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)

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.
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PMID:Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein. 136 54

Among the solid tumors of childhood and adolescence, osteosarcoma (OS) represents the most prominent example of efficient aggressive chemotherapy with secondary surgical therapy. A specific subclassification of the tumor is indispensable and must include recent results of cell biology. The co-distribution of different collagen types I-VI reflects the diverse differentiation of osteosarcoma cells, supporting the concept of a pluripotent mesenchymal cell to be the stem cell of the tumor. In contrast, osteonectin (SPARC) may not be considered as a reliable marker for osteosarcoma. The experience of special proteins being secreted by osteosarcoma cells is rather limited. Detailed molecular biological studies are still lacking. A loss of alleles on chromosome 17, particularly in the defined region 17p 13, can be observed in more than 75% of all OS, suggesting the contribution of a tumor suppressor gene, p53, located in that region. It is a 53 kd nucleophosphoprotein binding the major transforming protein, the large T antigen of Simian Virus 40. Immunohistological results showed positive staining with the antibody Pab 240 in 13 of 18 cases. In one osteoblastic OS, a novel splice mutation resulting in a fusing of exon 5 directly to exon 7 was detected. RB1 gene is also of major importance for the tumorigenesis of OS. The multidrug resistance (mdr) is associated with a membrane-bound channel-forming transport protein, the P-glycoprotein. It is a conserved plasma membrane component of about 170 kd. Both the human isoforms mdr 1 and mdr 3 are localised in the long arm of chromosome 7.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:New aspects of cell biology in osteosarcoma. 747 79

Bodipy-verapamil has been tested as a fluorescent substrate for P-glycoprotein-mediated transepithelial secretion in MDCK-C5A epithelia. Net transepithelial secretion (Jnet) of [3H]vinblastine from basal-to-apical surfaces of monolayer epithelia is inhibited by taxotere, verapamil and Bodipy-verapamil primarily by a reduction in basal to apical vinblastine (Jb-a) transport. Bodipy-verapamil is itself subject to transepithelial net secretion by MDCK-C5A epithelia; at 5 microM a Jnet of -310 +/- 32 nmol cm-2 h-1 (n = 3) was observed. When MDCK-C5A cells are grown to form enclosed cysts in hydrated collagen gel. Bodipy-verapamil is accumulated within the cyst lumen showing that epithelial P-glycoprotein function may be used to target substrates to renal cysts.
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PMID:Targeted delivery of a substrate for P-glycoprotein to renal cysts in vitro. 761 39

Previous studies reported that, in the absence of drug exposure, multidrug resistance, including resistance to Adriamycin (ADR), could develop in primary rat hepatocyte cultures (B. Carr, Proc. Am. Assoc. Cancer Res., 29:1158, 1988). However, the hepatocytes in that report were cultured on plastic without the benefit of an extracellular matrix (ECM). Because the ECM regulates hepatic gene expression, we have critically evaluated in primary cultures of rat hepatocytes how the ECM affects hepatic ADR resistance, the level of the drug efflux transporter associated with MDR, P-glycoprotein (pgp), and transport of a prototypical pgp substrate, vincristine. Hepatocytes cultured on type I collagen (Vitrogen) had greater resistance to ADR toxicity accompanied by parallel increases in the level of pgp mRNA, decreased drug accumulation, and enhanced drug efflux when compared with the hepatocytes maintained on the basement membrane matrix Matrigel. The development of ADR resistance coincided with the time course of increased pgp mRNA but was not coincident with the time course of expression of either the placental isozyme of glutathione S-transferase or P-450 reductase, proteins associated with MDR in some resistance models. Southern blot analysis revealed neither gross changes in pgp gene structure or gene copy number to account for the increase in pgp RNA levels for hepatocytes cultured on Vitrogen. ECM also regulated xenobiotic-inducible expression of hepatic pgp, since chemotherapeutic agents, including vincristine and colchicine, induced pgp mRNA exclusively in hepatocytes cultured on Vitrogen. The critical matrix proteins in Matrigel responsible for regulation of pgp were determined by the selective addition of its components to the culture environment. The presentation of the individual matrix elements as a rigid substratum to the hepatocyte did not decrease pgp mRNA. In contrast, the presentation to the same hepatocytes of either laminin or type IV collagen in a nonrigid state (solubly in the medium) selectively decreased hepatocellular pgp mRNA. We conclude that primary rat hepatocytes develop ADR resistance with time in culture due to increased expression of pgp and that ECM proteins represent endogenous physiological modulators of both basal and chemotherapeutically inducible expression of hepatic P-glycoprotein.
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PMID:Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes. 842 4

