Multidrug resistance-associated protein in acute myeloid leukemia: No impact on treatment outcome

Drug resistance remains a major problem in the treatment of patients with acute myeloid leukemia (AML). Expression of the MDR1 gene in leukemic cells was shown previously to be associated with worse clinical outcome of the patients. The multidrug resistance-associated protein (MRP) has been shown recently to be another protein causing the multidrug resistance phenotype in cell lines, but its impact on clinical outcome in patients with AML remains to be proven. To determine the clinical significance of MRP in patients with de novo AML, we have studied the MRP expression in leukemic cells and its association with both response to induction chemotherapy and survival of the patients. MRP gene expression was determined by immuno-cytochemistry (n = 80) by means of the monoclonal antibodies QCRL-1 and QCRL-3. MRP expression was low, intermediate, and high in 19, 55, and 26% of the patients, respectively. High MRP expression was independent of age and sex of the patients, WBC count, and percentage of blasts. However, high MRP expression was more frequent in the FAB M5 subtype as compared to the other subtypes. MRP expression had no impact on clinical outcome. The complete remission rates were 65, 68, and 63% for patients with low, intermediate, and high expression, respectively. Overall survival was also independent of MRP expression. In contrast, patients with P-glycoprotein-positive AML had lower complete remission rates and shorter durations of survival. These data indicate that MRP is expressed in patients with de novo AML but, in contrast to P-glycoprotein, does not predict for outcome of induction chemotherapy or survival.     

The role of MDR-1 in refractory lymphoma

Although lymphoma is one of the few solid tumours for which chemotherapy can be curative, the treatment of refractory lymphoma remains a major clinical problem. P-glycoprotein (Pgp), the drug efflux pump encoded by the MDR-1 gene is associated with multidrug resistance in several laboratory models of drug resistance, and a number of investigators have attempted to establish a role for Pgp in refractory lymphoma. Despite a considerable variability in the results of these studies investigating Pgp expression in lymphoma, the preponderance of the data suggests that Pgp may at least in part account for drug resistance in this disease. Several clinical trials using Pgp modulating compounds have attempted to reverse the drug resistant phenotype of refractory lymphoma. These studies, although difficult to interpret because of the effect of Pgp modulators on chemotherapeutic drug pharmacokinetics, also suggest a role for Pgp in mediating drug resistance in a subset of patients with refractory lymphoma. Studies with newer Pgp modulating agents with phase III designs will be needed before Pgp modulation can be considered for incorporation into routine oncologic practice.     

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.     

Screening for diabetic retinopathy: the utility of nonmydriatic retinal photography in Egyptian adults

BACKGROUND AND OBJECTIVE: Drug resistance has become a major cause of treatment failure in patients with acute leukemia. P-glycoprotein (Pgp), which is associated with the multidrug resistance (MDR) phenotype, has been reported to be an important predictor of treatment outcome. The aim of this study was to analyze the value of Pgp expression in bone marrow or peripheral blood as a predictor of the response to remission induction chemotherapy as well as the duration of remission in patients with de novo acute myeloid leukemia (AML). DESIGN AND METHODS: We examined the expression of Pgp in 82 patients with de novo AML using an immunocytochemical assay with the C219 monoclonal antibody. RESULTS: Twenty-seven of the 82 patients (33%) were C219-positive in from 1% to 100% of their cells. Thirteen cases (16%) showed a positive reaction in more than 50% of the leukemic cells. Only hyperleukocytosis was significantly associated with higher expression of Pgp. Although 8 of the 13 cases (62%) with more than 50% of cells having Pgp expression were CD34-positive, this association was not statistically significant. A univariate analysis of resistance to induction therapy showed a significantly higher resistance rate in patients with increased Pgp expression (P = 0.01) as well as in those patients with decreased reactivity to myeloperoxidase. The multivariate analysis revealed the independent prognostic value of Pgp expression. C219 reactivity did not have an influence on remission duration. INTERPRETATION AND CONCLUSIONS: Our data indicate that P-glycoprotein expression is a reliable marker of resistance to induction treatment in patients with de novo AML.     

