Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells

P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vinblastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min). MDCK-5'NU/- cells did not display functional expression of MDR. In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors. This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot. Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity. Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype. In conclusion: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.     

Mechanisms of multidrug resistance in tumor cells and analytical methods for its detection

We reviewed mechanisms of multidrug resistance (MDR) phenotype in tumor cells and evaluated analytical methods for detection of clinical MDR. A well-recognized mechanism of MDR phenotype is the induction and increased expression of P-glycoprotein (P-gp) which is a 170 kDa cellular transmembrane protein encoded by a multidrug-resistance 1 gene (MDR1) and works as a drug efflux pump. Cellular MDR phenotype through P-gp/MDR1 can be detectable at protein level by: (1) using immunohistochemical method, flow cytometric assay and Western blot analysis with monoclonal antibodies against human P-gp, and (2) measuring Rhodamine 123 dye-efflux as a functional assay of P-gp. Molecular knowledge and recent technical progress enable to determine MDR1 gene expression by RT-PCR-based analytical methods as well as conventional quantification methods of gene expression such as Northern blot analysis. In the evaluation of P-gp/MDR1 expression in clinical samples, in which amount of materials was limited, utilization of simple and sensitive methods like competitive RT-PCR assay might be efficacious for its quantitative detection in clinical laboratories. Evidences which showed the positive correlation between the expression of P-gp/MDR1 and clinical resistance or refractoriness of tumor cells to anticancer drugs involved in MDR have been accumulated and support the clinical importance of its detection to circumvent resistance with alternate use of non-MDR drugs.     

Flow cytometric analysis of P-glycoprotein function using rhodamine 123

The MDR1 gene product, P-glycoprotein (P-gp), works as a transmembrane efflux pump for several cytotoxic products, representing a major cause for cancer treatment failure. Rhodamine 123 (Rh123), a low toxic fluorescent probe commonly used to assess mitochondrial bioenergetics in living cells, has also been used to measure the efflux activity of P-gp in both normal and malignant cells. Analysis of variation in cellular fluorescence by measuring the rates of Rh123 influx and efflux, together with the effect of mdr reversing agents, allows the investigation of drug-resistant phenotypes in cancer samples. We have studied the functional activity of P-gp in human leukemic cell lines using flow cytometry, taking into consideration that variables such as Rh123 cytotoxicity, culture conditions, cell membrane integrity, as well as the effect of specific P-gp modulators, can impair the resolution of the Rh123-efflux measurements. The studies show that: (1)optimal non-cytotoxic concentrations of Rh123 which allow appropriate color compensation are in the range of 50-200 ng/ml; (2) life-gating allows accurate measurement on the 50% average rate of Rh123 efflux; (3) relative efficiency of P-gp inhibitors was PSC-833 > cyclosporin A > verapamil; and (4) the presence or absence of fetal calf serum had no effect on the bioavailability of chemosensitizer agents, with the exception of serum-free experiments, which showed a significant decrease in P-gp activity under the presence of PSC-833 (P = 0.05). Hence, we recommend this experimental strategy for clinical practice better to study the cellular drug resistance phenotype.     

The multidrug resistance in human leukemias. Minireview

Leukemia/lymphoma cells, clinically refractory to therapy are often associated with expression of P-glycoprotein (P-gp), which is encoded by the multidrug resistance (MDR) gene, mdr1. Cell lines expressing mdr1 exhibit resistance to several structurally unrelated lipophilic drugs, such as anthracyclines, vinca alkaloids, and epopodophyllotoxins. This MDR can be conferred to drug-sensitive cells mdr1 cDNA transfer. In resistant cells, MDR is characterized by overexpression of P-gp and by the enhanced efflux, and P-gp fluorescence probe, rhodamine 123 (Rh 123). This can be circumvented by addition of certain non-cytotoxic drugs, such as verapamil and cyclosporin A.     

Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay

BACKGROUND: Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1). However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression. PURPOSE: Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression). METHODS: P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated). We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump. Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured. Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases. RESULTS: Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors. Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression. Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug. MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26. MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion. CONCLUSIONS: These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs. This enhanced activity does not seem to be associated with more aggressive disease. Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening. IMPLICATIONS: Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.     

