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Double-label fluorescence microscopy was used to demonstrate the efflux activity of the multidrug transporter in single cultured cells. NIH3T3 cells expressing a transfected MDR1 gene (NIH3T3-MDR) were treated with vinblastine or daunomycin. The accumulation of vinblastine was monitored by examining the morphology of tubulin in cells, using immunofluorescence. Overnight treatment of drug-sensitive cells caused disassembly of microtubules and formation of paracrystals; the absence of vinblastine effects was evident by the presence of intact microtubules. Daunomycin accumulation was detected in nuclei using the inherent fluorescence of the drug with rhodamine epifluorescence microscopy. Drug efflux in multidrug-resistant cells was inhibited with verapamil. When multidrug-resistant cells were treated overnight in vinblastine, an effect of 0.5 microM vinblastine on microtubules was seen only in the presence of verapamil. Similarly, when cells were treated with daunomycin, this drug accumulated in nuclei only when verapamil was present. When cells incubated with vinblastine and verapamil were washed free of drugs, they did not accumulate daunomycin in a subsequent incubation, indicating that the multidrug transporter was still active; this occurred even though the morphologic effects of vinblastine persisted. Cells incubated with vinblastine alone showed an immediate inhibition of efflux activity when verapamil was subsequently added with daunomycin. These results show that the efflux activity of the multidrug transporter can be rapidly manipulated by agents such as verapamil, despite a prior history of drug treatment, and that the effects of inhibition of the transporter are rapidly reversible.

The multidrug transporter: rapid modulation of efflux activity monitored in single cells by the morphologic effects of vinblastine and daunomycin.