Coordinated Induction of MRP/GS-X Pump and g-GlutamylcysteineSynthetase by Heavy Metals in Human Leukemia Cells*

(Received for publication, November 27, 1995, and in revised form, March 6, 1996)

Toshihisa Ishikawa‡§, Jia-Ju Bao¶, Yoshiaki Yamane¶, Kunihiro Akimaru‡, Karl Frindrich‡,Christine D. Wright‡, and M. Tien Kuo¶

From the ‡Section of Molecular Therapeutics, Department of Experimental Pediatrics and ¶Section of Eucaryotic CellResearch, Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center,Houston, Texas 77030

We recently reported that GS-X pump activity, as as-sessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellularglutathione (GSH) levels were remarkably enhanced incis-diamminedichloroplatinum(II) (cisplatin)-resistanthuman leukemia HL-60 cells (Ishikawa, T., Wright, C. D.,and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085–29093).Now, using Northern hybridization and RNase protec-tion assay, we provide evidence that the multidrug re-sistance-associated protein (MRP) gene, which encodesa human GS-X pump, is expressed at higher levels incisplatin-resistant (HL-60/R-CP) cells than in sensitivecells, whereas amplification of the MRP gene is not de-tected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reducedMRP mRNA levels, but these levels could be induced torise within 30 h by cisplatin and heavy metals such asarsenite, cadmium, and zinc. The increased levels ofMRP mRNA were closely related with enhanced activi-ties of ATP-dependent transport of leukotriene C4(LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibitedATP-dependent LTC4 transport, suggesting that theMRP/GS-X pump transports both LTC4 and the GS-Ptcomplex. Expression of g-glutamylcysteine synthetasein the cisplatin-resistant cells was also co-inducedwithin 24 h in response to cisplatin exposure, resultingin a significant increase in cellular GSH level. The re-sistant cells exposed to cisplatin were cross-resistant tomelphalan, chlorambucil, arsenite, and cadmium. Theseobservations suggest that elevated expression of theMRP/GS-X pump and increased GSH biosynthesis to-gether may be important factors in the cellular metab-olism and disposition of cisplatin, alkylating agents, andheavy metals.

Development of drug resistance in tumor cells is a significantobstacle to long-term, sustained patient response to chemother-

apy. There is accumulating evidence that active export of an-ticancer drugs from cells is one of the major mechanisms ofdrug resistance. The GS-X pump1,2 has been shown to elimi-nate a potentially cytotoxic glutathione-platinum (GS-Pt) com-plex from tumor cells, thereby modulating glutathione (GSH)-associated resistance to cisplatin (1). The GS-X pump isfunctionally overexpressed in cisplatin-resistant human leuke-mia HL-60 cells (HL-60/R-CP), in which the cellular GSH levelis substantially enhanced (2).The GS-X pump is an ATP-dependent export pump for or-

ganic anions such as cysteinyl leukotrienes, glutathione disul-fide (GSSG), glutathione S-conjugates, and glucuronide conju-gates, and certain organic anions such as methotrexate. Itplays a physiologically important role in inflammation, oxida-tive stress, xenobiotic metabolism, and tumor drug resistance(3–5). Although the kinetic properties and substrate specificityof this novel transporter have been studied, only recently hasits molecular nature been identified. Studies by Muller et al. (6)and Leier et al. (7) have provided important evidence thatoverexpression of the multidrug resistance-associated protein(MRP) gene in human cancer cells results in increased ATP-dependent GS-conjugate transport, thus demonstrating thatthe MRP gene product is a human GS-X pump (8). Moreover,the yeast cadmium factor (YCF1) gene from Saccharomycescerevisiae has been identified on the basis of its ability to confercadmium resistance (9). The YCF1 gene encodes an ATP-bind-ing cassette protein with extensive sequence homology to hu-manMRP (9). More importantly to our present study, however,this gene product was recently found to be a vacuolar GS-Xpump in yeast cells.3 Thus, MRP and YCF1 gene products aremembers of theGS-X pump family occurring in both the animaland plant kingdoms. Based on those recent findings, we haveexamined in this study the expression of the MRP gene inHL-60/R-CP cells as well as its potential role in cellular resist-ance to heavy metals.A recent publication by de Vries et al. (10) pointed out that

* This study was supported in part by NCI, National Institutes ofHealth Research Grants R01 CA60486 (to T. I.) and CA56846 (toM. T. K.) and research grants from the International Life SciencesInstitute Research Foundation, Washington, D. C. (to T. I.). The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.We sincerely dedicate this study to Dr. Alton Meister for his great

contribution to glutathione research in biochemistry and cancer biology.Dr. Meister died on April 6, 1995.§ To whom correspondence and reprint requests should be addressed:

Dept. of Medicinal Biology, Central Research, Pfizer Inc., 5-2 Taketoyo,Aichi 470–23, Japan. Tel.: 81-569-72-8526; Fax: 81-569-72-8524; E-mail: ishikawa@pfizer.co.jp.

1 We use the term “MRP/GS-X pump” to specify the transporterprotein encoded by the human multidrug resistance-associated protein(MRP) gene (15, 45, 52) and characterized by its ATP-dependent, pri-mary active transport of glutathione S-conjugates across the cell mem-brane (3). Recent studies of transfection of the MRP gene into cancercells and functional analysis have provided evidence of the direct linkbetween the MRP gene and a human GS-X pump (6, 7).

