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)

Jpn J Cancer Res. 1999 Apr;90(4):439-47. doi: 10.1111/j.1349-7006.1999.tb00767.x.

Abstract

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 multidrug resistance-associated protein (MRP1), whereas the latter is resistant to cis-diamminedichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P-glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was overexpressed 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 C4 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 Km and Vmax values for DNP-SG uptake were 2.56 and 1.43 microM, 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-17beta-D-glucuronide (E(2)17betaG) 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 (Km=2.33 microM, Vmax=34 pmol/min/mg protein) was much more extensive than that in the case of KCP-4 (Km=5.5 microM, Vmax=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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / genetics*
  • Antineoplastic Agents / therapeutic use*
  • Carrier Proteins / genetics*
  • Cisplatin / therapeutic use
  • Drug Resistance, Multiple / genetics*
  • Drug Resistance, Neoplasm / genetics*
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Membrane Transport Proteins
  • Multidrug Resistance-Associated Proteins
  • Multigene Family*
  • Substrate Specificity
  • Tumor Cells, Cultured

Substances

  • ATP-Binding Cassette Transporters
  • Antineoplastic Agents
  • Carrier Proteins
  • Membrane Transport Proteins
  • Multidrug Resistance-Associated Proteins
  • glutathione transporter
  • Cisplatin