Estrogen memory effect in human hepatocytes during repeated cell division without hormone.

Transient stimulation of target tissues by sex steroids can cause long-lasting changes that may facilitate or alter responses to subsequent hormonal treatment. How these altered characteristics are propagated during cell division in the absence of the stimulating hormone is unknown. The human hepatocarcinoma cell line HepG2 was used as a model to examine the effects of estrogen on the synthesis of serum apolipoproteins in vitro. Treatment with low concentrations of estrogen for 24 to 48 hours resulted in long-lasting alterations in the kinetics with which the cells responded to subsequent stimulation with estrogen. Manifestation of this memory effect was correlated quantitatively with the induction and propagation of a moderate-affinity, nuclear, estrogen-binding protein with the characteristics of a type II estrogen receptor. The data indicate that transient exposure of these cells to estrogen can induce changes in their response characteristics and composition of nuclear proteins that are inherited by daughter cells grown in the absence of hormone for more than ten generations.

Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.

The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

A multidrug-resistance protein (MRP)-like transmembrane pump is highly expressed by resting murine T helper (Th) 2, but not Th1 cells, and is induced to equal expression levels in Th1 and Th2 cells after antigenic stimulation in vivo.

A transmembrane pump for organic anions was identified in resting murine T helper (Th) 2, but not Th1 lymphocyte cell clones, as revealed by extrusion of a fluorescent dye. Dye extrusion inhibition studies suggested that the pump may be the multidrug-resistance protein (MRP). The different expression of the pump in resting Th1 and Th2 cell clones correlated with their respective levels of MRP mRNA. The pump was inducible in Th1 cells by antigenic stimulation in vitro leading to equal expression in activated Th1 and Th2 cell clones. This suggested that dye extrusion might allow the detection of Th2 (resting or activated) or of activated Th1 cells ex vivo based on a functional parameter. To test this, mice were infected with Leishmania major parasites to activate L. major-specific T cells of either Th1 (C57BL/6 mice) or Th2 (BALB/c mice) phenotype: 2-3% of CD4+ lymph node T cells of both strains of mice extruded the dye, defining a cell subset that did not coincide with subsets defined by other activation markers. Fluorescence-activated cell-sorting revealed that the lymphokine response (Th1 or Th2, respectively) to L. major antigens was restricted to this dye-extruding subset.

Cloning and characterization of chicken YB-1: regulation of expression in the liver.

A cDNA expression library constructed from day 9 embryonic liver was screened with a previously identified protein binding site in the flanking region of the liver-specific, estrogen-dependent avian apoVLDLII gene. Two of the clones isolated were shown to encode the chicken homolog of the Y-box binding protein, YB-1 (dbpb), which we have designated chkYB-1. This protein was originally identified in avian extracts by virtue of its ability to bind to two reverse CCAAT motifs in the Rous sarcoma virus enhancer. Since its identification, additional nucleic acid binding properties have been ascribed to its homologs, or closely related proteins, in other species. We have determined the sequence of chkYB-1, investigated its ability to bind to sites known to be involved in tissue-specific expression in the liver, and examined factors influencing its hepatic expression. These studies have demonstrated that the level of chkYB-1 mRNA in the liver decreases steadily throughout embryogenesis and for several weeks posthatching until adult levels are attained. We present several lines of evidence that YB-1 expression in the liver is positively associated with DNA synthesis or cell proliferation. Its binding characteristics indicate that the protein can interact specifically with a number of binding sites for liver-enriched or specific factors. In addition, although it is not particularly asymmetric in terms of base composition, we find a marked preference in binding to the pyrimidine-rich strand of these sites regardless of the presence or polarity of an intact CCAAT box. The increased levels of expression of YB-1 during proliferation combined with its binding characteristics suggest that it may be involved in the reduced expression of liver-specific genes observed at early stages of development or during liver regeneration.

Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins.

