ACCELERATED DISCOVERY

Atypical Multidrug Resistance: Breast Cancer Resistance Protein Messenger RNA Expression in Mitoxantrone-Selected Cell Lines

Douglas D. Ross, Weidong Yang, Lynne V. Abruzzo, William S. Dalton, Erasmus Schneider, Hermann Lage, Manfred Dietel, Lee Greenberger, Susan P. C. Cole, L. Austin Doyle

Affiliations of authors: D. D. Ross, University of Maryland Greenebaum Cancer Center, Baltimore, Department of Medicine, Division of Hematology/Oncology, University of Maryland School of Medicine, and Baltimore Veterans Medical Center, Department of Veterans Affairs; W. Yang, University of Maryland Greenebaum Cancer Center; L. V. Abruzzo, University of Maryland Greenebaum Cancer Center and Department of Pathology, University of Maryland School of Medicine; W. S. Dalton, Moffitt Cancer Center, University of South Florida, Tampa; E. Schneider, Wadsworth Center, New York State Department of Health, Albany; H. Lage, M. Dietel, Institute of Pathology, University Hospital Charité, Humboldt University, Berlin, Germany; L. Greenberger, Wyeth-Ayerst Research, Pearl River, NY; S. P. C. Cole, Cancer Research Laboratories, Queen's University, Kingston, ON, Canada; L. A. Doyle, University of Maryland Greenebaum Cancer Center and Department of Medicine, Division of Hematology/Oncology, University of Maryland School of Medicine.

Correspondence to: Douglas D. Ross, M.D., Ph.D., Greenebaum Cancer Center of the University of Maryland, Rm. 9-015, Bressler Research Bldg., 655 West Baltimore St., Baltimore, MD 21201 (e-mail: DROSS{at}umcc01.umcc.ab. umd.edu). Reprint requests to: Douglas D. Ross or L. Austin Doyle, Greenebaum Cancer Center of the University of Maryland, Rm. 9-015, Bressler Research Bldg., 655 West Baltimore St., Baltimore, MD 21201.


    ABSTRACT
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone. METHODS: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively. RESULTS: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma(S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079),and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells. CONCLUSIONS: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines.



    INTRODUCTION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transport-mediated resistance to anticancer drugs has been a subject of active investigation for a number of years. Currently, two members of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily of transport proteins, P-glycoprotein (Pgp) and multidrug resistance protein (MRP; also known as multidrug resistance-associated protein), are known to cause cultured tumor cell lines to become resistant to multiple anticancer drugs (1,2). However, when the drug mitoxantrone is used as the selecting agent, the resulting drug-resistant cell lines frequently do not overexpress Pgp or MRP, despite a demonstrable reduction in drug accumulation (3-8). This result suggests that a novel transport-mediated drug resistance mechanism(s) is recruited in response to selection with mitoxantrone. The unique mitoxantrone-selected phenotype is characterized by the following: resistance to mitoxantrone, doxorubicin, and daunorubicin; an ATP-dependent reduction in drug accumulation; and sensitivity to vinca alkaloids, paclitaxel, and cisplatin. The drug resistance phenotype typically associated with mitoxantrone selection has also been observed in human breast carcinoma MCF-7/AdrVp cells, which were not selected with mitoxantrone but were selected with doxorubicin in the presence of verapamil, an inhibitor of Pgp (9). Despite the method of selection, MCF-7/AdrVp cells are considerably more resistant to mitoxantrone than to doxorubicin (10).

Recently, MCF-7/AdrVp cells were found to overexpress a novel ABC transport protein designated breast cancer resistance protein, or BCRP (11). Transfection of BCRP complementary DNA (cDNA) into drug-sensitive MCF-7 cells conferred resistance to mitoxantrone, daunorubicin, and doxorubicin but not to vinca alkaloids, paclitaxel, or cisplatin. In addition, lower intracellular accumulation and retention of daunorubicin and an ATP-dependent enhancement of the efflux of rhodamine 123 were observed in the transfected cells (11). Hence, transfection with BCRP cDNA reproduced in MCF-7 cells the drug resistance phenotype typical of mitoxantrone-selected cell lines. This finding suggests that BCRP may be responsible for the novel transport mechanism observed in the mitoxantrone-selected cell lines. This study was undertaken to test the prevalence of BCRP overexpression in mitoxantrone-selected cell lines.


    MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell lines. The cell lines used and the conditions under which they were cultured are given in Table 1Go and references listed therein. The S1M1-3.2 colon carcinoma cells were derived from S1 cells (a subclone of human colon carcinoma cell line LS174T) by selection for growth in increasing concentrations of mitoxantrone until a final concentration of 3.2 µM was achieved. The HL-60/MX2 leukemia cells were purchased from the American Type Culture Collection (Manassas, VA) and were maintained in culture as described previously (12).


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Table 1. Characteristics of selected multidrug-resistant human cancer cell lines

 
Northern blot hybridization. Total cellular RNA was used for northern blot hybridization analysis in all cases except for H209 or H69 cells, where poly A+ RNA was used. RNA extraction and northern blotting were performed by standard techniques as described previously (11). A 795-base-pair (bp) fragment of the 3' end of the 2418-bp BCRP cDNA (GenBank database accession number AF098951) was used as the hybridization probe after labeling with [32P]deoxycytidine triphosphate ("Prime-a-Gene" labeling kit; Promega Corp., Madison, WI) as described previously (11). To control for variations in sample loading, the blots were stripped, then rehybridized with 32P-labeled ß-actin or 18S RNA probes as described previously (11). The levels of messenger RNA (mRNA) expression in different cell lines were compared by an arbitrary grading system (Table 1)Go based on visual determination of signal intensities.

Southern blot hybridization. Genomic DNA was isolated from the MCF-7 cell lines, digested with EcoRI or BamHI, and separated by 0.8% agarose gel electrophoresis. After staining with ethidium bromide, the DNA was transferred and fixed to a nitrocellulose filter by use of standard techniques (13). The filter was hybridized with the 32P-labeled 795-bp BCRP probe described above for northern blot analysis.

Northern or Southern blots were prepared by collaborating authors (S. P. C. Cole, W. S. Dalton, M. Dietel, H. Lage, and E. Schneider) who maintain mitoxantrone-selected and other multidrug-resistant cell lines in their laboratories; the blots were then probed for BCRP expression in the laboratories of D. D. Ross and L. A. Doyle at the University of Maryland Greenebaum Cancer Center.


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The drug-resistant cell lines used and their characteristics with respect to the degree of resistance that they exhibit and expression (relative to parental cells) of mRNAs encoding the multidrug resistance transporters Pgp, MRP, and BCRP are summarized in Table 1.Go

All of the mitoxantrone-selected sublines derived from human breast carcinoma MCF-7 cells overexpressed BCRP mRNA relative to its expression in parental MCF-7 cells. BCRP-positive cell lines included MCF-7/Mitox cells (3) (Fig. 1, A;Go lane 2); MCF-7/MXPR cells, partial revertants of MCF-7/MX (5); and two sublines of MCF-7/MX (MCF-7/MXRS250 and MCF-7/MXRS600) (Mitox = MX = RNOV [see below] = mitoxantrone) that were reselected with higher concentrations of mitoxantrone (6) (Fig. 1, BGo; lanes 4-6). The MCF-7/MX cells (Fig. 1, BGo; lanes 4-6) appeared to express amounts of BCRP mRNA comparable to or greater than those expressed by the MCF-7/AdrVp1000 cells (Fig. 1, BGo; lane 10) from which BCRP was originally isolated (Adr = Adriamycin®) = doxorubicin, and Vp = verapamil) (11). In contrast to BCRP mRNA expression in the mitoxantrone-selected cell lines, BCRP mRNA was not overexpressed in MCF-7 cells selected with methotrexate [MCF-7/MTX (13)], etoposide [MCF-7/VP (14)], or doxorubicin [MCF-7/Adr (15)], which derive resistance, at least in part, from the overexpression of dihydrofolate reductase (DHFR), MRP, or Pgp, respectively (Fig. 1, B;Go Table 1Go). Mitoxantrone-selected human breast carcinoma MDA-MB-231RNOV cells, which are less resistant to mitoxantrone than the MCF-7 sublines, demonstrated only slightly elevated levels of BCRP mRNA compared with the levels seen in the parental cell line (Fig. 1, CGo; lanes 3 and 4).





