[CANCER RESEARCH 42, 1655-1660, May 1982]. 0008-5472/82/0042-OOOOS02.00. Establishment of Methotrexate-resistant Human Acute Lymphoblastic.
[CANCER RESEARCH 42, 1655-1660, May 1982] 0008-5472/82/0042-OOOOS02.00
Establishment of Methotrexate-resistant Human Acute Lymphoblastic Leukemia Cells in Culture and Effects of Folate Antagonists1 Taisuke Ohnoshi, Takao Ohnuma2, Isao Takahashi, Kevin Scanlon3, Barton A. Kamen, and James F. Holland Departments of Neoplastia Disease [T. Oi., T. Oa., I. T., J. F. H.] and Medicine (Oncology) ¡K.S., J. F. H.¡,Mt. Sinai School of Medicine, and Departments of Pediatrics and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510. [B. A. K.]
ABSTRACT A human acute lymphoblastic T-cell line, MOLT-3, was fed with Roswell Park Memorial Institute Medium 1640-10% fetal bovine serum-antibiotics, containing increasing concentrations of methotrexate (MIX). After 10 months of feeding, the cells became resistant to 10~7 M MTX; resistance was not reversed when the cells were placed in the original MTX-free medium. At 10~7 M MTX, the concentration which produced complete inhibition of the parent MOLT-3 cell growth, the resistant cells were not inhibited at all. On a 50% inhibitory concentration basis, the resistant cells were approximately 30-fold more resistant to MTX. The parent MOLT-3 and the resistant line had the same doubling time of approximately 36 hr. There were no differences in light microscopic morphology. MOLT-3 pro duced loose colonies on 0.5% agar enriched with fetal bovine serum, whereas the colonies of the resistant line were tightly packed. The development of resistance was accompanied by a 4- to 5-fold decrease in [3H]MTX transport (MOLT-3/MTX,). Kinetic analysis of MTX uptake showed that the resistant subline did not have an altered Km for MTX (6.6 /IM) but had a decreased Vma)< of about 20% of the parent cell line. These data suggest that either the number of folate transport sites or the turnover rate of these sites has been reduced in the MTXresistant cell line. Dihydrofolate reducÃ-ase was only minimally elevated in the resistant cell line. The MTX-resistant cell line was cross-resistant to dichloromethotrexate. The sensitivity of the resistant line to the substituted 2,4-diaminoquinazoline and pyrimidine compounds, 2,4-diamino-5-methyl-6-{(3',4',5'-trimethoxyanilino) methyl] quinazoline (JB-11 ) and 2,4-diamino5-(3',4'-dichlorophenyl)-6-methylpyrimidine, increased more than 3-fold. While leucovorin equally reversed the MTX effects on the parent and resistant cells, leucovorin reversal of 2,4diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino) methyl] quiazoline and 2,4-diamino-5-{3',4'-dichlorophenyl)-6-methylpyrimidine effects was limited only to the parent cell line. 2,4diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino) methyl] quina zoline or 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine plus leucovorin might prove to be unique in treating patients with acute lymphoblastic leukemia when the leukemic cells develop transport resistance to MTX.
1 Supported by USPHS research Grants CA-25865 from the National Cancer Institute and ROI-AM-16690-06 from the National Institute of Arthritis, Metabo lism, and Digestive Diseases. NIH, Bethesda Md.; by the Chemotherapy Foun dation, Inc., New York. N. Y.; by the United Leukemia Fund. Inc., New York, N. Y.; and by the T. J. Marteli Memorial Foundation, New York, N. Y. 2 To whom requests for reprints should be addressed, at the Department of Neoplastic Diseases, Mt. Sinai School of Medicine. One Gustave L. Levy Place, New York. N. Y. 10029. 3 Leukemia Society Fellow. Received April 20, 1981 ; accepted January 20, 1981.
