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R G Deeley

Publications and source records attributed to R G Deeley.

At least 37 records · Page 2Linked to original sources
PubMed2000

The multidrug resistance protein 1: a functionally important activation marker for murine Th1 cells.

Previously, we described the expression of an energy-dependent pump in resting murine Th2 (but not resting Th1) cells which extruded the fluorescent dye Fluo-3. After stimulation with Ag and APCs, Th1 cells also expressed this pump. Furthermore, expression of the murine multidrug resistance protein 1 (mrp1) correlated with the presence of the pump. In this study, we report that Fluo-3 is indeed transported by murine mrp1 or its human ortholog MRP1, as revealed by transfection of HEK 293 cells with mrp1 or MRP1 cDNA. Like antigenic activation, IL-2 dose-dependently enhanced the Fluo-3-extruding activity in murine Th1 cells. Although TNF-alpha and IL-12 by themselves only weakly enhanced Fluo-3 extrusion, each of them did so in strong synergism with IL-2. An Ab directed against mrp1 was used to quantify the expression of mrp1 protein in T cells at the single-cell level. Like the Fluo-3 pump, mrp1 protein expression was enhanced by IL-2. Immunohistochemical studies using confocal laser microscopy indicated that mrp1 is localized mainly at the plasma membrane. In addition, protein expression of mrp1 was induced in Vbeta8+CD4+ T cells 12 h after in vivo application of Staphylococcal enterotoxin B. Finally, mrp1 was functionally relevant during the activation process of Th1 cells, because T cell activation could be suppressed by exposure of cells to the mrp1 inhibitor MK571. Thus, we present mrp1 as a novel, functionally important activation marker for Th1 cells and short-term in vivo activated CD4+ T cells, whereas its expression seems to be constitutive in Th2 cells.

ATP-Binding Cassette Transporters↗
PubMed2000

Simultaneous quantitation of topoisomerase II alpha and beta isoform mRNAs in lung tumor cells and normal and malignant lung tissue.

Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIalpha and topo IIbeta. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II alpha and beta mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II alpha and beta mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo II alpha mRNA levels (12-fold) and topo IIbeta mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II alpha and beta mRNAs were similar. However, mean topo IIalpha mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIbeta mRNA levels were similar in both tumor and normal lung. Topo II alpha and beta mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIbeta mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIalpha and topo IIbeta mRNA levels feasible in large numbers of clinical samples.

Antigens, Neoplasm↗
PubMed2000

Cloning and promoter analysis of the chicken interferon regulatory factor-3 gene.

Interferon regulatory factors (IRFs) are a family of DNA-binding proteins involved in mediating the cellular response to interferons (IFNs) and viral infection. Although extensively studied in mammals, IRFs of other vertebrates have been less well characterized. Previously, we cloned chicken interferon regulatory factor-3 (chIRF-3) mRNA, which is rapidly and transiently induced by double-stranded (ds)RNA. The chIRF-3 mRNA encodes a protein distinct from any known mammalian IRF. Here, we show that chIRF-3 is activated additively by type I and type II IFNs. To delineate the sequence elements required to regulate chIRF-3 expression, we cloned chlRF-3 and 0.48 kb of 5' flanking sequence. Computer analysis of the proximal promoter revealed three putative binding sites for nuclear factor (NF)-kappaB, two overlapping interferon-stimulated response elements (ISREs), and an interferon gamma activating sequence (GAS). The presence of both GAS and ISRE consensus sequences in the chIRF-3 promoter is unique among IRF family members. Both type I and II IFNs, as well as dsRNA and IRF-1, trans-activate the promoter in short-term transfection experiments. Mutational analysis of the promoter demonstrated that the putative NF-kappaB binding sites are needed for stimulation by dsRNA but not by either type I or type II IFN and that both the overlapping ISREs and GAS are required for full induction by type I or type II IFN.

Animals↗
PubMed2000

Verapamil stimulates glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1).

