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. 1982 Mar;2(3):275–285. doi: 10.1128/mcb.2.3.275

Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells.

W F Flintoff, M K Weber, C R Nagainis, A K Essani, D Robertson, W Salser
PMCID: PMC369786  PMID: 6287233

Abstract

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

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Selected References

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

  1. Albrecht A. M., Biedler J. L., Hutchison D. J. Two different species of dihydrofolate reductase in mammalian cells differentially resistant to amethopterin and methasquin. Cancer Res. 1972 Jul;32(7):1539–1546. [PubMed] [Google Scholar]
  2. Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
  3. Alt F. W., Kellems R. E., Schimke R. T. Synthesis and degradation of folate reductase in sensitive and methotrexate-resistant lines of S-180 cells. J Biol Chem. 1976 May 25;251(10):3063–3074. [PubMed] [Google Scholar]
  4. BERNS K. I., THOMAS C. A., Jr ISOLATION OF HIGH MOLECULAR WEIGHT DNA FROM HEMOPHILUS INFLUENZAE. J Mol Biol. 1965 Mar;11:476–490. doi: 10.1016/s0022-2836(65)80004-3. [DOI] [PubMed] [Google Scholar]
  5. Biedler J. L., Albrecht A. M., Hutchison D. J., Spengler B. A. Drug response, dihydrofolate reductase, and cytogenetics of amethopterin-resistant Chinese hamster cells in vitro. Cancer Res. 1972 Jan;32(1):153–161. [PubMed] [Google Scholar]
  6. Biedler J. L., Spengler B. A. Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science. 1976 Jan 16;191(4223):185–187. doi: 10.1126/science.942798. [DOI] [PubMed] [Google Scholar]
  7. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  8. Bostock C. J., Clark E. M., Harding N. G., Mounts P. M., Tyler-Smith C., van Heyningen V., Walker P. M. The development of resistance to methotrexate in a mouse melanoma cell line. I. Characterisation of the dihydrofolate reductases and chromosomes in sensitive and resistant cells. Chromosoma. 1979;74(2):153–177. doi: 10.1007/BF00292270. [DOI] [PubMed] [Google Scholar]
  9. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [DOI] [PubMed] [Google Scholar]
  10. Chang S. E., Littlefield J. W. Elevated dihydrofolate reductase messenger RNA levels in methotrexate-resistant BHK cells. Cell. 1976 Mar;7(3):391–396. doi: 10.1016/0092-8674(76)90168-9. [DOI] [PubMed] [Google Scholar]
  11. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  12. Dolnick B. J., Berenson R. J., Bertino J. R., Kaufman R. J., Nunberg J. H., Schimke R. T. Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells. J Cell Biol. 1979 Nov;83(2 Pt 1):394–402. doi: 10.1083/jcb.83.2.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. FISCHER G. A. Detective transport of amethopterin (methotrexate) as a mechanism of resistance to the antimetabolite in L5178Y leukemic cells. Biochem Pharmacol. 1962 Dec;11:1233–1234. doi: 10.1016/0006-2952(62)90200-9. [DOI] [PubMed] [Google Scholar]
  14. Fanshier L., Garapin A. C., McDonnell J., Faras A., Levinson W., Bishop J. M. Deoxyribonucleic acid polymerase associated with avian tumor viruses: secondary structure of the deoxyribonucleic acid product. J Virol. 1971 Jan;7(1):77–86. doi: 10.1128/jvi.7.1.77-86.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Farrell S. A., Worton R. G. Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids. Somatic Cell Genet. 1977 Sep;3(5):539–551. doi: 10.1007/BF01539124. [DOI] [PubMed] [Google Scholar]
  16. Flintoff W. F., Davidson S. V., Siminovitch L. Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells. Somatic Cell Genet. 1976 May;2(3):245–261. doi: 10.1007/BF01538963. [DOI] [PubMed] [Google Scholar]
  17. Flintoff W. F., Essani K. Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate. Biochemistry. 1980 Sep 2;19(18):4321–4327. doi: 10.1021/bi00559a027. [DOI] [PubMed] [Google Scholar]
  18. Flintoff W. F., Weber M. Selection of wild-type revertants from methotrexate-resistant cells containing an altered dihydrofolate reductase. Somatic Cell Genet. 1980 Jul;6(4):517–528. doi: 10.1007/BF01539153. [DOI] [PubMed] [Google Scholar]
  19. Gupta R. S., Flintoff W. F., Siminovitch L. Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. Can J Biochem. 1977 Apr;55(4):445–452. doi: 10.1139/o77-062. [DOI] [PubMed] [Google Scholar]
  20. HAKALA M. T., ZAKRZEWSKI S. F., NICHOL C. A. Relation of folic acid reductase to amethopterin resistance in cultured mammalian cells. J Biol Chem. 1961 Mar;236:952–958. [PubMed] [Google Scholar]
  21. Hänggi U. J., Littlefield J. W. Altered regulation of the rate of synthesis of dihydrofolate reductase in methotrexate-resistant hamster cells. J Biol Chem. 1976 May 25;251(10):3075–3080. [PubMed] [Google Scholar]
  22. Hänggi U. J., Littlefield J. W. Isolation and characterization of the multiple forms of dihydrofolate reductase from methotrexate-resistant hamster cells. J Biol Chem. 1974 Mar 10;249(5):1390–1397. [PubMed] [Google Scholar]
