Experiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination.
Stimulation of [3H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested.
Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal.
Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation.
The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined.
hepatocytes DNA synthesis serum platelets growth factors
This is a preview of subscription content, log in to check access.
Bucher, N. L. R.; McGowan, J. A. Regeneration:regulatory mechanisms. Wright, E.; Alberti, K. G. M. M.; Karran, S.; Millward-Sadler, G. eds. Liver and biliary disease. London: W. B. Saunders; 1979: 210–227.Google Scholar
Richman, R. A.; Claus, T. H.; Pilkis, S. J.; Friedman, D. L. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 73: 3589–3493; 1976.PubMedCrossRefGoogle Scholar
Armato, U.; Draghi, E.; Andreis, P. G. Effects of purine cyclic nucleotides in the growth of neonatal rat hepatocytes in primary tissue culture. Exp. Cell Res. 105: 337–347; 1977.PubMedCrossRefGoogle Scholar
Leffert, H. L.; Moran, T.; Boorstein, R.; Koch, K. S. Procarcinogen activation and hormonal control of cell proliferation in differentiated primary adult rat liver cell cultures. Nature 267: 58–61; 1977.PubMedCrossRefGoogle Scholar
Leffert, H. L.; Moran, T.; Sell, S.; Skelly, H.; Ibsen, K.; Mueller, M.; Arias, I. Growth state-dependent phenotype of adult hepatocytes in primary monolayer cultures. Proc. Natl. Acad. Sci. USA 75: 1834–1838; 1978.PubMedCrossRefGoogle Scholar
Koch, K. S.; Leffert, H. L. Growth control of differentiated adult rat hepatocytes in primary culture. Ann. N. Y. Acad. Sci. 349: 111–127; 1980.PubMedCrossRefGoogle Scholar
Laishes, B. A.; Williams, G. M. Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12: 821–832; 1976.PubMedGoogle Scholar
Savage, C. R.; Bonney, R. S. Extended expression of differentiated function in primary cultures of adult liver parenchymal cells maintained on nitrocellulose filters. I. Induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase. Exp. Cell Res. 114: 307–315; 1978.PubMedCrossRefGoogle Scholar
Sirica, A. E.; Richards, W.; Tsukada, Y.; Sattler, C. A.; Pitot, H. C. Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc. Natl. Acad. Sci. USA 76: 283–287; 1979.PubMedCrossRefGoogle Scholar
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.PubMedCrossRefGoogle Scholar
Hayashi, I.; Sato, G. H. Replacement of serum by hormones permits growth of cells in a defined medium. Nature 259: 132–134; 1976.PubMedCrossRefGoogle Scholar
Strain, A. J.; McGowan, J. A.; Bucher, N. L. R. Stimulation of DNA synthesis in primary adult rat hepatocytes by sera from normal and partially hepatectomized rats (abstr.). In Vitro 16: 225; 1980.Google Scholar
Strain, A. J.; McGowan, J. A.; Bucher, N. L. R. Role of serum and platelets in stimulation of DNA synthesis in primary cultures of adult rat hepatocytes (abstr.). Gastroenterology 79: 1056; 1980.Google Scholar
Waymouth, C. Preparation of medium MAB 87/3 for primary cultures of epithelial cells. TCA Manual 3: 521–525; 1976.CrossRefGoogle Scholar
Leffert, H. L.; Paul, D. Studies in primary cultures of differentiated fetal liver cells. J. Cell Biol. 52: 559–568; 1972.PubMedCrossRefGoogle Scholar
Acosta, D.; Anuforo, D.; Smith, R. Primary monolayer cultures of postnatal rat liver cells with extended differentiated functions. In Vitro 14: 428–436; 1978.PubMedCrossRefGoogle Scholar
Seglen, P. O. Preparation of isolated rat liver cells. Prescott, D. M. ed. Methods in cell biology. Vol. 13. New York: Academic Press; 1976: 29–83.Google Scholar
Munro, H. N.; Fleck, A. The determination of nucleic acids. Glick, D. ed. Methods of biochemical analysis. Vol. 14. New York: John Wiley; 1966: 113–176.CrossRefGoogle Scholar
Prescott, D. M. Autoradiography with liquid emulsion. Prescott, D. M. ed. Methods in cell physiology. Vol. 1. New York: Academic Press; 1964: 365–370.Google Scholar
Burton, K. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 315–322; 1956.PubMedGoogle Scholar
Ross, R.; Nist, C.; Kariya, B.; Rivest, M. J.; Raines, E.; Callis, J. Physiological quiescence in plasma-derived serum: influence of platelet-derived growth factor on cell growth in culture. J. Cell Physiol. 97: 497–508; 1978.PubMedCrossRefGoogle Scholar
Bucher, N. L. R.; Malt, R. A. Regeneration of liver and kidney. Boston: Little, Brown; 1971: 161–176.Google Scholar
Ross, R.; Glomset, J.; Kariya, B.; Harker, L. A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cellsin vitro. Proc. Natl. Acad. Sci. USA 71: 1207–1210; 1974.PubMedCrossRefGoogle Scholar
Scher, C. D.; Shepard, R. C.; Antoniades, H. N.; Stiles, C. D. Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim. Biophys. Acta 560: 217–241; 1979.PubMedGoogle Scholar
Kohler, N.; Lipton, A. Platelets as a source of fibroblast growth-promoting activity. Exp. Cell Res. 87: 297–301; 1974.PubMedCrossRefGoogle Scholar
Westermark, B.; Wasteson, A. A platelet factor stimulating human normal glial cells. Exp. Cell Res. 98: 170–174; 1976.PubMedCrossRefGoogle Scholar
Eastment, C. T.; Sirbasku, D. A. Platelet-derived growth factor(s) for a hormone-responsive rat mammary tumor cell line. J. Cell Physiol. 97: 17–28; 1978.PubMedCrossRefGoogle Scholar
Hara, Y.; Steiner, M.; Baldini, M. G. Platelets as a source of growth-promoting factor(s) for tumor cells. Cancer Res. 40: 1212–1216; 1980.PubMedGoogle Scholar
Altman, P. L.; Dittmer, D. S. eds. Biology data book. 2nd ed. Vol. 3. Bethesda: Federation of American Societies for Experimental Biology; 1974: 1854.Google Scholar
Holley, R. W.; Kiernan, J. A. “Contact inhibition” of cell division in 3T3 cells. Proc. Natl. Acad. Sci. USA 60: 300–304; 1968.PubMedCrossRefGoogle Scholar
Rubin, H. Growth regulation in culture of chick embryo fibroblasts. Wolstenholme, G. E. W.; Knight, J. eds. Growth control in cell cultures (Ciba Foundation Symposium) London: Churchill Livingston; 1971: 127–145.Google Scholar
Leffert, H. L.; Weinstein, D. B. Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration. J. Cell Biol. 70: 20–32; 1976.PubMedCrossRefGoogle Scholar
Onda, H.; Yoshikawa, J. Studies on the regulatory mechanisms of cell division.α-1-Acid glycoprotein as a hepatic-specific mitosis inhibitory protein in regenerating rat liver. Gann 66: 227–235; 1974.Google Scholar
Sikas, G.; Cook, R. T. Isolation of a low molecular weight inhibitor of [3H]TdR incorporation into hepatic DNA. Exp. Cell Res. 102: 422–425; 1976.CrossRefGoogle Scholar
Zetter, B. R.; Antoniades, H. N. Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin. J. Supramol. Struct. 11: 361–370; 1979.PubMedCrossRefGoogle Scholar