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J Lankelma

Publications and source records attributed to J Lankelma.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Reversal of multidrug resistance by valinomycin is overcome by CCCP.

Reversal of P-glycoprotein-mediated multidrug resistance by valinomycin is overcome by the proton ionophore, CCCP. This effect, a complete suppression of the 5- to 10-fold valinomycin-induced reversal ("re-reversal"), exhibits a sharp extracellular potassium concentration ([K+(0)]) dependence. It is observed at [K+(0)] > 2-4 mM and not at [K+(0)] greater than or equal to 2 mM, in the case of the fluorescent substrates rhodamine 123 and daunorubicin. The fact that "re-reversal" is detected only for the combination of CCCP with valinomycin raises the possibility that a direct interaction between these ionophores may explain the phenomenon. We show spectroscopic evidence of such an interaction, with a [K+(0)]-dependence similar to that of the "re-reversal." These data suggest that the reversal of P-glycoprotein activity by valinomycin can be compromised by anionic compounds such as CCCP due to complex formation. More generally, molecular interactions involving P-glycoprotein substrates or reversing agents may significantly affect drug accumulation in multidrug resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells.

This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells. Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp. In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time. For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)). The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content. At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil. When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations. The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Multidrug resistance-modifying components in human plasma with potential clinical significance.

P-Glycoprotein (P-gp) and multidrug resistance protein (MRP) are plasma membrane associated proteins which can confer multidrug resistance (MDR) to cancer cells by lowering the intracellular amount of drug. Although clinical trials with MDR-reverting agents have been initiated, not much attention has been paid to blood components which may modulate the activity of P-gp or MRP. The present investigation was performed to identify and characterize blood components which may influence the drug content and the drug cytotoxicity of MDR cells. Human plasma, from healthy volunteers, was tested for its effects on the daunorubicin (DNR) accumulation and cytotoxicity in the MDR cell lines SW-1573/2R160 (2R160) and GLC4/ADR containing P-gp and MRP, respectively. The data were compared to the effects observed in wild-type cells. MDR-modifying plasma components were isolated by extraction procedures and characterized using ultrafiltration, high-performance liquid chromatography (HPLC) and mass spectrometry. An increase in the proportion of plasma in the culture medium led to a reduction of the ratio between the DNR content of wild-type and corresponding MDR cells. At 100% plasma we observed an increase in the cellular DNR content of 2R160 cells, which was 10-30% (median 18%) of the maximum possible increase induced by well-known MDR-reverting agents, such as verapamil (for GLC4/ADR cells: 10-20%, median 15%). The DNR cytotoxicity in MDR cells also increased with an increasing amount of plasma included in the culture media. There was neither an increase in the cellular DNR content nor an effect on the DNR cytotoxicity in wild-type cells. Plasma extract analysis by HPLC showed a major peak which increased the DNR content of MDR cells. The HPLC column retention time of this fraction was identical to that of a standard of cortisol and it was further confirmed to be cortisol using mass spectrometry. Moreover, inclusion of a standard of cortisol in culture media induced a similar effect. We analyzed the data for one of the plasma pools and found that blood cortisol was responsible for the MDR-modulating effect only for 35% of the effect of 100% plasma. Other plasma components were responsible for the remaining modulation effect on MDR cells. In conclusion, the DNR pumping activity of P-gp and MRP is inhibited by human plasma, resulting in 10-30% of the maximum possible increase in cellular drug content. Based on cellular pharmacokinetic calculations this percentage will most likely increase at clinical levels of drug resistance (reaching 40-50%). In one sample blood cortisol accounted for 35% of the effect of plasma on the DNR content in MDR 2R160 cells. These data show the need for additional studies to test plasma samples for their MDR modulating effects before the administration of MDR-reverting agents in chemotherapy. The data suggest that the effectiveness of chemotherapeutic drugs may be enhanced when administered in accordance with the circadian peak of endogenous corticoids.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

The P-glycoprotein-mediated relative decrease in cytosolic free drug concentration is similar for several anthracyclines with varying lipophilicity.