Multidrug resistance in tumor cells is often associated with reduced drug accumulation resulting from increased expression of the 190-kDa multidrug resistance protein 1 (MRP1) or the 170-kDa P-glycoprotein. However, unlike P-glycoprotein, MRP1 is a primary active transporter of many conjugated organic anions, including the cysteinyl leukotriene LTC(4). Moreover, agents such as verapamil that reverse P-glycoprotein-mediated resistance are often poorly, or not at all, effective in MRP1-overexpressing cells. In the present study, we investigated the effects of verapamil on MRP1-mediated transport processes. We found that verapamil inhibited LTC(4) transport into inside-out membrane vesicles prepared from MRP1-transfected cells in a competitive manner, but only in the presence of reduced glutathione (GSH) or its nonreducing S-methyl derivative. In the presence of 1 mM GSH, the apparent K(i) for verapamil was 1.2 microM, and in the presence of 100 microM verapamil, the apparent K(i) for GSH was 77 microM. Verapamil itself was not transported by MRP1 in either intact cells or membrane vesicles. However, verapamil strongly stimulated MRP1-mediated GSH uptake by membrane vesicles in a concentration-dependent and osmotically sensitive manner that was inhibitable by MRP1-specific monoclonal antibodies. In the presence of 100 microM verapamil, the apparent K(m) and V(max) for GSH uptake were 83 microM and 55 pmol mg(-1) min(-1), respectively. It is proposed that the variable ability of verapamil to modulate MRP1-mediated resistance in different cell lines may be more closely linked to its effect on the GSH status of the cells than on its ability to inhibit the MRP1 transporter itself.

ATP-Binding Cassette Transporters↗
PubMed1999

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.

ATP-Binding Cassette Transporters↗
PubMed1999

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.

ATP-Binding Cassette Transporters↗
PubMed1999

Localization of a substrate specificity domain in the multidrug resistance protein.

Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C(4) and the cholestatic estrogen, 17beta-estradiol 17(beta-D-glucuronide) (E(2)17betaG). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E(2)17betaG. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E(2)17betaG transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments of the COOH-terminal third of the mouse protein by replacement of either amino acids 959-1187 or 1188-1531 with those of MRP produced proteins capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C(4) with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E(2)17betaG with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E(2)17betaG.

ATP-Binding Cassette Transporters↗
PubMed1999

Monoclonal antibodies that inhibit the transport function of the 190-kDa multidrug resistance protein, MRP. Localization of their epitopes to the nucleotide-binding domains of the protein.

Multidrug resistance in tumor cells is often accompanied by overexpression of multidrug resistance protein (MRP), a 190-kDa transmembrane protein that belongs to the ATP-binding cassette superfamily of transport proteins. MRP mediates ATP-dependent transport of a variety of conjugated organic anions and can also transport several unmodified xenobiotics in a glutathione-dependent manner. To facilitate structure-function studies of MRP, we have generated a panel of MRP-specific monoclonal antibodies (mAbs). Four of these mAbs, QCRL-2, -3, -4, and -6, bind intracellular conformation-dependent epitopes, and we have shown that they can inhibit the transport of several MRP substrates. Binding competition and immunoprecipitation assays indicated that mAbs QCRL-4 and -6 probably recognize the same detergent-sensitive epitope in MRP, whereas mAbs QCRL-2, -3, and -4 each bind distinct, non-overlapping epitopes. Fab fragments inhibit transport as effectively as the intact mAbs, suggesting that inhibition results from direct interactions of the mAbs with MRP. Immunodot blot and immunoprecipitation analyses revealed that the minimal regions of MRP sufficient for full reactivity of mAbs QCRL-2 and -3 are amino acids 617-858 and 617-932, respectively, which encompass the NH2-proximal nucleotide-binding domain (NBD). In contrast, the epitope bound by mAb QCRL-4 localized to amino acids 1294-1531, a region that contains the COOH-proximal NBD. However, none of the mAbs inhibited photolabeling of intact MRP with 8-azido-[alpha-32P]ATP. This suggests that rather than preventing nucleotide binding, the mAbs inhibit transport by interfering with substrate binding or by trapping MRP in a conformation that does not allow transport to occur. Our results also demonstrate for the first time that the NBDs of MRP can be expressed as soluble polypeptides that retain a native conformation.

