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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 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 /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- 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 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- 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% , 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. generate replication-competent retroviruses [17], and was re- vealed to be negative. Determination of 5'Nu activity Two other MDCK transfected cell lines were used in this study: The activity of ecto-5'Nu was determined on intact cells using a one obtained by transfection with human MDR1 (kind gift frommethod adapted from Gentry and Olson [22] as previously Dr. M.M. Gottesman [18]) and the other transfected with thedescribed [23]. Briefly, after removal of culture medium, cells Na/So4 cotransport, Na/Sil (kind gift from Dr. J. Biber [19]). were washed once with HBSS containing 1 m levamisole, and All cell lines were grown in humidified atmosphere at 37°Cthen incubated at 37°C in the same solution (500 j.d/well) with containing 5% C02, in a medium made of a 1:1 (vol/vol) mixture[14C]5'-AMP (0.02 jICi/mi), unlabeled 5'-AMP (at adequate of 1-lam's F12 and Dulbecco's modified Eagle's medium contain-concentrations) and 20 jIM dp which abolishes cellular uptake of ing 15 mvi Hepes, 21.5 m'vi HCO3, 1 mrvi Na pyruvate, 1% of MEM generated adenosine [141. For determination of nonspecific activ- amino acid mixture, 4 m'vi L-glutamine, 50 U/mI penicillin, 50ity, i0— M a methylene adenosine diphosphate (a/3MADP) was ig/ml streptomycin, 50 JiM Na selenite and 5% newborn calfadded in one well for each condition. At this concentration, serum. Medium was changed three times a week and cells wereaMADP totally inhibits 5'Nu activity [7] since the activity is not suhcultured weekly. The splitting ratio was I to 2, 4 or 6 fordifferent from medium alone. After five minutes of incubation, MDCK-5'NU/-, MDCK-5'NU/i and MDCK-5'NU/+ cells, re-450 1d of supernatant were mixed with 100 jtl of 0.15 M ZnSO4 spectively. All experiments were performed on confluent cellsand 100 jil of 0.15 M Ba(OH)2. The mixture was vigorously shaken after four days of culture. and centrifuged (15000 )< g; 2 mm) in order to separate adenosine from 5'-AMP. Supernatants containing adenosine were counted Rhodamine 123 accumulation in a liquid scintillation counter and cells were lysed by 0.5% Triton Confluent cells grown in 24 well trays were loaded for 18 hoursX100 (500 jil/well) to determine protein content. in the culture medium with 10 jLM rhodamine (R123) with or The activity of 5'Nu in the supernatant was determined by the without dipyridamole (dp) or other MDR inhibitors. Cells weresame method except that reagents were added directly in the Ledoux et al: Ecto-5'-Nu promotes P-gp expression 955

