Methotrexate Cross-Resistance in a Mitoxantrone-Selected Multidrug-Resistant MCF7 Breast Cancer Cell Line Is Attributable to Enhanced Energy-Dependent Drug Efflux1

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[CANCER RESEARCH 60, 3514–3521, July 1, 2000] Methotrexate Cross-Resistance in a Mitoxantrone-selected Multidrug-resistant MCF7 Breast Cancer Cell Line Is Attributable to Enhanced Energy-dependent Drug Efflux1

Erin L. Volk, Kristin Rohde, Myung Rhee, John J. McGuire, L. Austin Doyle, Douglas D. Ross, and Erasmus Schneider2 Wadsworth Center, New York State Department of Health, Albany, New York 12201 [E. L. V., K. R., M. R., E. S.]; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, New York 12201 [E. L. V., E. S.]; Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 [J. J. M.]; Greenbaum Cancer Center of the University of Maryland, Baltimore, Maryland 21201 [L. A. D., D. D. R.]; Department of Medicine, Division of Hematology/Oncology, University of Maryland School of Medicine, Baltimore, Maryland 21201 [L. A. D., D. D. R.]; and Baltimore Veterans Medical Center, Department of Veterans Affairs, Baltimore, Maryland 21201 [D. D. R.]

ABSTRACT resistance has been shown to be caused by decreased uptake attributable to the absence (2) of or defects (5, 6) in RFC1, reduced poly- Cellular resistance to the antifolate methotrexate (MTX) is often caused glutamylation either through decreased FPGS activity and/or expres- by target amplification, uptake defects, or alterations in polyglutamyla- sion (7), or enhanced ␥-GH activity and/or expression (8), and DHFR tion. Here we have examined MTX cross-resistance in a human breast carcinoma cell line (MCF7/MX) selected in the presence of mitoxantrone, overexpression (9) or mutation (10–12). an anticancer agent associated with the multidrug resistance (MDR) In contrast, a putative role for drug efflux in MTX resistance, phenotype. Examination of protein expression and enzyme activities possibly mediated by an active transport mechanism, is less clear. showed that MCF7/MX cells displayed none of the classical mechanisms of Although evidence for an ATP-dependent MTX efflux mechanism MTX resistance. They did, however, exhibit an ATP-sensitive accumula- has accumulated over the years (13–17), its molecular identity has not tion defect accompanied by reduced polyglutamylation. Although the yet been established. Recently, loss of a folate efflux pump was kinetics of drug uptake was similar between parental and resistant cells, implicated in resistance to the antifolate pyrimethamine, although the the resistant cells exhibited increased energy-dependent drug efflux. This effect seems to be indirect through aberrations in the natural folate suggested the involvement of an ATP-binding cassette (ABC) transporter. pools (18). No evidence for the overexpression of a MTX-specific However, cells transfected with the breast cancer resistance protein (BCRP)—the ABC transporter known to be highly overexpressed in efflux protein as the cause of MTX resistance has thus far been MCF7/MX cells and to confer mitoxantrone resistance (D. D. Ross et al., reported. J. Natl. Cancer Inst. 91: 429–433, 1999)—were not MTX resistant, which A large number of ABC transport proteins have been identified (19, suggested that this transporter is not involved in MTX cross-resistance. 20). These proteins comprise an ever-growing family of efflux pumps, Moreover, members of the MRP protein family of transport proteins, some of which have been implicated in the MDR phenotype. MDR which had previously been implicated in MTX resistance, were not found occurs when cells selected for resistance to one compound develop to be overexpressed in the MCF7/MX cells. Thus, our data suggest that a cross-resistance to other structurally and functionally unrelated drugs. novel MTX-specific efflux pump may be involved in this unusual cross- The prototype of this class of proteins, Pgp, was first identified by resistance phenotype. Juliano and Ling (21) in 1976, and was found to produce resistance to drugs such as doxorubicin, MX, Taxol, and vincristine through its INTRODUCTION action as a drug efflux pump. Overexpression of Pgp was also shown to be associated with MTX resistance in CEM/MTX cells (22), MTX3 is a chemotherapeutic agent that is commonly used for the although cross-resistance to MTX does not appear to be a universal treatment of leukemias and lymphomas, as well as of solid tumors characteristic of cells that overexpress Pgp/MDR1 (23). Another such as breast cancer (1). This drug is a member of the antifolate class group of transporters found to be involved in the MDR phenotype are of compounds that exerts its cytotoxicity by blocking the intracellular the MRPs (24), some of which were shown to efflux various com- pathway whereby thymidylate and de novo purine biosynthesis occur. pounds conjugated to glutathione, glucuronide, or sulfate (25) and to MTX enters the cell via the RFC1 (2) and, once inside the cell, cause resistance to compounds such as doxorubicin, VP-16, and becomes polyglutamylated by the enzyme FPGS (3). Polyglutamyla- vincristine (26). However, cross-resistance to MTX has not been tion of the drug causes its retention within the cell and, therefore, shown. Recently, several reports have been published that demon- increases the likelihood that MTX will bind to its target enzyme, strate the potential for some of the MRP family members to actively DHFR (4). Alterations in each of these steps in the intracellular transport MTX and to cause resistance (27, 28), albeit only under metabolic pathway of MTX can lead to cellular resistance. MTX certain specific conditions, and, hence, their role in acquired MTX resistance remains unclear. Received 9/13/99; accepted 4/28/00. The costs of publication of this article were defrayed in part by the payment of page Here, we investigated the cause of MTX cross-resistance in MCF7 charges. This article must therefore be hereby marked advertisement in accordance with breast cancer cells selected in the presence of 600 nM MX (MCF7/ 18 U.S.C. Section 1734 solely to indicate this fact. MX), a topoisomerase II inhibitor. These cells exhibit a typical MDR 1 This work has been supported in part by NIH Grants CA72455 (to E. S.), CA25933 (to J. G.), and CA43500 (to J. J. M.); Roswell Park Cancer Institute Core Grant CA16056 phenotype in that they display an ATP-dependent drug accumulation (to J. J. M.); and a Veterans Affairs merit review grant (to D. D. R.). defect and are highly resistant to not only the drug for which they 2 To whom requests for reprints should be addressed, at Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509. Phone: (518) 474-2088; Fax: (518) 474-1850; were selected (MX) but also to other structurally unrelated drugs such E-mail: [email protected]. as the camptothecin derivatives TPT and CPT-11 (29). MX resistance 3 The abbreviations used are: MTX, methotrexate; ABC, ATP binding cassette; BCRP, in MCF7/MX cells is attributable to the overexpression of the recently breast cancer resistance protein; Pgp, P-glycoprotein; RFC, reduced folate carrier; TS, thymidylate synthase; DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate synthe- identified ABC half-transporter BCRP (30, 31), which has also been tase; ␥-GH, ␥-glutamyl hydrolase; LSC, liquid scintillation counting; MDR, multidrug called MX-resistance protein (MXR; Ref. 32) or placental ABC resistance; MRP, multidrug resistance-associated protein; MX, mitoxantrone; NE, noneffluxable; TPT, topotecan; DDATHF, 5,10-dideazatetrahydrofolate; WT, wild type; VP- protein (ABC-P; Ref. 33). In contrast, MTX resistance in these cells 16, etoposide. has yet to be characterized. In the present study, we further define this 3514

