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.


CONCLUSIONS
(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.
Ledoux
S
S
INSERM U 426, Faculté Xavier-Bichat, Université Denis-Diderot, Paris, France.
Leroy
C
C
Siegfried
G
G
Prie
D
D
Moullier
P
P
Friedlander
G
G
D015720:5'-Nucleotidase / Q000276:immunology / Q000378:metabolism*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003078:Colchicine / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020112:Rhodamine 123; D012235:Rhodamines / Q000493:pharmacokinetics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123; D015720:5'-Nucleotidase; D003078:Colchicine