Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This article represents the first evidence that the renal secretion of the commonly used drug, digoxin, is mediated by P-glycoprotein. In this study, it was demonstrated that digoxin is a substrate of P-glycoprotein, and the mechanism of a clinically important drug interaction, such as digoxin-quinidine, was elucidated. Human P-glycoprotein was expressed on the apical membrane of the porcine kidney epithelial cell line, LLC-PK1 by transfecting with human MDR1 cDNA. The expression and function of P-glycoprotein were confirmed by Southern and Western blotting, RNase protection assay, immunostaining and transporting activity for vinblastine. The transepithelial transport of [3H]digoxin was measured across the cell monolayers grown on microporous polycarbonate membrane filters. The transfectant cells exhibited markedly greater basal-to-apical transport and less apical-to-basal transport than the host cells, and the former was 8-fold greater than the latter. The augmented transepithelial transport resulted from the increased efflux from cells to apical side. This oriented transport was inhibited by the presence of 20 microM vinblastine, quinidine or verapamil. The rate of efflux to the apical side was 2-fold greater than that to the basal side. Quinidine inhibited the efflux to the apical side but did not affect transport into the basal side. These findings demonstrate that digoxin is transported by human P-glycoprotein, which is a previously undiscovered drug transport system in the kidney other than organic cation and anion transport systems, and suggest a molecular mechanism for the renal tubular secretion of digoxin as well as clinically important digoxin-quinidine interaction via P-glycoprotein.
 ...
PMID:Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). 135 20

Drug resistance is one of the major impediments to the treatment of advanced neuroblastoma. Two neuroblastoma cell lines established from the same patient before (KP-N-AY) and after (KP-N-AYR) chemotherapy are described. Both cell lines were established from bone-marrow metastases of a 2 1/2-year-old patient with stage IV neuroblastoma. Chromosomal analysis, catecholamine assessment and the surface membrane phenotype of these cell lines confirmed that the tumors were of neuroblastoma origin. Compared with the KP-N-AY cell line, the KP-N-AYR line had decreased N-myc amplification but increased N-myc expression. An in vitro sensitivity test using a clonogenic assay showed the KP-N-AYR cell line to be 3.0-fold resistant to adriamycin and 2.7-fold resistant to cis-platinum as compared with the KP-N-AY cell line. The expression of the multi-drug-resistance gene (MDR1) was not observed in either cell line by the ribonuclease protection assay. The KP-N-AY cell line revealed only faint MDR1 RNA by the polymerase chain reaction, whereas the KP-N-AYR cell line had no expression of the MDR1 gene. The level of glutathione-S-transferase-pi was significantly higher in the KP-N-AYR cell line than in the KP-N-AY cell line. These findings suggest that the development of clinical drug resistance may be associated with the enhanced glutathione-S-transferase-pi activity but not with MDR1 gene expression.
 ...
PMID:Different drug sensitivity in two neuroblastoma cell lines established from the same patient before and after chemotherapy. 200 54

Multidrug-resistance (MDR) genes are induced in the liver of rodents treated with a variety of foreign chemicals and hepatocarcinogens. It has been reported that 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD) might increase hepatic MDR transcripts in the Fischer rat and the C57BL/6 (B6) inbred mouse strain having the high-affinity aromatic hydrocarbon (Ah) receptor, but not in the DBA/2 (D2) strain having the low-affinity Ah receptor. These intriguing results suggest that TCDD might activate MDR gene expression by way of an Ah receptor-mediated signal transduction pathway. We have attempted to confirm these data in four inbred mouse strains: two (B6 and BALB/c) having the high-affinity Ah receptor, and two (D2 and AKR) having the low-affinity Ah receptor. The RNase protection assay was used to distinguish between the MDR1, MDR2, and MDR3 mRNAs. TCDD treatment at high (100 micrograms/kg) and low (1 mu/kg) doses, a time course from 6 to 96 hr of TCDD treatment, progeny from the B6D2F1 x D2 backcross, and transcriptional run-on experiments were performed. The Cyp1a-1 (cytochrome P1450) and Nmo-1 [NAD(P)H:menadione oxidoreductase] genes, two members of the TCDD-inducible [Ah] battery, were used as positive controls. We were unable to detect significant coinduction of MDR1, MDR2, or MDR3 mRNA with CYP1A1 mRNA or with Cyp1a-1 or Nmo-1 transcription under any conditions. Therefore, we conclude that any effects that TCDD might have on MDR expression must be substantially different from TCDD effects on genes known to be induced via the Ah receptor.
 ...
PMID:Murine mdr-1, mdr-2, and mdr-3 gene expression: no coinduction with the Cyp1a-1 and Nmo-1 genes in liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin. 206 18