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. MTX-SPECIFIC EFFLUX IN A MCF7 BREAST CANCER CELL LINE cell line by examining classical mechanisms of MTX resistance as Judes Children’s Research Hospital, Memphis, TN). SDS-Page and Western well as the potential role of ATP-dependent drug efflux as the cause blots for FPGS were performed as described previously (41). of this unusual cross-resistance. ␥-GH Activity Assay. Cells were grown for 72 h and were then extracted with 0.1 M Tris/HCl (pH 6.0) containing 0.1% Triton X-100, 0.1 M 2-mercaptoethanol, and protease inhibitors. The supernatant was used for ␥-GH assays with 100 ␮M MTXGlu (4-NH -10-CH PteGlu ) as substrate and 15 min of MATERIALS AND METHODS 3 2 3 3 incubation at 37°C (42). Activity is expressed as nmol/min/mg protein. Cell Culture and BCRP Transfection. The human breast cancer cell line DHFR Activity Assay. DHFR activity was assayed by the modified MCF7/WT, its drug-resistant variants MCF7/MX (34), MCF7/MTX (35), method of Mathews and Huennekens (43). The cell extracts were incubated in MCF7/VP (36), and BCRP-transfected MCF7 (31), and MDA-MB231 cells the presence of 0.066 mM dihydrofolate (FH2) and 0.1 mM NADPH in 0.05 M were cultured in IMEM medium (Richter’s modification) supplemented with potassium phosphate (pH 7.0)/0.1 M KCl buffer at 30°C. The decrease of the 10% fetal bovine serum and 0.01 g/liter ciprofloxacin at 37°C in a humidified absorbance was traced at 340 nm. One unit of DHFR activity is defined as ␮mol of FH reduced to FH per min. atmosphere of 5% CO2. Cloning of BCRP and transfection of MCF7 cells were 2 4 described previously (31). Human breast carcinoma MDA-MB231 cells were FPGS Activity. FPGS activity was assayed by the previously described transfected as described for MCF7 cells (31). The specific clones used in this method of McGuire et al. (44). Cells were grown for 72 h and then extracted study were clone 8 (MCF7/BCRP) and clone 23 (MDA-MB231/BCRP). Note into 0.5 M Tris-HCl (pH 8.85)/0.2 M 2-mercaptoethanol. Extracts containing that the MCF7/WT and the MCF7 cells used for transfection are two different FPGS were incubated at 37°C in the presence of 0.25 mM MTX substrate and sublines of the MCF7 cell line that have been kept separately in tissue culture 4mM [3H]glutamate (specific activity 24 Ci/mmol; NEN Life Science Prod- for many years. ucts, Boston, MA). Radiolabeled glutamates were then separated from unin- Cytotoxicity Assay. Drugs were stored at Ϫ20°C or at 4°C as the follow- corporated glutamate on DEAE cellulose columns and were quantitated using ing stock solutions: 6 mM MX in water (Sigma, St. Louis, MO), 10 mM MTX LSC. Activity is expressed as nmol MTX-Glu(n) formed per hour per mg in slightly basic sodium phosphate (Sigma), 100 mM VP-16 in DMSO (Sigma), protein. 10 mM TPT in water (Smith Kline Beecham Pharmaceuticals, King of Prussia, MTX Accumulation. Steady-state MTX accumulation was measured by PA), 18 mM DDATHF in DMSO (Eli Lilly and Company, Indianapolis, IN), growing MCF7/WT and MCF7/MX cells for 96 h, followed by incubation in 3 54 mM trimetrexate in DMSO (provided by Drs. Fry and Jackson of Warner serum-free IMEM for an additional 24 h. At this time, 2 ␮M [ H]MTX (specific 5 Lambert Laboratory, Ann Arbor, MI), 115 ␮M 5-fluorouracil in DMSO (Sig- activity 1.89 ϫ 10 dpm/nmol; Moravek Biochemicals, Brea, CA) was added ma), 27 mM metoprine in DMSO (Burroughs Wellcome, Research Triangle, for 3, 6, and 24 h. After the cells were lysed and neutralized, total MTX NC), 20 mM AG337 in DMSO (Aguron), and 5.4 mM Tomudex in water accumulation was measured by LSC. (ZD1694; Zeneca Pharmaceuticals, Wilmington, DE). Trypsinized cells (0.1 Short-term MTX accumulation in MCF7/WT and MCF7/MX cells was ml/well) were seeded in 96-well microtiter plates at a concentration of 2,500 determined in cells grown in 6-well plates to 70% confluency, that were then cells/ml for MCF7/WT; 5,000 cells/ml for MCF7/MX and MCF7/VP; and washed in prewarmed PBS (containing 2 mM each CaCl2 and MgCl2, without 10,000 cells/ml for MCF7/MTX, MCF7/BCRP, and MDA-MB231/BCRP. EDTA) and incubated for 15 min in either ATP-sustaining control medium (1 After 24 h of incubation at 37°C, cells were treated by adding 0.1 ml of drug g/liter glucose in PBS) or ATP-depletion medium (50 mM deoxyglucose, 15 solution with increasing concentration. Corresponding controls received me- mM sodium azide, and 1 mM dinitrophenol). Cells were then exposed to 10 ␮M dium instead of drug solution. Toxicity of the drugs was determined by [3H]MTX (specific activity 2.2 ϫ 105 dpm/nmol) in either control ATP- sulforhodamine B (SRB) assay as described previously (36, 37). sustaining conditions or