THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 27, Issue of September 25, pp. 20116-20125,1993 Printed in U. S. A.

Glutathione-associated cis-Diamminedichloroplatinum(I1) Metabolism and ATP-dependent Efflux from Leukemia Cells MOLECULAR CHARACTERIZATION OF GLUTATHIONE-PLATINUM COMPLEX AND ITS BIOLOGICAL SIGNIFICANCE*

(Received for publication, December 29, 1992, and in revised form, May 14, 1993)

Toshihisa IshikawaS and Francis Ali-Osman From the DeDartment of Emerimental Pediatrics. the University of Texas, M. D. Anderson Cancer Center, Houston, Te&s 77030

, I

Accumulating evidence suggests a critical role of intracellular glutathione in tumor cell resistance to alkylating agents. The present study provides evidence for the direct interaction between cis-diamminedichlo- roplatinum(I1) (cisplatin) and glutathione (GSH) both in a cell-free system, as well as in L1210 murine leu- kemia cells. We have isolated the reaction product and identified it by a combination of high performance liquid chromatography and atomic absorption spec- troscopy. Stoichiometric analysis showed a 2:l molar ratio of GSH/cisplatin for the reaction. The molecular mass assessed by mass spectroscopy was 809 Da, cor- responding to a GS-platinum chelate complex, bis-(glu- tathionat0)-platinum. The GS-platinum complex was detected in L12 10 leukemia cells incubated with 20 p~ cisplatin. The intracellular content of the GS-platinum complex reached a maximal level after 12 h, corre- sponding to about 60% of the intracellular platinum content. Thus, formation of the GS-platinum complex is considered a significant part of the cellular metabo- lism of cisplatin. The GS-platinum was found to inhibit cell-free protein synthesis in a rabbit reticulocyte ly- sate system using both chloramphenicol acetyltrans- ferase mRNA and poly(A) mRNA from HL-60 human promyelocytic leukemia cells (ICso = 190 p~ the GS- platinum complex). Elimination of the GS-platinum complex from tumor cells may represent an important mechanism which reduces the intracellular accumula- tion of the platinum complex. Using plasma membrane vesicles prepared from L1210 cells, the transport of the GS-platinum complex across the plasma membrane was found to be an ATP-dependent process (apparent K , values: 49 pM, ATP; 110 pM, GS-platinum com- plex). The ATP-dependent transport of the GS-plati- num complex was inhibited by vanadate (ICso = 35 pM) as well as by S-(2,4-dinitrophenyl)-glutathione, leu- kotriene C4, and GSSG, but not by doxorubicin, dau- norubicin, or verapamil. The ATP-dependent glutathi- one S-conjugate export pump, “GS-X pump” (Ishikawa, T. (1992) Trends Biochem. Sci. 17, 463-468), is sug- gested to play a role in the elimination of the GS- platinum complex from tumor cells.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This study is dedicated to Professor Helmut Sies (Diisseldorf, Germany) on the occasion of his 50th birthday.

$To whom correspondence and reprint requests should be ad- dressed: P. 0. Box 169, Dept. of Experimental Pediatrics, the Uni- versity of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

Glutathione (GSH) has a variety of physiologically impor- tant functions in cellular defense and metabolism, including modulation of thiol-disulfide status of cellular proteins, pro- tection of cells from oxidative stress, detoxication of electro- philic compounds, and synthesis and transport of biologically active, endogenous substances (1-5). Moreover, GSH inter- acts with a wide range of drugs. Recent studies show that GSH is a critical determinant in the tumor cell resistance to alkylating agents, such as cisplatin,’ L-phenylalanine mus- tard, and bifunctional nitrosoureas (6-9).

Cisplatin is an effective antitumor agent in the treatment of such varied human cancers as those of brain, head and neck, ovary, testicle, and bladder (see Refs. 10 and 11, for review). The antitumor activity of this agent is attributed primarily to its ability to form DNA-platinum adducts (12- 16). Despite its clinical effectiveness, cellular drug resistance is a significant obstacle to long term, sustained patient re- sponse to cisplatin-based therapy. Several potential biochem- ical and molecular mechanisms of cisplatin resistance have hitherto been identified, which include decreased intracellular accumulation of cisplatin (17, 18), elevated cellular GSH (19) and metallothionein content (20), and increased DNA repair (21). Importantly, the cytotoxicity of cisplatin has been shown to be significantly enhanced by depletion of cellular GSH in some tumor lines (22, 23). GSH can quench DNA-platinum monoadducts before their conversion to cytotoxic DNA cross- links (24), or GSH may form a complex (or complexes) with cisplatin, thereby reducing the amount of intracellular cispla- tin available for interaction with DNA (25, 26). Although accumulating evidence supports a significant role of GSH in tumor cisplatin resistance, the exact molecular mechanisms involved in the resistance are not fully understood. The pres- ent study addresses the reaction of cisplatin with GSH and provides direct evidence for the formation and molecular structure of the GS-platinum complex both in a cell-free system and in murine leukemia L1210 cells.

Furthermore, the evidence that the GS-platinum complex is formed in tumor cells exposed to cisplatin raises questions as to the biological activity and metabolic fate of the complex. In particular, if the GS-platinum complex is cytotoxic, then its elimination will be critical for cell survival, and this might provide an answer, at least in part, for the unknown metabolic

The abbreviations used are: cisplatin, cis-diamminedichloropla- tinum(I1); GS-platinum, bis-(g1utathionato)-platinum (11); DNP-SG, S-(2,4-dinitrophenyl)-glutathione; LTC,, leukotriene C4; GS-Xpump, the ATP-dependent glutathione S-conjugate export pump; AMP- PCP, adenosine 5’-(P,y-methylene)triphosphate; AMP-PNP, adeno- sine 5’-(P,y-imino)triphosphate; CAT, chloramphenicol acetyltrans- ferase; HPLC, high performance liquid chromatography; PBS, phos- phate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

20116

GSH-associated Cisplatin Metabolism and Export 20117

link between the increased intracellular GSH level and the decreased accumulation of platinum in cisplatin-resistant tu- mors. Many metals have been reported to be eliminated into bile and urine by a process that involves complex formation of the metals with GSH and the subsequent active membrane transport of the glutathione-metal complexes (27, 28). It has also been demonstrated that a variety of divalent organic anions, including GSSG, glutathione S-conjugates and cystei- nyl leukotrienes, are eliminated into bile. Recently, using plasma membrane vesicles, we have provided evidence that the efflux of those compounds is an ATP-dependent process mediated by the GS-X pump (as), and that this ATP-depend- ent export pump is localized in the plasma membranes of different organs and cell types (see Ref. 5 , for recent review). Based on our previous studies, we have hypothesized that the GS-X pump would play a role in the elimination of the GS- platinum complex from tumor cells. In this study, we have examined this hypothesis and provide evidence that the GS- platinum complex, which potentially inhibits protein synthe- sis, is exported from L1210 leukemia cells via the ATP- dependent export pump. This is the first report that demon- strates the physiological importance of the GS-X pump in the elimination of cellular metabolites of anticancer drugs from tumor cells.