2 The abbreviations used are: GS-X pump, ATP-dependent glutathi-one S-conjugate export pump; MRP, multidrug resistance-associatedprotein; YCF1, yeast cadmium factor in S. cerevisiae; ltpgpA, Leishma-nia tarentolae P-glycoprotein homolog gene; GS-Pt, bis-(glutathionato)-platinum(II); GS-conjugate, glutathione S-conjugate; g-GCS, g-glu-tamylcysteine synthetase; LTC4, leukotriene C4; RT-PCR, reversetranscriptase-polymerase chain reaction; kb, kilobase(s).

3 Li, Z.-S., Szczypka, M., Lu, Y.-P., Thiele, D. J., and Rea, P. A. (1996)J. Biol. Chem. 271, 6509–6517.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 25, Issue of June 21, pp. 14981–14988, 1996© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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the overexpression of the MRP/GS-X pump per se does notnecessarily result in resistance to anticancer drugs (10). If so,then the ultimate effect of the export pump would most likelyrequire other fundamentally important factors, one being thebiosynthesis of GSH. Through the conjugation reaction withcellular GSH, electrophilic organic compounds as well as heavymetals are converted to organic anions; thus, the multivalentnegative charge is apparently crucial for the recognition ofsubstrates by the MRP/GS-X pump (3, 11).Cellular GSH is synthesized by g-glutamylcysteine synthe-

tase (g-GCS) and GSH synthetase. The first reaction, catalyzedby g-GCS, is a rate-limiting step in overall GSH biosynthesis,and the cellular GSH level is substantially regulated by g-GCS(12). Furthermore, correlation between cisplatin resistance andexpression of g-GCS has been demonstrated in human ovariancancer cells (13). In HL-60/R-CP cells, cisplatin-resistant leu-kemia cells established in our laboratory, the cellular GSHlevel was 7- to 8-fold higher than in sensitive cells. To gainmore insight into the mechanism responsible for this, we ex-amined the expression of g-GCS in the cisplatin-resistant and-sensitive cells. We present strong evidence that the g-GCSexpression was remarkably induced by cisplatin within 24 hand that the cellular GSH level was concomitantly increased inthe cisplatin-resistant but not the sensitive cells.The present study demonstrates for the first time that both

the MRP/GS-X pump and g-GCS are induced by cisplatin andheavy metals. Their coordinate induction is likely to be animportant determinant for the acquired, cellular resistance tocisplatin, heavy metals, and alkylating agents.

MATERIALS AND METHODS

Biochemicals and Chemicals—GSH, GSSG, ATP, creatine phos-phate, creatine kinase, phenylmethylsulfonyl fluoride, glutathione re-ductase, and a random-primed labeling kit were purchased from Boeh-ringer Mannheim (Mannheim, Germany). [3H]GSH and [3H]LTC4 wereobtained from DuPont NEN. [32P]dCTP was from Amersham. Cisplatinwas obtained from Bristol-Myers Co. (Evansville, IN). RPMI 1640 me-dium, fetal calf serum, gentamicin, chlorambucil, melphalan, sodiumarsenite, cadmium chloride, and zinc acetate were from Sigma. Allother chemicals were of analytical grade.Cell Culture—Human myelocytic leukemia HL-60 cells (ATCC No.

CCL240), obtained from the American Type Culture Collection (Rock-ville, MD), were maintained in a humidified chamber (37 °C, 5% CO2) inRPMI 1640 medium supplemented with glutamine (2 mM), 10% (v/v)heat-inactivated fetal calf serum, and gentamicin (50 mg/ml). The cellnumbers were determined by trypan blue dye exclusion and countingwith a hemacytometer and were kept at 1.5 3 105 cells/ml by passagingevery 5 days. A cisplatin-resistant subline, named HL-60/R-CP, wasestablished by maintaining HL-60 cells in the presence of cisplatin(Bristol-Myers Squibb, Seattle, WA), as described previously (2). Forthe induction experiments, HL-60/R-CP cells were maintained in thecisplatin-free medium for 1 month and subsequently treated with cis-platin or heavy metals for various lengths of time.Determination of Cell Sensitivity to Anticancer Drugs and Heavy

Metals—Cells (1.5 3 105 cells/ml) were incubated in 100 ml of culturemedium containing melphalan, chlorambucil, cadmium chloride, or so-dium arsenite at different concentrations in 96-well plates in a humid-ified tissue culture chamber (37 °C, 5% CO2). After 72 h, the number ofsurviving cells was determined.Determination of GS-X Pump Activity in Plasma Membrane Vesi-

cles—Plasma membrane vesicles were prepared from HL-60 and HL-60/R-CP cells as described previously (2) and stored at 285 °C until use.Frozen stocked membrane vesicles were thawed quickly at 37 °C andstored on ice until used. Of the total membrane vesicles, 42 to 46% wereinside-out as assessed by sialidase accessibility assay (14). The stand-ard incubation medium for the assay of GS-X pump activity in thepreparation contained plasma membrane vesicles (50 mg of protein), 10nM [3H]LTC4, 0.25 M sucrose, 10 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 1mM ATP, 10 mM creatine phosphate, and 100 mg/ml creatine kinase ina final volume of 110 ml. The reaction was started by adding [3H]LTC4

to the incubation medium. The reaction was carried out at 37 °C, andthe amount of [3H]LTC4 incorporated into the vesicles was measured bya rapid filtration technique as described previously (14).

