Activity Of Multidrug Resistance Protein Research Articles

Expression of c‐kit and Sca‐1 and their relationship with multidrug resistance protein 1 in mouse bone marrow mononuclear cells

P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) are members of the ATP-binding cassette (ABC) family of transporter proteins. Both molecules are membrane-associated, energy-dependent efflux pumps with different substrate selectivity and they may play a role in the activation, differentiation and function of haematopoietic cells. Mouse haematopoietic cells are characterized by the expression of the cell surface molecules c-kit and Sca-1. Herein, the presence and activities of Pgp and MRP1 in mouse bone marrow mononuclear cells (BMMC) and their relationship with the proteins c-kit and Sca-1 were evaluated. Pgp and MRP activities were measured based on the extrusion of rhodamine 123 (for Pgp) and Fluo-3 (for MRP). Cell populations were assessed by cytometry using anti-c-kit and anti-Sca1 antibodies. Pgp activity was present in 5% of BMMC while 50% of BMMC cells showed MRP activity. These findings agreed with the proportion of cells expressing the MRP1 surface molecule (51.3 +/- 4.17%). About 14% of BMMC were positive for c-kit and/or Sca-1 (9.3% c-kit- Sca-1+, 4.2% c-kit+ Sca-1- and 0.9% c-kit+ Sca-1+). Among these subpopulations only c-kit- Sca-1+ cells presented Pgp activity (21.36%). On the other hand, MRP activity was present in all three subpopulations. Most cells (82.5%) of the c-kit+ Sca-1- subpopulation presented MRP1 activity compared to only 54.1% of c-kit+ Sca-1+ and 38.8% of c-kit- Sca-1+. This study demonstrates the expression and activity of MRP1 in BMMC. While only a small proportion of precursor cells had Pgp activity, MRP1 activity was present among different subpopulations of precursor cells. Further studies are necessary to establish the role of these transporters in haematopoietic cells.

Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis

MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>>>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.

Cyclooxygenase-2 Rescues Rat Mesangial Cells from Apoptosis Induced by Adriamycin via Upregulation of Multidrug Resistance Protein 1 (P-Glycoprotein)

Cyclooxygenase-2 (COX-2) is constitutively expressed in restricted subpopulations of kidney cells, where it presumably acts as an antiapoptotic factor. In conditions that are characterized by inflammation, COX-2 expression also has been described in glomerular mesangial cells (GMC), where COX-2 is not expressed constitutively. It was shown previously that adenovirus-mediated gene transfer of COX-2 into rat GMC led to increased expression and activity of multidrug resistance protein 1 (MDR-1), a membrane transporter that functions as an efflux pump for chemotherapeutic drugs, including Adriamycin (ADR). In ADR nephrotoxicity, a pathologic change in glomeruli could be partially explained by ADR-mediated changes in GMC. Here it is demonstrated that ADR (also known as doxorubicin; 1 microg/ml) induced apoptosis in 15.3 +/- 2.2% of GMC, whereas after adenovirus-mediated COX-2 expression, only 6.6 +/- 0.4% of ADR-treated cells underwent apoptosis. This protective effect was nullified by treatment with NS398, specific COX-2 inhibitor. ADR efflux is greater in COX-2-overexpressing cells, when compared with control, which is attributed to the increased MDR-1 expression. Addition of PSC833, the specific MDR-1 inhibitor, completely abolished the protective effect of COX-2 overexpression and increased the level of apoptosis in GMC that were exposed to ADR. These data suggest that COX-2 protects GMC from ADR-mediated apoptosis via transcriptional upregulation of MDR-1 and that induced COX-2 expression would lessen ADR nephrotoxicity.

Functional Importance of Three Basic Residues Clustered at the Cytosolic Interface of Transmembrane Helix 15 in the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg1138, Lys1141, and Arg1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg1138 and Arg1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[32P]ADP. Mutation of Lys1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter.

