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Drug Efflux Pumps (drug + efflux_pump)
Selected AbstractsFunctional role of the linker region in purified human P-glycoproteinFEBS JOURNAL, Issue 13 2009Tomomi Sato Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a ,linker region' of , 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the kcat values both with substrate (ksub) and without substrate (kbasal), but decreased the ksub/kbasal values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [ksub/Km(D)] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp. [source] Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membraneFEBS JOURNAL, Issue 23 2007Ronit Regev The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement. The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween-20, Triton X-100 and Cremophor EL modulated MDR by inhibition of Pgp-mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp-mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface-active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip-flop, occurs. [source] Differences in multidrug resistance phenotype and matrix metalloproteinases activity between endothelial cells from normal brain and gliomaJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Anthony Régina Abstract Endothelial cells (ECs) are new targets for tumor therapy. In this work, we purified endothelial cells from intracerebral and subcutaneous experimental gliomas as well as from normal brain in order to define some of the phenotypical differences between angiogenic and quiescent brain vasculature. We show that the multidrug resistance genes encoding drug efflux pumps at the brain endothelium are expressed differently in normal and tumoral vasculature. We also show that ECs from gliomas present increased activity of gelatinase B (MMP9), key enzyme in the angiogenic process. Importantly, we observe a different phenotype between ECs in the intracerebral and subcutaneous models. Our results provide molecular evidence of phenotypic distinction between tumoral and normal brain vasculature and indicate that the EC phenotype depends on interactions both with tumor cells and also with the microenvironment. [source] Genome-wide analysis of the general stress response in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 4 2001Chester W. Price Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the ,B transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce ,B activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to 33P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which ,B expression was controlled by an inducible promoter. On the basis of their significant ,B -dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the ,B regulon. Of these genes, 30 were known previously or inferred to be ,B dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible ,B recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the ,B -dependent messages for seven representative genes. For all seven, the 5, end of the message lay near typical ,B recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover. [source] Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cellsNMR IN BIOMEDICINE, Issue 6 2010Tariq Shah Abstract A direct correlation exists between increased choline kinase (Chk) expression, and the resulting increase of phosphocholine levels, and histological tumor grade. To better understand the function of Chk and choline phospholipid metabolism in breast cancer we have stably overexpressed one of the two isoforms of Chk-, known to be upregulated in malignant cells, in non-invasive MCF-7 human breast cancer cells. Dynamic tracking of cell invasion and cell metabolism were studied with a magnetic resonance (MR) compatible cell perfusion assay. The MR based invasion assay demonstrated that MCF-7 cells overexpressing Chk-, (MCF-7-Chk) exhibited an increase of invasion relative to control MCF-7 cells (0.84 vs 0.3). Proton MR spectroscopy studies showed significantly higher phosphocholine and elevated triglyceride signals in Chk overexpressing clones compared to control cells. A test of drug resistance in MCF-7-Chk cells revealed that these cells had an increased resistance to 5-fluorouracil and higher expression of thymidylate synthase compared to control MCF-7 cells. To further characterize increased drug resistance in these cells, we performed rhodamine-123 efflux studies to evaluate drug efflux pumps. MCF-7-Chk cells effluxed twice as much rhodamine-123 compared to MCF-7 cells. Chk-, overexpression resulted in MCF-7 human breast cancer cells acquiring an increasingly aggressive phenotype, supporting the role of Chk-, in mediating invasion and drug resistance, and the use of phosphocholine as a biomarker of aggressive breast cancers. Copyright © 2010 John Wiley & Sons, Ltd. [source] | ||||||||||