We have used a new methodology to measure the activity of P-glycoprotein (P-gp) in multidrug-resistant (MDR) tumor cells. This activity leads to a lower cytosolic concentration and a lower cytotoxicity of the classical anthracyclines, daunorubicin (DNR), and doxorubicin (DOX). It has been reported that the anthracycline idarubicin (IDA), which is more lipophilic, has a higher clinical efficacy in acute myeloid leukemias (AML) than DNR and DOX. In our study, the aim was to determine for a series of anthracyclines how variations in the passive drug influx rate as well as the P-gp-mediated drug pumping rate affect their cytosolic free drug concentrations and how these parameters are related to drug cytotoxicity. We selected six anthracyclines: DOX, DNR, epidoxorubicin (EPI), IDA, cyano-morpholino-doxorubicin (CMD), and carminomycin (CAR), ordered according to their increasing octanol/PBS buffer concentration ratios, respectively. To measure the passive permeation coefficient, the P-gp-mediated drug pumping rate, and the cytosolic free drug concentration, we used a flow-through system in which cells were exposed to a flowing medium containing drugs. We used the MDR P-gp-containing cell line KB8-5. It was shown that the passive drug permeation coefficient as well as the drug pumping rate of P-gp increased with increasing lipophilicity in this series of anthracyclines. The cytosolic free drug concentration was lowered by P-gp to a similar extent in KB8-5 cells for all drugs tested (40-50% of the extracellular drug concentration). CMD, IDA, and CAR had lower IC50 values and lower resistance factors in comparison to DOX, DNR, and EPI. Verapamil reversed the resistance for all anthracyclines tested. In conclusion, for several anthracyclines the activity of P-gp leads to a similar relative decrease in the cytosolic free drug concentration; consequently, the reported lower resistance factor of IDA compared to that of DNR is not due to the inability of P-gp to export IDA from cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Correlation between functional and molecular analysis of mdr1 P-glycoprotein in human solid-tumor xenografts.

The contribution of P-glycoprotein (Pgp) to multidrug resistance in human solid tumors is generally estimated from bulk mRNA measurements or immunohistochemistry, while direct measurement of the effect of Pgp on intracellular drug concentrations has not been pursued. We investigated the feasibility and sensitivity of a method for probing Pgp-mediated drug transport in cells isolated from solid tumors, using xenograft models. Human tumor xenografts (XG) were grown by s.c. injection of Pgp-expressing cell lines 2780AD, BRO/mdr1 and KB8-5. Tumor uptake of doxorubicin (DOX) after administration of DOX to the mice was determined. XG from untreated mice were enzymatically dissociated. The effect of the Pgp modulator bepridil on steady-state cellular daunorubicin (DNR) and vincristine (VCR) accumulation and chemosensitivity of these XG cells was compared with its effects in the cell lines (CL). mdr1 mRNA and Pgp (by flow cytometry) were measured. Also, the dependence on intracellular ATP concentration, [ATP]i, of the modulator effect was determined in intact KB8-5 cells. The results showed that i.v. administration of DOX to the mice led to lower DOX levels in the Pgp-expressing XG than in the "sensitive" XG, suggesting the presence of an in vivo functional Pgp in these XG tumor models. Dissociated, viable XG cells appeared to have ATP levels sufficient to sustain Pgp-ATPase-coupled drug transport. This was inferred from experiments using KB8-5 CL, which showed half-maximal inhibition of DNR transport at an [ATP]i of 1 to 2 mM. The effect of bepridil on DNR and VCR accumulation and chemosensitivity in the XG cells was in accordance with the XG expression of mdr1/Pgp. In KB8-5 XG cells, Pgp function was hardly detectable, in accordance with decreased mdr1/Pgp expression in vivo. In conclusion, Pgp activity can be determined in freshly dissociated XG human tumor cells. The results obtained with the more necrotic KB8-5 XG may represent some of the interpretation problems arising when low levels of Pgp expression occur within a heterogeneous cell population, such as may be expected in clinical human tumors. Also our results indicate that Pgp activity may be impaired in vivo at [ATP]i below 2 mM, which are realistic values for human solid tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Multidrug resistance proteins and other drug transport-related resistance to natural product agents.

