(CANCER RESEARCH 51. 3677-3681, July 15. 1991]

Role of the Na+,K+-Adenosine Triphosphatase in the Accumulation of cis-Diamminedichloroplatinum(II) in Human Ovarian Carcinoma Cells'

Paul A. Andrews,2 Stephen C. Mann,3 Hung H. Huynh, and Kathleen D. Albright

Cancer Center and Department of Medicine, University of California, San Diego, La Jolla, California 92093

ABSTRACT

We examined the importance of the Na+,K+-ATPase in cisplatin

(DDP) accumulation in 2008 human ovarian carcinoma cells and describechanges in the Na*,K*-ATPase in DDP-resistant cells with DDP accumulation defects. Approximately 50% of DDP accumulation was inhib-itable by ouabain. DDP accumulation into 2008 cells could be maximallyinhibited when cells were preincubated with ouabain for l h prior to DDPexposure. The half-maximal inhibition was obtained with 0.13 JIMouabain. Similar inhibition of DDP accumulation was obtained when theNa*,K*-ATPase was blocked by ATP depletion or by incubating cells inK*-free medium. This same percentage of DDP accumulation was Na*dependent and varied directly with Na+ concentration. These effects on

DDP accumulation could be detected as early as 1 min after the impositionof Q-trans conditions, strongly suggesting that the inhibition was due tomodulation of a drug influx step. The Na+,K+-ATPase in 2008/DDP cellshad a similar A',, for ouabain binding and 36% less Na*,k Wll'asc

molecules/mg of protein than 2008 cells. 2008/DDP cells were 2.3 ±0.2(SE, n = 3) fold cross-resistant to ouabain in a continuous exposureclonogenic assay. Despite these changes in the Na+,K+-ATPase, the netbasal Na*,K*-ATPase activity was the same in sensitive and DDP-resistant cells as determined by ouabain-inhibitable ""Kl>+influx. Thebasal NU* levels were also similar in the sensitive and resistant cells.These data suggest that DDP accumulation is partially Na+ dependentand that, therefore, the Na*,K+-ATPase which maintains the Na* gra

dient may play an important role in determining how much DDP enterscells. Whether there is a causal link between the changes in the Na+,K+-

ATPase in DDP-resistant cells and their DDP accumulation defect is notyet known.

INTRODUCTION

A variety of cell types with acquired resistance to DDP4

exhibit decreased accumulation of DDP as part of their resistantphenotype (1). The appearance of decreased DDP accumulationseems to happen at an early stage in the development ofresistance and in many cell types can account for the low levelsof resistance found after selection with DDP begins (1-4). Ithas been assumed for many years that DDP enters cells by apassive diffusion process; however, the exact mechanisms involved have not been completely defined and may differ betweenfunctionally different cell types. Considering the prevalence ofdecreased DDP accumulation as part of the DDP-resistant

phenotype, and the need to define DDP accumulation mechanisms in order to understand better the cellular pharmacologyof DDP, we have been studying the mechanisms whereby DDP

Received 10/25/90; accepted 5/6/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'Supported by Grant CA-23100 from the National Cancer Institute. GrantCH-417 from the American Cancer Society, and Grant 100-R107 from Bristol-Myers, Co. This work was conducted in part by the Clayton Foundation forResearch. California Division. P. A. A. is a Clayton Foundation Investigator.

2To whom requests for reprints should be addressed, at Georgetown Univer

sity, 4 Research Court, Rockville. MD 20850.3Clayton Foundation Fellow.4The abbreviations used are: DDP, a'5-diamminedichloroplatinum(II): PBS,

phosphate-buffered saline consisting of. per liter: 8 g NaCl, 0.2 g KCI. 1.15 gNa2HPO4, and 0.2 g KH2PO4; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesul-fonic acid.

enters 2008 human ovarian carcinoma cells and how thesemechanisms have been altered in accumulation-deficient and -resistant sublines (2, 5-7).