ATP-depleting conditions for 0, 2, 5, 10, 20, and 30 Cell Membrane Preparation. Cells were seeded at a density of 1,000,000 min. Three successive washes in ice-cold PBS were used to stop drug accu- cells/10-cm dish. After 72 h, or at 70% confluence, cells were washed twice mulation. Cells were lysed overnight in 0.2 N NaOH and were then neutralized with PBS and then scraped into PBS. Samples were sonicated (three 10-s with 0.2 N HCl, and radioactivity in the cells was quantitated by LSC. Values bursts at maximal setting), followed by centrifugation for 10 min at 1000 ϫ g were normalized to total protein content to determine pmol MTX accumulated at 4°C. The supernatant was diluted 1:4 into 10 mM Tris/HCl (pH 7.5) and 250 per mg cell protein. mM sucrose, and the membrane proteins were sedimented by centrifugation at The kinetics of MTX accumulation was assayed in MCF7/WT and 100,000 ϫ g for 30 min. The pellet was dissolved in 62.5 mM Tris/HCl (pH MCF7/MX cells as follows. Cells were grown to confluency in 6-well plates 6.8) and 1% CHAPS, sonicated (three 10-s bursts at maximal setting), and and were washed once in prewarmed PBS, followed by incubation for 2 min 3 solubilized for 30 min on ice. An equal volume of 2ϫ sample buffer [0.1 M in [ H]MTX ranging in concentration from 0.5 to 10 ␮M. As previously Tris/HCl (pH 6.8) and 4% DTT] was added. The protein concentration was described, uptake was terminated by washing the plates three times in ice-cold measured by Bradford assay (38). Samples were dispensed into aliquots and PBS, and radioactivity in the cell lysates was counted by LSC. Values were stored at Ϫ80°C. normalized to protein content, and uptake velocity was calculated as pmol/ Western Blotting. Western blots were generated using 100 ␮g of whole mg/min. These values along with MTX concentration were plotted as a double cell or cytoplasmic cell extracts or 50 ␮g of cell membrane preparations. reciprocal Lineweaver-Burke plot, and approximate Kt and Vmax values were Proteins were fractionated on polyacrylamide running gels (12% for DHFR, calculated from linear regression curves. RFC, and ␥-GH and 7.5% for MRPs) with a 4% stacking gel containing 8 M MTX Efflux. Six-well plates containing cells at 70% confluency were urea. Multimark (Novex, San Diego, CA) and/or broad range markers (Bio- washed in PBS and incubated for 15 min in either ATP-sustaining control Rad, Hercules, CA) were used as molecular weight standards. Proteins were medium (1 g/liter glucose in PBS) or ATP-depletion medium (50 mM deoxy- transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) glucose, 15 mM sodium azide, 1 mM dinitrophenol). Cells were then loaded 3 using wet electrophoretic transfer. TBST [20 mM Tris/HCl (pH 7.5), 0.137 M with 10 ␮M [ H]MTX in either control or depletion medium for 20 min. Efflux NaCl, and 0.01% Tween 20] was used for washing the blot. The nonspecific was examined by replacing the drug solution with the corresponding drug-free binding sites were blocked with Blotto (5% nonfat milk in TBST) overnight. medium for 0, 2, 5, 10, 20, and 30 min of efflux at 37°C. The cells were then The membrane was then incubated with primary antibody for2horovernight washed in ice-cold PBS, lysed, the remaining radioactivity was determined by and with the appropriate secondary antibody for 1 h. Chemiluminescence using LSC, and the protein concentration was determined as previously described. SuperSignal Substrate (Pierce, Rockford, IL) was used for detection. To strip The pool of noneffluxable MTX was determined by depleting cells of folate the blot, the membrane was incubated in stripping buffer [62.5 mM Tris/HCl by incubating in folate-free RPMI for 24 h followed by incubation in 2 ␮M (pH 6.8), 0.2% SDS, and 0.69% ␤-mercaptoethanol) for 30 min at 65°C, [3H]MTX for an additional 24 h. Cells were then washed in ice-cold PBS and followed by brief washing in TBST. The following antibodies were obtained: either immediately lysed yielding total MTX values or incubated in prewarmed anti-DHFR (Research Diagnostics Inc., Flanders, NJ); anti-␥-GH (Dr. Thomas folate-free RPMI in the absence of drug for a 1-h efflux period, which gave Ryan, Wadsworth Center, Albany, NY; Ref. 39); anti-RFC1 (Dr. Jeffrey measurements of the noneffluxable MTX pool. After cell lysis, radioactivity Moscow, University of Kentucky Medical Center, Lexington, KY; Ref. 40); was determined by LSC. anti-MRP1, -2, -3, and -5 (Dr. Rik Scheper, Department of Pathology, Free MTX Polyglutamylation. The long-term polyglutamate profile of MTX Hospital, Amsterdam, the Netherlands); and anti-MRP4 (Dr. John Schuetz, St. was measured after 24 h of exposure to 2 ␮M [3H]MTX. Polyglutamates were 3515