cDNA Probes for MRP and g-GCS—An MRP-cDNA probe (2.86 kb)was prepared from total RNA of human mononuclear cells by RT-PCRusingMRP-specific primers: forward primer (225–245), 59-TGCCTTGG-GATTTTTGCTGTG; backward primer (3085–3066), 59-CGATCCCTT-GTGAAATGCCC. The PCR reaction consisted of 34 cycles of 94 °C for30 s, 55 °C for 60 s, and 72 °C for 160 s. The resulting RT-PCR productwas ligated with the pCRTMII vector (Invitrogen) and amplified usingOne ShotTM INVaF9 competent cells (Invitrogen). The DNA sequence ofthe insert was identical with the partial sequence (225–3085) of theMRP cDNA (15). The 2.86-kb insert was excised by EcoRI digestion,purified, and used for Northern and Southern hybridizations.A cDNA probe for the catalytic subunit of g-GCS was prepared from

total RNA of HL-60 cells by RT-PCR using forward primer 59-GCTG-CATCTCCCTTTTACCGAG and backward primer 59-TGGCAACTGT-CATTAGTTCTCCAG. The 0.88-kb PCR product had a sequence iden-tical with the partial cDNA sequence (841–1723) of g-GCS (16). ThePCR product was ligated with the pCRTMII vector and amplified usingOne ShotTM INVaF9 competent cells. The 0.88-kb insert was excised byEcoRI digestion, purified, and used for Northern hybridization.Sequence Analysis—DNA sequences of the PCR products ligated with

pCRTMII vector were determined using a DNA sequence analyzer (Ap-plied Biosystems Model 373A). Sequencing reactions utilized Seque-nase (U. S. Biochemical Corp.) and synthetic oligonucleotide primerscorresponding to T7, SP6, and internal sequences of MRP or g-GCS.Northern Hybridization—Total cellular RNA was prepared by acid

guanidinium thiocyanate-phenol-chloroform extraction from samples of1 3 107 cells as described by Chomczynski and Sacchi (17). The RNA (10mg/lane as determined by absorbance at 260 nm) was fractionated byelectrophoresis in 1.0% (w/v) agarose gels containing formaldehyde (18)and transferred to Nytran membranes (Schleicher & Schuell). Themembranes were then baked at 80 °C for 2 h. 32P-Labeled DNA probeswere prepared by a random-primer labeling method. Hybridizationwith the DNA probe (1 3 106 cpm/ml) was performed at 42 °C for 24 hin a mixture containing 5 3 SSPE (750 mM sodium chloride, 5 mM

EDTA, and 50 mM sodium phosphate, pH 7.4), 50% formamide, 5 3Denhardt’s solution, 0.1% SDS, 10% dextran sulfate, and 200 mg/mldenatured salmon sperm DNA. After hybridization, the membrane waswashed in 0.1% SDS-2 3 SSC (300 mM sodium chloride and 30 mM

sodium citrate, pH 7.0) at room temperature for 20 min and subse-quently in 0.1% SDS-0.1 3 SSC (15 mM sodium chloride and 1.5 mM

sodium citrate, pH 7.0) at 55 °C for 30 min. The membranes were thenexposed to Kodak X-Omat AR films at 285 °C using intensifying screens.Southern Hybridization—Ten mg each of genomic DNA from HL-60

and HL-60/R-CP cells were digested with restriction endonucleaseEcoRI, separated by 1% agarose gel electrophoresis, and transferred toa nitrocellulose membrane. Hybridization was carried out according toprocedures described previously (18) using the 32P-labeled 2.86-kbMRPcDNA as a probe. Hybridization signals were detected by a Phosphor-Imager (Molecular Dynamics, Sunnyvale, CA).RNase Protection Assay—A 293-nucletotide fragment of MRP cDNA

spanning nucleotides 44 to 336 from the translation start site wassynthesized by RT-PCR using the following primers: forward primer59-GGGAATTCTGGGACTGGAATGTCACG (EcoRI site is underlined)and backward primer 59-CGGGATCCAGGAATATGCCCCGACTTC(BamHI site). The PCR product was digested with EcoRI and BamHIand cloned into EcoRI/BamHI sites of the pCRTMII vector. The result-ant plasmid DNA was linearized with XbaI, and the antisense RNAprobe was synthesized using SP6 RNA polymerase. To prepare theprobe for detecting g-GCS mRNA level, pCR 0.88 g-GCS cDNA waslinearized with PstI, and the antisense probe was synthesized usingSP6 RNA polymerase. Either 20 mg (forMRP and g-GCS probes) or 1 mg(for 18 S rRNA probe) of total RNA from HL-60/R-CP cells was hybrid-ized with 32P-labeled antisense RNA probes (2 3 105 cpm) and subjectedto the RNase protection assay as described previously (19).Determination of Cellular Level of Total Glutathione (GSH 1

