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Overexpression of Ecto-5′-Nucleotidase Promotes P View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 52 (1997), pp. 953—961 Overexpression of ecto-5 '-nucleotidase promotes P-glycoprotein expression in renal epithelial cells SEVERINE LEDOUX, CHRISTINE LEROY, GRALDINE SIEGFRIED, DOMINIQUE PRIE, PHILIPPE MOULLIER, and GJRARD FRIEDLANDER INSERM U 426, and Department of Physiology, Faculté Xavier-Bichat, Université Denis-Diderot, Paris, and Laboratoire de Thérapie Genique, CHI] Hôtel-Dieu, Nantes, France Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein ex- doxorubicin and epidophyllotoxin [1, 2] out of the tumoral cells pression in renal epithelial cells. P-glycoprotein (P-gp), responsible for and thus prevents drug intracellular accumulation and toxicity. multidrug resistance (MDR) of tumoral cells, is also expressed in apical P-gp has been detected in a variety of normal cells including membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush epithelial cells from small intestine, colon, biliary canalicules and border membranes, could be instrumental in the expression of MDR kidney proximal tubules [11.Inepithelial cells, especially in renal phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vin- proximal tubular cells, P-gp has been detected in apical mem- blastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in branes [1]. This localization suggests a role of P-gp in the which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/—- cells with a low 5'Nu activity <2 extrusion of molecules to the tubular lumen, but the precise pmol/mg protein/mm) and MDCK-5'NU/+ cells, which expressed a high function and the endogenous substrate(s) of P-gp in normal cells level of 5'Nu (Vr,,ax 150 18.5pmol/mg protein/mm). MDCK-5'NU/— remains unknown. It has been suggested that P-gp could be either cells did not display functional expression of MDR. In MDCK-5'NU/+ a chloride channel or a channel regulator involved in cell volume cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/— cells and was dramatically enhanced by P-gp inhibitors. regulation [1, 2], or a nucleotide channel [3]; however, the little This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their information available concerning P-gp regulation is controversial resistance to coichicine (measured by LDH release and MTT assay) as [2, 4—61. compared to MDCK-5'NU/— and was accounted for by increased mem- Inthe kidney, ecto-5'-nucleotidase (5'Nu) is also expressed brane expression of P-gp assessed by Western blot. Neither AMP nor mainly in the apical brush border membranes of proximal tubular adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with of3-methylene-adenosine-diphos- cells [7]. The primary function of this plasma membrane-bound phate (aMADP) or with a blocking anti-5'Nu antibody (1E9) did not ecto-enzyme is to catalyze the dephosphorylation of AMP into blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu adenosine in the extracellular medium [7, 8]. Other functions of antibody 5F/F9, which did not block the enzymatic site, induced a decrease ecto-5'Nu have been described in nonrenal cells, such as enhance- of P-gp activity. Further, incubation of MDCK-5'NU!— cells with condi- tioned medium from MDCK-5'NU/+ cells, which contained significant ment of neurite differentiation [91 or proliferation [8, 10], and amounts of released 5'Nu, induced MDR phenotype. In conclusion: (i) these effects seem partially independent from its enzymatic activ- expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in ity. renal epithelial cells by enhancement of P-gp expression; (ii) this effect Some reports have pointed out a correlation between 5'Nu does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu confered P-gp activity to 5'Nu negative cells. expression and outcome of neoplasia, especially lymphoma [11]. Furthermore, Ujhazy et al have suggested that there is a link between 5'Nu and P-gp expression [12, 13]. These authors have found that tumoral cell lines overexpressing P-gp frequently exhibited high 5'Nu expression. As a working hypothesis, they P-glycoprotein (P-gp) is an adenosine 5'-triphosphate (ATP)-proposed that, in MDR cell lines, 5'Nu serves as an accessory dependent plasma membrane efflux pump, a member of the ATPmolecule, by providing ATP precursors for ATP-dependent trans- binding cassette (ABC) transporter superfamily. Overexpressionporters. However, the possibility that the expression of one of of P-gp in turnoral cells confers a multidrug-resistance (MDR)these proteins promotes the expression of the other was not phenotype. P-gp pumps cytotoxic drugs such as ymca alkaloids,evaluated, On the other hand, the co-localization of the two proteins in the