Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine the role the multiple drug-resistance (MDR 1) gene plays in chronic lymphocytic leukemia (CLL), we measured the expression of the MDR 1 gene in 30 patients with this disease. A rapid, highly sensitive, and nonradioactive technique based on the polymerase chain reaction (PCR) was used for that purpose. In this technique, called differential PCR, the target (MDR 1) and a reference gene (beta 2-microglobulin) are co-amplified by PCR from random hexamer-primed cDNA in the same reaction vessel. The level of target gene expression is reflected in the ratio between the intensities of the two resulting PCR product bands, as measured by high-performance liquid chromatography (HPLC). MDR 1 gene expression was detectable in 29/30 (97%) patients with CLL, with a median expression level of 0.36 U (human placenta = 1 U). There was no correlation between expression of the MDR 1 gene and clinical stage, time from diagnosis, absolute lymphocyte count, several lymphocyte surface markers, or prior treatment in the patients analyzed. Immunocytochemical studies of the same material using the monoclonal antibody C219 showed a very low or undetectable expression of the P-glycoprotein in the lymphocytes of all patients studied, whereas granulocytes were significantly more immunoreactive. We conclude that the level of expression of the MDR 1 gene in CLL is generally low, that the removal of granulocytes is important in studies of expression of MDR 1 mRNA in CLL, and that differential PCR provides a rapid and reliable method for quantifying the amount of a specific mRNA, even in very small samples of total RNA.
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PMID:PCR-determined expression of the MDR1 gene in chronic lymphocytic leukemia. 136 Aug 21

Expression of the multidrug resistance efflux pump P-glycoprotein (Pgp) was measured in a series of AML patients using two flow cytometry methods. Transport function was assessed by measuring the modulating effect of the Pgp inhibitor cyclosporin A (CsA) on the cellular accumulation of daunorubicin, and Pgp antigen expression by surface immunofluorescence using the MRK-16 antibody. Both methods showed a wide range of values for Pgp expression between individual patients, but in contrast to a series of cell lines expressing Pgp there was no correlation between antigen expression and transport function in the clinical samples. As previously reported for chronic lymphocytic leukemia (CLL), pretreatment with neuraminidase markedly improved MRK-16 staining in some cases, indicating that abnormal glycosylation can cause epitope masking in AML blasts. Because experience with cell lines shows that Pgp expression is a continuous variable which correlates with the level of drug resistance, rather than the 'positive' or 'negative' which are frequently reported by clinical flow cytometry laboratories, we used a calibration procedure to estimate the actual number of Pgp molecules expressed in the AML samples. Despite the additional refinements of neuraminidase treatment and antigen quantification, the correlation between Pgp antigen expression and daunorubicin accumulation remained extremely weak (r = 0.11; P = 0.63). It is suggested that the assay for transport function can detect molecules that affect daunorubicin accumulation but are antigenically distinct from classical P-glycoprotein. Heterogeneity of multidrug resistance efflux pumps might in part explain the relatively weak prognostic significance of immunofluorescence detection of Pgp in AML patients.
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PMID:Discordant P-glycoprotein antigen expression and transport function in acute myeloid leukemia. 747 79

To optimize the immunohistochemical detection of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp) in chronic lymphoid disorders, the authors compared the sensitivity of three different monoclonal antibodies (MoAb) directed against P-gp (C219, JSB-1, and MRK 16) by using the APAAP technique on four tissue preparations obtained from lymphoid tumors: Cryostat sections, ModAMEX processed sections, frozen cytospin preparations, and fresh cytospin preparations. Tumor samples were obtained from patients with previously treated chronic lymphocytic leukemia (6 cases) or non-Hodgkin's malignant lymphoma (4 cases). Lymph nodes (n = 9), spleen (n = 3), and blood (n = 5) were analyzed. JSB-1 MoAb detected P-gp in 4 of 12 cases (33.3%) on either frozen sections or ModAMEX processed sections, and in 6 of 17 cases (35.3%) on frozen cytospin preparations. The sensitivity of JSB-1 was significantly improved when fresh cytospin preparations were used with an incidence of P-gp positive samples as high as 70.6% (P < .05). C219 MoAb was unreactive with lymphoid cells whatever the technique used, whereas this antibody stained stromal cells. MRK 16 MoAb was equally reactive to JSB-1 on fresh cytospin preparations, but unreactive when the other preparations were used. The specificity of JSB1 MoAb was confirmed by both Western blot analysis and Rhodamine 123 efflux assay. The authors used JSB-1 MoAb on fresh cytospin smears prepared from 28 CLL patients. Overall incidence of P-gp positive cases was 39.2%. Univariate analysis showed that P-gp expression was correlated with prior therapy, refractoriness to treatment, Rai stratification, and time of tissue storage after diagnosis. The authors recommend the use of JSB-1 on fresh cytospin preparations for the immunocytochemical detection of P-gp in chronic lymphoid disorders.
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PMID:Optimization of immunohistochemical detection of P-glycoprotein in chronic lymphoid disorders. 780 2

