Background: Various mechanisms can contribute to cellular resistance to doxorubicin. These include expression of the multidrug transporter P-glycoprotein (product of the mdr1 gene [also known as PGY1]), Mrp (multidrug resistance-associated protein), the p110 major vault protein, altered glutathione metabolism and altered levels or activity of topoisomerase II (Topo II). We reported recently that single-step treatment of human MES-SA sarcoma cells with 40 nM doxorubicin resulted in selection of spontaneous mutants at a rate of 1.8 x 10(-6) per cell generation. All individually selected mutants manifested the multidrug-resistant phenotype, related to activation of the mdr1 gene. Purpose: Luria and Delbruck fluctuation analysis was performed with MES-SA cells to determine the mutation rate and the nature and mechanisms of resistance after single-step selection with doxorubicin in the presence of the cyclosporin PSC 833, a potent modulator of multidrug resistance. Methods: Ten flasks mere seeded with 2000 cells/flask and grown to confluent populations of approximately 8 x 10(6) cells. After reseeding in 96-well plates, the populations mere treated with 40 nM doxorubicin and 2 mu M PSC 833 for 3 weeks. Surviving colonies were scored, individually harvested, and propagated. The drug-resistant phenotype mas assessed by the tetrazolium dye (MTT) cytotoxicity assay and by monitoring cellular glutathione content and radiolabeled drug accumulation. Coupled reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate mdr1, MRP, Topo II alpha, and Topo II beta gene expression. Topo II, P-glycoprotein, and p110 levels were examined by immunoblotting or immunocytochemistry. Topo II activity was assessed by decatenation of kinetoplast DNA, and etoposide-induced cleavable complex formation was studied by the potassium-sodium dodecyl sulfate precipitation assay. Results: Mutations were detected at a rate of 2.5 x 10(-7) per cell generation. Analysis of variance indicates that spontaneous mutations, rather than changes in cellular function, conferred resistance to doxorubicin and PSC 833. None of the isolated clones expressed mdr1 messenger RNA or P-glycoprotein, and none exhibited an increase in MRP expression. No alterations mere found in cellular glutathione content, intracellular accumulations of daunorubicin and etoposide, levels of p110 protein, or levels of Topo II beta transcripts. However, a significant decrease in Topo II alpha messenger RNA and protein mas found in ail examined clones, as well as decreased Topo II catalytic activity and reduced cleavable complex formation in the presence of etoposide. Conclusions: PSC 833 co-selection reduced the mutation rate for doxorubicin-selected resistance by 10-fold and suppressed the emergence of mdr1 mutants. Survival of cells exposed to doxorubicin and PSC 833 occurs by selection of spontaneously arising mutants that exhibit altered Topo II alpha expression. Implications: Our results suggest that treatment with multidrug resistance modulators such as PSC 833 together with multidrug resistance-related cytotoxins may suppress the activation of mdr1 and prevent the emergence of resistant cancer cell clones with the multidrug-resistant phenotype.