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New York, N. Y. 70029,
INTRODUCTION The development of resistance to MTX4 has been studied in animal and human tumors (see review, Refs. 1 and 4). An elevated level of the target enzyme, dihydrofolate reducÃ-ase (3, 5), and/or impaired membrane transport of drug (21) have been shown to be major causes of resistance in human tumors. In order to overcome the MTX resistance, 2 approaches can be considered: (a) the use of high-dose MTX with LV rescue where a significant amount of MTX can enter cells by passive diffusion (8, 29) rather than through the classical saturable temperature-sensitive process of active transport (15, 29); (o) the use of folate antagonists which differ from MTX for their entry into the cells (20). We have established a MTX-resistant subline of MOLT-3, a human lymphoblastic leukemia cell line, by continuous expo sure to increasing concentrations of MTX in culture medium. Also, we have observed that the major mechanism of MTX resistance of the subline is transport related, and we have designated this cell line MOLT-3/MTX,. Using this cell line, we have evaluated the effects of various folate antagonists with and without LV. MATERIALS
AND METHODS
The cell line used was of human acute lymphoblastic leukemia, MOLT-3, with T-cell characteristics (17). It was maintained as a sus pension in culture flasks (Falcon Plastics, Oxnard, Calif.) containing RPMI 1640 Medium (Grand Island Biological Co., Grand Island, N. Y.) supplemented with 10% FBS (Grand Island Biological Co.) and anti biotics (22, 28). Cells were fed with fresh culture medium 3 times a week. Effects of folate antagonists on cell growth were determined by growth inhibition assay, as described previously (28). The cells were in an exponential growth phase and had over 90% cell viability. Drugs tested in growth inhibition assays were MTX and LV (Lederle Labora tories, Pearl River, N.Y.), DCM (obtained from the National Cancer Institute, Bethesda, Md.), TMQ (JB-11 ; obtained from Dr. J. Plowman, Drug Evaluation Research, the National Cancer Institute), DDMP (Burroughs-Wellcome Co., Research Triangle Park, N. C.; Lot 58167, obtained through the courtesy of Dr. S. Waxman), 5-fluorouracil (Roche Laboratories, Nutley, N. J.), 1-/?-r>arabinofuranosylcytosine (The Up john Co., Kalamazoo, Mich.), Escherichia coli asparaginase (Merck, Sharp & Dohme, West Point, Pa.), cisplatin (Bristol Laboratories, Syr acuse, N. Y.), and daunorubicin (obtained from the National Cancer Institute). MTX and DCM were dissolved in 0.9% NaCI solution. DDMP was initially dissolved in a small quantity of 0.1 N HCI, and TMQ was dissolved in pure ethanol before being further diluted with 0.9% NaCI solution. Preliminary studies showed that the vehicles used (similarly * The abbreviations used are: MTX, methotrexate; LV, leucovorin; FBS, fetal bovine serum; DCM, dichloromethotrexate; TMQ, 2,4-diaminc-5-methyl-6[(3',4',5'-trimethoxyanilino)methyl]quinazoline (JB-11); DDMP, 2,4-diamino-5(3',4'-dichlorophenyD-6-methylpyrimidine; EBSS, Earl's balanced salt solution; 5-MeFH«,5-methyltetrahydrofolate;
ID»,50% inhibitory concentration.
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T. Ohnoshi et al. diluted HCI or ethanol in the culture medium) did not influence the cell growth. For all experiments, serial drug solutions were freshly prepared just before use. Ten-mi cell suspensions of the parent cell line and subline were
The cells were washed 3 times by centrifugation for 3 sec in a Beckman Microfuge B, and then the cell pellet was dissolved in 0.5 ml of NCS (Amersham) overnight at 37°.The samples were counted in a Beckman Model 250 liquid scintillation counter at 30% counting efficiency. The results were calculated as a net uptake as measured by the total uptake minus the diffusion value at 0°. Efflux was similarly measured after
prepared in individual culture tubes (no. 3033; Falcon) at an initial cell density of 1.5 x I05/ml, to which 0.1 ml of drug solutions at graded concentrations was added. The cells were incubated at 37°. Three
diluting an aliquot of the cells into 20-fold excess drug-free medium.