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. MTX-SPECIFIC EFFLUX IN A MCF7 BREAST CANCER CELL LINE extracted, separated by high-performance liquid chromatography and quanti- Table 2 DHFR, ␥-GH, and FPGS activities tated using LSC as described previously (8). Enzyme activities were determined as described in “Materials and Methods.” The data represent the means of at least two separate experiments with range or SEs. ␥ RESULTS DHFR activity -GH activity FPGS activity Cell lines (mU/mg protein) (nmol/min/mg protein) (nmol/h/mg protein) Drug Resistance. MCF7/MX cells were previously shown to be MCF-7/WT 1.90 1.1 1.2 Ϯ 0.31 (1.7, 2.1) (1.2, 1.0) highly and relatively specifically resistant to MX and TPT (29, 34), MCF-7/MX 2.45 1.5 1.4 Ϯ 0.24 presumably because of the overexpression of BCRP (30). During our (2.2, 2.7) (1.2, 1.8) work to characterize this cell line further, we observed that MCF7/MX MCF-7/VP 5.05 0.4 NDa (6.6, 3.5) (0.4, ND) cells were also 150-fold cross-resistant to the antifolate MTX (Table MCF-7/MTX 40.55 1.95 0.8 Ϯ 0.07 1), a drug not normally associated with MDR phenotypes. To eluci- (34.3, 46.8) (1.9, 2.0) date the mechanism for this unusual cross-resistance, we tested sev- a ND, not determined. eral additional antifolate drugs with different modes of entry and different intracellular targets for their cytotoxic effects on MCF7/MX and parental MCF7/WT cells (Table 1). As controls, we used MTX- showed clear overexpression of DHFR as expected. Thus, it seemed selected MCF7/MTX and etoposide-selected MCF7/VP cells, which unlikely that MTX cross-resistance in MCF7/MX cells was attribut- are known to overexpress DHFR and MRP1, respectively (35, 36). As able to DHFR overexpression. shown previously, MCF7/MX cells were highly resistant to the anti- Accumulation of MTX. Reduced drug accumulation has been topoisomerase II drug MX and the anti-topoisomerase I drug TPT and shown in some systems to cause MTX resistance (45), and, therefore, somewhat cross-resistant to another anti-topoisomerase II drug, VP- intracellular drug accumulation was measured. As shown in Fig. 2A, 16. As expected, MCF7/MTX cells were highly resistant to MTX but, steady-state levels of total MTX in MCF7/MX cells were one-half to surprisingly, also showed some cross-resistance to both MX and one-third of those in the parental cells, and short-term MTX accumu- VP-16, whereas MCF7/VP cells were resistant to VP-16 and MX. In lation was also decreased in MCF7/MX cells relative to MCF7/WT contrast, only little, if any, cross-resistance was observed with the cells (Fig. 2B). After 20 min of MTX exposure, accumulation was other antifolate drugs tested, with the exception of an approximately approximately 30% lower in the resistant cell line. This reduction 5-fold resistance to AG337 in MCF7/MX cells. Thus, it seems that the seemed to be ATP dependent; depletion of ATP in MCF7/MX cells mechanism responsible for the MTX cross-resistance in MCF7/MX resulted in increased drug accumulation to levels comparable with cells is rather specific for this particular antifolate. those in MCF7/WT cells, whereas little effect on MTX accumulation DHFR. Overexpression of the target enzyme DHFR has been was seen in the parental cell line. To determine whether the differ- shown to be one of the main mechanisms for MTX resistance (9). To ences in accumulation were caused by alterations in the initial rate of investigate whether resistance of MCF7/MX cells to MTX could be MTX uptake, cells were exposed to various drug concentrations for 2 attributable to the same mechanism, the activity of DHFR was deter- min. Approximate Kt and Vmax values were inferred from the double mined (Table 2). As expected (35), 22-fold higher DHFR activity was reciprocal plot of initial uptake rate and are as follows (Fig. 2C): the ϭ Ϯ ␮ ϭ Ϯ ␮ ϭ Ϯ found in MCF7/MTX cells. In contrast, no substantial difference KtWT 6.8 1.5 M; KtMX 5.4 2.3 M; VmaxWT 17.0 0.9 ϭ Ϯ could be measured in the DHFR activity of MCF7/MX and MCF7/VP pmol/mg/min; and VmaxMX 16.6 2.8 pmol/mg/min. Thus, it cells compared with MCF7/WT cells. These results were confirmed appears that the reduced MTX accumulation observed in MCF7/MX by Western blot using a polyclonal anti-DHFR antibody (Fig. 1). cells is not likely to be attributable to differences in drug uptake Neither in MCF7/MX cells nor in MCF7/VP cells could a substantial because parental and resistant cells exhibited very similar kinetic difference be detected in the expression level of the 22 kDa DHFR profiles. protein compared with MCF7/WT cells, whereas MCF7/MTX cells This conclusion is further supported by the results from Western

