[CANCER RESEARCH 43, 4211-4215, September 1983]

Inhibition of Amino Acid Transport by c/s-Diamminedichloroplatinum(ll)Derivatives in L1210 Murine Leukemia Cells1

K. J. Scanlon,2 R. L. Safirstein, H. Thies, R. B. Gross, S. Waxman, and J. B. Guttenplan

Cancer Chemotherapy Foundation Laboratory, Division of Medical Oncology [K. J. S., R. B. G., S. W.], Division of Nephrology, Department of Medicine [R. L S., H. T.],and Departments of Neoplastic Disease [K. J. S.] and Biochemistry [K. J. S.], Mount Sinai School of Medicine, New York 10029, and Department of Biochemistry [J. B.G.¡,New York University Dental Center, New York, New York 10010

ABSTRACT

The uptake of c;s-diamminedichloroplatinum(ll) (cisplatin) has

been studied in the L1210 murine lymphoid leukemia cell line.Labeled cisplatin and its aquated derivatives were resolved byhigh-performance liquid chromatography on a strong cationic

exchange column. After 10 min of incubation of cisplatin with thecells, the major portion of the non-protein-bound platinum was

in the form of cisplatin. However, a portion of this platinum wasconverted with time to a derivative which coeluted with themonoaquo derivative of cisplatin. With the appearance of thisderivative, there was a concomitant inhibition of sodium-dependent amino acid transport as measured by the uptake of amino-

isobutyric acid and methionine. Furthermore, the exposure ofL1210 cells to a preparation of predominantly aquated product(s)of cisplatin inhibited amino acid uptake following a brief (2-min)

incubation, whereas measurable inhibition of amino acid uptakeby cisplatin required a longer preincubation period. This inhibitionof aminoisobutyric acid and methionine was dependent on theconcentration of platinum. Aminoisobutyric acid and methioninewere shown to be concentrated in L1210 cells in the presenceof sodium ions, and competition experiments suggest similaruptake systems. Since L1210 cells are methionine-auxotrophic

leukemic cells, inhibition of essential amino acid transport bycisplatin may be a mechanism of cytotoxic action.

INTRODUCTION

The antineoplastic agent cisplatin3 is one of a group of platinum

coordination complexes that was first shown by Rosenberg efal. (18) to possess antitumor activity against L1210 murinelymphoid leukemia cells. The antitumor effect of cisplatin isbelieved to involve its binding with DMA bases (17, 29), but theprecise mechanism of its cytotoxicity is far from established.Many other sites of cell-drug interaction may well play a role

(16). An interaction of cisplatin with plasma membrane constituents is suggested by: (a) the effects of cisplatin on the movementof sodium, chloride, and urea across frog skin epithelium (27);(b) the binding of cisplatin to segments of mammalian tubules(26) and renal tissue slices (19); and (c) inhibition by cisplatin ofthe membrane-specific enzyme, sodium-potassium ATPase (8).

Additional effects of cisplatin on the renal transport of water andurea (20) make it highly likely that cisplatin may have other cellmembrane effects.

1This work is supported by NIH Grants AM-16690, CA 28,683, and CA 19,023

and the Chemotherapy Foundation, Inc.2Fellow of the Leukemia Society of America. To whom requests for reprints

should be addressed.'The abbreviations used are: cisplatin, c/s-dichlorodiammineplatinum(ll); HPLC,

high-performance liquid chromatography; AIB, aminoisobutyric acid; PtAMzAo/*,diaquodiammineplatinum; PtAm2CIAq+, monoaquomonochlorodiammineplatinum.

Received August 30,1982; accepted June 8,1983.

Many of the reactions of cisplatin with macromolecules areaccelerated if the drug has undergone prior aquation (12). It hasbeen suggested that aquation of cisplatin intracellularly is animportant step in its cell toxicity (17). The relative proportions ofcisplatin and its derivatives in tumor cells, however, have notbeen studied.