Effects of Acid and Lactone Forms of Eight HMG-CoA Reductase Inhibitors on CYP-Mediated Metabolism and MDR1-Mediated Transport

With the growing clinical usage of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), the number of reports concerning serious drug-drug interaction has been increasing. Because recent studies have shown that conversion between acid and lactone forms occurs in the body, drug-drug interaction should be considered on both acid and lactone forms. Thus, we investigated the inhibitory effects of acid and lactone forms of eight statins, including one recently withdrawn, cerivastatin, and two recently developed, pitavastatin and rosuvastatin, on cytochrome P450 (CYP) 2C8, CYP2C9, and CYP3A4/5 metabolic activities and multidrug resistance protein 1 (MDR1) transporting activity. The inhibitory effects of statins on CYP metabolic activities and MDR1 transporting activity were investigated using human liver microsomes and MDR1-overexpressing LLC-GA5-COL150 cells, respectively. The acid forms had minimal inhibitory effects on all CYP activities tested, except for fluvastatin on CYP2C9-mediated tolbutamide 4-hydroxylation (IC50 = 1.7 microM) and simvastatin on CYP3A4/5-mediated paclitaxel 3-hydroxylation (12.0 microM). Lactone forms showed no or minimal inhibitory effects on CYP2C8, CYP2C9, and CYP2C19 activities, except for rosuvastatin on the CYP2C9 activity (20.5 microM), whereas they showed stronger inhibitory effects on the CYP3A4/5 activity with the rank order of atorvastatin (5.6 microM), cerivastatin (8.1 microM), fluvastatin (14.9 microM), simvastatin (15.2 microM), rosuvastatin (20.7 microM), and lovastatin (24.1 microM). Pitavastatin and pravastatin had little inhibitory effect, and a similar order was found also for testosterone 6beta-hydroxylation. MDR1-mediated transport of [3H]digoxin was inhibited only by lactone forms, and the rank order correlated with that of inhibitory effects on both CYP3A4/5 activities. Inhibitory effects on MDR1 activity, and on both CYP3A4/5 activities, could be explained by the lipophilicity; however, a significant correlation was found between the lipophilicity and inhibitory effects on CYP2C8-mediated paclitaxel 6alpha-hydroxylation. We showed the difference between the acid and lactone forms in terms of drug interaction. The lipophilicity could be one of the important factors for inhibitory effects. In the case of statins, it is important to examine the effects of both forms to understand the events found in clinical settings, including the pleiotropic effects.

MRP1 mutated in the L 0 region transports SN-38 but not leukotriene C 4 or estradiol-17 (β- d-glucuronate)

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that confers multidrug resistance on tumor cells. Much convincing evidence has accumulated that MRP1 transports most substances in a GSH-dependent manner. On the other hand, several reports have revealed that MRP1 can transport some substrates independently of GSH; however, the importance of GSH-independent transport activity is not well established and the mechanistic differences between GSH-dependent and -independent transport by MRP1 are unclear. We previously demonstrated that the amino acids W 261 and K 267 in the L 0 region of MRP1 were important for leukotriene C 4 (LTC 4) transport activity of MRP1 and for GSH-dependent photolabeling of MRP1 with azidophenyl agosterol-A (azidoAG-A). In this paper, we further tested the effect of W222L, W223L and R230A mutations in MRP1, designated dmL 0MRP1, on MRP1 transport activity. SN-38 is an active metabolic form of CPT-11 that is one of the most promising anti-cancer drugs. Membrane vesicles prepared from cells expressing dmL 0MRP1 could transport SN-38, but not LTC 4 or estradiol-17 (β- d-glucuronate), and could not be photolabeled with azidoAG-A. These data suggested that SN-38 was transported by a different mechanism than that of GSH-dependent transport. Understanding the GSH-independent transport mechanism of MRP1, and identification of drugs that are transported by this mechanism, will be critical for combating MRP1-mediated drug resistance. We performed a pairwise comparison of compounds that are transported by MRP1 in a GSH-dependent or -independent manner. These data indicated that it may be possible to predict compounds that are transported by MRP1 in a GSH-independent manner.

