Isolation and Preliminary Characterization of an Adriamycin-Resistant Murine Fibrosarcoma Cell Line1
Total Page：16
File Type：pdf, Size：1020Kb
(CANCER RESEARCH 43. 2216-2222. May 1983] Isolation and Preliminary Characterization of an Adriamycin-resistant Murine Fibrosarcoma Cell Line1 Raffaella Giavazzi,2 Eric Scholar, and Ian R. Hart Cancer Metastasis and Treatment Laboratory, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701 ABSTRACT MATERIALS AND METHODS A variant cell line (UV-2237-ADMR) resistant to the anthracy- Tumor Lines. The UV-2237 fibrosarcoma, syngeneic to C3H/HeN cline antibiotic Adriamycin (doxorubicin) was selected in vitro mammary tumor virus-negative mice, was a gift of Dr. Margaret L. Kripke, from the murine UV-2237 fibrosarcoma tumor cell line. Resis National Cancer Institute-Frederick Cancer Research Facility (24). The tance to Adriamycin proved to be a stable characteristic of the parent line was maintained in tissue culture in CMEM (Flow Laboratories, UV-2237-ADMR line, whether the line was grown in vivo or in Inc., McLean, Va.). The so-called "universal fuser line" bearing a dominant marker for vitro. The UV-2237-ADMR line also exhibited increased resistance resistance to ouabain and a recessive marker for resistance to 6- to A/-trifluoroacetyladriamycin-14-valerate, daunorubicin, actino- thioguanine was selected from UV-2237 after mutagenesis with ethyl- mycin D, amsacrine, mitomycin C, vinblastine, and vincristine but methanesulfonate. This line, UV-2237RR, was maintained in CMEM con not to bleomycin. Cell-cell hybridization studies showed that the taining 3 mw ouabain and 6-thioguanine (10 ^g/ml) and was the gift of Adriamycin resistance is an incompletely dominant trait. Uptake Dr. Maria Cifone, NCI-Frederick Cancer Research Facility. and efflux studies with [14C]Adriamycin indicated that the resis Drugs. The antitumor agents ACT D, ADM, AMSA, BLEO, DNR, VBL, tance exhibited by the UV-2237-ADMR line was due to both and VCR were provided by Dr. J. Douros, National Products Branch, reduced uptake of the drug and an increased active efflux. 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, INTRODUCTION N. Y. BLEO, DNR, MIT C, and VCR were dissolved in distilled water; AMSA One of the most significant p'roblems in cancer chemotherapy was dissolved in 1 mM lactic acid, ACT D in 95% ethanol, AD 32 in 0.9% is the rapid emergence of variant subpopulations of tumor cells NaCI solution containing 10% Tween 80, and ADM in 0.9% NaCI solution. All drugs were dissolved immediately before use, except ADM, which that are resistant to a particular drug or combination of drugs. was stored at -20° in stock solutions of 1 mg/ml for periods of up to 2 The anthracycline antibiotic ADM3 (doxorubicin) is commonly weeks. Working concentrations were prepared in CMEM; control me used because it exhibits considerable cytotoxic activity against dium contained equal volumes of drug-free diluent. a broad spectrum of solid tumors and leukemias (4-6, 11). [14C]ADM (92 ^Ci/mg) was a gift from Dr. Federico Arcamone, Far- Patients frequently develop tumor cells that are resistant to ADM, mitalia Carlo Erba, Milan, Italy. but the cause of this resistance is unknown. Culture Conditions. All tumor cell lines were maintained on plastic To study the mechanisms of drug resistance, many investi and subcultured weekly by harvesting cells with 0.25% trypsin-0.02% gators have selected in vivo or in vitro mammalian cell lines that EDTA. CMEM was modified according to the cell phenotype as described in "Results." The cell lines were routinely examined for and found free are resistant to levels of ADM that are normally cytotoxic (3, 9, of Mycoplasma, reovirus type 3, pneumonia virus of mice, K virus, 17, 22, 28, 37, 38). Most of these tumor lines have been either Theiler's virus, Sendai virus, minute virus of mice, mouse adenovirus, leukemias or sarcomas grown in the ascites form; there is very mouse hepatitis virus, lymphocyte choriomeningitis virus, ectromelia little information in the literature regarding the selection of variant virus, and lactate dehydrogenase virus (M. A. Bioproducts, Walkersville, lines from solid tumors. As part of our work on developing Md.). experimental therapies of transplantable tumors, we needed to Determination of Cell Survival by Colony Formation. Dose-iesponse obtain a solid murine tumor that is resistant to ADM. In this initial curves of the different tumor lines to the various agents were established report, we describe the isolation and partial characterization of by plating 800 tumor cells into 60-mm tissue culture dishes (Falcon a variant of the UV-2237 fibrosarcoma (24) that exhibits such Plastics, Oxnard, Calif.; triplicate dishes/drug dose) containing 5 ml of behavior. 