MATERIALS AND METHODS

Biochemicals, Enzymes, and Cells-GSH, GSSG, ATP, creatine phosphate, creatine kinase, and phenylmethylsulfonyl fluoride were purchased from Boehringer Mannheim (Mannheim, Germany). Cis- platin was from Bristol-Myers co . (Evansville, IN). Rabbit reticulo- cyte lysate and chloramphenicol acetyltransferase (CAT) mRNA were purchased from Life Technology, Inc. Human HL-60 promyelocytic leukemia poly(A) mRNA was from Clontech Laboratories (Palo Alto, CA). [35S]Methionine and [3H]GSH were from Du Pont-New England Nuclear. Acivicin, AMP-PCP, AMP-PNP, doxorubicin, daunorubi- cin, verapamil, sialidase from Clostrium perfringens, iodoacetic acid, RPMI 1640 medium, fetal calf serum, and gentamycin were from Sigma. QAE-Sephadex and low molecular weight protein standards for SDS-PAGE were from Pharmacia-LKB (Uppsala, Sweden). [3H] DNP-SG was prepared according to Ishikawa (29). All other chemi- cals were of analytical grade. The wild-type L1210 murine leukemia cell line was obtained from the American Type Culture Collection (Rockville, MD.) and maintained in our laboratories in RPMI 1640 medium supplemented with 10% fetal calf serum.

UV Spectral Analysis and Kinetics of GS-Platinum Complex For- mation-GSH (3.33 mM) was incubated with 1.67 mM cisplatin in 15 ml of PBS (154 mM NaCl and 10 mM sodium phosphate, pH 7.4) a t 37 "C. At different time points (0, 1, 2, 4, 6 ,8 , 12, 24, and 48 h), a 20- pl aliquot of the reaction medium was withdrawn and diluted in 980 pl of PBS. The absorption spectrum of the diluted sample was determined by scanning spectrophotometry over a wavelength range of 200-400 nm in a Beckman DU-65 spectrophotometer.

Isolation of GS-Platinum Complex by Anion-exchange Chromatog- raphy-The GS-platinum complex was isolated from the reaction mixture by anion-exchange chromatography. A mixture of GSH (3.33 mM) and cisplatin (1.67 mM) was incubated in PBS at 37 "C for 48 h, after which the reaction mixture was applied to a QAE-Sephadex anion-exchange column (bed volume, 1 ml) equilibrated with 10 mM Tris-HC1, pH 7.5. The column was washed with 5-10 ml of deionized water.The GS-platinum complex hound to the anion-exchange col- umn was eluted with 0.2 M HC1. The eluent was collected and analyzed by atomic absorption spectroscopy or dried in a Speed Vac concen- trator (Savant, Farmingdale, NY) for mass spectroscopic analysis.

Atomic Absorption Analysis of GS-Platinum Complex-The plati- num content of the samples containing GS-platinum complex was determined by flameless atomic absorption spectroscopy (30). Briefly, aliquots of the eluents from the ion-exchange column were diluted l:lO, 1:100, and 1:lOOO in PBS and analyzed in an atomic absorption spectrometer (Varian Spectra AA 300) equipped with a graphite tube atomizer set at an atomization temperature of 2800 "C. A 4-10-mA hollow cathode lamp current was used, and signal detection was at 265.9 nm with a slit width of 0.2 nm. A signal integration time of 5 s was used, and background correction was performed with a deuterium

lamp. In each experiment, platinum content was determined in trip- licate measurements.

Mass Spectroscopic Analysis of GS-Platinum Complex-The chro- matographically purified and dried GS-platinum complex (yellow powder) was dissolved in 50 pl of HZO. A 0.5-pl aliquot of the solution was then mixed with 0.5 pl of sinapic acid (10 mg/ml) dissolved in acetonitrile/HzO/trifluoroacetic acid (70:30:0.1% (v/v)) solution and dried under infrared light. The matrix-assisted laser desorption of the sample was analyzed with a Lasermat mass analyzer (Finnigan Mat, Manchester, United Kingdom) (31). A pulsed laser beam (NdYAG pumped dye-laser, 330 nm, 10-65 mJ) was focused on the sample matrix, and the resultant ions were accelerated in an electric field of 20 kV. 'H N M R Analysis of GSH and GS-Platinum Complex-Proton

NMR spectra were obtained a t 25 "C a t 200 MHz with a Bruker WH 200 F T spectrometer. Sample volumes of 0.5 ml were used in 5-mm NMR tubes, which contained 5 mM GSH or GS-platinum complex dissolved in 'HZO. Chemical shifts were calculated from the external reference sodium 4,4-dimethyl-4-silapentane-l-sulfonate.

HPLC Analysis of GS-Platinum Complex-140 p1 of the acidic sample (GS-platinum complex in 0.2 M HC1 eluted from the QAE- Sephadex column or L1210 cell extract in 7.5% perchloric acid) was mixed with 40 pl of 100 mM iodoacetic acid and then neutralized by the addition of KHCO, powder. The sample was treated with 1% 1- fluoro-2,4-dinitrobenzene as described previously (32-34) and ana- lyzed by HPLC using a Waters HPLC system (type 700 WISP/6OOE pump) equipped with a 3-aminopropyl column (Customsil 132-204, Custom LC, Houston, TX). Mobile phase A contained 80% methanol, and mobile phase B 2.4 M sodium acetate, 45% (v/v) acetic acid, and 40% (v/v) methanol. Following injection of the derivative sample, the mobile phase was maintained at 97% A + 3% B for 5 min and subsequently run on a 20-min linear gradient to 70% A + 30% B to separate GSH and GSSG. This separation was then followed by running on a 20-min hyperbolic gradient to 20% A + 80% B. The flow rate was 1 ml/min throughout the chromatography. Dinitro- phenyl derivatives of GSH, GSSG, and GS-platinum complex were monitored a t 365 nm with a scanning photodiode array detector (Waters 991). The eluent was collected every 1 min, and platinum was detected by atomic absorption spectroscopy.

