Analysis of chloride conductive pathway in human cell lines according to MRP1 protein expression

Abstract

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ABC transporters which are involved in drug resistance might also exhibit a chloride channel-related function as it is the case for MDR1-Pglycoprotein which is associated with cell swelling-activated chloride channels. We therefore investigated whether MRP1 (Multidrug Resistance-associated Protein 1) which transports a wide range of conjugated organic anions out of cells and confers drug resistance might be associated with a chloride channel. Three human carcinoma cell lines which did not express MDR1 transcript and exhibited different MRP1 protein levels as assessed by Western blot were investigated for chloride conductive pathway by using a halide-sensitive SPQ fluorescent dye assay. This method measures the rate of chloride transport as the rate of fluorescence change of intracellular SPQ induced by the replacement of chloride by nitrate in external solutions. A cell was considered to display a chloride conductive pathway when the maximum fluorescence change after replacing chloride by nitrate was > 0.1. Two experimental protocols were used as cells were exposed to isotonic and hypotonic solutions to assess a basal chloride conductive pathway and a swelling-activated chloride conductive pathway, respectively. For each cell line, fluorescence changes were measured in at least 80 cells. HeLa cells exhibited the highest MRP1 level (2.3 arbitrary units, a.u.) and T47D cells the lowest (0.16 a.u.) while Capan 2 cells had an intermediate level (1.4 a.u.). The percentage of cells exhibiting change in fluorescence > 0.1 in isotonic conditions was higher for HeLa cells (40%) than for T47D cells (23%) and this difference was statistically significant (p=0.005). An intermediate percentage value was found for Capan 2 (34%). The difference in percentages between HeLa and T47D cells was even stronger in hypotonic conditions (82% versus 46%, p<0.0001) with an intermediate value for Capan 2 (59%). Thus, the highest percentage of cells with change in fluorescence > 0.1 was observed for the cell line with the highest MRP1 level while the lowest percentage was observed for the cell line with the lowest MRP1 level. These results suggest that MRP1 might be associated with a chloride channel and especially a swelling-activated chloride channel.