supernatant and 5'Nu activity was mesured after 20 minutes of Table 1. 5Nu activity and R123 accumulation (with or without dp) in incubation. MDCK cell lines MDCK-5'NTJ/- MDCK-5'NU/i MDCK-5NU/+ Western blot 5'Nu activity For the preparation of membrane fractions, cells cultured in pmollmgprot/min <2 54.0 11.5 150.0 18.5° petri dishes were washed twice with cold PBS (10 msi Na2HPO4, KmpM — 396±45 459±30 1.4 mrvi KH2PO4, 137 mivi NaCl, 2.7 mrvi KCI), pelleted, and there R123 accumulation homogeneized by sonication in 300 jxl PBS containing 2 mM FAU/mg protein control 10.55 1.54 4.550.25c 0.31 ()03h ethylenediaminetetraacetic acid (EDTA) and 1 mrvi phenylmeth- dp 11.99 1.83 7.00 034d 2.11 026w ylsulforyl fluoride (PMSF). Plasma membrane fractions were The results are expressed as mean SCM of 3 different experiments for obtained by ultracentrifugation of homogenates (100,000 g for 30 5'Nu parameters and 20 different experiments for R123 accumulation mm at 4°C). The pellets were dissolved by sonication in lysis buffer ap < 0.01 vs. MDCK-5'NU/i (100 mivi Tris pH 7.4, 300 msi NaCl, 2 mst EDTA, 1% Triton hp < 0.001 vs. MDCK-5'NU/i X-100, 0.1% SDS, 5 mi iodoacetamide, 1 jLM leupeptin, 1 mM °P < 0.001 vs. MDCK-5'NU/-- PMSF). Protein contents were determined by the method of dp < 0.001 vs. control Bradford [20] and sodium dodecyl sulfate—polyacrilamide gel electrophoresis (SDS-PAGE) was performed as described [241. Then, the proteins were transferred onto a nitrocellulose mem-dp had no effect on R123 accumulation, at any concentration brane. The nonspecific sites were blocked by incubation at roomtested, in MDCK-5'NU/—. temperature for one hour in PBS—Tween 0.1% (PBST) with For the subsequent experiments, we chose the concentration of 7.5% nonfat dairy milk powder. The membrane was then incu-20 jiM dp that induced a sevenfold increase in R123 accumulation bated overnight at 4°C with the specific monoclonal anti-P-gpin MDCK-5'NU/+. antibody C219 (0.75 jig/mI; Valbiotech, Paris, France) in the same The accumulation of 3H-VBL, another substrate of P-gp [18, milk solution. Specific antigen-antibody complexes were detected31, 32], was measured in MDCK-5'NU/— and MDCK-5'NU/+. by enhanced chemoluminescence (ECL kit, Amersham, Les Ulys,As for R123 accumulation, MDCK-5'NU/— retained more 3H- France) after incubation for two hours in PBST-7.5% milkVBL than MDCK-5'NU/+ and dp increased significantly 3H- containing horseradish peroxidase secondary antibody. VBL retention only in the MDCK-5'NU/+ cell line (Fig. 2). As a control, we verified that transfection of MDCK cells with Data presentation another apical membrane protein did not affect P-gp activity. We R123 accumulation was expressed as fluorescence arbitraryused a MDCK cell line transfeeted with the Na/SO4 cotransporter (NaSi-1; gift from Dr. J. Biber [19]). These cells did not express units per mg of protein (FAU/mg protein), 3H-VBL accumulation as pmol/mg protein. 5'Nu activity was expressed as nanomoles of5'Nu activity and their P-gp activity was not enhanced as com- adenosine per mg protein per minute for cells and nanomoles ofpared to their appropriate controls (R123 accumulation: 0.54 0.05 vs. 0.37 0.04 FAU/mg protein; 3H-VBL accumulation: adenosine per well per minute for supernatants. Cytotoxic assays were expressed as percent of control. 2.45 0.47 vs. 1.19 0,04 pmol/mg protein, N =3). Results were presented as mean SEM of 3 to 20 differentEffect of P-gp inhibitors on R123 accumulation experiments (N) in which triplicates were obtained. Statistical analyses were performed by unpaired t-test or by one In order to verify that the pharmacological properties of P-gp in way or two way analysis of variance (ANOVA) when appropriate.MDCK cells resembled those described in other cell lines, we tested the effect of other MDR inhibitors on R123 accumulation RESULTS in MDCK-5'NU/+ cells: 20 J.LM amiodarone [33], 2.5 jiM BIBW 22 [30], i0 M verapamil [1, 21, 20 jiM reserpin [34], 10 M 5'Nu activity cyclosporine A [2] increased significantly R123 accumulation. In Three MDCK cell lines with different 5'Nu activities werecontrast, 200 jIM genistein [35], a specific inhibitor of multidrug obtained: MDCK-5'NU/— with a very low activity, MDCK-5'NU/irelated protein (MRP), had no effect on R123 retention (Table 2). with a moderate activity and MDCK-5'NU/+ with a high activity (Table 1). The very low 5'Nu activity in MDCK-5'NU/-- did notCytotoxicity of coichicine allow us to calculate the apparent Km value. Affinities were not The cytotoxicity of colchicine depends on cellular accumulation different in the two other cell lines (Table 1). of the drug and is inversely proportional to the expression of P-gp that actively extrudes this drug [2, 36]. Determination of cytotox- R123 and 3H-VBL accumulations icity either by LDH release or by MTT, after incubation of R123 is a good substrate for MDR-associated P-gp [25—281.MDCK-5'NU/— or MDCK-5'NU/+ with increasing concentra- Cellular retention of this dye was therefore used in numeroustions of colchicine, was used as another test to probe the activity studies to evaluate the activity of P-gp. Agents such as dp thatof P-gp. block P-gp have been found to increase the retention of Ri 23 only As shown in Figure 3, the results were similar with the two in cells expressing P-gp [29, 30]. The intensity of R123 accumu-cytotoxic assays. MDCK-5'NU/+ were more resistant to eolchi- lation was in the following order: MDCK-5'NU/-- > MDCK-cine than MDCK-5'NU/—, since the toxicity was concentration- 5'NU/i> MDCK-5'NU/+ (Table 1). dependent in MDCK-5'NU/+ (P < 0.001 in 1 way analysis of As shown in Figure 1, dp enhanced R123 accumulation in avariance) while it was already maximal with the smallest tested concentration-dependent manner in MDCK-5'NU/+. In contrast,concentration of coichicine in MDCK-5'NU/—. Dipyridamole 956 Ledowc et al: Ecto-5'-Nu promotes P-gp expression