The stimulation of chicks or embryos with estrogen results in transient, hepatic expression of the vitellogenin gene, as well as long-term, propagatable alterations in the rapidity with which the gene can be reactivated. We examined the possibility that nuclear, type II estrogen-binding sites are involved in this long-term change in response characteristics. We demonstrate that the primary induction kinetics of type II sites in embryos and chicks correlated with the expression of the vitellogenin gene and that once their induction was triggered by estrogen, they accumulated, were propagated, and persisted for months after withdrawal of the hormone. We also show that their accumulation in the embryo was accompanied by prolonged expression of both the vitellogenin and very low-density apolipoprotein II genes, in the absence of elevated levels of type I receptor, and that the type II sites, like the classical receptor, appear to be preferentially associated with active or potentially active chromatin. Finally, we describe a regulatory mechanism, tested by computer modelling, that simulated the behavioral characteristics of these nuclear estrogen-binding sites and which may explain their role in mediating the long-term effects of estrogen.

Developmental regulation of specific protein interactions with an enhancerlike binding site far upstream from the avian very-low-density apolipoprotein II gene.

Expression of the avian very-low-density apolipoprotein II (apoVLDLII) gene is completely dependent on estrogen and restricted to the liver. We have identified binding sites for nonhistone nuclear proteins located between -1.96 and -2.61 kilobases. One of these sites, located at -2.6 kilobases (designated site 1), was found to span an MspI site that becomes demethylated between days 7 and 9 of embryogenesis, the stage of development at which competence to express the apoVLDLII gene begins to be acquired. Levels of the factor(s) involved were high at day 7 of embryogenesis, decreased two- to threefold by days 9 to 11, and continued to decline more slowly until hatching. Furthermore, the mobility of the complex formed underwent a well-defined shift between days 11 to 13 embryogenesis. Methylation interference studies showed that modification of the outer guanosines of the MspI site resulted in marked inhibition of the formation of the protein-DNA complex. Competition studies, fractionation of nuclear extracts, and tissue distribution indicated that the factor was not the avian homolog of hepatocyte nuclear factor 1, nuclear factor 1, or CCAAT/enhancer-binding protein (C/EBP). However, site 1 could complete for binding to an oligonucleotide, previously shown to be recognized by C/EBP, in a nonreciprocal fashion. These studies demonstrate that the sequence recognized by the protein includes a C/EBP consensus sequence but that elements in addition to the core enhancer motif are essential for binding.

DNA binding and transcription activation by chicken interferon regulatory factor-3 (chIRF-3).

Interferon regulatory factors (IRFs) are a family of transcription factors involved in the cellular response to interferons and viral infection. Previously we isolated an IRF from a chicken embryonic liver cDNA library. Using a PCR-based binding site selection assay, we have characterised the binding specificity of chIRF-3. The optimal binding site (OBS) fits within the consensus interferon-stimulated response element (ISRE) but the specificity of chIRF-3 binding allows less variation in nucleotides outside the core IRF-binding sequence. A comparison of IRF-1 and chIRF-3 binding to ISREs in electrophoretic mobility shift assays confirmed that the binding specificity of chIRF-3 was clearly distinguishable from IRF-1. The selection assay also showed that chIRF-3 is capable of binding an inverted repeat of two half OBSs separated by 10-13 nt. ChIRF-3 appears to bind both the OBS and inverted repeat sites as a dimer with the protein-protein interaction requiring a domain between amino acids 117 and 311. In transfection experiments expression of chIRF-3 strongly activated a promoter containing the OBS. The activation domain was mapped to between amino acids 138 and 221 and a domain inhibitory to activation was also mapped to the C-terminal portion of chIRF-3.

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione.

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.

Detection of the M(r) 190,000 multidrug resistance protein, MRP, with monoclonal antibodies.

MRP is a M(r) 190,000 integral membrane phosphoglycoprotein that is overexpressed in some drug-selected resistant cell lines and has been shown to cause multidrug resistance in transfected cells. Five murine hybridoma cell lines (QCRL-1, QCRL-2, QCRL-3, QCRL-4, and QCRL-6) have been generated which secrete monoclonal antibodies (MAbs) that react specifically with membrane proteins of MRP-overexpressing, multidrug-resistant, drug-selected H69AR cells and MRP-transfected HeLa cells (T5) but not the respective parental (H69) and vector-transfected (C1) cells. The ability of three of these MAbs (QCRL-1, QCRL-2, and QCRL-3) to selectively immunoprecipitate a M(r) 190,000 protein from 35S-labeled H69AR and T5 membranes indicates that these MAbs are specific for MRP. MAb QCRL-1 is also capable of detecting the low levels of MRP present in revertant H69PR cells by immunoblot analysis. Indirect immunofluorescence analyses show that MAbs QCRL-1, QCRL-2, and QCRL-3) strongly and differentially react with fixed T5 and H69AR cells but not with unfixed cells, suggesting that these MAbs recognize intracellular MRP epitopes. The availability of reagents for the specific and sensitive immunodetection of MRP should greatly facilitate biological and clinical studies of this novel drug resistance protein.

Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs.

Amplification of the gene encoding multidrug resistance-associated protein (MRP) and overexpression of its cognate mRNA have been detected in multidrug-resistant cell lines derived from several different tumor types. To establish whether or not the increase in MRP is responsible for drug resistance in these cell lines, we have transfected HeLa cells with MRP expression vectors. The transfectants display an increase in resistance to doxorubicin that is proportional to the levels of a M(r) 190,000, integral membrane protein recognized by anti-MRP antibodies. The transfectants are also resistant to vincristine and VP-16 but not to cisplatin. The results demonstrate that MRP overexpression confers a multidrug resistance phenotype similar to that formerly associated exclusively with elevated levels of P-glycoprotein.

Inwardly rectifying K+ channels and volume-regulated anion channels in multidrug-resistant small cell lung cancer cells.

Studies of multidrug-resistant H69AR cells which overexpress the multidrug resistance-associated protein, compared with drug-sensitive parental H69 cells and revertant H69PR cells, revealed an inwardly rectifying K+ channel current (conductance, 231 pS/pF) and increased volume-regulated anion current (limiting conductance, 2 nS/pF). The anion current was selective for Cl- ions and sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (0.1-1 mM) but ATP was not required for initial current activation even in excised patch experiments. K+ current reversal potential varied 52 mV/10-fold change in the external K+ concentration and current was blocked by BaCl2 (0.1-1 mM). The results indicate that overexpression of multidrug resistance-associated protein is accompanied by increases in both K+ channel and volume-regulated Cl- channel current in the multidrug-resistant cell line H69AR.

Localization of a novel multidrug resistance-associated gene in the HT1080/DR4 and H69AR human tumor cell lines.

Two doxorubicin-selected human tumor cell lines, H69AR and HT1080/DR4, display a multidrug resistance phenotype but do not overexpress P-glycoprotein. Recently, a 6.5-kilobase mRNA encoding a novel member of the ATP-binding cassette superfamily of transport proteins, designated multidrug resistance-associated protein (MRP), has been identified in the H69AR cell line. In the present study, the levels of MRP mRNA were found to be 14-fold higher in HT1080/DR4 cells relative to sensitive HT1080 cells. Southern blotting indicates that gene amplification contributes to the overexpression of MRP in HT1080/DR4 cells. Using a 4-kilobase MRP complementary DNA probe, MRP genes were localized to 2-5 chromosomes bearing homogeneously staining regions and to multiple double minute chromosomes in H69AR cells. Resistant H69AR cells also contained a new der(16) with a structural aberration affecting 16p13.1, the normal cellular locus of the MRP gene. The MRP probe hybridized to two small homogeneously staining regions (hsr) in HT1080/DR4 cells including hsr(7)(p12p15). MRP localization was restricted to the normal cellular locus, 16p13.1, in the parental H69 and HT1080 cells and the drug-sensitive H69PR revertant cells. Our data provide combined evidence that amplification of the MRP gene is associated with the expression of drug resistance in selected solid tumor cell lines.

Location of a protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRP-specific monoclonal antibody QCRL-1.

