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

(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 -20Â° 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 37Â°for 8 days at which time the medium was discarded, and the colonies were fixed, stained with mÃ©thylÃ¨nebluein 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 37Â°. 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

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 37Â°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 37Â°.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 Â¡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. tubes (Corning Glass Works, Corning, N. Y.). Following preloading of the In Vivo Growth of the UV-2237-ADM" Line. We determined that the cells with [14C]ADM prepared as described previously, the cells were ADM-resistant phenotype was maintained after in vivo growth by ex pelleted, resuspended in glucose solution, and incubated at 37Â°. At amining tumor cells that had been recovered after 42 days of growth in appropriate times. 100->il aliquots of cells were removed (triplicate sam the subcutis of nude mice of BALB/c origin. Five mice were given s.c. ples per time point) and added to tubes (Falcon; Model 2058) containing injections of 1 x 10s UV-2237-ADMH cells, and 5 mice were given s.c. ice-cold 10 mW sodium azide in phosphate-buffered saline. The cells injections of 1 x 10s UV-2237 parent cells. Six weeks later, all of the were then pelleted by centrifugation. The supernatant was aspirated off, mice were killed; tumors were excised, and single-cell suspensions were and the tubes were inverted and allowed to drain for 2 to 3 hr, after prepared (2 sequential 15-min exposures to 0.25% collagenase and which the cell pellets were lysed with 100 n\ of 0.2% sodium dodecyl 0.01% DNase). These suspensions were plated into 100-mm tissue sulfate and treated as described above. Essentially, the results obtained culture dishes in CMEM and grown as monolayer cultures for 1 week. with these assays were the same as those obtained with the monolayer Seven days later, the cells were harvested and tested in the colony cultures. 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 RESULTS cells/ml. Two hundred-/ul aliquots of these cell suspensions were added Isolation of the ADM-resistant Line. Early experiments to the wells of 96-well Microtest III tissue culture plates (Falcon), and these plates were incubated overnight at 37Â°. showed that a concentration of 0.1 /jg ADM per ml was required to inhibit completely the formation of colonies of the UV-2237 For uptake assays, the culture supernatant was aspirated off, the cells were washed once with phosphate-buffered saline, 100-/J aliquots of parent line (Chart 1). The resistant variant line was initially |'4C]ADM (20 MQ/ml in CMEM) were added to the wells, and the plates selected by plating 5 x 105 viable UV-2237 parent cells (viability were incubated at 37Â°. At the times indicated in "Results," the drug solution was aspirated off, cells were washed once with phosphate- buffered 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 Chromosome Cell line no." UV-2237 parent 42 Â±3Â° UV-2237RR 37 Â±3 UV-2237-ADM" 36 Â±3 10 UV-2237"" x UV-2237 parent 73 Â±4 Adriamycin Concentration (..g/mli UV-2237nR x UV-2237-ADM" CI3 63 Â±4 UV-2237"" x UV-2237-ADM" CI4 Chart 1. Survival of the UV-2237 parent and UV-2237-ADM" cell lines plated in 66 Â±5 various concentrations of ADM. â€¢,theUV-2237-ADM" line selected in the presence UV-2237"" x UV-2237-ADM" CI5 75 Â±5 of ADM (0.1 Â¿ig/ml);O, the UV-2237 parent at the time of selection; A, the UV- a Chromosome counts were obtained from at least 30 individual metaphase 2237 parent maintained in tissue culture for a period required to obtain resistant spreads. variants; and A, the UV-2237-ADM" line maintained in ADM (1.0M9/ml). .no 6 Mean Â±S.D. colonies observed at higher concentrations of the drug.

MAY 1983 2217

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. R. Giavazzi et al. determined by trypan blue exclusion) per dish into a total of fifty Stability of Resistance. The stable nature of ADM resistance 100-mm tissue culture dishes, each containing 25 ml of CMEM both in vitro and in vivo is shown in Chart 3. The UV-2237-ADMR per 0.1 ng of ADM per ml. After 3 weeks of continuous cultivation line maintained in drug-free medium for 6 weeks (6 passages) in this medium, 8 individual colonies developed. These colonies was less resistant to ADM than was the UV-2237-ADMR line were isolated with the aid of a cloning ring and were grown in maintained in 1.0 ng ADM per ml (Chart 3A). However, further fresh medium containing 0.1 //g ADM per ml to mass culture continuous cultivation of the cell line in drug-free medium for before being frozen. A representative plating experiment with periods of more than 5 months did not lead to any further one of these clones is illustrated in Chart 1; it showed that the decrease in these levels of resistance (data not shown). In vivo cell line derived from this first selection was approximately 10 cultivation of the UV-2237 parent and UV-2237-ADM" tumor times more resistant to the effects of ADM than was the UV- lines in the subcutis of nude mice for 6 weeks showed that the 2237 parent line. recovered UV-2237-ADM" line maintained a 10-fold increase in One of these clones was replated into CMEM containing, 0.1 resistance to the effects of ADM as compared to the recovered Mg ADM per ml in a 100-mm dish and then was plated in UV-2237 parent line (Chart 38). successive passages into 0.2, 0.5, and 1.0 ng of ADM per ml of Cross-Resistance of the UV-2237-ADM" Cell Line to Other medium. This cell line was labeled UV-2237-ADMR and was Agents. We tested the resistance of the UV-2237-ADM" line to maintained in CMEM containing 1.0 ^9 ADM per ml; Chart 1 several agents with different mechanisms of action. Represen shows that the UV-2237-ADMR cell line was approximately 100 tative results are presented in Chart 4. There was cross-resis times more resistant to ADM than was the parent cell line. In the tance to the closely related analogues AD 32 and DNR; there presence of 1.0 fig ADM per ml, the UV-2237-ADMR line was was cross-resistance also to ACT D, AMSA, MIT C, and the morphologically indistinguishable from the UV-2237 parent line Vinca alkaloids VBL and VCR. No cross-reactivity to BLEO was maintained in CMEM containing no ADM (Fig. 1). detected in these assays (Chart 4). The growth-inhibiting effects of ADM and the resistance of the Cell-to-Cell Hybridization Studies. To test for the dominant UV-2237-ADMR line to such effects were confirmed by in vitro or recessive nature of the ADM-resistant trait, cells of the UV- growth rate experiments (Chart 2). When grown in CMEM, the 2237-ADMR line were hybridized with ADM-sensitive cells of the UV-2237 parent line had a doubling time of approximately 18 hr; UV-2237RR line, which bears the dominant marker for ouabain when this cell line was cultivated for 7 days in the presence of resistance and the recessive marker for the absence of hypo- ADM (0.1 or 1.0 Mg/ml), the cells stopped growing and eventually xanthine phosphoribosyl transferase. The hybrids were re- died. In marked contrast, the UV-2237-ADM" line, which had a doubling time of 19 hr when grown in CMEM, was totally unaffected by the incorporation of 0.1 ^g ADM per ml into the growth medium; incorporation of 1.0 ^g ADM per ml into the culture medium increased the doubling time to 29 hr. 10- 5 x io6r

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0.01 0.1 1 10 Adriamycin Concentration (fig/ml) Days Chart 3. Stability of ADM resistance, A, in vitro retention of ADM resistance. Chart 2. Growth curve of the UV-2237 parent and UV-2237-ADM" cell lines. O, The UV-2237-ADM" line was cultured in ADM (1.0 Â»ig/ml)(â€¢)orin drug-free medium the UV-2237 parent in CMEM; A, in the presence of ADM (0.1 jig/ml); and D, in for 2 weeks (â€¢)and6 weeks (A); O, the UV-2237 parent line. B, in vivo retention the presence of ADM (1.0 jig/ml). â€¢.the UV-2237-ADM" line in CMEM; 4, in the of ADM resistance. UV-2237 parent (O) and UV-2237-ADM" cells (A) were plated presence of ADM (0.1 *ig/ml); and â€¢Â¡nthe presence of ADM (1.0 fig/ml). Each Â¡nmedium containing ADM following s.c. growth in nude mice for 6 weeks; â€¢,the point represents the mean of triplicate cell numbers. S.D. < Â±10%. UV-2237-ADM" line cultured Â¡nADM (1.0 ^g/ml) for the same period.

