Isolation and Preliminary Characterization of an Adriamycin-resistant Murine Fibrosarcoma Cell Line1

A variant cell line (UV-2237-ADMR) resistant to the anthracycline antibiotic Adriamycin (doxorubicin) was selected in vitro from the murine UV-2237 fibrosarcoma tumor cell line. Resis tance to Adriamycin proved to be a stable characteristic of the UV-2237-ADMR line, whether the line was grown in vivo or in vitro. The UV-2237-ADMR line also exhibited increased resistance to A/-trifluoroacetyladriamycin-14-valerate, daunorubicin, actinomycin D, amsacrine, mitomycin C, vinblastine, and vincristine but not to bleomycin. Cell-cell hybridization studies showed that the Adriamycin resistance is an incompletely dominant trait. Uptake and efflux studies with [14C]Adriamycin indicated that the resis tance exhibited by the UV-2237-ADMR line was due to both reduced uptake of the drug and an increased active efflux. INTRODUCTION One of the most significant p'roblems in cancer chemotherapy is the rapid emergence of variant subpopulations of tumor cells that are resistant to a particular drug or combination of drugs. The anthracycline antibiotic ADM3 (doxorubicin) is commonly used because it exhibits considerable cytotoxic activity against a broad spectrum of solid tumors and leukemias (4-6, 11). Patients frequently develop tumor cells that are resistant to ADM, but the cause of this resistance is unknown. To study the mechanisms of drug resistance, many investi gators have selected in vivo or in vitro mammalian cell lines that are resistant to levels of ADM that are normally cytotoxic (3, 9, 17, 22, 28, 37, 38). Most of these tumor lines have been either leukemias or sarcomas grown in the ascites form; there is very little information in the literature regarding the selection of variant lines from solid tumors. As part of our work on developing experimental therapies of transplantable tumors, we needed to obtain a solid murine tumor that is resistant to ADM. In this initial report, we describe the isolation and partial characterization of a variant of the UV-2237 fibrosarcoma (24) that exhibits such behavior. 1Research sponsored by the National Cancer Institute, Department of Health and Human Services, under Contract N01-CO-75380 with Litton Bionetics, Inc. 2 Recipient of a fellowship for advanced professional training from the Italian Labor Department and the European Economic Community. On leave of absence from Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy. To whom requests for reprints should be addressed. 3The abbreviations used are: ADM, Adriamycin (NSC 1213127); CMEM, Eagle's minimal essential medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, and L-glutamine; ACT D, actinomycin D (NSC 3053); AMSA, amsacrine (NSC 249992); BLEO, bleomycin (NSC 125066); DNR, daunorubicin (NSC 82151); VBL, vinblastine (NSC 49842); VCR, vincristine (NSC 67574); AD 32, A/-trifluoroacetyladriamycin-14-valerate (NSC 246131); MIT C, mitomycin C (NSC 26980); HAT medium, hypoxanthine (0.1 mw)-aminopterin (4 x 10~7 M)-thymidine (3.6 x 10"5 M)-glycine (3 x 10~* M). Received September 13, 1982; accepted February 3, 1983. MATERIALS AND METHODS Tumor Lines. The UV-2237 fibrosarcoma, syngeneic to C3H/HeN mammary tumor virus-negative mice, was a gift of Dr. Margaret L. Kripke, National Cancer Institute-Frederick Cancer Research Facility (24). The parent line was maintained in tissue culture in CMEM (Flow Laboratories, Inc., McLean, Va.). The so-called "universal fuser line" bearing a dominant marker for resistance to ouabain and a recessive marker for resistance to 6thioguanine was selected from UV-2237 after mutagenesis with ethylmethanesulfonate. This line, UV-2237RR, was maintained in CMEM con taining 3 mw ouabain and 6-thioguanine (10 ^g/ml) and was the gift of Dr. Maria Cifone, NCI-Frederick Cancer Research Facility. Drugs. The antitumor agents ACT D, ADM, AMSA, BLEO, DNR, VBL, and VCR were provided by Dr. J. Douros, National Products Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Md. AD 32 was a gift from Dr. M. Israel, Sidney Farber Cancer Institute, Boston, Mass. MIT C was obtained from Bristol Laboratories, Syracuse, N. Y. BLEO, DNR, MIT C, and VCR were dissolved in distilled water; AMSA was dissolved in 1 mM lactic acid, ACT D in 95% ethanol, AD 32 in 0.9% NaCI solution containing 10% Tween 80, and ADM in 0.9% NaCI solution. All drugs were dissolved immediately before use, except ADM, which was stored at -20° in stock solutions of 1 mg/ml for periods of up to 2 weeks. Working concentrations were prepared in CMEM; control me dium contained equal volumes of drug-free diluent. [14C]ADM (92 ^Ci/mg) was a gift from Dr. Federico Arcamone, Farmitalia Carlo Erba, Milan, Italy. Culture Conditions. All tumor cell lines were maintained on plastic and subcultured weekly by harvesting cells with 0.25% trypsin-0.02% EDTA. CMEM was modified according to the cell phenotype as described in "Results." The cell lines were routinely examined for and found free of Mycoplasma, reovirus type 3, pneumonia virus