The human MDR1 gene encoding P-glycoprotein, an energy-dependent drug-efflux pump, was initially isolated from a multidrug-resistant KB carcinoma cell. When a 3 kb genomic sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR1 gene was compared to the equivalent fragment from KB cells, the MDR1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR1 gene, it creates a single base mismatch between the 5' KB genomic fragment previously used for RNase protection analysis of MDR1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR1 RNA levels are as high in these tumors as in the multidrug-resistant cell line, KB-8-5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR1 mRNA levels and mRNA initiation sites in clinical samples.
 ...
PMID:Detection of multidrug resistance (MDR1) gene RNA expression in human tumors by a sensitive ribonuclease protection assay. 248 65

Expression of specific genes at the level of mRNA can be studied using techniques such as Northern blot, slot/dot blot, RNase protection assay, in situ hybridisation and RT-PCR. In this article these methods of analysis are compared; RT-PCR offers higher levels of specificity and sensitivity than traditional methods of RNA analysis and as such has become the method of choice for the study of gene expression. The RT-PCR technique is described in detail with sections dealing with RNA extraction, choice of primers (including the use of cDNA sequence data bases), PCR and RT-PCR protocols in addition to the limitations of the method. The study of one particular mRNA transcript (MDR1) using RT-PCR is discussed in detail. Recently described methods for quantitation of PCR products are discussed. Quantitative PCR would appear to offer a method of studying gene expression in a more extensive way than has been possible to date.
 ...
PMID:The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells. 750 67

Considerable evidence has accumulated indicating that overexpression of P-glycoproteins encoded by the multidrug-resistance (mdr) genes is responsible for the development of collateral resistance to a number of structurally and functionally dissimilar cytotoxic compounds in animal cells. There are three mdr genes (mdr1, mdr2, and mdr3) in the mouse genome and two (MDR1 and MDR2) in the human genome; however, only two mouse genes (mdr1 and mdr3) and one human gene (MDR1) can confer multidrug resistance upon transfection into otherwise drug-sensitive cells. Using RNase protection assay we report here that the steady-state levels of mdr1 and mdr3 messenger RNA were elevated in mouse hepatoma cells treated with dexamethasone (Dex); whereas no induction of mdr2 gene was found. Western blot analyses using anti-mdr1 and anti-mdr3 antibodies revealed that the encoded proteins appeared to be increased, but at much reduced levels. The induction was time and Dex concentration dependent. Nuclear run-on experiments demonstrated that the induction was at least in part by transcriptional control. The induction apparently required new protein synthesis since no increases in mdr1 and mdr3 transcripts was found when cultured cells were simultaneously treated with Dex and cycloheximide. Neither mdr1 nor mdr3 gene was induced in the Dex-treated nonhepatoma cell lines, LMtk- and NIH3T3. Similarly, MDR1 messenger RNA levels were elevated in the Dex-treated human hepatoma line, HepG2, but not in the nonhepatoma, HeLa. This study demonstrated that the hormonal regulation of mdr gene expression is gene and cell type specific.
 ...
PMID:Modulation of multidrug resistance gene expression by dexamethasone in cultured hepatoma cells. 810 93

Human cells can become multidrug resistant (MDR) by an increase in the activity of the MDR1 P-glycoprotein or by other, as yet unknown mechanisms, referred to as non-P-glycoprotein mediated MDR (non-Pgp MDR). S. P. C. Cole et al. [Science (Washington DC), 258: 1650-1654, 1992] recently reported that in two cell lines non-Pgp MDR was associated with the overexpression of a new putative membrane transporter gene, MRP. Using an RNase protection assay we have analyzed the expression of MRP in non-Pgp MDR sublines of the human lung cancer cell lines SW-1573 (non-small cell lung cancer) and GLC4 (small cell lung cancer). In all of ten SW-1573 derived lines examined the MRP mRNA level was equal to that in the parental line, whereas MRP was 25-fold overexpressed in a resistant subline of GLC4. We conclude that overexpression of MRP cannot account for all forms of non-Pgp MDR.
 ...
PMID:Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines. 846 91

