Basis of Observed Resistance of L1210 Leukemia in Mice

[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

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 ÃŸ- 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% COÃ®-90%air atmosphere stock (6). As a test for preexisting TG-resistant cells, each well at a constant temperature of 37Â°.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. â€”¿ 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 /

RESULTS LI2IO Resistance to Furine Analogs. Our initialexperimentswere en on development of resistance to MP (and TG) alone.This is O because MP and TG are primarily responsible, in applications IO of the 6 drugs, for delaying lethality of L1210 cells. On offset schedules, resistance to all 6 drugs and also resistance to both these purine analogs emerged simultaneously after only about 4 mouse passages, and sensitivity to the other 4 drugs was LI2IO/TG retained (12). Thus, appearance of resistance of L1210 cells to these analogs alone was sufficient to create resistance to IO 20 30 the 6-drug combinations. Incubation Time ( min ) Since MP and TG are similar and both are activated by Chart 2. HGPRT activity in Li 210 O) and L1210/TG (â€¢)cells.The assays conversion to nucleotides, it is likely that cells resistant to one were performed as described in "Materials and Methods." Inosinic acid produced drug would also be resistant to the other. L1210/TG cells by the 2 cell types is plotted as pmol/105 cells versus incubation time.

4530 CANCER RESEARCH VOL. 41

Table1 Fraction of TG-resistant cells in L1210 Results are sums of pairs of experiments. No. of inocu lated cells/ TG (fig/ Total no. of wells of wells of TG-re- sistantcells"1000.5 cells/wellsL1210Cell type well ml) with growth170withgrowth100 0 170 L1210 50 0 177 175 9899100 L1210L1210/TG 10016425 0 163170

L1210/TG 5010025 174 172 99 L1210/TGL1210/TG 163384 16246 9912

+ L12100/99) L1210/TG + U 210(1/99) 50 174 46 26 0.6 L1210/TG0(1/99)L1210 + L121 1002550100170 188384 1611 850.3 0.90.010

L1210 360 2 0.6 0.011 L121025 394No. 6% 1.5% 0.015 * Calculated from Poisson distribution. of a 96-well plate was inoculated with a small number of cells, then growth was estimated periodically by visual observation (Table 1). Controls (minus drug) showed growth in nearly all wells, inoculated with as few as 25 original cells. TG-resistant cells grew in all wells containing TG (1 /ig/ml). A mixture made of one TG-resistant cell per 100 TG-sensitive Controls cells showed growth at approximately the expected frequency -Drug in the presence of TG. The average number of TG-resistant E cells per well can be calculated from the number of wells *v showing no growth in the presence of TG, using the null case of the Poisson distribution: P(0) = e~", where P(0) is the fraction of wells showing no growth with TG present, and n is the average number of resistant cells in a well. Dividing n by LI2IO or LI2IO/TG the number of cells added to each well gives the fraction of MTX resistant cells in the population. Growth of L1210 in the presence of TG was seen rarely, in only about 1% of wells inoculated with 100 cells each (Table 1). This result indicates that about 1 in 104 of the original cells LI2IO/TG + HMT were TG resistant, a fraction also arrived at from the Poisson distribution. This test does not rule out the possibility of these Days Growth resistant cells rarely arising by mutation during the experiment. Charts. Sensitivity of L1210/TG cells in HMT medium. L1210 (3) and L1210/TG cells (â€¢)wereput into culture at 10s cells/ml. The media contained However, there is little if any growth of sensitive cells in the no drug O. â€¢¿),MTX(0.045 fig/ml; 7, V), or MTX (0.045 /ig â€¢¿ml)plushypoxan presence of TG (see Chart 1), and so there was little opportunity thine (12 fig/ml) plus thymidine (5 fig/ml; II. â€¢¿). for mutants to arise. Other data presented below argue against mutants arising during culture plus TG. ative) cells are highly sensitive to HMT (Chart 3). TG-sensitive The presence of 10 4 resistant cells in an L1210 population cells survive in HMT. Both lines are killed by MTX in the is consistent with in vivo results. A mouse given 106 L1210 absence of hypoxanthine and thymidine. L1210/HMT cells cells and daily injections of LD,0 of TG for 6 days died in about were injected into groups of mice, without and with TG treat 12 days. Mice given 200 TG-resistant cells and TG also died ment (Table 2). The mice given injections of L1210/HMT cells in 12 days, a result consistent with about 100 TG-resistant lived for 21 days if treated with TG, a remarkably longer survival cells in 10" (e.g., 10~4) in the L1210 population being respon in contrast to mice given injections of the original L1210 cells sible for death. which survived about half as long. Both kinds of L1210 cells A critical test of this hypothesis that preexisting TG-resistant killed the mice in about 9 days in the absence of TG. Therefore, cells are responsible for the appearance after the first mouse the lethality of the original L1210 culture in mice given TG was passage of a resistant population is to study lethality of an due to the initial presence and proliferation of TG-resistant L1210 population from which TG-resistant cells have been cells. removed. To obtain a culture low in TG-resistant cells, the Although greatly increased survival of L1210/HMT-injected original L1210 cells (2 x 10s/ml) were cultured in complete mice given TG for 6 days was found, no cures were achieved. HMT supplemented with MTX (0.045 /ig/ml), hypoxanthine (12 It is unlikely that death resulted from outgrowth of a few fig/ml), and thymidine (5 /ig/ml), which is like the hypoxan- surviving TG-resistant cells in the L1210/HMT population, thine-amethopterin-thymine medium usually used to counter- since even one TG-resistant cell kills a mouse in about 15 select HGPRT-negative mutants (8). TG-resistant (HGPRT-neg- days. Possibly TG-resistant mutant cells arose within the in-