GSSG)—A cell suspension (a total of 1 3 107 cells) was withdrawn fromthe cell culture and centrifuged at 40 3 g for 5 min at 4 °C. Theprecipitated cells were resuspended in 10 ml of ice-cold phosphate-buffered saline (0.9% (w/v) NaCl containing 10 mM potassium phos-phate, pH 7.4) and again centrifuged at 400 3 g for 5 min. The resultingcell pellet was resuspended in 750 ml of phosphate-buffered saline.From the cell suspension, a 500-ml aliquot was taken, mixed with 300 mlof 20% perchloric acid, and homogenized at 4 °C with an ultrasonicator.After centrifugation at 16,000 3 g for 5 min, a 200-ml aliquot of theresulting supernatant was withdrawn and neutralized by K2HCO3. Theconcentration of total glutathione (GSH 1 GSSG) in the neutralizedsample was determined according to the method of Tietze (20) with amodification described in Ref. 21.

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RESULTS

Expression of the MRP/GS-X Pump in HL-60/R-CP Cells—We previously reported that the GS-X pump is responsible fortransporting the GS-Pt complex in human HL-60 cells and thatGS-X pump activity is significantly enhanced (4- to 5-fold) incisplatin-resistant cells (2). Our recent RT-PCR study hasshown that the MRP gene encoding a human GS-X pump isexpressed at high levels in HL-60/R-CP cells (5). To confirm thefinding, we performed Northern hybridization and RNase pro-

tection assays using an MRP-specific probe (provided by Drs.Cole and Deeley; see Ref. 15) that encodes the 39 region (1-kb)sequence of MRP cDNA. MRP mRNA of about 8.0 kb wasobserved in both HL-60/R-CP and HL-60 cells; however, thelevel was remarkably (about 5-fold) higher in the resistant cellsthan in the sensitive cells (Fig. 1A). RNase protection assayalso showed increased expression of MRP in HL-60/R-CP cells(data not shown). On the other hand, the Southern hybridiza-tion of the EcoRI-digested genomic DNA from HL-60 and HL-60/R-CP cells with the MRP-specific probe (2.86-kb) (see “Ma-terials and Methods”) exhibited four hybridized bands at 3.7,4.1, 9.0, and 13.0 kb, and no amplification of theMRP gene wasobserved in HL-60/R-CP cells (Fig. 1B). These data suggesttranscriptional up-regulation of the MRP gene and/or en-hanced stability of its transcript in the cisplatin-resistant cells.Induction of MRP/GS-X Pump by Cisplatin and Heavy Met-

als—Expression of the MRP gene in HL-60/R-CP cells wasreversible and dependent on cisplatin. MRP mRNA level re-markably decreased when the cells were maintained withoutcisplatin for 1 month. GS-X pump activity, assessed by ATP-dependent transport of LTC4 in plasma membrane vesicles,decreased from 2.51 6 0.21 pmol/mg of protein/10 min to 0.306 0.04 pmol/mg of protein/10 min after the cisplatin-free incu-bation. On the other hand, when the cells were re-exposed to 20

FIG. 1. Detection of MRP gene expression in HL-60 and HL-60/R-CPcells by Northern blot hybridization (A). Hybridization of cellular RNA(10 mg for each lane) was performed with the probe encoding the 39region (1 kb) of the MRP cDNA (15). Detection of the MRP and g-GCSgenes in genomic DNA from HL-60 and HL-60/R-CP cells by Southernblot hybridization (B). 10 mg each of DNA was digested with EcoRI,separated by agarose gel (1%) electrophoresis, and then hybridized withthe 32P-labeled 2.86-kb probe encoding the partial MRP cDNA (225–3085) (a) or g-GCS cDNA (841–1723) (b) sequences. DNA was stainedby ethidium bromide (c).

FIG. 2. Effect of cisplatin on MRP transcript levels in HL-60/R-CP cells. HL-60/R-CP cells, which had been maintained in the cis-platin-free medium for 1 month, were incubated with 20 mM cisplatin forthe specified times (0, 6, 12, 18, 24, and 30 h). Total cellular RNA wasextracted from 1 3 106 cells and fractionated by 1% agarose gel elec-trophoresis. Northern blot hybridization (A) was performed using the32P-labeled 2.86-kbMRP cDNA probe. The relative level ofMRPmRNAwas determined using a scanning densitometer and normalized to thelevel at 0 h.