2218 CANCER RESEARCH VOL. 43

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Concentration (MM) Concentration (t g/ml) Concentration (MM) Chart 4. Survival response of UV-2237 parent and UV-2237-ADMB cells to anticancer drugs. O, UV-2237 parent; â€¢.UV-2237-ADM". covered in CMEM containing 3 mw ouabain and HAT medium. 100 The number of metaphase chromosomes of the parent lines was between 36 and 42, whereas that of the hybrids was 63 to 75 (Table 1). The dose-response curves of the ADM-sensitive UV- 2237RR line, the UV-2237-ADMR line maintained in drug-free 10-1 culture for 5 weeks, and 3 independently derived clones resulting from hybridization between these 2 cell types are presented in Chart 5. Also shown in this chart is the dose response of a hybrid resulting from the fusion of cells from the ADM-sensitive UV- 2237RRtumor and the UV-2237 parent tumor. 10-2 The UV-2237-ADMR line was maintained in drug-free medium for 5 weeks in order to serve as the appropriate control for the hybrid clones that were isolated and grown in medium that did not contain ADM. Chart 5 shows that the UV-2237-ADM" line 10-3 â€ž, exhibited the expected behavior for the cell line maintained in 0.01 0.1 1 Adriamycin Concentration { .q/nil) this manner and that resistance to the effects of ADM was Chart 5. Survival of somatic cell hybrids grown in the presence of ADM. G, UV- approximately 10 times that of the ADM-sensitive UV-2237RR. 2237"" cell line; O, UV-2237-ADMR cell line; A, UV-2237"" x UV-2237 parent; â€¢ The plating efficiency of all 3 independently derived hybrid clones UV-2237RH x UV-2237-ADM" clone 3; A, UV-2237"" x UV-2237-ADM" clone 4; and â€¢UV-2237"" x UV-2237-ADMR clone 5. , no colonies observed at was midway between these 2 phenotypes, suggesting that ADM higher concentrations of the drug. resistance is an incompletely dominant trait. The hybrid line derived from the fusion of the UV-2237 parent line and the UV- of 2 or 20 Mg/ml. In the first 10 min, uptake by the UV-2237- 2237RR line (UV-2237RR x UV-2237 parent) was no more resis ADMR cells was very similar to that by UV-2237 cells. However, tant to ADM than were the 2 individual lines. the resistant line rapidly achieved a near-plateau of incorporation Uptake and Efflux Studies. Results from a typical uptake such that, by 60 min, there was a 3-fold and by 120 min a 6-fold experiment are presented in Chart 6. Identical curves were difference in incorporation between the 2 cell lines (Chart 6). obtained whether the [UC]ADM was added at a concentration Efflux experiments consistently showed a difference in the

MAY 1983 2219

7 line (17), from leukemias (22, 28, 38), or from ascites-grown tumor lines (9, 37). Apart from brief mentions of a murine neu 6 roblastoma (2) and a murine sarcoma (34), there are no other

M reports on the isolation of such variants from a solid tumor line. Ã¯ 5 o Resistance to ADM is often associated with decreased drug & uptake (8, 14, 16, 29), which may be related to alterations in permeability of the cell membrane (32) or glycoprotein content of the cell (15) or may be the consequence of increased active efflux of the drug from the cell (18, 19). The UV-2237-ADM" cell line exhibited cross-resistance to a wide range of chemotherapeutic agents with dissimilar chemical structures and mechanisms of action; these agents include ACT D, AD 32, AMSA, DNR, MIT C, VBL, and VCR (Chart 4). 20 40 60 80 100 120 Reciprocal cross-resistance between ADM and DNR, which Incubation Time (min) bears a hydrogen atom in the acetyl moiety instead of a hydroxyl Chart 6. Uptake of [MC]ADM by the UV-2237 parent line (O) or by the UV-2237- group, has been well described in experimental tumors (7, 9,19, ADM" line (A). Results shown are for ADM at a concentration of 20 >ig/ml. Each 21, 32, 37). Cross-resistance has also been reported with AD 32 point, mean for triplicate wells. S.D. < Â±10%. (21), an A/-acylated derivative, although AD 32 does not bind to DNA (33), unlike the other anthracycline derivatives. Cross- 100 resistance to the Vinca alkaloids (2, 7, 21) and the nonanthra- cycline DNA-intercalating agents ACT D and AMSA (3, 21, 39) has also been associated with ADM-resistant lines. In contrast to several other studies (21, 31), however, we found cross- resistance to the alkylating agent MIT C. The basis for this f 50 O observed cross-resistance is, at the present time, unknown. This type of pleiotropic cross-resistance has been associated with a membrane alterations resulting in reduced permeability (1). In S. deed, Ling and his colleagues have shown that Chinese hamster ovary cells selected for resistance to colchicine have a drastic 10 15 20 25 30 35 40 45 reduction in permeability to not only that drug but also a number Incubation Time (min) of apparently unrelated agents (25-27). Therefore, a membrane Chart 7. Efflux of [MC]ADM from UV-2237 cells (O) or UV-2237-ADM" cells (A). mutation may be responsible for the resistance of the UV-2237- Results, each point representing the mean from triplicate samples, are presented ADMR line to some drugs. However, this is unlikely to be the as the percentage of radiolabeled drug remaining associated with the cells. One hundred % values were obtained by leaving glucose solution on cells for 5 sec total explanation. Pretreatment of the 2 cell lines with the meta only. S.D. < Â±10%. bolic inhibitor sodium azide leads to equal uptake of [14C]ADM, suggesting that passive diffusion alone is not sufficient to ac rates of efflux of the radiolabeled drug, although these differ count for the observed differences between UV-2237 and UV- ences were not always as large as those obtained in the exper 2237-ADM" lines. Equally, the efflux experiments indicate that iment illustrated in Chart 7. The UV-2237-ADM" line was better the UV-2237-ADM" cells are able to actively export ADM at a able to actively export the [14C]ADM than was the more sensitive faster rate than are the sensitive UV-2237 cells. These findings UV-2237 line. are consistent with observations made earlier by other workers who used different cell lines (18, 19). We are currently investi gating the exact basis of the resistance exhibited by the UV- DISCUSSION 2237-ADM" line. ADM binds and intercalates with double- We have isolated and propagated in vitro a variant line of the stranded DNA with relatively high efficiency, and many investi murine UV-2237 fibrosarcoma that is resistant to the cytotoxic gators agree that the cytotoxic activity of this drug is mediated action of ADM. This resistance is maintained in the absence of through this interaction (13, 23, 36). A recent report has sug ADM, whether the cells are grown in vitro or in vivo. Cells of the gested that some of the cytotoxic activity of ADM may be a UV-2237-ADMR variant appeared normal when maintained in 1.0 consequence of interactions at the cell surface without any Â¿