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.
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PMID:A non-P-glycoprotein-mediated mechanism of vincristine transport which is affected by resistance modifiers and present in chemosensitive cells. 791 47

The multidrug resistance (MDR) phenotype has been demonstrated to be related to the overexpression of P-glycoprotein, a 170 kDa transmembrane efflux pump. We studied P-glycoprotein expression in 40 patients with chronic B-cell leukemias by FACS analysis using MoAb c219, which recognizes both the MDR1 and the MDR3 gene product. We found significantly elevated P-glycoprotein expression in these patients as compared with normal controls. Patients who had received previous chemotherapy regimens containing MDR-related drugs showed significantly higher P-glycoprotein expression with MoAb c219 than those patients who had been untreated. Northern blot analysis of MDR1 and MDR3 gene expression in 32 of the patients gave a similar result: in the analysis of total RNA four of six patients (66%) pretreated with either vinca alkaloids or anthracyclines were MDR1 positive as opposed to 6 of 26 (23%) who had no treatment or treatment without these agents. In contrast, MDR3 expression was found more frequently (63%), but was randomly distributed in the differently treated groups. Increasing the sensitivity level by analysis of enriched mRNA (polyA+RNA) led to the detection of MDR1 and MDR3 expression all B-CLL patients. We conclude that a basic elevated P-glycoprotein expression is intrinsic in CLL cells, which is possibly upregulated under chemotherapy. This might be responsible for initial and acquired chemotherapy resistance in CLL patients. Follow-up of the B-CLL patients over 46 months showed that the median survival time for MDR1+ patients was 19 months as opposed to 46 months for MDR1- patients (p < 0.01). There was no statistical difference in survival between MDR3+ and MDR3- patients. In the MDR1+ group, eight of nine patients had developed resistance to the therapy with MDR-related drugs. The expression of MDR1 might, therefore, predict treatment failure with MDR-related drugs and be a negative prognostic factor.
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PMID:Multidrug resistance phenotype in patients with chronic lymphocytic leukemia as detected by immunofluorescence (FACS) and northern blot analysis. 791 54

Reduced drug accumulation may be one reason for intrinsic drug resistance in chronic lymphatic leukemia of the B-cell type (B-CLL). Immunophenotyped leukemic human B-cells from 38 cases of B-CLL were characterized for P-glycoprotein (PGP) content. In all, 30 cases of B-CLL were additionally analyzed for further parameters: accumulation of daunorubicin (DNR, n = 20) and rhodamine 123 (Rh123, n = 30) in both the presence and the absence of verapamil (VRP). Also, 16 cases of B-CLL were analyzed for vincristine (VCR) accumulation with or without VRP. Concerning the relative expression of PGP, these 16 cases of B-CLL were representative for the whole group of 30 cases. Spontaneous accumulation of Rh123 and VCR varied over a wide range: accumulation of Rh123, by a factor of 11.8; accumulation of VCR, by a factor of 9.7; and accumulation of DNR, by a factor of 3.6. VRP modulated the accumulation of RH123 in 16/30 cases (53%), that of VCR in 69% of cases, and that of DNR in 11% of cases. The maximal VRP-mediated increases in accumulation amounted to factors of 1.3 for DNR, 2.3 for Rh123, and 7.8 for VCR. Spontaneous drug accumulation did not correlate with the extent of chemomodulation. The amount of PGP in B-CLL cells (all cases studied were PGP-positive) did not correlate with drug accumulation or with the extent of VRP-mediated chemomodulation. Thus, high expression of PGP is only partially responsible for defective drug accumulation in B-CLL. Only the degree of chemomodulation by VRP is predictive for the extent of the PGP-related functional drug accumulation defect. Thus, in B-CLL, PGP-independent drug accumulation defects seem to be as important as those mediated by PGP. The extent of this drug accumulation defect varies for the different drugs in the following order VCR > Rh123 > DNR. The relevance of PGP-mediated and -independent drug accumulation defects in vivo may depend to a large extent on the drug being used and on the individual cell type. Both types of defect in drug accumulation are of high importance when regimens include VCR a drug commonly used in second-line chemotherapy of B-CLL. Both defects in drug accumulation may be responsible for intrinsic VCR resistance in B-CLL.
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PMID:Chemomodulation of drugs involved in multidrug resistance in chronic lymphatic leukemia of the B-cell type. 791 20