A B 30 4

3 2 0.20 0. 2 I10 I Fig. 1. Effect of dp concentration on R123 1 accumulation. (A) MDCK-5 'N UI— and (B) MDCK-5'NU/+ cells were incubated for 18 ______hourswith 10 p.M R123 and various 0 I, 0 concentrations of dp. Results are expressed as 0 10 20 50 0 10 20 50 means SCM of 3 experiments and tested by one way analysis of variance (P < 0.001 for dp,/IM dp, LM MDCK-5'NU/+, NS for MDCK-5'NU/--).

30 Table 2. Effect of various P-gp inhibitors and MRP inhibitor on R123 accumulation in MDCK-5'NU/+ R123 accumulation Inhibitors FA U/mg protein P 20 Control 0.66 0.14 Dipyridamole 20 jxM 4.35 0.75 <0.001 e Amiodarone 20 I.LM 3.08 1.15 <0.05 BIBW 22 20 p.M 5.86 1.68 <0.0 Verapamil i0' M 6.03 1.01 <0.001 0 Reserpin 20 p.M 6.39 1.63 <0.001 E Cyclosporine A io- M 9.35 1.14 <0.001 10 *** Genistein 200 p.M 1.18 0.18 NS Values arc means SEM of 3 to 6 different experiments.

0 MDCK-5'NU!— rrMDCK-5NU/-i- deaminase and AMP did not affect 3H-VBL accumulation (Fig. Fig. 2. 3H.YBL accumulation in MDCK-5'NU/— and MDCK.5'NU/+ 4). cells. Cells were incubated during one hour without (E) or with (•) 20 jLM Effect of anti-5'Nu antibodies on P-gp activity dp. Results are expressed as means SEM of 3 experiments. 555P < 0.001 vs. MDCK-5'NU/--, ***p < 0.001 vs. MDCK-5'NU/+ without dp. Two monoclonal antibodies were tested on 5'Nu and P-gp activities: 1E9 (kind gift from Dr. L. Thompson) raised against human 5'Nu [10, 38] and 5F!F9 (kind gift from Dr. B. Kaissling) raised against rat 5'Nu [39, 40]. 1E9 antibody (15 p.g/ml added 96 (Dp) had no toxic effect alone and did not modify cytotoxicity ofhr and again 48 hr before the experiments in order to compensate coichicine in MDCK-5'NU/--. In contrast, dp reversed the drugantibody degradation) induced an 87.5% inhibition of 5'Nu resistance in MDCK-5'NU/+ (P < 0.01 in 2-way analysis ofactivity in MDCK-5'NU/+ but did not affect P-gp activity mea- variance). The 5'Nu inhibitor, aI3MADP, did not enhance cyto-sured by R123 or 3H-VBL accumulation (Table 3). In contrast, toxicity of coichicine. 5F/F9 antibody (1/200) had no effect on 5'Nu activity but signif- icantly inhibited P-gp activity (Table 3). As a control, we tested Relationship between 5'Nu enzymatic function and P-gp the effect of 5F/F9 antibody on P-gp activity in MDCK cells that activity did not express 5'Nu. In these cells, 5F/F9 antibody, at the dilution To evaluate whether enzymatic activity of 5'Nu affected P-gpof 1/200, had no effect on P-gp activity (R123 accumulation: activity, we studied the effect of AMP, the main substrate of 5'Nu0.75 0.04 vs. 0.85 0.06 FAU/mg protein, NS; 3H-VBL [7, 8], adenosine, the product of 5'Nu, adenosine deaminaseaccumulation: 0.98 0.39 vs. 1.04 0.21 pmol/mg protein, NS, which converts adenosine into inosine [37], and c43MADP whichN =3). inhibits 5'Nu activity [7]. R123 accumulation in MDCK-5'NU/+ was not modified afterEffect of MDCK-5'NU/+ conditioned medium on 96 hours of incubation with AMP or adenosine at any concentra- MDCK-5'NU/-— cells tion tested between iO and 10 M (0.470.2 and 0.450,03 Supernatants of MDCK-5'NU/— and MDCK-5'NU!+ grown FAU/mg protein in the presence of iO M AMP or iO Mfor three days in usual medium (with or without aMADP iO adenosine, respectively, vs. 0,31 0.05 FAU/mg protein underM for MDCK-5'NU/+) were collected, centrifuged (3000 rpm; 10 basal conditions) or with iO M af3MADP or 1 mU/ml adenosinemm) and filtered (0.22 /xm). MDCK-5'NU/— were then cultured deaminase (Fig. 4). Along the same line, aMADP, adenosineduring four days with these conditioned media or with usual Ledoux et a!: Ecto-5'-Nu promotes P-gp expression 957