Table 1 IC50 values of MCF7/WT cells and relative resistance of the selected sublines Ϯ The cell lines were exposed to the drugs for 7 days. IC50 values (means SE) are from three or more separate experiments, each performed in triplicate. Fold-resistance of MCF7 cell lines

Drug Target Mode of entry Polyglutamylation MCF7/WT MCF7/MX MCF7/MTX MCF7/VP

MTX ␮M DHFR RFC ϩ 0.18 Ϯ 0.03 RFa 1.0 151 1822 3.7 Metoprine ␮M DHFR Diffusion Ϫ 4.5 Ϯ 0.8 1.0 1.4 2.7 1.3 Trimetrexate ␮M DHFR Diffusion Ϫ 44.8 Ϯ 6.7 1.0 1.24 0.56 0.76 AG337 ␮M TS Diffusion Ϫ 1.23 Ϯ 0.3 1.0 5.4 1.6 1.4 b 5-FU ␮M TS Diffusion ϩ 0.008 Ϯ 0.001 1.0 1.1 2.2 2.1 Tomudex ␮M TS RFC ϩ 0.84 Ϯ 0.23 1.0 0.21 1.01 0.73 DDATHF ␮M GAR-TF RFC ϩ 2.7 Ϯ 0.4 1.0 0.6 2.4 0.94 MX nM Topoisomerase II Diffusion Ϫ 0.40 Ϯ 0.07 1.0 1395 32.8 47.9 VP-16 ␮M Topoisomerase II Diffusion Ϫ 0.05 Ϯ 0.009 1.0 56.3 10.3 225 TPT nM Topoisomerase I Diffusion Ϫ 3.0 Ϯ 0.20 1.0 500 3.6 5.2 a RF, relative resistance factor, obtained by dividing the IC50 values of the resistant cells by that of the parental cells. b 5-FU, 5-fluorouracil; GAR-TF, glycinamide ribonucleotide transformylase. 3516

Glu2–6 species was approximately equal, in MCF7/MX cells, there was a gradual decrease in long-chain MTX-Glu(n) forms, with no detectable levels of MTX-Glu5 or -Glu6. To further examine the possible role of MTX polyglutamylation in MTX resistance, we

Fig. 1. Western blot analyses of ␥-GH, DHFR, FPGS, and RFC in WT and resistant MCF7 cells. Whole cell (for DHFR), cytoplasmic (for FPGS and ␥GH) extracts or membrane preparations (for RFC1) of MCF7/WT, MCF7/MX, MCF7/VP, and MCF7/ MTX cells were electrophoresed on a polyacrylamide gel and transferred onto a membrane as described in “Materials and Methods.” Antibody-specific signals were detected by enhanced chemiluminescence. A representative picture from at least two different gels per protein is shown. blots using a polyclonal antibody against the RFC to determine the expression of RFC1 in MCF7 cells (Fig. 1). RFC1, a transporter with a molecular mass of 58 kDa, appeared slightly overexpressed in the drug-resistant MCF7 sublines tested compared with MCF7/WT and, therefore, did not seem to be involved in the reduced drug accumulation and MTX cross-resistance found in the MX-selected cells. Fig. 3. Polyglutamylation of MTX in MCF7/WT and MCF7/MX cells. A, MTX MTX Polyglutamylation. Decreased polyglutamylation leading to polyglutamylation in MCF7/WT (black bars) and MCF7/MX (gray bars). After 24 h of accumulation of MTX, the various polyglutamate chain lengths were separated by decreased drug retention has been demonstrated to cause MTX resist- high-performance liquid chromatography and quantitated as described in “Materials and ance. Therefore, we analyzed the polyglutamylation pattern of MTX Methods.” Data are the means of two separate analyses. B, NE MTX pools in MCF7/WT at 24 h and found that the distribution of the various MTX-polyglu- and MCF7/MX cells were measured after a 24 h accumulation period. Total cellular MTX was measured immediately (total) or after a 1-h efflux (NE). The amount of NE drug is tamate species was different in MCF7/WT and MCF7/MX cells (Fig. expressed as percent of total and represents the average of three independent experiments .P Ͻ 0.03 ,ء .(3A). Although in MCF7/WT cells the amount of each of the MTX- performed in triplicate (mean Ϯ SE

