Skip to main content
SpringerLink
Log in

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

  • Regular Papers
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Allegre, J. C.; Lippman, M. E. Growth of a human breast cancer cell line in serum-free hormone-supplemented medium. Cancer Res. 38:3823–3829; 1978.

    Google Scholar 

  2. Barnes, D.; Sato, G. Growth of a human mammary tumour cell line in a serum-free medium. Nature 281:388–389; 1979.

    Article  PubMed  CAS  Google Scholar 

  3. Barnes, D.; Sato, G. Serum-free cell culture: a unifying approach. Cell 22:649–655; 1980.

    Article  PubMed  CAS  Google Scholar 

  4. Barnes, D.; Sirbasku, D. A.; Sato, G. H. eds. Cell culture methods for molecular and cell biology. Methods of preparation of media, supplements and substrata for serum-free animal cell culture, vol. 1. New York: Alan R. Liss, Inc.; 1984.

    Google Scholar 

  5. Barnes, D.; Sirbasku, D. A.; Sato, G. H. eds. Cell culture methods for molecular and cell biology. Methods for serum-free culture of cells of the endocrine system, vol. 2. New York: Alan R. Liss, Inc.; 1984.

    Google Scholar 

  6. Barnes, D.; Sirbasku, D. A.; Sato, G. H. eds. Cell culture methods for molecular and cell biology. Methods for serum-free culture of epithelial and fibroblastic cells, vol. 3. New York: Alan R. Liss, Inc.; 1984.

    Google Scholar 

  7. Barnes, D.; Sirbasku, D. A.; Sato, G. H., eds. Cell culture methods for molecular and cell biology. Methods for serum-free culture of neuronal and lymphoid cells, vol. 4. New York: Alan R. Liss, Inc.; 1984.

    Google Scholar 

  8. Bottenstein, J.; Hayashi, I.; Hutchins, S., et al. The growth of cells in serum-free hormone-supplemented media. Methods Enzymol. 58:94–109; 1979.

    PubMed  CAS  Google Scholar 

  9. Bradford, M. N. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976.

    Article  PubMed  CAS  Google Scholar 

  10. Danielpour, D.; Riss, T. L.; Ogasawara, M., et al. Growth of MTW9/PL2 rat mammary tumor cells in hormonally defined serum-free media. In Vitro 24:42–52; 1988.

    CAS  Google Scholar 

  11. Darbre, P. D.; Curtis, S.; King, R. J. B. Effects of estradiol and tamoxifen on human breast cancer cells in serum-free culture. Cancer Res. 44:2790–2793; 1984.

    PubMed  CAS  Google Scholar 

  12. Daughaday, W. H.; Kapadia, M.; Mariz, I. Serum somatomedin binding proteins: physiologic significance and interference in radioligand assay. J. Lab. Clin. Med. 109:355–363; 1987.

    PubMed  CAS  Google Scholar 

  13. Dickson, R. B.; Bates, S. E.; McManaway, M. E., et al. Characterization of estrogen-responsive transforming activity in human breast cancer cell lines. Cancer Res. 46:1707–1713; 1986.

    PubMed  CAS  Google Scholar 

  14. Dickson, R. B.; Huff, K. K.; Spencer, E. M., et al. Induction of epidermal growth factor-related polypeptides by 17β-estradiol in MCF-7 human breast cancer cells. Endocrinology 118:138–142; 1986.

    PubMed  CAS  Google Scholar 

  15. Dickson, R. B.; McManaway, M. E.; Lippman, M. E. Estrogen-induced factors of breast cancer cells partially replace estrogen to promote tumor growth. Science 232:1540–1543; 1986.

    Article  PubMed  CAS  Google Scholar 

  16. Engle, L. W.; Young, N. A.; Tralka, T. S., et al. Establishment and characterization of three new continuous cell lines derived from human breast carcinomas. Cancer Res. 38:3352–3364; 1978.

    Google Scholar 

  17. Esch, F.; Baird, A.; Ling, N., et al. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc. Natl. Acad. Sci. USA 82:6507–6511; 1985.

