Summary
A quantitative study was made of the cytotoxicity of methotrexate (MTX) for nontransformed and transformed NIH 3T3 cells in the presence and absence of leucovorin. The study was preceded by an analysis of the growth rates of the cells at low and high population density combined with low and high concentrations of calf serum (CS). The reduced maximal growth rates of the transformed cells at low population densities relative to the nontransformed cells reinforced earlier evidence that heritable damage involving chromosome aberrations drives the process of transformation. When small numbers of transformed cells are cocultured with a large excess of nontransformed cells in the assay for transformed foci, the transformed cells were more readily killed by MTX than the nontransformed cells. The selectivity was increased when leucovorin (folinic acid) was present in the medium. The selective killing of the transformed cells actively multiplying in foci was most pronounced when the background of nontransformed cells had become confluent and their growth was inhibited. However, selectivity has also been demonstrated when transformed and nontransformed cells are growing at their maximum rates at low density despite the lower growth rate of the transformed cells under these conditions. The sensitivity of transformed cells in pure culture to MTX was lower during the first 3 d of subculture than in the following 6 d but decreased to zero a few d after net growth had ceased. The nontransformed cells were more susceptible to killing by MTX in Dulbecco’s modified Eagle’s medium (DMEM) than in MCDB 402, but the transformed cells were sensitive to MTX in both media. The high selectivity of MTX for transformed over nontransformed cells in MCDB 402 results from the presence of 1.0 µM leucovorin (5-formyltetrahydrofolate), a reduced form of the folic acid present in most other culture media. When leucovorin was added to DMEM with its high concentration of folic acid, the resistance to MTX of both nontransformed and transformed cells was greatly increased, but the selectivity of MTX for transformed cells was almost entirely lost. The results indicate that leucovorin protects nontransformed cells against concentrations of MTX that kill transformed cells, but the protection is dependent on the relative amounts of leucovorin to folic acid in the medium. The relative sensitivities of transformed and nontransformed cells in our system to MTX when both cell types are exhibiting their characteristic differential in growth behavior is similar to that described for tumor and normal cells in vivo. Since the unregulated growth behavior of the transformed, tumor-producing cells is efficiently and quantitatively measured in this system, it can be used to develop general principles of treatment and resolve questions of cytotoxic mechanism.
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References
Allegra, C. J. Antifolates. In: Chabner, B. A.; Collins, J. M., ed. Cancer chemotherapy. Principles and practices. Philadelphia: J. B. Lippincott; 1990:110–153.
Bertino, J. R.; Romanini, A. Folate antagonists. In: Holland, J. F.; Frei, E., ed. Chemotherapeutic agents. Philadelphia: Lea & Febiger; 1993:698–711.
Chow, M.; Koo, J. J.; Ng, P.; Rubin, H. Random, population-wide genetic damage induced in replicating cells treated with chemotherapeutic drugs. Mutation Res. 413:251–264; 1998.
Chow, M.; Rubin, H. Evidence for cellular aging in long term confluent cultures: heritable impairment of proliferation, accumulation of age pigments and their loss in neoplastic transformation. Mech. Ageing Dev. 89:165–184; 1996.
Chow, M.; Rubin, H. Irreversibility of cellular aging and neoplastic transformation: a clonal analysis. Proc. Natl. Acad. Sci. USA 93:9793–9798; 1996.
Chow, M.; Rubin, H. Selective killing of preneoplastic and neoplastic cells by methotrexate with leucovorin. Proc. Natl. Acad. Sci. USA 95:4550–4555; 1998.
Clive, C.; Johnson, K. O.; Spector, J. F. S.; Batson, A. G.; Brown, M. M. M. Validation and characterization of the L5178Y/TK+/− mouse lymphoma mutagen system. Mutat. Res. 59:61–108; 1979.
Cook, J. D.; Cichowitz, D. J.; George, S.; Lawler, A.; Shane, B. Mammalian folypoly- “α”-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues of folate homeostasis. Biochemistry 26:530–539; 1987.
Frei, E.; Jaffe, N.; Tattersall, M. H. N.; Pitman, S.; Parker, L. New approaches to cancer chemotherapy with methotrexate. Seminars in Medicine of the Beth Israel Hospital, Boston. New Engl. J. Med. 846–851; 1975.
Galivan, J.; Nimec, Z. Effects of folinic acid on hepatoma cells containing methotrexate polyglutamates. Cancer Res. 43:551–555; 1983.
Goldin, A.; Venditti, J. M.; Kline, I.; Mantel, N. Eradication of leukaemic cells (L1210) by methotrexate and methotrexate plus citrovorum factor. Nature (Lond) 212:1448–1550; 1966.
Goldman, I. D.; Matherly, L. H. Biochemical factors in the selectivity of leucovorin rescue: selective inhibition of leucovorin reactivation of dihydrofolate reductase and leucovorin utilization in purine and pyrimidine biosynthesis by methotrexate and dihydrofolate polyglutamates. Folates and folic acid antagonists in cancer chemotherapy. Natl. Cancer Instit. Monogr. 5:1987.
Hozier, J.; Sawyer, J.; Clive, D.; Moore, M. Chromosome 11 aberrations in small colony L5178Y TK-/- mutants early in their clonal history. Mutation Res. 147:237–242; 1985.
Johnson, L. F.; Fuhrman, C. L.; Abelson, H. T. Resistance of resting 3T6 mouse fibroblasts to methotrexate cytotoxicity. Cancer Res. 38:2408–2412; 1978.