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 méthylènebluein 50% methanol, and counted. 1Research sponsored by the National Cancer Institute, Department of Health Relative plating efficiencies were calculated as and Human Services, under Contract N01-CO-75380 with Litton Bionetics, Inc. 2 Recipient of a fellowship for advanced professional training from the Italian Mean no. of colonies in treated dishes Labor Department and the European Economic Community. On leave of absence x 100 from Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy. To whom Mean no. of colonies in control dishes requests for reprints should be addressed. 3 The abbreviations used are: ADM, Adriamycin (NSC 1213127); CMEM, Eagle's The UV-2237-ADMR line was always subcultured once and grown to minimal essential medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, and L-glutamine; ACT D, actinomycin D (NSC partial confluency in ADM-free CMEM before being tested. 3053); AMSA, amsacrine (NSC 249992); BLEO, bleomycin (NSC 125066); DNR, Cell-to-Cell Hybridization. Cells were hybridized as described by daunorubicin (NSC 82151); VBL, vinblastine (NSC 49842); VCR, vincristine (NSC Davidson and Gerald (10). Briefly, 1 x 106 UV-2237-ADM" cells and 1 x 67574); AD 32, A/-trifluoroacetyladriamycin-14-valerate (NSC 246131); MIT C, 106 UV-2237RB cells were plated into a single 60-mm Petri dish containing 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). 4 ml of CMEM, and the cells were cocultivated at 37°. After 24 hr of Received September 13, 1982; accepted February 3, 1983. incubation, the culture medium was aspirated off, and the cells were 2216 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. ADM-resistant Solid Tumor Line treated with 2 ml of 50% (w/v) polyethylene glycol (M, 1000) in serum- background, and these values were subtracted from all subsequent free medium for 1 min at room temperature to induce fusion. The values. To determine the cell number, cells from 3 or 4 replicate wells/ polyethylene glycol solution was then removed, and the cells were cell line were harvested with 100 p\ of 0.25% trypsin-0.02% EDTA and washed 4 times with Hanks' balanced salt solution, refed with CMEM, counted using a hemocytometer. Results are expressed as cpm/106 and incubated for a further 24 hr. The cells were then harvested with cells. trypsin-EDTA, resuspended in serum-free medium, and plated (4x10" For the efflux experiments, the culture supernatants were aspirated cells/plate) into 100-mm tissue culture dishes (Falcon) containing CMEM off, and 100 n\ of phosphate-buffered saline containing 10 mw sodium plus 3 mM ouabain plus HAT medium. Two to 3 weeks after initiation, azide were added to the cultures, which were then incubated at 37°for growing colonies were isolated with a cloning ring, grown in the selection 10 min. This supernatant was then aspirated off, 100-/J aliquots of medium, and karyotyped to confirm their hybrid nature. Three independ [14C)ADM (20 Mg/ml in phosphate-buffered saline-10 mw sodium azide) ent isolates were obtained to test the nature of ADM resistance; their were added to each culture, and the cells were incubated for 45 min at karyotypes are presented in Table 1. Control hybrid lines were obtained 37°.Following this incubation, the drug solution was aspirated off and by fusing UV-2237nR with the unselected UV-2237-parent line. Control replaced with 100 ¡t\of phosphate-buffered saline containing 1 g of plating experiments with both UV-2237RR and UV-2237-ADM" lines that glucose per liter. At the indicated times (see "Results"), this glucose had been cultured in separate dishes demonstrated that no growth solution was aspirated off. Then, the adherent cells were lysed and occurred in dishes containing 3 mwi ouabain plus HAT medium, even harvested as described for the uptake experiments and assayed for the when 2.5 x 105 cells were plated per dish. retained radioactivity. Results are expressed in terms of the percentage In Vitro Growth Curve. Tumor cells from the different lines were of radioactive drug retained; 100% values were obtained by allowing the seeded into 60-mm dishes (5 x 104 cells/dish) containing 5 ml of the glucose solution to remain for only 5 sec. In some experiments, the 2 appropriate medium, as described in "Results." Every 24 hr, cells from cell lines were harvested, each was adjusted to 2 x 106 cells/ml in 10 triplicate cultures were harvested with 0.25% trypsin-0.02% EDTA, and rnw sodium azide in phosphate-buffered saline, and the efflux experi the number of viable trypan blue-excluding cells was determined using a ments were conducted on bulk cultures incubated in 50-ml centrifuge hemocytometer.