In the present studies, we have examined the effects ofcisplatin on the transport of amino acids in order to assesscisplatin-membrane interaction. The membrane transport sys

tems were analyzed for their sensitivity to cisplatin because ofthe important consequences of blocking concentrative sodium-

dependent methionine transport which may prevent optimal tumor cell growth. L1210 cells were chosen because of their knownsensitivity to cisplatin (17,18) and their established requirementfor exogenous methionine (11). Most mammalian cells have atleast 3 neutral amino acid transport systems, the A, L, and ASCsystems. These systems are responsible for the cellular transportof amino acids. The alanine or A system, which is dependent onextracellular sodium, is specific for the transport of the nonme-

tabolizable substrate methylaminoisobutyric acid (1). The leucineor L system is characterized by its lack of dependence onextracellular sodium and its specificity for the nonmetabolizablesubstrate 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (1, 2).

The ASC system has been less well characterized. Its propertiesmay vary between different cell types (3, 14). The ASC systemhas been shown to make a major contribution to concentrativecapacity of the neutral amino acid transport systems in mammalian cell lines (25). Recently, surprising differences in substratespecificity for the ASC system have been demonstrated betweennormal rat hepatocytes and the methionine-auxotrophic hepa-

toma cell line, HTC (9). L1210 cells and stimulated humanlymphocytes are auxotrophic for methionine (11,13). The uptakeof methionine in these cells was inhibited by methotrexate, a2,4-diamino antagonist (21, 24).

Because of the possible enzymatic or chemical conversions ofcisplatin in the cell cytosol, both the intracellular and extracellularforms of platinum were examined by HPLC. Studies were designed to correlate the presence of particular intracellular cisplatin species with inhibition of amino acid transport (23).

MATERIALS AND METHODS

Reagents. u-[1-14C]Methionine (50 mCi/mmol) was purchased fromAmersham (Arlington Heights, III.). [1-14C]AIB (51.6 ^Ci/mmol) was pur

chased from New England Nuclear (Boston, Mass.).Nonradioactive cisplatin was obtained from the Drug Synthesis and

Chemistry Branch, National Cancer Institute, Bethesda, Md. [195mPt]-

Cisplatin in 0.9% NaCI solution was made available by Dr. J. D. Hoescheleand Dr. F. F. Knapp, Jr., at Oak Ridge National Laboratories, Oak Ridge,Tenn. (10). [195nlPt]Cisplatin was received with a specific activity of

approximately 145 mCi/mmol, but due to its short half-life (4.02 days)

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K. J. Scanion et al.

the specific activity declined rapidly. PtAm2Aq22* was prepared by incubating cisplatin (0.1 HIM) in water at pH 4.0 for 48 hr at 24°with a 10%

molar excess concentration of AgNO3 (equivalent to the total chlorideplus twice the cisplatin concentration). PtAm2CIAq+ was prepared byincubating cisplatin (0.1 rriM) in water (pH 4.0) for 48 hr at 24°. The

species formed under these conditions were pH dependent, at equilibrium, the resultant mixture contained approximately equal portions ofPtAm2CIAq* and PtAm2Aq22+ and a small (<10%) amount of cisplatin.

These resultant compounds were used as standards against which theplatinum-containing compounds in samples of the cell supematants and

medium were compared. Retention times shortened as the column aged,and standards were therefore injected before each analysis.

HPLC was performed on a system constructed from the followingcomponents: pump, Eldex B-100S; injector, Altex Model 210; and aWhatman PXS-10 SCX (4 mm x 25 cm) strong cationic exchangecolumn.4 The column was eluted with 0.03 M ammonium formate (pH

3.5) or 0.03 M potassium phosphate (pH 3.5) buffers at a flow rate of 1ml/min. Chart 1 gives the Chromatographie separation achieved by thissystem when cisplatin and its aquated forms were injected and eluted.The aquated derivatives were retained on the column longer than wascisplatin. The order in the retention times is presumably a result of thegreater positivity and consequent binding to anionic sites on the columnby aquated species. Recovery of PtAm2Aq22'1'was 70 to 80%, which

was consistently lower than that for cisplatin and mixtures of aquatedspecies (<90%). The diaquo species may have bound irreversibly to thecolumn. Fractions were collected every 0.4 min and counted in 4.5 ml ofBudget-Solv (Research Products International Corp., Elk Grove, III.) using

a Packard (Downers Grove, III.) liquid scintillation counter. Incubationmedia and cell supernatant were analyzed by HPLC. Recovery of 195mpt

activity was 95 to 100% for media and about 85% for cell supematants.L1210 Cell and Growth Media. Stock suspension cultures of L1210

cells were grown in Fischer's medium (Grand Island Biological Co.)