A B 150 150

100 100 0C C) 50 50 7

0 0 0 25 50 100 0 25 50 100

c Coichicine,ng/mI D Colchicine, ng/mI 1200 1200 Fig. 3. Cytotoxicity of coichicine in MDCK- 0 5'NU/— and MDCK-5'NU/+ cells. Cytotoxicity 800 800 0C was measured after 72 hours of incubation by 0 LDH release A, B) and MTT assay (c,D)in 0 400 400 MDCK-5'NU/- (A, C) and MDCK-5'NU/+ (B, D) cells without (I) or with 20 !.LM dp (0) or i0 M a/3MADP (A). Results are shown as 0 0 means SCM of 3 experiments and tested by analysis of variance (P < 0.00 1 for ct MDCK- 0 25 50 100 5'NU/+ vs. Ct MDCK-5'NU/—, P < 0.01 for dp vs. ct in MDCK-5'NU/+, for both cytotoxic Coichicine, ng/mI Colchicine, ng/mI assays).

A

8 0

6 prot a 4

a Fig. 4. Effect of AMP, adenosine deaminase

pmot'mg and aI3MADP on P-gp activity in MDCK-S'NU/ 2 + cells. Cells were incubated during 96 hours ro without (LII)orwith i0- M csJ3MADP (),1 U/mI adenosine deaminase (U) or i0 M AMP ______I-'

0 o (11:1) prior to determination of R123 0 20 accumulation (A) and 3H-VBL accumulation (B). Results are shown as means SEM of 3 dp,iM experiments.

medium as a control. Membrane-bound 5'Nu activity in MDCK- Table 3. Effect of two different anti-5'Nu monoclonal antibodies on 5'NU/— was identical whatever the medium: 0.240.14, 0.26 5'Nu and P-gp activities 0,26 and 0.11 0.16 nmol/mg protein/mill for cells incubated with 3H-VBL conditioned medium from MDCK-5 'NU/+, MDCK-5 'NU/— and Antibodies S'Nu activity R123 accumulation accumulation with control medium, respectively. This result rules out a possible lE9 12.50 2.50a 108.50 25.00 119.50 3.50 tranfer of MDCK-5NU/+ cells into the wells containing MDCK- 5F/F9 (1/800) — 127.75 16.94 162.00 -54.02 5'NU/— cells. 5'Nu activity, measured in medium from MDCK-5F/F9 (1/400) 204.75 70.93 282.75 89.92 5'NU/+ cells, was significantly higher than in that from MDCK- SF/F9 (1/200) 155.00 27.00 574.40 172.39k' 860.25 3251 5'NU/— cells and was not altered by four days of incubation with Results are expressed as percent of control (S'Nu activity: 5.80 2.16 MDCK cells (Table 4). pmol/mg protein/mm, R123 accumulation 0.39 0.17 FAU/mg proteio, As shown in Figure 5, MDCK-5'NU/-— cells grown in MDCK-3H-VBL accumulation 1.16 0.63 pmol/mg protein) and arc mean SEM 5'NU/+ conditioned medium displayed a significant decrease ofof 3 different experiments. Statistical analysis was performed on absolute values. R123 accumulation when compared with those grown in MI)CK- P < 0.05 vs. control 5'NU/— conditioned medium. l)p, but riot rsMADP, reversed the effect of MDCK-5'NU/+ conditioned medium. Incubation of MDCK-5'NU/— during four days with 4 mU/mIcontrol, NS) despite a similar 5'Nu activity in the supernatant of 5'Nu from Crotalus Atrox venom did not influence R123than in conditioned medium from MDCK-5'NU/+ (1.50 0.53 accumulation (15.78 5.11 vs. 14.84 2.09 FAU/mg protein invs. 1.80 0.14 nmol/well/min, NS). 958 Ledouxet a!: Ecto-5'-Nu promotes P-gp expression