In vitro effect of GF120918, a novel reversal agent of multidrug resistance, on acute leukemia and multiple myeloma cells

Resistance to chemotherapy in multiple myeloma (MM) and acute myeloid leukemia (AML) is frequently caused by multiple drug resistance (MDR), characterized by a decreased intracellular drug accumulation. MDR is associated with expression of P-glycoprotein (P-gp). GF120918, an acridine derivative, enhances doxorubicin cell kill in resistant cell lines. In this study, the effect of GF120918 on MDR cell lines and fresh human leukemia and myeloma cells was investigated. The reduced net intracellular rhodamine-123 (Rh-123) accumulation in the MDR cell lines RPMI 8226/Dox1, /Dox4, /Dox6 and /Dox40 as compared with wild-type 8226/S was reversed by GF120918 (0.5-1.0 microM), and complete inhibition of rhodamine efflux was achieved at 1-2 microM. This effect could be maintained in drug-free medium for at least 5 h. GF120918 reversal activity was significantly reduced with a maximum of 70% in cells incubated with up to 100% serum. GF120918 significantly augmented Rh-123 accumulation in vitro in CD34-positive acute leukemia (AML) blasts and CD38-positive myeloma (MM) plasma cells obtained from 11/27 de novo AML and 2/12 refractory MM patients. A significant correlation was observed between a high P-gp expression and GF120918 induced Rh-123 reversal (P=0.0001). Using a MRK16/IgG2a ratio > or = 1.1, samples could be identified with a high probability of GF120918 reversal of Rh-123 accumulation. In conclusion, GF120918 is a promising MDR reversal agent which is active at clinically achievable serum concentrations.     

Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux

Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a quiescent hematopoietic stem cell (HSC) population in mouse bone marrow, which provides stable, long-term hematopoiesis after transplantation. Rh-123 labels mitochondria with increasing intensity proportional to cellular activation, however the intensity of staining also correlates with the multidrug resistance (MDR) phenotype, as Rh-123 is a substrate for P-glycoprotein (P-gp). To address the mechanisms of long-term repopulating HSC discrimination by Rh-123, mouse bone marrow stem and progenitor cells were isolated based on surface antigen expression and subsequently separated into subsets using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function. We determined the cell cycle status of the separated populations and tested for HSC function using transplantation assays. Based on blocking studies using MDR modulators, we observed little efflux of Rh-123 from HSC obtained from young (3- to 4-week-old) mice, but significant efflux from HSC derived from older animals. A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function. Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or membrane potential indicated that mitochondrial activation is more important than either mitochondrial mass or MDR function in defining HSC in young mice. This conclusion was supported by morphologic studies of cell subsets separated by Rh-123 staining.     

Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.     

Regulation of the function of P-glycoprotein by epidermal growth factor through phospholipase C

Many multidrug-resistant (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp). However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear. Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC. Treatment of the human MDR breast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20-50%. The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Treatment of MDR cells with EGF also had detectable effects on P-gp function. For example, following incubation of MCF-7/AdrR cells with ECF, we observed a consistent decrease in total vinblastine (VBL) accumulation. Kinetic analysis revealed this change to be due to an increase in membrane efflux. The latter was measured by the initial uptake velocity, which was inhibited by EGF. VBL uptake measured at 0-320 sec was inhibited by 20-40%, which was associated with a similar increase in VBL efflux. EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells. These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC.     

Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells

The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane. In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines. Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region. Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus. The same observations have been obtained on a primary culture from a metastasis of human melanoma. Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined. MRP1 also showed Golgi-like localization. The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic. P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells. On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.     