We previously reported differences in the accumulation ofDDP in DDP-sensitive and -resistant human ovarian carcinomacells (2). As part of those studies, we discovered that a portionof DDP accumulation was energy dependent and ouabain in-hibitable. These findings were intriguing in light of strongevidence that DDP accumulation was not carrier mediated, i.e.,accumulation was not saturable or competitively inhibited withanalogues (6, 7). To explain these results, our objective in thesestudies was to define the role of the Na+,K*-ATPase (EC

3.6.1.37), the specific target of ouabain, on DDP accumulationin 2008 cells.

MATERIALS AND METHODS

Drugs and Chemicals. DDP (clinical formulation) was obtained fromthe Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). DDP pure powderwas obtained from Alfa Products (Danvers, MA). [195mPt]DDP was

obtained from Oak Ridge National Laboratories (Oak Ridge, TN) (8).3-O-[m«//i>'/-'H]D-glucose(79 Ci/mmol), ['HJouabain (15.7 Ci/mmol),and *6RbCl (0.37 Ci/mmol) were obtained from NEN Research Products (Boston, MA). The 22NaCI (90 Ci/mmol) was obtained from

Amersham Corp. (Arlington Heights, IL). Ouabain octahydrate, sodium azide, 3-0-methyl-D-glucose, choline chloride, and choline bicarbonate were obtained from Sigma Chemical Co. (St. Louis, MO). RPMI1640 powdered medium without glutamine, glucose, NaHCOs, NaCl,and KCI was obtained from Flow Laboratories, Inc. (McLean, VA).

Cell Lines. The 2008 cell line, established from a patient with serouscystadenocarcinoma of the ovary, was used in these studies (9). TheDDP-resistant cells were generated as previously described by monthlyselection with 1 ^M DDP (9). These cells are designated 2008/DDP.Cells were grown as monolayers in RPMI 1640 medium supplementedwith 10% heat-inactivated fetal bovine serum, 2 mivi freshly addedglutamine, 100 units/ml penicillin, and 100 jig/ml streptomycin. Cultures were equilibrated with humidified 5% COj in air at 37°C.Cells

were routinely tested at 6-week intervals with a Gen-probe Mycoplasmadetection kit (Fisher, Tustin, CA). All studies were done with Myco-plasma-negati\e cells. Clonogenic assays on plastic were conducted as

previously described (9).Cell Volume Determinations. The intracellular water space was de

termined in parent and resistant cells with 3-0-[merA>'/-:lH]D-glucose

by the method of Kletzien et ai. (IO).DDP Accumulation. For DDP accumulation studies, parental and

resistant cells were seeded into 6-well tissue culture plates or 100-mmtissue culture dishes, depending on the specific activity of the ("5mPt]DDP. After 3-4 days, when the plates approached confluency, themedium was aspirated and replaced with RPMI 1640 medium containing the indicated concentrations of ["5mPt]DDP at 37"C. The dishes

were immediately returned to the incubator. At appropriate time points,the plates were removed from the incubator, the medium was aspirated,and the cells were washed 4 times with PBS at 4°C(Oxoid, Columbia,

MD). For 100-mm plates, 3-ml of l N NaOH was added, and the cellswere allowed to digest overnight. An aliquot was removed for thedetermination of protein content by the method of Bradford (11), and2.5 ml was either placed in test tubes and counted with a Trak 1191gamma counter (Tracor Analytic, Elk Grove Village, IL) or mixed with

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Na».K*-ATPaseAND OSPLATIN ACCUMULATION

8 ml of 3a70B scintillation fluid (RPI Corp., Mount Prospect, IL) and1 ml of 3.5 M acetic acid and counted on a Beckman LS-230 betacounter (Beckman, Fullerton, CA) with the channel windows set towide open.

