The expression of
P-glycoprotein (P-gp) in
tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that
BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human
lymphoma cells in a dose-dependent fashion. Using a photoactive analog of
BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-
morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa
protein in
drug-resistant cells immunoprecipitated with P-gp-specific
monoclonal antibodies. The photolabeling of P-gp by [3H]azido-
BIBW22 BS was specific and saturable. Furthermore,
BIBW22 BS,
vinblastine, and
verapamil, but not
colchicine, inhibited the photolabeling of P-gp by [3H]azido-
BIBW22 BS.
Drug binding studies showed that membranes from MDR cells bound more
BIBW22 BS than parental
drug-sensitive cells, and this binding was inhibited with
vinblastine and, to a lesser extent, with
uridine. However,
drug transport studies demonstrated that
BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly,
BIBW22 BS was shown to accumulate more in resistant cells. Also,
BIBW22 BS accumulation in
drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]
vinblastine seen in the same MDR cells. A comparison of [3H]azido-
BIBW22 BS or [3H]
azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8
protease showed differences in the photolabeled
peptides. Taken together, the results of this study show that
BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from
drug-resistant cells.