Skip to main content
Log in

Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor

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

Summary

Induction of hepatocyte DNA synthesis in culture by cyproterone acetate (CPA), a potent hepatomitogen in vivo, was studied. Adult rat hepatocytes were grown on collagen gels in primary culture for 3 to 10 d. Epidermal growth factor (EGF) was used as a model inducer to establish appropriate culture conditions. (a) In serum-free medium EGF stimulated a wave of DNA synthesis in 10 to 30% of the hepatocytes. CPA had only a weak effect. (b) Increasing concentrations of newborn bovine serum (NBS) at 5 to 95% progressively inhibited the stimulatory effect of EGF. A similar inhibition was obtained by adding bovine serum albumin; 20% NBS, however, had a slightly stimulatory effect on the induction of DNA synthesis by CPA. (c) Portal rat serum (RS) at concentration of 5 to 95% markedly stimulated DNA synthesis, a plateau being reached between 20 and 95%. EGF had a distinct enhancing effect on DNA synthesis in the presence of 5 and 20% RS but not at 50 and 95%. CPA stimulated DNA synthesis in the presence of 20, 50, and 95% RS in a synergistic way. (d) Mitoses were found after treatment with EGF or with CPA. These results show that CPA can induce DNA synthesis in cultured hepatocytes and that RS contains factors facilitating the response to CPA.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Argyris, A. S.; Magnus, D. R. The stimulation of liver growth and demethylase activity following phenobarbital treatment. Dev. Biol. 17:187–201; 1968.

    Article  PubMed  CAS  Google Scholar 

  2. Berry, M. N.; Friend, D. S. High yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43:506–520; 1969.

    Article  PubMed  CAS  Google Scholar 

  3. Bieri, F.; Bentley, P.; Waechter, F. et al. Use of primary cultures of adult rat hepatocytes to investigate mechanisms of action of nafenopin, a hepatocarcinogenic peroxisome proliferator. Carcinogenesis 5:1033–1039; 1984.

    Article  PubMed  CAS  Google Scholar 

  4. Bronstad, G. O.; Sand, T.; Christoffersen, T. Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes. Biochem. Biophys. Acta 763:58–63; 1983.

    Article  PubMed  CAS  Google Scholar 

  5. Bucher, N. L. R.; Swaffield, M. N. Synergistic action of glucagon and insulin in regulation of hepatic regeneration. Adv. Enzyme Regul. 13:281–293; 1975.

    PubMed  CAS  Google Scholar 

  6. Bursch, W.; Schulte-Hermann, R. Synchronization of hepatic DNA synthesis by scheduled feeding and lighting in mice treated with the chemical inducer of liver growth ahexachlorocyclohexane. Cell Tissue Kinet. 16:125–134; 1983.

    PubMed  CAS  Google Scholar 

  7. Chambard, M.; Gabrion, J.; Mauchamp, J. Influence of collagen gel on the orientation of epithelial cell polarity: Follicle formation from isolated thyroid cells and from preformed monolayers. J. Cell Biol. 91:157–166; 1981.

    Article  PubMed  CAS  Google Scholar 

  8. Draghi, E.; Armato, U.; Andreis, P. G. et al. The stimulation by epidermal growth factor (urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones. J. Cell. Physiol. 103:129–147; 1980

    Article  PubMed  CAS  Google Scholar 

  9. Ehrmann, R. L.; Gey, G. O. The growth of cells on a transparent gel of reconstituted rat-tail collagen. J. Natl. Cancer Inst. 16:1375–1403; 1956.

    PubMed  CAS  Google Scholar 

  10. Enat, R.; Jefferson, D. M.; Ruiz-Opazo, N. et al. Hepatocyte proliferation in vitro: Its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc. Natl. Acad. Sci. USA 81:1411–1415; 1984.

    Article  PubMed  CAS  Google Scholar 

  11. Friedman, D. L.; Claus, T. H.; Pilkis, S. J. et al. Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes. Exp. Cell Res. 135:283–290; 1981.

    Article  PubMed  CAS  Google Scholar 

  12. Glinos, A. D.; Gey, G. O. Humoral factors involved in the induction of liver regeneration in the rat. Proc. Soc. Exp. Biol. Med. 80:421–425; 1952.

