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)

Abstract

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.

Interactions of the Human Multidrug Resistance Proteins MRP1 and MRP2 with Organic Anions

“…Concerning the role of MRP1 and MRP2 in cytostatic drug resistance, it has been documented in several experiments that both proteins are able to transport unconjugated hydrophobic drugs and anions (Feller et al, 1995;Versantvoort et al, 1995;Holló et al, 1996), but GSH modifies this transport, most likely via a cotransport mechanism (Loe et al, 1996b(Loe et al, , 1998Deeley and Cole, 1997;Evers et al, 1998). In experiments to be reported elsewhere, we found that vinblastine and GSH produced a synergistic stimulation of the MRP2-ATPase (manuscript under preparation).…”

Differential Multidrug Resistance-Associated Protein 1 through 6 Isoform Expression and Function in Human Intestinal Epithelial Caco-2 Cells

“…The efflux of free calcein (the fluorescent form) is then determined after washing away excess calcein-AM. Whereas calcein AM is a substrate for MDR1 and MRP, calcein is not a substrate for MDR1, thus allowing specific MRP efflux activity to be determined (Versantvoort et al, 1995;Essodaigui et al, 1998).…”

“…We have then quantified the functional expression of MRP-like activity with the fluorescent probe calcein (Versantvoort et al, 1995;Essodaigui et al, 1998) and probed for functional differences in MRP-like activities at apical and basal membranes with a range of pharmacological tools. T84 cells, a model of human colonocytes that lack MRP2 expression Simmons, 2001, 2002), are used in comparison.…”

Vincristine-induced expression of P-glycoprotein in MOLM-13 and SKM-1 acute myeloid leukemia cell lines is associated with coexpression of nestin transcript

“…Nestin→ GAPDH→ (Versantvoort et al 1995). Thus, in P-gp-positive cells, the efflux activity of this transporter blocks calcein/AM entry and antagonizes the intracellular retention of fluorescent calcein.…”