When ["*mPt]DDP was not available, accumulation studies were

conducted with unlabeled DDP. Cells were seeded into 100-mm tissueculture plates, and when the cells approached confluency, the mediumwas replaced with 5 ml of RPM1 1640 containing 100 MMDDP dilutedfrom clinical vials. After a 1-h incubation, the medium was aspirated,and the monolayers were washed once with 0.05% trypsin/0.53 m.MEDTA at 4°Cand then trypsinized with 5 ml of trypsin/EDTA for 6min at 37°C.The trypsinized cells were added to 10 ml of completemedium at 4°Cand centrifugea for 3 min at 500 x g. The cell pelletwas resuspended in 10 ml of PBS at 4°Cand centrifuged again. The

cell pellet was then resuspended in 1.0 nil of 0.9% saline and sonicatedfor 30 s at a power setting of 3 and a 30% duty cycle (5 W pulsed)(Sonifier 450; Branson, Danbury, CT). One hundred p\ was removedand mixed with 100 M' of l N NaOH for protein determination. Theremaining cell lysate was then analyzed for platinum by atomic absorption spectroscopy using a Perkin Elmer 373 atomic absorption spectro-photometer equipped with a 2200 graphite furnace (Perkin Elmer.Norwalk, CT). The following heating program was used: 90°Cfor 50s, ramp to 1300°Cin 10 s and hold for 30 s, 2500°Cunder maximum

power for 7 s. No differences in the results were noted between accumulation studies conducted with radiolabeled DDP versus unlabeledDDP: the method used is indicated in the figure legends.

Short-term accumulation experiments were conducted as describedpreviously (6). Briefly, subconfluent monolayers of cells were generatedin 60-mm tissue culture plates. Cells were washed twice with RPMI1640 or the modified RPMI 1640. RPMI 1640 at 37°Ccontaining 500

MMDDP dissolved directly in the medium from pure powder was thenadded to the plates and incubated for 1-10 min. The media were thenaspirated and the cells rapidly washed 4 times with PBS at 4°C.The

cells were scraped with a rubber policeman into the residual PBS(approximately 0.2 ml). The sample was sonicated, an aliquot removedand mixed with an equal volume of l N NaOH for protein determination, and the platinum content of the remainder was determined byatomic absorption spectrometry.

Ouabain Binding. For ouabain-binding studies, two 100-mm platesof subconfluent cells were washed with PBS and then scraped into 1.25ml of a K+-free buffer at 37°Cconsisting of 140 mM NaCl-1.5 mM

MgCl2-3.0 m.M CaClrlO mM glucose-10 mM Tris-HCI, pH 7.4. Ali-quots (0.225 ml) of this cell suspension were mixed with an equalvolume of the same buffer containing ['Hjouabain and incubated for 90min at 37°C.Duplicate 0.2-ml aliquots were then collected onto pre-

wetted 0.45 MMHAVVP filters (Millipore Corp.. Bedford, MA) on avacuum manifold and rinsed 5 times with 1 ml of PBS at 4°C.Back

ground binding to cells and filters was determined from suspensionsthat contained 1000-fold excesses of cold ouabain and was subtractedfrom the total counts. The filters were digested overnight in 4 ml ofscintillation fluid and then counted in an LS 1801 liquid scintillationcounter (Beckman). A 100-Mlaliquot of the unlabeled cell suspensionwas digested in an equal volume of l NNaOH and was used to determineprotein by the Bradford method (11). Data from 3 separate experimentswere averaged and then submitted to Scatchard analysis. Scatchardanalysis was conducted with Pharmacologie Calculation System software (Microcomputer Specialists, Philadelphia. PA).

