Regulation of volume-sensitive Cl À channels in multi-drug resistant MCF7 cells Matthieu Marin * , Agne `s Poret, Ge ´raldine Maillet, Franc ¸ois Leboulenger, Frank Le Foll Laboratory of Ecotoxicology UPRES-EA 3222, IFRMP 23, University of Le Havre, 25 rue Philippe Lebon, 76058 Le Havre cedex, France Received 30 June 2005 Available online 18 July 2005 Abstract The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR À ), resistant (MDR + ), and reversed resistant (MDR REV ) phenotypes. Western blot analysis showed that incubation of cells with doxo- rubicin induced P-gp expression in a reversible manner. Exposure of MDR + cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents (I Cl-swell ) in MDR À , MDR + , and MDR REV MCF7 cells. While verapamil inhibited I Cl-swell in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I Cl-swell uniquely in MDR + . Intracellular dialysis of MDR + cells with C219 anti-P-gp antibody abolished the sensitivity of I Cl-swell to doxorubicin and led to a response pattern very close to that of MDR À cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I Cl-swell in resistant MCF7. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Multi-drug resistance; P-glycoprotein; Volume-sensitive chloride channels; Cell volume; MCF7; Patch-clamp; Whole-cell configuration; Regulatory volume decrease; MTT; Calcein-AM Multi-drug resistance (MDR) 1 is a phenotype associ- ated to breakdown of cancer treatment and character- ized by resistance to a broad spectrum of chemotherapeutic agents belonging to different pharma- cological classes [1–3]. The major cause of MDR pheno- type is the overexpression of a transmembrane phosphoglycoprotein, the P-glycoprotein (P-gp) [4]. The human P-gp, a 170 kDa product of the human mdr1 gene, is a member of the ATP-binding cassette (ABC) superfamily of transporters. This family encom- passes many other proteins, for instance, a chloride channel such as the cystic fibrosis transmembrane regu- lator (CFTR), regulatory subunits associated to ion channels such as the sulfonylurea receptor SUR1, and even a bacterial histidine transporter [5,6]. The P-gp acts as a weakly specific carrier responsible for the energy-de- pendent efflux of a number of structurally and functionally unrelated hydrophobic compounds, including anti-cancer agents (doxorubicin, vincristine) and fluorescent dyes (calcein-AM or rhodamine B) [7,8], causing a drastic reduction in accumulation of the chemicals in the cytosol. One of the most crucial regulatory processes in living cells is volume regulation. A large number of cellular mechanisms are influenced by changes in cell volume such as receptor recycling, excitability and contraction, 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.010 * Corresponding author. Fax: +33 232 744314. E-mail address: matthieu.marin@univ-lehavre.fr (M. Marin). 1 Abbreviations: MDR, multi-drug resistance; P-gp, P-glycoprotein; ABC, ATP binding cassette; CFTR, cystic fibrosis transmembrane regulator; SUR1, sulfonylurea receptor 1; RVD, regulatory volume decrease; AVD, apoptotic volume decrease; VSOR, volume-sensitive outward rectifying; MXR, multi-xenobiotic resistance; DIDS, 4,4 0 - diisothiocyanostilbene-2,2 0 -disulfonate; VRP, verapamil; Doxo, doxorubicin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diph- enyltetrazoliumbromide; CSP A, cyclosporine A; I Cl-swell , swelling- activated chloride current; MRP, multi-drug resistance-associated protein; BCRP, breast cancer resistance protein. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 334 (2005) 1266–1278 BBRC