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)

Transfer of the multidrug resistance-1 (MDR1) gene to hemopoietic cells for myeloprotection against cytostatic agents is a new and rapidly developing field in "cancer gene therapy." Before clinical application, safety and efficacy criteria need to be met. The retroviral producer cell lines and the retroviral supernatant need to be tested for replication-competent retrovirus and contamination with adventitious agents. The cell source needs to contain sufficient hemopoietic cells with repopulating ability. We used CD34(+)-selected mobilized peripheral blood progenitor cells (PBPC) for MDR1 transductions in order to obtain a favorable vector to target cell ratio. An analysis of 249 patients who had undergone PBPC harvesting revealed that primarily solid tumor and non-Hodgkin's lymphoma patients are eligible for CD34+ selection. They can be expected to retain sufficient CD34+ cells for rapid and sustained engraftment after myeloablative therapy if the CD34+ cell loss (approximately 50%) during the procedure is taken into account. Clinical MDR1 gene therapy protocols focus on these two patient groups. Next we characterized MDR1 gene transfer into lineage-committed and primitive hemopoietic cells. Provirus-specific polymerase chain reactions showed a high efficiency gene transfer into colony-forming-units granulocyte-macrophage and long-term culture cells. The level of the conferred P-glycoprotein expression was estimated by fluorescence-activated cell sorting analysis to be up to 3 log above mock-transduced controls. The cobblestone area forming cell assay, which is a stroma-dependent long-term culture assay measuring frequencies of stem cell subsets in a limiting-dilution set-up, allowed demonstration of sustained expression of the MDR1 gene in the progeny of primitive hemopoietic cells. This is a favorable basis for a clinical MDR1 gene therapy trial.
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PMID:Retroviral transfer of the multidrug resistance-1 gene into lineage-committed and primitive hemopoietic cells. 874 95

Transfer of the multidrug resistance-1 (MDR1) gene into hematopoietic progenitor cells may reduce myelotoxicity of MDR1-related cytotoxic agents and therefore allow dose intensification. Mobilized peripheral blood progenitor cells (PBPC) can be obtained in ample quantity and are a suitable target cell population. CD34-selected PBPC samples (n = 6) were transduced with cell-free supernatant (SNT) of a cell line producing recombinant retrovirus containing the human MDR1 gene. Limiting-dilution long-term cultures were employed that allow continuous monitoring of stroma-adherent cobblestone areas (CA) and comparison of their frequency in a 5-log range over time. MDR1 provirus integration in CA-containing wells followed single-hit kinetics. According to Poisson statistics, proviral DNA was contained in 22% of unselected cobblestone area-forming cells (CAFC) at week 6, which represent primitive hematopoietic precursors. In comparison, 1.0 +/- 0.44% (mean +/- SEM) of week-6 CAFC were expressing P-glycoprotein at sufficient levels to convey vincristine resistance, suggesting low expression of the retroviral vector or splicing of the vector-drived mRNA in hematopoietic progenitor cells. Next we analyzed lineage-committed progenitors. The proviral DNA was detectable in 20-66% of colony-forming units granulocyte-macrophage (CFU-GM) while corresponding percentages (25-52%) of CD34+ PBPC were in the S/G2M phase of the cell cycle at the end of the transduction period. The proportion of vincristine-resistant CFU-GM was similar to the CAFC data and no significant differences were found between various MDR1-SNT transduction schedules whereas MDR1 co-cultivation, which served as a positive control, yielded significantly higher proportions of resistant colonies (5.3 +/- 1.4%, IL-3, 96 hr, p < or = 0.05). Assessment of rhodamine-123 (Rh-123) efflux in the myelo-monocytic progeny of MDR1-transduced cells mirrored the colony assay results in the SNT and co-cultivation groups. Less culture effort was required in the Rh-123 assay and functional characterization of the transferred P-glycoprotein was possible using cyclosporin A. Further development toward an effective MDR1 gene therapy should be facilitated by the CAFC assay, which allows estimation of the retroviral gene transfer frequency into primitive hematopoietic cells, and by the Rh-123 assay, which permits tractable side-by-side assessments of numerous MDR1 transduction protocols or different MDR1-SNT lots.
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PMID:Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123. 879 46