days later, the viable cell number was counted by the trypan blue dye exclusion method; dose-response curves were obtained by calculating the percentage of viable cells in drug-treated tubes as compared to
RESULTS
those in control tubes without drugs. To determine the reversal effects of LV against folate antagonists, equimolar concentrations of LV (0.1 ml) and each drug (0.1 ml) at graded concentrations were added successively to 10 ml of the cell suspension. After 3 days of incubation at 37°,the protective effect of LV was determined by comparing dose-
Development of the MTX-resistant Cell Line, MOLT-3/ MTXi. A resistant subline was developed by growing MOLT-3 cells in the continuous presence of MTX. The initial MTX concentration used, 5 x 10~9 M, was raised by 1.5- to 2-fold
response curves with and without LV. All experiments were carried out in triplicate and repeated at least twice. For the morphological study, cells were examined under light mi croscopy after Wright-Giemsa staining. Cells were also plated on
successively. With increase in MTX concentration, the cell growth rate was temporarily decreased; it took 2 to 4 months until the growth rate returned to that of the parent cell. The process was then repeated until the MTX concentration reached 10~7 M. At this stage, the MTX-resistant cells were
replica plates (16) containing 0.5% agar in Roswell Park Memorial Institute Medium 1640 enriched with 10% FBS and incubated at 37° in 5% CO. in humidified air. Cell volumes of the parent and resistant cell lines were measured by the Coulter Counter Channelyser (Coulter Electronics, Hialeah, Fla.) and were calibrated using standard spheres with diameters of 5.02, 10.02, and 14.76pm (Coulter). For the determination of dihydrofolate reducÃ-ase, the cells were washed twice in Dulbecco's phosphate-buffered saline (Grand Island Biological Co.). The cell pellets were then frozen and thawed 3 times in 0.05 M Tris buffer (pH 7.5), containing albumin (1 mg/ml) and 0.15 M KCI. The 20,000 x g supernatant was subjected to enzyme assay by 2 methods. First was the labeled dihydrofolate method (10). The incubation mixture contained 50 mw phosphate buffer (pH 6.0), 0.1 rriM NADPH, 0.04 mw [3H]dihydrofolate (Amersham/Searle, Arlington Heights, III. 35,000 to 45,000 cpm), 0.2 M KCI, and approximately 8 to 64 ng of protein in a final volume of 200 pi. The reaction was initiated by the addition of NADPH at 37° and terminated in a 0°bath by the addition of 25 pi of 0.11 M folate. The folate and dihydrofolate were precipitated by the addition of 25 pi of 0.17 M zinc sulfate in 25% acetic acid. The appropriate blanks contained either all components except NADPH or all components except enzyme in which case enzyme was added immediately prior to terminating the reaction. Blank values were subtracted from the values obtained from complete assay mix tures to yield the data presented in this paper. The reaction mixtures were centrifuged at 17,000 x g for 10 min. Aliquots of 100 pi of the supernatant were added to 5 ml of ACS (Amersham) and analyzed for radioactivity. The second method was MTX-binding capacity assay (14). For this study, [3H]MTX (6 to 12 Ci/nmol; Amersham) was purified on DEAEcellulose as needed (13). The reaction was carried out at 4°in a total
fed in the same concentration of MTX for 3 months and then returned to the original MTX-free medium and subjected to characterization studies. The parent line and the resistant subline had the same doubling time of approximately 36 hr. There were no differences in morphology. On the replica plate, the parent MOLT-3 produced loose colonies, whereas the colonies of the resistant line were tightly packed. The cell volume of the parent and resistant cells was equal. This subline seemed to be a stable mutant. Thus, during the 16-month observation period, MTX resistance of the subline in the MTXfree medium had not changed. Inhibitory effects of MTX on the parent MOLT-3 and the resistant MOLT-3/MTX, cells deter mined by cell growth inhibition assay are illustrated in Chart 1. At the MTX concentration of 10~7 M, the viable cell growths of the parent cells were completely inhibited, whereas the growth of the resistant cells was uninhibited. The ID50s of MTX were 10~8 M for the parent cells and 3 x 10~7 M for the resistant cells. Thus, on an ID50 basis, the MOLT-3/MTX, cells were approximately 30-fold more resistant to MTX than were the parent cells.
