Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multidrug resistance 1 (MDR1) gene encodes a M(r) 170,000 membrane glycoprotein termed P-glycoprotein, which catalyzes the energy-dependent efflux of multiple anticancer agents. We investigated the activation of the MDR1 gene promoter by UV light irradiation in human cancer KB cells after both transient and stable transfection assays of the MDR1 promoter fused to the chloramphenicol acetyltransferase (CAT) gene. Following exposure to UV irradiation, CAT gene expression was about 20-fold increased. A series of promoter dissection analyses showed that two elements extending from -136 to -76 of the 5' flanking sequence and from +1 to +121 of the sequence downstream from the initiation site were required for the stress induction of MDR1 promoter activity. Gel shift assays showed that the specific DNA binding activities of the transacting protein to the MDR1 promoter were augmented in nuclear extracts from the cells treated with UV irradiation. A DNA sequence, an inverted CCAAT box, was identified that specifically bound to this protein, and mutation of this sequence abolished the binding of this protein. Two guanines in the inverted CCAAT box were found to be critical, as methylation of these guanines abrogated the binding. Nuclear run-on assay demonstrated that the transcription level was increased about 5-fold. These results suggest that the activation of the MDR1 promoter may result from transcriptional rather than posttranscriptional events. These studies will provide the basis for understanding the regulatory mechanism for appearance of the drug-resistant phenotype during cancer chemotherapy.
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PMID:Enhanced expression of the human multidrug resistance 1 gene in response to UV light irradiation. 846 53

Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited and acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a bicistronic cassette in which the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) a multidrug efflux pump, and the human glucocerebrosidase (GC) gene are transcriptionally fused. Transduction of human Gaucher fibroblasts with this recombinant virus allowed coordinate expression of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expression of GC, which paralleled the stringency of drug selection. Thus, selection of the genetically modified Gaucher fibroblasts in 1 microgram/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzyme deficiency present in Gaucher cells. Moreover, by simultaneously inhibiting the P-gp pump, it was possible to use much lower concentrations of colchicine to select for high-level expression of MDR1 and GC. Thus, selection with colchicine at 5 ng/ml in combination with the P-gp inhibitors verapamil or PSC 833 produced a complete correction of the GC deficiency in the CA2-transduced fibroblasts. These combination regimens, already in clinical use for the treatment of multidrug-resistant malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebrosidase when transcriptionally fused to the MDR1 gene.
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PMID:Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy. 893 30

Expression of the drug efflux pump P-glycoprotein, encoded by the multidrug resistance (MDR1) gene, has been identified as an impediment to successful chemotherapy of neoplastic diseases. More recently, its potential use for gene therapy has been analyzed. Expression of a full-length MDR1 cDNA in hematopoietic cells renders them resistant to various anticancer drugs, as first shown in a transgenic mouse model. Similarly, mouse hematopoietic progenitor cells in bone marrow or peripheral blood are protected from the toxicity of anticancer chemotherapy by retroviral transduction of the MDR1 gene. Furthermore, cells engineered to express P-glycoprotein survived after the administration of cytotoxic drugs, indicating that the gene could function as a selectable marker in vivo. Recently, MDR1 transduction into isolated pluripotent hematopoietic stem cells has been demonstrated. Clinical studies on MDR1 gene transfer into hematopoietic cells of cancer patients are being planned. Transfer of the MDR1 gene into hematopoietic precursor cells may allow the introduction and selection of otherwise non-selectable genes in bone marrow. The ability to select transduced cells can circumvent the low transduction efficiency that has hampered efficient gene therapy. Recently, fusion genes in which the MDR1 cDNA is fused to genes that correct genetic disorders have been constructed to facilitate gene therapy of inherited metabolic disorders.
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PMID:Transfer of the MDR1 (multidrug resistance) gene: protection of hematopoietic cells from cytotoxic chemotherapy, and selection of transduced cells in vivo. 938 59

