Differences in Substrate Specificity among Glutathione Conjugates (GS‐X) Pump Family Members: Comparison between Multidrug Resistance‐associated Protein and a Novel Transporter Expressed on a Cisplatin‐resistant Cell Line (KCP‐4)

The substrate specificity of primary active transporters expressed on two kinds of human epidermoid KB‐3‐1 derived cell lines, C‐A500 and KCP‐4, was examined; the former expresses multi‐drug resistance‐associated protein (MRP1), whereas the latter is resistant to cis‐diammine‐dichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P‐glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was over‐expressed on KCP‐4. Membrane vesicles isolated from C‐A500 and KCP‐4, but not from KB‐3‐1, exhibited the ATP‐dependent uptake of glutathione conjugates (GS‐X) such as leukotriene C(4) and 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG), indicating the presence of GS‐X pumps on these cells. The uptake of these GS‐X by membrane vesicles from C‐A500 was approximately twice that in the case of KCP‐4. Kinetic analysis indicated that the K(m) and V(max) values for DNP‐SG uptake were 2.56 and 1.43 μM, and 570 and 160 pmol/min/mg protein for C‐A500 and KCP‐4, respectively. In marked contrast, significant ATP‐dependent uptake of glutathione‐platinum complex was observed only in membrane vesicles from KCP‐4, but not those from KB‐3‐1 and C‐A500. The transport properties of estradiol‐17β‐d‐glucuronide (E(2)17βG) were also different between the two cell lines. This was reflected in the findings that the ATP‐dependent uptake of this conjugated metabolite in membrane vesicles from C‐A500 (K(m)= 2.33 μM, V(max)= 34 pmol/min/mg protein) was much more extensive than that in the case of KCP‐4 (K(m)= 5.5 μM, V(max)= 35 pmol/min/mg protein), and that comparable uptake was observed between KCP‐4 and KB‐3‐1. Overall, a clear difference in substrate specificity among GS‐X pump family members expressed on resistant tumor cells was demonstrated.