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Fig. 1. Northern blot hybridizations of breast cancer resistance protein (BCRP) messenger RNA (mRNA). The blots were probed with a 795-base-pair fragment of BCRP complementary DNA labeled with [32P]deoxycytidine triphosphate (upper panels). To control for variation in sample loading, the blots were stripped and reprobed for ß-actin or 18S RNA, as indicated in the figure (bottom panels). Pgp = P-glycoprotein; MRP = multidrug resistance protein; Mitox = MX = RNOV = MR = mitoxantrone resistant; Adr = Adriamycin® = doxorubicin; Vp = verapamil; VP = V = etoposide; RDB = daunorubicin resistant; MTX = methotrexate; DHFR = dihydrofolate reductase. A) Northern blot hybridization of mRNA from human breast carcinoma MCF-7 cells, human myeloma 8226 cells, and related drug-resistant cell lines. Lane 1 = MCF-7; lane 2 = MCF-7/Mitox; lane 3 = 8226; lane 4 = 8226/MR20. B)Northern blot hybridization of mRNA from MCF-7 cells, human colon carcinoma S1 cells, and related drug-resistant cell lines. Lane 1 = S1/M1-3.2; lane 2 = S1; lane 3 = MCF-7; lane 4 = MCF-7/MXPR; lane 5 = MCF-7/MXRS250; lane 6 = MCF-7/MXRS600; lane 7 = MCF-7/VP (overexpresses MRP); lane 8 = MCF-7/Adr (overexpresses Pgp); lane 9 = MCF-7/MTX (overexpresses DHFR); lane 10 = MCF-7/AdrVp1000 (overexpresses BCRP). C) Northern blot hybridization of mRNA from human colon carcinoma HT29 cells, human breast carcinoma MDA-MB-231 cells, human fibrosarcoma EPF86-079 cells, human gastric carcinoma EPG85-257 cells, human pancreatic carcinoma EPP85-181 cells, and related drug-resistant cell lines. Lane 1 = HT29; lane 2 = HT29RNOV; lane 3 = MDA-MB-231; lane 4 = MDA-MB-231RNOV; lane 5 = EPF86-079; lane 6 = EPF86-079RNOV; lane 7 = EPG85-257; lane 8 = EPG85-257RNOV; lane 9 = EPG85-257RDB (overexpresses Pgp); lane 10 = EPP85-181; lane 11 = EPP85-181RNOV; lane 12 = EPP85-181RDB (overexpresses Pgp).

 
Amplification of the BCRP gene was detected in MCF-7/MXPR cells (Fig. 2;Go lanes 2 and 8), MCF-7/MXRS250 cells (Fig. 2;Go lanes 3 and 9), and MCF-7/MXRS600 cells (Fig. 2;Go lanes 4 and 10). No BCRP gene amplification was observed in etoposide-selected, MRP-overexpressing MCF-7/VP cells (Fig. 2;Go lanes 5 and 11) or in methotrexate-selected, DHFR-amplified MCF-7/MTX cells (Fig. 2;Go lanes 6 and 12).