Fig. 2. Accumulation of MTX into MCF7/WT and MCF7/MX cells. A, time course for the steady-state accumulation of [3H]MTX by MCF7/WT (f) and MCF7/MX (F) cells. Total MTX accumulation was measured at 0, 3, 6, and 24 h as described in “Materials and Methods.” B, time course for the short-term accumulation of [3H] MTX by MCF7/WT (f, Ⅺ) and MCF7/MX (F, E) cells in the presence (f, F) and after depletion (Ⅺ, E) of ATP. Accumulation of total MTX was measured at 0, 2, 5, 10, 20, and 30 min as described in “Materials and Methods.” C, an inverse reciprocal plot of a 2-min MTX uptake over a concentration range of 0.5 to 10 ␮M in MCF7/WT (f) and MCF7/MX (F) cells. The graph indicates the mean Ϯ SE of three experiments performed in triplicate. The diagonal lines represent the linear regressions across the data points. 3517

cells were essentially unaffected by energy depletion. Under these conditions, there was no difference in drug efflux between the two cell lines. Thus, these data suggested the presence of an ATP-dependent efflux mechanism, possibly an ABC transporter. BCRP. Because the ABC transporter BCRP is known to be highly overexpressed in MCF7/MX cells and is presumably responsible for the resistance to MX (30, 31), its possible role in MTX resistance was examined. Two sets of BCRP- and control-transfected human breast cancer cells were used. Overexpression of BCRP in the transfected cells was approximately 5- to 10-fold as compared with the respective control transfectants (Ref. 31 and data not shown). By comparison, BCRP expression in the MCF7/MX cells is at least 100-fold higher than in MCF7/WT cells. To explore its potential role in MTX resistance, cytotoxicity assays were performed with BCRP-transfected cells. As shown in Table 3, the BCRP transfectants were not cross- resistant to MTX; MCF7/BCRP (clone 8) cells were 0.71-fold resist- Fig. 4. MTX efflux from MCF7/WT and MCF7/MX cells. After 20 min of incubation ant relative to the pcDNA3 control transfectants, whereas MDA- 3 in the presence of 10 ␮M [ H]MTX (external concentration), cells were washed and MB231/BCRP (clone 23) cells were 1.45-fold resistant, differences incubated in drug-free PBS. To evaluate the role of ATP, cells were preincubated for 15 that were not statistically significant. As expected, transfected MCF7 min in 50 mM deoxyglucose, 15 mM sodium azide, and 1 mM dinitrophenol, followed by 20-min drug uptake in the same depletion medium. Efflux was measured by determining and MDA-MB231 cells were 23- and 89-fold, respectively, resistant the amount of drug remaining in the cells after 0, 2, 5, 10, 20, and 30 min of incubation to MX. Interestingly, BCRP transfection seemed to have only a in drug-free PBS. The data are represented as the relative amount of MTX remaining in MCF7/WT (f, Ⅺ) and MCF7/MX (F, E) cells in the presence (f, F) or absence (Ⅺ, E) modest effect on TPT sensitivity, an effect that did not quite reach of ATP and are the average of three experiments Ϯ SEs. statistical significance, which raised the question of whether BCRP can indeed confer cross-resistance to this drug. In conclusion, these data suggest that BCRP overexpression alone is not sufficient for measured the noneffluxable MTX pools in both MCF7/WT and MTX cross-resistance in MCF7/MX cells. MCF7/MX cells. After a 24-h exposure to 2 ␮M MTX, the fraction of MRP Expression. MTX has recently been shown to be a substrate noneffluxable drug in MCF7/MX compared with MCF7/WT cells was for transport by several members of the MRP family of ABC trans- significantly smaller (Fig. 3B). Thus, both the absence of long-chain porters (27, 28, 46–48). The