K* Influx. The Na+,K*-ATPase activity in parental and DDP-resist-ant cells was determined by measuring '"'Rb* influx as a marker for K*influx. Subconfluent cells in 24-well tissue culture dishes were prein-cubatcd for l h at 37°Cin Hcpes buffer containing 200 MMouabain or

in Hepes buffer alone (10 mM glucose, 5 mM KCI, 1 mM MgCI3, 1 mMCaCI2, 10 mM Hepes-HCI, and 123 mM NaCI and adjusted to pH 7.4with 1 M Tris base). The medium was then replaced with 0.2 ml ofHepes buffer containing "'Rb* ( 1MCi/ml) at room temperature. In some

experiments, the buffer also contained 200 MMbumetanide, an inhibitorof the NaCI, KCI cotransporter. At appropriate times, triplicate wellswere quickly aspirated and washed 4 times with PBS at 4°C.The cells

were digested in 1 ml of l N NaOH overnight. An 0.8-ml aliquot wasmixed with acidified Ecolite scintillation fluid (ICN Biomedicai, Inc.,

Costa Mesa, CA) and counted on a scintillation counter. Protein wasdetermined in parallel wells digested with l N NaOH (II). The rate ofS6Rb*uptake was determined over a period of 10 min at room temperature, during which time the 8<vRb*influx was linear (data not shown).Na*.K*-ATPase activity was taken as the ouabain-inhibitable "'Rb*

influx.Na* Measurements. Sodium concentrations inside cells were deter

mined in double-label experiments with "Na+ and 3-O-[methyl-3H]n-

glucose as a marker of intracellular water space (10, 12). Cells wereseeded into 6-well tissue culture dishes. When the monolayers approached subconfluency, the medium was replaced with I ml of RPMI1640 containing 1 MCi/ml "Na* and 5 imi S-O-Imef/yY-'HlD-glucose

(1 MCi/ml). The plates were returned to the incubator. In some wells,ouabain was added after 2 h to give a 200 MMfinal concentration. At 3h the media were quickly aspirated and the wells washed 4 times withPBS at 4°C.The cells were digested with NaOH and analyzed forprotein and radioactivity as described above for 86Rb+. Radioactivity

was determined with a Rackbeta 1209 scintillation counter (PharmaciaLKB Nuclear Inc., Gaithcrsburg, MD) with the windows set at 5-250for detection of [3H] and 250-980 for detection of "Na. Spillover fromthe "Na channel to the |'H] channel was <2%, and in all cases was less

than the natural background.Statistics. All values are reported as means ±SE.

RESULTS

We originally observed that a 30-min exposure to 200 n\\ouabain caused an approximate 25% decrease in the DDPaccumulated over the following hour (2). We have subsequentlyexamined the time course of this inhibition. Fig. 1 shows thatthe inhibition was maximized when 2008 cells were incubatedwith ouabain for approximately l h prior to DDP exposure.The accumulation in ouabain-treated cells was 53 ±7% ofcontrol (n = 8) after a 1-h ouabain pretreatment (Table 1).Ouabain was maintained in the medium during the course ofthe DDP exposure. Longer preincubation times actually reversed the inhibition and led to a slight stimulation of DDPaccumulation. To determine whether the inhibition was due toan effect on cell volume, we measured the changes in theintracellular water space during the course of the experiment.No significant changes in cell volume were found during ouabain treatment (Fig. 1). The reason for the increasing accumulation after 1.5 h was unknown but was probably a result ofouabain toxicity. However, even though no cells survived thisbrief exposure to ouabain as assessed by clonogenic assay,trypan blue was excluded from cells after a 2.5- to 4-h exposure

0 1Pre-lncubation

o

Fig. 1. Effect of ouabain preincubation time on DDP accumulation. 2008 cellswere treated with 200 nM ouabain for the amount of time indicated before additionof 10 H.M["imPt|DDP. Incubation was continued for an additional l h with

ouabain present: •.the amount of platinum/nig protein. Points, means of twoexperiments: control accumulation was 88 pmol/mg protein. Right ordinate (•),effects of these treatments on cell volume. Points, mean values from 5 experiments, bars, SE.