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Fig. 2. Southern blot hybridization of genomic DNA from human breast carcinoma MCF-7 cells and drug-resistant sublines. DNA was isolated, digested with EcoRI or BamHI, separated by 0.8% agarose gel electrophoresis, transferred, and fixed to a nitrocellulose filter. The filter was probed with a 795-base-pair fragment of breast cancer resistance protein complementary DNA that was labeled with [32P]deoxycytidine triphosphate (top panel). Ethidium bromide staining of the agarose gels prior to nitrocellulose filter transfer demonstrated approximate equivalency of sample loading (bottom panel). MRP = multidrug resistance protein; MX = mitoxantrone; VP = etoposide; MTX = methotrexate; DHFR = dihydrofolate reductase; Kb = kilobase. Lanes 1 and 7 = MCF-7; lanes 2 and 8 = MCF-7/MXPR; lanes 3 and 9 = MCF-7/MXRS250; lanes 4 and 10 = MCF-7/MXRS600; lanes 5 and 11 = MCF-7/VP (overexpresses MRP); lanes 6 and 12 = MCF-7/MTX (overexpresses DHFR).

 
Human myeloma 8226/MR20 cells are capable of sustained growth in 200 nM mitoxantrone (4) and also demonstrated overexpression of BCRP compared with BCRP expression in parental 8226 cells (Fig. 1, A;Go lanes 3 and 4). The expression of BCRP mRNA in 8226/MR20 cells was less than the amount of BCRP mRNA expressed in MCF-7/Mitox cells (Fig. 1, A;Go lane 2); however, this may be because the latter cell line is more resistant to mitoxantrone (1208-fold, Table 1Go) than is the former (36-fold, Table 1Go).

Overexpression of BCRP mRNA was also observed in human colon carcinoma S1/M1-3.2 cells (resistant to 3.2 µM mitoxantrone) (Fig. 1, B;Go lane 1), compared with parental S1 cells (Fig. 1, B;Go lane 2). Another human colon carcinoma cell line selected with mitoxantrone, HT29RNOV, displayed overexpression of BCRP compared with the BCRP expression in parental HT29 cells (Fig. 1, C;Go lanes 1 and 2).

None of the small-cell lung cancer cell lines tested overexpressed BCRP (data not shown), including those selected with mitoxantrone (H209/MX2 and H209/MX4) (Table 1).Go It is of note that these mitoxantrone-selected lines showed only a low degree of resistance to the drug and also have no demonstrable overexpression of Pgp or MRP. Etoposide-selected H209/V6 cells appear to derive their resistance from a mutated topoisomerase II (16); H69/AR cells, selected with doxorubicin, markedly overexpress MRP (17).

The human gastric carcinoma-derived resistant EPG85-257RNOV cell line was developed by stepwise selection with mitoxantrone (8). Compared with parental cells, EPG85-257RNOV cells have no detectable overexpression of Pgp or MRP; they display decreased mitoxantrone accumulation and cross-resistance to anthracyclines, but they retain sensitivity to cisplatin and vinca alkaloids (8,18). Northern blot analysis indicated elevated levels of BCRP mRNA in EPG85-257RNOV cells compared with the levels in the parental EPG85-257 cells (Fig. 1, C;Go lanes 7 and 8). In contrast, a Pgp-overexpressing subline selected with daunorubicin, EPG85-257RDB, did not overexpress BCRP relative to its expression in parental EPG85-257 cells (Fig. 1, C;Go lane 9).

BCRP mRNA was not overexpressed in human pancreatic carcinoma EPP85-181 cells selected in mitoxantrone (EPP85-181RNOV) or daunorubicin (EPP85-181RDB) (19) (Fig. 1, C;Go lanes 10-12). The baseline expression of BCRP in the parental EPP85-181 pancreatic carcinoma cells appeared to be lower than that in the parental EPG85-257 gastric carcinoma cells (Fig. 1, C;Go lanes 7 and 10).

Mitoxantrone-selected EPF86-079RNOV human fibrosarcoma cells displayed overexpression of BCRP mRNA compared with the BCRP mRNA expression in parental EPF86-079 cells (Fig. 1, C;Go lanes 5 and 6).