    Article  PubMed  CAS  Google Scholar 

  18. Floyd, J. C.; Fajan, S. S.; Conn, J. W., et al. Stimulation of insulin secretion by amino acids. J. Clin. Invest. 45:1487–1502; 1966.

    PubMed  CAS  Google Scholar 

  19. Furlanetto, R. W.; DiCarlo, J. N. Somatomedin-c receptors and growth effects in human breast cells maintained in long-term tissue culture. Cancer Res. 44:2122–2128; 1984.

    PubMed  CAS  Google Scholar 

  20. Furlanetto, R. W.; Underwood, L. E.; Van Wyk, J. J., et al. Estimation of somatomedin-c levels in normals and patients with pituitary disease by RIA. J. Clin Invest. 60:648–657; 1979.

    Google Scholar 

  21. Grinnell, F.; Hays, D. G.; Minter, D. Cell adhesion and spreading factor. Exp. Cell Res. 110:175–190; 1977.

    Article  PubMed  CAS  Google Scholar 

  22. Ham, R. G. Formulation of basal nutrient media. In: Barnes, D.; Sirbasku, D. A.; Sato, G. H., eds. Methods for preparation of media, supplements, and substrata for serum-free animal cell culture, vol. 1. New York: Alan R. Liss, Inc.; 1984:3–21.

    Google Scholar 

  23. Hayashi, I.; Sato, G. H. Replacement of serum by hormones permits growth of cells in defined medium. Nature 259:132–134; 1976.

    Article  PubMed  CAS  Google Scholar 

  24. Hintz, R. L.; Liu, F. A radioimmunoassay of insulin-like growth factor II specific for the c-peptide region. J. Clin. Endocrinol. Metab. 54:442–446; 1982.

    Article  PubMed  CAS  Google Scholar 

  25. Ikeda, T.; Danielpour, D.; Sirbasku, D. A. Characterization of a sheep pituitary derived growth factor for rat and human mammary tumor cels. J. Cell. Biochem. 25:213–229; 1984.

    Article  PubMed  CAS  Google Scholar 

  26. Ikeda, T.; Liu, Q-F.; Danielpour, D., et al. Identification of estrogen-inducible growth factors (estromedins) for rat and human mammary tumor cells in culture. In Vitro 18:961–979; 1982.

    Article  PubMed  CAS  Google Scholar 

  27. Ikeda, T.; Sirbasku, D. A. Purification and properties of a mammary/uterine/pituitary tumor cell growth factor from pregnant sheep uterus. J. Biol. Chem. 259:4049–4064; 1984.

    PubMed  CAS  Google Scholar 

  28. Imagawa, W.; Tomooka, Y.; Nandi, S. Serum-free growth of normal and tumor mouse mammary epithelial cells in primary culture. Proc. Natl. Acad. Sci. USA 79:4074–4077; 1982.

    Article  PubMed  CAS  Google Scholar 

  29. Imai, Y.; Leung, C. K. H.; Friesen, H. G., et al. Epidermal growth factor receptors and the effect of epidermal growth factor on growth of human breast cancer cells in long-term tissue culture. Cancer Res. 42:4394–4398; 1982.

    PubMed  CAS  Google Scholar 

  30. Kano-Sueoka, T.; Errick, J. E. Effects of phosphoethanolamine and ethanolamine on growth of mammary carcinoma cells in culture. Exp. Cell Res. 136:137–145; 1981.

    Article  PubMed  CAS  Google Scholar 

  31. Kano-Sueoka, T.; Cohen, D. M.; Yamaizumi, Z., et al. Phosphoethanolamine as a growth factor of a rat mammary carcinoma cell line. Proc. Natl. Acad. Sci. USA 76:5741–5744; 1979.

    Article  PubMed  CAS  Google Scholar 

  32. Keydar, I.; Chen, L.; Karby, S., et al. Establishment and characterization of a cell line of human breast cancer carcinoma origin. Eur. J. Cancer 15:659–670; 1979.

