GS-X Pump Is Functionally Overexpressed in cis-Diamminedichloroplatinum(I1)-resistant Human Leukemia HL-60 Cells and Down-regulated by Cell Differentiation*

The ATP-dependent glutathione S-conjugate export pump, named GS-X pump, has been shown to eliminate a potentially cytotoxic glutathione-platinum (GS-Pt) com- plex from tumor cells, thereby modulating glutathione (GSH)-associated resistance to cis-diammined~chloro- platinum(I1) (cisplatin) (Ishikawa, T., and Ali-Osman, F. J. BioZ. Chem. 268, 20116-20125). The present study provides evidence that the GS-Xpump is functionally overexpressed in cisplatin-resistant human promy- elocytic leukemia HL-60 (HL-BO/R-CP) cells, in which the cellular GSH level was substantially enhanced. Indeed, the rate of ATP-dependent transport of the GSWPt complex, measured with plasma membrane vesicles, was about 4-fold greater in HL-GO/R-CP cells than in HL-60 cells. Three membrane proteins with apparent molecu- lar masses of 200,110, and 70 kDa were overexpressed in HLSO/RCP cells, whereas P-glycoprotein (MDR1) was not immunologically detected in the membrane preparations from resistant and sensitive cells. Unlike in HL-60 cells, increased numbers of intracellular vesicles were observed in the cytoplasm of HL-GO/R-CP that the fluorescent glutathione Sconjugate accumulated in intracellular vesicles of the cisplatin-resistant cells in an energy-dependent manner. to to vesicle- excretion of GSH-drug conjugates from cells. chromatography. The standard incubation medium for the transport experiment contained the membrane vesicles (50 pg of protein), 200 p~ [%IGS.F't. complex, 0.25 M sucrose, 10 m~ Tris-HC1, pH 7.4, 10 m~ MgCl,, 10 m~ creatine phosphate, and 100 pdml creatine kinase and either 1 m~ (0) or 0 m~ (.)ATP, in a final volume of 110 pl. The reaction was started by adding the L3H1GS.Pt complex to the incubation medium. The reaction was carried out at 37 "C, and the r3KIGS.Pt complex in- corporated into the vesicles was measured by a rapid filtration technique. Data are expressed as mean * S.E. (n 3).

~ ~~ ~~ ~~ one S-conjugates, cysteinyl leukotrienes, and certain organic anions from normal and cancer cells (1). The function of the GS-X pump is critically linked to a variety of biological phenomena, such as oxidative stress, detoxification, inflammation, and modulation of cell proliferation (see Ref. 2 for recent review). The export of glutathione S-conjugates from cells is important not only in interorgan metabolism of glutathione (GSH) but also in reducing the intracellular accumulation of potentially cytotoxic GSH conjugates. Thus, the GS-X pump is called the "phase 111" detoxification system for biotransformation of endo-and xenobiotics (2). Accumulating evidence suggests that cellular GSH is a critical determinant in the tumor cell resistance to chemotherapeutic agents, such as nitrogen mustards, chloroethyl nitrosoureas, and cisplatin (see Refs. 3-6 for review). Evidence that several GSHdrug conjugates are potentially cytotoxic suggests that the elimination of GSH-drug conjugates from tumor cells is an important factor for the cellular toxicity of anticancer drugs (7). We have recently shown that cisplatin reacts with intracellular GSH and that the resulting glutathione-platinum (GSPt) complex is actively exported from leukemia cells via the GS-X pump (7). Since the GSPt complex is a potential inhibitor for protein synthesis, the function of the GS-X pump is considered to modulate the resistance of human cancers to cisplatin (7,8).
Cisplatin is an effective antitumor agent for treating various human cancers of the brain, head and neck, ovary, testicle, and bladder (9). Its antitumor activity is attributed primarily to its ability to form DNA-platinum cross-link adducts (10,11). Despite its clinical effectiveness, cellular drug resistance is a significant obstacle to a long term, sustained patient response to cisplatin-based therapy. Intracellular GSH would be a significant determinant in the resistance and cytotoxicity of cisplatin (12,13); however, its exact molecular mechanisms are not fully understood. In the present study, we examined the role of the GS-X pump in cisplatin resistance, using a cisplatin-resistant variant of human leukemia HL-60 cells as a model system. Here we provide evidence that the GS-X pump is functionally overexpressed in cisplatin-resistant HL-60 cells and that the GS-X pump plays a significant part in vesicle-mediated excretion of GSH-drug conjugates from resistant cells.