The occurrence of multidrug resistance (MDR) is one of the main obstacles in the successful chemotherapeutic treatment of cancer. MDR cell lines are resistant to the so-called naturally occurring anti-cancer drugs, such as anthracyclines, Vinca alkaloids and epipodophyllotoxins, but are not cross-resistant to alkylating agents, antimetabolites and cisplatin. So far, three separate forms of MDR have been characterized in more detail: classical MDR, non-Pgp MDR and atypical MDR. Although all three MDR phenotypes have much in common with respect to cross-resistance patterns, the underlying mechanisms certainly differ. Atypical MDR is associated with quantitative and qualitative alterations in topoisomerase II alpha, a nuclear enzyme that actively participates in the lethal action of cytotoxic drugs. Atypical MDR cells do not overexpress P-glycoprotein, and are unaltered in their ability to accumulate drugs. In this review we will focus on classical and non-Pgp MDR. The molecular mechanism of classical and non-Pgp MDR is transcriptional activation of membrane-bound transport proteins. These transport proteins belong to the ATP-binding cassette (ABC) superfamily of transport systems. The classical MDR phenotype is characterized by a reduced ability to accumulate drugs, due to activity of an energy-dependent uni-directional, membrane-bound, drug-efflux pump with broad substrate specificity. The classical MDR drug pump is composed of a transmembrane glycoprotein (P-glyco-protein-Pgp) with a molecular weight of 170 kD, and is, in man, encoded by the so-called multidrug resistance (MDR1) gene. Typically, non-Pgp MDR has no P-gly-coprotein expression, yet has about the same cross-resistance pattern as classical MDR. This non-Pgp MDR phenotype is caused by overexpression of the multidrug resistance-associated protein (MRP) gene, which encodes a 190 kD membrane-bound glycoprotein (MRP). MRP probably works by direct extrusion of cytotoxic drugs from the cell and/or by mediating sequestration of the drugs into intracellular compartments, both leading to a reduction in effective intracellular drug concentrations. For the classical MDR phenotype, evidence is accumulating that it plays a role indeed, in clinical drug resistance, especially in some hematological malignancies (acute myeloid leukemia, multiple myeloma and non-Hodgkin's lymphoma) and solid tumors (soft tissue sarcomas and neuroblastoma). The association of MRP with clinical drug resistance has not been elaborated, yet, and studies on MRP expression in human cancer have just begun. We found that overexpression of MRP, as determined by RNase protection assay as well as by immunohistochemistry, occurs in several human cancers, among which are cancer of the lung, esophagus, breast and ovary, and leukemias. Further studies are indicated to establish whether elevated MRP expression at diagnosis is an unfavorable prognostic factor for clinical outcome of chemotherapy.
 ...
PMID:Molecular mechanisms of multidrug resistance in cancer chemotherapy. 888 Aug 78

P-glycoproteins are members of a large superfamily of transport proteins (the 'traffic ATPases') that utilize ATP to translocate a wide range of substrates across biological membranes. Using a PCR-based approach, and degenerate oligonucleotides corresponding to conserved motifs, two 300-bp cDNA fragments (pBMDR1 and pBMDR2) with a significant sequence similarity to mammalian P-glycoproteins were amplified from barley (Hordeum vulgare) root poly A+ RNA and used as probes to screen a barley root cDNA library. A single full-length clone pHVMDR2 coding for a polypeptide of 1232 residues (c. 134 kDa) was isolated. Comparison of this barley sequence with Arabidopsis ATPGP1 and human MDR1 and MDR3 P-glycoprotein sequences showed that the barley cDNA has 44%, 37% and 38% amino acid (aa) identity, respectively, with these sequences, and conserved structural features. RNase protection analysis showed that HVMDR2 mRNA is expressed at low levels in both barley roots and leaves. Southern blot analyses indicated that there is a small multigene family related to P-glycoproteins in barley. Possible functions for these barley P-glycoproteins are discussed.
 ...
PMID:Cloning and characterisation of a novel P-glycoprotein homologue from barley. 935 56

Overexpression of P-glycoprotein (Pgp) or MDR1 mRNA has been shown to be a negative prognostic factor for clinical outcome in acute myeloid leukemia (AML). However, resistance to chemotherapy also occurs in the absence of Pgp overexpression. Therefore, besides Pgp expression, we have assessed the expression of MRP, a novel drug transporter gene, along with the functional multidrug-resistant (MDR) phenotype of leukemic cells. These MDR parameters are correlated with clinical outcome in individual patients. We found functional changes in fresh leukemic cells from de novo or relapsed patients similar to those reported for tumor cell lines with the MDR phenotype. These changes were reduced drug accumulation as assessed with radiolabeled doxorubicin (factor 1.6), daunomycin (factor 1.13), and vincristine (factor 1.6) in patients who were refractory to the combination treatment of 1-beta-D-arabinofuranosylcytosine (ara-C) and daunomycin or mitoxantrone as opposed to patients who had complete responses. Also, the intracellular distribution of doxorubicin fluorescence (nuclear/cytoplasmic ratio), as assessed with laser scan microscopy, was reduced 1.4-fold in blasts from refractory patients. Based on historically known clinical response to single-agent daunomycin or ara-C in the group of responding de novo AML patients, we have set a threshold level such that a defined part of the samples that had the highest drug accumulation or nuclear to cytoplasmic ratios were above this threshold value. This allowed discrimination between patients responding to daunomycin from those who were refractory to this drug. By using this threshold level, in the refractory group clinical resistance corresponded with high sensitivity with a resistant phenotype. A similar threshold was set for the data of the in vitro ara-C sensitivity test. By combining both assays for all individual patients, clinical refractoriness as well as sensitivity could be predicted with high accuracy. There appeared to be no stringent relationship between the functional MDR phenotype with expression of either Pgp (fluorescence-activated cell sorting analysis) or MRP mRNA (RNase protection). However, by combining both parameters the functional MDR phenotype correlated with the overexpression of either one or both of the parameters in 94% of the samples studied. It is concluded that this combined overexpression in conjunction with functional changes for MDR drugs and ara-C reveal a correlation of MDR phenotype with clinical resistance to combination chemotherapy in AML patients and hereby may adequately predict clinical MDR in individual AML patients.
 ...
PMID:Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-beta-D-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia. 981 90


1 2 Next >>