brush border of normal epithelial cells, and their common potential implication in nucleotidc metabolism, suggest that a functional interaction between 5'Nu and P-gp could also Key words: P-glycoprotcin, niultidrug resistance, ecto-5'Nu, transport, exist in normal cells. cytotoxicity. Because 5'Nu activity was shown to modulate renal sodium! Received for publication August 19, 1996 phosphate cotransport that is expressed in brush border mem- and in rcvised form May 30, 1997 branes [14], we asked the question of whether 5'Nu could also Accepted for publication June 2, 1997 modulate the activity of P-gp, another apical transporter, in renal © 1997 by the International Society of Nephrology tubule cells. To answer this question, we transfected in a stable 953 954 Ledoux et al: Ecto-5'-Nu promotes P-gp expression manner MDCK cells, which were deprived of the endogenousthen washed twice with ice-cold buffer (buffer T) containing 137 enzyme, with human 5'Nu. The results show that overexpressionmM NaC1, 5.4 mrvi KC1, 1.2 mM MgSO4, 14 ms'i Tris-HC1, pH 7.4, of 5'Nu induced P-gp expression, and that catabolism of AMP1 mM CaCl2 and 40 jIM dp. Cells were then lysed with 500 jrl per into adenosinc is not responsible for the emergence of MDRwell of 0.5% Triton X100 and incubated during two hours in the phenotype. dark at 4°C. Aliquots of 50 jtl of lysate were transferred to a cuvette with 2 ml of distilled water. R123 was excited at 495 nm METHODS and fluoresence emission was measured at 535 nm in a F2000 Reagents Hitachi spectrofluorometer (Hitachi Corp). Proteins were deter- mined in each well by the Bradford technique modified for Polybrene (hexadimethrine bromide), Rhodamine 123 (R123),microtitration [20]. csJ3 methylene adenosine diphosphate (a/3MADP), coichicine, MIT, levamisole, dipyridamole (dp) and other P-gp inhibitors3H-vinblastine accumulation were obtained from Sigma (Saint-Quentin Fallavier, France). After removal of the culture medium, confluent cells in 24 well Geneticin G-418 sulphate (G418), Hank's balanced saline solu-plates were washed with Hank's balanced saline solution (HBSS) tion and all culture products were purchased from Gibco BRLcontaining 15 mM Hepes and 0.1% glucose, pH 7.4. Cells were (Cergy Pontoise, France). [3H]vinblastine (3H-VBL) and [14Cj5'-then incubated in a humidified atmosphere at 37°C for one hour AMP were provided by Amersham (Les Ulis, France). with the solution of HBSS containing 0.1% BSA, 37 nM 3H- vinbiastine (3H-VBL) with or without 20 jIM dp. Then cells were Cell lines washed three times with ice-cold buffer T and lysed with 300 jIl of 5'Nu-expressing MDCK were obtained using a recombinant0.5% Triton X100. Lysates (250 jIl) were added to the scintillation amphotrophic retroviral vector harboring the human 5'Nu cDNAliquid (2 ml) and radioactivity was quantified in a scintillation under the control of the Moloney long terminal repeat (LTR).counter (EGG instruments; Wallac Berthold Bioanalytical, Evry, The vector was derived from the LXSN backbone [15] in whichFrance). the neomycin cDNA is placed under the SV40 promoter, down- stream from the 5'Nu cassette. Retroviral particles were gener-Cytotoxicity assays ated from the 4rCRIP packaging cell line [16], and individual Cells were seeded in 24 well plates. After the first 24 hours, cells clones were isolated and tested for their ability to transfer 5'Nuwere exposed to 0, 25, 50, or 100 ng/ml of coichicine with or activity onto MDCK. without dp. At 96 hours, cytotoxicity was determined either by Retroviral infections were realized as described [15]. Briefly,lactate dehydrogenase (LDH) release in the medium or by transduced MDCK were obtained after a two hours incubationviability assay based on mitochondrial integrity using MiT [21]. with the retroviral supernatant including polybrene (8 jtg/ml). LDH activity was determined in the cells and in the supernatant Amplification of genetically modified MDCK clones required thewith the LDH Enzyiine kit (BioMérieux, Marcy l'étoile, France). presence in the culture media of 500 tg/ml of G418 (based onThe ratio LDH in supernatant/LDH in supernatant + cells was control untransduced MDCK). Subsequently, three 5'Nu-express-calculated for each well. For the MIT assay, cells were incubated ing MDCK clones were obtained expressing different levels ofin humidified atmosphere at 37°C and 5% CO2 for one hour with exogenous enzyme activity: MDCK-5'NU/— (low), MDCK-0.5 mg/mi of MTT and then the medium was removed. The 5'NU/i (intermediate), and MDCK-5'NU/+ (high). intracellular MTT-formazan complex was solubilized in 400 d of Each transduced MDCK clone was tested for its ability todimethyl sulfoxide and absorbance was measured at 570 nm.
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