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)

The cells from approximately 70% patients with acute myeloblastic leukaemia exhibit autonomous growth characteristics in vitro, which have been associated with a poor response to therapy. We have previously shown that leukaemic cells with autonomous growth characteristics express high levels of bcl-2 and are relatively resistant to apoptosis. As bcl-x(L) is a bcl-2-related gene with anti-apoptotic activity which also confers resistance to cytotoxic drugs we have studied its expression in AML in relation to cellular growth characteristics and to the expression of P-glycoprotein. Cells from 15 patients were studied. Immunoblotting demonstrated bands at 31 kDa corresponding to bcl-x(L) from the cells of all patients. Bcl-x(S) was not detected in any sample. Using standardised, quantitative flow cytometry, bcl-x(L) expression ranged from 0.25 x 10(5) to 4.24 x 10(5) bound FITC molecules, (median 1.35 x 10[5]). AML blasts with autonomous growth in vitro expressed more bcl-x(L) (median 1.76 x 10[5]) than those which did not (median 0.86 x 10(5), P=0.01). Quantitative bcl-x(L) expression strongly correlated with that of P-glycoprotein, also measured by quantitative flow cytometry using the MRK16 antibody (r=0.95, P < 0.001), but not with MRPr1. These results provide a further explanation for the poor prognosis associated with autonomous in vitro growth of AML blasts and illustrate that these cells may coexpress different modalities of resistance to cytotoxic drug therapy involving both anti-apoptotic pathways (bcl-x(L), bcl-2) and classic multidrug resistance (MDR1). The implication of these findings is that the use of agents to reverse MDR1 function in AML may be unsuccessful in the absence of strategies to reduce resistance to apoptosis.
Leukemia 1997 Jul
PMID:Bcl-x(L) is heterogenously expressed by acute myeloblastic leukaemia cells and is associated with autonomous growth in vitro and with P-glycoprotein expression. 920 73

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.
Leukemia 1997 Jul
PMID:Multidrug resistance in acute leukemia: a comparison of different diagnostic methods. 920 93

Immunocytochemical detection of the expression of the MRP gene and the MDR1 gene in clinical specimens might be affected by several factors. Thus, we studied the impact of monoclonal antibodies, sample source (peripheral blood vs bone marrow) and disease status on the expression of multidrug resistance-associated protein (MRP) as well as P-glycoprotein (P-gp) in leukemic cells of patients with acute myeloid leukemia (AML). MRP expression was determined by means of anti-MRP antibodies (QCRL-1, QCRL-3, QCRL-1/QCRL-3 or MRPr1). In the case of P-gp, monoclonal antibodies C219 and MRK16 were used. High MRP expression ranged from 5 to 35% and high P-gp expression from 5 to 14% of the specimens. A fair correlation between results obtained with QCRL-1/QCRL-3 and those obtained with MRPr1, as well as a moderate correlation between C219 and MRK16, were seen. MRP and P-gp expression of peripheral blood blasts were similar to those of bone marrow blasts in the majority of cases. The degrees of MRP expression at the time of diagnosis were also similar to the degrees of expression at relapse, albeit an analysis of sequential MRP expression in 13 patients indicated an increase of expression at relapse in six patients as compared to the time of diagnosis.
Leukemia 1997 Jul
PMID:Immunocytochemical detection of the multidrug resistance-associated protein and P-glycoprotein in acute myeloid leukemia: impact of antibodies, sample source and disease status. 920 94

The clinical relevance of multidrug resistance (MDR)-related proteins in childhood acute lymphoblastic leukemia (ALL) is largely unknown. The diversity of techniques, fixation methods, storage of cells (fresh or cryopreserved) etc, may contribute to discrepancies observed between several studies. We therefore optimized the detection of P-glycoprotein (P-gp), MDR-associated protein (MRP) and lung resistance-related protein (LRP) by immunocytochemistry and flow cytometry in childhood ALL cells. Thirteen fixation methods were compared using six antibodies in both immunocytochemistry and flow cytometry. The optimal fixation for P-gp (C219, MRK16), MRP (MRPr1) and LRP (LRP56) was a mixture of 2% (v/v) formaldehyde solution and acetone incubated for only 10 s at room temperature (FAc). For MRP recognized by MRPm6, the optimal fixation condition was acetone for 5 min at room temperature in immunocytochemistry, and methanol for 15 min at -20 degrees C in flow cytometry. P-gp staining by 4E3 was strongly antibody batch-dependent; on cytospins FAc fixation was optimal, but inconclusive data were obtained by flow cytometry. The optimized fixation conditions on fresh samples revealed a day-to-day variation in staining (both increasing and decreasing) in one third of the immunocytochemical tests. In flow cytometry the day-to-day variation in the fluorescence index was -1 +/- 22%. In both techniques, staining was comparable between fresh and cryopreserved cells. We recommend the use of the above mentioned fixation methods in order to study the clinical relevance of P-gp, MRP and LRP in childhood ALL.
Leukemia 1997 Jul
PMID:Optimal immunocytochemical and flow cytometric detection of P-gp, MRP and LRP in childhood acute lymphoblastic leukemia. 920 95