Table 4. 5'NU activity in different conditioned media

5'Nu activity nmo!/well/min MDCKNU/+ Control MDCK5'NU/- MDCKNU/+ +OI3MADP Before incubation 0.21) 0.01 0.130.03 1.800.06a 0.000.00 NS After incubation 0.16 0.17 1.620.40 0.000.00 5'NU activity was measured in usual medium (control) or in conditioned medium from MDCK-5'NU/— and MDCK-5'NU/+ cells without or with 104uaMADP,before or after 4 days of incubation with MDCK-5'NU/---. Values are means SCM of 3 experiments. aP< 0.001 vs. control; NS, all values after incubation are not significantly different from those before incubation.

30

20 175 162 —

(A) (B) (C) (D) (E) 0 Fig.6. Western blot of P-gp expression. Membranes of MDCK-5'NUI-- CT MDCK MDCK MDCK cells under control conditions i4), MDCK-5'NU/—- cells after 96 hours incubation with conditioned medium from MDCK-5'NU/+ (B), and -5NU/— -5NU/+ -5'NU/+ MDCK-5'NU/+ cells (C) were hybridized with C219 monoclonal anti +ct3MADP P-gp antibody. HeLa cells (D) and a MDCK cell line transfected with Medium human MDR1 (F) were used as negative and positive controls, respec- Fig.5. Effect of conditioned media on P-gp activity in MDCK-5'NU/— tively (representative picture of 3 experiments). cells. MDCK-5'NU/— were incubated during 96 hours with usual medium (CT), conditioned medium from MDCK-5'NU/-- or from MDCK- 5 'NU/+,with or without io- M aJ3MADP prior to determination of R123 accumulation in the absence (E) or the presence ( of 20 jxM dp. Results by enhancement of P-gp expression; (ii) induction of P-gp expres- are shown as means SCM of 3 experiments (***P < 0.001 vs. MDCK- sion did not result from the enzymatic activity of 5'Nu; and (iii) 5'NU/— medium, 5P < 0.05 vs. MDCK-5'NU/+ medium without dp). supernatant of cells that express 5'Nu confered P-gp activity to 5'Nu negative cells. MDCK cells, a widely used model of renal epithelial cells [18, Western blot 32], were chosen for transfection of human ecto-5'Nu because To acertain that the differences in MDR activity betweenexpression of the endogenous enzyme was undetectable. That MDCK-5'NU/+ and MDCK-5'NU/--- cells result from differenttransfection conferred ecto-5'Nu activity is assessed by the fol- levels of P-gp expression, we tested membrane fractions bylowing: (i) degradation of 5'-AMP into adcnosine occurred in the Western blot with the specific monoclonal anti-P-gp antibodyextracellular medium; (ii) the apparent Km values are close to C219. As shown in Figure 6, P-gp could be detected in MDCK-those reported in glomerular cells [41]; (iii) csf3MADP, a specific 5'NU/— and MDCK-5'NU/+ cells with the C219 antibody, as wellinhibitor of membrane-bound 5'Nu [7], abolished 5'Nu activity in as in a MDCK cell line transfected with human MDRI used as atransfected cells, (iv) immunofluorescent studies revealed that positive control (kind gift from Dr. M.M. Gottesman [18]). No5'Nu was predominantly expressed in the apical membrane of P-gp expression was detected in HeLa cells devoided of functionalMDCK cells grown on permeable supports (not shown). MDR activity (as assessed by R123 and 3H-VBL accumulation, MDR activity is enhanced in 5'Nu transfected cells. (i) Both data not shown). Expression of the protein was much morerhodamine and vinhiastine, two dissimilar and well-documented abundant in MDCK-5'NU/+ than in MDCK-5'NU/— cells. Mem-P-gp substrates [18, 25—28, 31, 32], were accumulated to a much brane expression of P-gp in MDCK-5'NU/— cells was enhancedlower extent in MDCK-5'NU/+ cells than in the parental coun- by incubation with conditioned medium from MDCK-5'NU/+terparts. (ii) Dipyridamole, known to antagonize P-gp activity [29, cells (in the same conditions described for R123 accumulation30], increased accumulation of these substrates only in MDCK- studies). 5'NU/+ cells. (iii) Moreover, MDCK-5'NU/+ cells were more resistant than MDCK-S'Nu/— to the cytotoxic effect of coichicine, DISCUSSION another P-gp substrate [2, 361. (iv) In this study, the pattern of In the present study, we show that: (i) expression of humaninhibition of R123 extrusion is consistent with previous observa- 5'Nu in MDCK cells induced multidrug resistance (MDR) activitytions [4, 42] that kidney preferentially express P-gp rather than Ledoux et al: Ecto-5' -Nu promotes P-gp expression 959

