Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1993 Apr;91(4):1289–1294. doi: 10.1172/JCI116327

Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

K N Chung 1, Y Saikawa 1, T H Paik 1, K H Dixon 1, T Mulligan 1, K H Cowan 1, P C Elwood 1
PMCID: PMC288097  PMID: 7682567

Abstract

A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels of hFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of [3H]methotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity.

Full text

PDF

Images in this article

1291Image
on p.1291
1292Image
on p.1292
1292Image
on p.1292
1292Image
on p.1292
1292Image
on p.1292

Selected References

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

  1. Antony A. C., Kane M. A., Portillo R. M., Elwood P. C., Kolhouse J. F. Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells. J Biol Chem. 1985 Dec 5;260(28):14911–14917. [PubMed] [Google Scholar]
  2. Assaraf Y. G., Schimke R. T. Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7154–7158. doi: 10.1073/pnas.84.20.7154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brigle K. E., Westin E. H., Houghton M. T., Goldman I. D. Characterization of two cDNAs encoding folate-binding proteins from L1210 murine leukemia cells. Increased expression associated with a genomic rearrangement. J Biol Chem. 1991 Sep 15;266(26):17243–17249. [PubMed] [Google Scholar]
  4. Campbell I. G., Jones T. A., Foulkes W. D., Trowsdale J. Folate-binding protein is a marker for ovarian cancer. Cancer Res. 1991 Oct 1;51(19):5329–5338. [PubMed] [Google Scholar]
  5. Coney L. R., Tomassetti A., Carayannopoulos L., Frasca V., Kamen B. A., Colnaghi M. I., Zurawski V. R., Jr Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Cancer Res. 1991 Nov 15;51(22):6125–6132. [PubMed] [Google Scholar]
  6. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  7. Deutsch J. C., Elwood P. C., Portillo R. M., Macey M. G., Kolhouse J. F. Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells. Arch Biochem Biophys. 1989 Nov 1;274(2):327–337. doi: 10.1016/0003-9861(89)90446-3. [DOI] [PubMed] [Google Scholar]
  8. Elwood P. C., Kane M. A., Portillo R. M., Kolhouse J. F. The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells. J Biol Chem. 1986 Nov 25;261(33):15416–15423. [PubMed] [Google Scholar]
  9. Elwood P. C. Molecular cloning and characterization of the human folate-binding protein cDNA from placenta and malignant tissue culture (KB) cells. J Biol Chem. 1989 Sep 5;264(25):14893–14901. [PubMed] [Google Scholar]
  10. Fan J., Vitols K. S., Huennekens F. M. Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells. J Biol Chem. 1991 Aug 15;266(23):14862–14865. [PubMed] [Google Scholar]
  11. Flintoff W. F., Nagainis C. R. Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding. Arch Biochem Biophys. 1983 Jun;223(2):433–440. doi: 10.1016/0003-9861(83)90607-0. [DOI] [PubMed] [Google Scholar]
  12. Frei E., 3rd, Rosowsky A., Wright J. E., Cucchi C. A., Lippke J. A., Ervin T. J., Jolivet J., Haseltine W. A. Development of methotrexate resistance in a human squamous cell carcinoma of the head and neck in culture. Proc Natl Acad Sci U S A. 1984 May;81(9):2873–2877. doi: 10.1073/pnas.81.9.2873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Galivan J. 5-Methyltetrahydrofolate transport by hepatoma cells and methotrexate-resistant sublines in culture. Cancer Res. 1981 May;41(5):1757–1762. [PubMed] [Google Scholar]
  14. Henderson G. B. Folate-binding proteins. Annu Rev Nutr. 1990;10:319–335. doi: 10.1146/annurev.nu.10.070190.001535. [DOI] [PubMed] [Google Scholar]
  15. Henderson G. B., Tsuji J. M., Kumar H. P. Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate. J Membr Biol. 1988 Mar;101(3):247–258. doi: 10.1007/BF01872839. [DOI] [PubMed] [Google Scholar]