We examined the multidrug resistance (MDR) P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. With biotinylated MRK16 and a Streptavidin-RED670 (SA-RED670) conjugate, we succeeded in the detection of a small amount of P-gp on these cells. In normal bone marrow cells, the percentage of P-gp positive CD34+CD33-, CD34+CD33+ and CD34-CD33+ cells were 12.2 (2.2% (mean +/- standard deviation), 6.3 +/- 3.1% and 1.4 +/- 0.9%, respectively. By the more precise list-mode analysis, myeloid lineage cells showed continuously regressing P-gp expression as they maturated from CD34+CD33- to CD34-CD33+ cells. In AML cells at diagnosis, CD34+ CD33- cells expressed P-gp strongly, CD34+CD33+ cells moderately, and CD34-CD33+ cells weakly, showing the same tendency as observed in normal BM cells. Blast cells from acute promyelocytic leukemia (APL), which mainly expressed CD34-CD33+ but no detectable CD34+CD33- at diagnosis, expressed less amount of P-gp than the other subtypes of AML. P-gp expression on these three phenotypes increased in relapsed cases, especially on the CD34+CD33- subpopulation.
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PMID:[Multidrug resistance in acute leukemia]. 882 70

Resistance to chemotherapy in multiple myeloma (MM) and acute myeloid leukemia (AML) is frequently caused by multiple drug resistance (MDR), characterized by a decreased intracellular drug accumulation. MDR is associated with expression of P-glycoprotein (P-gp). GF120918, an acridine derivative, enhances doxorubicin cell kill in resistant cell lines. In this study, the effect of GF120918 on MDR cell lines and fresh human leukemia and myeloma cells was investigated. The reduced net intracellular rhodamine-123 (Rh-123) accumulation in the MDR cell lines RPMI 8226/Dox1, /Dox4, /Dox6 and /Dox40 as compared with wild-type 8226/S was reversed by GF120918 (0.5-1.0 microM), and complete inhibition of rhodamine efflux was achieved at 1-2 microM. This effect could be maintained in drug-free medium for at least 5 h. GF120918 reversal activity was significantly reduced with a maximum of 70% in cells incubated with up to 100% serum. GF120918 significantly augmented Rh-123 accumulation in vitro in CD34-positive acute leukemia (AML) blasts and CD38-positive myeloma (MM) plasma cells obtained from 11/27 de novo AML and 2/12 refractory MM patients. A significant correlation was observed between a high P-gp expression and GF120918 induced Rh-123 reversal (P=0.0001). Using a MRK16/IgG2a ratio > or = 1.1, samples could be identified with a high probability of GF120918 reversal of Rh-123 accumulation. In conclusion, GF120918 is a promising MDR reversal agent which is active at clinically achievable serum concentrations.
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PMID:In vitro effect of GF120918, a novel reversal agent of multidrug resistance, on acute leukemia and multiple myeloma cells. 894 33