The term multidrug resistance is defined in this article as cellular resistance to anticancer agents due to a decreased concentration of active drug at the target sites that is caused by increased metabolism or altered transport or routing of the active drug species. Resistance related to alterations in the drug targets or apoptotic pathways is not discussed. Until recently multidrug resistance was associated almost exclusively with p-glycoprotein (Pgp)-overexpression. However, other non-Pgp-related mechanisms have been tracked down. It has been shown that transfection of the gene that encodes a novel drug transport protein, the multidrug resistance protein, induces cross-resistance for many multidrug resistance drugs as well as active transport of daunorubicin from tumor cells. Surprisingly, it has also been found that multidrug resistance protein mediates transport of negatively charged species that are not classic multidrug resistance drugs, such as leukotriene C4 and other glutathione conjugates as well as negatively charged dyes. It was therefore suggested that multidrug resistance protein is identical with the multispecific organic anion transporter. The transport rate of several positively charged drugs (vincristine, rhodamine-123, daunorubicin) by multidrug resistance protein appeared to be dependent on the cellular glutathione levels. Multidrug resistance protein seems to be constitutively expressed in normal tissues at a low level with few tissues having higher expression. Multidrug resistance protein overexpression in in vitro-selected MDR cell lines occurs relatively frequently in lung cancer and leukemia cell lines and often precedes Pgp overexpression. Differential expression has been demonstrated in tumor samples, which suggests a role in resistance to chemotherapy in at least certain tumor types. Modulation studies of multidrug resistance protein activity are still scarce. Other non-Pgp, non-multidrug resistance protein multidrug resistance mechanisms probably exist but have not been identified at the molecular level as yet.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Daunorubicin efflux against a concentration gradient in non-P-glycoprotein multidrug-resistant lung-cancer cells.

Multidrug-resistant, human non-small-cell lung carcinoma SW-1573/2R120 (2R120) cells, not containing the drug efflux pump P-glycoprotein (Pgp), have reduced initial daunorubicin (DN) accumulation rates and decreased cellular steady-state drug concentrations. Previously we found indications of the presence of a plasma membrane "vacuum cleaner", pumping DN directly from the membrane, and reported evidence of active DN pumping using digitonin. Further evidence of active DN pumping is now provided via a different methodology and the active drug pump flux is estimated. Cells were exposed to a flowing medium containing the cytotoxic agent DN. After reaching a steady state, in which net DN uptake equals net DN efflux, high concentration pulses of vincristine (VCR) were injected into the flowing medium. A rapid increase in cellular DN content was observed, while only a minimal effect was seen in SW-1573 wild-type cells. After passage of the VCR pulse, the extra accumulated DN was effluxed against a concentration gradient. Upon increasing the VCR concentration, a maximum pump inhibition was reached which was similar to the effect of cellular energy depletion. Similar effects were observed for Pgp-containing SW-1573/2R160 (2R160) cells as well as non-Pgp MDR human small-cell lung carcinoma GLC4/ADR cells. With increasing extracellular DN concentrations, saturation of the VCR-induced DN influx was observed (DN medium concentration 2.5 microM at 1/2 Vmax). At an extracellular DN concentration of 5 microM, higher concentrations of VCR were needed to reach the maximum effect in 2R120 cells than at 0.5 microM DN. This is an indication of competitive interaction between DN and VCR for the putative DN efflux system. In summary, we found indications of inhibition of active DN efflux by VCR and DN efflux against a concentration gradient in non-Pgp MDR 2R120 and GLC4/ADR cells. These features are consistent with the presence of a multidrug transporter, different from Pgp, in the plasma membrane of these cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Competitive inhibition by genistein and ATP dependence of daunorubicin transport in intact MRP overexpressing human small cell lung cancer cells.

In several multidrug resistant tumor cell lines without overexpression of P-glycoprotein (non-Pgp MDR), a decreased accumulation of drugs has been shown to contribute to resistance. We have recently reported that daunorubicin (DNR) accumulation was decreased in the multidrug resistance-associated protein overexpressing GLC4/ADR non-Pgp MDR small cell lung cancer cell line due to an enhanced energy-dependent efflux which could be inhibited by the isoflavonoid genistein. The purpose of this work was 2-fold: (i) to investigate the mechanism by which genistein inhibits the DNR efflux in the GLC4/ADR cells; and (ii) to characterize the dependence of DNR transport on ATP concentration in intact GLC4/ADR cells. The active transport of DNR in GLC4/ADR cells appeared to be a saturable process with an apparent Km of DNR of 1.4 +/- 0.4 microM. Genistein increased the apparent Km value of DNR, suggesting that this agent is a competitive inhibitor of DNR transport. These data provide additional evidence that energy-dependent DNR transport in GLC4/ADR cells is a protein-mediated process. In addition, genistein decreased cellular ATP concentration in a dose-dependent manner in sensitive as well as in resistant cells. Marked inhibition of DNR transport activity in intact GLC4/ADR cells was found when cellular ATP concentration was decreased below 2 mM by sodium azide or 2-deoxy-D-glucose. Thus, since DNR transport in intact GLC4/ADR is already inhibited at modest cellular ATP depletion, a limitation in ATP supply might open ways to make MDR cells more susceptible to drug toxicity.