Furthermore, we investigated the effect of cell differentiation on the activity of the GS-X pump to examine a potential link between the functional overexpression of the GS-X pump and cell proliferation. The HL-60 leukemia cell line (14) provides a useful model system for studying the role of proto-oncogenes in drug resistance as well as in cellular proliferation and differ-Me,SO, dimethyl sulfoxide; GS.Pt, bis-(g1utathionato)-platinum (11); LTC,, leukotriene C,; monochlorobimane, syn-(C1CH2,CH,)-1,5-diazabicyclo-[3.3.0]-oda-3,6-dione-2,8-dione; M R P , multidrug resistance-related protein; PBS, phoaphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TPA, 12-O-tetradecanoylphorbol-13-acetate. in Cisplatin-resistant Leukemia Cells entiation. Several mutations in specific proto-oncogenes have been identified in HL-60 cells (15). In particular, the c-myc gene is amplified 8-30-fold and highly expressed (16,17), and this is associated with N-ras gene activation (18) and deletion of t h e p53 gene (19). HL-60 cells differentiate along the granulocytic pathway when they are treated with dimethyl sulfoxide (20, 21) or retinoic acid (22), and along the monocytic pathway when treated with phorbol esters (23) and vitamin D analogs (24). In the present study, the activity of the GS-X pump was found to be down-regulated by cell differentiation. Based on our results, we discuss a possible relationship between the expression of the GS-X pump and cell proliferation.
MATERIALS AND METHODS Biochemicals, Enzymes, and Cells-GSH, GSSG, ATP, creatine phosphate, creatine kinase, phenylmethylsulfonyl fluoride, l-y-glutamyl-3carboxyl-4-nitroanilide, and a random-primed labeling kit were purchased from Boehringer Mannheim (Mannheim, Germany Cell Culture-The wild type of human promyelocytic leukemia HL-60 cells (ATTC No. CCL240) from the American Type Culture Collection (Rockville, MD) were maintained in RPMI 1640 medium supplemented with glutamine, 10% (v/v) heat-inactivated fetal calf serum, and gentamycin (50 pglml) in a humidified atmosphere of 5% CO, in air. Cells (1.5 x lo5 celllml) were passaged every 5 days. The number of cells was determined in a hemocytometer by trypan blue-dye exclusion.
A cisplatin-resistant subline, named HL-GO/R-CP, was established by maintaining HL-60 cells in the presence of cisplatin over 10 months. Briefly, during the first 4 months, cells were grown in the presence of cisplatin at increasing concentrations of 0.5-5 PM and then cultured with 10 p~ cisplatin for the following 3 months. The surviving cells were further maintained in the presence of 17 PM cisplatin for another 3 months. The resulting resistant cells were about 10-fold more resistant to cisplatin than the parent cells (see "Results").
Determination of Cell Sensitiuity to Anticancer Drugs-Cells (1.5 x lo5 cells/ml) were incubated in 100 pl of the culture medium containing cisplatin or doxorubicin at different concentrations in 96-well plates in a humidified tissue-culture chamber (37 "C, 5% CO,). After 72 h, the number of surviving cells was counted.
Determination of Cellular Leuel of Total Glutathione (GSH + GSSG)-A cell suspension (a total of 1 x lo7 cells) was withdrawn from the cell culture and centrifuged at 40 x g for 5 min at 4 "C. The precipitated cells were resuspended in 10 ml of ice-cold phosphate-buffered saline (PBS) and again centrifuged at 40 x g for 5 min. The resulting cell pellet was resuspended in 750 pl of PBS. From the cell suspension, a 500-pl aliquot was taken, mixed with 300 pl of 20% perchloric acid, and homogenized at 4 "C with an ultrasonicator. After centrifugation at 16,000 x g for 5 rnin, a 200-pl aliquot of the resulting supernatant was withdrawn and neutralized by &HCO,. The concentration of total glutathione (GSH + GSSG) in the neutralized sample was determined according to Tietze (25) with a modification described previously (26). Assay of Glutathione S-Bansferase and Myeloperoxidase Actiuities-250 p1 of the cell suspension in PBS was treated with 0.3% Triton X-100 at 4 "C. The activity of glutathione S-transferase in the sample was determined according to the method of Habig et al. 127) with 1 m M l-chloro-2,4-dinitrobenzene and 1 m M GSH being used as substrates. Myeloperoxidase activity was determined with 100 p~ hydrogen peroxide and 1 m M guaiacol according to the method of Kimura and Yamazaki (28).