MRP. R123 accumulation was increased by cyclosporine A and In contrast with 1E9 antibody, 5F/F9 antibody, which did not verapamil, which are both inactive on MRP, but not by genistein,affect the enzymatic activity of 5'Nu, inhibited P-gp activity. This a specific MRP inhibitor [351.(v)That enhancement of MDRresult suggests that another epitope of the enzyme is involved in activity does not result from a nonspecific response to thethe regulation of P-gp expression. Indeed, it has been suspected transfection procedure is attested by the fact that overexpressionthat 5'Nu plays roles independent from the conversion of AMP of Na/SO4 cotransporter, another apical protein, has no effect oninto adenosine, such as tyrosine activation, involvement in R123 and 3H-VBL accumulations. lymphocyte proliferation, binding of compounds of the extracel- This enhancement of MDR activity in MDCK-5'NU/+ cells islular matrix or neurite differentiation [8—10, 45—47]. related to the induction of P-gp expression, as demonstrated by The observation that conditioned medium from MDCK- Western blot with the anti-P-gp antibody C219. It has been5'Nu/+ cells induced P-gp expression in MDCK-5'Nu/— suggests recently reported [431thatthis antibody could recognize proteinsthat 5'Nu released in the supernatant could also enhance P-gp other than P-gp. However, these proteins had molecular weightsexpression. It is noteworthy that the presence of 5'Nu in the distinct from that of P-gp. The fact that the molecular weight ofsupernatant of MDCK-5'NU/+ cells could be demonstrated. the protein detected with C219 in MDCK-5'NU/+ cells was veryAgain, the enzymatic activity of 5'Nu did not play a major role in close to that of human P-gp expressed in a MDCK cell linethis context since (i) aMADP did not prevent P-gp activity, and transfected with MDRI argues strongly against this hypothesis.(ii) the addition of soluble 5'Nu from snake venom, which has an Even more importantly, results of Western blot analysis didenzymatic activity close to that of the ecto-enzyme but is struc- parallel those of functional studies of P-gp. The molecular weightturaly different [8], did not elicit 5'Nu activity. Along the same of P-gp in MDCK-5'NU/— cells was slightly higher than inline, it has been suggested that 5'Nu, after its release from the MDCK-5'NU/+ cells. This difference could be accounted for byplasma membrane, can interact with distant sites of fixation and different levels of glycosylation that have been frequently de-induce cellular effects, such as neurite differentiation [8, 9]. From scribed [1, 2, 241.Itis noteworthy, however, that this change inour experiments, however, the possibility that other soluble molecular weight is not mandatory for the activity of P-gp,factors, yet to be identified, are involved in the induction of P-gp inasmuch as conditioned medium from MDCK-5'NU/+ cellsactivity cannot be definitly ruled out. enhanced P-gp activity in MDCK-5'NU/— cells without affecting A link between 5'Nu and P-gp expression was first described in the molecular weight of the protein. tumoral cells [12, 13]. Our results, obtained in non-tumoral renal These data are in agreement with previous studies that haveepithelial cells, suggest that such a link could exist in normal cells. suggested a link between 5'Nu and P-gp expression. Overexpres-Several lines of evidence argue in favor of a physiological inter- sion of both proteins is considered to be a serious prognosticaction between the two proteins. They have a very similar tissue factor in