Skip to main content
SpringerLink
Log in

Purification and identification of transferrin as a major pituitary-derived mitogen for MTW9/PL2 rat mammary tumor cells

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

Summary

Transferrin was identified as a major tissue-derived growth factor for MTW9/PL2 rat mammary tumor cells. Mitogenic activity was assayed by the ability to stimulate the increase in number of MTW9/PL2 cells over 4 d in Dulbecco's modified Eagle's medium containing only 15 mM HEPES, 2 mM glutamine, and 50 μg/ml gentamicin. This growth-promoting activity was purified from ammonium sulfate precipitates of phosphate buffered saline extracts of porcine pituitaries using DEAE-Sepharose, chromatofocusing, molecular sieve chromatography and reverse phase high performance liquid chromatography. Pig pituitary mitogen (PPM) migrated as a single band at molecular weight 78 000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, eluted from chromatofocusing at multiple pH values near 6.3, exhibited an absorption maximum at 465 nm which was diminished by removal of iron, showed a characteristic salmon-pink color in aqueous solution, and was similar in amino acid composition to previously reported values for porcine transferrin. Purified PPM similar to commercially available human transferrin (ED50=160 to 350 ng/ml). We have concluded that using serum-free assay conditions with MTW9/PL2 cells, transferrin was a major source of the mitogenic activity present in extracts of porcine pituitary.

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. Allegra, 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.

    PubMed  CAS  Google Scholar 

  2. Antoniades, H. N. Human platelet-derived growth factor (PDGF): purification of PDGF-I and PDGF-II and separation of their reduced subunits. Proc. Natl. Acad. Sci. USA 78:7314–7317; 1981.

    Article  PubMed  CAS  Google Scholar 

  3. Assoian, M. A.; Komoriya, A.; Meyers, C. A., et al. Transforming growth factor-β in human platelets: identification of a major storage site, purification and characterization. J. Biol. Chem. 258:7155–7160; 1983.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  5. Barnes, D. W.; Sato, G. H. Growth of a human breast cancer cell line in serum-free medium. Nature 281:388–389; 1979.

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

  7. Barnes, D.; Sirbasku, D. A.; Sato, G. H. editors. 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 

  8. Barnes, D.; Sirbasku, D. A.; Sato, G. H. editors. 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 

  9. Barnes, D.; Sirbasku, D. A.; Sato, G. H. editors. 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 

  10. Bottenstein, J.; Hayashi, I.; Hutchings, S., et al. The growth of cells in serum-free hormone supplemented media. Methods Enzymol. 58:44–109; 1979.

    Article  Google Scholar 

  11. Carpenter, G.; Cohen, S. Epidermal growth factor. Ann. Rev. Biochem. 48:193–216; 1979.

    Article  PubMed  CAS  Google Scholar 

  12. Danielpour, D.; Sirbasku, D. A. Fibroblast growth factor-like activity as a major autocrine mitogen secreted by MTW9/PL2 rat mammary tumor cells. J. Cell Biol. 103(2):11a; 1986.

    Google Scholar 

  13. Davis, R. J.; Czech, M. P. Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor. EMBO J. 5:653–658; 1986.

    PubMed  CAS  Google Scholar 

  14. Eagle, H. Nutrition needs of mammalian cells in tissue culture. Science 122:501–504; 1955.

    Article  PubMed  CAS  Google Scholar 

  15. 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 62:6507–6511; 1985.

    Article  Google Scholar 

  16. Gregory, H. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257:325–327; 1975.

    Article  PubMed  CAS  Google Scholar 

  17. Ham, R. G. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci. USA 53:288–293; 1965.

    Article  PubMed  CAS  Google Scholar 

  18. Ham, R. G.; McKeehan, W. L. Development of improved media and culture conditions for clonal growth of normal diploid cells. In Vitro 14:11–22; 1978.

    Article  PubMed  CAS  Google Scholar 

  19. Hamilton, W. G.; Ham, R. G. Clonal growth on Chinese hamster cell lines in protein-free media. In Vitro 13:537–547; 1977.

    Article  PubMed  CAS  Google Scholar 

  20. Hashizume, S.; Kuroda, K.; Murakami, H. Identification of lactoferrin as an essential growth factor for human lymphocyte cell lines in serum-free medium. Biochim. Biophys. Acta. 763:377–382; 1983.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  22. Hudson, B. G.; Ohno, M.; Brockway, W. J., et al. Chemical and physical properties of serum transferrins from several species. Biochemistry 12(6):1047–1053; 1973.

    Article  PubMed  CAS  Google Scholar 

  23. Ii, I.; Kimura, I.; Ozawa, E. Myotropic protein from chick embryo extract: its purification, identity to transferrin, and indispensability for avian myogenesis. Dev. Biol. 94:366–377; 1982.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  25. 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.