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2'-7'-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation.

Analysis of the drug resistance profile of multidrug resistance protein 7 (ABCC10): resistance to docetaxel.

The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH(2)-terminal) membrane-spanning domain is present. Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents. We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17beta-estradiol 17-(beta-d-glucuronide). However, the drug resistance profile of the pump has not been determined. In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed. MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9-13-fold). In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3-4-fold). Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid. These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.

Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1).

Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins. In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions. MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH2-terminal MSD1. MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys332 and Asp336. We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys332 mutants, exhibits a selective decrease in leukotriene C4 (LTC4) transport, associated with substantial changes in both Km and Vmax and LTC4 binding. The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R. In addition, nonconservative substitutions of TM6-associated Lys319 and Lys347 resulted in a selective decrease in GSH transport. Of eight other charged residues in or proximal to TM7 to TM11 that were investigated, nonconservative substitutions of three of them [Lys396 (TM7), Asp436 (TM8), and Arg593 (TM11)] caused a substantial and global reduction in transport activity. However, unlike TM6 Asp336, wild-type transport activity could be reestablished in these MRP1 mutants by conservative substitutions. We conclude that MSD2-charged residues in or proximal to TM6, TM7, TM8, and TM11 play critical but differential roles in MRP1 transport activity and substrate specificity.

Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites.

Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

The drug pump multi-drug resistance protein 1 (MRP1) is induced upon T cell activation and its inhibition blocks T cell function

Rationale We observed that activated T cells have increased activity of MRP1 by virtue of rapid extrusion of its fluorescent substrates. We asked whether MRP1 is expressed during T cell activation and whether it might be a target for modulation of the T cell response. Methods We tested for expression and function of MRP1 on adult human PBMC using RT-PCR and fluorescent dye extrusion studies, respectively. PBMC were stimulated with the superantigen TSST-1 (10 ng/ml) and the chemokine SDF-1 in the absence and presence of the MRP1 inhibitor MK-571, and tested for inhibition of T cell function. Results MRP1 is selectively expressed on memory T cells, compared with naïve and cord blood T cells, and is up-regulated during activation. MK-571 blocks TSST-1-induced morphologic changes and the expression of the activation marker CD69, and of IFN-γ, TNF-α, IL-10, and IL-2, in a dose dependent manner. Activation blockade was reversible upon withdrawal of MK-571. MRP1 inhibition also blocked calcium flux in CD4 T cells in response to T cell receptor stimulation with anti-CD3, and calcium flux and chemotaxis in response to stimulation with the chemokine SDF-1. Conclusions MRP1 may be a target for reversible inhibition of T cell activation, and may provide insight into signaling pathways necessary for T cell function.

Multidrug resistance protein attenuates gemtuzumab ozogamicin–induced cytotoxicity in acute myeloid leukemia cells

AbstractGemtuzumab ozogamicin (GO) is a novel immunoconjugate therapy for acute myeloid leukemia (AML). P-glycoprotein (Pgp) confers resistance to GO and is associated with a worse clinical response. To address whether multidrug resistance protein (MRP) affects GO susceptibility, we characterized Pgp, MRP1, and MRP2 expression in CD33+ cell lines and CD33+ AML samples and analyzed the effect of the Pgp inhibitor cyclosporine (CSA) and the MRP inhibitor MK-571 on GO-induced cytotoxicity. MRP1, but not MRP2, expression correlated with MRP activity. MK-571 enhanced GO-induced cytotoxicity in Pgpnegative/MRP-positive NB4 and HL-60 cells. CSA, but not MK-571 alone, restored GO susceptibility in Pgp-positive/MRP-positive TF1 cells; however, MK-571 enhanced cytotoxicity in the presence of CSA. All patient samples exhibited MRP activity, and 17 of 23 exhibited Pgp activity. CSA increased GO-induced cytotoxicity in 12 Pgp-positive samples, whereas MK-571 alone was effective in only one sample with minimal Pgp activity. In 3 Pgp-positive/MRP-positive samples, MK-571 enhanced GO-induced cytotoxicity in the presence of CSA. Thus, MRP1 may attenuate susceptibility to GO. This effect was comparatively less than that for Pgp and required the inhibition of Pgp for detection in cells that coexpressed both transporters. Because MK-571 and CSA failed to affect cytotoxicity in a portion of Pgp-positive/MRP-positive AML samples, additional resistance mechanisms are likely important.