Determination of GS-Platinum Complex and Total Platinum Con- tents in L1210 Cells-Cisplatin (at a final concentration of 20 p ~ ) was added to 100 ml of a suspension of L1210 cells (4 x IO6 cells/ml) in RPMI 1640 medium containing 10% fetal calf serum. After 3, 6,9, 12, and 24 h, 10 ml of the cell suspension was withdrawn and centrifuged a t 40 X g for 5 min. The pelleted cells were resuspended in 10 ml of ice-cold PBS and again centrifuged at 40 X g for 5 min. The resulting cell pellet was resuspended in 500 pl of PBS. A 250 pl- aliquot of this cell suspension was taken, mixed with 150 p1 of 20% perchloric acid, and homogenized. After centrifugation a t 16,000 X g for 5 min, the resulting supernatant (7.5% perchloric acid extract) was stored at -20 "C or used immediately in HPLC and atomic absorption analysis of GS-platinum complex, as described above.

Total platinum content in L1210 cells was determined according to Siddik et al. (30). Briefly, the cell suspension in PBS (230 pl) was centrifuged a t 16,000 x g for 5 min. The resulting cell pellet was mixed with 500 p1 of hyamine hydroxide and kept at 50-60 "C overnight. The sample was then acidified with HC1. Platinum was determined by atomic absorption spectroscopy, as described above. Protein concentration in the cell suspension (20 pl in PBS) was determined according to Lowry et al. (35).

Determination of Protein Synthesis in L1210 Cells-The activity of protein synthesis in L1210 cells was determined based on the rate of [35S]methione incorporation into cellular proteins. Cells (1 X lo6/ ml) were incubated with or without 20 p~ cisplatin in a total volume of 10 ml of RPMI 1640 medium containing 10% fetal calf serum. At different time points of the incubation (Le. 3, 6, 9, 12, 18, and 24 h), 1 ml of the cell suspension was taken and mixed with 0.17 pCi of [35S]methione. After 1-h incubation a t 37 "C under 5% CO,, the cells were collected by centrifugation (200 X g for 5 min, a t 4 "C) and then resuspended in 1 ml of ice-cold PBS. An aliquot of 50 p1 of the cell suspension was kept for protein determination, and the rest (950 pl) was centrifuged at 200 X g for 5 min and the resulting cell pellet was treated with 1 ml of 10% trichloroacetic acid. Acid-precipitated pro- teins were collected by membrane filtration (Millipore GS filter membrane, 0.22-pm pore size). The filter membrane was washed with 5 ml of 5% trichloroacetic acid, and the radioactivity remaining on the filter membrane was determined in a scintillation counter.

In Vitro Translation-This was performed according to the man-

20118 GSH-associated Cisplatin Metabolism and Export ufacturer's protocol (Life Technology, Inc.). 300 ng of mRNA of CAT or poly(A) mRNA from HL-60 cells was incubated with 25 pCi of ["'S]methionine a t 30 "C in a total volume of 30 pl of the rabbit reticulocyte lysate containing 8 p~ hemin, 50 pg/ml calf-liver tRNA, 19 amino acids (50 PM each), 25 mM HEPES, pH 7.4, 1.1 mM MgCl,, 40 mM KCl, 23 mM NaC1, 100 mM potassium acetate, 310 p~ CaC12, 620 p M EGTA, 1.6 p M EDTA, 150 p M dithiothreitol, 10 mM creatine phosphate, 20 pg/ml creatine kinase, and the GS-platinum complex (0,50,100,200, or 500 p ~ ) . At different incubation times, the reaction was terminated by transferring the samples onto ice.

To determine the amount of [35S]methionine incorporated into synthesized proteins, a 2-pl aliquot of the incubation medium was withdrawn and diluted in 200 p1 of ice-cold distilled water. 2 pl of the diluted sample was spotted on a Whatman 111 filter paper. The filter paper was placed in 100 ml of 10% trichloroacetic acid for 10 min. Subsequently, it was washed twice with 5% trichloroacetic acid and rinsed with distilled water. After the filter paper was dried, the radioactivity was determined in a liquid scintillation counter.

For SDS-PAGE analysis of the synthesized proteins according to Laemmli (36), the incubation medium (28 pl) was mixed with 10 p1 of SDS-PAGE sample buffer containing 250 mM Tris/glycine, pH 6.5, 40% glycerol, 2% SDS, 20% 2-mercaptoethanol, and 0.004% bromphenol blue. SDS-PAGE was performed using 12% acrylamide gels. After the electrophoresis, the gels were treated with En3Hance (Du Pont-New England Nuclear), dried in a gel dryer, and exposed to x-ray films (Kodak X-OMAT) a t -70 "C.

Preparation of Plasma Membrane Vesicles from LIZ10 Cells-In each preparation, L1210 cells (5 X 10' cells) were harvested by centrifugation and suspended in 50 ml of ice-cold PBS. After centrif- ugation a t 40 X g for 5 min, the cell pellet was diluted 40-fold with a hypotonic buffer (0.5 mM sodium phosphate, pH 7.0, 0.1 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride). The cell lysate was then centrifuged at 100,000 X g for 40 min, and the resulting pellet was suspended in the hypotonic buffer and homogenized with a Potter- Elvehjem homogenizer. The crude membrane fraction will be layered over 38% sucrose solution and centrifuged a t 100,000 X g for 30 min. The turbid layer at the interface was collected, suspended in 250 mM sucrose containing 10 mM Tris-HC1, pH 7.4, and centrifuged at 100,000 X g for 30 min. The membrane fraction was collected and resuspended in a small volume (50-100 p l ) of 250 mM sucrose con- taining 10 mM Tris-HC1, pH 7.4. Vesicles were formed by passing the suspension through a 27-gauge needle, frozen in liquid N,, and stored at -70 "C until used. Protein concentration was determined according to Lowry et al. (35). Sialidase accessibility for the determination of inside-out vesicles was examined as described previously (29).