of mice, K virus, Theiler's virus, Sendai virus, minute virus of mice, mouse adenovirus, mouse hepatitis virus, lymphocyte choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (M. A. Bioproducts, Walkersville, Md.). Determination of Cell Survival by Colony Formation. Dose-iesponse curves of the different tumor lines to the various agents were established by plating 800 tumor cells into 60-mm tissue culture dishes (Falcon Plastics, Oxnard, Calif.; triplicate dishes/drug dose) containing 5 ml of CMEM with varying drug concentrations. Cultures were incubated at 37°for 8 days at which time the medium was discarded, and the colonies were fixed, stained with méthylà ̈neblue in 50% methanol, and counted. Relative plating efficiencies were calculated as Mean no. of colonies in treated dishes Mean no. of colonies in control dishes x 100 The UV-2237-ADMR line was always subcultured once and grown to partial confluency in ADM-free CMEM before being tested. Cell-to-Cell Hybridization. Cells were hybridized as described by Davidson and Gerald (10). Briefly, 1 x 106 UV-2237-ADM" cells and 1 x 106 UV-2237RB cells were plated into a single 60-mm Petri dish containing 4 ml of CMEM, and the cells were cocultivated at 37°. After 24 hr of incubation, the culture medium was aspirated off, and the cells were 2216 CANCER RESEARCH VOL. 43 Research. on January 12, 2018. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from ADM-resistant Solid Tumor Line treated with 2 ml of 50% (w/v) polyethylene glycol (M, 1000) in serumfree medium for 1 min at room temperature to induce fusion. The polyethylene glycol solution was then removed, and the cells were washed 4 times with Hanks' balanced salt solution, refed with CMEM, and incubated for a further 24 hr. The cells were then harvested with trypsin-EDTA, resuspended in serum-free medium, and plated (4x10" cells/plate) into 100-mm tissue culture dishes (Falcon) containing CMEM plus 3 mM ouabain plus HAT medium. Two to 3 weeks after initiation, growing colonies were isolated with a cloning ring, grown in the selection medium, and karyotyped to confirm their hybrid nature. Three independ ent isolates were obtained to test the nature of ADM resistance; their karyotypes are presented in Table 1. Control hybrid lines were obtained by fusing UV-2237nR with the unselected UV-2237-parent line. Control plating experiments with both UV-2237RR and UV-2237-ADM" lines that had been cultured in separate dishes demonstrated that no growth occurred in dishes containing 3 mwi ouabain plus HAT medium, even when 2.5 x 105 cells were plated per dish. In Vitro Growth Curve. Tumor cells from the different lines were seeded into 60-mm dishes (5 x 104 cells/dish) containing 5 ml of the appropriate medium, as described in "Results." Every 24 hr, cells from triplicate cultures were harvested with 0.25% trypsin-0.02% EDTA, and the number of viable trypan blue-excluding cells was determined using a hemocytometer. In Vivo Growth of the UV-2237-ADM" Line. We determined that the ADM-resistant phenotype was maintained after in vivo growth by ex amining tumor cells that had been recovered after 42 days of growth in the subcutis of nude mice of BALB/c origin. Five mice were given s.c. injections of 1 x 10s UV-2237-ADMH cells, and 5 mice were given s.c. injections of 1 x 10s UV-2237 parent cells. Six weeks later, all of the mice were killed; tumors were excised, and single-cell suspensions were prepared (2 sequential 15-min exposures to 0.25% collagenase and 0.01% DNase). These suspensions were plated into 100-mm tissue culture dishes in CMEM and grown as monolayer cultures for 1 week. Seven days later, the cells were harvested and tested in the colony formation assay as described. Uptake and Efflux Studies. UV-2237 and UV-2237-ADM" lines were harvested as described and adjusted in CMEM to give 2.5 x 106 viable cells/ml. Two hundred-/ul aliquots of these cell suspensions were added to the wells of 96-well Microtest III tissue culture plates (Falcon), and these plates were incubated overnight at 37°. For uptake assays, the culture supernatant was aspirated off, the cells were washed once with phosphate-buffered saline, 100-/J aliquots of |'4C]ADM (20 MQ/ml in CMEM) were added to the wells, and the plates were incubated at 37°. At the times indicated in "Results," the drug solution was aspirated off, cells were washed once with phosphatebuffered saline, and the remaining adherent cells were lysed with 100 p\ of 0.2% sodium dodecyl sulfate. The lysates were harvested and added to 5 ml of scintillation fluid (PCS; Amersham/Searle Corp., Arlington Heights, III.) in glass vials, and radioactivity was monitored in a Beckman /i-scintillation counter. The cpm obtained with replicate cultures to which the [14C]ADM solution had been added for 10 sec were treated as Table 1 Karyotypes of parent and hybrid cell lines Cell line Chromosome no." UV-2237 parent UV-2237RR UV-2237-ADM" UV-2237"" x UV-2237 parent UV-2237nR x UV-2237-ADM" CI3 UV-2237"" x UV-2237-ADM" CI4 UV-2237"" x UV-2237-ADM" CI5 42 ±3°