s iOOF
volume of 1.2 ml. To 0.9 ml of 50 mw phosphate buffer (pH 6.0), containing 1 mg albumin and 0.1 pmol NADPH per ml, were added 0.1 ml standard MTX solution containing 2.2 to 90.0 pmol/ml and 0.1 ml [3H]MTX containing about 1.5 pmol MTX. The reactants were mixed, and the incubation was started by the addition of 0.1 ml of the supernatant with serial dilutions (binding capacity of 1 to 1.2 pmol MTX, enough to bind 70 to 80% of the (3H]MTX). After gently shaking to ensure mixing, the reaction mixture was incubated for 10 min at 4° in the dark. The incubation was terminated by the addition of charcoal coated with dextran. For the study of MTX and 5-MeFH4 uptake, the parent and resistant cells were washed twice and resuspended in EBSS (Grand Island Biological Co.) at a cell concentration of 2 x 107/ml. After preincubation of the cells for 15 min at 37°,[3H]MTX or 5-[14C]-MeFH4 (58pCi/ mmol; Amersham) at graded concentrations was added to the incuba tion medium to initiate the reaction. At various time intervals (0 to 15 min), the reaction was terminated by pipetting out a 0.2-ml aliquot of cell suspension into a 15-fold dilution of ice-cold 0.9% NaCI solution.
1656
05
l
5
METHOTREXATE
IO
(xlOJM)
50 ICO
Chart 1. Dose-response curves of the parent and resistant cell lines to MTX by growth inhibition assay. Cells were allowed to grow in the presence of the indicated concentrations of MTX in culture tubes for 3 days. Numbers of viable cells were counted by the trypan blue dye exclusion method, and dose-response curves of each cell line were obtained by calculating the percentage of viable cells in drug-tested tubes as compared to those in control tubes containing no drug. Points, mean values of 3 experiments, each with triplicate determinations, S.D.
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Antifolates on a MTX-resistant Dihydrofolate ReducÃ-ase Activity and Radioactive MTX Uptake. To clarify the mechanism of MTX resistance of the subline, dihydrofolate reducÃ-ase activity and intracellular trans port of [3H]MTX were compared with the parent cell line. The results of the enzyme assay are summarized in Table 1. At a MTX concentration of 5 x 10~9 M, extracts from both cells exhibited tight-binding kinetics; i.e., both enzymes were in hibited stoichiometrically when the cell supernatant was preincubated with MTX prior to addition of the folate substrate. Either the amount of dihydrofolate reducÃ-ase ([3H]MTX assay) or the functional activity ([3H]dihydrofolate assay) was only minimally elevaled in Ihe resislanl cells. The 30-fold differences in the sensitivily belween parenl and resislant cells cannot be accounted for on Ihe basis of increased dihydrofolale reduc Ã-asealone. Results of the MTX transport in EBSS are shown in Chart 2. The MTX transport was linear for al least 15 min in Ihe parenl cell line. Initial uptake sludies (15 min) revealed lhal Ihe resisl anl cells Iransported MTX 75% less lhan did the sensitive cells at MTX concenlralions varying up lo 25 (IM. The MTX efflux sludies revealed similar properties for bolh sensilive and re sislanl cell lines (Chart 3). Kinelic analysis of Ihe inilial uptake of MTX al various con cenlralions (2.5 lo 10 /¿M) revealed a similar Km of 6.6 /¿M for bolh cell lines (Chart 4). The Vmaxvalues, however, are 12.5 and 1.7 nmol/2.5 min/107 cells for Ihe parenl cell line and Ihe
Human Leukemia Cell Line
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Chart 9. Protective effects of LV on TMQ-induced growth inhibition in the original and resistant cells. Points, mean values of 2 or 3 experiments, each with triplicate determinations (see legend of Chart 1). CANCER
RESEARCH
VOL. 42
Antifolates on a MTX-resistant
Human Leukemia Cell Line
but different rates of initial velocity. The possibility of a second transport site has been proposed in a murine lymphoma cell
DDMP (xlCT8M) LEUCOVORIN(xlCT8M) Chart 10. Protective effects of LV on DDMP-induced growth inhibition in the original and resistant cells. Points, mean values of 2 or 3 experiments, each with triplicate determinations (see legend of Chart 1).
