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. 1987 Jul;84(14):4860–4864. doi: 10.1073/pnas.84.14.4860

Toxicity of folic acid analogs in cultured human cells: a microtiter assay for the analysis of drug competition.

D S Roos, R T Schimke
PMCID: PMC305205  PMID: 3496598

Abstract

We have used a microtiter assay to study the toxicity of various folate analogs in a series of cultured human cell lines that exhibit different degrees of resistance to methotrexate, an inhibitor of dihydrofolate reductase. These cells retain their sensitivity to the lipophilic antifolate BW301U despite the amplification of dihydrofolate reductase genes. Because the cell lines under investigation grow very slowly and have poor plating efficiencies in unconditioned medium, an assay was developed that relies on cell proliferation rather than colony formation as a measure of toxicity. This approach is easily generalized to provide a rapid and inexpensive assay of drug competition. Two-dimensional studies indicate that methotrexate and BW301U show differences in patterns of toxicity, competition, and rescue by folinic acid, suggesting that the two drugs act on different targets. Further applications of the microtiter assay to the analysis of multidrug interactions are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anagnou N. P., O'Brien S. J., Shimada T., Nash W. G., Chen M. J., Nienhuis A. W. Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification, and a novel form of polymorphism. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5170–5174. doi: 10.1073/pnas.81.16.5170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown P. C., Beverley S. M., Schimke R. T. Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines. Mol Cell Biol. 1981 Dec;1(12):1077–1083. doi: 10.1128/mcb.1.12.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown P. C., Tlsty T. D., Schimke R. T. Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea. Mol Cell Biol. 1983 Jun;3(6):1097–1107. doi: 10.1128/mcb.3.6.1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen M. J., Shimada T., Moulton A. D., Cline A., Humphries R. K., Maizel J., Nienhuis A. W. The functional human dihydrofolate reductase gene. J Biol Chem. 1984 Mar 25;259(6):3933–3943. [PubMed] [Google Scholar]
  5. Chen T. R. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1977 Feb;104(2):255–262. doi: 10.1016/0014-4827(77)90089-1. [DOI] [PubMed] [Google Scholar]
  6. Chou T. C., Talalay P. Generalized equations for the analysis of inhibitions of Michaelis-Menten and higher-order kinetic systems with two or more mutually exclusive and nonexclusive inhibitors. Eur J Biochem. 1981 Mar 16;115(1):207–216. doi: 10.1111/j.1432-1033.1981.tb06218.x. [DOI] [PubMed] [Google Scholar]
  7. Duch D. S., Edelstein M. P., Bowers S. W., Nichol C. A. Biochemical and chemotherapeutic studies on 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW 301U), a novel lipid-soluble inhibitor of dihydrofolate reductase. Cancer Res. 1982 Oct;42(10):3987–3994. [PubMed] [Google Scholar]
  8. Grivsky E. M., Lee S., Sigel C. W., Duch D. S., Nichol C. A. Synthesis and antitumor activity of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine. J Med Chem. 1980 Mar;23(3):327–329. doi: 10.1021/jm00177a025. [DOI] [PubMed] [Google Scholar]
  9. Haber D. A., Schimke R. T. Unstable amplification of an altered dihydrofolate reductase gene associated with double-minute chromosomes. Cell. 1981 Nov;26(3 Pt 1):355–362. doi: 10.1016/0092-8674(81)90204-x. [DOI] [PubMed] [Google Scholar]
  10. Hoy C. A., Salazar E. P., Thompson L. H. Rapid detection of DNA-damaging agents using repair-deficient CHO cells. Mutat Res. 1984 Oct;130(5):321–332. doi: 10.1016/0165-1161(84)90018-9. [DOI] [PubMed] [Google Scholar]
  11. Iland H. J., Laszlo J., Sedwick W. D. Paradoxical effect of BW 301U, a lipophilic antifolate, on methotrexate-inhibitable deoxyuridine incorporation by human hematopoietic cells. Cancer Res. 1985 Aug;45(8):3962–3968. [PubMed] [Google Scholar]
  12. LOEWE S. Antagonisms and antagonists. Pharmacol Rev. 1957 Jun;9(2):237–242. [PubMed] [Google Scholar]
  13. Masters J. N., Yang J. K., Cellini A., Attardi G. A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA. J Mol Biol. 1983 Jun 15;167(1):23–36. doi: 10.1016/s0022-2836(83)80032-1. [DOI] [PubMed] [Google Scholar]
  14. Nunberg J. H., Kaufman R. J., Chang A. C., Cohen S. N., Schimke R. T. Structure and genomic organization of the mouse dihydrofolate reductase gene. Cell. 1980 Feb;19(2):355–364. doi: 10.1016/0092-8674(80)90510-3. [DOI] [PubMed] [Google Scholar]
  15. Rath H., Tlsty T., Schimke R. T. Rapid emergence of methotrexate resistance in cultured mouse cells. Cancer Res. 1984 Aug;44(8):3303–3306. [PubMed] [Google Scholar]
  16. Schimke R. T. Gene amplification in cultured animal cells. Cell. 1984 Jul;37(3):705–713. doi: 10.1016/0092-8674(84)90406-9. [DOI] [PubMed] [Google Scholar]
  17. Sedwick W. D., Hamrell M., Brown O. E., Laszlo J. Metabolic inhibition by a new antifolate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methyl-pyrido[2,3-d]pyrimidine (BW3O1U), an effective inhibitor of human lymphoid and dihydrofolate reductase-overproducing mouse cell lines. Mol Pharmacol. 1982 Nov;22(3):766–770. [PubMed] [Google Scholar]
  18. Sirotnak F. M., Moccio D. M., Kelleher L. E., Goutas L. J. Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant L1210 clonal cell lines derived in vivo. Cancer Res. 1981 Nov;41(11 Pt 1):4447–4452. [PubMed] [Google Scholar]
  19. Takebe H., Miki Y., Kozuka T., Furuyama J. I., Tanaka K. DNA repair characteristics and skin cancers of xeroderma pigmentosum patients in Japan. Cancer Res. 1977 Feb;37(2):490–495. [PubMed] [Google Scholar]
  20. Taylor I. W., Slowiaczek P., Friedlander M. L., Tattersall M. H. Selective toxicity of a new lipophilic antifolate, BW301U, for methotrexate-resistant cells with reduced drug uptake. Cancer Res. 1985 Mar;45(3):978–982. [PubMed] [Google Scholar]

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