The metabolism of diclofenac--enzymology and toxicology perspectives.

Diclofenac is a nonsteroidal anti-inflammatory drug bearing a carboxylic acid functional group. As a result, the metabolism of diclofenac in humans partitions between acyl glucuronidation and phenyl hydroxylation, with the former reaction catalyzed primarily by uridine 5'-diphosphoglucuronosyl transferase 2B7 while the latter is catalyzed by cytochrome P450 (CYP)2C9 and 3A4. Further hydroxylation of diclofenac glucuronide was shown to occur in vitro with recombinant CYP2C8, which may be of clinical significance in terms of defining major metabolic routes involved in the elimination of diclofenac in humans. The 4'-hydroxylation of the drug appears to represent a feature reaction for CYP2C9 catalysis, and this regioselective oxidation is presumably dictated by interactions of the carboxylate moiety of the substrate with a putative cationic residue of the enzyme. Several other residues of CYP2C9 were identified in studies with site-directed mutants that influence substrate binding affinity and specificity, including Arg97, Phe114, Asn289 and Ser286. The 5-hydroxylation of diclofenac is subject to CYP3A4 cooperativity elicited by quinidine. In this case, enhancement by quinidine of diclofenac metabolism in vitro was attributed to increases in the V(max) with little contribution from changes in the K(m) value. These cooperative interactions in recombinant systems, however, appeared to be influenced by enzyme host membranes of various cDNA-directed expressing CYP3A4. Nevertheless, the in vivo significance of CYP3A cooperativity was demonstrated in a pharmacokinetic study in monkeys, wherein the hepatic clearance of diclofenac increased 2-fold when quinidine was co-administered. Therapeutic use of diclofenac is associated with rare but sometimes fatal hepatotoxicity characterized by delayed onset of symptoms and lack of a clear dose-response relationship. The toxicity has consequently been categorized as metabolic idiosyncrasy. In this regard, the acyl glucuronide of the drug was demonstrated to be reactive and capable of covalent modification of cellular proteins, with covalent binding to liver proteins in rats depending on the activity of multidrug resistance protein 2, a hepatic canalicular transporter. One of the modified proteins was identified as dipeptidyl peptidase IV. Formation of protein adducts also was evident following the oxidative metabolism of diclofenac catalyzed by CYP enzymes. The reactive intermediates in this case were presumably diclofenac 1',4'- and 2,5-quinone imines, both of which were trapped by conjugation with glutathione and identified as glutathione adducts. These same glutathione adducts were detected in rats as well as in human hepatocytes treated with diclofenac, and a corresponding mercapturic acid derivative was identified in urine from patients administered the drug. It is conceivable that the acyl glucuronide and benzoquinone imines derived from diclofenac modify proteins covalently and thereby produce toxicity in susceptible patients via either direct disruption of critical cellular functions or elicitation of immunological responses.

Subcellular localization and activity of multidrug resistance proteins.

The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.

Bioflavonoid stimulation of glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1).