    PubMed  CAS  Google Scholar 

  13. Hasegawa, K.; Namai, K.; Koga, M. Induction of DNA-synthesis in adult rat hepatocytes cultured in a serum-free medium. Biochem. Biophys. Res. Commun. 95:243–249; 1980.

    Article  PubMed  CAS  Google Scholar 

  14. Hasegawa, K.; Watanabe, K.; Koga, M. Induction of mitosis in primary cultures of adult rat hepatocytes under serum-free conditions. Biochem. Biophys. Res. Commun. 104:259–265; 1982.

    Article  PubMed  CAS  Google Scholar 

  15. Hoffmann, V.; Schulte-Hermann, R. The regulative role of food consumption in the induction of rat liver cell proliferation by drugs and environmental pollutants. Arch. Toxicol. Suppl. 2:457–461; 1979.

    PubMed  Google Scholar 

  16. Kallenbach, M.; Roome, N. O.; Schulte-Hermann, R. Kinetics of DNA synthesis in feeding-dependent and independent hepatocyte populations of rats after partial hepatectomy. Cell Tissue Kinet. 16:321–332; 1983.

    PubMed  CAS  Google Scholar 

  17. Leffert, H. L.; Koch, K. S.; Moran, T. et al. Hormonal control of rat liver regeneration. Gastroenterology 76:1470–1482; 1979.

    PubMed  CAS  Google Scholar 

  18. Leffert, H. L.; Koch, K. S.; Rubalcava, B. et al. Hepatocyte growth control: in vivo approach to problems of liver regeneration and function. Natl. Cancer Inst. Monogr. 48: 1978:87–101.

    PubMed  Google Scholar 

  19. Leffert, H. L.; Moran, T.; Boorstein, R. et al. Procarcinogen activation and hormonal control of cell proliferation in differentiated primary adult rat liver cell cultures. Nature 267:58–61; 1977.

    Article  PubMed  CAS  Google Scholar 

  20. Levin, W. G.; Ord, M. G.; Stocken, L. A. Some biochemical changes associated with nafenopin-induced liver growth in the rat. Biochem. Pharmacol. 26:939–942; 1977.

    Article  Google Scholar 

  21. Lie, S. O. Growth promoting effect of serum from portal blood on liver cells in vitro. J. Clin. Invest. 2:163–168; 1972.

    Article  CAS  Google Scholar 

  22. Marceau, N.; Noel, M.; Deschenes, J. Growth and functional activities of neonatal and adult rat hepatocytes cultured on fibronectin-coated substratum in serum-free medium. In Vitro 18:1–11; 1982.

    PubMed  CAS  Google Scholar 

  23. McGowan, J. A.; Strain, A. J.; Bucher, N. L. R. DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP. J. Cell. Physiol. 108:353–363; 1981.

    Article  PubMed  CAS  Google Scholar 

  24. Michalopoulos, G.; Cianciulli, H. D.; Novotny, A. R. et al. Liver regeneration studies with hepatocytes in primary culture. Cancer Res. 42:4673–4682; 1982.

    PubMed  CAS  Google Scholar 

  25. Michalopoulos, G.; Pitot, H. C. Primary culture of parenchymal liver cells and collagen membranes. Exp. Cell Res. 94:70–78; 1975.

    Article  PubMed  CAS  Google Scholar 

  26. Nakamura, T.; Tomita, Y.; Ichihara, A. Density-dependent growth control of adult rat hepatocytes in primary culture. J. Biochem. 94:1029–1035; 1983.

    PubMed  CAS  Google Scholar 

  27. Nakamura, T.; Yoshimoto, K.; Nakayama, Y. et al. Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell-cell contact and cell membranes. Proc. Natl. Acad. Sci. USA 80:7229–7233; 1983.

    Article  PubMed  CAS  Google Scholar 

  28. Radke, M.; Schulte-Hermann, R.; Junge, U. Induction of hepatic DNA synthesis in rats with streptozotocin-induced diabetes. Hepatogastroenterology 30:54–57; 1983.

    CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  30. Richman, R. A.; Claus, T. H.; Pilkis, S. J. et al. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 73:3589–3593; 1976.

    Article  PubMed  CAS  Google Scholar 

  31. Russel, W. E.; McGowan, J. A.; Bucher, N. L. R. Partial characterization of a hepatocyte growth factor from rat platelets. J. Cell. Physiol. 119:183–192; 1984.