The aim of the study was to test whether fractionated (weekly) idarubicin administration to multiply pretreated leukemia patients is effective and tolerable for outpatient treatment, and whether idarubicin alone can overcome P-glycoprotein (P-gp)-related resistance. P-gp was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. P-gp. expression was characterized as a percentage of P-gp-positive blasts. Additionally, the function of P-gp was determined with the rhodamine-123 (R-123) accumulation test in combination with or without verapamil and expressed as the R123 accumulation ratio. Fractionated idarubicin (12 mg/m2/week) was given to 36 acute myelogenous leukemia (AML) patients, 12 acute lymphoblastic leukemia (ALL) patients, and eight chronic myelogenous leukemia (CML) patients in blast crisis. Furthermore, 11 AML and four ALL patients were treated with fractionated daunorubicin at a dose of 50 mg/m2/week. All patients had been pretreated with drugs inducing P-gp-related resistance including daunorubicin and/or doxorubicin or vindesine (CML patients). Of 71 pretreated patients, 51 (72%) had a P-gp value between 25 and 98%. Six of these patients with increased P-gp expression had a nonpumping P-gp; four of them were CD34 positive. Of 51 patients with increased P-gp expression, 30 (59%) were CD34 positive. With regard to idarubicin monotherapy, overall response was 33/56 (59%) patients, and 23/33 (70%) responding patients showed a P-gp expression between 25 and 95%. All idarubicin-responding patients with high P-gp expression before treatment showed a clear reduction of P-gp-positive blasts. No patients with P-gp expression between 34 and 85% treated with fractionated daunorubicin showed response or reduction of P-gp-positive blasts in bone marrow. This study demonstrates that P-gp-related resistance can be overcome in multiply pretreated leukemia patients with idarubicin alone, and that the protocol used here is tolerable for outpatient treatment.
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PMID:Idarubicin monotherapy in multiply pretreated leukemia patients: response in relation to P-glycoprotein expression. 906 74

The MDR1 gene is of prognostic significance in acute myeloid leukemia (AML). The relationship of this gene to surface markers largely remains unclear. Therefore, we have studied the association of MDR1 gene expression with the expression of specific surface markers in AML. MDR1 RNA expression of leukemic cells was determined by slot blot analysis. Expression of P-glycoprotein and surface markers (CD7, CD13, CD19, CD34, HLA-DR, TdT, blood group H) was assessed by immunocytochemistry. MDR1 RNA (n = 79) and P-glycoprotein (n = 52) expression were detected in 63% and 63% of the patients, respectively. CD7, CD13, CD19, CD34, HLA-DR, TdT and blood group H were positive in 17%, 84%, 0%, 51%, 82%, 11% and 11% of the patients. MDR1 RNA or P-glycoprotein expression were not associated with the expression of either CD7, CD13, CD19, CD34, TdT or blood group H. However, P-glycoprotein expression was more frequent in HLA-DR positive than in HLA-DR negative patients (47% versus 10%, p = 0,04). Consistent with the latter finding, patients with intermediate or high MDR1 RNA expression expressed HLA-DR more frequently than patients with negative or weak MDR1 RNA expression (96% versus 76%, p = 0,03). In conclusion, MDR1 gene expression of AML cells was independent of surface markers except HLA-DR.
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PMID:Relationship between MDR1 gene and surface markers in acute myeloid leukemia. 906 14

We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34+ AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34- and CD34+ AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34+ cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34+ but not in CD34- cells. However, DNR sequestration was not observed in normal CD34+ cells. Finally, our results show that DNR is sequestered in organelles in CD34+ AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines.
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PMID:Altered intracellular distribution of daunorubicin in immature acute myeloid leukemia cells. 913 56

Accurate measurement of P-glycoprotein (P-170) expression in clinical samples still remains a controversial issue. In this study tumor cell P-170 expression was assessed in 29 patients suffering from acute leukemia (17 acute myeloid leukemia (AML) and 12 acute lymphoblastic leukemia (ALL)) using three different techniques: flow cytometry measuring rhodamine 123 (Rh123) efflux (functional level), immunocytochemistry (protein level) and RT-PCR (mRNA level). Rh123 efflux was detectable in 10/29 (34%) of all cases, in 9/17 (53%) of AML and in 1/12 (8%) of ALL samples. In AML patients a significant association of CD34 expression and P-170 activity was observed (P < 0.02). All AML patients with the FAB subtype M5 were Rh123 negative (P < 0.007). Cytospin preparations were analyzed for staining with monoclonal antibodies JSB1 and MM4.17. Eight of 16 (50%) AML and 0/9 (0%) ALL cases expressed the multidrug resistance (MDR) protein assessed by JSB1. With MM4.17 87% of AML and 50% of ALL patients were scored positive. Agreement between both antibodies was found in only 13/23 (57%) samples. Extracted RNA from 12 patients was analyzed by RT-PCR to evaluate the expression of MDR1 and multidrug resistance-associated protein (MRP) mRNA. An increased level of MDR1 mRNA was detectable in 4/7 AML and 0/5 ALL cases. MRP expression was found in 3/7 AML and 0/5 ALL patients. Comparison of Rh123 assay and immunocytochemistry revealed a very good correlation when using MoAb JSB1 (P < 0.004) but not with MM4.17 (not significant (NS)). JSB1 also showed a much better association with the PCR results (P < 0.05) than MM4.17 (NS). Finally, we compared the results of the functional Rh123 assay and RT-PCR and observed a high correlation for Rh123/MDR1 (r = 0.819, P < 0.001) but low for Rh123/MRP (r = 0.562, NS). We conclude that measurement of Rh123 efflux and immunocytochemical staining of cytospin preparations with JSB1 allows the accurate monitoring of P-170 expression in acute leukemia. The simplicity of these two MDR assays suggests their use for routine MDR screening.
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PMID:Multidrug resistance in acute leukemia: a comparison of different diagnostic methods. 920 93