Multidrug resistance protein (MRP) is a Mr 190,000 integral membrane phosphoglycoprotein which has been shown by transfection studies to confer multidrug resistance. We have previously raised and characterized a panel of MRP-specific monoclonal antibodies (MAbs) which detect distinct epitopes in the MRP molecule (D. R. Hipfner et A, Cancer Res., 54. 5788-5792, 1994), and, in the present study, we have identified the epitope of one of these, MAb QCRL-1. Immunoblot analysis of MRP fragments generated by digestion with formic acid or trypsin suggested that the MAb QCRL-1 epitope was located in the region connecting the two halves of MRP. Subsequent analyses of a series of truncated bacterial glutathione S-transferase fusion proteins containing segments of human MRP further localized the MAb QCRL-1 epitope to a region encompassing amino acids 903-956. Similar experiments with an analogous segment of murine MRP demonstrated that MAb QCRL-1 was highly specific for the human protein. The reactivity of MAb QCRL-1 with a series of overlapping hexapeptides and heptapeptides within this region identified the human MRP-specific heptapeptide SSYSGDI (corresponding to amino acids 918-924) as the epitope, and this peptide was shown to specifically inhibit MAb QCRL-1 binding to MRP. The results of these studies confirm that this epitope has a cytoplasmic location consistent with the topology of MRP predicted from hydrophobicity analyses. These experiments also revealed the presence of a number of protease-sensitive sites on either side of the MAb QCRL-1 epitope in the cytoplasmic domain connecting the two halves of MRP. Future epitope-mapping studies with other MRP-specific MAbs win provide additional insights into the topology of MRP, and may help to identify functionally important regions of this protein. Moreover, definition of the epitope recognized by MAb QCRL-1 as well as the other MAbs will facilitate the use of these reagents for immunohistological studies of MRP expression in drug-resistant tumors.

Sequential coexpression of the multidrug resistance genes MRP and mdr1 and their products in VP-16 (etoposide)-selected H69 small cell lung cancer cells.

Resistance to drugs included in the multidrug-resistance phenotype has been attributed to overexpression of either mdr1 or MRP genes and their products in numerous cell lines, while coexpression, to our knowledge, has not previously been reported in the same cells. Human small cell lung cancer H69/VP cells were developed by continuous incubation in increasing doses of VP-16. In reverse transcription-PCR assays we found over-expression of both mdr1 and multidrug-resistance protein (MRP) genes, and immunoblots showed both elevated P-glycoprotein and MRP in H69/VP cells. Double immunocytochemical staining demonstrated the expression of both MRP and P-glycoprotein in the same cells, indicating that the observations do not result from the selection of two independent clones. Examination of early passages of H69/VP cells showed that overexpression of MRP mRNA occurred prior to mdr1. Thus, cell lines and clinical samples in the future should be tested for both mdr1/P-glycoprotein and MRP since a positive result for one of the phenotypes does not preclude the existence of the other.

Characterization of the M(r) 190,000 multidrug resistance protein (MRP) in drug-selected and transfected human tumor cell.

Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types. The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated [S. P. C. Cole et al., Science Washington, DC), 258: 1650-1654, 1992]. Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vinca alkaloids, and epipodophyllotoxins (C. E. Grant et al., Cancer Res., 54: 357-361, 1994). Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation characteristics, relative resistance to certain drugs, and MRP mRNA:protein ratio. Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known. We have utilized polyclonal anti-MRP-peptide antibodies to compare MRP post-translational modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells. These studies establish that MRP in both the transfected and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000. No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells. Analyses of the subcellular distribution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein. The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles. The relatively high proportion of MRP associated with these fractions in H69AR cells may contribute to the lack of an observable accumulation defect in these cells when compared with the transfectants.

Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein.

Failure of chemotherapy associated with expression of the multidrug resistance protein p170 frequently occurs in retinoblastoma (RB). Despite using cyclosporine, which inhibits p170 and improves our chemotherapy results, rare failures occur. In nonmetastatic primarily enucleated RBs, we show expression of p170 in 3 of 18 samples and expression of multidrug resistance protein (MRP), the second protein associated with resistance to chemotherapy, in 1 of 18 samples. All three RBs that failed chemotherapy without cyclosporine expressed MRP with p170. All three RBs that were enucleated immediately when chemotherapy failed despite the addition of cyclosporine expressed only MRP. One RB enucleated 2 years after failing chemotherapy with cyclosporine, despite radiation and salvage chemotherapy, expressed both p170 and MRP. Two metastatic RBs that expressed both p170 and MRP at diagnosis and at recurrence failed chemotherapy without cyclosporine, whereas one metastatic RB that expressed neither protein was cured by chemotherapy without cyclosporine. MRP may result in failure of chemotherapy despite the elimination of p170-expressing clones by cyclosporine.

Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells.