In order to assess the clinical role of the MDR1 gene in chronic lymphocytic leukemia (CLL), we determined its expression in the leukemic cells of 39 patients with CLL and compared this with other clinical and laboratory parameters. MDR1 RNA expression was detected in 29 patients. MDR1 RNA transcripts were independent of age, treatment status of the patients and the clinical stage of CLL, but correlated with the white blood cell count and MDR2 RNA transcripts. Expression of the tumor suppressor gene p53 was found in 30 out of 37 patients and was associated with MDR1 RNA expression (P < 0.001). Immunocytochemistry using the monoclonal antibody C219 was performed in 38 patients, and in 28 cases, more than 5% of the leukemic cells were found to express cell surface P-glycoprotein. P-glycoprotein expression correlated with the expression of MDR1 RNA (P = 0.048).
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PMID:MDR1 gene expression in chronic lymphocytic leukemia. 791 28

The sensitivities of AML and BCLL blasts to daunorubicin have been determined, using an in vitro (MTT) assay of resistance, and compared with the sensitivities of normal haemopoietic populations and cells of the multidrug-resistant, T-lymphoid line CEM VLB100; The role of the drug-efflux pump, P-glycoprotein, was determined by adding the 'modifier' cyclosporin and by measuring numbers of P-glycoprotein positive cells by immunofluorescence. ID50s for 17 cases of de novo AML varied from 5 to 300 ng/ml giving a median of 105 ng/ml which was similar to the median of 11 normal marrow mononuclear cell preparations (80 ng/ml) but considerably less than the median ID50 of eight blood lymphocyte samples (3500 ng/ml). ID50s for five relapsed and two refractory AML samples ranged from 27 to 240 ng/ml, well within the de novo range: we had obtained presentation samples for two of these and, in both cases, ID50s were lower at relapse. ID50s, however, were raised in seven marrow mononuclear cell populations taken soon after remission induction (ID50 for remission MNC and normal MNC = 200 and 80 ng/ml, respectively); this may reflect either a property of regenerating populations, or an activation of cellular resistance mechanisms following chemotherapy. ID50s for 17 cases of BCLL ranged from 7 to 200 ng/ml with a median of 48 ng/ml which was significantly lower than the ID50 of AML blasts or of blood lymphocytes. Cyclosporin induced less than two-fold reductions in ID50s of blood lymphocytes, marrow mononuclear cells and de novo AML and BCLL blasts despite giving log reversals in resistance in the CEM VLB100 line. This reflected numbers of P-glycoprotein positive cells in our samples, which were high in CEM VLB100 but low in fresh normal or leukaemic cell suspensions. For both de novo AML and BCLL groups, however, the change in ID50, on addition of cyclosporin, was significant. These data imply a minor role for P-glycoprotein in drug resistance of leukaemic blasts. Nevertheless, there was a positive correlation between daunorubicin ID50s in de novo AML and time to remission which confirms that in vitro chemosensitivity assays can provide a useful measure of in vivo resistance.
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PMID:In vitro drug resistance in acute myeloid and chronic B-lymphocytic leukaemic blasts and in normal blood and marrow populations. 793 44

Fifteen samples from 11 patients suffering from chronic lymphocytic leukaemia (CLL; 5 untreated, 6 chemotherapeutically treated) were analysed for their individual gene expression of the multidrug resistance (MDR) associated genes encoding mdr1/P-glycoprotein, mrp, and topoisomerase II alpha/beta-isoenzymes by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. The expression of glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as standard. Thereby, we generally found high mdr1- and mrp-, but low topoisomerase II alpha-mRNA levels. While mdr1 levels of the CLL samples were mostly found to be in the range of values measured in the T-lymphoblastoid, P-glycoprotein MDR cell line CCRF VCR 100, mrp levels were usually found to be 2-4-fold higher compared therewith. This might represent a multifactorial MDR in CLL. In contrast to the low or even absent topoisomerase II alpha gene expression, however, the expression of the topoisomerase II beta gene was generally high in the CLL lymphocytes exceeding the value observed in the cell line CCRF VCR 100 up to 5-fold. mdr1 gene expression correlated significantly with mrp gene expression in samples from patients having received chemotherapy (rs = 0.5833, P < 0.05, n = 10). In two patients the follow-up analysis revealed combined increases in mdr1- and mrp-gene expression levels in the course of the disease.
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PMID:High mdr1- and mrp-, but low topoisomerase II alpha-gene expression in B-cell chronic lymphocytic leukaemias. 795 50

A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp. Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL. The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively. When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004). The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.
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PMID:Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3. 809 47


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