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[CANCER RESEARCH 41, 4529-4534, November 1981] 0008-5472/81 /0041-OOOOS02.00 Basis of Observed Resistance of L1210 Leukemia in Mice Methotrexate, 6-Thioguanine, 6-Methylmercaptopurine Riboside, 6-Mercaptopurine, 5-Fluorouracil, and 1-/?-D-Arabinofuranosylcytosine Administered in Different Combinations1 Kami Kim, William J. Blechman, Veronica G. H. Riddle, and Arthur B. Pardee Sidney Farber Cancer Institute and Pharmacology Department, Harvard Medical School, Boston, Massachusetts 02115 [K. K., W. J. B., V. G. H. R, A. B. P.] ABSTRACT They found that these 6 drugs in combination were more effective in prolonging mouse lifetime than was any single drug. Schmid et al. (Cancer Treat. Rep., 60: 23-27, 1976) re They also could delay the resistance to the individual drugs ported rapid emergence of resistance of L1210 leukemia cells with 6-drug combinations. Schmid ef al. (12) administered the in mice to two schedules of six antimetabolites and much drugs either simultaneously (at % LD,,, each for 6 days,) or slower development of resistance to a third schedule. Such sequentially for 6 days (as one drug daily at LD,,>,)in2 different rapid development of resistance to six drugs presents a striking "offset" sequences. L1210 cells were transferred to other puzzle, and one whose solution gives some insights into the mice shortly before death. Strong drug resistance, observed basis for general emergence of drug resistance. Our approach as a decrease of mouse survival to well below those times was to examine the consequences of applying these drugs initially obtained, occurred with simultaneous administration of singly or in pairs and, from the results, to infer interactions in the 6 drugs at mouse transfer generation 31, whereas resist six-drug combinations. ance to 2 sequential schedules of 6 drugs appeared in only 5 6-Thioguanine (TG) and 6-mercaptopurme are the key drugs or 3 generations. since, as shown by Schmid et al., resistance of leukemic cells These strikingly rapid emergences of resistance to multiple appeared to six-drug combinations at the same time as did drug therapy, and the major effects of scheduling do not appear resistance to the purine analogs; sensitivity to the other drugs to have been experimentally followed up. They have been remained. We demonstrated that cells which emerged were commented upon at length by workers at the Southern Re resistant to both of the purine analogs, owing to a deficiency of search Institute (13). The aim of the present work was to find the activating enzyme hypoxanthine-guanine phosphoribosyl- out why rate of appearance of resistance is so different under transferase. different drug schedules and why it occurs so rapidly with TG resistance arose in the presence of TG because of an some schedules. The results and conclusions are relevant to overgrowth of TG-resistant mutants that were present as one cell in 10* in the original L1210 population. L1210 cultures the general problem of emergence of drug resistance. were prepared free of TG-resistant mutants. With these cells, TG administered shortly after inoculation was very effective in MATERIALS AND METHODS delaying their death. The cells that finally grew out were still TG sensitive. In Vivo Experiments. The methods of Schmid et al. (12) were used. Simultaneous treatment with all the drugs greatly delayed Experimental groups consisted of 8 male C57BL/6 x DBA/2 F, (hereafter called B602F,) mice 5 to 6 weeks old weighing 20 to 30 g appearance of TG resistance in vivo and in vitro. Methotrexate obtained from The Jackson Laboratory, Bar Harbor, Maine. The original alone was responsible for this result, owing to its ability pref L1210 cells, maintained in male DBA/2 mice, were those used rou erentially to kill TG-resistant cells. The other three drugs were tinely at the Sidney Farber Cancer Institute. not effective in delaying TG resistance. Methotrexate was In vivo experiments involved repeated tumor passage and drug effective only if it was added daily; one large injection was challenge with a variety of antimetabolites. Each mouse was inoculated ineffective. Therefore, TG and methotrexate added daily for 6 i.p. with 106 L1210 cells. Twenty-four hr later, and for 6 successive days (simultaneous schedule) was the most effective drug days thereafter, each mouse was injected with drugs. The numbers of regimen tested. mice surviving were counted daily. When a mouse was heavily laden with tumor, 106 cells were transferred to the next generation. The transfer time varied with the nature of the 6-day drug treatment. INTRODUCTION Toxicity experiments with each drug were run on tumor-free mice to verify that no more than 10% of the mice were killed with a given drug Several