A mitoxantrone-selected subline of human acute myeloid leukemia HL-60 cells [HL-60/MX2 (12)] did not overexpress BCRP mRNA (data not shown). However, these resistant cells do not display the typical phenotype displayed by mitoxantrone-selected cells that overexpress BCRP, since HL-60/MX2 cells do not have diminished accumulation of mitoxantrone and are cross-resistant to etoposide. HL-60/MX2 cells have altered catalytic activity of DNA topoisomerase II and reduced levels of topoisomerase II alpha and beta proteins (12).


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Elevated expression of the novel ABC transporter BCRP is a common feature of many cancer cell lines selected with mitoxantrone and is consistently associated with a phenotype that includes high-level resistance to mitoxantrone, lower resistance to anthracyclines, and sensitivity to vinca alkaloids, paclitaxel, and cisplatin. ATP-dependent export of mitoxantrone, anthracyclines, and rhodamine 123 has been observed in several BCRP-positive, mitoxantrone-resistant cell lines and BCRP-transfected breast cancer cells (7,11). Cross-resistance to topoisomerase I-directed agents has been reported in some mitoxantrone-resistant cell lines (20), but resistance to these agents has not yet been confirmed in BCRP-transfected cells.

The BCRP peptide has the characteristics of a "half-transporter," having only a single ATP-binding domain and a single lipophilic region containing transmembrane domains (11). Hence, it is possible that the most efficient transmembrane conductance channel contains BCRP as a dimer or multimer either with itself or with another heretofore undescribed "half-transporter" (11). Although the transfection studies suggest that the enforced overexpression of BCRP cDNA is sufficient to confer drug resistance to the transfected cells (11), it is possible that another transporter(s) may be involved in a dimeric or a multimeric complex with BCRP, which may contribute to the resistance of the mitoxantrone-selected cell lines. The identification of other transporters that may participate in the BCRP transmembrane conductance channel is currently under active investigation in our laboratory.

The finding of elevated expression of BCRP mRNA in the human colon carcinoma S1M1-3.2 cells suggests that BCRP is the "non-Pgp, non-MRP" drug transporter manifested by this multidrug-resistant cell line. This is of particular importance because of the recent report (7) of a novel and specific inhibitor of the transporter identified in S1M1-3.2 cells. This inhibitor, fumitremorgin C, does not reverse resistance in cells that overexpress Pgp or MRP. In preliminary studies in our laboratory, fumitremorgin C is able to enhance the accumulation and inhibit the efflux of BBR 3390 (an aza-anthrapyrazole drug that is effluxed by BCRP) in BCRP-transfected MCF-7 cells [data not shown; (11)]. Further development of fumitremorgin C is warranted for use in functional assays of BCRP activity and possibly as a clinical adjunct to chemotherapy, should BCRP protein/activity levels be shown to be elevated in human cancers.

BCRP expression is associated with multidrug resistance in mitoxantrone-selected cell lines derived from human breast, gastric, and colon cancers, as well as from fibrosarcoma and multiple myeloma cells. High levels of BCRP expression are not associated with strong expression of MRP or Pgp in mitoxantrone-selected cell lines or in multidrug-resistant cell lines known to overexpress MRP or Pgp.

In conclusion, our study indicates that elevated expression of BCRP is frequently observed in mitoxantrone-selected cell lines derived from human multiple myeloma and a number of solid tumors. Considered together with results from BCRP transfection studies (11), these data demonstrate that BCRP is a novel ABC protein responsible for mediating resistance to mitoxantrone and other important chemotherapeutic agents in a wide variety of human cancer cell lines.


    NOTES
 
L. Greenberger owns stock in, and is an employee of, American Home Products, which fully owns Wyeth-Ayerst Research, the company that manufactures and sells mitoxantrone.

Supported in part by Public Health Service grant CA52178 (D. D. Ross) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by a merit review grant (D. D. Ross) from the Department of Veterans Affairs.


    REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received January 13, 1999; accepted January 27, 1999.


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