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Table 2 Effect of selection in HMT medium on in vivo TG treatment OÃ•L1210 cells TG (V4 LDio) was given daily for 6 days. The results are for duplicate experi ments with 8 animals/group. No cures were achieved. Range of % in- death Median day Mean day creased life Cell type Drug (days) of death of death span L1210L1210L1210/HMTL1210/HMTTGTG7-118-138-1215-31812,91021812,91020,2250,4100, E ^ 126 â€”¿ O) O jected mice. To determine the drug resistance characteristics of the cells that killed these mice, the cells were tested in culture. These cells were still sensitive to TG (1 /ig/ml; Chart 4). They were also sensitive to MTX (0.045 /tg/ml), a concen tration that stopped growth of MTX-sensitive but not MTX- resistant cells. TG-resistant cells did not appear in vivo in large numbers during this experiment, since these would have been observed to grow. Thus, despite elimination of apparently all TG-resistant cells, TG was unable to kill enough of the sensitive Days Growth tumor cells. Death appears to be caused by outgrowth in vivo Chart 4. Retention of TG sensitivity of LI210/HMT cells after passage of a very few surviving TG-sensitive cells during the 15 days through mice given TG. L1210/HMT cells (10e) were injected, and 'A LD10 of TG was given daily for 6 days thereafter. Cells were collected shortly before death following the 6 days of drug treatment (Table 2). and (2 x 10'Vml) put into culture without drug (O), plus TG (1 fig/ml; A), or plus MTX Specifically Delays TG Resistance. Development of MTX (0.045 fig/ml; V). Cell counts were made daily thereafter. resistance to TG was greatly delayed (from 1 until 28 mouse Table 3 passages) when all 6 drugs were given in the combination Development of resistance to TG in presence of another drug mode of scheduling (12). MTX is particularly lethal to TG- L1210 cells were used for passage 1. One drug at LD ; or 2 drugs at 0.5 LD,,, resistant cells in culture (when given plus hypoxanthine and were given daily for 6 days starting 1 day after i.p. injection of 10Â°tumor cells. thymidine which rescue MTX-sensitive cells). Therefore, simul Cells taken shortly before death were taken from passage 1 animals treated with 2 drugs and injected into passage 2 animals that were given drugs as shown on taneous administration of MTX and TG should kill both TG- the same line of the table. resistant cells with MTX and also TG-sensitive cells with TG. Mean survival time (days) This was the case: simultaneous TG and MTX (for 6 days) prolonged mouse life span (passage 1) to 20 days (Table 3). Passage 2 uiner drug "A"MTX "A"8 This survival is considerably longer than without drugs or with +TG12 drug8 TG15 +TG20 either drug alone. Also, cells taken from mice treated with both MTX and TG remained as sensitive in vivo to TG or MTX plus 14 20 TG as were the original cells; mice inoculated with these cells MMPR 14 17 8 8 11 ara-C 16 18. 17 8 8 15 and again treated with TG survived for 15 days, and with both FU+ 16"A" 20,20No 9+ 9"A" 17 drugs for 20 days, as before (Table 3, passage 2). Thus, MTX prevented development of TG resistance. Each of the other 3 drugs increased the life span of mice given L1210 cells (12). An additional increase in life span was seen when each of these drugs was given together with TG (Table 3, passage 1). However, resistance to TG developed in the presence of each of these drugs plus TG, as contrasted to cells exposed to both MTX and TG (Table 3, passage 2). These mice only survived 8 to 9 days, either in the presence or absence of TG. The cells apparently were still somewhat sen 0> sitive to ara-C or FU (plus TG); possibly, some MMPR resist U ance had developed (see also Tables 3 and 4 in Ref. 12). We conclude that the most important drug in the simultaneous schedule is MTX, which kills TG-resistant cells and thereby permits TG to kill almost all remaining cells. Tissue culture experiments were performed next to test the conclusion that only MTX prevented development of TG resist ance in vivo. Cells taken from mice exposed to TG plus another drug were cultured in media with and without TG or another Days Growth drug. Chart 5 illustrated a typical example of the resistance Chart 5. TG resistance and ara-C sensitivity of L1210 cells taken from mice exposed to both TG and ara-C. The cells collected from mice exposed to both characteristics of tumor cells obtained from mice exposed to drugs, each given daily at '.,.LD,,, for 6 days, were put into culture at 2 x 10'Vml TG plus ara-C. These cells grew as well in the presence of TG in the presence of no drug (O), TG (1 fig/ml; A), or ara-C (10 fig/ml; O). Cell as in its absence, showing that ara-C did not prevent TG counts were made daily thereafter.

