A kinetic study of Rhodamine123 pumping by P-glycoprotein

The MDR1 P-glycoprotein (P-gp) actively extrudes a wide variety of structurally diverse cytotoxic compounds out of the cell, is widely expressed in the epithelial cells of kidney, liver and intestine, and in the endothelial cells of brain and placenta, and plays an important role in drug resistance. We measured the accumulation of Rhodamine 123 (Rho123), a substrate of P-gp, into a drug sensitive and a drug resistant strain of the human leukemia cell line K562, as function of Rho123 concentration. With the aid of a mathematical transformation, we used the accumulation of Rho123 into the sensitive cells as a surrogate measure for the internal concentration of the probe in the resistant cells, and were thus able to measure the kinetic parameters of drug efflux pumping by P-gp. Drug pumping was half-saturated at an external Rho123 concentration of 7.2E-06+/-1.1E-06 M, and displayed a co-operative behaviour with a Hill number of 1.94+/-0.32. Verapamil could be shown to inhibit Rho123 efflux uncompetitively.     

A kinetic study of Rhodamine123 pumping by P-glycoprotein

The MDR1 P-glycoprotein (P-gp) actively extrudes a wide variety of structurally diverse cytotoxic compounds out of the cell, is widely expressed in the epithelial cells of kidney, liver and intestine, and in the endothelial cells of brain and placenta, and plays an important role in drug resistance. We measured the accumulation of Rhodamine 123 (Rho123), a substrate of P-gp, into a drug sensitive and a drug resistant strain of the human leukemia cell line K562, as function of Rho123 concentration. With the aid of a mathematical transformation, we used the accumulation of Rho123 into the sensitive cells as a surrogate measure for the internal concentration of the probe in the resistant cells, and were thus able to measure the kinetic parameters of drug efflux pumping by P-gp. Drug pumping was half-saturated at an external Rho123 concentration of 7.2E-06 ± 1.1E-06 M, and displayed a co-operative behaviour with a Hill number of 1.94 ± 0.32. Verapamil could be shown to inhibit Rho123 efflux uncompetitively.     

Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

七叶皂苷对P-糖蛋白功能的影响

目的:探讨七叶皂苷时P-糖蛋白功能的影响.方法:构建稳定表达P-糖蛋白的LLC-PK1细胞系,以real-time RT-PCR和Western Blotting分析P-糖蛋白基因mRNA和蛋白表达,共聚焦显微镜观察P-糖蛋白细胞定位,流式细胞术检测细胞内罗丹明123荧光强度.结果:(1)P-糖蛋白在LLC-PK1细胞中稳定高表达;(2)转染细胞中P-糖蛋白定位在细胞膜上;(3)七叶皂苷抑制P-糖蛋白功能.细胞内罗丹明123荧光强度增加123%,但其抑制效果是维拉帕米的30%.结论:七叶皂苷抑制P-糖蛋白功能,但其抑制效果弱于维拉帕米.     

Influence of combinations of digitonin with selected phenolics,terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of β-carotene (40 μM) and sanguinarine (1 μM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5 μM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p < 0.01) and 46% (p < 0.01) in Caco-2, and to 61% (p < 0.05) and 1% (p < 0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Elevation of P-glycoprotein function by a catechin in green tea

The ABC transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of and exposure to lipophilic and amphipathic drugs, carcinogens, toxins, and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since dietary chemicals in green tea (and several other foods) appear to exert anticarcinogenic effects by an unknown mechanism, the constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. We characterized several green tea catechins for their interaction with P-gp and their specific effects on P-gp export activity of several marker substrates. Some of these compounds inhibit the active efflux of the fluorescent markers LDS-751 (LDS) and rhodamine 123 (Rho) with low potency. Remarkably, others of these catechins facilitate the P-gp-mediated transport of LDS without affecting daunorubicin (DNR) transport or Rho. Moreover, (-)epicatechin, though an inhibitor of Rho transport, can significantly enhance the active net transport of another P-gp marker substrate, LDS. This result indicates that (-)epicatechin may bind to and activate an allosteric site that enhances P-gp overall function or efficiency. Such a mechanism of heterotropic allosteric enhancement of P-gp could serve as chemoprotective to many cells and contribute to the purported anticarcinogenic effect of green tea consumption.     

