Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance.

Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.

[1]  John D. Allen,et al.  A mutation hot spot in the Bcrp1 (Abcg2) multidrug transporter in mouse cell lines selected for Doxorubicin resistance. , 2002, Cancer research.

[2]  P. A. Rea,et al.  Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport. , 2001, Cancer research.

[3]  T. Litman,et al.  Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. , 2001, Cancer research.

[4]  T. Litman,et al.  A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). , 2001, Biochimica et biophysica acta.

[5]  F H Hausheer,et al.  Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF120918. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[6]  A. Klein-Szanto,et al.  Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells. , 2000, Journal of the National Cancer Institute.

[7]  M. Kuo,et al.  BCRP/MXR/ABCP expression in topotecan-resistant human breast carcinoma cells. , 2000, Biochemical pharmacology.

[8]  L. Doyle,et al.  Methotrexate cross-resistance in a mitoxantrone-selected multidrug-resistant MCF7 breast cancer cell line is attributable to enhanced energy-dependent drug efflux. , 2000, Cancer research.

[9]  E. Hudson,et al.  The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). , 2000, Journal of cell science.

[10]  T. Litman,et al.  The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane. , 2000, Biochemical and biophysical research communications.

[11]  T. Fojo,et al.  Amplification of 4q21–q22 and the MXR gene in independently derived mitoxantrone‐resistant cell lines , 2000, Genes, chromosomes & cancer.

[12]  T. Litman,et al.  Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR. , 1999, Cancer letters.

[13]  J. Schellens,et al.  Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. , 1999, Cancer research.

[14]  J. Wijnholds,et al.  The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. , 1999, Cancer research.

[15]  I. Goldman,et al.  Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis. , 1999, Molecular pharmacology.

[16]  G. Peters,et al.  Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2. , 1999, Cancer research.

[17]  Hiroshi Suzuki,et al.  Characterization of the Transport Properties of Cloned Rat Multidrug Resistance-associated Protein 3 (MRP3)* , 1999, The Journal of Biological Chemistry.

[18]  M Dietel,et al.  Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. , 1999, Journal of the National Cancer Institute.

[19]  L. Doyle,et al.  A multidrug resistance transporter from human MCF-7 breast cancer cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  S. Rabindran,et al.  Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C. , 1998, Cancer research.

[21]  Vincenzo,et al.  A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. , 1998, Cancer research.

[22]  G. Peters,et al.  A Structurally Altered Human Reduced Folate Carrier with Increased Folic Acid Transport Mediates a Novel Mechanism of Antifolate Resistance* , 1998, The Journal of Biological Chemistry.

[23]  F. Sirotnak,et al.  A Single Amino Acid Difference within the Folate Transporter Encoded by the Murine RFC-1 Gene Selectively Alters its Interaction with Folate Analogues , 1998, The Journal of Biological Chemistry.

[24]  F. Sirotnak,et al.  Posttranscriptionally Mediated Decreases in Folylpolyglutamate Synthetase Gene Expression in Some Folate Analogue-resistant Variants of the L1210 Cell , 1997, The Journal of Biological Chemistry.

[25]  M. Saxena,et al.  Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 Cells. , 1996, Biochemical pharmacology.

[26]  F. Sirotnak,et al.  Structural preferences among folate compounds and their analogues for ATPase-mediated efflux by inside-out plasma membrane vesicles derived from L1210 cells. , 1995, Biochemical pharmacology.

[27]  P. Johnston,et al.  Characterization of cross-resistance to methotrexate in a human breast cancer cell line selected for resistance to melphalan. , 1995, Biochemical pharmacology.

[28]  K. Cowan,et al.  A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells. , 1994, The Journal of biological chemistry.

[29]  M. Yu,et al.  Allelic variation in the dihydrofolate reductase gene at amino acid position 95 contributes to antifolate resistance in Chinese hamster cells. , 1993, Cancer research.

[30]  C. Swanson,et al.  Decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan. , 1993, British Journal of Cancer.

[31]  C. Cucchi,et al.  Methotrexate and gamma-tert-butyl methotrexate transport in CEM and CEM/MTX human leukemic lymphoblasts. , 1993, Biochemical pharmacology.

[32]  Ying Wang,et al.  Acquisition of Resistance to Antifolates Caused by Enhanced γ-Glutamyl Hydrolase Activity , 1993 .

[33]  F. Sirotnak,et al.  Retentiveness of methotrexate polyglutamates in cultured L1210 cells. Evidence against a role for mediated plasma membrane transport outward. , 1993, Biochemical pharmacology.

[34]  K. Cowan,et al.  Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. , 1992, Cancer research.

[35]  F. Sirotnak,et al.  Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles. , 1992, The Journal of biological chemistry.

[36]  J. Trent,et al.  Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line. , 1991, British Journal of Cancer.

[37]  F. Sirotnak,et al.  The issues of transport multiplicity and energetics pertaining to methotrexate efflux in L1210 cells addressed by an analysis of cis and trans effects of inhibitors. , 1991, Cancer research.

[38]  M. Dietel,et al.  Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257. , 1990, Cancer research.

[39]  D. Scudiero,et al.  New colorimetric cytotoxicity assay for anticancer-drug screening. , 1990, Journal of the National Cancer Institute.

[40]  A. Fojo,et al.  Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein. , 1990, The Journal of biological chemistry.

[41]  D. Banerjee,et al.  Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate-resistant Chinese hamster ovary cell line Pro-3 MtxRIII. , 1990, The Journal of biological chemistry.

[42]  B. Schweitzer,et al.  Amplification of a polymorphic dihydrofolate reductase gene expressing an enzyme with decreased binding to methotrexate in a human colon carcinoma cell line, HCT-8R4, resistant to this drug. , 1989, The Journal of biological chemistry.

[43]  J. Trent,et al.  Cytogenetic and phenotypic analysis of a human colon carcinoma cell line resistant to mitoxantrone. , 1988, Cancer research.

[44]  J. Bertino,et al.  Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. , 1978, The Journal of biological chemistry.

[45]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[46]  A. Long,et al.  A human cell line from a pleural effusion derived from a breast carcinoma. , 1973, Journal of the National Cancer Institute.

[47]  I. Goldman.  Transport energetics of the folic acid analogue, methotrexate, in L1210 leukemia cells. Enhanced accumulation by metabolic inhibitors. , 1969, The Journal of biological chemistry.

[48]  T. Litman,et al.  Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[49]  T. Litman,et al.  Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. , 1999, Cancer research.

[50]  K. Cowan,et al.  Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance. , 1994, Cancer research.

[51]  Scott,et al.  Energy-dependent Efflux of Methotrexate in L 1210 Leukemia Cells , 2022 .