Northern Hybridization-Total cellular RNA was prepared by the acid guadinium thiocyanate-phenol-chloroform extraction method from samples of 1 x 10' cells as described by Chomczynski and Sacchi (29).
The RNA (10 pgnane as determined by absorbance at 260 nm) was fractionated by electrophoresis in 1.0% (w/v) agarose gels containing formaldehyde and transferred to Nytran membranes (Schleicher & Schuell). The membranes were then baked at 80 "C for 2 h. A 1.4-kilobase C-myc genomic DNAfragment containing exon 3 and a 0.7-kilobase p-actin fragment (Oncor, Gaithersburg, MD) was used to generate 32P-labeled DNA probes by a random-primed labeling method.
Hybridization with the DNA probe (1 x lo6 counts/min/ml) was performed at 42  The membranes were exposed to Kodak X-Omat AR films at -85 "C using intensifymg screens. Fluorescence Microscopy-Cells were incubated with 20 p~ monochlorobimane (30) at 37 "C for 20 min. Subsequently, the cells were washed with ice-cold Hanks'balanced salt solution and incubated in the monochlorobimane-free medium at 37 "C. Next, the cells were placed on glass slide plates and observed without fmation under an Olympus Fluorescence Microscope, Vanox AH-2, equipped with a 20UG1 excitation filter and a 17L420 barrier filter. The photographs were taken with Kodak Ektachrome ASA 400 film.
Flow Cytometry-The relative fluorescence intensity of cells was measured with an EPICS Elite flow cytometer (Coulter Corp. Hialeah, FL) equipped with an air-cooled helium-cadmium laser for excitation and an 470-nm long-pass filter for emission. The emission intensity was converted from log fluorescence to linear fluorescence intensity. Every observation, at least 1 x lo4 cells were subjected to the flow cytometv.
Preparation of Plasma Membrane Vesicles from HL-60 Cells-In each preparation, HL60 cells (5 x 10' cells) were harvested by centrifugation and suspended in 50 ml of ice-cold PBS. After centrifugation at 40 x g for 5 min, the cell pellet was diluted 40-fold with a hypotonic buffer (0.5 m~ sodium phosphate, pH 7.0,O.l m~ EGTA, and 0.1 m~ phenylmethylsulfonyl fluoride). The cell lysate was then centrihged at 100,000 x g for 30 min, and the resulting pellet was suspended in the hypotonic buffer and homogenized with a Potter-Elvehjem homogenizer. The crude membrane fraction was layered over 38% sucrose solution and centrifuged at 100,000 x g for 30 min. The turbid layer at the interface was collected, suspended in 250 m~ sucrose containing 10 m~ Tris-HC1, pH 7.4, and centrifuged at 100,000 x g for 20 min. The membrane fraction was collected and resuspended in a small volume (50-100 pl) of 250 m~ sucrose containing 10 m~ Tris-HC1, pH 7.4. Vesicles formed by passing the suspension through a 27-gauge needle were frozen in liquid N, and stored at -85 "C until used. Sialidase accessibility for the determination of inside-out vesicles was examined as described previously (31).
Measurement of Marker Enzyme Actiuities-y-Glutamyl transferase activity was determined according to Tate and Meister (32) using l-yglutamyl-3-carboxyl-4-nitroanilide and glycylglycine. The reaction was measured spectrophotometrically at 407 nm ( E = 9.9 mM-l x cm") (33). 5'-Nucleotidase, alkaline phosphatase, and succinate dehydrogenase were assayed according to Golish et al. (34). Alkaline phosphodiesterase activity was determined according to Edelson and Erbs (35) using pnitrophenyl thymidine-5'-monophosphate acetate. The reaction was measured by following the liberation ofp-nitrophenylate at 400 nm ( E = 12.0 mM-I x cm") (36). P-Glucuronidase activity was determined according to Brittinger et al. (37) using phenolphthalein-glucuronic acid, where phenolphthalein generated from the reaction was spectrophotometrically measured at 550 nm (E = 26.6 mwl x cm") (38). All the assays were performed at 37 "C. Protein concentration was determined according to the protocol of the BCA protein assay provided by Pierce.