containing 10% horse serum (Flow Laboratories) and equilibrated with5% CO2 in air at 37°. Cultures were maintained in the presence of

antibiotics (penicillin G, 62.9 mg/liter; streptomycin, 100 mg/liter). Logarithmic-phase cells, grown in 100 to 200 ml suspension culture, wereharvested for transport studies when a cell density of 5 x 105/ml was

attained. Under such conditions, L1210 displayed an average generationtime of 12 hr. Cultures were established from frozen Mycoplasma-ireestocks at 3-month intervals.

Uptake of Cisplatin. The uptake of [195mPt]cisplatinwas determinedby incubating L1210 cells (5 x 107 cells/ml) in a Cross and Taggart

buffer, containing 1 rriM CaCl. 7 mw phosphate (pH 7.4), 10 HIM sodiumacetate, 36 rriM KCI, and 77 mw NaCI (4). [195mPt]Cisplatin was stable inthis buffer at 37°for 60 min.

The reaction was stopped by pipeting an aliquot (200 n\) into a 20-fold dilution of ice-cold 0.9% NaCI solution, and the aliquot was centri-fuged. The cell pellet was washed 3 times with ice-cold 0.90% NaCI

solution, then dissolved in 0.5 ml NCS (Amersham), solubilized overnightat 37°,and counted.

Incubation Media for Amino Acid Transport Studies. The sodium-dependent amino acid transport was performed in Earie's balanced salt

solution which contains 116 mM NaCI (IM-1 ). The sodium-independenttransport was studied in NaCI-free Earie's balanced salt solution contain

ing 150 rnw choline chloride (IM-2). The net sodium-dependent aminoacid transport was determined by subtracting the sodium-independenttransport from the total sodium-dependent transport (IM-1 transport -IM-2 transport = net sodium-dependent transport). Earie's balanced salt

solution (IM-1) was used because it supported maximum rates of amino

acid uptake (21). Cisplatin also inhibited amino acid uptake in the Crossand Taggart media.

Amino Acid Transport Measurements. Logarithmic-phase cells wereharvested by centrifugation, washed 3 times with each specific incubationmedium, and resuspended in the incubation medium to a final density of

4 R. Safirstein, M. Daye, and J. Guttenplan. Mutagenic activity and identification

of excreted platinum in human and rat urine after cisplatin administration, submittedfor publication.

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Chart 1. HPLC of [195mPt]cisplatin (PMmjCy (A) and aquated derivatives ofcisplatin (B and C). Chromatography was performed using a Partisil SCX-1 0 column(4 mm inside diameter x 25 cm long) with a mobile phase consisting of 0.03 Msodium acetate buffer (pH 3.5) at a flow rate of 1.0 ml/min. Fractions were collectedat 0.4-min intervals. A. Chromatograph of [19SmPt]cisplatin. Twenty /il of a solutioncontaining [195mR]cisplatin (0.14 mg/ml) in 0.9% NaCI solution (specific activity,0.25 pCi/mg) was injected. B, Chromatograph of a 75-jil solution of cisplatin (10~*M) in water, incubated for 48 hr at 23°.Prior to incubation, cisplatin in 0.9% NaCI

solution was injected onto the column eluted with water (pH 4.0) and collected onice. This was done to separate cisplatin from NaCI in the solvent. Chloride elutedbefore cisplatin. Fractions free of chloride were added to a solution of unlabeledcisplatin to achieve a final concentration of 10~* M. The 3 species present arecisplatin, PtAm2CIAq*, and PtAn^qz2*. C, Chromatograph of a cisplatin solution(10~4 M) incubated with AgNO3 for 48 hr at room temperature and centrifuged; 20

u\ were injected and eluted as above.