ATP-Binding Cassette Transporters↗
PubMed1999

Determinants of the DNA-binding specificity of the Avian homeodomain protein, AKR.

AKR (Avian Knotted-Related) was the first example of a vertebrate homeodomain protein with a highly divergent Ile residue at position 50 of the DNA-recognition helix. The protein was cloned from a liver cDNA expression library of a day-9 chick embryo by virtue of its ability to bind to the F' site in the proximal promoter of the avian apoVLDLII gene. Expression of the apoVLDLII gene is completely estrogen dependent, and mutation or deletion of the F' site decreases estrogen inducibility 5- to 10-fold. Subsequent data indicated that AKR is capable of repressing the hormone responsiveness of the apoVLDLII promoter, specifically through binding to F'. Involvement of the F' site in the hormone-dependent activation of apoVLDLII gene expression, as well as AKR-mediated repression, strongly suggests that both positive and negative regulatory factors interact with this site. Although several mammalian proteins have now been isolated whose homeodomains share many of the structural features of AKR, including the Ile at position 50, little is known of their functions in vivo or the identities of the genes they regulate. Consequently, the elements through which they exert their effects and the structural determinants of their binding specificities remain largely uncharacterized. In this study, we defined the sequence specificity of binding by AKR using polymerase chain reaction-assisted optimal site selection and determined the affinity with which the protein binds to both the optimized site and the F' site. Additionally, we generated a three-dimensional model of the AKR homeodomain binding to its optimized site and probed the validity of the model by examining the consequences of mutating amino acid residues in recognition helix 3 and the N-terminal arm on the binding specificity of the homeodomain. Finally, we present evidence that the F' site itself may act as an estrogen response element (ERE) when in the vicinity of imperfect or canonical EREs and that AKR can repress hormone inducibility mediated via this site.

Animals↗
PubMed1999

Expression of multidrug resistance protein-related genes in lung cancer: correlation with drug response.

Recently, cDNAs have been identified that encode four human proteins (MRP2-5) with structural similarity to the multidrug resistance protein (MRP). Preliminary studies have shown that levels of mRNAs encoding MRP2, MRP3, and MRP5, are increased in some drug-selected cell lines, but the correlation of MRP2-5 mRNA levels with drug resistance has not been examined. Using a collection of small cell lung cancer (SCLC) and non-SCLC patient samples and unselected cell lines established from patients at various stages of treatment, we examined the expression of MRP2, MRP3, MRP4, and MRP5, as well as MDR1 and MRP, by PCR. The levels of individual mRNAs were correlated with the sensitivity of these cell lines to doxorubicin (DOX), vincristine, VP-16, and cis-diamminedichloroplatinum(II), as determined by a modified MTT assay. Using both SCLC and non-SCLC cell lines, we confirmed the previously observed correlation of MRP mRNA levels with resistance to DOX (B. G. Campling et al., Clin. Cancer Res., 3:115-122, 1997) and found a strong correlation of MRP3 mRNA levels with resistance of the cell lines to DOX. In addition, the mRNA levels of both MRP and MRP3 correlated with resistance of the cell lines to vincristine, VP-16, and cis-diamminedichloroplatinum(II). These findings are consistent with the suggestion that MRP3, like MRP, may contribute to the drug resistance phenotype of lung cancer cells.

ATP-Binding Cassette Transporters↗
PubMed1998

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Multidrug resistance genes (MRP) and MDR1 expression in small cell lung cancer xenografts: relationship with response to chemotherapy.