has been related to elevated levels of dihydrofolate reducÃ-ase (3, 5), structurally altered dihydrofolate reductase (2), or an impairment in the membrane transport of MTX (21, 24). In the present study, although dihydrofolate reductase levels of the resistant cell line MOLT-3/MTX, were slightly elevated (1.4fold in [3H]dihydrofolate assay) as compared to that of the parent cells, it does not appear to be of sufficient magnitude to be the primary site of resistance. The turnover numbers for MOLT-3 and MOLT-3/MTX, are within the range of those in fresh human tumor dihydrofolate reductase 385 ±20.5 Based on the data that there was a more than 7-fold decrease in the initial uptake of MTX into the resistant cells, that Km values were similar, and that there was a lack of significant difference in the efflux patterns of the drug between sensitive and resistant cell lines, it is concluded that the development of resistance to MTX is associated with a decreased ability to transport the drug. The morphological differences in the colonies between the parent and resistant cells on the replica plate support the possibility that the membrane characteristics had indeed changed. We observed that the efflux of MTX appeared to be much more rapid than the influx. No complete explanation is available for this observation, but the finding is consistent with that made on murine L1210 leukemia cells (25). The parent MOLT-3 cells transported 5-MeFH4 at an initial rate more than 2-fold faster than that of MTX, while the Km values for both of these substrates are similar. In L1210 cells, 5-MeFH4 and MTX share a common transport site, and the Km and V,™« are similar. These differences between mouse and human cells may help to discriminate the cytotoxic differences of MTX seen between the 2 cell lines. As to why this occurs, there are at least 2 possibilities: (a) Although the Kms are similar for 5-MeFH4 and for MTX in human cells, the specificity of binding to the carrier protein may be different, which may account for a less efficient initial uptake of MTX into the human cells and a possible site of alteration for developing resistance to MTX; (b) a possibility exists that there is an additional transport site for 5-MeFH4 uptake that may have a similar Km 5 B. A. Kamen, J. R. Berlino, M. White, and P. Nyler, unpublished data. Human tumor specimens tested include 3 with Wilms' tumor, 2 with acute lymphoblastic leukemia, and one each with Burkitt's lymphoma and hepatoma.
MAY 1982
line (11). With regard to the apparent discrepancy between the 4- to 5-fold difference in uptake and the 30-fold difference in growth inhibition assay for the parent and resistant cells, the following observations can be made, (a) Uptake studies were performed over a 15-min period in EBSS, which is used routinely for transport studies of low-molecular nutrients (6), whereas growth inhibition assays were compared after 3 days of incu bation in the continuous presence of MTX in the medium. It has been indicated that the intracellular exchangeable MTX levels must be excessive for MTX to inhibit cell growth effec tively (9, 26). (b) Although labeled MTX had been purified on the day of the experiment, it cannot be completely ruled out that a part of the radioactivities transported into the resistant cells may in fact be some labeled breakdown products of MTX (14). This question may be answered by dialysis and/or by Chromatographie procedures. Development of MTX-resistant human cell lines by gradient MTX feeding has been reported (21 , 24). In the former case, where the parent cell line was derived from the spleen of a patient with spherocytic anemia, cells became resistant due to an elevated dihydrofolate reductase, an impaired membrane transport, or both. The latter case, wherein the cell line was derived from leukemic blasts of a patient with acute lympho blastic leukemia, was shown to have an impaired membrane transport. Bertino (1 ) has suggested the possibility of effective cell kill for MTX-resistant cells by virtue of impaired transport by utiliz ing folate antagonists that were more efficiently transported by the mutant line. As an example of this phenomenon, Walker carcinoma 256 resistant to MTX was shown to be highly sensitive to triazinate (Baker's antifol) (27). In addition, Rosowsky ef al. (24) have indicated that CEM/MTX cells, which were resistant to MTX by impaired transport, showed collateral sensitivity to DDMP as well as di-n-butyl ester of MTX. TMQ (2, 7) and