Mechanisms of resistance to daunorubicin in Ehrlich ascites tumor cells.

Uptake and binding of daunorubicin were studied in a sensitive (EHR 2) and in a resistant (EHR 2/DNR+) subline of Ehrlich ascites tumor cells. At steady state, the celhmedium ratio of daunorubicin was about 10-fold higher in EHR 2 than in EHR 2/DNR + . The bindings of daunorubicin to cell homogenate of the two cell lines were equal when the concentration at equilibrium was below 0.7 fig/ml, whereas homogenate of EHR 2 bound signifi cantly more daunorubicin at higher concentrations. Based on the binding affinity for cell homogenate, the cyto plasm imedium ratio at steady state was estimated to be below 0.15 in EHR 2/DNR+, while the ratio in EHR 2 was 0.6 to 1.0. Omission of glucose together with the addition of sodium azide resulted in a considerable increase in drug uptake and in an equalization of the cytoplasm: medium gradient in EHR 2/DNR + . If glycolysis was re stored by the addition of glucose to cells treated with sodium azide and loaded with daunorubicin, an uphill exodus of the drug was induced in EHR 2/DNR+. In both cell lines omission of glucose together with sodium azide increased the initial rate of uptake. How ever, the increment was three times as high for EHR 21 DNR+ as for EHR 2. In the medium without glucose but with sodium azide, the influx followed simple saturation kinetics in both cell lines, indicating carrier-mediated transport. As a consequence of a lower Vraax,the influx was significantly lower in EHR 2/DNR+ than in EHR 2. The data indicate that lower drug uptake in cells resistant to daunorubicin may be a result of at least three different mechanisms: a lower influx, a higher active extrusion, and a lower affinity for intracellular binding sites.