Synthesis of 3H-labeled GS-Platinum Complex-Dithiothreitol was first removed from the [2-3H]glycine-labeled GSH sample (Du Pont- New England Nuclear) by extraction with ethyl acetate under acidic condition (10 mM HCl, pH 2.2). 1 mM l3H]GSH was incubated with 0.5 mM cisplatin in PBS a t 37 "C for 96 h under N, gas. The incubation medium was then applied onto a QAE-Sephadex column (bed volume, 1 ml) equilibrated with 10 mM Tris-HC1, pH 7.4, and the column was subsequently washed with 10 ml of distilled water. The retained [3H]GS-platinum complex was eluted with 0.2 M HC1 and then dried in a Speed Vac concentrator (Savant, Farmingdale, NY). The [3H]GS-platinum complex was dissolved in 10 mM Tris- HC1, pH 7.4, containing 0.25 M sucrose, and the concentration of GS- platinum was determined spectrophotometrically (e280 = 8.05 mM" cm"; see "Results"). The recovery was 97.3% as estimated from the initial radioactivity of the l3H]GSH sample.

Measurement of fH]GS-Platinum Complex Uptake by Plasma Membrane Vesicles Prepared from LIZ10 Cells-Frozen stocked mem- brane vesicles were thawed quickly a t 37 "C and stored on ice until used. The standard incubation medium contained plasma membrane vesicles (50 pg of protein), 200 p~ ['HIGS-platinum complex, 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4, 10 mM MgCl,, 1 mM ATP, 10 mM creatine phosphate, and 100 pg/ml creatine kinase in a final volume of 110 pl. The reaction was started by adding [3H]GS-platinum complex to the incubation medium. The reaction was carried out at 37 "C, and [3H]GS-platinum complex incorporated into the vesicles was measured by rapid filtration technique according to Ishikawa (29).

RESULTS

Reaction of Cisplatin with GSH: Formation of GS-Platinum Complex-During the incubation of cisplatin with GSH in PBS at 37 "C, the color of the reaction mixture gradually

turned from colorless to yellow. This was accompanied by a spectral change in which a broad absorption band in the wavelength range between 200 and 400 nm steadily increased (Fig. 1). Despite this broad absorbance range, peak absorbance occurred at 280 nm and increased over the 24-h incubation period. This 280-nm absorbance peak was not observed for incubates of GSH alone or cisplatin alone and thus was specific for the GS-platinum complex.

The stoichiometry for the GSH-cisplatin reaction deter- mined at partial molar ratios (GSH/cisplatin) of 0.5-4 is shown in Fig. 2. The absorbance at 280 nm (Ata0), representing the GS-platinum complex, increased with increasing GSH/ cisplatin ratios. A maximum Atso was attained at a GSH/ cisplatin molar ratio of 2; ratios greater than 2 did not result in increased Atso, indicating that 2 mol of GSH are involved in the reaction with 1 mol of cisplatin. The molar extinction coefficient at 280 nm (tZa0) was determined to be 8.05 mM" cm".

Isolation of GS-Platinum Complex-Because cisplatin is uncharged at physiological pH, it is not retained on QAE- Sephadex in the anion-exchange column chromatography (Fig. 3A) . In contrast, after incubation with GSH, cisplatin was converted to an anionic compound, which was tightly absorbed onto the anion-exchange matrix. As shown in Fig. 3B, the adsorbed reaction product could be eluted from the column with 0.2 M HCl, and the elution profile monitored at 280 nm was virtually superimposable on the curve obtained from platinum concentration of the collected fractions.

Mass Analysis of GS-Platinum Complex-The anionic com- pound, GS-platinum complex, eluted from the QAE-Sephadex column was further analyzed by mass spectroscopy. As shown

1 .o 1-

0 Wavelength (nrn)

FIG. 1. UV spectral change during the reaction of cisplatin with GSH. Cisplatin (1.67 mM) was incubated with 3.33 mM GSH in PBS a t 37 "C. At different times indicated in the figure, a 20-p1 aliquot was withdrawn from the incubation medium and diluted with 980 pl of PBS. The spectra were detected as described under "Mate- rials and Methods." The time course of the absorbance a t 280 nm is presented in the inset.

0 1 2 3 4 [GSH]/pisplatin]

FIG. 2. Stoichiometry for the reaction of cisplatin with GSH. Cisplatin (1.67 mM) was incubated a t 37 "C with different concentrations of GSH for 24 h under the conditions described in Fig. 1. A 20-4 aliquot of the incubate was diluted with 980 pl of PBS. Absorbance a t 280 nm of the diluted sample was plotted against the concentration ratio of GSH to cisplatin.

GSH-associated Cisplatin Metabolism and Export 20119

0.4

0 2 - I

E 0 ;

2

0 m

0.4 E 2

0.2

0 2 4 6 8 1 0

Elutlon Volume (ml)

FIG. 3. Anion-exchange column chromatography of cispla- tin ( A ) and GS-platinum complex ( B ) . Cisplatin (1.67 mM) was incubated without ( A ) or with ( B ) 3.33 mM GSH in PBS at 37 “C for 48 h as described in Fig. 1. 1 ml of the samples were applied onto QAE-Sephadex columns (1 ml of bed volume) equilibrated with 10 mM Tris-HC1, pH 7.5. The columns were washed with 5 ml of distilled water and subsequently with 5 ml of 0.2 M HC1. A fraction of eluent was collected every 0.5 ml. Platinum in each fraction was measured by atomic absorption spectroscopy as described under “Materials and Methods”; the concentration of platinum in the eluent is represented by the open circles. For the photometric assay, 50 pl of the eluent from each fraction was diluted with 950 p1 of PBS, and the absorbance at 280 nm was measured (solid circles).

o J, I 500 600 700 800 900 1000 1100 1200

Mass (m/z)

FIG. 4. Mass spectrum of GS-platinum complex. Matrix-as- sisted laser desorption mass spectroscopy of GS-platinum complex was performed as described under “Materials and Methods.” The molecular structure deduced from the observed molecular mass, 809 Da, is presented in the figure.

in Fig. 4, a single peak was detected at a molecular mass of 809 Da. This molecular mass corresponds to that of a GS- platinum chelate complex with a structure as presented in the inset of Fig. 4. Such a structure is also in agreement with the stoichiometric data, in which 2 mol of GSH complexed with 1 mol of platinum.

‘ H NMR Spectrum of GS-Platinum Complex--In contrast to that of GSH (Fig. 5 A ) , the ‘H NMR spectrum of the GS- platinum complex (Fig. 5B) exhibited broadened signal lines. Disappearance of fine structures of the signals was remarka- ble. In particular, the protons bound to cysteinyl a- and p- carbons, which are conformationally adjacent to the platinum ion (see inset of Fig 4), appeared to be strongly affected, suggesting an effect from the central platinum in the molecule. The signals of the protons of glutamyl a-, p-, and y-carbons as well as glycinyl a-carbon, were slightly shifted toward the hyper-magnetic field; they were 4.41, 2.00, 2.39, and 3.80 parts/million, respectively.