5 x 106 cells/ml. Cell viability after cisplatin (50 ^M) exposure for 30 min

was decreased a maximum of 15% as determined by trypan blueexclusion, using a hemocytometer. For each transport measurement,cells were first incubated in the appropriate medium for 15 min at 37°

with or without cisplatin. At zero time, a portion of the cell suspensionwas withdrawn and added to the incubation medium with the labeledamino acid. At timed intervals, 200 ¿ilsamples were withdrawn. The cellswere immediately separated from the incubation medium by centrifugation through a layer of silicone:mineral oil into perchloric acid, as described below. This technique is similar to the method of Wohlhueter efal. (28).

In the present experiments, a series of 400-jul conical polyethylene

tubes (Beckman) were prepared by layering 100 n\ of silicone (No. 550;Dow-Corning Corp.):mineral oil (8.4:1.6, v/v, respectively) over 50 n\ of7% (v/v) perchloric acid. Each 200-Ml aliquot of cells withdrawn from the

incubation medium was carefully and rapidly layered on top of thesilicone:mineral oil. The tube was immediately centrifuged at 11,600 x gfor 5 sec in a Beckman microfuge B.

At the completion of an uptake experiment, an aliquot from theperchloric acid fraction was placed in 6 ml of Aquasol II (Amersham) andcounted for 10 min in a Beckman LS-250 liquid scintillation counter (22).

The data presented in the charts are the means of 3 experiments whichincluded all the drug treatments. In these representative experiments,the standard error was less than 10% of the mean.

RESULTS

There was a correlation between increasing concentrations ofcisplatin outside and inside the cell as seen in Chart 2. This

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Cisplatin-Membrane Interactions

uptake of cisplatin was rapid and reached an apparent steadystate during the first 10 to 15 min except at the highest concentrations. To establish the stability of the drug during the incubation period, cisplatin was incubated in the Cross and Taggartbuffer, and HPLC analysis revealed very little change in thecomposition of the platinum-containing species for up to 30 minat 37°(Chart 3A). Cisplatin was less stable in the Earle's bal

anced salt solution; the same intracellular intermediates wereformed in both media (results not shown).

Once inside the cell, a portion (25 to 35%) of the cisplatin wasconverted to a species comigrating with PtAm2CIAq+ (Chart 3ß,

Peaks 2 and 3). Platinum in the incubation medium was almostcompletely in the cisplatin form.

The L1210 cells were treated with cisplatin for 30 min andtransferred to a drug-free medium after 2 washes. After 60 min,

the cells retained 25% of the total labeled cellular platinum.Analysis of the labeled drug released from the cells into the drug-free media during the 60-min incubation revealed only the cispla

tin form (Chart 3C). Other forms may have effluxed from the cell;however, in the presence of high concentrations of chloride in

I5 30 45 60TIME (minutes)

Chart 2. Uptake of [195mPt]cisplatin(C/sPf) into L1210 cells. Cells (5 x 107/ml)

were incubated in a Cross and Taggart buffer with several time points over 60 minand with increasing concentrations (MM)of labeled drug for 60 min. Cells werewashed in 0.9% NaCI solution and counted as described in "Materials and Methods." The background (zero time) was subtracted from these results.

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ChartS. Analysis by HPLC of [195mPt]cisplatinand its aquation products in

Cross and Taggart uptake media and L1210 supernatant. HPLC was performedusing a 0.03 M KH2P04 elution buffer (pH 3.5) at a flow rate of 1 ml/min. A,chromatogram of cisplatin in Cross and Taggart incubation media for 30 min at37°.B, cells incubated for 30 min with cisplatin washed (3 times) in 0.9% NaCIsolution and suspended in 0.5 ml of 7% trichloroacetic acid, and the filtered (0.45-nm Millipore filter) supernatant then chromatographed; C, chromatogram of thecell-freemedia after 60 min. The 3 species present coelute. Peak 1, cisplatin; Peak2, PtAm2CIAq*;Peak 3,

the buffer, these forms may have been reconverted back tocisplatin.