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NaMC*-ATPase AND CISPLATIN ACCUMULATION

Table 1 Effect ofouabain and medium cations on DDP accumulation in 2008cells

100

Treatment200

MMouabain200 MMouabainK* free''

Low NaLow Na*Pretreatment

timeI

h1h5

minAccumulation

time60min*

1 min'1 h'

60 min*1 minf%of

control"53

±760 ±1558 ±746 ±1344 ±8No.8

3353

°Mean ±SE of separate experiments conducted with duplicate or triplicate

plates.* Determined with 100 MMDDP.c Determined with 500 MMDDP.' RPMI medium with KCI replaced by NaCI.' Determined with 10 MM["""PtJDDP.•'RPMI1640 medium with NaCI replaced by choline chloride and NaHCO3

replaced by choline bicarbonate. Residual Na* concentration was 6.7 mM.

to 200 iiM ouabain, indicating that this increase was not due togeneralized permeabilization of the cells (data not shown).

With a l h preincubation, the effect of ouabain on DDPaccumulation was linear with ouabain concentrations up to atleast 0.100 UM (Fig. 2). The maximum inhibition of approximately 45% was achieved at 0.500 ^M; only a slight additionalincrease in inhibition was found up to 200 ^M. The half-maximal inhibition occurred at 0.130 n\i.

The effect of ouabain on the accumulation of platinum incells exposed to DDP for l h does not provide informationconcerning whether the result was due to changes in drugtransport, drug metabolism, or intracellular sequestration. Accordingly, short-term DDP accumulation experiments wereconducted after a 1-h preincubation of cells in 200 MMouabain.Fig. 3A shows that ouabain decreased DDP accumulation asearly as 1 min after exposure to DDP. Accumulation at 1 mindeclined to 60 ±15% of controls (n = 3), suggesting that theeffect was on a drug influx step (Table 1). For comparison, wealso examined the accumulation of DDP in energy-depletedcells. Fig. 3B shows that using glucose-free medium containing10 mM azide also caused an approximate 50% decrease in DDPaccumulation that was apparent at 1 min. Although the onlyknown action of ouabain is inhibition of the Na+,K+-ATPase,we confirmed that the Na+,K+-ATPase was the locus of the

effect ofouabain on DDP accumulation by blocking the activityof the Na+,K+-ATPase with a K+-deficient medium which prevents the pump from cycling. As with ouabain, a K+-free medium decreased DDP accumulation to 58 ±7% (n = 3) ofcontrols (Table 1).

The Na+,K+-ATPase maintains the potassium and sodium

gradients and the associated electrochemical potential acrossplasma membranes. Inhibition of the Na+,K*-ATPase with

ouabain destroys these gradients. To determine whether theNa+ gradient was necessary for DDP accumulation, we con

ducted studies in RPMI 1640 medium in which the NaCI wasreplaced with equimolar choline chloride, and the NaHCOj

0 100 200 300 400 500Ouabain Concentration (nM)

Fig. 2. Effect ofouabain concentration on DDP accumulation. 2008 cells wereincubated l h with the indicated concentration of ouabain prior to the additionof 10 MM|"!l"Pt]DDP. Incubations were then continued an additional l h with

ouabain present. No further inhibition was found up to 200 MMouabain. Controlaccumulation was 93 pmol/mg protein.

was replaced with equimolar choline bicarbonate. DDP accumulation in low-Na+ medium was 46 ±13% (n = 5) of controls

at l h and 44 ±8% (n = 3) of controls at 1 min (Table 1). Fig.4 shows the effect of Na+ concentration on DDP accumulation.DDP accumulation increased steadily with Na+ concentration.

Since a functioning Na+,K+-ATPase affects a significant por

tion of DDP accumulation and since our DDP-resistant cellsare deficient in DDP accumulation, we asked whether 2008/DDP cells had an alteration in their Na+,K+-ATPase. The rateof [3H]ouabain binding to 2008 and 2008/DDP cells is shown

in Fig. 5/4. The 2008/DDP cells bound ouabain more slowlythan 2008 cells, and the maximal binding at saturation wasless. Scatchard analysis of these data (Fig. 5B) indicated thatthe KD was 27 nM in 2008 cells and 29 nM in 2008/DDP cells.There was 0.72 pmol/mg protein of the Na+,K+-ATPase on the

surface of 2008 cells, and this decreased by 36% to 0.46 pmol/mg protein in 2008/DDP cells. The 2008/DDP cells were foundto be cross-resistant to ouabain as indicated by continuousexposure clonogenic assay (Fig. 6). 2008/DDP cells that wereapproximately 3-fold resistant to DDP were 2.3 ±0.2 (n = 3)cross-resistant to ouabain as determined by the concentrationcausing 50% inhibition of colony formation.