1 1 I 1 I I

GSH

B GS-Pl Complex

5 4 3 2 1 Chemical Shift (ppm)

FIG. 5. ‘H NMR spectra of GSH ( A ) and GS-platinum com- plex ( B ) . The NMR spectra were obtained at 200 MHz in a Bruker WH 200 F T spectrometer as described under “Materials and Meth- ods.”

1.5 I 2 2 I 3

6 i0 40 Retention Time (min)

FIG. 6. Detection of GS-platinum complex by HPLC and atomic absorption spectroscopy. A mixture of dinitrophenyl-de- rivatives of GS-platinum complex (4 pmol), GSSG (4 pmol), and GSH (8 pmol) was subjected to HPLC as described under “Materials and Methods.” GSH, peak 1; GSSG, peak 2; GS-platinum complex, peak 3. The eluent was collected every 1 min, and the platinum concentra- tion was determined by atomic absorption spectroscopy.

HPLC Analysis of GS-Platinum Complex-The reaction product of cisplatin and GSH was derivatized using l-fluoro- 2,4-dinitrobenzene and subjected to HPLC. Fig. 6 is a graph of the typical HPLC elution profiles for GSH (peak I ) , GSSG (peak Z ) , and the GS-platinum complex (peak 3 ) . The reten- tion times were 13, 20, and 39 min, respectively. The GS- platinum peak (peak 3 ) was identified by analysis of the platinum content of the fractions.

Formation of GS-Platinum Complex in LIZ10 Cells-Using this HPLC technique, we have examined the formation of the GS-platinum complex in L1210 cells. The cells were incubated with 20 ~ L M cisplatin for 0, 3, 6, 9, 12, and 24 h and the GS- platinum complex in the cell extract was analyzed by the combination of HPLC and atomic absorption spectroscopy. Fig. 7 shows the HPLC trace ( A ) and the intracellular con- tents of the GS-platinum complex ( B ) as well as GSH (C) in

20120 GSH-associated Cisplatin Metabolism a n d Export

I 20 40 Retention Time (min)

i Time (h) i2

0 12 24 Time (h)

FIG. 7. Intracellular GS-platinum complex and GSH levels in L1210 cells incubated with cisplatin. A , HPLC trace of the cell extract obtained after 12 h of incubation. 2,4-Dinitrophenyl derivatives of the cell extract were separated as described under “Materials and Methods” and in Fig. 6. The amount of GS-platinum complex in the eluent was determined by atomic absorption spectros- copy. B, time courses of intracellular levels of the GS-platinum complex (open triangles) and total platinum (solid triangles) in L1210 cells incubated with 20 pM cisplatin. C , time courses of intracellular GSH level in L1210 cells incubated with (open circles) or without (solid circles) 20 p~ cisplatin. Data are expressed mean * S.E., n = 3.

L1210 cells. The intracellular content of the GS-platinum complex reached its maximal level (0.91 +. 0.14 nmol x mg protein”, n = 3) after 12 h, corresponding to 60% of the intracellular platinum level (1.50 * 0.38 nmol X mg protein”, n = 3) (Fig. 7B). Interestingly, in the cells incubated with cisplatin, intracellular GSH level was elevated about &fold and reached its maximal level (103 & 18 nmol x mg protein“, n = 3) after 9 h. The GSH content in control cells did not significantly change over this time period (Fig. 7C).

Effect of Cisplatin on Protein Synthesis in L1210 Cells-In L1210 cells incubated with 20 pM cisplatin, [“S]methione incorporation rate was significantly decreased during a period of 6-24 h, whereas in the control cells the rate was almost constant (Fig. 8A) . Although no remarkable difference was observed in the protein pattern between the control and cisplatin-treated cells (Fig. 8B, Coomassie staining), the au-

40-

20-

0- , 0 12 24

Time (h)

r’ 30.

20

Coornassie [%]Met Staining Incorporation

FIG. 8. Effect of cisplatin on protein synthesis in L1210 cells. A , time courses of the rate of [“S]methionine incorporation into cellular proteins in L1210 cells incubated with 20 pM cisplatin (open circles) and in the control cells (solid circles). [”S]Methionine incorporation was determined as described under “Materials and Methods.” Data are expressed as mean k S.E., n = 3. B, SDS-PAGE and [3sS]methionine autoradiography of the cellular proteins of the control cells (lane I ) and the cells exposed to cisplatin (lane 2) . After 12 h of incubation with or without 20 p~ cisplatin, the cells were incubated with [“‘S]methionine a t 37 “C for 1 h as described under “Materials and Methods.” 70 pg of the cellular proteins were applied to SDS-PAGE and stained with Coomassie Blue R250. [35S]Methio- nine incorporated into the proteins was detected by autoradiography.

toradiography clearly demonstrates that de novo synthesis of a number of cellular proteins were affected in the cisplatin- incubated cells (Fig. 8B, [”Slmethione incorporation).

Inhibition of in Vitro Protein Synthesis by GS-Platinum Complex-The biological activity of the GS-platinum complex was examined with an in vitro translation system. Fig. 9 shows the time courses of the translation of chloramphenicol ace- tyltransferase (CAT) mRNA in the presence and absence of the GS-platinum complex, demonstrating an inhibitory effect of the complex on the protein synthesis. The inhibition by the GS-platinum complex was dose dependent with a half- inhibitory concentration (ICso) of 190 p~ (Fig. 10). Such inhibition was observed not only for CAT mRNA but also for poly(A) mRNA from HL-60 human leukemia cells (data not shown). I t is noteworthy that the inhibition resulted in a decrease in the amount of synthesized CAT protein with no alterations of its molecular size (Fig. 1OA). This indicates that the inhibition occurred at an initiation step of the trans- lation process, rather than in the elongation process.

ATP-dependent Transport of GS-Platinum Complex across Plasma Membrane of L1210 Cells-Active elimination of the

GSH-associated Cisplatin Metabolism and Export

+ CATmRNA

/ None

10 20 30 Time (min)

FIG. 9. Inhibition of in vitro protein synthesis by the GS- platinum complex. 300 ng of CAT mRNA was incubated with [''SI methionine in 30 pl of rabbit reticulocyte lysate in the absence (solid circles) and presence (open circles) of the GS-platinum complex (500 p M ) a t 30 "C for 5, 10, 20, and 30 min as described under "Materials and Methods." As control, ["S]methionine was incubated in the rabbit reticulocyte lysate without mRNA and the GS-platinum com- plex (solid triangles). Radioactivity incorporated into the synthesized proteins was counted in a liquid scintillation counter.