Synthesis of 3H-Labeled GS.Platinum Complex-The 3H-labeled GS.R complex was synthesized using [2-3Hlglycine-labeled GSH and cisplatin as described previously (7). The resulting L3H1GS.Pt complex was dissolved in 10  tion was carried out at 37 "C and the amount of [3HlGS.Pt complex incorporated into the vesicles was measured by a rapid filtration technique as described previously (31). Electrophoresis and Immunoblotting-SDS-PAGE was carried out according to the method of Laemmli (39) using 10%-or 12%-acrylamide gels. After electrophoresis, proteins were either stained by Coomassie Blue R-250 or electrophoretically transferred to ImmobilonTM-P membranes (Millipore, Bedford MA) in a solution containing 25 m~ Ws, 192 m M glycine, pH 8.3, at 4 "C with a constant current of 150 mA for 8 h, according to the method of 'Ibwbin et al. (40). The active site of the protein-transferred membrane was blocked by 3% (w/v) gelatin in 0.9% NaCl containing 10 m~ Tris-HC1, pH 7.7, and 0.05% (v/v) Tween 20 at 37 "C for 2 h. The membrane was then incubated with a rabbit polyclonal antibody raised against P-glycoprotein (Oncogene Science, Manhasset, N Y ) in 0.9% NaCl, 10 m M Tris-HC1, pH 7.4, 1% gelatin, 0.05% Tween 20 at 37 "C for 2 h. After washing the membrane with the same buffer, the membrane was incubated with goat anti-(rabbit-1gG)horseradish peroxidase conjugate (Oncogene Science) in the same buffer at 37 "C for 1.5 h. The peroxidase reaction on the membrane was allowed to proceed at room temperature using a peroxidase-substrate DAB kit (Vector, Burlingame CA).
Plasma Membrane Overexpressing P-glycoprotein-Murine CUD/ VCR1 cells, which overexpress P-glycoprotein (411, were kindly provided by Dr. M. Tien Kuo (Department of Molecular Pathology, M. D. Anderson Cancer Center, Houston). The cells had been established by maintaining murine CUD cells with increasing concentrations (up to 1 pg/ml) of vincristine (41). The plasma membrane from the cells was prepared as described for HL-60 cells (see above). ATP-dependent Dansport of GS.Pt Complex in Cisplatin-resistant and -sensitive Cells-In our previous study, the transport of the GSPt complex across the plasma membrane of murine leukemia L1210 cells was found to be an ATP-dependent process mediated by the GS-X pump (7). In order to determine the ATP-dependent transport of the GSPt complex in cisplatin-sensitive and -resistant cells, plasma membrane vesicles were prepared from HL-60 and HL-GO/R-CP cells by sucrose-density gradient centrifugation. The enrichment of the plasma membrane in the preparations was examined by measuring the specific activities of marker enzymes (Table I). The specific activities of plasma membrane enzymes, i.e. y-glutamyl transferase, alkaline phosphatase, alkaline phosphodiesterase, and 5'-nucleotidase, in the membrane preparations were 20-27-fold higher than those in the cell homogenate, whereas the specific activity of succinate dehydrogenase, a mitochondrial enzyme, was about 10% of the cell homogenate. As indicated by the activity of p-glucuronidase, the lysozome membrane was slightly contaminated in the plasma membrane preparations. Taken together, these results support a high enrichment of the plasma membrane in the membrane preparations from both HL-60 and HL-GOB-CP cells. Based on the sialidase accessibility, 45-55% of the total population of the membrane vesicles was estimated to be inside-out. Fig. 2 demonstrates the time courses of transport of the 3H-labeled G S P t complex into membrane vesicles prepared from HL-GO/R-CP and HL-60 cells. The rate of the ATP-dependent transport of the GSPt complex was rbout 4-fold greater in the membrane vesicles from HL-GO/R-CP cells than in those from HL-60 cells, whereas the apparent K,,, value for the GSPt complex was unchanged (130 w for the membrane vesicle preparations from resistant and sensitive cells). In addition, ATP-dependent transport of LTC,, an endogenous substrate of the GS-X pump (11, was also 5-fold higher in the membrane vesicles from HL-GO/R-CP cells than in those from HL-60 cells (data not shown). These results suggest that the GS-X pump was functionally overexpressed in the cisplatin-resistant cells.

