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)

P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4). Phenotypic mutants corresponding to these loci have not yet been described. Two of the genes, pgp-1 and pgp-3, were analyzed in detail. They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far. Intra-species divergence of P-glycoprotein genes is more pronounced in C. elegans than in mammals. Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3. pgp-1 consists of 14 exons, pgp-3 of 13. The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes. pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes. pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends. Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types. Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA. Thus, in C. elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.
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PMID:The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences. 136 May 40

We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells. With low frequency we obtained stable, MDR3-expressing clones. Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated. Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance. The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D. We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy. Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones. Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs. We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting. Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein. In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.
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PMID:Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies. 167 38

Overexpression of P-glycoprotein genes is a well-established cause of one form of multidrug resistance. P-glycoproteins are plasma membrane proteins containing two ATP-binding sites and twelve putative transmembrane segments. P-glycoproteins are thought to act as ATP-dependent drug efflux pumps, actively extruding a range of structurally different, hydrophobic drugs from the cell. This simple model can account for the properties of multidrug resistant cells, even those that seem to require more complex explanations. The structure and function of P-glycoprotein genes has been studied in mammals and in several lower eukaryotes. These studies are helping to delineate the range of drugs that can be transported by P-glycoproteins; the genetic mechanisms that can lead to elevated cellular P-glycoproteins levels; and the evolution of the versatile and prolific P-glycoprotein gene family. The physiological function of the human P-glycoproteins encoded by the MDR1 and MDR3 (or MDR2) genes remains a matter of speculation.
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PMID:Multidrug resistance mediated by P-glycoproteins. 168 Apr 93

We determined the expression levels of the mdr1 and mdr3 multidrug-resistance genes (also known as PGY1 and PGY3, respectively) in peripheral blood cells from 69 adult patients with acute and chronic leukemias, using an RNase protection assay. Expression of mdr1 was found in samples from patients with acute nonlymphocytic leukemia (13 of 17), chronic myelocytic leukemia (CML, chronic phase, 10 of 10; blast crisis, three of four), acute lymphocytic leukemia (ALL, eight of 11), B-cell chronic lymphocytic leukemia (B-CLL, 17 of 17), hairy cell leukemia (HCL, one of two), and T-cell prolymphocytic leukemia (one of one), but not in B-cell prolymphocytic leukemia (B-PLL, 0 of seven). Expression of mdr3 was only detected in samples from B-cell lymphocytic leukemias: CML, lymphoid blast crisis (one of one), B-cell ALL (two of two), B-CLL (17 of 17), B-PLL (seven of seven), and HCL (two of two). In vitro drug uptake studies by on-line flow cytometry showed that in leukemia cells expressing either mdr1 or mdr3, the steady-state accumulation of daunorubicin could be significantly increased by addition of cyclosporine and, to a lesser extent, by verapamil. Because cyclosporine and verapamil are known as inhibitors of the mdr1-encoded P-glycoprotein drug-efflux pump, and because the mdr1 and mdr3 genes are highly homologous, our data suggest that the mdr3 gene encodes a functional drug pump in B-cell lymphocytic leukemias. The results of this study may have implications for clinical therapy for acute or chronic leukemias expressing the mdr1 or mdr3 gene, in particular, treatment with combinations of cytotoxic drugs plus agents that reverse multidrug resistance. Since mdr1 and mdr3 are frequently expressed in untreated as well as treated leukemia, such combination therapy should be considered for untreated patients as well as treated patients.
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PMID:Expression of mdr1 and mdr3 multidrug-resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine. 197 61

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.
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PMID:Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. 197

Two genes, MDR1 and MDR3, constitute the human P-glycoprotein gene family. To examine the evolutionary relationship between the three known classes of mammalian P-glycoprotein genes, we have cloned the MDR3 gene and compared its structure with that of the human MDR1 and the mouse mdr1 (mdr1b) genes analyzed by other groups. The MDR3 gene contains 28 exons and 27 of these contain coding sequences for the two homologous halves of the protein that correlate with functional domains. This structure is virtually identical to that of the human MDR1 gene and the mouse mdr1 (mdr1b) gene, indicating that the exon/intron structure was fixed before the duplication events that generated different classes of P-glycoproteins, but after the P-glycoproteins diverged from related genes, like the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has an entirely different exon/intron structure. The four alternatively spliced transcripts of the MDR3 gene arise from alternative splicing of exons 23 and 26. Our analysis of DNA clones covering about 120 kilobases (kb) of the human MDR locus, including the entire MDR3 gene (74 kb) and the intergenic region between both genes (34 kb), combined with pulsed-field gel electrophoresis data shows that the human MDR locus covers about 230 kb. In contrast to the mouse mdr genes, both human genes are transcribed in the same direction (MDR3 located downstream of MDR1). The CpG-rich sequences marking the 5' ends of both genes are hypomethylated to different extents in different cell lines. Hypomethylation roughly correlates with transcriptional activity.
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PMID:Structure of the human MDR3 gene and physical mapping of the human MDR locus. 200 63