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mM cisplatin, the level ofMRP mRNA (8.0 kb in size) increasedsignificantly (7-fold) (Fig. 1A). Fig. 2B shows the time course ofMRP mRNA levels during cisplatin incubation. Interestingly,MRP was also induced by arsenite, cadmium, and zinc, whenthe cells were incubated with these metals at noncytotoxicconcentrations for 48 h. Fig. 3 demonstrates the increasedlevels of MRP mRNA detected by Northern hybridization (A)and by RNase protection assay (B).Fig. 4A shows a time course of zinc-mediated induction of

MRP in HL-60/R-CP cells. MRP mRNA level increased about7-fold after a 48-h exposure to zinc acetate (200 mM). To exam-ine functional induction of the MRP/GS-X pump, the activityof ATP-dependent LTC4 transport was determined usingplasma membrane vesicles prepared from HL-60/R-CP cells

before and after the exposure to zinc. Fig. 4B shows timecourses of LTC4 uptake by the plasma membrane vesicles,demonstrating a remarkable increase in the GS-X pump activ-ity after zinc exposure. The specific activity of the GS-X pump

FIG. 3. Induction of MRP/GS-X pump by heavy metals. HL-60/R-CP cells, which had been maintained in the cisplatin-free medium for1 month, were incubated with 20 mM cisplatin (Cis-Pt), 20 mM sodiumarsenite (As), 50 mM cadmium chloride (Cd), or 200 mM zinc acetate (Zn)for 48 h. MRP mRNA was detected by Northern blot hybridization (A)and RNase protection assay (B).

FIG. 4. Induction of MRP/GS-X pump by zinc acetate in HL-60/R-CP cells. A, HL-60/R-CP cells, which had been maintained in thecisplatin-free medium for 1 month, were incubated with 200 mM zincacetate for the specified times (0, 12, 24, 36, and 48 h). Total cellularRNA was extracted from 1 3 106 cells. The transcripts were detected byRNase protection assay using MRP-specific (293 base pairs) and 18 SrRNA-specific (80 base pairs) oligonucleotides. B, GS-X pump activityin the plasma membrane preparations form cisplatin-free and zinc-treated HL-60/R-CP cells. Plasma membrane vesicles were preparedfrom HL-60/R-CP cells that had been maintained in cisplatin-free me-dium for 1 month (Ç, å) and from the cells which were incubated with200 mM zinc acetate for 48 h (E, ●). Uptake of LTC4 by plasma mem-brane vesicles in the presence of ATP (open symbols) or in the absenceof ATP (closed symbols) was determined as described under “Materialsand Methods.” C, effect of cisplatin and the GS-Pt complex on ATP-de-pendent transport of LTC4 in the plasma membrane vesicles preparedfrom the zinc-treated HL-60/R-CP cells. ATP-dependent transport ofLTC4 was determined by incubating the plasma membrane vesicleswith 10 nM LTC4 in the presence of cisplatin (Cis-Pt) or the GS-Ptcomplex (GS-Pt). Data are expressed as mean 6 S.E. (n 5 3).

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increased from 0.30 6 0.04 pmol/mg of protein/10 min to 1.95 60.11 pmol/mg of protein/10 min (n 5 3), which is consistentwith the elevated levels of MRP mRNA (Fig. 5A).It would be of importance to examine whether the zinc-

induced MRP/GS-X pump had an affinity for the GS-Pt com-plex. We previously reported that transport of the GS-Pt com-plex is an ATP-dependent process and inhibited by LTC4,

GSSG, and S-(2,4-dinitrophenyl)glutathione, suggesting thatthe GS-X pump exports the GS-Pt complex from cells (2). Fig.5C demonstrates that LTC4 transport was dose dependentlyinhibited by the GS-Pt complex but not by cisplatin. The GS-Ptcomplex inhibited GS-X pump activity 50% at a concentrationof about 150 mM, which is similar to the Km value (130 mM) forthe complex (2). Thus, these results strongly suggest that theheavy metal-inducibleMRP/GS-X pump transports both LTC4and the GS-Pt complex.Induction of g-GCS by Cisplatin in HL-60/R-CP Cells—

Biosynthesis of cellular GSH is a key factor in the function oftheMRP/GS-X pump as well as in the overall metabolism anddisposition of cisplatin in cancer cells. In HL-60/R-CP cellsmaintained with cisplatin, the intracellular GSH level was 7.526 0.74 nmol/106 cells, about 7-fold higher than in HL-60 cells(1.15 6 0.12 nmol/106 cells) (2). After the incubation mediumwas replaced with cisplatin-free medium, the intracellularGSH level in the resistant cells decreased to 2.01 nmol/106

cells, with a half-time of 5 days. This suggests a reversibleregulation of intracellular GSH levels.As Fig. 5 illustrates, g-GCS, which is a rate-limiting enzyme

of cellular GSH biosynthesis, was induced by cisplatin in HL-60/R-CP cells. Prior to this experiment, cells had been main-tained in the cisplatin-free medium for 1 month. The cisplatin-

FIG. 6. Changes of cellular GSH level in HL-60 and HL-60/R-CPcells exposed to cisplatin. A, cells were exposed to 20 mM cisplatin for24 h as described in Fig. 5. At specified time points (0, 1, 2, 6, 12, 18, and24 h), cells (1 3 107) were withdrawn from the incubation medium, andthe levels of GSH in the cells were determined. Cellular GSH levels inHL-60 and HL60/R-CP cells at 0 h were 1.15 and 2.01 nmol/106 cells,respectively. Data are expressed as mean values of duplicate experi-ments. B, cellular GSH levels in HL-60/R-CP cells treated with differentconcentrations of cisplatin. HL-60/R-CP cells were incubated withcisplatin at concentrations of 0, 10, 20, and 40 mM for 12 h and thencellular GSH levels were determined. Data are expressed as mean 6S.D. (n 5 3).

