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 specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.
Leukemia 1995 Aug
PMID:Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis. 764 31

We looked for bcl-2 protein expression by immunocytochemistry on bone marrow slides from 51 cases of myelodysplastic syndrome (MDS), of whom 25 received some form of chemotherapy. Forty-six of them had at least 20% bcl-2 positive blasts and the median percentage of positive blasts was 80%, whereas myeloid cells beyond blasts were always negative. No correlation was found between bcl-2 expression and the FAB type of MDS, CD34 expression and P-glycoprotein expression. A strong correlation between weak bcl-2 expression and the presence of a p53 mutation detected by SSCP analysis and direct sequencing was found. Response to chemotherapy (intensive chemotherapy or low-dose Ara-C) and survival were not significantly influenced by the intensity of bcl-2 expression in blasts, although there was a trend for better response to chemotherapy and longer survival in patients with strong bcl-2 expression. This trend was no longer found, however, if patients with a p53 mutation were excluded. Our findings show that blasts from a majority of MDS cases have bcl-2 expression and that strong bcl-2 expression is not associated with a poor prognosis. The correlation between weak bcl-2 expression and p53 mutation suggests a possible downregulation of bcl-2 gene expression by mutated p53, the mechanism of which remains to be established.
Leukemia 1995 Apr
PMID:bcl-2 expression in myelodysplastic syndromes and its correlation with hematological features, p53 mutations and prognosis. 772 10

In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp). The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ). All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression. Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1. When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833. In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels. Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines. MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells. Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp. With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype. Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.
Leukemia 1995 May
PMID:Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines. 776 42

The purpose of our investigations was to measure P-glycoprotein (P-170) activity in blast cells of 35 adults with acute myeloid leukemia (AML), and 24 children and adults with acute lymphoblastic leukemia (ALL) at time of diagnosis. Studies were based on a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123 (Rh123), which is transported from the cell by the P-170 pump. Dual-fluorescence staining with Rh123 and phycoerythrin-labeled monoclonal antibodies allowed selective measurement of Rh123 efflux in blast cells. Samples were scored positive when the fraction of blast cells showing Rh123 efflux exceeded 10% after a 120-min incubation. Activity of P-170 was observed in 19 (54%) of the 35 AML cases and was completely blocked in the presence of multidrug resistance inhibitors. Efflux activity was significantly higher in CD34-positive AML samples (p < 0.02). All AML patients with the FAB-subtype M5 (n = 5) lacked Rh123 pumping activity (p < 0.03). The complete remission rate in response to induction chemotherapy was significantly higher for Rh123-negative (11/13, 85%) than for Rh 123-positive AML patients (4/15, 27%) (p < 0.007). At a median follow-up of 9 months overall survival was significantly shorter for Rh123-positive than for Rh123-negative patients (p < 0.05). In contrast to AML, we could detect Rh123 efflux in only two (8%) out of 24 ALL cases. The immunological subtypes of these two positive cases was of B-ALL and pre-T-ALL. Bone marrow cryostat sections from 13 AML and five ALL patients were further analyzed for staining with monoclonal antibodies MM4.17 and JSB1. Ten of 13 AML and two of five ALL cases expressed the MDR protein. Our results indicate that there is a rather low frequency of P-170 pumping activity in ALL compared with AML. Further, functional activity of P-170 contributes to chemoresistance in de novo AML.
Leukemia 1995 Feb
PMID:Low frequency of activity of P-glycoprotein (P-170) in acute lymphoblastic leukemia compared to acute myeloid leukemia. 786 74

Expression of the multidrug resistance gene mdr1 is reported to be an important determinant of responsiveness to therapy and survival in some cancers. Many different methods have been used to evaluate mdr1 expression in these studies. This paper compares four methods for determination of mdr1 expression. We studied the mdr1 gene expression in 36 freshly established cell lines from 28 children with acute lymphoblastic leukemia (16 T-ALL, six BCP-ALL, two B-ALL (L3), two biphenotypic leukemias, two Burkitt's lymphomas). Leukemic specimens were obtained at the time of diagnosis in 16 cases, and after chemotherapy in 20 cases. In all the samples, mdr1 mRNA was measured by slot blotting and reverse transcriptase polymerase chain reaction (rt-PCR), and the presence of the mdr1 product, P-glycoprotein, was detected by immunohistochemistry with the MRK-16 monoclonal antibody. In situ mdr1 RNA hybridization was performed in 30 cases. Complete agreement was noted between all the techniques in 14 cases (39%). Results differed on a single test result in another 39% of the cases. These 78% of cases were considered assessable, and the consensus result was presumed to be correct. By this consensus criterion, immunohistochemistry yields both false negative (11%), and false positive (11%) results. RNA slot blotting has a high (21%) false positive rate. In situ mRNA hybridization and rt-PCR have the highest concordance, 80%. The 28 patients from whom these cell lines were derived appear to represent a very poor prognosis group, since there are only two patients (with Burkitt's lymphoma) who are long-term survivors. Nonetheless, a complete clinical response to therapy was correlated with absence of mdr1 expression in assessable cases (p = 0.04). These four methods of determining mdr1 expression often yield discordant results. Therefore, the use of at least two methods for evaluating mdr1 expression is advisable. Rt-PCR is recommended because of its relative simplicity and specificity. This should be supplemented by a technique (immunohistochemistry or flow cytometry) able to detect heterogeneity of P-glycoprotein expression among cells.
Leukemia 1994 Feb
PMID:Mdr1 gene expression in childhood acute lymphoblastic leukemias and lymphomas: a critical evaluation by four techniques. 790 44

