Decreased Anthracycline Uptake Precedes Increased Murine Erythroleukemia Cells under Adriamycin Selection: Sequential Emergence of Distinct Resistance Phenotypes in

First-step Adriamycin (doxorubicin)-resistant mutants of the murine erythroleukemia cell line PC4 were cloned from Adriamycin-containing (10 ng/ml) methylcellulose at a frequency of 3 x 10~4.They demonstrated 1.6to 2.4-fold stable resistance to Adriamycin. Most were cross-resistant to etoposide, but not to vincristine, and were without enhanced expression of mdr genes, which code for P-glycoproteins. Two different murine erythroleukemia cell lines, PC4 and (71). were passaged in suspension culture into stepwise increasing amounts of Adriamycin. No high-level resistant mutants were isolated de nova, cells initially displayed low-level resistance to Adriamycin and etoposide. Two stepwise doublings of the drug concentration were needed before PC4 cells acquired vincristine resistance, but there was no detectable overexpression of mdr or a change in anthracycline uptake. In a subsequent doubling of Adriamycin concen tration, the cells showed a further increase in resistance to all three drugs and now a decreased anthracycline accumulation. However, there was still no detectable increase in mdr expression as judged by Northern analysis of poly(A)+ enriched RNA and Western blot analysis of mem brane proteins. Only after a fourth doubling of Adriamycin concentration did the cells demonstrate enhanced expression of mdr and P-glycoprotein. Equivalent mutants of C7D were selected, but generally at lower Adria mycin concentrations. Verapamil partially lowered resistance, but failed to restore parental susceptibility in any mutant; it caused an increased uptake in those mutants showing decreased anthracycline accumulation, including those that did not overexpress mdr. This study demonstrated different resistance phenotypes among mutants appearing spontaneously under stepwise drug selection; mutants with vincristine resistance and decreased anthracycline uptake preceded those associated with overexpression of P-glycoprotein.