The problem of tumor cell drug resistance remains a barrier to the successful treatment of many neoplastic diseases. Problems of tumor cell heterogeneity and expression of multiple mechanisms of drug resistance complicate treatment strategies. Indeed, even that form of resistance to natural product anticancer agents, multidrug resistance (MDR), can have multiple mechanisms. Compounding these problems is the use of different methodologies and different reagents to assess expression of the most widely studied form of MDR, that due to increased expression of the MDR1 gene and its product, P-glycoprotein (Pgp). In this paper, we discuss problems associated with assay variability and accurate measurement of markers of drug resistance, and summarize consensus findings of the St Jude Workshop on methods to detect Pgp in tumors.
Leukemia 1997 Jul
PMID:Methods to detect P-glycoprotein and implications for other drug resistance-associated proteins. 920 98

This paper summarizes experimental data and theoretical considerations, that are important for the measurement of P-glycoprotein (Pgp) function in acute myeloid leukemia (AML). The data are presented in subdivisions based on the techniques used, which will facilitate finding specific information. Based on our extensive experience with Pgp analysis, which includes radioactive assays, flow cytometry and fluorescence microscopy, we recommend a flow cytometry-based assay, that measures the effect of 2 microM PSC 833 on rhodamine 123 (R123) accumulation as the most practical and sensitive functional Pgp test. In combination with the flow cytometric measurement of Pgp using an antibody against an extracellular epitope (eg MRK16), this offers a sensitive and reproducible method for Pgp detection in AML, which is also rapid and practical. Furthermore, an R123 accumulation assay is specific for Pgp, because R123 is transported much less efficiently by the multidrug resistance protein (MRP) than by Pgp. Another probe of similar sensitivity and specificity is 3,3'-diethyloxacarbocyanine iodide. Alternatively, especially for the analysis of small numbers of cells (for example sorted subpopulations of leukemic cells), convenient and sensitive procedures are being developed by using DNA-binding Pgp substrates which remain fixed in the nuclei of the cells upon formaldehyde exposure for quantitative fluorescence laser scanning microscopy with image analysis. Less experimental data have been published to establish the optimal conditions for dual parameter flow cytometry (Pgp function, in eg Pgp+ or CD34+ cells). However, laboratories with flow cytometry experience will be able to implement this useful option to analyze subpopulations of cells.
Leukemia 1997 Jul
PMID:Theoretical and practical considerations for the measurement of P-glycoprotein function in acute myeloid leukemia. 920 99

Multidrug resistance (MDR) is one of the major obstacles to successful cancer chemotherapy. Since P-glycoprotein (P-gp) encoded by the MDR-1 gene plays a key role in MDR, many P-gp-specific monoclonal antibodies (MAbs) have been generated for characterization and analysis of P-gp. Among those antibodies, MRK16 has been widely used not only for elucidation of the mechanisms of P-gp-mediated MDR but also for diagnostic and therapeutic studies. Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established by us and may offer a useful tool to quantitate low levels of P-gp expression. This article describes the characteristics of the antibodies against P-gp and discuss the diagnostic implications of the antibodies.
Leukemia 1997 Jul
PMID:Monoclonal antibodies specific for P-glycoprotein. 920