    Article  Google Scholar 

  32. Rutenburg, A. M.; Kim, H.; Fischbein, J. W. et al. Hitochemical and ultrastructural demonstration of g-glutamyl transpeptidase activity. J. Histochem. Cytochem. 17:517–526; 1969.

    PubMed  CAS  Google Scholar 

  33. Sattler, C. A.; Michalopoulos, G.; Sattler, G. L. et al. Ultrastructure of adult rat hepatocytes cultured on floating collagen membranes. Cancer Res. 38:1539–1549; 1978.

    PubMed  CAS  Google Scholar 

  34. Schulte-Hermann, R. Induction of liver growth by xenobiotic compounds and other stimuli. Crit. Rev. Toxicol. 3:97–158 1974.

    CAS  Google Scholar 

  35. Schulte-Hermann, R. Two-stage control of cell proliferation induced in rat liver by a-hexachlorocyclohexane. Cancer Res. 37:166–171; 1978.

    Google Scholar 

  36. Schulte-Hermann, R. Reactions of the liver to injury: adaptation. In: Farber, E.; Fisher, M. M., eds. Toxic injury of the liver, part A: New York: Marcel dekker, Inc.; 1978;385–444.

    Google Scholar 

  37. Schulte-Hermann, R.; Hoffman, V.; Parzefall, W. et al. Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers. II. Induction of growth. Chem. Biol. Interact. 31:287–300; 1980.

    Article  PubMed  CAS  Google Scholar 

  38. Schulte-Hermann, R.; Landgraf, H. Circadian rhythm of cell proliferation in rat liver: synchronization by feeding habits. Z. Naturforsch. 29c:421–424; 1974.

    Google Scholar 

  39. Schulte-Hermann, R.; Ohde, G.; Schuppler, J. et al. Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane, steriod compounds, and nafenopin. Cancer Res. 41:2556–2562; 1981.

    PubMed  CAS  Google Scholar 

  40. Schulte-Hermann, R.; Schuppler, J.; Timmermann-Trosiener, I. et al. The role of growth of normal and preneoplastic cell populations for tumor promotion in rat liver. Environ. Health Perspect. 50:185–194; 1983.

    Article  PubMed  CAS  Google Scholar 

  41. Schulte-Hermann, R.; Timmermann-Trosiener, I.; Schuppler, J. promotion of spontaneous preneoplastic cells in rat liver as a possible explanation of tumor production by nonmutagenic compounds. Cancer Res. 43:839–844; 1983.

    PubMed  CAS  Google Scholar 

  42. Seglen, P. O. Preparation of isolated rat liver cells. In: Prescott, D. M., ed. Methods in cell biology, vol. 13. New York: Academic Press; 1976:29–83.

    Google Scholar 

  43. Sirica, A. E.; Richards, W.; Tsukada, Y. et al. Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc. Natl. Acad. Sci. USA 76:283–287; 1979.

    Article  PubMed  CAS  Google Scholar 

  44. Strain, A. J.; McGowan, J. A.; Bucher, N. L. R. Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s). In Vitro 18:108–116; 1982.

    Article  PubMed  CAS  Google Scholar 

  45. Sudweeks, A. D.; Hill, R. B. Control of liver cell replication by albumin need. J. Cell Biol. 34:404–406; 1967.

    Article  PubMed  CAS  Google Scholar 

  46. Wachstein, M.; Meisel, E. On the histochemical demonstration of glucose-6-phosphatase. J. Histochem. Cytochem. 4:592; 1956.

    Google Scholar 

  47. Williams, G. M.; Bermudez, E.; Scaramuzzino, D. Rat hepatocyte primary cultures III. Improved dissociation and attachment techniques and enhancement of survival by culture medium. In Vitro 13:809–817; 1977.

    Article  PubMed  CAS  Google Scholar 

  48. Wittich, U. Wirkung von alpha-Hexachlorcyclohexan auf das wachstum der Leber und die Aktivität des fremdstoff-metabolisierenden Monooxygenase-systems der Lebermikrosomen während der Perinatal-und Säugeperiode der Ratte. Thesis, Philipps-University Marburg: 1980.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

This study was supported by Gesellschaft für Strahlen-und Umweltforschung mbH, München, Germany.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Parzefall, W., Galle, P.R. & Schulte-Hermann, R. Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor. In Vitro Cell Dev Biol 21, 665–673 (1985). https://doi.org/10.1007/BF02620920

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02620920

Key words

Navigation