GS-platinum complex from tumor cells is proposed as a potentially significant mechanism for reducing intracellular accumulation of the cytotoxic complex. Fig. 11 shows the time course of uptake of "-labeled GS-platinum complex by plasma membrane vesicles prepared from L1210 leukemia cells. The transport of the GS-platinum complex into the plasma membrane vesicles was stimulated by ATP. 47 & 4 % (mean & S.E., n = 3) of the total membrane vesicle population was inside-out as assessed by the sialidase accessibility. As shown in Fig. 12, the amount of the GS-platinum complex taken up by vesicles was markedly decreased by increasing osmolarity of the extravesicular medium with sucrose, strongly suggesting that the complex was actually transported into the intravesicular space. The ATP-dependent transport was not affected by acivicin, a known inhibitor of y-gluta- myltransferase, indicating the accumulation of the GS-plati- num complex into the vesicles, not the decomposed product of the complex through the action of y-glutamyltransferase. Furthermore, the GS-platinum complex transported into the vesicles was identified by HPLC (Fig. 13). 97% of total radio- activity incorporated into vesicles was ascribed to the GS- platinum complex. Contamination of GSSG was about 3%; this may be due to dissociation of the GS-platinum complex and subsequent oxidation of GSH. In addition, platinum remaining on filter membrane after rapid filtration was de- tected by atomic absorption spectroscopy. These data confirm the uptake of the GS-platinum complex by plasma membrane vesicles from L1210 cells.

Fig. 14 shows the effect of GS-platinum and ATP concen- trations on the rate of GS-platinum complex uptake. The apparent K , values for the GS-platinum complex and for ATP were estimated to be 110 and 48 p ~ , respectively. The ATP-dependent transport of the GS-platinum complex was vanadate-sensitive. Half-maximal inhibition was observed a t a concentration of 35 p~ vanadate. Table I summarizes the nucleotide specificity of the GS-platinum complex uptake. ATP analogues, i.e. AMP-PCP and AMP-PNP, were not effective, suggesting that hydrolysis of y-phosphate is essen-

A 1 2 3 4 5

94 -- . ._.. 67 - 43 -

2 30-

m

x f

20 - 14 -

20121

0 0.1 GS-Pt Complex (o), Cisplatin (0 ) (mW

FIG. 10. Dose dependence in the inhibition of in vitro pro- tein synthesis by GS-platinum complex. 300 ng of CAT mRNA was incubated with [:"S]methionine in 30 p1 of rabbit reticulocyte lysate in the presence of the GS-platinum complex a t concentrations of 0,50, 100, 200, and 500 p~ a t 30 "C for 30 min. A, aliquots of the samples were applied to SDS-PAGE. The gel was treated with En'Hance and exposed to an x-ray film. Lane 1 , control; lane 2, 50 pM; lane 3, 100 pM; lane 4, 200 pM; lane 5, 500 pM GS-platinum complex. B, radioactivity incorporated into the synthesized CAT protein in the presence of cisplatin (solid circles) or the GS-platinum complex (open circles) was counted in a liquid scintillation counter. Data are expressed as mean S.E., n = 3.

tial for the uptake. Other nucleotide triphosphates, i.e. CTP, GTP, TTP, and UTP, could be replaced for ATP, however, their efficiency was less than that of ATP. The ATP-stimu- lated uptake of the GS-platinum complex was inhibited by GSSG, DNP-SG, and LTC4, substrates for the ATP-depend- ent glutathione S-conjugate export pump (GS-X pump) (Table 11). The extent of the inhibition by LTC, is highest among these three different compounds. On the other hand, doxorubicin, daunorubicin, and verapamil, which are sub- strates for P-glycoprotein, did not inhibit the ATP-dependent uptake of the GS-platinum complex.

DISCUSSION

Molecular Structure of GS-Platinum Complex-Recent studies suggest a critical role of GSH in mechanisms of tumor- cell resistance to alkylating agents, such as cisplatin (19, 22- 26, 37, 38), L-phenylalanine mustard (22, 23, 39), cyclophos- phamide (40,41), and chloroethylnitrosoureas (9,32,42). This has been attributed, in part, to the ability of GSH to inactivate compounds and to quench the DNA cross-link precursors that these compounds produce. Despite the significant role of GSH in determining the resistance of tumor cells to alkylators, the actual structure of drug-GSH complexes has been character- ized for only a few of these agents (24-26,43).

In the present study, we have examined the kinetics and stoichiometry of the formation of a GS-platinum complex in

20122 GSH-associated Cisplatin Metabolism and Export

0

T

I I

20 40 Time (min)

FIG. 11. Time course of [3H]GS-platinum complex uptake by inside-out plasma membrane vesicles prepared from L1210 cells. Plasma membrane vesicles from L1210 cells (50 pg of protein) were incubated 200 p~ [3H]GS-platinum complex at 37 “C in the absence (solid circles) or the presence (open circles) of 1 mM ATP in 110 p1 of the incubation medium containing 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4, 10 mM MgCl,, 10 mM creatine phosphate and creatine kinase (100 pg/ml). Uptake of GS-platinum complex was determined as described under “Materials and Methods.” Data are expressed as mean & S.E., n = 3.