overexpression of an MRP protein, polyglutamates as well as the decreased pool of noneffluxable MTX therefore, might confer MTX resistance in MCF7/MX cells. To ex- in the resistant relative to the parental cells are consistent with altered amine this possibility, Western blot analyses of MRPs 1–5 were polyglutamylation playing a role in the accumulation defect associ- performed (Fig. 5). However, no evidence for overexpression was ated with MTX resistance in MCF7/MX cells. found for any of the MRP proteins in MCF7/MX cells compared with FPGS and ␥-GH. The formation of MTX polyglutamates depends MCF7/WT cells (Fig. 5). Thus, it seems unlikely that these transport on the cellular level of substrates and the function of two enzymes, proteins are responsible for MTX resistance in MCF7/MX cells. FPGS and ␥-GH. FPGS catalyzes the addition of glutamates, whereas ␥ -GH catalyzes their removal. Thus, reduced FPGS and/or increased DISCUSSION ␥-GH activity could be responsible for the low levels of long-chain MTX-Glu formation. Therefore, the activities of both FPGS and Cellular resistance to MTX is known to occur at various steps in the ␥-GH were measured (Table 2). Cell extracts from MCF7/MX cells intracellular metabolic pathway of this antifolate. Intracellular access showed no significant difference in either FPGS or ␥-GH activity of MTX is mediated by the RFC; mutant or absent carriers have been compared with MCF7/WT cells. In contrast, ␥-GH activity was slightly increased (1.7-fold) and FPGS activity was slightly decreased Table 3 IC values for BCRP-transfected breast tumor cell lines in the MTX resistant cell line MCF7/MTX, whereas ␥-GH activity in 50 Both MCF7 and MDA-MB231 breast cancer cells were transfected with either an the MCF7/VP cells was substantially lower. These results were con- empty vector control (pcDNA3) or BCRP and were exposed to varying concentrations of firmed by Western blot using polyclonal antibodies against the re- MX, MTX, or TPT in a continuous exposure cytotoxicity assay. Mean values represent Ϯ IC50s from three or more experiments SE and significance was calculated using a spective human proteins (Fig. 1). MCF7/MX and MCF7/WT cells two-tailed t test. showed little difference in the expression levels of FPGS and ␥-GH, ␥ MX MTX TPT whereas there was a slight increase in the level of -GH in MCF7/ Cell lines nM ␮M nM MTX cells, in agreement with the activity data. Interestingly, there MCF7/pcDNA3 appeared to be a clear reduction in ␥-GH protein in MCF7/VP cells, Mean 3.1 7.3 16.6 which was also reflected in the activity. Thus, it seems unlikely that SE Ϯ0.63 Ϯ2.8 Ϯ4.6 Fold resistance 1.00 1.00 1.00 the reduced formation of MTX polyglutamates in MCF7/MX cells MCF7/BCRP was attributable to an alteration in these enzymes. Mean 72.2 5.2 37.1 MTX Efflux. To examine whether the decreased accumulation of SE Ϯ10.1 Ϯ1.6 Ϯ8.4 Fold resistance 23.2 0.71 2.24 MTX observed in MCF7/MX cells was attributable to enhanced P 0.005 0.52 0.071 efflux, cells were preloaded with radiolabeled MTX, washed, and MDA-MB231/pcDNA3 Mean 0.15 0.49 15.0 incubated for 0, 2, 5, 10, 20, or 30 min. As shown in Fig. 4, SE Ϯ0.02 Ϯ0.012 Ϯ2.8 MCF7/MX cells exhibited increased MTX efflux relative to Fold resistance 1.00 1.00 1.00 MCF7/WT cells. To determine whether this was an energy-dependent MDA-MB231/BCRP Mean 13.4 1.05 32.4 phenomenon, the cells were depleted of ATP before measuring drug SE Ϯ2.9 Ϯ0.36 Ϯ9.4 efflux. As shown in Fig. 4, in the absence of ATP, MCF7/MX cells Fold resistance 89.1 1.45 2.1 retained more MTX than in the presence of ATP, whereas MCF7/WT P 0.002 0.24 0.12 3518

