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)

Cross-resistance to anticancer drugs, termed multidrug resistance (MDR), is functionally associated with the expression of a plasma membrane, energy-dependent, drug efflux pump termed P-glycoprotein (PGP), the product of the mdr1 gene. We have shown previously that MCF-7 breast carcinoma cells transfected with the human mdr1 gene (BC-19 cells) exhibit greater MDR when stably transfected with protein kinase C alpha (PKC alpha). We now demonstrate that transfection of BC-19 cells with the gamma isoform of PKC (BC-19/PKC gamma cells), which is not normally present in BC-19 cells, does not confer increased resistance to doxorubicin, despite a 19-fold increase in PKC activity. All of the increased PKC activity is accounted for by PKC gamma and it is rapidly down-regulated by phorbol dibutyrate, within 15 min of treatment. Endogenous PKC alpha and PKC epsilon activities are not affected by phorbol dibutyrate. The cytotoxicity of doxorubicin was similar in BC-19/neo or BC-19/PKC gamma cells after either 2-hr or continuous drug exposure, and co-treatment with phorbol dibutyrate increased resistance to doxorubicin 4-fold in both cell lines. Phosphorylation of PGP was similar in both cell lines and drug accumulation was not affected by overexpression of PKC gamma. These results demonstrate that transfection of PGP-expressing cells with an atypical isoform of PKC does not confer increased MDR, and they suggest that the regulation of PGP is phenotype specific with respect to the isoform of PKC.
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PMID:Role of protein kinase C in the modulation of multidrug resistance: expression of the atypical gamma isoform of protein kinase C does not confer increased resistance to doxorubicin. 136 42

Protein kinase C (PKC) is a Ca++- and phospholipid-dependent protein kinase that plays an important role in signal transduction pathways that regulate cell growth. Tumor cells selected for a multidrug resistant (MDR) phenotype often express elevated levels of PKC activity. To directly test whether PCK overexpression can produce an MDR phenotype, we studied rat embryo fibroblasts that were infected with the full-length cDNA clone RP58 encoding the beta I form of rat brain PKC. The PKC-beta I gene recipient R6-PKC3 cells are stable, overproduce PKC, and express an elevated level of PKC activity. R6-PKC3 cells exhibited significant resistance to adriamycin, actinomycin D, vinblastine, and vincristine but not to 5-fluorouracil. Intracellular accumulation of adriamycin, vinblastine, and vincristine was decreased in the R6-PKC3 cells, but this was not associated with an altered level of P-glycoprotein expression. Moreover, the reduction in drug accumulation appeared to be a consequence of a decreased rate of drug uptake. The data indicate that overexpression of PKC in rat fibroblasts produces an MDR phenotype without altering P-glycoprotein expression.
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PMID:Stable expression of a cDNA encoding rat brain protein kinase C-beta I confers a multidrug-resistant phenotype on rat fibroblasts. 162 23

Cross-resistance to anticancer drugs, termed multidrug resistance (mdr), has been functionally associated with the expression of a plasma membrane energy-dependent efflux pump, termed P-glycoprotein, the product of the mdr1 gene. When MCF-7 breast carcinoma cells were transfected with the human mdr1 gene (BC-19 cells), they expressed levels of P-glycoprotein equivalent to those of cells selected for resistance to doxorubicin (MCF-7/ADR) but exhibited 10- to 50-fold less resistance to doxorubicin and vinblastine. We have now demonstrated that when BC-19 cells were stably transfected with protein kinase C alpha (PKC alpha), resistance to doxorubicin and vinblastine was increased; wild-type MCF-7 cells transfected with PKC alpha did not exhibit any change in drug resistance. Increased resistance in PKC alpha-transfected BC-19 cells was associated with enhanced PKC activity and phosphorylation of P-glycoprotein and decreased drug accumulation. The PKC activator, phorbol dibutyrate, further increased resistance to doxorubicin and stimulated P-glycoprotein phosphorylation. These results demonstrate that transfection of P-glycoprotein-expressing cells with PKC resulted in increased mdr and that PKC may have served as an important modulator of this process.
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PMID:Transfection with protein kinase C alpha confers increased multidrug resistance to MCF-7 cells expressing P-glycoprotein. 167 75

