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Prevalence of Multidrug Resistance Related to Activation of the mdr1 Gene in Human Sarcoma Mutants Derived by Single-Step Doxorubicin Selection¹

Oncology and Clinical Pharmacology Divisions, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5306

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10⁶ cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II and IIß genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M_r 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5–6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10^-6 per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topolsomerase II or IIß, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 µM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study 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 selected by DOX in these resistant clones.

¹ Supported in part by American Cancer Society Grant DHP-76E and NIH Grant CA 52168 (to B. I. S.); the Ho Tim, Stanley Ho, and Li Ka-Shing Medical Fund of Stanford University School of Medicine (to G. C.); l'Association pour la Recherche sur le Cancer, la Ligue Nationale Contre le Cancer, and the Philippe Foundation, Inc. (to J-P. J.); and NIH Grant CA 42551 (to W. H. F.).

² To whom reprint requests should be addressed, Oncology Division, Department of Medicine, Room M-211, Stanford University Medical Center, Stanford, CA 94305.

Received 3/31/94. Accepted 7/20/94.

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