4532 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1981 American Association for Cancer Research. Multiple Drug Resistance of L1210 Leukemia resistance from appearing. These cells were still sensitive to must eradicate the maximum number of nenoplastic cells while ara-C, being unable to grow in the presence of this drug. The minimizing trauma to the host. The problem is acute because same experiments were done with cells obtained from mice the low chemotherapeutic indices of most anticancer drugs exposed to TG plus other drugs (Table 4). Only MTX prevented makes eradication of even slightly resistant cells impossible at the emergence of TG-resistant cells; ara-C, MMPR, and FU permissible drug doses. Drugs used in combination should were ineffective. Drugs other than MTX decreased the net effectively check growth of most resistant cells since, if the growth of L1210 cells in vivo and thereby delayed lethality. But mechanisms of resistance to 2 drugs differ, resistance to one they must be equally inhibitory for TG-sensitive and TG-resist drug should occur independently of resistance to another drug. ant cells, since the population did not change resistance to TG. Therefore, the probability of a cell being doubly resistant should Nor apparently is the sensitivity of L1210 cells to the second be very low, and the use of drugs in combination should result drug great enough to select strongly for cells resistant to that in negligible growth of resistant cells. Combinations of drugs drug (12). can cure animals bearing up to 100 times as many injected One of the most puzzling results of Schmid ef al. (12) was leukemia cells as are curable with optimum doses of any single the great efficiency of the simultaneous schedule in delaying drug in the combination (2). Combination therapy is also more resistance to TG as compared to the sequential schedules. effective against human tumors than are many agents used Since the total amounts of drugs given were the same in all 3 alone (3). schedules, and TG-resistant cells are extraordinarily vulnerable In experiments with animal neoplasms, however, rapid selec to MTX in culture, the inadequacy of the sequential schedules tion of cells with permanent resistance to 2-, 3-, or more drug was surprising. combinations has been demonstrated (12, 13). Because of Scheduling effects of MTX alone on survival of L1210-in- constraints of host toxicity, drugs having the capacity to kill jected mice were therefore studied (Table 5). Six daily doses resistant cells are unable to do so effectively enough to prevent of VeLD10of MTX were much more effective in prolonging life overgrowth of tumor cells. The basis for cancer chemotherapy than one LD10dose on Day 1, which appeared almost ineffec and the emergence of resistance has been extensively studied tive. Six (daily) LD1(Idoses were also effective, but the drug in mouse systems by Skipper, Schabel, and their colleagues, was toxic at this level. MTX was even more effective against so that there are quite clear ideas of the factors involved (13). TG-resistant cells, particularly if given in 6 doses of % LD,,> Resistance can develop in a population owing to the overgrowth each. One LD,,, dose was quite ineffective against even these of preexisting rare mutant cells (6). Also, the progression of highly sensitive L1210/TG cells. Depending on the frequency tumors quite rapidly gives rise to cells with drug resistance (4). of its application, MTX is potentially lethal to TG-resistant cells. It is, however, surprising that resistance rapidly emerges to It was much more effective when given daily in small doses as therapies involving 6 commonly used antineoplastic antimetab- in the simultaneous schedule than when given once as in the olites (12). An understanding of these results provides insights sequential schedules. to some factors basic to appearance of resistance. Extending these experiments, we find that overgrowth of TG- DISCUSSION resistant cells is a major factor in the failure of the 6 drugs to eliminate L1210 cells. Approximately one in 10''"cells in our Resistance to anticancer drugs is a major factor preventing laboratory line was initially resistant to TG. Deficiency in the the cure of cancer. An effective chemotherapeutic regimen enzyme HGPRT, which converts TG to its cytotoxic form, was responsible for the resistance to TG and also MP, a common Table 4 Development of TG resistance in vivo mechanism of resistance to both TG and MP. Because so many Cells (1 x 105) per ml obtained after in vivo passages 1 (Table 3) were put tumor cells were initially TG resistant, complete resistance was into culture and grown for 7 days. Final cell counts per ml are given. quickly observed. (Note that the same results would have been observed for most mice a few days later if only 10~6 of the (xNo in culture cells had initially been resistant.) TG/ig/ml)233 The remaining 4 drugs were effective (in the "simultaneous" Cells1TG from passage drug038 + MTX schedule) mainly because of one, MTX. This drug exerts a TG + MMPR 36 potent lethality upon TG-resistant cells and prevents them from TG + ara-C 33 34 developing into a population that kills the animal in the pres TG + FUCells/ml 361CT5)+ 36 ence of TG and MP. Many lines of cells resistant to purine