Adenosine Triphosphate↗
PubMed1994

The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump.

The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant. The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient. Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells. We conclude that MRP is a plasma membrane drug-efflux pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles.

Most of the multidrug resistant human tumor cell lines overexpress the MDR1 gene product P-glycoprotein (P-gp) which is believed to function as an energy-dependent drug efflux pump. Here we describe a novel method that allows the kinetic characterization of P-gp-mediated active drug transport. This method is based on the fluorescence quenching of anthracyclines transported into DNA-loaded plasma membrane vesicles. The uptake of daunorubicin (DNR) into the plasma membrane vesicles was saturable in terms of the extravesicular DNR concentration with a Km of 1.5 +/- 0.1 microM. This transport occurred by a cooperative process with a Hill coefficient close to 2 for DNR. A model is discussed in which P-gp pumps two molecules of drug per turnover.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The multidrug-resistance-reverser verapamil interferes with cellular P-glycoprotein-mediated pumping of daunorubicin as a non-competing substrate.

We examined P-glycoprotein-mediated verapamil transport, using two drug-sensitive and multi-drug resistant cell-line couples, i.e. A2780, 2780AD and SW-1573, SW-1573/1R500. The interaction of 3H-labeled verapamil with cells was measured using a flow-through system. The verapamil-containing medium was pumped over the cells and monitored on-line for radioactivity. In the P-glycoprotein-expressing cells, verapamil accumulation was increased by vinblastine and some known multidrug resistant (MDR) modifiers. Subsequent removal of these modifiers caused release of verapamil into the medium against a verapamil concentration gradient. In this manner, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein. Using the flow-through system, we also exposed the cells to flowing culture medium containing daunorubicin, and measured the inhibition of daunorubicin efflux by verapamil. We found that, although the active efflux of daunorubicin was maximally blocked by verapamil short-term, longer-term active efflux of daunorubicin resumed. At a daunorubicin concentration in the flowing medium of 5 microM, increasing the verapamil concentration resulted in the same short-term effects, but in a significantly longer period of a maximal inhibition of daunorubicin efflux from the cells. At a daunorubicin concentration of 20 microM, increasing the verapamil concentration affected neither the short-term nor the long-term effects. These and other observations are in agreement with a model in which daunorubicin and verapamil are non-competing substrates for P-glycoprotein. In conclusion, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein and that verapamil and daunorubicin are non-competing substrates for P-glycoprotein. Consequently, the effectiveness of verapamil as an MDR antagonist may be compromised because it is extruded by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

A plasma membrane 'vacuum cleaner' for daunorubicin in non-P-glycoprotein multidrug-resistant SW-1573 human non-small cell lung carcinoma cells. A study using fluorescence resonance energy transfer.

A multidrug resistant (MDR) human non-small cell lung carcinoma cell line, SW-1573/2R120 (2R120), not containing the drug-efflux pump P-glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane. Earlier, reduced initial DN-uptake rates and lower cellular DN steady-state concentrations were found for this cell line, when it was compared to the SW-1573 wild-type cell line. This finding was an indication for the presence of another cellular drug-efflux pump. However, we found similar DN-efflux rates in drug-free medium for the two cell lines, while for Pgp-containing MDR SW-1573/2R160 (2R160) cells the efflux rate was increased compared to wild-type cells. In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence-resonance-energy transfer. For this purpose, the plasma-membrane probe 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was chosen since, because of the overlap between the emission spectrum of TMA-DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA-DPH to DN. Cells were loaded with TMA-DPH and, after addition of 10 microM DN, the TMA-DPH fluorescence was quenched. Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp-containing) cells and in the SW-1573 wild-type cells (21 +/- 1% and 20 +/- 2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14 +/- 1%). This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells. We interpret these data to be the result of a 'vacuum-cleaner' pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.

Biological Transport↗