLEOSIDE TRANSPORT IN ANIMAL CELLS

WT 80-5D2NBMPR U ODPA A A

NBMPR CONC. (yM) DPA CONC. (pM)

FIG. 3. Ability of wild-type (WT) and 80-5D2 cells to surviveHAT medium in the absence and presence of inhibitors of nucleo-side transport. The abilities of WT and 80-5D2 cells to grow in thepresence of 0.5 mM hypoxanthine-0.4 ,M amethopterin-16 ,uMthymidine were ascertained in the absence or presence of increasingconcentrations of either NBMPR or dipyridamole (DPA). Theresults are plotted as a percentage of growth in the absence of HATmedium (control growth) as a function of the concentration of theinhibitor of nucleoside transport. These results are from one of threevirtually identical experiments.

ity and ability to accumulate nucleosides of 80-5D2 cellswere unaffected by NBMPR, the number of cell surfacebinding sites for [3H]NBMPR on mutant cells was comparedwith that on wild-type cells. Previously characterized nucle-

oside transport-deficient cells have been shown to lackNBMPR binding sites (1, 5). The 80-5D2 cells were com-pletely deficient in high-affinity NBMPR binding sites underconditions which permitted their detection in wild-type cells(Fig. 4).

Nucleoside and nucleobase transport measurements. Thelack of NBMPR binding sites and the resistance of 80-5D2cells to multiple nucleosides which are metabolized bybiochemically distinct metabolic pathways suggested that80-5D2 cells might possess a defect in their nucleosidetransport system. Therefore, the abilities of wild-type andmutant cells to transport a variety of nucleosides over shorttime intervals were compared. The 80-5D2 cells were 85 to90% deficient in their ability to transport 2 ,uM concentra-tions of cytidine, thymidine, deoxyguanosine, deoxyadeno-sine, and adenosine (Fig. 5). NBMPR (10 ,uM) virtuallyabolished adenosine transport in wild-type cells, whereasresidual transport of adenosine by 80-5D2 cells was onlypartially inhibited by 10 puM NBMPR (Fig. 5). Conversely,10 puM dipyridamole inhibited adenosine transport by 80-5D2and wild-type cells to equivalent degrees.Although 80-5D2 cells possessed a defect in their nucleo-

side transport system, they still transported nucleosides at ameasurable rate. We therefore measured the effects ofincreasing concentrations of nonradiolabeled nucleoside on[3H]adenosine transport in parental and 80-5D2 cells. Figure6 depicts the results of an experiment in which nonradiola-beled nucleosides interfered with adenosine transportequally in wild-type and 80-5D2 cells. Hypoxanthine (1 mM),however, inhibited [3H]adenosine entry into 80-5D2 cells toa greater extent than it inhibited its entry into parentalcells.

Previous studies indicated that 80-5D2 cells, despite theirnucleoside transport deficiency, transport hypoxanthine

WT

Sites X 40,000/cellKd 3x 10-10M

80 -5D2

1.2--

1.)NBMPR (nM)

FIG. 4. Quantitation ofNBMPR binding sites on wild-type (WT)and 80-5D2 cells. The number of high-affinity NBMPR binding sitesand their affinity for NBMPR were determined for WT and 80-5D2cells as described in Materials and Methods. Cells (7 x 106) of eachtype were exposed to increasing nanomolar concentrations of['H]NBMPR for 20 min in the absence or presence of 20 ,uMnonradioactive NBMPR. The amount of [3H]NBMPR bound was

taken to be a measure of nonspecifically bound NBMPR andsubtracted from the amount of radioactivity associated with the cellsin the absence of nonradiolabeled NBMPR. The depicted experi-ment is one of numerous experiments that always gave identicalresults.

50

2w

0ob-

E

H 2500~U,z

tr

w

0.6.D Ja

0- C'

TF

FL

I

CYD dTHD dGUO dADO ADO ADO ADO(+ DPA) (+NBMPR)

FIG. 5. Nucleoside transport in wild-type and 80-5D2 cells. Theabilities of wild-type (open bars) and 80-5D2 (hatched bars) cells totransport cytidine (CYD), thymidine (dTHD), deoxyguanosine(dGUO), deoxyadenosine (dADO), and adenosine (ADO) weredetermined as described in Materials and Methods. The effects of 10F.M dipyridamole (DPA) and 10 FM NBMPR on adenosine transportwere also determined for wild-type and 80-5D2 cells. Transportmeasurements were always carried out with 2 F.M radioactiveligand. The results depicted are those of a single experiment,versions of which were repeated multiple times.