In tumor cells, the human multidrug resistance protein 1 (MRP1), confers resistance to a broad spectrum of anticancer agents. MRP1 is also expressed in many normal tissues where it acts as an ATP-dependent transporter of organic anions. Reduced glutathione (GSH) is transported by MRP1 with very low affinity, and certain MRP1 substrates are transported in association with this tripeptide. Previous studies have shown that various dietary flavonoids stimulate the ATPase activity of MRP1 and inhibit transport of its conjugated organic anion substrates but are poor reversers of MRP1-mediated drug resistance. In contrast, many of the same flavonoids markedly stimulate GSH transport by MRP1. In the present study, we found that stimulation of GSH transport in inside-out MRP1-enriched membrane vesicles by apigenin, naringenin, genistein, and quercetin was maximum at a concentration of 30 microM. Apigenin was the most efficacious of the four bioflavonoids, showing a maximal 6-fold increase over basal levels of GSH transport. The apparent K(m) and V(max) for GSH uptake in the presence of 30 microM apigenin were 116 microM and 666 pmol mg(-1) min(-1), respectively. Chemosensitivity assays with control-transfected and MRP1-transfected HeLa cell lines showed that the IC(50) values for apigenin, naringenin, genistein, and quercetin were similar, demonstrating that overexpression of MRP1 does not confer resistance to these bioflavonoids. Our results suggest that flavonoids stimulate MRP1-mediated GSH transport by increasing the apparent affinity of the transporter for GSH but provide no evidence that a cotransport mechanism is involved.

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

"Pros and Cons" on How to Measure Multidrug Resistance in Leukemias

Drug resistance is one of the most significant challenges in the treatment of various types of malignancies, however most of the experimental and clinical data in multidrug resistance (MDR) has been obtained in leukemias. MDR is the term that describes innate or acquired resistance of tumor cells to a wide range of anticancer drugs. As its presence determines treatment outcome in several forms of leukemias, it is imperative that clinical laboratories provide the most useful data on its expression. Here, a brief review is provided on the pathomechanism and diagnostics of MDR. From the diagnostic point of view it is fortunate that MDR proteins display similar effluxing activity towards many dissimilar agents some of which can be used in fluorescent assays. These tests mimic the real clinical problem i.e. the extrusion activity of MDR proteins towards xenobiotics. Thus, we believe that functional assays when carried out in a standardized way and particularly combined with labeling for various surface markers can be recommended as a front-line test in MDR measurement.

Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia.

Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD). In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up. Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML. A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity. Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations. In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10). This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.

Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia.

The multidrug resistance proteins (MRPs) MRP1, MRP2, MRP3, MRP5 and P-glycoprotein (P-gp) act in concert with each other to give a net resultant pump function in acute myeloid leukemia (AML). The aim of the present study was to analyze the activity of these proteins, which might be upregulated at relapse as compared with de novo AML due to clonal selection. The mRNA expression and activity of P-gp and the MRPs were determined with RT-PCR and flow cytometry, in conjunction with phenotype, as measured with the monoclonal antibodies CD34, CD38 and CD33, in 30 paired samples of de novo and relapsed AML. P-gp and MRP activity varied strongly between the cases (rhodamine 123 efflux-blocking by PSC833: 5.4+/-7.7, and carboxyfluorescein efflux-blocking by MK-571: 4.3+/-6.7, n = 60). P-gp and MRP activity were increased in 23% and 40% of the relapse samples, and decreased in 30% and 20% of the relapse samples, respectively (as defined by a difference of >2 x standard deviation of the assays). Up- or downregulation of mRNA expression was observed for MDR1 (40%), MRP1 (20%), MRP2 (15%), MRP3 (30%), and MRP5 (5%). Phenotyping demonstrated a more mature phenotype in 23% of the relapsed AML cases, and a more immature phenotype in 23% of the relapses, which was independent of the karyotypic changes that were observed in 50% of the studied cases. P-gp and MRP activity correlated with the phenotypic changes, with higher P-gp and MRP activities in less mature cells (r = -0.66, P < 0.001 and r = -0.31, P = 0.02, n = 58). In conclusion, this study shows that P-gp and MRP activity are not consistently upregulated in relapsed AML. However, P-gp and MRP activities were correlated with the maturation stage as defined by immune phenotype, which was observed to be different in 46% of the relapses.

Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2′,7′-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.