Marker enzyme activities in the plasma membrane preparations from HL-60 and HL-GOIR-CP cells
Marker enzyme activities of the plasma membrane preparations from HL60 and HL-GO/R-CP cells were determined as described under "Materials and Methods." The specific activities are expressed as mean S.E., n = 3. In parenthesis, the relative enrichment is shown as the ratio of the specific activity of the corresponding enzyme in the plasma membrane preparations over the specific activity in the homogenates. Alterations in Membrane Proteins in Cisplatin-resistant Cells-SDS-PAGE of the plasma membrane preparations from HL-60 and HL-GO/R-CP cells demonstrated that at least three membrane proteins with apparent molecular masses of 200, 110, and 70 kDa, respectively, were overexpressed in the cisplatin-resistant cells (Fig. 3A). Immunoblot analysis showed that P-glycoprotein was not expressed in the cisplatin-resistant cells, whereas it was clearly expressed in the plasma membrane from vincristine-resistant CllD cells (CllDNCR), as the positive control (Fig. 3B).
Morphological Alterations in Cisplatin-resistant Cells: Intracellular Vesicles-Besides those biochemical changes, HL-601 R-CP cells exhibited a detectable morphological alteration characterized by increased numbers of intracellular vesicles (Fig. 4). Whereas the sensitive parent cells contained only a few vesicles in the cytoplasmic space, resistant cells contained five to eight or even more. Cisplatin is conjugated with intracellular GSH to form the GS-Pt complex (7), and the intracellular GSH level was significantly increased in the HL-GOR-CP cells of this study. This result (Fig. 4) therefore implied a potential link between the formation in the present intracellular vesicles and the GSH-associated biotransformation of cisplatin in HL-601 R-CP cells. Based on our current knowledge, it could be hypothesized that the GS-Pt complex formed in cells accumulates in the vesicles and is subsequently excreted via exocytosis and that the GS-X pump is supposed to be involved in the transport of the GS-Pt complex from the cytosol space into the vesicles. Biological Function of Intracellular Vesicles in Cisplatin-resistant Cells-We have tested the above stated hypothesis by fluorescence microscopy. Monochlorobimane (301, a noduorescent compound, is specifically conjugated with GSH in the cell by the action of glutathione S-transferases, and the resulting glutathione S-conjugate exhibits intense fluorescence (excitation at 370-385 run; emission at 477-484 nm) (42, 43). Since the GS-X pump has a broad substrate specificity toward a variety of glutathione S-conjugates (1, 21, the compound made A it possible to study the GS-X pump-mediated transport process by pursuing the fluorescence under a microscope. Fig.  5A provides direct evidence that the glutathione S-conjugate accumulates in intracellular vesicles in HL-GO/R-CP cells.

GS-X Pump in Cisplatin-resistant Leukemia Cells
The vesicular accumulation of the glutathione-bimane conjugate was inhibited by ATP depletion. Incubation of the resistant cells with 20 1.1~ potassium cyanide and 10 m~ sodium fluoride resulted in a decrease in the accumulation of the fluorescence in the intracellular vesicles, and most fluorescence remained in the cytosol and nuclear spaces almost homogeneously (Fig. 5B, left). Under this condition, the intracellular ATP level was 610% of the control level. On the other hand, treatment of HL-GO/R-CP cells with monensin, an inhibitor of vesicular trafficking out of the trans-Golgi complex, enhanced vesicular fluorescence intensity as well as the size of the intracellular vesicles (Fig. 5B, right). Thus, these results strongly suggest that the vesicular accumulation of the glutathione Sconjugate is ATP dependent and is an important part of the overall process of glutathione S-conjugate excretion.
The accumulation of fluorescence in the intracellular vesicles was more prominent in HL-GO/R-CP cells than in HL-60 cells. Fig. 6 shows the fluorescence microscopy results of the sensitive and resistant cells at 0, 45, and 90 min. Just after the incubation with monochlorobimane, the fluorescence was detected almost homogeneously within the cells (time, 0 min; Fig.  6). m e r 45 min, the accumulation of fluorescence in the vesicles was observed in about 50% of the total resistant cells, and after 90 min, it was detected in almost 100% of the cells. It is very important to note that the fluorescence intensity of HL-GO/R-CP cells decreased dramatically during this period (0-90 min), although the initial fluorescence intensity in the resistant cells was even higher than that in HL-60 cells because of the higher intracellular GSH content (Fig. 6). This is more clearly shown by flow cytometry as a shift of the fluorescence intensity peak (Fig. 7). In the sensitive parent cells, on the other hand, the fluorescence intensity and its intracellular distribution was only slightly changed during the same period (0-90 min) (Figs. 6 and 7). Thus, these data strongly suggest that the cisplatin-resistant cells excrete the glutathione S-conjugate more effectively than the sensitive parent cells.