  23. Jackson R. C., Niethammer D. Acquired methotrexate resistance in lymphoblasts resulting from altered kinetic properties of dihydrofoltate reductase. Eur J Cancer. 1977 Jun;13(6):567–575. doi: 10.1016/0014-2964(77)90118-9. [DOI] [PubMed] [Google Scholar]
  24. Kaufman R. J., Brown P. C., Schimke R. T. Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5669–5673. doi: 10.1073/pnas.76.11.5669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kellems R. E., Alt F. W., Schimke R. T. Regulation of folate reductase synthesis in sensitive and methotrexate-resistant sarcoma 180 cells. In vitro translation and characterization of folate reductase mRNA. J Biol Chem. 1976 Nov 25;251(22):6987–6993. [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  29. Lewis J. A., Kurtz D. T., Melera P. W. Molecular cloning of Chinese hamster dihydrofolate reductase-specific cDNA and the identification of multiple dihydrofolate reductase mRNAs in antifolate-resistant Chinese hamster lung fibroblasts. Nucleic Acids Res. 1981 Mar 25;9(6):1311–1322. doi: 10.1093/nar/9.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Littlefield J. W. Hybridization of hamster cells with high and low folate reductase activity. Proc Natl Acad Sci U S A. 1969 Jan;62(1):88–95. doi: 10.1073/pnas.62.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McBurney M. W., Whitmore G. F. Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell. 1974 Jul;2(3):173–182. doi: 10.1016/0092-8674(74)90091-9. [DOI] [PubMed] [Google Scholar]
  32. Melera P. W., Lewis J. A., Biedler J. L., Hession C. Antifolate-resistant Chinese hamster cells. Evidence for dihydrofolate reductase gene amplification among independently derived sublines overproducing different dihydrofolate reductases. J Biol Chem. 1980 Jul 25;255(14):7024–7028. [PubMed] [Google Scholar]
  33. Melera P. W., Wolgemuth D., Biedler J. L., Hession C. Antifolate-resistant chinese hamster cells. Evidence from independently derived sublines for the overproduction of two dihydrofolate reductases encoded by different mRNAs. J Biol Chem. 1980 Jan 25;255(2):319–322. [PubMed] [Google Scholar]
  34. Nakamura H., Littlefield J. W. Purification, properties, and synthesis of dihydrofolate reductase from wild type and methotrexate-resistant hamster cells. J Biol Chem. 1972 Jan 10;247(1):179–187. [PubMed] [Google Scholar]
  35. 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]
  36. Nunberg J. H., Kaufman R. J., Schimke R. T., Urlaub G., Chasin L. A. Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5553–5556. doi: 10.1073/pnas.75.11.5553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  38. Ray M., Mohandas T. Proposed banding nomenclature for the Chinese hamster chromosomes (Cricetulus griseus). Cytogenet Cell Genet. 1976;16(1-5):83–91. doi: 10.1159/000130559. [DOI] [PubMed] [Google Scholar]
  39. Schimke R. T., Kaufman R. J., Alt F. W., Kellems R. F. Gene amplification and drug resistance in cultured murine cells. Science. 1978 Dec 8;202(4372):1051–1055. doi: 10.1126/science.715457. [DOI] [PubMed] [Google Scholar]
  40. Sirotnak F. M., Kurita S., Hutchison D. J. On the nature of a transport alteration determining resistance to amethopterin in the L1210 leukemia. Cancer Res. 1968 Jan;28(1):75–80. [PubMed] [Google Scholar]
  41. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  42. Stanners C. P., Eliceiri G. L., Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52–54. doi: 10.1038/newbio230052a0. [DOI] [PubMed] [Google Scholar]
  43. Stavnezer J., Huang R. C., Stavnezer E., Bishop J. M. Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constatn region of kappa chain in the mouse. J Mol Biol. 1974 Sep 5;88(1):43–63. doi: 10.1016/0022-2836(74)90294-0. [DOI] [PubMed] [Google Scholar]
  44. Strohman R. C., Moss P. S., Micou-Eastwood J., Spector D., Przybyla A., Paterson B. Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures. Cell. 1977 Feb;10(2):265–273. doi: 10.1016/0092-8674(77)90220-3. [DOI] [PubMed] [Google Scholar]
  45. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  46. Wigler M., Perucho M., Kurtz D., Dana S., Pellicer A., Axel R., Silverstein S. Transformation of mammalian cells with an amplifiable dominant-acting gene. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3567–3570. doi: 10.1073/pnas.77.6.3567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Worton R. G., Ho C. C., Duff C. Chromosome stability in CHO cells. Somatic Cell Genet. 1977 Jan;3(1):27–45. doi: 10.1007/BF01550985. [DOI] [PubMed] [Google Scholar]
  48. Worton R., Duff C., Flintoff W. Microcell-mediated cotransfer of genes specifying methotrexate resistance, emetine sensitivity, and chromate sensitivity with Chinese hamster chromosome 2. Mol Cell Biol. 1981 Apr;1(4):330–335. doi: 10.1128/mcb.1.4.330. [DOI] [PMC free article] [PubMed] [Google Scholar]

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