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Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

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A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated.

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References

  1. Barcellos-Hof, M. H.; Bissell, M. J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105:223–235; 1989.

    Google Scholar 

  2. Blackburn, D. E.; Hobbs, A. A.; Rosen, J. M. Rat beta casein cDNA: sequence analysis and evolutionary comparisons. Nucleic Acid Res. 10:2295–2307; 1982.

    Article  PubMed  CAS  Google Scholar 

  3. Blum, J. L.; Wicha, M. S. Role of the cytoskeleton in laminin induced mammary gene expression. J. Cell. Physiol. 135:13–22; 1988.

    Article  PubMed  CAS  Google Scholar 

  4. Blum, J. L.; Zeigler, M. E.; Wicha, M. Extracellular matrix: structure, biosynthesis and role in mammary differentiation. In: Medina, D.; Kidwell, W.; Heppner, G., et al. eds. Cellular and molecular biology of mammary cancer. New York: Plenum Press; 1987;105–128.

    Google Scholar 

  5. Blum, J. L.; Zeigler, M. E.; Wicha, M. S. Regulation of mammary gene expression by extracellular matrix components. Exp. Cell Res. 173:322–340; 1987.

    Article  PubMed  CAS  Google Scholar 

  6. Chomczynski, P.; Qasba, P.; Topper, Y. J. Transcriptional and post-transcriptional roles of glucocorticoid in the expression of the rat 25,000 molecular weight casein gene. Biochem. Biophys. Res. Commun. 134:812–818; 1986.

    Article  PubMed  CAS  Google Scholar 

  7. Doppler, W.; Groner, B.; Ball, R. K. Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat β-casein gene promoter constructs in a mammary epithelial cell line. Proc. Natl. Acad. Sci. USA 86:104–108; 1989.

    Article  PubMed  CAS  Google Scholar 

  8. Durban, E. M.; Medina, D.; Butel, J. S. Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland developmentin vivo. Dev. Biol. 109:288–298; 1985.

    Article  PubMed  CAS  Google Scholar 

  9. Emerman, J. T.; Pitelka, D. R. Mainenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328; 1977.

    Article  PubMed  CAS  Google Scholar 

  10. Emerman, J. T.; Enami, J.; Pitelka, D. R., et al. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. USA 74:4466–4470; 1977.

    Article  PubMed  CAS  Google Scholar 

  11. Enami, J.; Nandi, S. Sensitive radioimmunoassay for a component of mouse casein. J. Immunol. Methods 18:235–244; 1977.

    Article  PubMed  CAS  Google Scholar 

  12. Engvall, E. Enzyme immunoassay ELISA and EMIT. In: Vunakis, H. V.; Langone, J. J., eds. Methods in enzymology, vol. 70. Immunological techniques, part A. New York: Academic Press; 1980:419–439.

    Chapter  Google Scholar 

  13. Engvall, E.; Jonsson, K.; Perlmann, P. Enzyme linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme labeled antigen and antibody-coated tubes. Biochim. Biophys. Acta 251:427–434; 1971.

    PubMed  CAS  Google Scholar 

  14. Ethier, S. P. Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture. In Vitro Cell. Dev. Biol. 22:485–490; 1986.

    PubMed  CAS  Google Scholar 

  15. Haeuptle, M.-T.; Suard, Y. L. M.; Bogenmann, E., et al. Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture. J. Cell Biol. 96:1425–1434; 1983.

    Article  PubMed  CAS  Google Scholar 

  16. Hahm, H. A.; Ip, M. M. Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors.In Vitro 26:791–802; 1990.

    CAS  Google Scholar 

  17. Hobbs, A. A.; Richards, D. A.; Kessler, D. J., et al. Complex hormonal regulation of rat casein gene expression. J. Biol. Chem. 257:3598–3605; 1982.

    PubMed  CAS  Google Scholar 

  18. Jahn, G. A.; Djiane, J.; Houdebine, L.-M. Inhibition of casein synthesis by progestagensin vitro: modulation in relation to concentration of hormones that synergize with prolactin. J. Steroid Biochem. 32:373–379; 1989.

    Article  PubMed  CAS  Google Scholar 

  19. Kleinman, H. K.; McGarvey, M. L.; Hassell, J. R., et al. Basement membrane complexes with biological activity. Biochemistry 25:312–318; 1986.

    Article  PubMed  CAS  Google Scholar 

  20. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.

    Article  PubMed  CAS  Google Scholar 

  21. Lee, E. Y. H.; Parry, G.; Bissell, M. J. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata. J. Cell Biol. 98:146–155; 1984.

    Article  PubMed  CAS  Google Scholar 

  22. Lee, E. Y. H. P.; Lee, W. H.; Kaetzel, C. S., et al. Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion. Proc. Natl. Acad. Sci. USA 82:1419–1423; 1985.

    Article  PubMed  CAS  Google Scholar 

  23. Levay-Young, B. K.; Bandyopadhya, G. K.; Nandi, S. Linoleic acid, but not cortisol, stimulates accumulation of casein by mouse mammary epithelial cells in serum-free collagen gel culture. Proc. Natl. Acad. Sci. USA 84:8448–8452; 1987.

    Article  PubMed  CAS  Google Scholar 

  24. Levine, J. F.; Stockdale, F. E. Cell-cell interactions promote mammary epithelial cell differentiation. J. Cell Biol. 100:1415–1422; 1985.

    Article  PubMed  CAS  Google Scholar 

  25. Li, M. L.; Aggeler, J.; Farson, D. A., et al. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc. Natl. Acad. Sci. USA 84:136–140; 1987.

    Article  PubMed  CAS  Google Scholar 

  26. Luna, L. G., editor. Methods for fats and lipids. Manual for histologic staining methods of the Armed Forces Institute of Pathology. New York: McGraw-Hill; 1983:140–142.

    Google Scholar 

  27. Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular cloning: a laboratory manual, ed. 1. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.

    Google Scholar 

  28. Medina, D.; Li, M. L.; Oborn, C. J., et al. Casein gene expression in mouse mammary epithelial cell lines: dependence upon extracellular matrix and cell type. Exp. Cell Res. 172:192–203; 1987.

    Article  PubMed  CAS  Google Scholar 

  29. Melton, D. A.; Krieg, P. A.; Rebagliati, M. R., et al. Efficientin vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035–7056; 1984.

    Article  PubMed  CAS  Google Scholar 

  30. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J. Immunol. Methods 65:55–63; 1983.

    Article  PubMed  CAS  Google Scholar 

  31. Ono, M.; Oka, T. The differential actions of cortisol on the accumulation of α-lactalbumin and casein in midpregnant mouse mammary gland in culture. Cell 19:473–480; 1980.

    Article  PubMed  CAS  Google Scholar 

  32. Quiocho, F. A.; Richards, F. M. Intermolecular cross linking of a protein in the crystalline state: carboxypeptidase-A. Proc. Natl. Acad. Sci. USA 52:833–839; 1964.

    Article  PubMed  CAS  Google Scholar 

  33. Ray, D. B.; Jansen, R. W.; Horst, I. A., et al. A complex non-coordinate regulation of α-lactalbumin and 25 K β-casein by corticosterone, prolactin and insulin in long-term cultures of normal rat mammary cells. Endocrinology 118:393–407; 1986.

    PubMed  CAS  Google Scholar 

  34. Reichlin, M.; Nisonoff, A.; Margoliash, E. Immunological activity of cytochrome C. III. Enhancement of antibody detection and immune response initiation by cytochrome c polymers. J. Biol. Chem. 245:947–954; 1970.

    PubMed  CAS  Google Scholar 

  35. Richards, J.; Larson, L.; Yang J., et al.Method for culturing mammary epithelial cells in a rat tail collagen gel matrix. J. Tissue Cult. Methods 8:31–36; 1983.

    Article  Google Scholar 

  36. Rocha, V.; Ringo, D. L.; Read, D. B. Casein production during differentiation of mammary epithelial cells in collagen gel culture. Exp. Cell Res. 159:201–210; 1985.

    Article  PubMed  CAS  Google Scholar 

  37. Rosen, J. M. Milk protein gene structure and expression. In: Neville, M. C.; Daniel, C. W., eds. The mammary gland. Development, regulation, and function. New York: Plenum Press; 1987:301–322.

    Google Scholar 

  38. Rosen, J. M.; Eisenstein, R. S.; Schlein, A. R. et al. Hormonal and cell-substratum regulation of milk protein gene expression at the posttranscriptional level. Prog. Cancer Res. Ther. 35:134–141; 1988.

    CAS  Google Scholar 

  39. Rosen, J. M.; Matusik, R. J.; Richards, D. A., et al. Multihormonal regulation of casein gene expression at the transcriptional and posttranscriptional levels in the mammary gland. Recent Prog. Horm. Res. 36:157–193; 1980.

    PubMed  CAS  Google Scholar 

  40. Rosen, J. M.; Woo, S. L. C.; Comstock, J. P. Regulation of casein messenger RNA during the development of the rat mammary gland. Biochemistry 14:2895–2903; 1975.

    Article  PubMed  CAS  Google Scholar 

  41. Salomon, D. S.; Liotta, L. A.; Kidwell, W. R. Differential response to growth factor by rat mammary epithelium plated on different collagen substrata in serum-free medium. Proc. Natl. Acad. Sci. USA 78:382–386; 1981.

    Article  PubMed  CAS  Google Scholar 

  42. Sanford, K. K.; Earle, W. R.; Evans, V. J., et al. The measurement of proliferation in tissue cultures by enumeration of cell nuclei. JNCI 11:773–795; 1950.

    Google Scholar 

  43. Shannon, J. M.; Pitelka, D. R. The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro. In Vitro 17:1016–1028; 1981.

    Article  PubMed  CAS  Google Scholar 

  44. Suard, Y. M. L.; Haeuptle, M.-T.; Farinon, E., et al. Cell proliferation and milk protein gene expression in rabbit mammary cell cultures. J. Cell Biol. 96:1435–1442; 1983.

    Article  PubMed  CAS  Google Scholar 

  45. Terada, N.; Wakimoto, H.; Oka, T. Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland. J. Steroid Biochem. 29:99–104; 1988.

    Article  PubMed  CAS  Google Scholar 

  46. Tonelli, Q. J.; Sorof, S. Induction of biochemical differentiation in three-dimensional collagen cultures of mammary epithelial cells from virgin mice. Differentiation 22:195–200; 1982.

    Article  PubMed  CAS  Google Scholar 

  47. Topper, Y. J.; Freeman, C. S. Multiple hormone interactions in the developmental biology of the mammary gland. Physiol. Rev. 60:1049–1106; 1980.

    PubMed  CAS  Google Scholar 

  48. Twentyman, P. R.; Luscombe, M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer 56:279–285; 1987.

    PubMed  CAS  Google Scholar 

  49. Vacca, L. L.; Lipids: Oil red O. In: Laboratory manual of histochemistry. New York: Raven Press; 1985:235–237.

    Google Scholar 

  50. Wicha, M. S.; Liotta, L. A.; Kidwell, W. R. Effects of free fatty acids on the growth of normal and neoplastic rat mammary epithelial cells. Cancer Res. 39:426–435; 1979.

    PubMed  CAS  Google Scholar 

  51. Wicha, M. S.; Lowrie, G.; Kohn, E., et al. Extracellular matrix promotes mammary epithelial growth and differentiationin vitro. Proc. Natl. Acad. Sci. USA 79:3213–3217; 1982.

    Article  PubMed  CAS  Google Scholar 

  52. Wiens, D.; Park, C. S.; Stockdale, F. Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and-independent phases of adipocyte-mammary epithelial cell interaction. Dev. Biol. 120:245–258; 1987.

    Article  PubMed  CAS  Google Scholar 

  53. Yoshimura, M.; Banerjee, M. R.; Oka, T. Nucleotide sequence of a cDNA encoding mouse beta casein. Nucleic Acids Res. 14:8224; 1986.

    Article  PubMed  CAS  Google Scholar 

  54. Yoshimura, M.; Oka, T. Hormonal induction of β-casein gene expression: requirement of ongoing protein synthesis for transcription. Endocrinology 126:427–433; 1990.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Grace Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, 666 Elm Street, 14263, Buffalo, New York

    Hillary A. Hahm, Margot M. Ip, Kathleen Darcy, Jennifer D. Black, Wendy K. Shea & Suzanne Forczek

  2. Laboratory of Molecular and Cellular Biology, National Institutes of Health, 20892, Bethesda, Maryland

    Masami Yoshimura & Takami Oka

Authors
  1. Hillary A. Hahm
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  2. Margot M. Ip
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  3. Kathleen Darcy
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  4. Jennifer D. Black
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  5. Wendy K. Shea
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  6. Suzanne Forczek
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  7. Masami Yoshimura
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  8. Takami Oka
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Additional information

This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.

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Hahm, H.A., Ip, M.M., Darcy, K. et al. Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions. In Vitro Cell Dev Biol 26, 803–814 (1990). https://doi.org/10.1007/BF02623622

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  • DOI: https://doi.org/10.1007/BF02623622

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