years ago, Schmid ef al. (12) studied the develop schedule. The LD,0 doses used were those administered by Schmid ef ment of resistance of L1210 leukemia in mice to combinations a/.; for TG, it was 2.9 mg/kg; for MTX, it was 3.4 mg/kg. When 2 of the antimetabolites MMPR,2 MP, TG, MTX, FU, and ara-C. drugs were used simultaneously, V?LD,,>of each drug was tested. Only animals that died of tumor were included in the final tabulation of data. Received May 4, 1981 ; accepted August 6, 1981. Drug-resistant Cells. To obtain L1210/TG cells, mice were injected 1 This investigation was supported by Grants CA 19864 and CA 22427 with L1210 cells and with TG as above, and at Day 11 or 12, 10e cells awarded by the National Cancer Institute, Department of Health, Education, and Welfare, and by The Friends of the Sidney Farber Cancer Institute. were transferred to another group of mice and TG treatments were 2 The abbreviations used are: MMPR, 6-methylmercaptopurine riboside; MP, 6-mercaptopurine; TG. 6-thioguanine; MTX, methotrexate, FU, 5-fluorouracil; ara-C, cy tosine arabinoside; LD, n.one-tenth lethal dose; L1210/'TG, TG-resistant hypoxanthine-methotrexate-thymidine medium; LI 2IO/HMT. HMT-selected L1210 cells; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; HMT, L1210 cells. NOVEMBER 1981 4529 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1981 American Association for Cancer Research. K. Kim et al. repeated. To ensure that resistance had developed, tumor cells from obtained in vivo (see "Materials and Methods") were tested in these mice were injected into a third group and treated with TG. Cells culture for sensitivity to MP (5 /ig/ml) or TG (1 jug/ml; Chart 1). from these mice were maintained in B602F, mice and also were placed These TG-resistant cells were cross-resistant to MP. in culture. They were stably and irreversibly resistant to TG indefinitely Resistance to TG is commonly due to loss of the enzyme without further TG treatment, as tested both m vivo and in vitro (see "Results"). HGPRT, which converts the purine to nucleotide (5). Assay for MTX-resistant cells were obtained from Linda Anderson of the Sid HGPRT of L1210/TG cells revealed no activity, as contrasted to the original TG-sensitive L1210 cells (Chart 2). Resistance ney Farber Cancer Institute. They were not inhibited in culture by MTX (0.045 fig/ml). to TG (and MP) was thus due to a deficiency in HGPRT. L1210 Growth in Culture. L1210 cells, aseptically removed from Consistent with a genetic change, TG resistance was retained the mouse peritoneum, were maintained in Eagle's minimum essential in the absence of the drug through several passages in culture medium (Grand Island Biological Co.) supplemented with 10% calf or in mice. serum, penicillin (100 units/ml), streptomycin (100 /ig/ml), and ß- Drug-resistant cells might arise as mutants during the exper mercaptoethanol (0.78 /ig/ml). The cultures were incubated in 75-sq iment, or they could have preexisted in the original L1210 cm Falcon T-flasks in a water-saturated 10% COî-90%air atmosphere stock (6). As a test for preexisting TG-resistant cells, each well at a constant temperature of 37°.Cells were plated at a density of 1.0 or 2.0 x 10s cells/ml and became confluent at approximately 4.0 x 10e cells/ml. Cell viability was periodically checked using trypan blue. Cell counts were performed using a Model 2, Coulter Counter. For growth in 96-well plates, cells were diluted in complete Eagle's IO6 minimum essential medium to the desired concentration. When used, TG was added to obtain a final concentration of 1 /ig/ml. Cells were pipetted into each well in a total volume of 0.1 ml. Visual counts were Controls LI2IO/TG conducted with a microscope to monitor cell growth for several days -Drug + TG or MP E subsequent to plating. Two to 3 weeks later, cultures were scored for ^ growth of cells. —¿ HGPRT Enzyme Assay. HGPRT activity was assayed (5) using a 0> crude cell extract prepared from 107 cells. Cells were centrifugea at O 1500 rpm for 7 to 10 min, the pellet was resuspended in 0.5 ml sucrose buffer (0.25 M sucrose-50 mw Tris-3 mw MgCI2-7 rriM /8-mercaptoeth- IO LI2IO anol), and the resuspended cells were disrupted with several pulses of + TG or MP 30 sec each with a Branson sonifier set at 1. The disrupted cells were centrifuged in a microfuge at 13,000 rpm for 2 min to remove mem brane and other debris. The supernatant was then used in the enzyme assay. The final incubation mix of 300 /<!consisted of 0.05 M phosphate buffer (pH 7.4), 5 mM MgCU 1 mw 5-phosphoribosyl 1-pyrophosphate I 2 3 sodium salt, and 20 /IM [3H]hypoxanthine (10 to 50 /iCi/,umol). Cell Days Growth extract (108 /il) was added to start the reaction.

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