    PubMed  CAS  Google Scholar 

  33. Kirkland, W. L.; Sorrentino, J. M.; Sirbasku, D. A. Control of cell proliferation. III. demonstration of the direct mitogenic effect of thyroid hormones on an estrogen-dependent rat pituitary tumor cell line. JNCI 56:1159–1164; 1976.

    PubMed  CAS  Google Scholar 

  34. Leland, F. E.; Danielpour, D.; Sirbasku, D. A. Studies of the endocrine, paracrine and autocrine control of mammary tumor cell growth. In: Sato, G. H.; Pardee, A. B.; Sirbasku, D. A., eds. Cell growth in hormonally defined media; Cold Spring Harbor conferences on cell proliferation, vol. 9. New York: Cold Spring Harbor Laboratory; 1982:741–750.

    Google Scholar 

  35. Leland, F. E.; Iio, M.; Sirbasku, D. A. Hormone dependent cell lines. In: Sato, G. H., ed. Functionally differentiated cell lines. New York: Alan R. Liss, Inc.; 1981:1–46.

    Google Scholar 

  36. Lippman, M. E.; Dickson, R. B.; Kasid, A., et al. Autocrine and paracrine growth factor regulation of human breast cancer. J. Steroid Biochem. 24:147–154; 1986.

    Article  PubMed  CAS  Google Scholar 

  37. Marquardt, H.; Todaro, G. J.; Henderson, L. E., et al. Purification and primary structure of a polypeptide with multiplication-stimulating activity from rat liver cell cultures. J. Biol. Chem. 256:6859–6865; 1981.

    PubMed  CAS  Google Scholar 

  38. Massague, J.; Czech, M. P. The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J. Biol. Chem. 257:5038–5045; 1982.

    PubMed  CAS  Google Scholar 

  39. Mather, J. P., ed. Mammalian cell culture. New York: Plenum Press; 1984.

    Google Scholar 

  40. Mormede, P.; Baird, A.; Pigeon, P. Immunoreactive fibroblast growth factor (FGF) in rat tissues: molecular weight forms and the effects of hypophysectomy. Biochem. Biophys. Res. Commun. 128:1108–1113; 1985.

    PubMed  CAS  Google Scholar 

  41. Myal, Y.; Shiu, R. P. C.; Bhaumick, B., et al. Receptor binding and growth-promoting activity of insulin-like growth factors in human breast cancer cells. Cancer Res. 44:5486–5490; 1984.

    PubMed  CAS  Google Scholar 

  42. Neufeld, G.; Gospodarowicz, D. Basic and acidic fibroblast growth factor interact with the same cell surface receptors. J. Biol. Chem. 261:5631–5637; 1986.

    PubMed  CAS  Google Scholar 

  43. Ogasawara, M.; Marquardt, H.; Sirbasku, D. A. Further purification of uterine derived growth factors for mammary tumor cells: partial amino acid sequence identification as a truncated form of IGF-I. Proc. Am. Assoc. Cancer Res. 28:58; 1987.

    Google Scholar 

  44. Ogasawara, M.; Sibasku, D. A. Use of serum-free defined conditions to characterize new uterine and pituitary derived growth factors for MCF-7 and T47D human mammary tumor cells. J. Cell Biol. 103 (pt 2):15a; 1986.

    Google Scholar 

  45. Oka, Y.; Orth, D. N. Human plasma epidermal growth factor/β-urogastrone is associated with platelets. J. Clin. Invest. 72:249–259; 1983.

    Article  PubMed  CAS  Google Scholar 

  46. Osborne, C. K.; Hamilton, B.; Titus, G., et al. Epidermal growth factor stimulation of human breast cancer cells in culture. Cancer Res. 40:2361–2366; 1980.

    PubMed  CAS  Google Scholar 

  47. Osborne, C. K.; Monaco, M. E.; Kahn, C. R., et al. Direct inhibition of growth and antagonism of insulin action by glucocorticoids in human breast cancer cells in culture. Cancer Res. 39:2422–2428; 1979.

