INFLUX OF DAUNORUBICIN IN MULTIDRUG-RESISTANT EHRLICH ASCITES TUMOR-CELLS - CORRELATION TO EXPRESSION OF P-GLYCOPROTEIN AND EFFLUX - INFLUENCE OF VERAPAMIL

Classic multidrug resistance is characterized by a decrease in the int racellular concentration of drugs in resistant cells as compared to se nsitive cells. This is correlated with the presence of P-glycoprotein in the membrane. P-glycoprotein is responsible for an active efflux of drug. In this study we investigated the correlation between P-glycopr otein and influx of daunorubicin. Four Ehrlich ascites tumour cell lin es selected in vivo for resistance to daunorubicin were investigated. The sublines EHR2/0.1, EHR2/0.2, passage no. 12 of EHR2/0.8, EHR2/0.4, and passage no. 72 of EHR2/0.8 were 6-, 6-, 5-, 33-, and 35-fold resi stant to daunorubicin, respectively. All sublines overexpressed P-glyc oprotein as determined with Western blot. Influx was measured over 40 sec. In glucose-enriched medium influx was significantly decreased in all but one of the resistant sublines. A correlation between P-glycopr otein, degrees of resistance, and influx was demonstrated in four subl ines. Comparing influx experiments with efflux experiments (Nielsen ei al., Biochem Pharmacol 1994, 47, 2125-2135) we found a linear relatio nship between influx and efflux in the resistant sublines (r = 0.97). Verapamil (5.5 mu M, 11.0 mu M) increased influx significantly in all resistant sublines, whereas the drug had no effect on sensitive cells. Verapamil (3.3 mu M) increased influx in the EHR2/0.8 (passage no. 72 ) subline to the level of sensitive cells. Comparing this result with efflux experiments, verapamil was found to increase influx preferentia lly. Depletion of energy (medium without glucose including Na+-azide) increased influx in all resistant sublines. In EHR2/0.4 and EHR2/0.8 ( passage no. 72) the influx, however, was still significantly decreased after depletion of energy. In these cells further addition of verapam il increased influx to the level of EHR2. These data were consistent w ith the hypothesis that P-glycoprotein effluxes drug directly from the plasma membrane.