Glutamate up-regulates P-glycoprotein expression in rat brain microvessel endothelial cells by an NMDA receptor-mediated mechanism

The accumulation of glutamate in the extracellular space in the central nervous system (CNS) plays a major part in ischemic and anoxic damage. In this study, we examined the effect of glutamate on the expression and activity of P-glycoprotein (P-gp) in rat brain microvessel endothelial cells (RBMECs) making up the blood-brain barrier (BBB). The level of P-gp expression significantly increased in RBMECs after the treatment of 100 microM glutamate. At this concentration, glutamate also enhanced rat mdr1a and mdr1b mRNA levels determined by RT-PCR analysis. Flow cytometry was used to study P-gp activity by analysis of intracellular rhodamine123 (Rh123) accumulation. Overexpression of P-gp resulted in a decreased intracellular accumulation of Rh123 in RBMECs. Glutamate-induced increase of intracellular reactive oxygen species (ROS) was observed by using the 2',7'-dichlorofluorescein (2',7'-DCF) assay. MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and ROS scavenger N-acetylcysteine obviously blocked ROS generation and attenuated the changes of both expression and activity of P-gp induced by glutamate in RBMECs. These data suggested that glutamate up-regulated P-gp expression in RBMECs by an NMDA receptor-mediated mechanism and that glutamate-induced generation of ROS was linked to the regulation of P-gp expression. Therefore, transport of P-gp substrates in BBB appears to be affected during ischemic and anoxic injury.     

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.     

Lobeline,a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells

Multidrug resistance (MDR) can limit efficacy of chemotherapy. The best studied mechanism involves P-gp (P-glycoprotein) mediated drug efflux. This study focuses on MDR reversal agents from medicinal plants, which can interfere with P-gp. Rhodamine 123 accumulation assay and flow cytometry analysis were employed to screen for P-gp dependant efflux inhibitors. Lobeline, a piperidine alkaloid from Lobelia inflata and several other Lobelia species, inhibited P-gp activity. MDR reversal potential of lobeline could be demonstrated in cells treated with doxorubicin in that lobeline can sensitize resistant tumor cells at non-toxic concentrations. However, lobeline cannot block BCRP (Breast Cancer Resistance Protein) dependent mitoxantrone efflux. Lobeline could be a good candidate for the development of new MDR reversal agents.     

Down-regulation of ABCB1 transporter by atorvastatin in a human hepatoma cell line and in human peripheral blood mononuclear cells

PURPOSE: The effect of atorvastatin, an HMG-CoA reductase inhibitor, on expression and activity of the drug transporter ABCB1 in HepG2 cells and peripheral blood mononuclear cells (PBMCs) was examined. METHODS: Localization and expression of ABCB1 in hepatocytes was examined by indirect immunofluorescence. Expression of ABCB1 mRNA and ABCB1 activity were examined in atorvastatin-treated and control cells and PBMCs using real-time PCR and Rhodamine 123 efflux assay. RESULTS: Immunohistochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi. Atorvastatin at 10 and 20 microM up-regulated ABCB1 expression resulting in a significant 1.4-fold increase of the protein levels. Treatment of HepG2 cells with 20 microM atorvastatin caused a 60% reduction on mRNA expression (p<0.05) and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p<0.01) by flow cytometry. Correlation was found between ABCB1 mRNA levels and creatine kinase (r=0.30; p=0.014) and total cholesterol (r=-0.31; p=0.010). CONCLUSIONS. Atorvastatin leads to decreased ABCB1 function and modulates ABCB1 synthesis in HepG2 cells and in PBMCs. ABCB1 plays a role in cellular protection as well as in secretion and/or disposition, therefore, inhibition of ABCB1 synthesis may increase the atorvastatin efficacy, leading to a more pronounced reduction of plasma cholesterol.     

Relevance of multidrug resistance 1 and P-glycoprotein to drug resistance in patients with systemic lupus erythematosus

Although corticosteroids and immunosuppressants are widely used for the treatments of various autoimmune diseases such as systemic lupus erythematosus (SLE), we often experience patients with SLE who are resistant to these treatments. P-glycoprotein (P-gp) of membrane transporters, a product of the multiple drug resistance (MDR)-1 gene, is known to play a pivotal role in the acquisition of drug resistance to chemotherapies in malignancy. However, the relevance of MDR-1 and P-gp to resting and activated lymphocyte, major targets of the treatments in autoimmune diseases, remains unclear. We found that peripheral lymphocytes in patients with SLE express P-gp on the surface and its expression is highly correlated with disease activity. P-gp on lymphocytes is induced by not only genotoxic stresses but also activation stimuli such as cytokines, resulting in active efflux of corticosteroids from cytoplasm of lymphocytes, resulting in drug-resistance and high disease activity. However, the addition of P-gp antagonists such as ciclosporin A and inhibitors of P-gp synthesis successfully reduce efflux of corticosteroids from lymphocytes in vitro and these results imply that P-gp antagonists and P-gp synthesis inhibitors could work in order to overcome drug-resistance in vivo. Therefore, we propose that the measurement of P-gp on lymphocytes is a useful marker to indicate drug resistance and requirement of antagonists and/or intensive treatments to overcome drug resistance in active SLE patients.     