TABLE IEffect of cisplatin, arsenite, cadmium, and zinc on cellular

GSH level and GS-X pump activityHL-60/R-CP cells were maintained in the cisplatin-free medium for 1

month (none) and then incubated with 20 mM cisplatin, 20 mM sodiumarsenite (As), 50 mM cadmium chloride (Cd), or 200 mM zinc acetate (Zn)for 48 h. Cellular GSH levels and GS-X pump activity (ATP-dependentLTC4 transport) in the plasma membrane preparations were deter-mined as described under “Materials and Methods.” Data are expressedas mean 6 S.D. (n 5 3).

Treatment Cellular GSH GS-X pump activity

nmol/106 cells (fold) pmol/mg protein/10 min (fold)

None 2.0 6 0.2 (1.0) 0.30 6 0.04 (1.0)Cisplatin 7.2 6 0.6 (3.6) 1.24 6 0.18 (4.1)As 8.1 6 0.4 (4.0) 1.62 6 0.15 (5.4)Cd 5.2 6 0.4 (2.6) 1.81 6 0.20 (6.0)Zn 5.5 6 0.5 (2.7) 1.95 6 0.11 (6.5)

FIG. 5.Changes of g-GCSmRNA level in HL-60 andHL-60/R-CPcells exposed to cisplatin. Cells were exposed to 20 mM cisplatin for24 h. At specified time points (0, 1, 2, 6, 12, 18, and 24 h), cells (1 3 107)were withdrawn from the incubation medium, and the levels of 4.0-kbg-GCS mRNA were determined. Prior to the experiment, HL-60/R-CPcells had been maintained in cisplatin-free medium for 1 month, asdescribed under “Materials and Methods.” The relative level of g-GCSmRNA was determined using a scanning densitometer and normalizedto the level of HL-60 cells at 0 h.

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resistant and -sensitive cells were then exposed to 20 mM

cisplatin for 24 h (Fig. 5A). It is important to note that theg-GCS mRNA level in HL-60/R-CP cells significantly increasedduring the incubation with cisplatin (Fig. 5). By contrast,g-GCS mRNA levels in HL-60 cells remained almost constantthroughout the incubation (Fig. 5A). No amplification of theg-GCS gene was detected in both HL-60 and HL-60/R-CP cells(Fig. 1B).Fig. 6A shows time courses of intracellular GSH levels in the

cisplatin-resistant and -sensitive cells during the incubationwith cisplatin. Intracellular GSH level in HL-60/R-CP cellsincreased up to 5.8 nmol/106 cells, corresponding to about 80%of the GSH level in cells continuously exposed to cisplatin. Onthe other hand, the cellular GSH level in the sensitive cells wasvirtually unchanged during the first 18-h incubation, and itbegan to decrease after 24 h owing to cell damage. The increasein cellular GSH level in the cisplatin-resistant cells is inti-mately linked with the increase of g-GCS mRNA level (Fig. 5).As shown in Fig. 6B, the increase of intracellular GSH level inHL-60/R-CP cells after a 12-h exposure to cisplatin was directlyproportional to cisplatin concentration up to 40 mM.Effect of Cisplatin and Heavy Metals on Cellular GSH and

GS-X Pump Activity—Table I demonstrates that cisplatin andheavy metals, i.e. arsenite, cadmium, and zinc, significantlyenhanced cellular GSH levels and GS-X pump activity in HL-60/R-CP cells. The results also suggest that induction of bothg-GCS andMRP/GS-X pump occurs in concert in the cisplatin-resistant cells.Cross-resistance of HL-60/R-CP Cells to Melphalan,

Chlorambucil, Cadmium, and Arsenite—Fig. 7 shows the sen-sitivity of HL-60 and HL-60/R-CP cells to melphalan, chloram-

bucil, cadmium, and arsenite and clearly demonstrates thecross-resistance of HL-60/R-CP cells to these alkylating agentsand heavy metals. As assessed on the basis of IC50 value, theextents of resistance of HL-60/R-CP cells were 3.2-, 2.6-, 2.9-,and 3.0-fold for melphalan, chlorambucil, cadmium, and arsen-ite, respectively. As previously reported (2), HL-60/R-CP cellswere about 10-fold more resistant to cisplatin than HL-60 cells.It is remarkable that cisplatin-resistant HL-60/R-CP cells grewnormally in the presence of cadmium at concentrations of up to100 mM, whereas the growth and viability of cisplatin-sensitiveHL-60 cells were greatly affected in that same concentrationrange (Fig. 7).

DISCUSSION

Induction of the MRP/GS-X Pump by Cisplatin and HeavyMetals—We have previously established HL-60/R-CP cells anddemonstrated an elevated GS-X pump activity in this cisplatin-resistant cell line. We also demonstrated that the ATP-depend-ent transport of the GS-Pt complex and LTC4 measured withplasma membrane vesicles was about 4- to 5-fold greater inHL-60/R-CP cells than in HL-60 cells. The Km value for theGS-Pt complex was estimated to be 130 mM (2), similar to thatfor GSSG, one of the endogenous substrates for the GS-X pump(21). Since our studies were published, several laboratorieshave reported that the human GS-X pump is encoded by theMRP gene (6–8). The present study demonstrates that thehuman MRP gene is expressed at higher levels in HL-60/R-CPcells. Furthermore, we showed that LTC4 and the GS-Pt com-plex mutually inhibit their ATP-dependent transport in plasmamembrane vesicles prepared from HL-60/R-CP cells (Fig. 4Cand Ref. 2). These results strongly suggest thatMRP and GS-X

