Fig. 2.

Simultaneous detection of Ho33342 and R123 transport. Flow cytometry was performed on the parental cell line K562 (bottom panels, human erythroleukemia) and the MDR1 transfectant K562-G185 (top panels). A gating strategy was used to analyze only singlet viable cells. Monoclonal antibody staining on separate aliquots revealed that parental K562 cells were ABCB1 (MDR1) negative and expressed a very small subpopulation of ABCG2+, low side scatter cells (0.08% of viable cells, top right). In contrast, the transfectant line K562-G158, which is maintained in the presence of 100 ng/mL vincristine, is uniformly positive for ABCB1, and expresses a small subpopulation of ABCG2+ cells identical to that of the parent cell line. Both cell lines were coincubated with Ho33342 and R123 in the absence and presence of MDR inhibitors. In the absence of inhibitors, only a minor subpopulation within the parental K562 exhibited the SP phenotype (3.36%), whereas almost all K562-G185 (ABCB1 transfected) cells transported Ho33342 (71% SP). Addition of 5 μM cyclosporin A (CsA) abrogated transport in both cell lines. Vincristine had no effect on either cell line, whereas the ABCG2 specific inhibitor, fumitremorgin (1 μM), only inhibited Ho33342 transport in parental K562 (native ABCG2 mediated transport) and had no effect on Ho33342 transport through ABCB1. Verapamil (50 μM, a MDR nonspecific inhibitor) blocked SP phenotype in both cell lines. R123 transport was limited to cells with ABCB1 expression (K562-G185 only), the parental K562 had no constitutive ABCB1 activity or expression. The R123 dull phenotype was reversed by the addition of either CsA or verapamil but was not blocked by the addition of ABCG2-specific inhibitor fumitremorgin.