Expression of the multidrug resistance (MDR) phenotype is an independent prognostic variable in acute myeloid leukemia. Approximately 43-57% of the patients have P-glycoprotein (P-gp) expression. A major drawback with the interpretation of P-gp data in AML is the lack of coherence with different analytical assays. We have focused our efforts of P-gp detection on flow cytometry using a dual technique of P-gp staining with antibodies for the extracellular epitope (MRK16) and a functional analysis of P-gp using the rhodamine efflux assay and the effect of P-gp inhibitors such as SDZ PSC 833. This technique was combined with the staining of lineage-specific antigens such as CD34, CD56 and c-kit. In this way, various subsets of AML cells can be identified such as MRK 16+/-, CD34+/- blasts. These cells can be sorted for further analysis, such as the molecular expression of P-gp and other pleiotropic drug resistance genes.
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PMID:Assays for the analysis of P-glycoprotein in acute myeloid leukemia and CD34 subsets of AML blasts. 920 6

Cells with multidrug resistance(MDR) phenotype express P-glycoprotein(P-gp) on cell membrane, which works as a drug-efflux pump with low selectivity. P-gp function can be determined microfluorometrically using the fluorescent dye rhodamine 123(Rh123), which is an artificial substrate for P-gp. In this study, we assessed P-gp function in human leukemia sublines of MOLT-3 with various magnitude of MDR phenotype using the Rh123-efflux assay. The MDR cells efficiently pumped out Rh123 outside cells in parallel with the magnitude of resistance to vincristine, while the parent MOLT-3 cells scarcely showed dye efflux. The P-gp function determined by the dye efflux assay was correlated with the degree of cell surface expression of P-gp measured by indirect flow cytometric analysis using MRK16 anti-P-gp antibody and with the amount of MDR1 mRNA (encoding P-gp) quantified by Northern blot analysis and by competitive reverse transcription-polymerase chain reaction (RT-PCR) assay. In the evaluation of 28 clinical samples obtained from patients with leukemias, 9 cases exhibited positive results Rh123-efflux. A good correlation of Rh123-efflux with MDR1 expression measured by competitive RT-PCR was observed in these samples. Since subpopulations of normal lymphocytes show low degree of P-gp function, the strict gating of leukemia cells was mandatory in the dye-efflux assay in clinical samples. Although leukemia cells could not be distinguished from normal lymphocytes in the conventional scattergram in some cases, additional staining of the former cells with specific monoclonal antibody such as CD34(labelled with PE-Cy5, a dye without interference with Rh123 fluorescence emission) enabled a selective analysis of a particular subpopulation. The Rh123 dye-efflux assay is a simple and sensitive method for the determination of P-gp expression and its function, and is particularly suitable for the analyses in the clinical laboratory.
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PMID:[Flow cytometric analysis of P-glycoprotein function by rhodamine 123 dye-efflux assay in human leukemia cells]. 931 Dec 64


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