In addition to the above-mentioned observations, just after incubation with monochlorobimane, HL-GO/R-CO cells exhibited a greater heterogeneity in the fluorescence intensity than HL-60 cells (0 min in Fig. 7). This suggests differences in single-cell GSH content and/or in the rate of glutathione Sconjugate efflux among the cells. Likewise, heterogeneity in single-cell GSH content was previously reported in cells obtained by disaggregation of a biopsy of human renal cell carcinoma (42). treated with TPA (23). However, it was not known whether the cisplatin-resistant cells established in this study maintain such properties or have lost, through the selection process, the biological response to those differentiation-inducing agents. Thus, the potency of HL-GO/R-CP cells for cell differentiation was examined as below.

Effect of Retinoic Acid, MeBO, and TPA on Cisplatin-resistant
When HL-GO/R-CP cells were incubated with retinoic acid, Me,SO, or TPA, the cells arrested their proliferation and, concomitantly, the mRNA level of c-myc decreased substantially. Northern blot analysis clearly demonstrated that the mRNA level of c-myc started to decrease 1 h after the onset of incubation with 1 PM all-trans-retinoic acid (Fig. SA). After 6 h, c-myc mRNA had diminished almost completely. The mRNA level of p-actin remained constant throughout the incubation period (up to 12 h) (data not shown). Similar results were also obtained by incubation with 1.3% (v/v) Me,SO. When the cells were incubated with 32 nM TPA, the c-myc expression level dropped remarkably between 2 and 6 h of the incubation period (Fig. 8B). The response of HL-GO/R-CP cells to these differentiation-inducing agents was virtually identical to that observed for HL-60 cells. Thus, these results confirm that the cisplatinresistant cells maintain the biological properties of the sensitive parent cells with respect to cell proliferation and differentiation.
Effect of Cell Differentiation on GS-X Pump Activity-Based on the results we obtained regarding cell differentiation, we examined the effect of differentiation on the activity of the GS-X pump in both HL-60 and HL-GO/R-CP cells. Cell differentiation was induced by retinoic acid, Me,SO, or TPA, as described above. After 72 h, the activity of ATP-dependent transport of the GS-Pt complex in the plasma membrane vesicles was determined. Table I1 summarizes the effect of the differentiation-inducing agents on the transport activity. After cell differentiation, the activity of ATP-dependent transport of G S P t complex in plasma membrane vesicles significantly decreased in both HL-60 and HL-GO/R-CP cells. In particular, in HL-60 cells, a remarkable decrease was observed with retinoic acid; the transport activity in the differentiated cells was 30% of that of the undifferentiated cells. This decrease was not due to different populations of inside-out vesicles in the preparations (inside-out vesicles, 52% for differentiated cells; 46% for undifferentiated cells).

DISCUSSION
Drug resistance is a biological response of tumor cells to chemotherapeutic agents and represents a constitution of different resistance determinants.
Identifylng and modulating these determinants is important for understanding the biological nature of drug resistance and solving the associated problems in human cancer chemotherapy. Recent studies of the multidrug resistance phenotype of tumor cells have led to the discovery of P-glycoprotein, a 170-kDa plasma membrane glycoprotein that mediates the efflux of anticancer drugs such as doxorubicin, vincristine, and taxol (44,45). The overexpression of this export pump in tumor cells has been found to be closely associated with several multidrug resistance phenotypes. More recently, another type of drug transporter, the multidrug resistance-related protein (MRP), has been identified in doxorubicin-resistant small cell lung cancer in humans (46). MRP mediates the transport of doxorubicin into specific extranuclear compartments (46). Intracellular GSH is a critical determinant in the drug resistance of human tumors, especially to alkylating anticancer agents such as nitrogen mustard, bifunctional nitrosoureas, and cisplatin (3-6). Increased cellular GSH confers tumor cell resistance to those agents, whereas depletion of cellular GSH results in a reversal of the cellular sensitivity. While much attention has hitherto been paid to the function of cellular GSH in the resistance mechanism, evidence is gradually accumulating to demonstrate the important role of the GS-X pump in modulation of GSH-associated drug resistance (47). Continuous elimination of GSH conjugates from cells is an important mechanism of reducing intracellular accumulation of glutathione S-conjugates. The G S P t complex generated from the conjugation reaction of cisplatin with intracellular GSH is potentially cytotoxic. The GS-X pump is proposed to play a major role in eliminating of the GSPt complex from cancer cells (7). The present study provides evidence that the GS-X pump is functionally overexpressed in the cisplatin-resistant variant of HL-60 cells, i.e. HL-GOB-CP. It is noteworthy that the enhanced activity of the GS-X pump in our study did not correlate with the cellular sensitivity to doxorubicin (Fig. 1) or vincristine (data not shown). Furthermore, P-glycoprotein (MDRI) was not immunologically detected in the plasma membrane preparation of HL-GO/R-CP cells (Fig. 3B). These results support our conclusion (48) that the GS-X pump is distinct from P-glycoprotein (MDR1).