Amino Acid Transport Studies. Amino acid transport in L1210cells was measured in Earle's balanced salt solution, since higher

rates of sodium-dependent amino acid transport were achieved

in this medium than in the Cross and Taggart buffer. AIB, anamino acid which is transported but not metabolized, and methionine were shown to inhibit the uptake of each other (Chart4). These competition experiments were measured at one timepoint where there was primarily unidirectional influx of the substrates AIB (1 min) and methionine (0.5 min).

Cisplatin Inhibition of Amino Acid Transport. AIB and methionine were transported by a sodium-dependent and a sodium-

independent system (Charts 5 and 6). The uptake of AIB andmethionine was greater in sodium than in choline chloride buffer.Cisplatin caused an inhibition of sodium-dependent AIB (500 /*M)

uptake which was dependent on the concentration of cisplatin(Chart 5). The accumulation of both methionine and AIB wasinhibited by concentrations of cisplatin as low as 10 /¿M(Chart6). There was little further inhibition of AIB and methionine uptakeby cisplatin at concentrations of 25 to 50 ^M. The addition ofcisplatin did not inhibit amino acid transport immediately. However, the longer the drug was incubated with the cells, the greaterwas the inhibition of transport. The maximum inhibition of aminoacid transport occurred following 10 to 30 min of cisplatin prein-cubation. This inhibition resulted in a decrease of the sodium-dependent AIB and methionine uptake by about 50%. Sodium-

100755025

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Chart 4. Amino acid competition experiments in L1210 cells. Initial (1-min)labeled AIB (500 I¡M)uptake was measured with increasing concentrations ofunlabeled methionine (MET) (1 to 50 HIM)(ft,). Initial (30-sec) labeled methionine(500 >iM)uptake was measuredwith increasingconcentrations of unlabeledAIB (1to 50 mM)(B).AIB (2.5 pmol/103cells/min)and methionine(0.3 pmol/103cells/min)uptake was measured and counted as described in "Materials and Methods." Thetransport medium was Earle's balancedsalt solution with 150 mMNaCI.

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Chart 5. Uptake of AIB in the presence and absence of cisplatin (C/s Pf) inEarle's balanced salt solution. Initial AIB (500 ^M) uptake was measured in thepresence of Earle's balanced salt solution containing sodium chloride or cholinechloride (Ch CI). Cells were preincubated at 37°for 15 min in the presence of 1,10, or 25 »iMcisplatin, and AIB uptake was measuredduring the first 3 min, in thepresenceof either sodiumchlorideor cholinechloride.See"Materialsand Methods"

for uptake and counting techniques.

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K. J. Scanion et al.

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Charte. Uptake of methionine in the presence and absence of cisplatin inEarte's balanced salt solution. Initial methionine (MET) (10 Õ<M)uptake was measured

in the presence of sodium chloride or choline chloride (Ch CI) at a concentration of150 HIM. Cells were preincubated at 37°for 15 min in the presence of 10, 25, or

50 tiM cisplatin. Methionine uptake was measured during the first 2 min in thepresence of either sodium chloride or choline chloride. See "Materials and Methods"

for uptake and counting techniques.

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Chart 7. Inhibition of AIB and methionine uptake by aquated cisplatin in Earte's

balanced salt solution. Initial uptake (1 min) of AIB (O) or methionine (•)wasmeasured in the presence of sodium chloride. Aquated cisplatin was prepared byincubating cisplatin in water at pH 4.0 for 48 hr at 37". HPLC revealed 3 species:cisplatin (18%), PtAm2CIAq* (63%), and PtArrizAcfc2* (at least 17%). Cells wereexposed for 2 min at 37°,washed, and resuspended in fresh media. Uptake of AIB(1.66 pmol/103 cells/min) and methionine (3.15 pmol/103 cells/min) without drug

was 100%, and this uptake is compared to uptake in the presence of the drug (1to 10 pu). Results are expressed as a percentage of the control. Data are themeans of 3 experiments that included all the drug concentrations. Bars, S.E. In thisrepresentative experiment, if bars are not shown, they are within the symbols.