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.
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PMID:Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells. 197 44

A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established. In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis. The mutant was 100-fold more resistant to OA in terms of effects on these parameters. Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant. Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type. Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells. The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels. The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid. Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells. These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.
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PMID:Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein. 751 66

Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect. P-glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300. Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect. Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2-fold greater than classic MDR modulators and Hill coefficients >> 2. In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells. In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P-glycoprotein and for the quantitative characterization of transporter modulation and regulation. The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation.
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PMID:Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation. 754 62

We compared the influence of exogenous N-ras oncogene and treatment with PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.
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PMID:Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function. 762 41

The newly identified drug transporter MRP is functionally linked to a multiple drug resistance independent from P-glycoprotein. Resistance modifiers for this type of MDR are rare at present. We analyzed the modulating effect of the highly selective bisindolylmaleimide PKC inhibitor GF 109203X on the MRP overexpressing human MDR sublines HL60/AR and GLC4/ADR. Applying a 72 hour MTT-assay we demonstrate a complete reversal of the vincristine resistance of HL60/AR cells. Adriamycin resistance of HL60/AR, or vincristine resistance of GLC4/ADR were partially reversed. Furthermore, rhodamine 123 efflux from HL60/AR was strongly modulated by GF 109203X. Since the PKC inhibitor did not significantly influence MRP gene expression at the mRNA level which was examined by cDNA-PCR, our results suggest either a direct interaction of the compound with MRP or/and an indirect influence on MRP activity via altering the phosphorylation status of the transporter.
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PMID:The specific bisindolylmaleimide PKC-inhibitor GF 109203X efficiently modulates MRP-associated multiple drug resistance. 781 10

Acquired resistance to chemotherapeutic drugs by tumor cells is an important obstacle to effective therapy of human malignancy. These resistance cell lines originated from human or rodent have been characterized by increased expression of MDR (Multidrug-resistance) gene and P-glycoprotein which plays as efflux pump of drugs from cells. These multidrug-resistance sublines also have been reported increased activities of protein kinases and glutathione S-transferase-pi. Although there have been extensive biophysical and biochemical characterization of the differences between parental lines and MDR tumor cell sublines, morphologic observations have been limited. In this study, filamentous cytoskeletons which involve many biological phenomena such as maintenance of cell morphology, mitosis, cellular movement, transport, and adhesion, were observed by confocal laser microscopy. To compare the expression of each cytoskeletons, fluorescent intensities of cells stained for each cytoskeletons were measured by confocal laser microscopic system. Utilizing this methodology, higher microtubular expression was observed in HL-60/ADR and K562/ADR than in their parental lines, but no significant differences of actin and vimentin were observed. Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal function. But little is known about the role of protein phosphorylation in cytoskeletal function. Since increased activities of PKC and PTK were detected in HL-60/ADR, the effect of PKC inhibitor, staurosporine (STR), or PTK inhibitor, genistein (GNS), on cell growth was detected. STR and GNS reduced the resistance to Adriamycin in HL-60/ADR. Furthermore, STR and GNS disrupted the filamentous structure of microtubules in HL-60/ADR, and suppressed the expression of microtubules to 37%, and 49%, respectively. In contrast, PKC activator, phorbol ester (TPA), caused stronger microtubular assembling in HL-60/ADR, and increased the expression of microtubules to 134%. Resulting from this study, it is likely that acquired MDR of HL-60 and K562 was associated with increased expression of microtubules, and microtubular assembling or disassembling was considered to be regulated in part by PKC and PTK.
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PMID:[Features of filamentous cytoskeletons in acquired multidrug-resistance of HL-60 human leukemia cell line]. 790 88

The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system. PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha. Photoaffinity labeling of membrane vesicles with [3H]azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha. In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220. Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity. These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.
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PMID:Modulation of P-glycoprotein by protein kinase C alpha in a baculovirus expression system. 791 39


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