I I

0 1 2 3 4

[Sucrose]’ (MI)

FIG. 12. Effect of osmolarity on ATP-dependent uptake of the GS-platinum complex by L1210 cell plasma membrane vesicles. Plasma membrane vesicles from L1210 cells (50 pg of protein) were incubated 200 p~ [3H]GS-platinum complex at 37 “C for 30 min in the absence or presence of 1 mM ATP in 110 p1 of the incubation medium containing 10 mM Tris-HC1, pH 7.4, 10 mM MgCl,, 10 mM creatine phosphate, creatine kinase (100 pg/ml), and sucrose (0 , 25,0.33, 0.50, or 1.00 M) . Uptake of GS-platinum complex was determined as described under “Materials and Methods.” ATP- dependent uptake was obtained from the difference in the radioactiv- ities incorporated into the vesicles in the presence and absence of ATP. Data are expressed as mean f S.E., n = 3.

a cell-free system and have clarified its structure. In the reaction with cisplatin, each GSH molecule acts as a bidentate chelating ligand, coordinating to platinum via cysteinyl sulfur and nitrogen atoms. This is supported by the NMR spectrum exhibiting broadened signal lines of the protons bound to cysteinyl a- and p-carbons (Fig. 5B) . These carbons are covalently bound to the sulfur and nitrogen atoms and con- formationally close to the platinum ion. Thus, those protons are likely to be more strongly affected by the platinum ion than the other protons. In addition, the molecular mass of

20 40 Retention Time (min)

FIG. 13. Identification of the GS-platinum complex incor- porated into plasma membrane vesicles. ATP-dependent uptake of (3H]GS-platinum complex by L1210 cell plasma membrane vesicles was performed as described under “Materials and Methods.” After rapid filtration, the filter membranes were treated with distilled water and centrifuged at 10,000 X g for 30 min. The resulting supernatant was concentrated in a Speed Vac concentrator and then treated with 1% l-fluoro-2,4-dinitrobenzene. The sample was analyzed by HPLC as described under “Materials and Methods.” The eluent was collected every 1 min, and the radioactivity was detected in a liquid scintillation counter.

0.25 GS-PI Complex (mM)

0.5

0 0.25 0.5 1.0 ATP (mM)

FIG. 14. Effect of GS-platinum complex (A) and ATP ( B ) concentrations on the rate of GS-platinum complex uptake into L1210 cell plasma membrane vesicles. A, the membrane vesicles (50 pg of protein) were incubated with [3H]GS-platinum complex (10, 25, 50, 100, 200, and 500 p M ) for 10 min in the absence (solid circles) or presence (open circles) of 1 mM ATP under the conditions described in Fig. 10. B, the membrane vesicles (50 pg of protein) were incubated with 200 p~ [3H]GS-platinum complex at 37 “C for 20 min in the presence of ATP (0, 20, 50, 100, 200, 500 pM and 1 mM). Uptake of GS-platinum complex was determined as described under “Materials and Methods.” Data are expressed as mean k S.E., n = 3.

the GS-platinum complex was determined to be 809 Da by mass spectroscopy (Fig. 4). The GS-platinum complex showed a broad spectrum of absorption in a wavelength range of 200 to 400 nm, and its molar extinction coefficient at 280 nm was computed as 8.05 mM-l cm”. The GS-platinum complex is

~ S H - ~ s s o c ~ a t e d ~ ~ s p l a t i ~ M e t a b o ~ ~ s m and Export 20123

TABLE I EJJect of various nucleotides o n the uptake of the GS-platinum

complex by L1210 plasma membrane vesicles L1210 plasma membrane vesicles (50 pg of protein) were incubated

with 200 PM ~3H]GS-platinum complex at 37 "C for 10 min in 110 pl of the incubation medium containing 10 mM Tris-HC1, pH 7.4, 250 mM sucrose, and 4 mM MgCl, in the presence of the indicated nucleotide (4 mM). Data are expressed as mean values of triplicate experiments.

Nucleotide GS-platinum uptake nmol/mg proteinfI0 min

None 0.32 ATP 1.51

0.33

CTP 0.38 GTP TTP

0.57 0.52

UTP 0.53

AMP-PCP AMP-PNP 0.33

TABLE I1 EJJect of GSSG, DNP-SG, LTC,, verapamil, doxorubicin, and

daunorubicin on ATP-stimulated GS-platinum uptake by L1210 plasma membrane vesicles

L1210 plasma membrane vesicles (50 fig of protein) were incubated with 200 p~ [3H]GS-platinum complex at 37 "C for 10 min as de- scribed under "Materials and Methods." The compounds were added into the incubation medium at the final concentrations indicated in the table. ATP-stimulated uptake was obtained from the difference in the radioactivities incorporated into the vesicles in the presence and absence of 1 mM ATP. Data are presented as mean values of triplicate measurements. "_ -___

Compound

None GSSG DNP-SG

LTC,

Doxorubicin Daunorubicin Verauamil

Conc.

irM

0 100 100 10 10

1 100 100 100

ATP-stimulated GS-platinum uptake

nmollmg proteinll0 min 1.26 0.56 0.15 0.78 0.11 0.43 1.16 1.20 1.31

% 100 44 12 62 9

34 92 95

104

stable in acidic or neutral aqueous solutions, and no remark- able change was detected in the UV spectrum after 1 month of storage at 4 "C.

We have provided here evidence for the formation of this GS-platinum complex in L1210 leukemia cells (Fig. 7). In a previous report on the subcellular distribution of cisplatin in rat liver and kidney, Sharma and Edwards (44) showed that a major part of cytosolic platinum was present as low molec- ular weight species (molecular weight <1,000). However, the nature of these complexes was not clarified. Other studies, using cell-free systems, have shown the products of the reac- tion of cisplatin with sulfur-containing nucleophiles, includ- ing cysteine and GSH, to be chelate complexes with platinum (25, 26) . We have extended these studies and have described a relatively simple method for the detection of the GS-plati- num complex that combines atomic absorption spectroscopy with HPLC separation of 2,4-dinitro~nzene derivatives of cell extracts. The optimal conditions for the separation of the GS-platinum complex from GSH and GSSG by HPLC have also been described.

Biological Activity of GS-Platinum Complex-It has been reported that about 1% of cisplatin reacts with genomic DNA, whereas most part of the drug interacts with proteins, RNA, and small thiol compounds (45). The present study shows that the GS-platinum complex was formed as a major metab-

olite in L1210 cells exposed to cisplatin (Fig. 7). The result is important to understanding GSH-associated cellular metab- olism of cisplatin, and it may contribute to the clarification of the role of GSH in tumor cell resistance (or sensitivity) to cisplatin, Protein synthesis was inhibited in the L E 1 0 cells exposed to cisplatin (Fig. 8). Although this could result from the inhibition of transcription at the sites of DNA-platinum cross-links, the evidence that the GS-platinum complex in- hibited in vitro translation of mRNA in the reticulocyte lysate system (Figs. 9 and 10) suggests an additional mechanism for the inhibition of protein synthesis by cisplatin in cells. Such inhibition of protein synthesis has also been reported previ- ously for a GSH-selenium chelate complex, selenodigluta- thione (46, 47). Accumulating evidence suggests that the GSH-selenium chelate complex is critically involved in selen- ite-mediated inhibition of tumor cell growth (48, 49). In the case of the GS-platinum complex, the inhibition of protein synthesis did not result in significant changes in the molecular weight of the synthesized proteins (Fig. lo), suggesting that the inhibition may occur at the initiation step of mRNA translation. This is consistent with the report that protein synthesis inhibition by GSSG (50, 51) could be ascribed to the inhibition of eukaryotic initiation factor 2 (52). The ICs0 value for the inhibition of in uitro translation of CAT mRNA by the GS-platinum complex was 190 PM (Fig. 10). Although this value is higher than that of GSSG (20-50 ,UM) (50, 51), accumulation of the complex in cells could result in intracel- lular concentrations equivalent to or even higher than the ICbo value and could Contribute to the toxicity of cisplatin.