independent transport of AIB or methionine appeared to be lessaffected by the presence of cisplatin. However, the sodium-

independent transport represented a smaller contribution of AIBand methionine transport than did sodium-dependent transport

in L1210 cells (Charts 5 and 6).Similar results with cisplatin inhibition of sodium-dependent

transport of AIB and methionine were observed in Cross andTaggart buffer where cisplatin was the only species found.Therefore, the small amount (<10%) of the specie(s) of cisplatinthat was found in the Earle's balanced salt solution could not

completely account for this effect on amino acid transport.Aquated Cisplatin Inhibition of Amino Acid Transport. Chart

7 summarizes the effect of the aquated cisplatin preparation onAIB and methionine transport. The mixture of aquated cisplatinused for these studies contained 63% PtAm2CIAq+ and equalportions of cisplatin and PtAm2Aq22+. Pure PtAm2Aq22+could not

be tested in this system since the nitrate used in its preparationinterfered with amino acid uptake. The L1210 cells were exposedto varying concentrations of the aquated cisplatin preparation (1to 10 fiM) for 2 min, and the cells were washed and resuspendedin drug-free media. The initial uptake of AIB and methionine wascompared in the presence or absence of PtAm2CIAq+. There

was about a 30% inhibition of AIB and methionine uptake as aresult of a 2-min preincubation with 10 /tw aquated cisplatin. Thisinhibition was most apparent in sodium-dependent transport

(data not shown) and was not reversible after washing the cellswith drug-free media.

DISCUSSION

Cisplatin has been shown to be a highly reactive compoundwith a potential to bind to most biological molecules (17). Thisstudy was initiated in order to determine whether cisplatin interactions were not confined to DMA only. Cisplatin selectivelyinhibited sodium-dependent amino acid uptake into L1210 cells,which was suggestive of a drug-membrane interaction. Thishypothesis is based in part on the inhibition of AIB, a nonmetab-

olized amino acid analogue. The fact that AIB and methionineare known to share similar transport systems (1-3) was also

suggestive for the L1210 cells (Chart 4). Since the concentrativecomponent for methionine uptake was blocked by cisplatin inthe methionine-auxotrophic L1210 cells, it is possible that this

event may contribute to the drug cytotoxicity for L1210 cells.Cisplatin has also been shown to share with other heavy metalsand their salts [uranyl nitrate (6), cadmium (7), and p-chloromer-

curibenzoate (6)] inhibitory effects on amino acid transport.L1210 cells have been shown previously to require an exog

enous source of methionine for optimal cell growth (11). Themethionine requirement cannot be eliminated by supplying sufficient quantities of the precursors for methionine biosynthesis, 5-

methyltetrahydrofolate and homocysteine (11, 22). Methioninesynthetase is present in the cell, and there are sufficient quantities of the enzyme to convert the precursors to methionine (22).The reason for the exogenous methionine requirement in L1210cells remains unclear. Nevertheless, the inhibition of methionineuptake into L1210 cells by cisplatin could be cytotoxic to thesemethionine-auxotrophic cells by a mechanism of methionine dep

rivation.The only species of platinum detected in the incubation me

dium during the amino acid uptake inhibition experiments wascisplatin. Once in the cell, however, cisplatin was partially converted to a compound coeluting with PtAm2CIAq+. This conver

sion would be favored by the low intracellular chloride ion concentration. The formation of this intracellular aquated cisplatinspecies corresponded in time to the inhibition of amino aciduptake. When aquated derivatives of cisplatin (10 MM)were used,an inhibition of at least 30% of AIB and methionine uptake in theL1210 cells occurred after only 2 min incubation. In contrast,cisplatin required a more prolonged period of incubation to obtainmeasurable inhibition of amino acid uptake. These results suggest that the inhibition of transport may be due to the formationof the aquated compound in the cell. The aquated species ofcisplatin (<10 (¿M)can bind irreversibly to the cell and inhibitamino acid transport (Chart 7). Extensive washing of the cellsdid not reverse this inhibitory effect of cisplatin. This concentration of cisplatin correlates with the pharmacological dose thatcan be achieved in the plasma (21.8 to 36 UM) (5). However,since cisplatin was the predominant species at both sides of theplasma membrane, a time-dependent direct interaction of cispla

tin and some component of the amino acid transport system(see below) cannot be excluded. Try pan blue studies on the cellviability also suggest that cisplatin (>50 ^M) with prolongedincubation (>45 min) may contribute to general membrane damage.