    PubMed  CAS  Google Scholar 

  26. 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 

  27. James, R.; Bradshaw, R. A. Polypeptide growth factors. Ann. Rev. Biochem. 53:259–292; 1984.

    Article  PubMed  CAS  Google Scholar 

  28. Kano-Sueoka, T. Growth of rat mammary tumor cells in serum-free hormone-supplemented medium. In: Barnes, D.; Sirbasku, D. A.; Sato, G. H., eds. Methods of serum-free culture of cells of the endocrine system, vol. 2. New York: Alan R. Liss, Inc., 1984:89–104.

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  30. Marquardt, H.; Hunkapiller, M. W.; Wood, L. E., et al. Transforming growth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells; amino acid sequence homology with epidermal growth factor. Proc. Natl. Acad. Sci. USA 80:4684–4688; 1983.

    Article  PubMed  CAS  Google Scholar 

  31. 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; homology with human insulin-like growth factor II. J. Biol. Chem. 256:6859–6865; 1981.

    PubMed  CAS  Google Scholar 

  32. Mather, J. P., editor. Mammalian cell culture. New York: Plenum Press; 1984.

    Google Scholar 

  33. Ogasawara, M.; Sirbasku, 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(2):15a; 1986.

    Google Scholar 

  34. Raines, E. W.; Ross, R. Platelet-derived growth factor. I. High yield purification and evidence for multiple forms. J. Biol. Chem. 257:5154–5160; 1982.

    PubMed  CAS  Google Scholar 

  35. Riss, T. L.; Marquardt, H.; Sirbasku, D. A. Purification of βTSH-like molecule from porcine pituitary that has mitogenic activity on mouse and human mammary cells. Program of the Fourth Decennial Tissue Culture Association Review Conference. Hershey, PA. 1986:27. (Abstract).

    Google Scholar 

  36. Riss, T. L.; Sirbasku, D. A. Growth and serial passage of COMMA-1D mouse mammary epithelial cells in hormonally defined serum-free medium. J. Cell Biol. 103(2):14a; 1986.

    Google Scholar 

  37. Roberts, A. B.; Frokik, C. A.; Anzano, M. A., et al. Transforming growth factors from neoplastic and non-neoplastic tissues. Fed. Proc. 42:2612–2626; 1983.

    Google Scholar 

  38. Roscoe, J. M.; Danielpour, D.; Riss, T. L. et al. Growth of the MTW9/PL2 rat mammary tunor cells in serum-free hormonally defined medium. J. Cell Biol. 103(2):14a: 1986.

    Google Scholar 

  39. Rubin, J. S.; Bradshaw, R. A. Preparation of guinea pig prostate epidermal growth factor. In: Barnes, D. W.; 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., 1984:139–145.

    Google Scholar 

  40. Sato, G. H.; Pardee, A. B.; Sirbasku, D. A. editors. Growth of cells in hormonally defined media. Cold spring harbor conferences on cell proliferation, vol. 9. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.

    Google Scholar 

  41. Savage, C. R., Jr.; Cohen, S. Epidermal growth factor and a new derivative: rapid isolation procedures and biological and chemical characterization. J. Biol. Chem. 247:7609–7611; 1972.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  43. Sirbasku, D. A. Hormone-responsive growth in vivo of a tissue culture cell line established from the MT-W9A rat mammary tumor. Cancer Res. 38:1154–1165; 1978.

    PubMed  CAS  Google Scholar 

  44. Sirbasku, D. A.; Leland, F. E.; Benson, R. H. Properties of a growth factor activity present in crude extracts of rat uterus. J. Cell. Physiol. 107:345–358; 1981.

    Article  PubMed  CAS  Google Scholar 

  45. Sirbasku, D. A.; Officer, J. B.; Leland, F. E. et al. Evidence of a new role for pituitary derived hormones and growth factors in mammary tumor growth in vivo and in vitro. 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. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982:763–778.

    Google Scholar 

  46. Summers, P. J.; Ashmore, C. R.; Lee, Y. B., et al. Stretch-induced growth in chicken wing muscles: role of soluble growth-promoting factors. J. Cell. Physiol. 125:288–294; 1985.

    Article  PubMed  CAS  Google Scholar 

  47. 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 

  48. Waymouth, C. Construction of tissue culture media. In: Rothblat, G. H.; Cristafalo, V. J., eds. Growth, nutrition and metabolism of cells in culture. New York: Academic Press, Inc., 1972:11–47.

    Google Scholar 

  49. Waymouth, C. Preparation and use of serum-free culture media. In: Barnes, D. W.; Sirbasku, D. A.; Sato, G. H., eds. Methods of preparation of media, supplements, and substrata for serum-free animal cell culture, vol. 1. New York: Alan R. Liss, Inc., 1984:23–68.

    Google Scholar 

  50. Zapf, J.; Froesch, E. R.; Humbel, R. E. The insulin-like growth factors (IGF) of human serum: chemical and biological characterization and aspects of their possible physiological role. Curr. Top. Cell Reg. 19:257–309; 1981.

    CAS  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

    Terry L. Riss & David A. Sirbasku

Authors
  1. Terry L. Riss
    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, and American Cancer Society grant BC-255.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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 Cell Dev Biol 23, 841–849 (1987). https://doi.org/10.1007/BF02620963

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02620963

Key words

Search

Navigation