hematologic malignancies [1, 11, 44]. In addition,distribution and, notably, both are expressed in brush border malignant cell lines selected with doxorubicin, in order to enhancemembranes of proximal tubular cells [1, 7]. Interraction between MDR expression, also overexpress ecto-5'Nu, in comparison toecto-5'Nu and an other member of ABC transporter family, the parental cells [12, 13]. It was suggested that this link was sup-sulfonylurea receptor (SUR), has been reported and could be ported by a metabolic interraction: 5'Nu could provide ATPrelevant in adaptation to hypoxia [48]. Finally, it can be stressed precursors to P-gp. However, the cause-effect relationship be-that both 5'Nu and P-gp are involved in nucleotide metabolism tween expression of the two proteins was not clarified. Our resultsand/or transport: 5'Nu is essential in the nucleotide salvage show, for the first time, that 5'Nu overexpression may induce P-gppathway while P-gp was reported, at least in one study, to behave expression. as an ATP channel [3]. The question then arose of the mechanism by which 5'Nu In conclusion, our results demonstrate that ecto-5'Nu overex- induced P-gp expression. We first evaluated the possibility thatpression increases membrane P-gp level and that this effect does 5'Nu enzymatic activity was instrumental in the emergence ofnot results from S'Nu enzymatic activity. The physiological signif- P-gp activity. Several lines of evidence argue against such aicance of such an interaction needs to be elucidated. Since mechanism. (i) Neither AMP nor adenosine, which are themodulators of 5'Nu have been recently reported [23, 49—51], substrate and the product of this enzyme, respectively [7], affectedthese modulators might indirectly affect P-gp expression in renal the activity of P-gp. (ii) Adenosine deaminase, which convertsepithelial cells and, thereby, affect the capacity of these cells to active adenosine into inactive mosine 137],oraI3MADP, whichextrude endogenous or exogenous P-gp substrates. blocks the convcrtion of AMP into adenosine [7], did not decrease P-gp activity. (iii) Finally, the blocking monoclonal anti-5'NuACKNOWLEDGMENTS antibody I E9 did not affect the P-gp activity. Our results contrast with those obtained by Ujhazy Ct al [12, 13], Séverinc Ledoux and Christine Leroy contributed equally to this work. who observed that n1MADP increased coichicine toxicity andThis study was supported by grants from INSERM, CNRS, Univcrsité Paris 7, Faculté X. Bichat, Fondation pour Ia Recherchc Médicale, and MDR activity assessed by R123 accumulation in cells overexpress-Lahoratoirc de Recherehes Physiologiqucs. ing 5'Nu and P-gp. The reasons for this discrepancy are not clear. However, it is noteworthy that in these studies CsMADP by itself Reprint requests to Gerard Friedlander, M.D., PhD, INSERM U 426, affected cell viability and its inhibitory effect on 5'Nu activity wasFacultC Xavier-Bichat, BP 416, 75870 Pari France. not quantified. It may therefore be that the effect of aMADP was independent from 5'Nu inhibition. Another possibility would APPENDIX be that P-gp regulation of tumoral cells is distinct for that of normal cells, as notably demonstrated for regulation by protein Abbreviations arc: P-gp, P-glycoprotcin; 5 'Nu, Ecto-5 '-nucleotidase; [5]. R123, rhodamine 123; [3HJVBL, [3Hlvinblastine; MDCK, Madin-Darhy 960 Ledouxet a!: Ecto-5'-Nu promotes P-gp expression canine kidney; MDR, multidrug resistance; aI3MADP, a13-methylene- 23. 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