FIG. 7. Sensitivity of HL-60 (●) and HL-60/R-CP (E) cells to melphalan, chlorambucil, cadmium, and arsenite. For each experiment,0.15 3 106 cells/ml were incubated with the applicable alkylating agent or heavy metal in 100 ml of the culture medium in 96-well plates in ahumidified tissue culture chamber (37 °C, 5% CO2). After 72 h, the cell density of surviving (trypan blue-negative) cells was counted with ahemacytometer. Cx is the density after treatment with melphalan, chlorambucil, cadmium, or arsenite at concentration x. Co is the cell density ofthe untreated control. Data are expressed as mean 6 S.D. (n 5 3).

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pump have similar, if not identical, substrate specificity. Re-cent studies by Fujii et al. (22) and Goto et al. (23) also showedthat GS-X pump activity was significantly enhanced in cispla-tin-resistant human epidermoid carcinoma KB, colonic cancerHCT8, and ovarian cancer A2780 cells. Collectively, these re-sults are consistent with the idea that the overexpressed GS-Xpump is responsible for eliminating the GS-Pt complex as wellas GS-conjugates from these cisplatin-resistant cells.Our present results also demonstrate that HL-60/R-CP cells

are cross-resistant to heavy metals (cadmium and arsenite) inaddition to alkylating agents, melphane and chlorambucil (Fig.7). Like cisplatin, these heavy metals react with GSH to formGSH chelate complexes. Thus, it is reasonable to speculate thatthe overexpressed MRP/GS-X pump in these cells is responsi-ble for detoxifying these heavy metals. Several recent findingssupport this contention. (i) The primary structure of the mam-malian MRP gene product deduced from its cDNA sequence isremarkably similar to that of YCF1, which is responsible forcadmium resistance in S. cerevisiae (9). Deletion of the YCF1gene results in defective transport of the glutathione-bimaneconjugate into vacuoles of yeast cells,3 suggesting that theYCF1 gene encodes a vacuolar GS-X pump in yeast cells andthat its MRP homolog functions as a transporter for GSH-metal complexes as well. (ii) The primary sequences of MRPand YCF1 genes are both remarkably similar to that of theltpgpA gene (24), which is a putative pump for As31-GSHcomplex (25) and confers arsenite resistance in Leishmania (26,27). (iii) Cole et al. (28) have suggested a potential role for theMRP gene in cellular resistance to some heavy metal anions,including arsenite and antimony. Furthermore, Zaman et al.(29) have recently reported that arsenite efflux from MRP-transfected cells was accompanied by a significant increase ofGSH efflux, suggesting that arsenite may be transported as aGSH-chelate complex by the same export pump. These results,taken together, strongly suggest that the mechanisms of elim-inating cytotoxic heavy metals via GSH conjugation are con-served throughout evolution.We would like to stress that overexpression ofMRP/GS-X is

not the only mechanism that is involved in cisplatin resistancein cancer cells. A number of cisplatin-resistant cell lines havefailed to display increased MRP/GS-X pump (30).4 Othermechanisms, such as removal of platinum-DNA adducts, maybe involved in these non-MRP/GS-X pump-overexpressingcells (Ref. 31 and references therein). Likewise, overexpressionof the MRP/GS-X pump does not necessarily have to result incisplatin resistance (28, 32, 33). Since formation of the GS-Ptcomplex is a prerequisite for the function of GS-X pump, onecan envision that intracellular levels of GSH would also play animportant role in the overall drug resistance mechanism (seebelow).Induction of g-GCS by Cisplatin—Glutathionation allows

substrate recognition by the MRP/GS-X pump. While GSHtransferases catalyze the conjugation reactions with electro-philic organic compounds, GSH spontaneously reacts withheavy metals to form GSH-metal chelate complexes (1, 34, 35).Increased cellular GSH propels the reaction of cytotoxic heavymetals with GSH to generate GSH-metal complexes, therebyproviding an important defense mechanism. Previous studydemonstrated that cisplatin resistance of cancer cells was oftenobserved with increased levels of intracellular GSH and highlevels of g-GCS expression (13). Transcriptional up-regulationof g-GCS gene expression was also reported for melphalan-resistant human prostate carcinoma cells (36). The presentstudy demonstrates that g-GCS was induced by cisplatin in

HL-60/R-CP cells and that cellular GSH levels were concomi-tantly increased in the cisplatin-resistant cells (Figs. 5 and 6).These results strongly suggest that replenishment of intracel-lular GSH is required for the elevated GS-X pump activity inthese drug-resistant variants. Furthermore, our results dem-onstrate that the cellular GSH status is dynamic and can bemodulated by extracellular stimuli. Rapid up-regulation ofg-GCS expression must therefore be a critical determinant forcellular tolerance to heavy metals and electrophilic compounds.