Radiochemotherapy-resistant blasts commonly cause treatment failure in acute myeloid leukemia (AML), and their resistance is due, in part, to overexpression of multidrug resistance (mdr) proteins. We reasoned that targeted delivery of protein synthesis inactivating toxins to leukemic blasts would reduce the cellular concentrations of relatively short half-life resistance proteins and sensitize the cells to cytotoxic drugs. To test this hypothesis, we employed human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GMCSF). The human AML cell line HL60 and its vincristine-resistant sublines, HL60Vinc and HL60VCR, were incubated in vitro for 24 h with varying concentrations of toxin. Doxorubicin was added for an additional 24 h, and cell cytotoxicity was assayed by thymidine incorporation and colony formation in semisolid medium. DT388-GMCSF sensitized HL60Vinc and HL60VCR but not HL60 to doxorubicin. Combination indices for three log cell kill varied from 0.2 to 0.3. In contrast, pretreatment with doxorubicin followed by toxins failed to show synergy. At least in the case of the vincristine-resistant cell lines, modulation of drug resistance correlated with reduction in membrane P-glycoprotein concentrations based on immunoblots with C219 antibody, flow cytometry with MRK16 antibody, and cell uptake of doxorubicin. These observations suggest clinical trials of combination therapy may be warranted in patients with refractory AML. Further, targeted toxins may represent a novel class of cell-specific modulators of drug resistance for a number of malignancies.
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PMID:Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF. 966 51

Drug resistance, both primary and acquired, is a major obstacle to advances in cancer chemotherapy. In vitro, multidrug resistance can be mediated by P-glycoprotein (PGY1), a cell surface phosphoglycoprotein that acts to efflux natural products from cells. PGY1 is encoded by the MDR1 gene located at 7q21.1. Overexpression of MDR1 has been demonstrated in many cancers, both in patient tumors and in cell lines selected with a variety of chemotherapeutic agents. Recent studies in drug-selected cell lines and patients samples have identified hybrid mRNAs comprised of an active, but apparently random, gene fused 5' to MDR1. This observation indicates that random chromosomal rearrangements, such as translocations and inversions, leading to "capture" of MDR1 by constitutively expressed genes may be a mechanism for activation of this gene following drug exposure. In this study, fluorescence in situ hybridization (FISH) using whole chromosome paints (WCP) and bacterial artificial chromosome (BAC)-derived probes showed structural rearrangements involving 7q in metaphase and interphase cells, and comparative genomic hybridization (CGH) revealed high levels of amplification at chromosomal breakpoints. In an adriamycin-selected resistant colon cancer line (S48-3s/Adr), WCP4/WCP7 revealed t(4;7)(q31;q21) and BAC-derived probes demonstrated that the breakpoint lay between MDR1 and sequences 500-1000 KB telomeric to it. Similarly, in a subline isolated following exposure to actinomycin D (S48-3s/ActD), a hybrid MDR1 gene composed of heme oxygenase-2 sequences (at 16p13) fused to MDR1 was identified and a rearrangement confirmed with WCP7 and a subtelomeric 16p probe. Likewise, in a paclitaxel-selected MCF-7 subline where CASP sequences (at 7q22) were shown to be fused to MDR1, WCP7 showed an elongated chromosome 7 with a homogeneously staining regions (hsr); BAC-derived probes demonstrated that the hsr was composed of highly amplified MDR1 and CASP sequences. In all three selected cell lines, CGH demonstrated amplification at breakpoints involving MDR1 (at 7q21) and genes fused to MDR1 at 4q31, 7q22, and 16p13.3. Finally, in samples obtained from two patients with drug refractory ALL, BAC-derived probes applied to archived marrow cells demonstrated that a breakpoint occurred between MDR1 and sequences 500-1000 KB telomeric to MDR1, consistent with a random chromosomal rearrangement. These results support the proposal that random chromosomal rearrangement leading to capture and activation of MDR1 is a mechanism of acquired drug resistance.
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PMID:Cytogenetic and molecular characterization of random chromosomal rearrangements activating the drug resistance gene, MDR1/P-glycoprotein, in drug-selected cell lines and patients with drug refractory ALL. 971 96

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.
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PMID:Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein. 993 1