The basal activity of the Na+,K+-ATPase was compared in2008 and 2008/DDP cells by measuring the influx of 86Rb+.No significant difference in total, ouabain-inhibitable, or bu-metanide-inhibitable 86Rb*influx was observed between the twocell types (Table 2). To determine whether the altered Na+,K+-ATPase generated an altered Na* gradient in 2008/DDP cells,we measured Na+ levels normalized to concomitant measure

ments of intracellular water space. No significant differencewas found under basal conditions. The 2008 and 2008/DDPcells maintained intracellular concentrations of 15.1 ±1.7 and12.9 ±2.3 mM Na+, respectively. Treatment with 200 ¿¿M

Fig. 3. A, effect of ouabain on short-termDDP accumulation. Cells were incubated withRPMI 1640 (•)or RPMI 1640 containing 200MMouabain (O) for l h prior to exposure to500 MMDDP. B, effect of ATP depletion onDDP accumulation. Cells were incubated withRPMI 1640 (•)or glucose-free RPMI 1640containing 10 mM sodium azide (O) for 30 minprior to exposure to 20 MM("5mPt]DDP. The

data are a composite of 2 separate experimentsconducted with different time ranges indicatedby the break.

600£- 500

(lu O.u< a

400

300

200

100

0.0

g

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t< o, «H

En 'c S

2.0 4.0

Time6.0(min)

10.0

B

20 30 40 K>

Time (min)60

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Na*.K*-ATPasc AND CISPLATIN ACCUMULATION

100-1

E E3« wu ri" 40-

E oci« 20

O 25 50 75 100125Na+ Concentration (mM)

Fig. 4. Effect of Na* concentration on short-term DDP accumulation. Points.

means of two separate experiments and represent the platinum accumulation Imin after exposure to 500 /¿MDDP. Cells were preincubated 5 min in theappropriate medium. Control accumulation was 184 pmol/mg protein.

ouabain for l h increased these values 8-fold to 121.8 ±5.5and 99.2 ±13.4 mM.

DISCUSSION

Our studies have shown that DDP accumulation in 2008human ovarian carcinoma cells is neither saturable nor competitively inhibited by structural analogues (6, 7). These datastrongly suggest that DDP transport is not carrier mediated inthese cells. However, DDP accumulation is modulated bycAMP, dependent on the membrane potential, partially ouabaininhibitable, partially energy dependent, and, as now shown,partially sodium dependent (2, 13, 14). Many of these observations point to a central role for the Na*,K*-ATPase in DDP

accumulation. The present studies were undertaken to examinethis role in more detail. Our data suggest that the ouabain effectwas due to a direct inhibition of the Na+,K+-ATPase anddissipation of the Na+ gradient which then affects DDP influx.

The inhibition of DDP accumulation by ouabain was maximized by increasing the preincubation time to 1 h. This wasconsistent with the slow association of ouabain with theNa*,K+-ATPase in K+-containing media (15, 16). The effect of

ouabain was linear with concentrations up to approximately0.2 MM.which was consistent with the range of the publishedKI, for ouabain binding to the Na+,K+-ATPase and the An

found by Scatchard analysis of ouabain binding to 2008 cells(16, 17). The effects of energy depletion, ouabain inhibition,and Na+ depletion on DDP accumulation were detectable as

early as 1 min after 500 //M DDP exposure. The detection ofdecreased accumulation 1 min after the imposition of Q-transconditions provides strong evidence that these effects on drugaccumulation were a result of inhibition of initial drug influxand not due to an effect on DDP metabolism, subcellulardistribution, or DDP efflux.