As indicated in Figs. 5 and 6, the GS-Xpump may play a key role in the accumulation of GSH-drug conjugates in the intracellular vesicles. Intracellular compartmentalization of drug metabolites is considered to be one of the significant mechanisms in drug resistance. In many cases, drug-resistant tumor cells are more vesicular than are sensitive cells, suggesting that intracellular vesicles may sequester drugs or drug metabolites in a nontoxic site and/or emux them by means of exocytosis (49). Using monochlorobimane, we demonstrated that accumulation of the fluorescent glutathione S-conjugate in intracellular vesicles was more prominent in HL-GO/R-CP cells than in HL-60 cells (Fig. 6). Moreover, the HL-GO/R-CP cells excreted the glutathione S-conjugate more effectively than the sensitive cells (Fig. 6). The vesicular accumulation was ATP dependent (Fig. 5B ), and the vesicular trafficking was inhibited by monensin (Fig. 5B). Based on these results, we hypothesize that, in resistant cells, glutathione S-conjugates of drugs are accumulated in intracellular vesicles by the GS-X pump and subsequently excreted by exocytosis (see Fig. 9 for schematic illustration). The accumulation of the glutathione S-conjugate of monochlorobimane in intracellular vesicles was also observed in cultured rat hepatocytes (43). The hepatic process is mediated by a canalicular multispecific organic anion transporter identical to the GS-X pump. Importantly, the vesicular accumulation does not occur in hepatocytes from mutant rats in which the hepatic export pump is functionally defective (43). In plant cells, on the other hand, it has recently been reported that glutathione S-conjugates of phytotoxic foreign compounds are transported into intracellular vacuoles. The transport into the vacuoles is mediated by a specific ATPase that is very similar to the GS-X pump in animal cells (50). Thus, it is conceivable that the GS-X pump and/or its similar transporters are involved in such subcellular compartmentalization of glutathione S-conjugates in animal as well as plant cells.
At present, the molecular structure of the GS-X pump is not known. In HL-GO/R-CP cells, three membrane proteins with apparent molecular masses of 200, 110, and 70 kDa, respectively, were found to be overexpressed. Kawai et al. (51) previously reported that a 200-kDa membrane glycoprotein is overexpressed in cisplatin-resistant sublines of murine lymphoma cells. The expression of the 200-kDa protein was correlated P of GSH-drug conjugates from cancer cells. DNA-reacting, electro-FIG. 9. Schematic illustration for vesicle-mediated excretion philic agents (X) are conjugated with cellular GSH to form GSH-drug conjugates (GS-X). The GSH-drug conjugates are subsequently transported into intracellular vesicles via the GS-X pump. The vesicles are fused with the plasma membrane, and GSH-drug conjugates are released from the cell by exocytosis. Through the fusion of the vesicular membrane with the plasma membrane, the GS-X pump is translocated to the plasma membrane.
with reduced accumulation of platinum in the lymphoma cells (511, but the function of the protein remains unknown. It has slowly become apparent that the GS-X pump plays a role in the elimination of a variety of glutathione-drug conjugates (1,2) as well as heavy metal-glutathione complexes (7, 52, 53) from cells. Heavy metal tolerance in fission yeast has recently been found to be closely related to the expression of an ATP-binding cassette-type vacuolar membrane transporter, named HMTl (54). Fission yeast strains harboring an HMT1-expressing multicopy plasmid also exhibited metal tolerance to cadmium, im-in Cisplatin-resistant Leukemia Cells plying a relationship between HMT1-mediated transport and compartmentalization of heavy metals (or heavy metal cornplexes) in vacuoles (54). In the fission yeast, cadmium is chelated with phyrochylatins (55) or cadmium-binding peptides (561, which are enzymatically synthesized from GSH (57). In mammmalian cells, the reaction of cadmium with GSH is a first-line defense mechanism (58, 59). Interestingly, HL-60/ R-CP cells were &fold more resistant to cadmium than HL-60 cells (IC5,, = 21 p~ HL-60 cells versus 110 p~ HL-6OR-CP cells).2 It is, therefore, tempting to speculate about a functional and structural relationship between HMTl in the fission yeast and the GS-Xpump in mammalian cells with respect to their role in the ATP-dependent transport of heavy metal-thiol complexes.