T

-J0CK

crz0(-

-J-Jw0

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w0

VOL. 6, 1986 1299

r-", --Fb

1300 ARONOW ET AL.

more efficiently than do wild-type cells (lb). A comparisonof the abilities of parental and 80-5D2 cells to transportadenosine and hypoxanthine as a function of concentrationat 37°C indicated that the mutation conferred opposite alter-ations in the maximal velocities of transport. The maximalvelocities of adenosine and hypoxanthine transport by80-5D2 cells were 40 and 170% of those obtained for wild-type cells (Fig. 7). The data indicated a fourfold increase inthe apparent Km for adenosine transport by 80-5D2 cells(Fig. 7). Consistent with their NBMPR-resistant growthphenotype, the hypoxanthine transport capacity of 80-5D2cells was much less sensitive to inhibition by NBMPR,whereas hypoxanthine transport was inhibited to an equiv-alent extent by dipyridamole in wild-type and mutant cells(Fig. 8). Moreover, high concentrations of nucleosides couldinhibit transport of hypoxanthine by 80-5D2 cells. Inosine,uridine, and thymidine, each at a concentration of 5 mM,inhibited hypoxanthine by 74, 55, and 80%, respectively.

A

0

4 450C

x300

C

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e69

c 150I..

loar

Km ("IM) Vmax(nmol/min/l09o*Wildtype 950±250 71 ±20080-5D2 570±200 123±50 /

20 30

Hypoxanthin(1 (uM)-1

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0

.E

E

z 120a-

(nz

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6

.

WT

*

80 - 5D2

B

go

E11%

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0

I,.

a

A

0<' 5 50 500 5000NUCLEOSIDE CONCENTRATION (pM)

FIG. 6. Competition for adenosine (ADO) transport by purinenucleosides and hypoxanthine by wild-type (WT) and 80-5D2 cells.The rates of [3H]ADO transport by WT and 80-5D2 cells weredetermined in the absence or presence of an excess of nonradiola-beled guanosine (V), deoxyguanosine (0), thymidine (A), cytidine(U), or hypoxanthine (0) as described in Materials and Methods.The concentration of [3H]ADO was 5 FM, whereas those ofnonradiolabeled competitors ranged from 5 ,uM to 5 mM.

Kmn(jM)*Wildtype 34±9

0 80-5D2 128 ± 32

so

Vmax(nmol/min/ 1 092008 ± 250

816± 140 Z

-1 -1

Adenosine1 (yM)FIG. 7. Lineweaver-Burk analyses of hypoxanthine and adeno-

sine transport by wild-type and 80-5D2 cells. The abilities ofwild-type and 80-5D2 cells to transport increasing concentrations ofhypoxanthine (panel A) and adenosine (panel B) were tested asdescribed in Materials and Methods.

The residual rate of hypoxanthine transport by 80-5D2 cellsin the presence of competing nucleosides was far below thatof wild-type cells and equivalent to the amount ofnucleobase transport observed for wild-type cells that doesnot appear to be mediated by the nucleoside transportfunction (lb).Another method by which alterations in transporters can

be demonstrated is by assessing sensitivities toward sulfhy-dryl reagents. p-Chloromercuribenzenesulfonate, a sulfhy-dryl reagent which has been used previously to demonstratealterations in the nucleoside transporter (1), inhibited trans-port of adenosine into 80-5D2 cells to a greater extent thaninto wild-type cells (Fig. 9). Nevertheless, the extent ofinhibition was less than that observed with the NBMPR-insensitive transporter of KAB5 cells, a previously charac-terized mutant S49 cell line which possessed both NBMPR-sensitive and NBMPR-insensitive transport components (1).

40 50

MOL. CELL. BIOL.