Renal impairment is known as a major toxic complication of cisplatin therapy. The extent of cellular uptake or retention of platinum in the renal tissue is suggested to be a critical determinant. Sharma and Edwards reported the high accu- mulation of platinum in the cytosol of rat kidney (44). They pointed out that localization of cellular platinum in the cytosol and the presence of a high proportion as non-protein bound species (molecular weight < 1,000) may be important factors in relation to the renal toxicity of cisplatin. Their suggestion would become even more important if the GS-platinum com- plex is identified in the kidney and its biological activity in the tissue is characterized. Using several resistant and sensi- tive variant cells derived from the L1210 cell line, Richon et al. (37) indicated that both decreased cellular accumulation of platinum and increased cellular GSH level are important in the development of resistance to cisplatin. Based on those studies, as well as on experimental data in this paper, we suggest that the formation of the chelate complex between platinum and GSH and the subsequent elimination of this complex from cells would be of biological importance.

Efflux of GS-PEatinum Complex from Cells-Many metals are eliminated into bile and urine (27). The complex forma- tion of these metals with GSH and the subsequent active membrane transport of the glutathione-metal complexes is assumed to be the underlying mechanism for their elimination (28). Recent studies have shown that chromium (VI), which induces DNA damage, forms a thiolate complex with GSH (53) and that arsenic, a cytotoxic metal ion, is excreted into bile as a GSH complex (54). Evidence that some glutathione- drug conjugates have biological activity (28, 55) strongly sug- gests that the elimination of such complexes could be a critical determinant of cellular response to the drugs.

In previous studies, our group and others have characterized an ATP-dependent export pump, GS-X pump, that transports a variety of divalent organic anions, including cysteinyl leu- kotrienes, glutathione S-conjugates and GSSG (see Ref. 56, for recent review). This GS-X pump is distinct from P-

20124 GSH-associated Cisplatin Metabolism and Export

lnhibnory En& on Protein Synthesis I GS-XPump

FIG. 15. Schematic illustration for intracellular reactions of cisplatin with GSH and DNA as well as for the elimination of GS-platinum complex. The antitumor activity of cisplatin is attributed primarily to the formation of DNA-platinum-DNA cross- links. GSH in the nucleus can quench DNA-platinum monoadducts before their conversion to the cross-links. In the cytoplasm, and probably in the nucleus as well, GSH reacts with cisplatin to form the GS-platinum chelate complex which is potentially active in the inhibition of protein synthesis. The GS-X pump localized in the plasma membrane eliminates the GS-platinum complex from the cell by an ATP-dependent manner.

glycoprotein (57 ) . Based on the structural homology of the GS-platinum complex to GSSG, as well as on the fact that GS-platinum is a divalent anion at the physiological pH,' it is hypothesized that the complex is eliminated from cells via the GS-X pump.

Direct evidence has been provided here for the ATP-de- pendent transport of the GS-platinum complex across the plasma membrane of L1210 cells (Figs. 11-14). The ATP- dependent transport was inhibited by typical substrates of the GS-Xpump, i.e. DNP-SG, GSSG, and LTC4, but not inhibited by doxorubicin, daunorubicin, or verapamil, which are sub- strates and mutual inhibitors for P-glycoprotein (Table 11). ATP-dependent uptake of [3H]DNP-SG (100 PM in the in- cubation medium) into the plasma membrane vesicles was inhibited by the GS-platinum complex with an ICso value of 150 PM (data not shown), suggesting a mutual competition between the GS-platinum and DNP-SG. Moreover, the nu- cleotide specificity of GS-platinum transport (Table I) and its kinetics for vanadate inhibition (ICso = 35 PM) are similar to those reported for ATP-dependent transport of glutathione S-conjugate mediated by the GS-X pump (29). Thus, based on these results, we conclude that the GS-X pump plays a role in export of the GS-platinum complex from L1210 cells.

Concluding Remarks-The present study addresses the bi- ological importance of the chelate complex formed between platinum and GSH, as well as the subsequent elimination of the chelate complex from cells. Fig. 15 depicts a scheme for the cellular metabolism of cisplatin and efflux of the GS- platinum complex. Intracellular GSH level in L1210 cells was found to be enhanced about &fold during incubation with 20 PM cisplatin (Fig. 7 C ) , suggesting that an increase in cellular GSH synthesis is part of the cellular response to cisplatin. A recent study of Godwin et al. (19) has shown that increased cellular GSH levels in cisplatin-resistant variants of human ovarian cancer cell lines are closely related to enhanced expression of mRNAs of both y-glutamylcysteine synthetase and y-glutamyltransferase, suggesting that the glutathione- associated metabolism of cisplatin would be a significant property of these cisplatin-resistant cells. In this context, it is of interest and importance to know whether the GS-X pump, which exports the GS-platinum complex, is overex- pressed in cisplatin-resistant tumor cells, and also to what extent the expression of the GS-X pump could contribute to

cellular resistance to cisplatin and other anticancer drugs that are conjugated with GSH.

Acknowledgments-We thank Drs. B. Seifert and A. Ballatore (Analytical Chemistry Center, The University of Texas Medical School) for support in the mass-spectroscopic analysis of the GS- platinum complex, as well as Dr. T. Madden (Department of Exper- imental Pediatrics, M. D. Anderson Cancer Center) for initial assist- ance in platinum measurement. We are particularly grateful to Dr. Z. H. Siddik (Department of Medical Oncology, M. D. Anderson Cancer Center) for helpful discussions and generous support in the determi- nation of platinum, as well as Drs. S. Khan and D. Farquar (Depart- ment of Medical Oncology, M. D. Anderson Cancer Center) for NMR analysis. Finally, we thank Alexander Renwick and Hui Wang for their excellent technical support in the HPLC analysis and Lois Craft for her assistance in preparing this manuscript.

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