    PubMed  CAS  Google Scholar 

  48. Raines, E. W.; Bowen-Pope, D. F.; Ross, R. Plasma binding proteins for platelet-derived factor that inhibit its binding to cell-surface receptors. Proc. Natl. Acad. Sci. USA 81:3424–3428; 1984.

    Article  PubMed  CAS  Google Scholar 

  49. Riss, T. L.; Sirbasku, D. A. Purification and identification of transferrin as a major pituitary derived mitogen for MTW9/PL2 rat mammary tumor cells. In Vitro 23:841–849; 1987.

    CAS  Google Scholar 

  50. Riss, T. L.; Ogasawara, M.; Karey, K. P., et al. Use of serum-free hormonally defined media to evaluate the effects of growth factors and inhibitors on proliferation of estrogen-responsive mammary and pituitary tumor cells in culture. J. Tissue Cult. Methods 10:133–150; 1986.

    Article  CAS  Google Scholar 

  51. Rowe, J. M.; Kasper, S.; Shiu, R. P. C., et al. Purification and characterization of a human mammary tumor-derived growth factor. Cancer Res. 46:1408–1412; 1986.

    PubMed  CAS  Google Scholar 

  52. Sato, G. H.; Pardee, A. B.; Sirbasku, D. A., eds. Growth of cells in hormonally defined media. Cold Spring Harbor conferences on cell proliferation, vol. 9. New York: Cold Spring Harbor Laboratory; 1982.

    Google Scholar 

  53. Shing, Y.; Folkman, J.; Sullivan, R., et al. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science 223:1296–1299; 1984.

    Article  PubMed  CAS  Google Scholar 

  54. Sirbasku, D. A. Estrogen-induction of growth factors specific for hormone-responsive mammary, pituitary and kidney tumor cells. Proc. Natl. Acad. Sci. USA 75:3786–3790; 1978.

    Article  PubMed  CAS  Google Scholar 

  55. Sirbasku, D. A.; Kirkland, W. L. Control of cell proliferation. IV. growth properties of a new cell line established from an estrogen-dependent kidney tumor of the syrian hamster. Endocrinology 98:1260–1272; 1976.

    Article  PubMed  CAS  Google Scholar 

  56. Sirbasku, D. A.; Leland, F. E. Estrogen-inducible growth factors; proposal of a new mechanism of estrogen-promoted tumor cell growth. In: Litwack, G., ed. Biochemical action of hormones, vol. 9. New York: Academic Press; 1982:115–140.

    Google Scholar 

  57. Smith, G. L. Somatomedin carrier proteins. Mol. Cell. Endocrinol. 34:83–89; 1984.

    Article  PubMed  CAS  Google Scholar 

  58. Soule, H. D.; Vazquez, J.; Long, A., et al. A human cell line from a pleural effusion from a breast carcinoma. JNCI 51:1409–1416; 1973.

    PubMed  CAS  Google Scholar 

  59. Thomas, K. A.; Rios-Candelore, M.; Fitzpatrick, S. Purification and characterization of acidic fibroblast growth factor from bovine brain. Proc. Natl. Acad. Sci. USA 81:357–361; 1984.

    Article  PubMed  CAS  Google Scholar 

  60. Van Wyk, J. J.; Graves, D. C.; Casella, S. J., et al. Evidence from monoclonal antibody studies that insulin stimulates deoxyribonucleic acid synthesis through the type I somatomedin receptor. J. Clin. Endocrinol. Metab. 61:639–643; 1985.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Texas Medical School, P. O. Box 20708, 77225, Houston, Texas

    Masami Ogasawara & David A. Sirbasku

Authors
  1. Masami Ogasawara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David A. Sirbasku
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ogasawara, M., Sirbasku, D.A. A new serum-free method of measuring growth factor activities for human breast cancer cells in culture. In Vitro Cell Dev Biol 24, 911–920 (1988). https://doi.org/10.1007/BF02623902

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02623902

Key words

Search

Navigation