The mechanism of inhibition of methionine transport by cisplatin was not examined in these studies, but several possibilities

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Cisplatin-Membrane Interactions

may be considered. Inhibition of methionine uptake may be dueto (a) drug binding to specific membrane carriers, (b) reductionof the sodium gradient across the plasma membrane drivingcellular uptake of amino acids, and (c) effects on intracellularprocesses which support uptake of amino acids; e.g., glutathionedepletion in lymphocytes has been shown to reduce amino aciduptake in these cells (15). Cisplatin can bind to membranevesicles, and it is conceivable that amino acid carrier moleculescould be altered by such binding. Such types of binding mightalter the function of these carriers. Regarding the second possibility of a diminished transmembrane sodium electrochemicalgradient, it has been reported that cisplatin inhibits the sodium-

potassium ATPase isolated from mammalian and amphibian cells(8), the activity of which is essential to maintain and generatetranscellular sodium gradients. Indeed, cisplatin had its greatestinhibitory effect on sodium-dependent amino acid uptake. This

is a rather discriminating interaction for cisplatin. Its importanceis not completely understood at this time, but it is currently thefocus of further experiments. Finally, since L1210 cells aremethionine-auxotrophic tumor cells, any perturbation of the me

thionine pools could be cytotoxic.

ACKNOWLEDGMENTS

We would like to thank Michele Pallai for her expert technical work on thispaper.

REFERENCES

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2. Christensen, H. N., Handlogten, M. E., Lam, l., Tager, H. S., and Zand, R. Abicyclic amino acid to improve discriminations among transport systems. J.Bid. Chem., 244: 1510-1520,1969.

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4. Cross, R. J., and Taggart, J. V. Renal tubular transport: accumulation of p-aminohippurateby rabbit kidney slices. Am. J. Physiol., 161:181-190,1950.

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6. Foulkes, E. C. Effects of heavy metals on renal aspartate transport and thenature of solute movement in kidney cortex slices. Biochim. Biophys. Acta,24Õ:815-822, 1971.

7. Foulkes, E. C., and Gieske, T. Specificity and metal sensitivity of renal aminoacid transport. Biochim. Biophys. Acta, 378: 439-445,1973.

8. Guarino, A. M., Miller, D. S., Amok), S. T., Pritchard, J. B., Davis, R. D.,Urbanek,M. A., Miller, T. J., and Utterst, C. L. Platínatetoxicity: past, presentand future prospects. Cancer Treat. Rep., 63:1475-1483,1979.

9. Handlogten, M. E., Garcia-Cancero,R., Lancaster, K. T., and Christensen, H.N. Surprising differences in substrate selectivity and other properties of sys

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14. Kilberg, M. S., Handlogten, M. E., and Christensen, H. N. Characteristics ofsystem ASC for transport of neutral aminoacids in the isolated rat hepatocyte.J. Biol. Chem., 256: 3304-3312,1981.

15. Novogrodsky, A., Nehring, R. E., Jr., and Meister, A. Inhibition of amino acidtransport into lymphoid cells by the glutamine analog i_-2-amino-4-oxo-5-chloropentanoate. Proc. Nati. Acad. Sei. U. S. A., 76. 4932-4935,1979.

16. Renshaw, E., and Thomson, A. J. Tracer studies to locate the site of platinumions within filamentousand inhibited cells of Escherichia coli. J. Bacterio!.,94:1915-1918,1967.

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SEPTEMBER 1983 4215

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1983;43:4211-4215. Cancer Res   K. J. Scanlon, R. L. Safirstein, H. Thies, et al.   Leukemia Cells-Diamminedichloroplatinum(II) Derivatives in L1210 Murine

cisInhibition of Amino Acid Transport by

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