Since the Na*,K+-ATPase appeared to play a pivotal role in

DDP accumulation, we hypothesized that resistant cells withdecreased DDP accumulation may have alterations in theirNa+,K+-ATPase. 2008/DDP cells were found to be cross-re

sistant to ouabain, suggesting that this was the case. DDP-resistant R 1.1 murine T-lymphoma cells with accumulationdefects have also been reported to be cross-resistant to ouabain,implying that this observation is not unique to 2008 cells (18,19). Scatchard analysis of ouabain binding indicated that theKD did not change in 2008/DDP cells, but the number ofNa+,K*-ATPase molecules had decreased 36%. To determineNa+,K+-ATPase activity, we measured K+ influx with 86Rb+.No change was found in either total K+ influx or K* influxthrough the Na+,K+-ATPase. This indicated that the Na+,K+-

ATPase basal activity was the same in parent and resistantcells. This was not unexpected in that HeLa cells with 10-folddifferences in Na+,K+-ATPase expression nonetheless have similar Na+,K+-ATPase activity (12). At present, we do not knowwhether the change in the Na+,K+-ATPase in resistant cells is

directly linked to the accumulation defect or why a change inNa+,K+-ATPase number without a change in activity shouldaffect DDP accumulation. The alteration in the Na+,K+-ATPase in 2008/DDP cells does not appear to result in a Na+

gradient that is any less steep than in 2008 cells. An obviousexplanation for our data is that a Na+-dependent DDP transporter exists and that destruction of the Na+ gradient either byincubation in low-Na+ medium or inhibition of the Na+,K+-ATPase (by ouabain, ATP depletion, or low-K+ medium) com

promises this DDP transport pathway. However, this explanation is contradicted by the inability to demonstrate the existenceof a carrier for DDP by saturation kinetics or analogue competition (6, 7). This Na+-dependent pathway appears to account

for approximately half of the total DDP accumulation in vitroat all concentrations studied. Whether Na+ dependent transport

also makes a significant contribution to DDP accumulation invivo remains to be determined.

We and others have not failed to notice that tissues with thehighest levels of Na+,K+-ATPase are also the ones associated

with the primary toxicities of DDP, i.e., nephro-, neuro-, andototoxicity. Recent findings strengthen this correlation in thatthe distribution of Na*,K+-ATPase along the nephron corre

lates with the foci of DDP toxicity (20), i.e., the proximalconvoluted tubule, the medullary and cortical ascending limbs,and the distal tubule all have higher levels of Na+,K+-ATPase

than other regions of the nephron (21). In addition, the locusof DDP damage in the inner ear is the outer hair cells in thecochlea (22). The cochlea is rich in Na+,K+-ATPase which

functions to maintains the steep ion gradients in the endolymph(22-25). The specificity of DDP toxicity for peripheral nervesand not the brain can be explained by the reasoning that,because of the blood-brain barrier, this is the only Na+,K+-ATPase-rich nerve tissue that is accessible to DDP. The

Fig. 5. Ouabain binding to suspended 2008(•)and 2008/DDP (A) cells. Cells were incubated with varying concentrations of |'H]oua-bain for 90 min in a K*-free buffer. A, binding

versus ouabain concentration. Points, mean values from 3 experiments; bars, SE. B, Scatchardplot of binding data. Correlation coefficientswere 0.972 and 0.946 for the 2008 and 2008/DDP cells, respectively.

0.8-1

•?S 0.8 -

0.4-

0.2-

0-0

0.03

• 0.02-

ea

0.01 -

O SO 100 150 200[3H]Ouibaln Concentration (nM)

3680

0.000.0 0.2 0.4 0.8 0.8Ouabain Bound (pmol/mg protein)

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NaMC-ATPase AND CISPLATIN ACCUMULATION

100

£(0

20 40 60 80 100Ouabain Concentration (nM)

Fig. 6. Cross-resistance of 2008/DDP cells to ouabain. Cytotoxicity wasdetermined by clonogenic assay with continuous exposure to ouabain. Plot showstypical experiment. 2008/DDP cells (•)were 2.3 ±0.2-fold («= 3) resistant toouabain relative to 2008 cells (•).