Kennedy, A. R.; Fox, M.; Murphy, G.; Little, J. B. Relationship between X-ray exposure and malignant transformation in C3H 10T1/2 cells. Proc. Natl. Acad. Sci. USA 77:7262–7266; 1980.
Kinzler, K. W.; Vogelstein, B. Lessons from hereditary colorectal cancer. Cell 87:159–170; 1996.
Li, J. C.; Kaminskas, E. Accumulation of DNA strand breaks and methotrexate toxicity. Proc. Natl. Acad. Sci. USA 81:5694–5698; 1984.
Liber, H. L.; Yandell, D. W.; Little, J. B. A comparison of mutation induction at the tk and hprt loci in human lymphoblastoid cells; quantitative differences are due to an additional class of mutations at the autosomal tk locus. Mutat. Res. 216:9–17; 1989.
Little, J. B.; Nagasawa, H.; Phennig, T.; Vetrous, H. Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of x-rays and alpha particles. Radiat. Res. 148:299–307; 1997.
Matherly, L. H.; Anderson, L. A.; Goldman, I. D. Role of the cellular oxidation-reduction state in methotrexate binding of dihydrofolate reductase and dissociation induced by reduced folates. Cancer Res. 44:2325–2330; 1984.
Matherly, L. H.; Barlowe, C. K.; Goldman, I. D. Antifolate polyglutamylation and competitive drug displacement at dihydrofolate reductase as important elements in leucovorin rescue in L1210 cells. Cancer Res. 46:588–593; 1986.
Matherly, L. H.; Fry, D. W.; Goldman, I. D. Role of methotrexate polyglutamylation and cellular energy metabolism in inhibition of methotrexate binding to dihydrofolate reductase by 5-formyltetrahydrofolate in Erlich ascites tumor cells in vitro. Cancer Res. 43:2694–2699; 1983.
Matherly, L. H.; Seither, R. L.; Goldman, I. D. The basis for antifolate cytotoxicity, selectivity and metabolic transformation: Effects on utilization of endogenous and exogenous folate cofactors. In: Powis, G., ed. Anticancer drugs: antimetabolite metabolism and natural anti-cancer agents. United Kingdom: Pergamon Press; 1994:139–177.
McCullough, K. D.; Coleman, W. R.; Smith, G. J.; Grisham, J. W. Age-dependent induction of hepatic tumor regression by the tissue microenvironment after transplantation of neoplastically transformed rat liver epithelial cells into the liver. Cancer Res. 57:1807–1813; 1997.
Mead, J. A. R.; Venditti, J. M.; Schrecker, A. W.; Goldin, A.; Kereszetzky, J. C. The effect of derivatives of folic acid on toxicity and antileukemic effect of methotrexate in mice. Biochem. Pharmacol. 12:371–383; 1963.
Moore, M. M.; Clive, D.; Howard, B. E.; Batson, G. A.; Turner, N. T. In situ analysis of trifluor thymidine-resistant (TFTr) mutants of L5178 Y/TK+/− mouse lymphoma cells. Mutat. Res. 151:147–159; 1985.
Pinedo, H. M.; Zaharko, D. S.; Bull, J. M.; Chabner, B. A. The reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides. Cancer Res. 36:4418–4424; 1976.
Poser, R. G.; Sirotnak, F. M.; Chello, P. L. Differential synthesis of methotrexate polyglutamates in normal proliferative and neoplastic mouse tissues in vivo. Cancer Res. 41:4441–4446; 1981.
Puck, T. T. Gamma-ray mutagenesis measurement in mammalian cells. Mutat. Res. 329:173–181; 1995.
Revell, S. H. Relationship between chromosome damage and cell death. In: Ishihara, T. Sasaki, M.S. eds. Radiation induced chromosome damage in man. New York: Allan R. Liss, Inc.; 1983:215–233.
Reznikoff, C. A.; Bertram, J. S.; Brankow, D. W.; Heidelberger, C. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res. 33:3239–3249; 1973.
Rubin, H.; Xu, K. Evidence for the progressive and adaptive nature of spontaneous transformation in the NIH 3T3 cell line. Proc. Natl. Acad. Sci. USA 86:1860–1864; 1989.
Rubin, H.; Yao, A.; Chow, M. Heritable, population-wide damage to cells as the driving force of neoplastic transformation. Proc. Natl. Acad. Sci. USA 92:4843–4847; 1995.
Rubin, H.; Yao, A.; Chow, M. Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density. Proc. Natl. Acad. Sci. USA 92:7734–7738; 1995.
Sirotnak, F. M.; Donsbach, R. C. Kinetic correlates of methotrexate transport and therapeutic responsiveness in murine tumors. Cancer Res. 36:1151–1158; 1976.
Sirotnak, F. M.; Moccio, D. M.; Dorick, D. M. Optimization of high-dose methotrexate with leucovorin rescue therapy in the L1210 leukemia and sarcoma 180 murine tumor models. Cancer Res. 38:345–353; 1978.
Smith, G. J.; Bell, W. N.; Grisham, J. W. Clonal analysis of the expression of multiple transformation phenotypes and tumorigenicity by morphologically transformed 10T1/2 cells. Cancer Res. 53:500–508; 1993.
Thacker, J. The nature of mutants induced by ionizing radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by x-rays or particles showing that the majority have deletions of all or part of the hprt gene. Mutat. Res. 160:267–275; 1989.
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Chow, M., Rubin, H. Quantitative aspects of the selective killing of transformed cells by methotrexate in the presence of leucovorin. In Vitro Cell.Dev.Biol.-Animal 35, 394–402 (1999). https://doi.org/10.1007/s11626-999-0114-5
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DOI: https://doi.org/10.1007/s11626-999-0114-5