Besides identifying the GS-Xpump molecule, it would also be important to understand how the expression of the GS-X pump gene or genes is regulated in drug-resistant and -sensitive tumor cells. A significant increase was observed in both the cellular GSH level and the activity of the GS-X pump in HL-601 R-CP cells (the present study).
The mRNA level of y-glutamylcysteine synthetase was also 3-&fold higher in HL-60iR-CP cells than in HL-60 cells.' y-Glutamyl transferase activity was about %fold higher in the resistant cells than the sensitive cells (Table I). As demonstrated in the cisplatin-resistant ovarian cancer cell line, the increased GSH level was associated with enhanced expression of mRNA levels of y-glutamylcysteine synthetase and y-glutamyl transferase (12). The latter enzyme catalyzes the first step of the catabolism of GSH conjugates exported from cells via the GS-X pump. Thus, it is suggested that the expression of GSH-metabolizing enzymes involved in biosynthesis, transport, and catabolism of GSH, is possibly regulated by certain master switches. For instance, heat shock reportedly leads to an increased expression of y-glutamylcysteine synthetase and reportedly enhanced the efflux of a glutathione S-conjugate, ,942, 4-dinitrophenyl)-glutathione, in K562 erythroid cells (60). Several heat shock proteins are induced by heavy metals and certain SH group reagents (611, including cisplatin (62). It is likely that the expression of some enzymes in GSH metabolism is modified by such stress-response elements. Studies in a variety of cisplatin-resistant cell lines have suggested a potentially important role of fos and ras oncogenes i n drug resistance (63)(64)(65). Elevated mRNA levels of c-fos and c-Haras oncogenes have been shown in cisplatin-resistant cell lines in vitro as well as in cell lines developed from patients failing cisplatin combination therapy (63)(64)(65). The observation that the gene of the GST n isozyme and thepgpl gene of P-glycoprotein contain an AP-1-binding site in the upstream regions suggests that FodJun might play a role in the coordination of gene expression of those proteins (66,67). It was also shown that a mutantp53 stimulated the promoter of P-glycoprotein (MDR1) in NIW3T3 cells (68). In our preliminary studies, however, no significant correlation was detected between the expression level of the c-fos gene and the cellular resistance of HL-6OR-CP cells or the activity of the GS-X pump.2 In addition, evidence that thep53 gene is deleted in HL-60 cells suggests that the p53 gene is not involved in the functional overexpression of the GS-X pump in HL-6OiR-CP cells.
Retinoic acid, Me'SO, and TPA inhibited c-myc expression in both HL-60 and HL-6OiR-CP cells t o lead to cell differentiation and, importantly, there was no significant difference in response between sensitive and resistant cells (Fig. 8). Therefore, HL-GOiR-CP cells virtually maintain the cell proliferation and differentiation properties of the parent cells. The present study has clearly shown that the activity of the GS-X pump in HL-60 and HL-6OR-CP cells decreased after cell differentiation induced by retinoic acid, Me2S0, and TPA (Table 11). This implies that the expression of the GS-X pump is directly or indirectly T. Ishikawa, unpublished results. related to cell proliferation. These findings become even more interesting when we consider facts that retinoic acid is widely used in the treatment of human cancers (69) and that TPA sensitizes cisplatin-resistant human ovarian carcinoma cells (70). In addition, the expression of the human GST T gene is down-regulated by retinoic acid (71).
We have first demonstrated that LTC, and its metabolites are endogenous substrates for the GS-X pump (72-74). LTC, production in leukemia cells was reported to be enhanced after terminal cell differentiation (75)(76)(77). The present finding, however, suggests that the regulation of the GS-Xpump expression in HL-60 cells is more closely associated with cellular detoxification rather than with leukotriene biosynthesis.