Table 5 Effects of MTX scheduling of death day day of increased CelltypeL1210L1210L1210L1210L1210/TGL1210/TGL1210/TGL1210/TGMTXVi(days)7-1112-1315-178-117-815-1917-199-10Medianof death8121798161810Meandeath8121698171810%lifespan9551031611413223

daysLD,,,LD,o for 6 for 6daysLD,,, 1% at Day

daysLD,ocLD,,,for 6 for 6daysLD,o at Day 1Range ' No cures were achieved. Pronounced toxicity. c Pronounced toxicity; 3 of 8 mice died due to MTX.

NOVEMBER 1981 4533

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1981 American Association for Cancer Research. K. Kim et al. analogs have been reported to be collaterally sensitive to MTX MTX may also have been more potent in several doses (7, 11, 14), and at least one of these lines is known to be because of its ability to arrest cells at the beginning of S phase HGPRT deficient (11). (10). The cells not in S at the time of exposure to MTX are Administration of TG with MMPR, ara-C, or FU was more arrested in S at the G,-S boundary. MTX can only kill the cells effective in prolonging mouse lifetime than was TG alone, or already in S; it is a "self-limiting" drug, and thus cells not in S any of the drugs singly. But these drugs, unlike MTX, were not are protected (1). After MTX disappears, these cells recover able to prevent TG resistance from developing rapidly. There by synthesizing dihydrofolate reductase and move into S. upon, the major effect of the combination was lost. That the 3 where they will be susceptible to the next MTX dose (9). other drugs in combination could have a major additional effect seems unlikely, and was not tested. ACKNOWLEDGMENTS The initial presence of TG-resistant cells was the major factor We thank Or. G. P. V. Reddy for performing assays for HGPRT. limiting treatment with TG or the 6 drugs. But it was not the only reason why mice inoculated with L1210 cells were not REFERENCES cured by TG. Even when TG-resistant cells were eliminated 1. Bhuyan, B. K., Fraser, T. J . Gray, L. G., Kuentzel. S. L , and Neil, G. L. from the inoculum, sensitive cells eventually proliferated and Cell-kill kinetics of several S-phase-specific drugs. Cancer Res., 33: 888- 894.1973. killed the mice. These final cells were TG sensitive, showing 2. Brockman. R. W. Mechanisms of resistance. In A. C. Sartorelli and D. G. mutation to resistance was rare. The 6-day treatment with TG Johns (eds ), Antmooplastic and immunosuppressive agents. Part I, pp. 352-410. Berlin Springer-Verlag, 1974. appears not to have killed all the L1210 cells. Surviving cells 3. Henderson, E. S., and Samaba, R. J. Evidence that drugs in multiple must multiply between drug doses, compensating to some combinations have materially advanced the treatment of human malignan extent for a rather small kill at each dose; the net kill per dose cies. Cancer Res., 29: 2272-2280. 1969. 4. Heppner, G. H , Dexter, 0. L., OeNucci. T., Miller. F. R.. and Calabresi. P. for 6 doses may have been insufficient. Alternatively, a few Heterogeneity in drug sensitivity among tumor cell subpopulations of a single L1210 cells may have been retained in some sanctuary in the mammary tumor. Cancer Res.. 38. 3758-3763, 1978. 