Overexpression of lung-resistance protein and increased P-glycoprotein function in acute myeloid leukaemia cells predict a poor response to chemotherapy and reduced patient survival

We investigated the role of the drug resistance-related proteins LRP, MRP and Pgp and the apoptotic suppressor, bcl-2, in relation to other clinical characteristics, with respect to response and survival in 91 patients with newly diagnosed AML, treated with standard chemotherapy. Multivariate analysis showed that poor response to chemotherapy was associated with increasing age (P=0.0004), LRP expression (P=0.0001) and Pgp function (P=0.015). The significant predictors of both leukaemia-free survival (LFS) and overall survival (OS) were LRP (LFS, P=0.01; OS, P=0.0001), Pgp function (LFS, P=0.0001; OS, P=0.0003) and cytogenetic abnormalities (LFS, P=0.0001; OS. P=0.0005). Patients with the lowest expression of LRP and Pgp function and favourable karyotype (group I) had an LFS of 30.2 months compared to 8 5 months in the group with the highest expression of LRP and Pgp and poor prognosis karyotype (group III, P=0.002). OS decreased from 75.4 months in group I to 7.9 months in group III patients (P <0.0001). Neither MRP nor bcl-2 were significantly associated with chemotherapy response and survival. Correlations were found between increasing expression of LRP and older age (P=0.05) and an unfavourable karyotype (P=0.005), but these variables were independent of each other in analysis of treatment response and patient survival. Our findings suggest that both LRP and Pgp are clinically relevant drug-resistance proteins and it may be necessary to modulate both LRP and Pgp functions in order to reverse the multidrug resistance phenotype in AML.     

Impact of exogenous growth factors on proliferation and chemosensitivity of minimal residual acute myeloid leukemia

The biological heterogeneity of AML makes growth factor augmentation of cell cycle-dependent chemotherapy unlikely to be successful for all patients. Patients whose leukemic cells empirically demonstrate cytokine-induced chemosensitization in vitro might benefit from the concurrent administration of growth factors during consolidation chemotherapy. We have explored the growth factor-dependence and response of primary bone marrow samples from patients with AML at diagnosis, remission, and relapse to determine whether minimal residual leukemia remains growth factor-responsive. Most cases of AML studied at all phases of treatment were growth factor-responsive. Growth factor response of occult remission clonogenic leukemic precursors (CFU-L) was usually concordant with their response at diagnosis. Occult CFU-L were markedly resistant to cytosine arabinoside (median LD99% 20 microM); preincubation with IL-3 or GM-CSF did not significantly improve their ara-C sensitivity. While occult remission CFU-L appear to remain growth factor-responsive, it appears unlikely that growth factor augmentation of consolidation chemotherapy will overcome the important problem of drug resistance of residual leukemia.     

Comparative effects of K+ channel modulating agents on contractions of rat intestinal smooth muscle

In the bench to bedside development of drugs to treat patients with cancer, the common guide to dose and schedule selection is toxicity to normal organs patterned after the preclinical profile of the drug. An understanding of the cellular pharmacology of the drug and specifically the cellular targets linked to the drug's effect is of substantial value in assisting the clinical investigator in selecting the proper dose and schedule of drug administration. The clinical development of ara-C for the treatment of acute myeloid leukemia (AML) provides a useful paradigm for the study of this process. An understanding of the cellular pharmacology, cytokinetics and pharmacokinetics of ara-C in leukemic mice showed substantial schedule-dependency. Exposure to high doses for a short duration (C x t) resulted in a palliative therapeutic outcome. In marked contrast, exposure to lower doses for a protracted period (c x T) was curative. Clinical use of ara-C in patients with AML patterned after the murine experience, c x T approach, has been of limited benefit in terms of long-term disease-free survival. Studies with human leukemia blasts from patients have shown that for the majority of patients, the initial rate-limiting step is membrane transport, the characteristics of which are substantially affected by extracellular drug concentration (dose). This pharmacologic impediment is eliminated with the blood levels attained during the infusion of gram doses (1-3 gm/m2) of the drug (high-dose ara-C, HiDaC) for shorter periods of time, a C x t approach. Clinical confirmation of these pharmacologic observations is evident in the therapeutic efficacy of HiDaC in patients with relapsed or SDaC-refractory acute leukemia. This is further emphasized by the significantly improved leukemia-free survival of patients with AML treated with HiDaC intensification during remission compared to those patients treated with milligram doses typical of SDaC protocols. Thus, the identification and monitoring of important parameters of drug action in tumors during the course of a clinical trial can be of substantial assistance in optimizing drug dose and schedule so as to attain the best therapeutic index.     

Theoretical and practical considerations for the measurement of P-glycoprotein function in acute myeloid leukemia

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.     

