On the relationship between the probenecid-sensitive transport of daunorubicin or calcein and the glutathione status of cells overexpressing the multidrug resistance-associated protein (MRP).

Cells exposed to calcein acetoxymethyl ester (calcein AM) in the growth medium become fluorescent following cleavage of calcein AM by cellular esterases to produce the fluorescent derivative calcein. It has previously been shown by others that multidrug resistant cells which overexpress P-glycoprotein accumulate much less fluorescent calcein than the corresponding parental cells. We have now examined the transport of calcein in multidrug resistant cells which overexpress an alternative transporter, the multidrug resistance-associated protein (MRP). Accumulation of calcein fluorescence was greatly reduced in the MRP-overexpressing human lung cancer cell lines COR-L23/R and MOR/R compared with their parental lines. Energy depletion resulted in a considerably increased accumulation in the resistant lines. Treatment of resistant cells with buthionine sulfoximine (BSO), which depletes cellular glutathione (GSH), did not affect calcein accumulation, in marked contrast to our previous results for daunorubicin or the fluorescent probe rhodamine 123. Genistein, verapamil, cyclosporin A and ouabain were also each able to modify, to some extent, accumulation of daunorubicin, whilst having essentially no effect on calcein accumulation. However, the organic anion transport inhibitor probenecid was able to increase accumulation of both calcein and daunorubicin in the resistant cells. Genistein and verapamil treatment preferentially reduced the GSH content of resistant cells, whilst probenecid did not. However, probenecid caused a clear decrease in release of GSH from resistant cells into the medium.