  16. Henderson G. B., Zevely E. M. Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of L1210 cells by treatment with an N-hydroxysuccinimide ester of [3H]methotrexate. J Biol Chem. 1984 Apr 10;259(7):4558–4562. [PubMed] [Google Scholar]
  17. Jansen G., Kathmann I., Rademaker B. C., Braakhuis B. J., Westerhof G. R., Rijksen G., Schornagel J. H. Expression of a folate binding protein in L1210 cells grown in low folate medium. Cancer Res. 1989 Apr 15;49(8):1959–1963. [PubMed] [Google Scholar]
  18. Jansen G., Westerhof G. R., Jarmuszewski M. J., Kathmann I., Rijksen G., Schornagel J. H. Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates. J Biol Chem. 1990 Oct 25;265(30):18272–18277. [PubMed] [Google Scholar]
  19. Kane M. A., Elwood P. C., Portillo R. M., Antony A. C., Kolhouse J. F. The interrelationship of the soluble and membrane-associated folate-binding proteins in human KB cells. J Biol Chem. 1986 Nov 25;261(33):15625–15631. [PubMed] [Google Scholar]
  20. Kane M. A., Elwood P. C., Portillo R. M., Antony A. C., Najfeld V., Finley A., Waxman S., Kolhouse J. F. Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells. J Clin Invest. 1988 May;81(5):1398–1406. doi: 10.1172/JCI113469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kane M. A., Portillo R. M., Elwood P. C., Antony A. C., Kolhouse J. F. The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein. J Biol Chem. 1986 Jan 5;261(1):44–49. [PubMed] [Google Scholar]
  22. Kane M. A., Waxman S. Role of folate binding proteins in folate metabolism. Lab Invest. 1989 Jun;60(6):737–746. [PubMed] [Google Scholar]
  23. Lacey S. W., Sanders J. M., Rothberg K. G., Anderson R. G., Kamen B. A. Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol. J Clin Invest. 1989 Aug;84(2):715–720. doi: 10.1172/JCI114220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matherly L. H., Czajkowski C. A., Angeles S. M. Identification of a highly glycosylated methotrexate membrane carrier in K562 human erythroleukemia cells up-regulated for tetrahydrofolate cofactor and methotrexate transport. Cancer Res. 1991 Jul 1;51(13):3420–3426. [PubMed] [Google Scholar]
  25. Matsue H., Rothberg K. G., Takashima A., Kamen B. A., Anderson R. G., Lacey S. W. Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6006–6009. doi: 10.1073/pnas.89.13.6006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Price E. M., Ratnam M., Rodeman K. M., Freisheim J. H. Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: involvement of a membrane receptor and a cytosolic protein. Biochemistry. 1988 Oct 4;27(20):7853–7858. doi: 10.1021/bi00420a040. [DOI] [PubMed] [Google Scholar]
  27. Sadasivan E., Rothenberg S. P. The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA. J Biol Chem. 1989 Apr 5;264(10):5806–5811. [PubMed] [Google Scholar]
  28. Schuetz J. D., Matherly L. H., Westin E. H., Goldman I. D. Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. J Biol Chem. 1988 Jul 15;263(20):9840–9847. [PubMed] [Google Scholar]
  29. Sherwood S. W., Assaraf Y. G., Molina A., Schimke R. T. Flow cytometric characterization of antifolate resistance in cultured mammalian cells using fluoresceinated methotrexate and daunorubicin. Cancer Res. 1990 Aug 15;50(16):4946–4950. [PubMed] [Google Scholar]
  30. Sirotnak F. M. Obligate genetic expression in tumor cells of a fetal membrane property mediating "folate" transport: biological significance and implications for improved therapy of human cancer. Cancer Res. 1985 Sep;45(9):3992–4000. [PubMed] [Google Scholar]
  31. Underhill T. M., Flintoff W. F. Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer. Mol Cell Biol. 1989 Apr;9(4):1754–1758. doi: 10.1128/mcb.9.4.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang C. H., Sirotnak F. M., Mines L. S. Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling. J Biol Chem. 1988 Jul 15;263(20):9703–9709. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES