PREVALENCE OF MULTIDRUG-RESISTANCE RELATED TO ACTIVATION OF THE MDR1 GENE IN HUMAN SARCOMA MUTANTS DERIVED BY SINGLE-STEP DOXORUBICIN SELECTION

Fluctuation analysis experiments were performed in the human sarcoma c ell line MES-SA to assess whether selection or induction mechanisms de termine resistance to doxorubicin (DOX), mutation rates, and the natur e of the surviving clones. Thirteen flasks were seeded with 2000 cells /flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harves ted. Clones were propagated and analyzed for drug resistance phenotype . Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measur ed by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M, 110,000 vesicular protein (p110) expression was detected by imm unohistochemistry. Analysis of variance supported the hypothesis of sp ontaneous mutations rather than induction conferring DOX resistance. A t this stringent level (5-6 log cell killing) of drug exposure, the mu tation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etopos ide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increa sed mRNA levels of mdr1 were observed in all 27 clones tested, includi ng at least 1 from each of the 13 populations. No alterations were fou nd in expression or level of topoisomerase II alpha or II beta mrp, gl utathione, and p110. Expression of P-glycoprotein was confirmed by flo w cytometry using the monoclonal antibody UIC2. In almost all tested c lones, decreased intracellular rhodamine-123 accumulation was modulate d by 2 mu M SDZ PSC 833, and the vinblastine resistance in all examine d clones was completely reversed by SDZ PSC 833 and verapamil. Our stu dy demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism sele cted by DOX in these resistant clones.