Intrinsic or acquired drug resistance is a major limiting factor of the effectiveness of chemotherapy. Increased expression of either the MRP gene or the MDR1 gene has been demonstrated to confer drug resistance in vitro. In this study, we examined MRP and MDR1 gene expression in a panel of 17 small cell lung cancers (SCLC) xenografted into nude mice from treated and untreated patients using an RT-PCR technique. For some of them, the outcome of the corresponding patients was known and we related MDR1/MRP expression with the xenograft response to C'CAV (cyclophosphamide, cisplatin, adriamycin and etoposide) combined chemotherapy. Fifteen (88%) of the 17 cases of SCLC were found to be positive for either MDR1 or MRP. MRP gene expression was present in 12 (71%) of 17 cases, whereas MDR1 gene expression was detected in eight (50%) of 16 cases. For six SCLC, the survival duration of patients differed, with three patients surviving for more than 30 months after therapy. Among these six turnours, five expressed MRP and/or MDR1. These six xenografts responded to the C'CAV treatment but a significant rate of cure was obtained in only three cases. No obvious relationship was observed between the response to this treatment and MRP or MDR1 expression. However, the remarkably high levels and frequency of MRP expression in some SCLC samples indicate that future developments in chemotherapy of this tumour type should anticipate that drugs which are substrates of MRP may be of limited effectiveness.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Multidrug resistance protein. Identification of regions required for active transport of leukotriene C4.

Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells. The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains. The role(s) of the third MSD of MRP and its related transporters is not known. To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4 (LTC4). We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane. However, all three modifications decreased LTC4 transport activity by at least 90%. Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD. In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP.

Resistance to multiple natural product drugs associated with reduced drug accumulation in human tumor cells may be conferred by either the 170 kDa P-glycoprotein or the 190 kDa multidrug resistance protein, MRP. Both MRP and P-glycoprotein belong to the large and ancient ATP-binding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identify. Unlike P-glycoprotein, MRP actively transports conjugated organic anions such as the cysteinyl leukotriene C4 and glutathione-conjugated aflatoxin B1. Transport of unconjugated chemotherapeutic agents appears to require cotransport of glutathione. MRP and several more recently discovered ABC proteins contain an additional NH2-proximal membrane-spanning domain not found in previously characterized ABC transporters. This domain, whose NH2-terminus is extracytosolic, is essential for MRP-mediated transport activity. This review summarizes current knowledge of the structural and transport characteristics of MRP which suggest that the physiologic functions of this protein could range from a protective role in chemical toxicity and oxidative stress to mediation of inflammatory responses involving cysteinyl leukotrienes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Epitope mapping of monoclonal antibodies specific for the 190-kDa multidrug resistance protein (MRP).

Inherent or acquired resistance to multiple natural product drugs in human tumour cells is often associated with increased expression of multidrug resistance protein (MRP), a 190-kDa integral membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. Both clinical and experimental investigations of MRP have been facilitated by several monoclonal antibodies (MAbs) generated against intracellular epitopes of the molecule. Recently, however, several new ABC transporters that are quite closely related to MRP have been identified, raising concerns about the specificity of the MRP-reactive MAbs. In the present study, we have mapped the epitopes of MAbs MRPr1 and MRPm6 to the decapeptides 238GSDLWSLNKE247 (located in the intracellular loop between the first and second membrane-spanning domains of MRP) and 1511PSDLLQQRGL1520 (located near the carboxy terminus of MRP) respectively. Alignment of the MRPr1 and MRPm6 epitope sequences with the comparable regions in mammalian ABC proteins most closely related to MRP indicates that, with the exception of murine mrp, the sequences are poorly conserved. We conclude that MAbs MRPm6 and MRPr1, together with MAb QCRL-1, which has previously been mapped to the heptapeptide 918SSYSGDI924, remain highly specific probes for detection of different regions of the MRP molecule.

ATP-Binding Cassette Transporters↗