Table 2 Na*,K*-ATPase activity as measured by A'* influx in cell monolayers

"Rb* influx was measured over IO min as a marker for K* movement. Values

are the means ±SE.

Cell2008

2008/DDPK*

influx

(nmol/mg/min)20.6±3.0 (8)°

20.8 ±3.5 (8)%

Inhibitable(%)Ouabain43

±5 (5)45 ±4 (5)Bumetanide42

±7 (3)49 ±8 (3)Passive156

°Value in parentheses, number of experiments.

Na*,K+-ATPase isoform may also be in an important factor in

determining DDP toxicity. The «lisoform is expressed at muchhigher levels in kidney, brain, and cochlea than in other tissues,whereas muscle expresses predominantly the «2isoform (24-

26).The connection between DDP toxicity and Na+,K+-ATPase

has been made before, and many studies have examined theeffect of DDP on Na+,K+-ATPase both in the kidney and theinner ear (22, 27-31). DDP, however, is an ineffective inhibitorof Na+,K+-ATPase either in vitro or in vivo, requiring highconcentrations to cause an affect if any (22, 27-31). We areproposing that DDP is particularly damaging to these tissues,not because it inhibits or platínalesthis key protein, but simplybecause the presence of high levels of the Na+,K+-ATPase «1

isoform may cause much higher levels of DDP to be broughtinto these cells. The converse, however, is not true since hepatictissue is virtually devoid of Na+,K+-ATPase, yet it accumulates

significant amounts of DDP (26, 32). Clearly, the connectionbetween Na+,K+-ATPase levels and DDP toxicity is more com

plicated than a simple correlation with DDP accumulation.This hypothesis would predict that in vivo modulators of renalNa+,K+-ATPase (such as adrenalectomy, potassium-rich diets,

and corticol steroids) or inhibitors such as cardiac glycosideswill modulate DDP nephrotoxicity and that cells with alteredNa+,K+-ATPase will have altered DDP accumulation. These

predictions can easily be tested.

REFERENCES

1. Andrews, P. A., and Howell, S. B. Cellular pharmacology of cisplatin:perspectives on mechanisms of acquired resistance. Cancer Cells, 2: 35-43,1990.

2. Andrews, P. A.. Velury, S., Mann, S. C.. and Howell. S. B. rá-Diammine-dichloroplatinum(II) accumulation in sensitive and resistant human ovariancarcinoma cells. Cancer Res., 48: 68-73. 1988.

3. Andrews, P. A., Murphy, M. P., and Howell, S. B. Characterization ofcisplatin-resistant COLO 316 human ovarian carcinoma cells. Eur. J. CancerClin. Oncol., 25: 619-625. 1989.

4. Andrews. P. A.. Jones, J. A., Varki, N. M., and Howell, S. B. Rapid

emergence of acquired c/i-diamminedichloroplatinumfll) resistance in an invivo model of human ovarian carcinoma. Cancer Commun.. 2:93-100. 1990.

5. Mann, S. C., Andrews, P. A., and Howell. S. B. Comparison of lipid content,surface membrane fluidity, and temperature dependence of m-diamminedi-chloroplatinum(II) accumulation in sensitive and resistant human ovariancarcinoma cells. Anticancer Res., A: 1211-1216, 1988.

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1991;51:3677-3681. Cancer Res   Paul A. Andrews, Stephen C. Mann, Hung H. Huynh, et al.   Ovarian Carcinoma Cells

-Diamminedichloroplatinum(II) in HumancisAccumulation of -Adenosine Triphosphatase in the+,K+Role of the Na

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