5. Johnson, G. G.. and Lutietieid, J. W. Assay of hypoxanthine-guanine phos- mouse, eventually to emerge and multiply. phoribosyltransterase in human Iibroblast lysates: mactivation of nucleotid- Scheduling of drugs was also a major factor influencing the ase. Anal. Btochem.. 92:403-410,1979. success of treatment (12). Increased dose at one time for cell 6. Law, L. W. Origin of the resistance of leukemic cells to folie acid antagonists. cycle phase-specific drugs like MTX and TG does not increase Nature (Lond ). 169. 628-629. 1952. 7. Law, L. W. Some aspects of drug resistance in neoplasms. Ann. N. Y. Acad. cell kill, whereas increased duration of treatment does so Sci.. 71:976-992. 1958. markedly (15). Corresponding to data of Schmid et al. with 6 8. Littlefleid. J. W. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science (Wash. D. C.), 145: 709-710,1964. drugs (12), in our experiments several doses of MTX eliminated 9. Neil. G. L., and Homan, E. R. The effect of dose Interval on the survival of tumor cells more effectively than did one large dose, given as Li 210 leukemic mice treated with ONA synthesis inhibitors. Cancer Res . the same total amount; the single-dose response curve for MTX 33:895-901, 1973. 10. Scher. C. D.. Stone, M. E., and Stiles. C. 0. Platelet-derived growth factor is also very shallow (13). The great disparity between the prevents Go growth arrest. Nature (Lond.). 287:390-392, 1979. effectiveness of the dose schedules is probably largely a result 11. Schmid, F. A., and Hutchison, D. J. Collateral sensitivity of resistant lines of of the rapidity with which MTX disappears from the serum of mouse leukemias L1210 and L5178Y. Cancer Res., 32: 808-812.1972. 12. Schmid, F. A., Hutchison, D. J., Otter. G. M , and Stock. C. C. Development mice (13). One dose of MTX would disappear rapidly and not of resistance to combinations of six antimetaboHtes In mice with LI 210 kill enough of the TG-resistant cells to prevent survivors from leukemia. Cancer Treat. Rep., 60:23-27. 1976. 13. Skipper, H. E. Cancer chemotherapy. Vol. 1 (Sponsor Series). Ann Arbor, taking over the population. Several doses would result in a Mich.: Monograph Publishing, 1978. greater net exposure time to MTX and greater cell kill. More of 14. Tomizawa, S., and Aronow, L. Studies on drug resistance in mammalian the TG-resistant cells would be eliminated, to account for the cells. II. 6-Mercaptopurine resistance in mouse flbroblasts. J. Pharmacol. Exp. Ther.. J28: 107-114, 1960. concomitant delay in the development of resistance to purine 15. Valenote, F.. and van Putten, L. Proliferation-dependent cytotoxicily of analogs. anticancer agents: a review. Cancer Res., 35: 2619-2630,1975.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1981 American Association for Cancer Research. Basis of Observed Resistance of L1210 Leukemia in Mice Methotrexate, 6-Thioguanine, 6-Methylmercaptopurine Riboside, 6-Mercaptopurine, 5-Fluorouracil, and 1- β -d-Arabinofuranosylcytosine Administered in Different Combinations

Kami Kim, William J. Blechman, Veronica G. H. Riddle, et al.

Cancer Res 1981;41:4529-4534.

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