Human MDR1 encodes an ATP-binding cassette transporter, P-glycoprotein, that mediates multiple drug resistance (MDR) to antitumor agents. It has been previously shown that photoaffinity drug-labeling sites reside within, or near, the last transmembrane loop of each cassette within P-glycoprotein (transmembrane domains (TM) 5-6 and 11-12). A genetic approach was used to determine if the drug-labeling site in the second cassette contains functionally important amino acids. Since human MDR3 is 77% identical to MDR1 but does not mediate MDR, the region from TM10 to the C terminus of MDR1 was replaced with the corresponding sequences from MDR3. The resultant chimeric protein was expressed but not functional. By using progressively smaller replacements, we show that replacements limited to TM12 markedly impaired resistance to actinomycin D, vincristine, and doxorubicin, but not to colchicine. The phenotype was associated with an impaired ability to photoaffinity label the chimeric P-glycoprotein with [125I]iodoaryl azidoprazosin. In contrast, replacement of the loop between TM11 and 12 appears to create a more efficient drug pump for actinomycin D, colchicine, and doxorubicin, but not vincristine. These results suggest that, similar to voltage-gated ion channels, amino acids within and immediately N-terminal to the last transmembrane domain of P-glycoprotein compose part of the drug-binding pocket and are in close proximity to photoaffinity drug-labeling domains.
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PMID:Functional evidence that transmembrane 12 and the loop between transmembrane 11 and 12 form part of the drug-binding domain in P-glycoprotein encoded by MDR1. 789 Jun 59

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.
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PMID:Characterization of the promoter region of the human MDR3 P-glycoprotein gene. 789 60

The multidrug resistance (MDR) phenotype has been demonstrated to be related to the overexpression of P-glycoprotein, a 170 kDa transmembrane efflux pump. We studied P-glycoprotein expression in 40 patients with chronic B-cell leukemias by FACS analysis using MoAb c219, which recognizes both the MDR1 and the MDR3 gene product. We found significantly elevated P-glycoprotein expression in these patients as compared with normal controls. Patients who had received previous chemotherapy regimens containing MDR-related drugs showed significantly higher P-glycoprotein expression with MoAb c219 than those patients who had been untreated. Northern blot analysis of MDR1 and MDR3 gene expression in 32 of the patients gave a similar result: in the analysis of total RNA four of six patients (66%) pretreated with either vinca alkaloids or anthracyclines were MDR1 positive as opposed to 6 of 26 (23%) who had no treatment or treatment without these agents. In contrast, MDR3 expression was found more frequently (63%), but was randomly distributed in the differently treated groups. Increasing the sensitivity level by analysis of enriched mRNA (polyA+RNA) led to the detection of MDR1 and MDR3 expression all B-CLL patients. We conclude that a basic elevated P-glycoprotein expression is intrinsic in CLL cells, which is possibly upregulated under chemotherapy. This might be responsible for initial and acquired chemotherapy resistance in CLL patients. Follow-up of the B-CLL patients over 46 months showed that the median survival time for MDR1+ patients was 19 months as opposed to 46 months for MDR1- patients (p < 0.01). There was no statistical difference in survival between MDR3+ and MDR3- patients. In the MDR1+ group, eight of nine patients had developed resistance to the therapy with MDR-related drugs. The expression of MDR1 might, therefore, predict treatment failure with MDR-related drugs and be a negative prognostic factor.
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PMID:Multidrug resistance phenotype in patients with chronic lymphocytic leukemia as detected by immunofluorescence (FACS) and northern blot analysis. 791 54

In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism. Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile. This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located. These and other results show the importance of the genetic approach for studying drug metabolism. MDR is not only caused by increased activity of Pgps. When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity. Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur. This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms. The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.
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PMID:Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals. 791 35


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