g-GCS exists as a holoenzyme in vivo, consisting of disso-ciable heavy and light subunits (37, 38). The heavy subunitexpresses the catalytic function whereas the light subunit,regulatory function. Recent transfection experiments using ex-pression vectors containing cDNA encoding these subunitsdemonstrated that, while co-transfecting both heavy and lightsubunits yielded high levels of intracellular GSH, transfectingeither subunit alone could produce moderate increases of GSH(39). While the levels of the regulatory subunit expression inHL-60/R-CP cells remain to be determined, our present resultsapparently are consistent with the notion that overexpressionof the heavy subunit is sufficient to increase GSH levels.Our HL-60/R-CP cells exhibit cross-resistance to other heavy

metals, e.g. cadmium, arsenite (Fig. 4). A recent report alsoshowed that a cisplatin-resistant human ovarian carcinomacell line exhibited cross-resistance to antimony and cadmium(40). However, these resistance profiles are slightly differentfrom those reported for the MRP-transfected cells (28, 29).These reports demonstrated that cells transfected with theMRP gene displayed resistance to doxorubicin, vincristine, ar-senite, and antimony, but not to cisplatin and cadmium,whereas our HL-60/R-CP cells show no cross-resistance todoxorubicin and vincristine (2). These results, taken together,suggest that overexpression of the MRP/GS-X pump per se isnecessary but not sufficient to generate a full spectrum of drugresistance. Multiple biochemical events such as reduction, de-glycosylation, and conjugation with GSH are considered to beinvolved in the cellular metabolism of anthracyclines andtransport of their metabolites via theMRP/GS-X pump (41). Inthis context, increased GSH biosynthesis may also be essentialfor acquired resistance to a wide spectrum of anticancer drugsand heavy metals.Mechanisms of Induction of the MRP/GS-X Pump and

g-GCS Gene Expression by Heavy Metals—One of the majorfindings presented in this communication is that genes encod-ing the MRP/GS-X pump and g-GCS can be induced by heavymetals. Coordinated regulation of these two genes have beenfound in g-GCS-transfected human small cell lung cancer cells(42), as well as in other drug-resistant variants and in tumorbiopsies.5 These results suggest that certain common factor(s)may be involved in the expression of these two genes. Althoughthe mechanisms underlying the concerted regulation of thesetwo genes are not known at present, possible mechanisms canbe speculated. In yeast, the GSH1 gene encoding g-GCS andthe YCF1 gene encoding the MRP homolog are coordinatelyregulated by yAP-1, which encodes yeast transcription factorAP-1 (43). Transcriptional activation of these genes mediatedby yAP-l is essential for cadmium tolerance in yeast cells (44).Induction of c-myc and c-jun expression in rat L6 myoblasts bycadmium has been reported (45), and we have found that levelsof c-jun transcript are increased in HL-60/R-CP cells (data notshown). Whether g-GCS and MRP/GS-X pump genes in mam-malian cells are also transcriptionally regulated by AP-1 re-mains to be determined. The promoter regions of human g-GCSand MRP genes have been characterized recently, and it is

4 M. T. Kuo, N. Savaraj, and T. Ishikawa, unpublished data. 5 M. T. Kuo, S. A. Curley, and T. Ishikawa, unpublished findings.

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noteworthy that cis-regulatory elements including AP-1 bind-ing sites have been identified for both of the genes (46–48).Several recent reports have demonstrated transient induc-

tions of drug resistance gene expression by cytotoxic com-pounds. The human MDR1 gene encoding P-glycoprotein hasbeen shown to be regulated by heat shock, arsenite, and cad-mium (49) as well as other cytotoxic compounds (50). Further-more, following exposure to several cytotoxic compounds, mostof which are known to be substrates for the multidrug trans-porter, mdr RNA levels in cultured rodent cells were found toincrease (51). Both transcriptional and post-transcriptionalmechanisms are apparently involved in this regulation (51).The present study showing that g-GCS and MRP/GS-X pumpcan be coordinately induced by anticancer drug cisplatin andheavy metals provide important information to the steadilyaccumulating evidence that various drug resistance genes canbe acutely induced upon drug treatments. These observationsmay have important clinical implications. Unlike cell culturestudies where drug-resistant variants are usually obtainedthrough long-term, continuous drug exposure, such transientinduction of drug resistance gene expression is considered to bemore relevant to the treatment protocols for cancer patients.Thus, understanding the mechanisms involved in transcrip-tional and/or post-transcriptional regulation of the expressionof MRP/GS-X pump and g-GCS genes may facilitate the de-velopment of novel approaches to control drug resistance incancer chemotherapy. These experiments are currently underway in our laboratories.

Acknowledgments—We thank Drs. Susan P. C. Cole and Roger G.Deeley (Queen’s University, Kingston, Canada) for kindly providing thecDNA probe for MRP. In addition, we thank Dr. Philip A. Rea (PlantScience Institute, University of Pennsylvania, Philadelphia, PA) for hisfruitful discussion regarding the function of the YCF1 gene.

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Christine D. Wright and M. Tien KuoToshihisa Ishikawa, Jia-Ju Bao, Yoshiaki Yamane, Kunihiro Akimaru, Karl Frindrich,

Heavy Metals in Human Leukemia Cells-Glutamylcysteine Synthetase byγ Pump and GS-X/MRPCoordinated Induction of

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