A simplified protocol for isolating brain microvessel endothelial cells (BMEC) from human cortex and culturing them on a thick collagen plug is described. This method results in the establishment of monolayers of BMEC that retain numerous properties indicative of the blood-brain barrier (BBB) phenotype, such as elevated transendothelial electrical resistance, attenuated paracellular flux of sucrose, peripheral actin filament distribution and asymmetric localization of the efflux peptide, P-glycoprotein, to the apical (luminal) BMEC surface. The novel 3-dimensional nature of this model system renders it ideally suitable for assaying such varied aspects of BBB physiology as solute transport, pathogen penetrance, leukocyte infiltration and tumor metastasis into the brain. Moreover, the fact that the system is derived from human brain allows for the study of pathogenetic mechanisms that may only be operative in humans.
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PMID:Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro. 854 2

Solitary stroma-invading tumor cells expressing the ATP-binding cassette transporter P-glycoprotein have been reported to be associated with a significantly higher incidence of vessel invasion and lymph node metastases. In contrast to P-gp-mediated multidrug resistance (MDR) which has become well characterized over the last decade, little is known about further morphological and functional alterations in drug-resistant tumor cells. Binding of malignant cells to components of the extracellular matrix mediated by beta 1 integrins has been suggested to play a substantial role in the metastatic cascade. We studied alterations of beta 1 integrin expression and in vitro adhesiveness to extracellular matrix proteins of the human renal carcinoma line Caki-1 in comparison to the vinblastine resistant sublines Caki-1/V1 and Caki-1/V10 (cultured in the presence of 1 ng/ml and 10 ng/ml vinblastine, respectively). Both VLA-1 and VLA-2 receptors were acquired by the Caki-1/V10 subline, whereas untreated and Caki-1/VI cells lacked surface expression of these antigens. VLA-6 was found to be decreased in the vinblastine-resistant sublines. Attachment of drug-resistant Caki-1/V1 and Caki-1/V10 cells to collagen type I was significantly increased when compared to parental cells (p < or = 0.005). Significant differences in the attachment to type IV collagen were observed between Caki-1/V10 and untreated cells (p < or = 0.045). Both Caki-1/V1 and Caki-1/ V10 cells exhibited increased adhesion to fibronectin when compared to cells of the untreated line (p < or = 0.04). Whether an aberrant expression of beta 1 integrin receptors in resistant cells in combination with altered tumor cell adhesiveness is caused by MDR induction or whether it is an epiphenomenon of cytotoxic stress is unknown. Future studies will be needed to characterize the clinical relevance of MDR-associated changes in tumor cells.
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PMID:Exposure to vinblastine modulates beta 1 integrin expression and in vitro binding to extracellular matrix molecules in a human renal carcinoma cell line. 903 Feb 41

Luminal membranes of the vascular endothelium were isolated from brain, heart and lungs by modification of their density. The presence of P-glycoprotein (P-gp) was detected by Western blotting in luminal membranes from the endothelium of the three tissues. Strong enrichment in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400-500-fold), indicates that P-gp is mainly located on the luminal side of the brain endothelium. Western blotting was also performed with antibodies directed against GLUT1, glial fibrillary acidic protein, adaptin, IP3R-3, integrins alphav and collagen IV as controls to determine whether the preparations were contaminated by other membranes. Strong enrichment of GLUT1 in brain capillary luminal membranes (9.9-fold) showed that the preparation consisted mainly of endothelial cell plasma membranes. Poor enrichment of glial fibrillary acidic protein (1.4-fold) and adaptin (2.4-fold) and a decreased level of IP3R-3, integrins alphav and collagen IV excludes the possibility of major contamination by astrocytes or internal and anti-luminal membranes. High levels of P-gp in the luminal membranes of brain capillary endothelial cells suggests that it may play an important role in limiting the access of anti-cancer drugs to the brain.
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PMID:P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. 929 Nov 29