The MDR1 gene product, P-glycoprotein (P-gp), works as a transmembrane efflux pump for several cytotoxic products, representing a major cause for cancer treatment failure. Rhodamine 123 (Rh123), a low toxic fluorescent probe commonly used to assess mitochondrial bioenergetics in living cells, has also been used to measure the efflux activity of P-gp in both normal and malignant cells. Analysis of variation in cellular fluorescence by measuring the rates of Rh123 influx and efflux, together with the effect of mdr reversing agents, allows the investigation of drug-resistant phenotypes in cancer samples. We have studied the functional activity of P-gp in human leukemic cell lines using flow cytometry, taking into consideration that variables such as Rh123 cytotoxicity, culture conditions, cell membrane integrity, as well as the effect of specific P-gp modulators, can impair the resolution of the Rh123-efflux measurements. The studies show that: (1)optimal non-cytotoxic concentrations of Rh123 which allow appropriate color compensation are in the range of 50-200 ng/ml; (2) life-gating allows accurate measurement on the 50% average rate of Rh123 efflux; (3) relative efficiency of P-gp inhibitors was PSC-833 > cyclosporin A > verapamil; and (4) the presence or absence of fetal calf serum had no effect on the bioavailability of chemosensitizer agents, with the exception of serum-free experiments, which showed a significant decrease in P-gp activity under the presence of PSC-833 (P = 0.05). Hence, we recommend this experimental strategy for clinical practice better to study the cellular drug resistance phenotype.
Leukemia 1997 Jul
PMID:Flow cytometric analysis of P-glycoprotein function using rhodamine 123. 920 1

P-glycoprotein-mediated multidrug resistance has emerged as one of the most attractive targets to improve anticancer therapy. The P-glycoprotein functions as an energy-dependent, membrane transport pump capable of decreasing the intracellular concentration of a broad range of chemotherapeutic agents. Pharmaceuticals which inhibit P-glycoprotein transport activity are currently being evaluated in clinical trials. Characterization of P-glycoprotein functional activity is critical in determining if these multidrug resistance reversal agents improve therapeutic responses of tumors expressing P-glycoprotein. In this report, we directly compare and characterize assays using rhodamine 123, dimethyloxadicarbocyanine iodide (DiOC2), [3H]daunorubicin and hexakis(2-methoxyisobutyl isonitrile)technetium(I) ([(99m)Tc]Sestamibi) as P-glycoprotein transport probes to quantitate functional activity. The accumulation of certain substrates is concentration dependent and the parameters which determine probe accumulation are impacted by the level of P-glycoprotein expression. In addition, higher concentrations of reversal agents are required to inhibit multidrug resistance in cell lines expressing higher levels of P-glycoprotein. Furthermore, the concentration of reversal agents required to inhibit completely P-glycoprotein transport activity is higher than generally recognized. Thus, the level of P-glycoprotein expression may confound intersample comparisons unless sensitive probes are used in combination with saturating concentrations of potent reversal agents. These results highlight the importance of carefully characterizing assay systems under uniform conditions to quantitate P-glycoprotein function.
Leukemia 1997 Jul
PMID:Characterization of functional assays of multidrug resistance P-glycoprotein transport activity. 920 2

Structure/functional studies previously reported showed that in a series of simple organic cations in which the charge is delocalized, an aromatic ring and a minimal degree of lipophilicity (log P > -1) were required for recognition by murine cells which express P-glycoprotein (p-gp)-mediated multidrug resistance (MDR). In the present report we find that 3H-octylpyridinium, the simple aromatic cation which has been shown to be preferentially toxic to MDR- as compared to MDR+ cells, accumulates 4.7-fold greater in the MDR- cell line. In contrast, we find that 3H-guanidinium which displays no selective toxicity between MDR+ and MDR- cells, shows no significant uptake differences between these two cell types. We also present data which demonstrate that other organic cations which contain aromatic rings, a minimal degree of lipophilicity (log P> -1) and carry a delocalized (Rho 123) or shielded (triphenylmethyl phosphonium) positive charge, also accumulate to a greater degree in MDR- vs MDR+ cells. Additionally, we find that human cells which express p-gp MDR, have similar requirements for recognition of these simple compounds. In fact, the sensitivity profiles of these compounds closely correlate between murine and human cell lines. It was also found that none of the series of simple organic compounds tested showed modulatory activity in MDR+ cells, as assayed by monitoring retention of Rho 123. Thus, the requirements for MDR recognition vs those for MDR modulation are clearly distinguished with these simple structured compounds. In comparison, the calcium channel antagonist, verapamil, and a calcium channel agonist, Bay K 8644, both showed modulatory activity by increasing Rho 123 retention in MDR+ cells, further supporting the interpretation that verapamil's modulation of MDR is unrelated to its action on calcium flux. Overall, the data presented here add further information for defining the structural requirements of compounds for their recognition by, or modulation of, human cells expressing p-gp-mediated MDR.
Leukemia 1997 Jul
PMID:Accumulation of simple organic cations correlates with differential cytotoxicity in multidrug-resistant and -sensitive human and rodent cells. 920 5


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