Synthesis,in silico and in vitro studies of new 1,4-dihydropiridine derivatives for antitumor and P-glycoprotein inhibitory activity

P-glycoprotein (P-gp) is one of the cell membrane pumps which mediate the efflux of molecules such as anticancer drugs to the extracellular matrix of tumor cells. P_gp is a member of the ATP-binding cassette (ABC) transporter family that is implicated in cancer multidrug resistance (MDR). Since MDR is a contributor to cancer chemotherapy failure, modulation of efflux pumps is a viable therapeutic strategy. In this study, new synthetic 1,4 dihydropiridine (DHP) derivatives containing thiophenyl substitution were tested as inhibitors of P-gp. Efflux assay was conducted to evaluate the intracellular accumulation of Rhodamine123 (Rh123) as a pump substrate. MTT assay, cell cycle analysis and in silico methods were also examined. Flow cytometric analysis revealed that synthetic DHP derivatives (15 µM) increased intracellular concentration of the substrate by 2–3 folds compared with verapamil as a standard P-gp inhibitor. MTT assay on EPG85-257P and its drug-resistant EPG85-257RDB cell line revealed antitumor effects (30–45%) for new DHP derivatives at 15 µM following 72 h incubation. However, MTT test on normal cell line showed negligible toxic effects. Finally combination of synthetic derivatives with doxorubicin showed that these compounds decrease IC50 of doxorubicin in resistant cell lines from 9 to 1.5 µM. Sub-G1 peak-related apoptotic cells showed a stronger effect of synthetic compounds at 5 µM compared with verapamil. Molecular dynamic results showed a high binding affinity between DHP derivative and protein at drug binding site. Findings of these biological tests indicated the antitumor activity and P-gp inhibitory effects of new 1,4-DHP derivatives.     

Quinoline derivative KB3-1 potentiates paclitaxel induced cytotoxicity and cycle arrest via multidrug resistance reversal in MES-SA/DX5 cancer cells

AIMS: The resistance to chemotherapeutic drugs is a major problem for successful cancer treatment. Multidrug resistance (MDR) phenotype is characterized by over-expression of P-glycoprotein (P-gp) on the cancer cell plasma membrane that extrudes drugs out of the cells. Therefore, novel MDR reversal agents are desirable for combination therapy to reduce MDR and enhance anti-tumor activity. Thus, the present study was aimed to evaluate the potent efficacy of novel quinoline derivative KB3-1 as a potent MDR-reversing agent for combined therapy with TAX. MAIN METHODS: MDR reversing effect and TAX combined therapy were examined by Rhodamine accumulation and efflux assay and Confocal immunofluorescence microscopy, Western blotting, TUNEL assay, and cell cycle analysis. KEY FINDINGS: The discovery of quinoline-3-carboxylic acid [4-(2-[benzyl-3[-(3,4-dimethoxy-phenyl)-propionyl]-amino]-ethyl)-phenyl]-amide (KB3-1) as a novel MDR-reversal agent. KB3-1 significantly enhanced the accumulation and retention of a P-gp substrate, rhodamine-123 in the P-gp-expressing MES-SA/DX5 uterine sarcoma cells but not in the P-gp-negative MES-SA cells at non-toxic concentrations of 1 microM and 3 microM. Similarly, fluorescence microscopy observation revealed that KB3-1 reduced the effluxed rhodamine-123 expression on the membrane of MES-SA/DX5 cells. Consistent with decreased P-gp pumping activity, confocal microscopic observation revealed that KB3-1 effectively diminished the expression of P-gp in paclitaxel (TAX)-treated MES-SA/DX-5 cells. Furthermore, Western blotting confirmed that KB3-1 reduced P-gp expression and enhanced cytochrome C release and Bax expression in TAX treated MES-SA/DX-5 cells. In addition, KB3-1 enhanced TAX-induced apoptotic bodies in MES-SA/DX5 cells by TdT-mediated-dUTP nick-end labeling (TUNEL) staining assay aswell as potentiated TAX- induced cytotoxicity, G2/M phase arrest and sub-G1 apoptosis in MES-SA/DX5 cells but not in MES-SA cells. Interestingly, KB3-1 at 3 microM had comparable MDR-reversal activity to 10 microM verapamil, a well-known MDR- reversal agent. SIGNIFICANCE: KB3-1 can be a MDR-reversal drug candidate for combination chemotherapy with TAX.     

Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (P_s) of passive diffusion and rate constants of active transport (k_T) by P-gp as a result of different experimental conditions. The P_s value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the k_T value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in k_T values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.     

Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells

The (−)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (−)CLA (1 or 5 μmol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (−)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 μmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (−)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 μmol/L had significantly lower intracellular uptake than (−)CLA with a ratio[(−)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 μmol/L. Besides, in the non-induced KB cells, (+)CLA(5 μmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (−)CLA(5 μmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (−)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs.     

Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells.

Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.