MX-selected cell line, MCF7/MX, that does not appear to be caused by any of the “classical” mechanisms described above. Overproduc- tion of the target enzyme DHFR does not seem to play a role in MTX resistance in MCF7/MX cells, because both enzyme activity as well as protein expression showed no significant differences between MCF7/MX and MCF7/WT cells. This conclusion is further supported by the lack of cross-resistance to other antifolates that target DHFR, such as metoprine (1.4-fold) and trimetrexate (1.2-fold). Interestingly, MCF7/MTX cells were moderately hypersensitive to trimetrexate, despite the overexpression of DHFR. We have not, however, further investigated the cause for this observation. The major differences between MCF7/MX and MCF7/WT cells, besides reduced MTX sensitivity, are the 3-fold reduction in steady- state accumulation accompanied by reduced formation and accumulation of long-chain MTX polyglutamates. This suggests that lower intracellular drug concentration is responsible for MTX resistance. Reduced accumulation could be attributable to either a quantitative or qualitative defect in uptake via RFC1, whereas reduced polyglutamylation could be attributable to alterations in FPGS and/or ␥-GH. However, neither of these mechanisms seems to play a role. The RFC is expressed at approximately equal levels in both the MTX-resistant and -sensitive cell lines. In further support of an intact and functional carrier is the lack of cross-resistance of MCF7/MX cells to other RFC1-dependent antifolate drugs such as DDATHF (0.6-fold) and Tomudex (0.2-fold). Moreover, MCF7/MX cells are Fig. 5. Western blot analyses of MRP protein expression in MCF7/WT and MCF7/MX not more sensitive to trimetrexate, a lipophilic antifolate that does not cells. Membrane preparations of MCF7/WT and MCF7/MX cells were electrophoresed on require RFC1 to gain cellular access. Cells that are MTX-resistant a polyacrylamide gel and transferred to a membrane as described in the “Materials and Methods.” Antibody-specific signals were detected by enhanced chemiluminescence. A because of RFC1 defects have been shown to be hypersensitive to representative picture from at least two different gels per protein is shown. trimetrexate (50). The accumulation kinetics data also provide evidence against a role of reduced uptake in MTX resistance because MCF7/WT and MCF7/MX cells have approximately the same trans- associated with MTX resistance (1). Qualitative defects in RFC1 port affinity and uptake velocity for MTX. The slight differences attributable to various point mutations leading to single amino acid observed between the two cell lines may be attributable to the com- substitutions have been shown to produce 12- to 200-fold resistance in mencement of efflux activity in the resistant cells. Sarcoma 180 cells and the human leukemia cell line CCRF-CEM (5, Both FPGS and ␥-GH proteins were expressed at similar levels 6, 45). Also, a generally direct relationship was observed between relative to sensitive cells, and, based on activity measurements, there RFC1 levels and MTX sensitivity (49, 50). Loss of RFC1 expression was no evidence for either decreased FPGS activity or hyperactivity resulted in increased MTX resistance that was reversible upon trans- of ␥-GH. Thus, the reduced formation of MTX polyglutamates in fection of a cDNA encoding mRFC1 into carrier deficient, MTX- MCF7/MX compared with MCF7/WT cells does not appear to be resistant ZR-75–1 breast cancer cells (2). MTX resistance has also attributable to an altered function of either of those enzymes. This is been shown to occur at the drug target level; both overexpressed further supported by the absence of MCF7/MX cross-resistance to and/or altered DHFR can cause resistance. A direct correlation has Tomudex (0.2-fold), a TS inhibitor that is dependent on the glutamy- been made between the acquisition of MTX resistance and the number lation activity of FPGS for cytotoxicity. of DHFR copies present in various cell lines, such as the MTX- Taken together, these data indicate that both MTX import and resistant AT-3000 and MCF7/MTX cell lines (9, 35). In addition, metabolism are intact and functional in MCF7/MX cells. Neverthe- DHFR mutations that result in an enzyme with reduced affinity for less, our data indicate that the critical factor in the MTX resistance of MTX have been observed in multiple MTX-resistant cell lines (12, MCF7/MX cells is reduced drug accumulation accompanied by de- 51). creased polyglutamylation. This observation is reminiscent of one Another important feature of MTX metabolism that is involved in previously reported by Moscow et al. (52), who showed that melpha- cellular resistance is polyglutamylation (7, 18). The addition of glu- lan-selected MelRMCF7 cells displayed MTX cross-resistance attrib- tamate residues onto MTX causes the drug to be retained within the utable to a lack of polyglutamylation associated with a decreased drug cell at cytotoxic concentrations. Thus, the balance between the en- accumulation. We show here that the reduced MTX accumulation zyme that catalyzes the addition of glutamates, FPGS, and the enzyme found in MCF7/MX cells could be attributable to an enhanced efflux that functions in the reverse reaction to cleave those glutamate resi- that is sensitive to ATP levels, analogous to what has been shown for dues from MTX, ␥-GH, determines the level of MTX polyglutamy- various natural product drugs. Although MTX is not a classical lation. Both enzymes have been directly linked to MTX resistance. MDR-drug, our data support the hypothesis that the presence of an Decreased FPGS activity and protein expression were shown to cause ABC transporter that may be specific for MTX is the cause of the folate analogue resistance in L1210 leukemic cell variants (7). Con- cross-resistance in the MCF7/MX cell line. An enhanced efflux mech- versely, H35D cells, a rat hepatoma cell line selected for DDATHF anism is also consistent with reduced polyglutamylation; if the drug is resistance, were found to exhibit increased activity of ␥-GH relative to rapidly removed from the cell, it does not become available as a sensitive parental cells (8). Thus, there are multiple mechanisms by substrate for FPGS. Here, we demonstrate a rapid drug efflux in which which tumor cells can acquire MTX resistance. 50% of the drug has been exported within the first 2 min of efflux. In the present report, we describe MTX cross-resistance in the Although the rate of efflux is faster than seen for many natural product 3519

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. MTX-SPECIFIC EFFLUX IN A MCF7 BREAST CANCER CELL LINE drugs, it is not without precedent inasmuch as previous studies have ACKNOWLEDGMENTS shown that VP-16 efflux via MRP1 in MCF7/VP cells occurred at a similarly rapid rate (53, 54). We thank Dr. John Galivan for experimental insight and critical review of the manuscript, and Cynthia Russell for performing the FPGS Western blots. Although it is not a classical mechanism of MTX resistance, there are other reports of ATP-dependent MTX efflux, and ABC transport proteins have been implicated. Data with L1210 cells demonstrated REFERENCES that energy depletion resulted in increased short-term drug uptake and reduced efflux, similar to results reported here (55). Furthermore, 1. Moscow, J. A. Methotrexate transport and resistance. Leuk. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. Methotrexate Cross-Resistance in a Mitoxantrone-selected Multidrug-resistant MCF7 Breast Cancer Cell Line Is Attributable to Enhanced Energy-dependent Drug Efflux

Erin L. Volk, Kristin Rohde, Myung Rhee, et al.

Cancer Res 2000;60:3514-3521.

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