Antifolate Resistance Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly Impaired Export of Folate and Cholate

Stark, Michal; Rothem, Lilah; Jansen, Gerrit; Scheffer, George L.; Goldman, I. David; Assaraf, Yehuda G.

doi:10.1124/mol.64.2.220

Cited by 38 publications

(24 citation statements)

References 29 publications

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“…Although the objective of this study was not to elucidate a mechanistic basis for the efficacy of the triple DMARD therapy, recent studies by our group (47) did indicate that CEM/SSZ cells displayed enhanced sensitivity to both chloroquine and dexamethasone. In the case of chloroquine, this may be related to downregulation in the expression of MRP-1 (see Figure 4), which, apart from a physiologic role of MRP-1 in folate homeostasis (53)(54)(55), is best known as a drug efflux pump that can export various drugs (52,66), including chloroquine (67,68). Down-regulation of MRP-1 may therefore lead to increased intracellular retention of chloroquine, resulting in enhanced efficacy.…”

Section: Discussionmentioning

confidence: 99%

“…At the level of the cell membrane, cellular folate homeostasis is regulated by at least 2 groups of transporters. The RFC mediates folate transport inward (29,32), while members of the MRP family (52), in particular MRP-1, control cellular export of folates (53)(54)(55). In order to assess the effects of long-term exposure to SSZ, we analyzed RFC protein expression and transport activity and MRP-1 protein expression in CEM/SSZ cells.…”

Section: Effects Of Ssz On Mtx Efficacymentioning

confidence: 99%

“…Interestingly, MRP-1 levels were markedly decreased in CEM/SSZ cells compared with CEM cells (Figure 4). It is conceivable that this down-regulation of MRP-1 is a response to minimize export of folates after inhibition of RFC-mediated uptake of natural folates by SSZ (53,55).…”

Section: Effects Of Ssz On Mtx Efficacymentioning

confidence: 99%

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Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Jansen¹,

Heijden²,

Oerlemans³

et al. 2004

Arthritis & Rheumatism

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Objective. To investigate whether interactions of sulfasalazine (SSZ) with reduced folate carrier (RFC), the dominant cell membrane transporter for natural folates and methotrexate (MTX), may limit the efficacy of combination therapy with MTX and SSZ in patients with rheumatoid arthritis. Conclusion. At clinically relevant plasma concentrations, interactions of SSZ with RFC provide a biochemical rationale for 2 important clinical observations: 1) the onset of (sub)clinical folate deficiency during SSZ treatment, and 2) the lack of additivity/synergism of the combination of SSZ and MTX when these diseasemodifying antirheumatic drugs are administered simultaneously. Thus, when considering use of these drugs in combination therapies, the present results provide a rationale both for the use of folate supplementation and for spacing administration of these drugs over time.

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Section: Discussionmentioning

confidence: 99%

Section: Effects Of Ssz On Mtx Efficacymentioning

confidence: 99%

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Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Jansen¹,

Heijden²,

Oerlemans³

et al. 2004

Arthritis & Rheumatism

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“…GLC-4/ADR cells 23 were established by doxorubicin stepwise selection of human small-cell lung GLC-4 cancer cells and were maintained in the presence of 1 M doxorubicin. Chinese hamster ovary (CHO) AA8 cells were grown in RPMI-1640 supplemented as above, whereas their pyrimethamine-resistant subline, Pyr R100 , 24 which has completely lost MRP1 expression, 25 was grown in the presence of 100 M pyrimethamine and 1 mM sodium pyruvate. All cell lines were cultured in RPMI-1640 supplemented with 10% fetal calf serum (Biological Industries, Beth-Haemek, Israel), 2 mM L-glutamine, penicillin and streptomycin.…”

Section: Cell Linesmentioning

confidence: 99%

“…In contrast, parental GLC4/WT, CHO AA8 and 2008/WT cells express low to moderate levels of MRP1. As a negative control, we used the MRP1 null CHO cell line, Pyr R100 , in which MRP1 expression was completely lost 25 following selection with the lipophilic antifolate, pyrimethamine. 24 Immunofluorescence flow cytometric analysis revealed that the A5 scFv antibody strongly reacted with viable ovarian carcinoma 2008/MRP1 and small cell lung carcinoma GLC4/ADR cells that overexpress MRP1 (Fig.…”

Section: Binding Of the A5 Scfv Antibody To Mrp1-expressing Mdr Tumormentioning

confidence: 99%

Targeting an extracellular epitope of the human multidrug resistance protein 1 (MRP1) in malignant cells with a novel recombinant single chain Fv antibody