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preexisting clones or as a result of in situ hybridization. The daunorubicin by sensitive and anthracycline-resistant sublines of P388 leuke stability of the ADM-resistant characteristic in vivo and the mia. Biochem. Pharmacol., 27: 2123-2130,1978. possession of a "universal fuser" line of UV-2237 should allow 19. Inaba, M., Kobayashi, H., Sakurai, Y., and Johnson, R. K. Active efflux of daunorubicin and Adriamycin in sensitive and resistant sublines of P388 us to further explore this possibility in vivo. leukemia. Cancer Res., 39: 2200-2203, 1979. 20. Janzen, H. W., Millman, P. A., and Thurston, O. G. Hybrid cells in solid tumors. Cancer (Phila.), 27: 455-459,1971. ACKNOWLEDGMENTS 21. Johnson, R. K., Chitnis, M. P., Embrey, W. M., and Gregory, E. B. In vivo characteristics of resistance and cross-resistance of an Adriamycin-resistant We thank Dr. Cora Bucana for preparing micrographs and Professor Federico subline of P388 leukemia. Cancer Treat. Rep., 62: 1535-1547, 1978. Arcamone, Farmitalia Carlo Erba. Milan, Italy, for supplying radiolabeled Adriamycin. 22. Johnson, R. K., Ovejera, A. A., and Goldin, A. Activity of anthracyclines against an Adriamycin (NSC-123127)-resistant subline of P388 leukemia with special emphasis on Cinerubin A (NSC-18334). Cancer Treat. Rep., 60:99-102,1976. REFERENCES 23. Kim, S. H., and Kim, J. H. Lethal effect of Adriamycin on the division cycle of HeLa cells. Cancer Res., 32: 323-325, 1972. 1. Baker, R. M., and Ling, V. Membrane mutants of mammalian cells in culture. 24. Kripke, M. L., Gruys, E., and Fidler, I. J. Metastatic heterogeneity of cells from Methods Membr. Biol., 9: 337-384,1978. an ultraviolet light-induced murine fibrosarcoma of recent origin. Cancer Res., 2. Baskin, F., Rosenberg, R. N., and Dev, V. Correlation of double-minute 38: 2962-2967, 1978. chromosomes with unstable multidrug cross-resistance in uptake mutants of 25. Lalande, M. E., Ling, V., and Miller, R. G. Hoechst 33342 dye uptake as a neuroblastoma cells. Proc. Nati. Acad. Sei. U. S. A., 78: 3654-3658,1981. probe of membrane permeability changes in mammalian cells. Proc. Nati. Acad. 3. Belli, J. A., and Harris, J. R. Adriamycin resistance and radiation response. Int. Sei. U. S. A., 78: 363-367, 1981. J. Radial. Oncol. Biol. Phys., 5. 1231-1234, 1979. 26. Ling, V., and Baker, R. Dominance of colchicine resistance in hybrid CHO 4. Blum, R. H., and Carter, S. K. Adriamycin: a new anticancer drug with cells. Somatic Cell Genet., 4: 193-200,1978. significant clinical activity. Ann. Intern. Med., 80: 249-259,1974. 27. Ling, V., and Thompson, L. H. Reduced permeability in CHO cells as a 5. Bonadonna, G., Beretta, G., Tancini, G., Brambilla, C., Bajetta, G., De Palo, mechanism of resistance to colchicine. J. Cell Physiol., 83: 103-116, 1974. M., De Lena, M., Fossati-Bellani, M., Gasparini, P., Valagussa, P., and Vero 28. Nishimura, T., Muto, K., and Tanaka, N. 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Fig. 1. a, UV-2237 parent cells maintained on plastic in CMEM; b, UV-2237-ADM" cells maintained on plastic in CMEM containing ADM (1

2222 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. Isolation and Preliminary Characterization of an Adriamycin-resistant Murine Fibrosarcoma Cell Line

Raffaella Giavazzi, Eric Scholar and Ian R. Hart

Cancer Res 1983;43:2216-2222.

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