The bacitracin resistance of Bacillus licheniformis, a producer of bacitracin, is mediated by the ABC transporter Bcr. Bacillus subtilis cells carrying bcr genes on high-copy number plasmids developed collateral detergent sensitivity, as did human cells with overexpressed multidrug resistance P-glycoprotein. Resistance against bacitracin and sensitivity of resistant cells to detergents were shown to be inseparable phenomena associated with the membrane part of Bcr transporter, namely protein BcrC. A fused protein, consisting of ATP-binding protein BcrA and membrane component BcrC was constructed. It resembled a half molecule of P-glycoprotein and was capable of providing a significant degree of antibiotic resistance and detergent sensitivity.
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PMID:The role of the bacitracin ABC transporter in bacitracin resistance and collateral detergent sensitivity. 1086 42

Derivatives of alpha-conidendrin, podophyllotoxin, and sikkimotoxin were prepared to evaluate the cytotoxic contributions of C-4 configuration and pendant and fused arene substitutions. Dimethyl-alpha-conidendryl alcohol (5), 9-deoxypodophyllol (6), and 9-deoxysikkimol (17) were dehydrated to their respective oxolane derivatives 4, 3, and 9. Diols 5 and 6 were converted via oxabicyclo[3.2.1]octanols 10 and 14 to target oxolanes 8 and 7 where C-4 had been inverted relative to that in 3 and 4. Cytotoxicities of the five oxolanes were determined in two drug-sensitive human leukemia and two multidrug-resistant cell lines expressing P-glycoprotein or multidrug-resistance associated protein (MRP). Changing the pendant arene configuration or replacing a m-methoxy by hydrogen resulted in a 100-fold cytotoxicity loss. Replacing a methylenedioxy group in the fused arene by two methoxy substituents reduced cytotoxicity by 10-fold. Drug-resistant cell lines were equally resistant to compounds 3, 4, 8, and 9 indicating that these four compounds do not serve as substrates of the transport proteins P-glycoprotein and MRP.
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PMID:Cytotoxic responses to aromatic ring and configurational variations in alpha-conidendrin, podophyllotoxin, and sikkimotoxin derivatives. 1117 Jun 27

The MDR1 gene product P-glycoprotein (Pgp) plays a key role in multidrug resistance of cancer cells. Pgp is an ATP-driven efflux pump that extrudes a variety of dissimilar hydrophobic cytotoxic compounds. P-glycoprotein overexpression results in multidrug resistance (MDR) of tumor cell lines in vitro as well as in cancer patients. To selectively target and eliminate MDR tumor cells, we have isolated a monoclonal antibody that specifically reacts with the first extracellular loop of the human Pgp. We have cloned the variable domain genes of this antibody and assembled a functional single-chain Fv fragment capable of specifically targeting various Pgp-expressing MDR carcinoma cells lines. Targeting and specific elimination of Pgp-dependent MDR human cancer cells was achieved by constructing a single-chain immunotoxin in which the scFv fragment was fused to a truncated form of Pseudomonas exotoxin (PE38). We conclude that recombinant Fv-immunotoxins or other Fv-based molecules armed with potent cytotoxins represent an effective tool in targeted cancer therapy aimed at specific elimination of MDR tumor cell sub-populations. Recombinant antibody fragments targeting MDR proteins such as Pgp may be also used for intracellular expression and consequent phenotypic knockout of MDR.
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PMID:Targeting multidrug resistant tumor cells with a recombinant single-chain FV fragment directed to P-glycoprotein. 1174 90

Heterocyclic phenazinecarboxamides were prepared by condensation of aminoheterocycles and 2-halo-3-nitrobenzoic acids, followed by reductive ring closure and amidation. They showed similar inhibition of paired cell lines that underexpressed topo II or overexpressed P-glycoprotein, indicating a non topo II mechanism of cytotoxicity and indifference to P-glycoprotein mediated multidrug resistance. Compounds with a fused five-membered heterocyclic ring were generally less potent than the pyrido[4,3-a]phenazines. A 4-methoxypyrido[4,3-a]phenazine (IC(50)s 2.5-26 nM) gave modest (ca. 5 day) growth delays in H69/P xenografts with oral dosing.
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PMID:Structure-activity relationships for pyrido-, imidazo-, pyrazolo-, pyrazino-, and pyrrolophenazinecarboxamides as topoisomerase-targeted anticancer agents. 1180 25


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