AC)_r-

Is

ALTERED NUCLEOSIDE TRANSPORT IN ANIMAL CELLS

0 1 2 3LOG CONCENTRATION (nM)

FIG. 8. Effects of NBMPR and dipyridamole on hypoxanthinetransport in wild-type and 80-5D2 cells. The effect of increasing logconcentrations ofNBMPR (U, 0) and dipyridamole (A, A) to inhibitthe ability of wild-type (U, A) and 80-5D2 (O, A) cells to transport50 FM hypoxanthine across the plasma membrane are depicted. Theresults are those of a single experiment, parts of which wererepeated several times with essentially identical results.

DISCUSSION

In these studies, we describe a novel S49 mutant cell line,80-5D2, which has lost a substantial proportion of its nucle-oside transport capability and all of its NBMPR bindingcapacity yet transports the nucleobase hypoxanthine moreeffectively than do wild-type parental cells. Two alternativehypotheses can be proposed to explain the unique biochem-ical phenotype of the 80-5D2 cell line. The first hypothesis ispredicated on the existence of multiple classes of carriers forthe same substrates. Mammalian cells are known to possessseveral forms of nucleoside transport that vary with respectto their kinetic parameters (4, 22), sodium dependence (17,27), and sensitivity to NBMPR (3, 22). Additionally, rodent(12, 24) and human (10) cells have been shown to possesstwo types of inosine transport system, the first a facilitateddiffusion mechanism and the second a purine nucleosidephosphorylase-dependent group translocation. The hypothe-sis that the complex phenotype of 80-5D2 cells can beattributed to alterations in more than one gene product mustbe considered. For instance, it is certainly possible that agenetic switching event or a double mutation could havecaused loss of expression of the wild-type transport functionwith simultaneous activation of an allele, leading to the80-5D2 transport phenotype. Such genetic switching eventshave been documented in the mammalian genome, as inswitching from fetal to adult hemoglobin. In selections forS49 variants with NBMPR-insensitive transporter functions,most of the cell lines had a normal complement of NBMPRbinding sites, whereas 10 to 20% of the NBMPR-insensitiveclones had lost 50 to 75% of their binding sites (1). These

latter results could also be explained by activation of asecond gene. However, the possibility of a gene activationevent accounting for the 80-5D2 phenotype does not alter theconclusion that a gene coding for nucleoside transport orthat a gene affecting a modifying system for nucleosidetransport is altered in 80-5D2 cells.A second explanation that can account for the complex

phenotype of the 80-5D2 cell line is that a single gene codingfor a membrane function that carries both nucleosides andhypoxanthine and binds NBMPR was altered in the mutantcell line. Several lines of evidence favor the existence of onlya single nucleoside transport function in wild-type S49 cells.First, we have generated 10 other independent nucleosidetransport-deficient cell lines in single-step selections, someof which have been described (la, lb, 11). Were S49 cells toexpress two genetic loci coding for nucleoside transportfunction, generation of nucleoside transport-deficient cellswould have been unlikely. Second, purine nucleoside phos-phorylase-deficient S49 cells transport nucleosides normally,indicating that the group translocation mechanism is notoperative in this cell line (30). Third, the possibility that the80-5D2 cell line arose from independent mutations in twogenes, one coding for nucleoside transport and one codingfor hypoxanthine, is improbable, since Aronow and Ullman(lb) have demonstrated that two other nucleoside transport-deficient S49 cell lines are markedly deficient in the ability totransport hypoxanthine, as well as the ability to transportadenine, and that nucleosides and hypoxanthine each com-petitively inhibit the transport of the other. Thus, the nucle-oside transport system of S49 cells, like that described inother cell lines (23) is capable of transporting nucleobases.Fourth, nucleosides inhibit translocation of hypoxanthine,and hypoxanthine inhibits adenosine entry in 80-5D2 cells.Fifth, since all the nucleoside transport-deficient cell linescharacterized thus far lack NBMPR binding sites, it is likelythat the gene that controls nucleoside and nucleobase trans-

Z00 \0

z0

0g 75-

0a-U)z 50-

wzEn- 25-0zwo ~~~~~~~~~~~x

X

0 0.05 0.1 02 05pCMBS (mM)

FIG. 9. Effects of p-chloromercuribenzenesulfonate (pCMBS)on adenosine transport by wild-type and mutant cells. The inhibitoryeffects of increasing concentrations of pCMBS on adenosine trans-port by wild-type (0), 80-5D2 (0), and NBMPR-treated KAB5 (X)cells were measured as described in Materials and Methods. KAB5cells are mutant S49 cells generated in a single step and, unlikeparental wild-type cells, possess both an NBMPR-sensitive and anNBMPR-insensitive nucleoside transport component.