1. The present work has examined the effects of short- (30 min) and long-term (14 h) exposure to cyclosporine A (CsA) on the uptake of L-DOPA, its decarboxylation to dopamine and the cellular extrusion of taken up L-DOPA and of newly-formed amine in monolayers of LLC-PK1 cells. 2. In the presence of benserazide (50 microM), L-DOPA was rapidly accumulated in LLC-PK1 cells (cultured in collagen-treated plastic) attaining equilibrium at 30 min of incubation. Non-linear analysis of the saturation curves revealed a Km of 113+/-16 microM and a Vmax of 5581+/-297 pmol mg(-1) protein 6 min(-1). 3. In the absence of benserazide, LLC-PK1 cells incubated with increasing concentrations of L-DOPA (10 to 500 microM) for 6 min accumulate newly-formed dopamine by a saturable process with apparent Km and Vmax values of 31+/-6 microM and 1793+/-91 pmol mg(-1) protein 6 min(-1), respectively. The fractional outflow of newly-formed dopamine was found to be 20%. Up to 200 microM of intracellular newly-formed dopamine, the outward transfer of the amine was found to be a non-saturable process. 4. Short-term exposure to CsA (0.3, 1.0 and 3.0 microg ml(-1)) was found not to change the intracellular concentrations of newly-formed dopamine, but increased the levels of dopamine in the incubation medium (143% to 224% increase) and the total amount of dopamine formed (31% to 59% increase). Long-term exposure to CsA (0.03 to 3.0 microg ml(-1)) reduced the total amount of dopamine (15% to 39% reduction) and the intracellular levels of the amine (11% to 56% reduction), without changing dopamine levels in the incubation medium. Both short- and long-term exposure to CsA resulted in a concentration-dependent increase in the fractional outflow of newly-formed dopamine. 5. Short-term exposure to CsA (3.0 microg ml(-1)) reduced the apical extrusion of intracellular L-DOPA by 15% (P<0.05), whereas long-term exposure to CsA reverted this effect and decreased its intracellular availability (15% reduction; P<0.05). 6. Detection of P-glycoprotein activity was carried out by measuring verapamil- or UIC2-sensitive rhodamine 123 accumulation. Both UIC2 (3 microg ml(-1)) and verapamil (25 microM) significantly increased the accumulation of rhodamine 123 in LLC-PK1 cells. A 30 min exposure to CsA was found not to affect the accumulation of rhodamine 123 in the presence of verapamil (25 microM), whereas a 14 h exposure to CsA was found to reduce the accumulation of rhodamine 123. 7. In conclusion, the increase and the reduction in the formation of dopamine after short- and long-term exposure to CsA, respectively, correlate with the effects of the immunosuppressant on the apical cell extrusion of taken up L-DOPA, suggesting the involvement of P-glycoprotein. The effects of CsA on the fractional outflow of newly-formed dopamine appear to be mediated by a different mechanism.
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PMID:Evidence for the involvement of P-glycoprotein on the extrusion of taken up L-DOPA in cyclosporine A treated LLC-PK1 cells. 948 49

A new semi-synthetic podophyllotoxin derivative, 4'-O-demethyl-4beta-2"-nitro-4"-fluoroanilino)-4-desoxypo dophyllotoxin (compound 1), an analog of GL-331 (compound 2), is a potent and broad-spectrum inhibitor of cultured human cancer and drug-resistant cell growth. In general, 4'-demethylepipodophyllotoxin analogs, including 2, exert anti-tumor activity by targeting the nuclear enzyme DNA topoisomerase II, but 1 is not an enzyme inhibitor. Unlike the cytotoxic activity of compound 2, cell killing by 1 is dose-limiting and a significant fraction of cells (30-40%) survive treatment. As an approach to investigate mechanism of action, 1-resistant A549 (human lung cancer) sub-lines were selected and characterized. Results of the work show that 1-resistant cells: (i) are moderately cross-resistant (2- to 3-fold) to various cytotoxic drugs via a P-glycoprotein-independent mechanism, (ii) have an altered growth habit, (iii) are deficient in normal attachment on plastic and collagen substrata, and (iv) have an altered plasma membrane protein composition including several proteins in the 140->200 kDa molecular mass range and a doublet of phosphoserine-containing proteins of about 135 kDa. Since 1 treatment of cells affects neither cellular attachment or membrane-protein phosphorylation, the changes observed in 1-resistant cells are interpreted as a survival response to drug action.
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PMID:Characterization of human lung cancer cells resistant to 4'-O-demethyl-4beta-(2"-nitro-4"-fluoroanilino)-4-desoxypodophyllotoxin , a unique compound in the epipodophyllotoxin antitumor class. 1075 59

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650Ml) and a paclitaxel-resistant variant (RPMI-2650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650, were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650Ml cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha(2), alpha(5), alpha(6), beta(1) and beta(4) integrin subunits, decreased expression of alpha(4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-2650Ml cells. Decreased expression of the alpha(2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype, but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells.
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PMID:Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness. 1133 31


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