Clinical and cytologic characteristics were correlated to immunologic markers in 154 patients with newly diagnosed acute myeloid leukemia (AML). The panel of monoclonal antibodies (MoAbs) was selected to identify differentiation-associated antigens of both the myeloid and the lymphoid lineages (CD13, CD33, CD14, CD15, CD7, CD34, CD10, HLA-DR, CD19, CD2, CD5, TdT). The expression of multidrug resistance P-glycoprotein (P-170) was also evaluated in 117 patients. Differences in antigenic expression was observed among the various French-American-British (FAB) subgroups. HLA-DR was poorly expressed on the blasts of acute promyelocytic leukemia (M3), and was always found in FAB M5. CD34 was detectable in all M0 cases and only in one M3 (p < 0.001). Lymphoid-associated antigens were positive in 74 cases (48.1%). In particular, CD7 was found in 49 patients (31.8%), and TdT in 30 (21.3%), 15 samples displaying coexpression of these two antigens. The incidence of CD7+ cases was particularly elevated in M0 and M5 AML (p = 0.005). It significantly correlated with the expression of CD34, HLA-DR, P-170 (p < 0.001, p = 0.018 and p = 0.034 respectively), and with a leukocyte count > 50 x 10(9)/l (p = 0.038). Sixty-nine (59%) samples demonstrated P-170 positivity. Again, this phenotype was particularly expressed in the poorly differentiated forms (M5, M0 and M1) and showed significant correlation with the immaturity markers CD34, CD7 and HLA-DR (p = 0.013, p = 0.022 and p = 0.001, respectively). Expression of individual antigens correlated with prognosis. Refractoriness to first line therapy was associated with CD7 expression (p = 0.002) and P-170 (p = 0.001). The CD7 marker was also significantly associated with a very low overall survival (p < 0.001) and continuous complete remission (p < 0.001). CD14 expression also significantly predicted lower survival rates (p = 0.033). The combination (CD7+ CD14+) identified a subset of patients with a particularly adverse outcome. The prognostic value of CD7 expression, alone or in combination with other markers, was confirmed in multivariate analysis.
Leukemia 1994 Mar
PMID:Prognostic value of cell marker analysis in de novo acute myeloid leukemia. 790 93

Taxol-resistant sublines of HL-60 myeloid leukemia cells (HL-60/TAX100 and HL-60/TAX1000) have been isolated in vitro by subculturing in progressively higher concentrations of taxol. HL-60/TAX100 and HL-60/TAX1000 cells are capable of continuous growth in the presence of 0.1 microM and 1.0 microM taxol, respectively, and the IC50 (50% growth inhibitory dose) values for taxol for the two sublines are 0.34 and 2.44 microM as compared to 3.1 nM for the parent HL-60 cells. HL-60/TAX100 and HL-60/TAX1000 cells display a variable degree of cross-resistance to taxotere, vincristine and doxorubicin, but are sensitive to the antimetabolite Ara-C. Both HL-60/TAX100 and HL-60/TAX1000 cells over-express MDR-1 m-RNA and the membrane efflux multidrug transporter P-glycoprotein (PGP), as determined by Western blot and immunofluorescence labeling with anti-PGP antibodies. Consequently, exposure of the taxol-resistant cells to [3H]taxol or daunomycin results in the accumulation of significantly lower levels of the two drugs. Co-treatment with cyclosporine (0.5 microgram/ml) or verapamil (10 microM) partially overcomes taxol resistance in HL-60/TAX1000 cells. Following treatment with clinically relevant concentration of taxol (1.0 microM for 24 h), HL-60 but not HL-60/TAX1000 cells display intracellular microtubular bundling, markedly enhanced accumulation of the cells in G2/M phase of cell-cycle and internucleosomal DNA fragmentation associated with apoptosis which is independent of bcl-2 gene expression. These taxol-resistant myeloid leukemia cells may serve as in vitro experimental models for examinating strategies which may have potential applicability for overcoming taxol resistance.
Leukemia 1994 Mar
PMID:Characterization of a human myeloid leukemia cell line highly resistant to taxol. 790 95