Semi-automated PCR method for quantitating MDR1 expression

The MDR1 gene is involved in drug resistance in many hematopoietic and solid tumors. The Quantitative PCR System 5000 (QPCR-5000; Perkin-Elmer) is a new instrument system that uses electrochemiluminescence to automatically quantitate polymerase chain reaction (PCR) products. A comparative study between radioactively labeled PCR (32P-PCR) and QPCR was performed to analyze the MDR1 gene expression in the drug-resistant (Doxorubicin) cell lines Dox40, Dox6, the parental cell line 8226/S, CEM Dox1 and three acute myeloid leukemia (AML) patient samples. Using the Dox40 and Dox6 resistant cell lines, we compared the sensitivities of QPCR and 32P-PCR. A strong signal was obtained from QPCR at 20 to 25 cycles (which is in the linear range for quantitation), while a weak signal was obtained using 32P-PCR at the same cycle number. Dilution experiments gave better precision with the QPCR than with the radioactive method. AML samples were studied with the MDR1-specific MAbs MRK16 and 4E3, and the efflux function was analyzed using Rh-123 retention in the absence or presence of verapamil. The three samples showed high (D = 0.79), medium (D = 0.52) and negative (D = 0.08) p-glycoprotein (P-gp) levels and correlated with efflux function. The MDR1/beta 2-M mRNA ratios for 32P-PCR were 0.41, 0.40 and 0.12, respectively, and were 0.127, 0.097 and 0.028, respectively, for QPCR. There were significant differences between the samples with high and medium P-gp levels comparing the two methods. Very low levels of MDR1 in CEM Dox1 cells could be detected only by QPCR. In conclusion, QPCR was found to be more reproducible, accurate and sensitive than 32P-PCR.     

P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.     

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells

The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins alpha, theta and epsilon; and (4) gene copy number of PKC-alpha and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-alpha and -theta than wild-type cells and possessed a different cellular localization of PKC-epsilon. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-alpha gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.     

Decreased activity of the multidrug resistance P-glycoprotein in acquired aplastic anaemia: possible pathophysiologic implications

To address a possible impairment of multidrug resistance mechanisms in acquired aplastic anaemia (AA), the functions of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were respectively assessed by rhodamine 123 (Rh123) and daunorubicin (DNR) efflux in peripheral blood lymphocytes from AA patients. The proportion of Rh123-effluxing T cells was significantly decreased in AA, relative to controls. Interestingly, these changes were also present in patients with AA in remission. Conversely, Rh123 efflux in B and natural killer (NK) cells and DNR efflux in peripheral blood lymphocytes were unchanged. These data indicated that P-gp activity was decreased in AA not only during the development of the disease, but also after remission, introducing a new concept on the pathophysiology of AA by suggesting that it may contribute to drug-induced injury to haemopoietic cells in some cases of AA, by increasing the proportion of susceptible cells.     

Abnormal expression of a 170-kilodalton P-glycoprotein encoded by MDR1 gene, a metabolically active efflux pump, in CD4+ and CD8+ T cells from patients with human immunodeficiency virus type 1 infection

Peripheral blood CD4+ and CD8+ T cells from 16 patients with HIV-1 infection, 8 each with CD4+ T cell counts of > 200/mm3 (group I) and with CD4+ T cell counts of < 200/mm3 (group II), and 8 age- and sex-matched controls, were examined for the expression of P-glycoprotein (P-gp), a 170-kDa phosphoglycoprotein encoded by the MDR1 gene, using dual-color flow cytometric analysis. The function of P-glycoprotein was assessed by the accumulation of rhodamine-123 (Rh123) dye in the presence or absence of cyclosporin A (which inhibits Rh123 efflux). A significantly increased proportion of CD4+ T cells from patients with HIV-1 infection expressed P-glycoprotein as compared to controls, resulting in a significantly increased ratio of the proportions of CD4+P-gp+/CD8+P-gp+ cells. The ratio of CD4+P-gp+/CD8+P-gp+ in group II patients was significantly higher (p = 0.02) than in group I patients, suggesting a progressive increase in P-gp expression with the advancement of HIV-1 infection. The proportions of CD4+P-gp+ and CD8+P-gp+ T cells did not differ significantly between those who received AZT and those who were not treated with AZT. Contrary to expectation, both CD4+ and CD8+ T cells from patients accumulated significantly more Rh123 as compared to controls. Furthermore, cyclosporin A failed to increase intracellular accumulation of Rh123 in CD4+ and CD8+ T cells from patients. These data suggest a functionally defective P-gp expression in HIV-1 infection that appears to increase with the progression of HIV-1 infection. A study of a large number of patients with HIV-1 infection is needed to determine the effects of opportunistic infection and antiretroviral therapy on the expression of P-gp and to determine whether the expression of P-gp could serve as another surrogate marker for the progression of HIV-1 infection.     

Internet resources for orthopaedic surgeons

PURPOSE: Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). METHODS: The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. RESULTS: At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in R123 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. CONCLUSIONS: Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.