DDMP (18, 20, 23) are lipid-soluble folate antagonists. These antifolates are known to enter cells by a mechanism different from MTX. Our observation that the MTX-resistant cells developed by a gradient MTX feeding were transport related and are more sensitive to TMQ and DDMP than are the parent cells is consistent with these reports. As a biochemical basis for the collateral sensitivity observed in the resistant cells, Rosowsky ef al. have suggested 2 pos sibilities: (a) the insufficient uptake of reduced folate present in culture medium containing 10% FBS; or (b) some structural alteration of plasma membrane which rendered it more perme able to lipid-soluble agents. In our reversal experiments using LV, the reversal effects of LV against equimolar MTX in the resistant cells, to which much higher MTX and LV were added than in the parent cells, were essentially proportional to those in the parent cells. This would indicate a competitive nature between MTX and LV on the membrane transport where they share a common carrier system in both cells (8, 19) and that the ability to transport reduced folate in the resistant cells was impaired to almost the same degree as was MTX. Our obser vations indicate, therefore, that the former possibility sug gested by Rosowsky ef al. is operative as a mechanism of the collateral sensitivity of resistant cells to TMQ and DDMP. The observation of the ability of TMQ and DDMP to effectively kill the MTX-resistant cells with impaired drug transport appears
1659
T. Ohnoshi et al. important in light of the fact that the initial event of the MIX resistance in human cancers is most probably the decreased ability of the drug to penetrate the cell membrane, rather than elevation of dihydrofolate reductase activity (15, 21). Furthermore, we believe that our observation that TMQ plus LV or DDMP plus LV selectively inhibited the MTX-resistant human tumor cells has important implications. This observation, previously made in the murine lymphoma cell line L5178Y (12), raises a possibility that selective cell kill of transport-resistant neoplasms can be accomplished in humans with little toxicity on host tissues. Thus, the resistant cells with an impaired common reduced folate-MTX transport system will not be pro tected by LV, whereas host normal cells with an intact reduced folate transport system would be protected effectively from the inhibitory effects of these antifols by using sufficient doses of LV. This thesis can be tested in humans.
10. 11.
12. 13.
14.
15.
16. 17.
ACKNOWLEDGMENTS We thank Drs. Joseph R. Berlino of Yale University School of Medicine and Samuel Waxman of Mt. Sinai School of Medicine for their advice and helpful suggestions on this work.
18. 19.
20.
REFERENCES 1. Berlino, J. R. Resistance of human tumors to cancer chemotherapeutic agents: an important research problem. Med. Pediat. Oncol., 5: 105-114, 1978. 2. Berlino, J. R.. and Sawicki. W. L. Potent inhibitory activity of trimethoxyquine (TMQ), "nonclassical" 2,4-diaminoquinazoline, on mammalian DMA synthe sis. Proc. Am. Assoc. Cancer Res., 18: 165, 1977. 3. Berlino, J. R., Sawicki, W. L., Cashmore. A. R., Cadman, E. C., and Skeel, R. T. Natural resistance to methotrexate in human acute nonlymphocytic leukemia. Cancer Treat. Rep., 61: 667-673, 1977. 4. Brockman, R. W. Mechanism of resistance to folie acid analogs. In: A. C. Salterelli and D. C. Johns (eds.), Antineoplastic and Immunosuppressive Agents, Vol. 1, pp. 358-367. Berlin: Springer-Verlag, 1975. 5. Chello, P. L., McQueen, C. A., DeAngelis, L. M., and Berlino, J. R. Elevation of dihydrofolate reductase, thymidylate synthetase, and thymidine kinase in cultured mammalian cells after exposure to folate antagonists. Cancer Res., 36: 2442-2449, 1976. 6. Oantzig, A. M., Adelberg, E. A., and Slayman, C. W. Properties of two mouse lymphocyte cell lines genetically defective in amino acid transport. J. Biol. Chem.. 245. 8988-8993. 1979. 7. Davoll, J., Johnson, A. M., and Oavies, H. J. Folate antagonists. 2. 2,4Diamino-6-(aralkyl and (heterocyclic) methyl)amino)quinazolines. a novel class of antimetabolites of interest in drug-resistant malaria and Chagas' disease. J. Med. Chem., 75. 812-826, 1972. 8. Goldman, I. D. The characteristics of membrane transport of amethopterin and the naturally-occurring folates. Ann. N. Y. Acad. Sci., Õ86: 400-422, 1971. 9. Goldman, I. D. Analysis of the cytotoxic determinants for methotrexate (NSC-
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