Effect on cell kill of addition of multidrug resistance modifiers cyclosporin A and PSC 833 to cytotoxic agents in acute myeloid leukaemia

Multidrug resistance (MDR) mediated by the drug efflux pump P-glycoprotein (Pgp), may cause remission failure and relapse in patients with acute myeloid leukaemia (AML) by extruding cytotoxic agents such as anthracyclines from leukaemic cells thus allowing them to survive. Cell line data suggest that reversal of MDR is possible using modifying drugs such as cyclosporin A (CSA) and its analogue PSC 833. We have investigated the effects on cell kill of the addition of CSA and PSC 833 to daunorubicin, idarubicin, mitozantrone, etoposide and cytarabine in 52 fresh cell samples from AML patients using an MTT assay. Pgp status was determined by using monoclonal antibodies JSB-1 and MRK-16 and by assessment of rhodamine efflux. Although overall each cytotoxic-modifier combination produced significant improvements in cell kill compared to cytotoxic alone (P values ranged from P < 0.001 to P = 0.017), modifiers also produced significant cytotoxicity in their own right, and no consistent difference was seen between responses in Pgp-positive and negative groups. Up to one in three Pgp-positive samples failed to show any improvement in cell kill with the addition of CSA or PSC 833, possibly owing to co-expression of alternative resistance mechanisms not affected by the MDR modifiers. The best responses were seen when PSC 833 was added to idarubicin, with 7 out of 22 Pgp-positive cases (32%) showing five-fold improvements in cell kill or better compared to idarubicin alone. Comparison of equimolar concentrations of the two modifiers in the Pgp positive group failed to show a significant difference in cell kill, though PSC 833 was markedly superior to CSA in a minority of highly responsive samples which demonstrated clear evidence of MDR reversal. Our in vitro data suggest that MDR modifiers such as CSA and PSC 833 could play an important role in the therapy of AML and indicate the need for prospective randomised trials to assess their clinical efficacy.     

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.     

In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.     

Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin

Intrinsic or acquired drug resistance is a major limiting factor of the effectiveness of chemotherapy. Increased expression of either the MRP gene or the MDR1 gene has been demonstrated to confer drug resistance in vitro. In this study, we examined MRP and MDR1 gene expression in a panel of 17 small cell lung cancers (SCLC) xenografted into nude mice from treated and untreated patients using an RT-PCR technique. For some of them, the outcome of the corresponding patients was known and we related MDR1/MRP expression with the xenograft response to C'CAV (cyclophosphamide, cisplatin, adriamycin and etoposide) combined chemotherapy. Fifteen (88%) of the 17 cases of SCLC were found to be positive for either MDR1 or MRP. MRP gene expression was present in 12 (71%) of 17 cases, whereas MDR1 gene expression was detected in eight (50%) of 16 cases. For six SCLC, the survival duration of patients differed, with three patients surviving for more than 30 months after therapy. Among these six turnours, five expressed MRP and/or MDR1. These six xenografts responded to the C'CAV treatment but a significant rate of cure was obtained in only three cases. No obvious relationship was observed between the response to this treatment and MRP or MDR1 expression. However, the remarkably high levels and frequency of MRP expression in some SCLC samples indicate that future developments in chemotherapy of this tumour type should anticipate that drugs which are substrates of MRP may be of limited effectiveness.     

Emergency day hospital assessments

RT-PCR method was used to detect the expression level of multi-drug resistance gene (MDR1) and multi-drug resistance associated protein (MRP) in patients with malignant blood disease, including 19 cases of acute leukemia and 6 cases of multiple myeloma. The results showed: expression level of MDR1 in patients with known clinical drug resistance elevated obviously and was significantly associated with clinical drug resistance (r = 0.612, P < 0.01), which indicated that expression of MDR1 was the main mechanism of clinical drug resistance. There is no obvious relationship between expression level of MDR1 and MRP (r = 0.035, P > 0.05).     

Activity of 2-fluoro-5-methylarabinofuranosyluracil against mouse leukemias sensitive to and resistant to 1-beta-D-arabinofuranosylcytosine

A new pyrimidine nucleoside, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil, previously has been shown to be active against the herpes group of viruses in vitro and in vivo. It is also active against mouse and human leukemic cells in culture and against mouse leukemias L1210, P388, and P815 in vivo. In contrast to other 1-beta-D-arabinofuranosylcytosine (ara-C) derivatives, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil, when given either i.p. or p.o., is highly active against lines of leukemias P815 and L1210 made resistant to ara-C. Against P815/ara-C and L1210/araC, it is more effective than is 5-azacytidine, a drug which has shown definite effectiveness in patients with acute leukemia whose disease has become resistant to ara-C. For these reasons, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil would seem to merit clinical trial in patients with acute nonlymphocytic leukemia whose disease has become resistant to ara-C.     

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.