The accumulation and cytotoxicity of vincristine (Vcr), etoposide (VP16), and daunorubicin (Dau) and effect of the resistance modifiers (RM) verapamil (Ver; 10 microM) and cyclosporin A (CyA; 3 microM) were studied in isolated rat cardiac myocytes, peripheral lymphocytes from seven patients with chronic lymphocytic leukemia (CLL), in the human leukemic cell line K562 and its two Vcr resistant mdr1 gene expressing sublines, K562/Vcr30, K562/Vcr150. Both RMs increased the accumulation and cytotoxic effect of Vcr and Dau in the resistant sublines. In K562 cells, lymphocytes from patients with CLL and rat cardiac myocytes, which all were mdr1 RNA negative RMs increased the cellular accumulation and potentiated the cytotoxic effect of Vcr but not that of Dau. K562/Vcr30 and K562/Vcr150 were cross resistant to Dau but not to VP16 and RMs had no effect on the cytotoxicity of VP16 in any of cell lines. The results indicate that chemosensitive cells also have a transport mechanism, not mediated by P-glycoprotein, which transports Vcr but not Dau and VP16. This suggests that addition of RMs to Vcr-containing chemotherapy may enhance the antineoplastic effect also by inhibition of non-P-glycoprotein mediated transport mechanisms.
Leukemia 1994 Jun
PMID:A non-P-glycoprotein-mediated mechanism of vincristine transport which is affected by resistance modifiers and present in chemosensitive cells. 791 47

Vincristine sensitive (L1210) and resistant (L1210/VCR) L1210 mouse leukemia cells were studied from morphological and histochemical point of view. The morphological and histochemical findings reflected differences in membrane structure and in physiological state of sensitive and resistant cells. Numerous villous projections and cytoplasmic protrusions of the cell surface as well as higher activity of membrane enzymes (5'-nucleotidase, ATPase, alkaline phosphatase) were found in vincristine resistant cells. It is assumed that in resistant cells these differences are connected with overexpression of membrane P-glycoprotein. Moreover, in resistant cells more condensed mitochondria were found after their exposure to vincristine. This finding can reflect a higher activity of these organelles in conditions when activity of P-glycoprotein is manifested and is in agreement with increased rate of oxygen consumption by resistant cells from 2.5 +/- 0.3 to 3.3 +/- 0.2 microliter/min.10(6) cells induced by vincristine.
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PMID:Characterization of morphological and histochemical changes induced by overexpression of P-glycoprotein in mouse leukemic cell line L1210. 791 60

We used a recently established stroma-supported tissue culture technique that allows long-term culture of acute lymphoblastic leukemia (ALL) cells to study 2-chloro-2'-deoxyadenosine (2CdA) cytotoxicity to leukemic lymphoblasts. In the 20 cases of ALL studied, the number of cells recovered after 7 days of culture on allogeneic stromal layers were 58-192% (median, 95.5%) of those originally seeded. In parallel cultures with 2CdA (100 nM), 74- > 99% (median, 97.5%) of leukemic lymphoblasts were killed. The cytotoxicity of 2CdA extended to all ten samples with either the t(9;22) (q34;q11) or 11q23 chromosomal abnormalities, karyotypes associated with an extremely poor outcome, as well as to two samples collected at the time of relapse. The effects of 2CdA were dose-dependent, and were due to triggering of apoptosis as shown by typical morphologic changes and occurrence of DNA fragmentation. Stromal layers were apparently not affected by 2CdA treatment, even when used at 1000 nM. We also tested 2CdA cytotoxicity to multidrug resistant subclones of the CCRF-CEM ALL cell line. CEM/VLB100 expresses P-glycoprotein, whereas CEM/VM-1 and CEM/VM-1-5 have topoisomerase II mutations that are associated with resistance to topoisomerase II inhibitors. Overexpression of P-glycoprotein or alterations in topoisomerase II did not protect cells from 2CdA cytotoxicity. We conclude that 2CdA is cytotoxic in most cases of ALL. The method used in this study may be applied to evaluate leukemic blast cell sensitivity to compounds with potential anti-leukemic activity, and to select patients for entry into clinical trials.
Leukemia 1994 Jul
PMID:Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs. II. Potent cytotoxicity of 2-chloro-deoxyadenosine in acute lymphoblastic leukemia. 791 12


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