We have previously identified and characterized a novel member of the ATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitive cells (Cole et al., Science (Washington DC), 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994). In the present study, we have transfected two different eukaryotic expression vectors containing MRP complementary DNA into HeLa cells to study the pharmacological phenotype produced exclusively by overexpression of human MRP. The drug resistance patterns of the two MRP-transfected cell populations were similar. They were characterized by a moderate (5- to 15-fold) level of resistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (< or = 3-fold) level of resistance to taxol, vinblastine, and colchicine. The transfectants were not resistant to 9-alkyl anthracyclines, mitoxantrone, or cisplatin. The MRP-transfected cells were also resistant to some heavy metal anions including arsenite, arsenate, and trivalent and pentavalent antimonials but were not resistant to cadmium chloride. Accumulation of radiolabeled vincristine was reduced by 45% in the MRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP. Rates of vincristine efflux did not differ greatly in the sensitive and resistant cells. The cytotoxic effects of vincristine and doxorubicin could be enhanced in a dose-dependent fashion by coadministration of verapamil. Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity. The degree of chemosensitization by verapamil and cyclosporin A was similar in MRP-transfected cells and in cells transfected with the vector alone, suggesting that sensitization involved mechanisms independent of MRP expression. Verapamil and cyclosporin A caused a modest increase in vincristine accumulation in the resistant cells but did not restore levels to those of the sensitive cells. Taken together, these data indicate that drug-resistant cell lines generated by transfection with MRP complementary DNA display some but not all of the characteristics of MRP-overexpressing cell lines produced by drug selection in vitro. They further demonstrate that the multidrug resistance phenotype conferred by MRP is similar but not identical to that conferred by P-glycoprotein and includes resistance to arsenical and antimonial oxyanions.

The LRP gene encoding a major vault protein associated with drug resistance maps proximal to MRP on chromosome 16: evidence that chromosome breakage plays a key role in MRP or LRP gene amplification.

A cDNA encoding the novel drug resistance gene, LRP (originally termed lung resistance-related protein), was isolated from HT1080/DR4, a 220-fold doxorubicin-resistant human fibrosarcoma cell line which displays a multidrug resistance phenotype and overexpresses the multidrug resistance protein (MRP) but does not overexpress P-glycoprotein encoded by the MDR1 gene. Using the full-length 2.8-kb cDNA probe, the gene for LRP was regionally localized to the 16p13.1-16p11.2 chromosomal segment in human metaphases. Dual color fluorescence in situ hybridization studies refined the localization of LRP to 16p11.2, a location approximately 27 cM proximal to MRP (16p13.1). Two color hybridization studies indicated that HT1080/DR4 fibrosarcoma cells contain amplification of both the MRP and LRP genes in a striking striped pattern in the homogeneously staining region, hsr(7)(p12p15). In contrast, only amplified MRP gene sequences were contained within the homogeneously staining region, hsr(18q). Amplification of LRP was not identified in any of seven other drug-resistant tumor cell lines characterized by 20-300-fold levels of doxorubicin resistance, including two cell lines known to overexpress LRP (SW1573/2R120 and GLC4/ADR). Amplified MRP gene sequences were identified in H69AR, GLC4/ADR, and HL-60/AR whereas only MDR1 gene amplification was observed in the S1B120 colon carcinoma cell line. These data indicate that although both the MRP and LRP genes map to the short arm of chromosome 16, they are rarely coamplified and are not normally located within the same amplicon. A key role for chromosome breakage in gene amplification is supported by the presence of non-random karyotypic anomalies near the MRP and LRP normal cellular loci.

ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.

Structural, mechanistic and clinical aspects of MRP1.

The cDNA encoding ATP-binding cassette (ABC) multidrug resistance protein MRP1 was originally cloned from a drug-selected lung cancer cell line resistant to multiple natural product chemotherapeutic agents. MRP1 is the founder of a branch of the ABC superfamily whose members (from species as diverse as plants and yeast to mammals) share several distinguishing structural features that may contribute to functional and mechanistic similarities among this subgroup of transport proteins. In addition to its role in resistance to natural product drugs, MRP1 (and related proteins) functions as a primary active transporter of structurally diverse organic anions, many of which are formed by the biotransformation of various endo- and xenobiotics by Phase II conjugating enzymes, such as the glutathione S-transferases. MRP1 is involved in a number of glutathione-related cellular processes. Glutathione also appears to play a key role in MRP1-mediated drug resistance. This article reviews the discovery of MRP1 and its relationships with other ABC superfamily members, and summarizes current knowledge of the structure, transport functions and relevance of this protein to in vitro and clinical multidrug resistance.