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

Abstract

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 ...

Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

“…The effects of mitochondrial inhibitors further support the observation that VDAC1 can be induced in response to ATP depletion and consequentially lead to apoptosis. Interestingly, inosine (IN) and sodium pyruvate (SP), which are routinely used to restore cellular ATP levels, 27, 28 rescue ATP depletion and partially inhibited myostatin-induced VDAC1 upregulation (Figure 5e) and apoptosis (Figure 5f). Combined, all of these results support the conclusion that ATP depletion is responsible for myostatin-induced VDAC1 upregulation and apoptosis.…”

Arsenic Triglutathione [As(GS)3] Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) Is Selectively Modified by Phosphorylation of Tyr920/Ser921 and Glycosylation of Asn19/Asn23

“…Indeed, some evidence has been reported, suggesting that the intracellular domains can influence the extracellular NH 2 -terminus of MRP1. For example, evidence from antibody binding to the NH 2 -terminus suggests that ATP-binding to the cytosolic NBDs of MRP1 results in a conformational change propagated to the NH 2 -terminal domain (Binyamin et al, 2005). Interestingly, reduced N-linked glycosylation of MRP1 has been reported in an oxaliplatin-selected ovarian carcinoma cell line relative to its parental cell line (Beretta et al, 2010).…”

Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy

“…Microvoltammetry was also used to measure the efflux of chemotherapeutic drugs from normal and MDR cancer cells on the single-cell level, with detection limits in the nanomolar range (11). Finally, flow cytometry has routinely been used to quantify and compare expression levels and activity of different ABC transporters that are linked to MDR (12)(13)(14). Although the transporter activity can be inferred from the drug retention in the cell, this method has been advanced by using the invariable drug retention on polymer beads as a standard in flow cytometry measurements (15).…”