Binyamin¹,

Assaraf²,

Haus-Cohen³

et al. 2004

Intl Journal of Cancer

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Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer. Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP-binding cassette (ABC) superfamily including multidrug resistance protein 1 -MRP1 (ABCC1). To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope. Using a phage display approach, we have isolated a recombinant singlechain Fv (scFv) antibody that specifically reacts with the extracellular N-terminus of the human MRP1. Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable Key words: multi drug resistance (MDR); scFv antibody; MRP1; phage displayCombination chemotherapy continues to play a major role in the treatment of various human malignancies. However, the efficacy of various chemotherapeutics has been limited by the frequent emergence of various anticancer drug resistance phenomena. The best-studied mechanisms of multidrug resistance (MDR) in malignant cells involve the overexpression of ATP-driven anticancer drug efflux pumps of the ABC superfamily. 1-3 These include the multidrug transporter, P-glycoprotein (Pgp), which extrudes various hydrophobic cytotoxic agents, as well as members of the multidrug resistance protein (MRP) family, currently comprising 9 exporters (MRP1 through MRP9). 3-5 MRP1 was discovered during a differential hybridization screen aimed at identifying mRNAs overexpressed in doxorubicin-resistant lung cancer cells that lack P-glycoprotein overexpression. 6 The 100 -200-fold MRP1 mRNA overexpression was a result of gene amplification. Analysis of the 1,531 amino acid sequence of MRP1 sequence identified this protein as a member of the ATP-binding cassette (ABC) superfamily of transporter proteins. 6 Importantly, transfection of the MRP1 cDNA conferred upon sensitive cells MDR to anthracyclines, Vinca alkaloids, etoposide, arsenical and antimonial oxyanions but not to cisplatinum and mitoxantrone. 7 These studies paved the way for the isolation of additional MRPs, MRP2 through MRP9, which have been shown to confer resistance to various toxic compounds that are either initially hydrophilic or that undergo intracellular conjugation to glutathione, sulfate and glucuronate. 5 Furthermore, another ABC transporter recently shown to mediate MDR in various tumor cells of epithelial origin is the breast cancer resistance protein (BCRP). 8 In order to study anticancer drug transporter proteins such as MRP1 in clinical specimens and to reveal their physiological functions, specific monoclonal antibodies (MAbs) were recently developed. 9 -11 Monoclonal antibodies against MDR proteins were used for 2 main purposes: a) detection and quantification of MRP and P-glycoprotein expression in drug-resistant cancer cells and b) reversal of MDR in cancer cells by abolishin...

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Probing ATP-dependent conformational changes in the multidrug resistance protein 1 (MRP1/ABCC1) in live tumor cells with a novel recombinant single-chain Fv antibody targeted to the extracellular N-terminus

2005

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The multidrug resistance protein 1 (MRP1/ABCC1) is an ATPdriven transporter that mediates the cellular extrusion of various chemotherapeutic agents. We have previously isolated a novel recombinant single-chain Fv antibody (A5scFv), which specifically targets the extracellular N-terminus of the human MRP1 expressed on the surface of live tumor cells. Fusion of A5scFv to Pseudomonas exotoxin revealed an immunotoxin that bound to the immobilized MRP1-derived peptide upon ELISA, but surprisingly failed to recognize MRP1 on the surface of live tumor cells. As these results suggested that the N-terminus of MRP1 has a limited accessibility to the extracellular space, we used the A5scFv antibody to probe for putative conformational changes that might occur in viable tumor cells upon ATP binding. A5scFv recognized viable MRP1-expressing cells with intact ATP pools, whereas ATP depletion resulted in the loss of A5scFv reactivity. Consistently, restoration of cellular ATP levels resulted in resumption of A5scFv binding to MRP1 in live tumor cells. Flow cytometric analysis confirmed that ATP-depleted cells accumulated significantly higher levels of the established substrate calcein AM, whereas after restoration of cellular ATP pools, cells displayed a much lower level of calcein AM accumulation. Moreover, pretreatment of MRP1-expressing cells with the membrane fluidizer benzyl alcohol resulted in a dramatic increase in A5scFv reactivity, suggesting that membrane fluidization results in the exposure of the N-terminus of MRP1 to the extracellular milieu. These results constitute the first extracellular probing of the putative conformational changes that MRP1 adopts in viable tumor cells upon ATP binding. Furthermore, although ATP binding occurs in the cytosolic nucleotide binding domains of MRP1, significant conformational changes are apparently propagated to the N-terminus residing at the extracellular compartment. ' 2005 Wiley-Liss, Inc.Several members of the large ATP-binding cassette (ABC) superfamily of transport proteins have the facility to translocate an extraordinarily diverse array of structurally dissimilar endogenous and exogenous substrates and their metabolites across cell membranes. 1,2 Among these are 3 important anticancer drug efflux transporters: the multidrug resistance protein 1 (MRP1/ ABCC1), 3,4 breast cancer resistance protein (BCRP/ABCG2) 5 and P-glycoprotein (Pgp/ABCB1). 1,2,6,7 Overexpression of these MDR efflux transporters results in an ATP-dependent decrease in drug accumulation in malignant cells. 1,2,6,7 Consequently, overexpression of MRP1, BCRP, or Pgp results in the acquisition of MDR to multiple anticancer drugs. Increased expressions of MRP1, Pgp and BCRP have been documented in hematologic malignancies and solid tumors, suggesting an important role for these transporters in the potential conferring of clinical drug resistance upon malignant cells. [5][6][7][8][9] Chemotherapeutic agents continue to be the most effective treatment for metastatic tumors. 7 However, the ability of malignant ...

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Antifolate Resistance Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly Impaired Export of Folate and Cholate

Cited by 38 publications

References 29 publications

Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Targeting an extracellular epitope of the human multidrug resistance protein 1 (MRP1) in malignant cells with a novel recombinant single chain Fv antibody

Probing ATP-dependent conformational changes in the multidrug resistance protein 1 (MRP1/ABCC1) in live tumor cells with a novel recombinant single-chain Fv antibody targeted to the extracellular N-terminus

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