VOL. 6, 1986 1301

1302 ARONOW ET AL.

port is also required for expression of high-affinity NBMPRbinding sites. Finally, although several different mammaliancell lines possess both NBMPR-sensitive and NBMPR-insen-sitive nucleoside transport systems (1, 3, 22), several groups

have demonstrated that nucleoside transport in S49 cells isobliterated by submicromolar concentrations of NBMPR (1,5). Thus, the wild-type S49 cell line, from which the 80-5D2cell line was derived, possesses only an NBMPR-sensitivenucleoside transport component.The residual transport capacity of 80-5D2 cells has al-

lowed genetic dissection of nucleoside transport functionthat cannot be probed with mutants totally deficient innucleoside transport. Several important conclusions con-

cerning the nucleoside transport function of S49 cells can bemade from the results of these studies. First, althoughnucleoside transport-deficient cells lack NBMPR bindingsites (1, 5), the residual capacity of 80-5D2 cells to transportnucleosides indicated that there does not necessarily exist a

one-to-one correlation between the number of NBMPRbinding sites and the number of nucleoside carrier sites.Based on studies indicating that the transport capacity oferythrocytes among mammalian species is linearly propor-

tional to the number of NBMPR binding sites and on theresults of competition studies between NBMPR and nucle-osides for either NBMPR binding or nucleoside transport (1,7, 15, 16), Jarvis and Young (15, 16) have proposed thatNBMPR and nucleosides bind to a common site on thenucleoside transport function. However, residual transportcapacity in cells completely deficient in NBMPR binding,coupled with the ability to generate S49 mutants, such as theKAB5 cell line, which have gained an NBMPR-insensitivetransport function (1, 9), provide strong genetic evidencethat the nucleoside carrier site and the NBMPR binding sitein S49 cells can be distinguished genetically. Second, 80-5D2cells, having lost their NBMPR binding sites, were refrac-tory to the pharmacological effects of NBMPR. Neverthe-less, by a variety of criteria, including growth sensitivity andability to transport, 80-5D2 cells had retained their sensitiv-ity to dipyridamole, indicating that the two inhibitors ofnucleoside transport, dipyridamole and NBMPR, interactwith different subsets of determinants on the transporter.

Interestingly, dipyridamole protected 80-5D2 cells from thetoxicity of certain nucleosides to a greater extent thanwild-type cells (Table 1). A likely explanation for this effectis that, in the presence of dipyridamole, the genetic andpharmacologic deficiencies in nucleoside transport in 80-5D2cells act in an additive fashion. Third, the residual transportin 80-5D2 cells has enabled us to determine that the geneticdefect in this nucleoside transport-deficient line did not

result in lack of binding capacity for nucleosides but ininability to translocate the permeant efficiently, since trans-port of nucleosides could be competed for by other nucleo-sides equally effectively in wild-type and 80-5D2 cells.Whether the inability of other nucleoside transport-deficientcell lines to transport nucleosides is due to defective bindingor translocation cannot be ascertained, since residual trans-

port is not retained. Finally, the role of the nucleosidetransport carrier in hypoxanthine entry was confirmed byvirtue of the altered, albeit greater, ability of 80-5D2 cells to

transport hypoxanthine. An augmented rate of hypoxanthinepermeation by 80-5D2 cells was not accompanied by an

increase in adenine transport (lb). Furthermore, hypoxan-thine transport in mutant cells, unlike that in wild-type cells,was insensitive to NBMPR inhibition yet retained sensitivityto inhibition by dipyridamole. In conclusion, the propertiesof this unique nucleoside transport-deficient cell line provide

strong evidence that nucleoside, hypoxanthine, NBMPR,and dipyridamole binding sites are composed of structuraldeterminants that can be distinguished genetically.

ACKNOWLEDGMENTSThis work was funded in total by Public Health Service grants RO

1 CA32580 and RO 1 CA42539 from the National Institutes of Health.B.U. is a recipient of a Research Career Development Award.

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