Advertisement

Virchows Archiv B

, 53:44 | Cite as

Expression and regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesions in rats

  • Gabriele Seelmann-Eggebert
  • Doris Mayer
  • Dieter Mecke
  • Peter Bannasch
Article

Summary

Glycogen phosphorylase (PHO) was demonstrated immunocytochemically and enzyme histochemically in cryostat sections of liver from rats treated for 7 weeks with N-nitrosomorpholine (120 mg/1 and 200 mg/1 drinking water) and from untreated controls. The activity and distribution of PHO protein were studied in normal liver and correlated with morphologically defined stages of hepatic tumour development. In normal liver the amount of enzyme protein, as visualized by the immunoperoxidase method using antibodies against phosphorylase, showed some heterogeneity within the liver lobule. The intralobular and intracellular distribution of PHO protein was the same as that of glycogen, namely coarse and granular in periportal hepatocytes and very fine in perivenular cells. In glycogen storage foci the amount of PHO protein was increased. In contrast, PHO activity was generally decreased. In other preneoplastic and neoplastic lesions such as mixed cell foci, neoplastic nodules and hepatocellular carcinomas, PHO protein was increased in all glycogen-loaded cells while PHO activity was reduced. In all glycogen-poor and basophilic cells, both PHO protein and PHO activity were decreased or absent. It was concluded that the decrease in PHO activity in glycogen storage foci was not the direct consequence of genetic changes leading to a loss in enzyme protein but was due to a defect in the cascade of phosphorylation processes resulting in active PHO. Alteration in gene expression leading to a loss of PHO protein was a late event in the process of hepatocarcinogenesis.

Key words

Glycogen phosphorylase Immunocytochemistry Enzyme histochemistry Hepatocarcinogenesis N-nitrosomorpholine 

Abbreviations

NNM

N-nitrosomorpholine

DAB

diaminobenzidine

PBS

phosphate-buffered saline

PHO

glycogen phosphorylase

PAP

peroxidase-anti peroxidase

SDS

dodecylsulfate. Na-salt

References

  1. Appleman MM, Krebs EG, Fischer EH (1966) Purification and properties of inactive liver phosphorylase. Biochemistry 5:2101–2107PubMedCrossRefGoogle Scholar
  2. Bannasch P (1986) Preneoplastic lesions as end points in carci- nogenicity testing. I. Hepatic preneoplasia. Carcinogenesis 7:689–695PubMedCrossRefGoogle Scholar
  3. Bannasch P, Mayer D, Hacker HJ (1980) Hepatocellular glyco- genosis and hepatocarcinogenesis. Biochim Biophys Acta 605:217–245PubMedGoogle Scholar
  4. Bannasch P, Hacker HJ, Klimek F, Mayer D (1984) Hepatocel- lular glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis. Adv Enzyme Regul 22:97–121PubMedCrossRefGoogle Scholar
  5. Bannasch P, Zerban H, Hacker HJ (1985) Foci of altered hepa- tocytes, rat. In: Jones TC, Mohr U, Hunt RD (eds) Mono- graphs on pathology of laboratory animals, digestive sys- tem. Springer, Berlin Heidelberg New York Tokyo, pp 10–30Google Scholar
  6. Cohen P (1980) Molecular aspects of cellular regulation, vol. 1. Recently discovered systems of enzyme regulation by re- versible phosphorylation. Elsevier/North HollandGoogle Scholar
  7. Ehemann V, Mayer D, Hacker HJ, Bannasch P (1986) Loss of adenylate cyclase activity in preneoplastic and neoplastic lesions induced in rat liver by N-nitrosomorpholine. Carci- nogenesis 7:567–573Google Scholar
  8. Friemel H (1980) Immunologische Arbeitsmethoden. Fischer, StuttgartGoogle Scholar
  9. Gilboe DP, Larson KL, Nuttall FQ (1972) Radioactive method for the assay of glycogen phosphorylase. Anal Biochem 47:20–27PubMedCrossRefGoogle Scholar
  10. Goris J, Waelkens E, Camps T, Merlevede W (1984) Regulation of protein phosphatase activity by the deinhibitor protein. Adv Enzyme Regul 22:467–484PubMedCrossRefGoogle Scholar
  11. Hacker HJ (1978) Histochemical demonstration of glycogen phosphorylase (EC 2.4.1.1) through the use of semiperme- able membranes. Histochemistry 58:289–296PubMedCrossRefGoogle Scholar
  12. Hacker HJ, Moore MA, Mayer D, Bannasch P (1982) Correla- tive histochemistry of some enzymes of carbohydrate metab- olism in preneoplastic and neoplastic lesions in the rat liver. Carcinogenesis 3:1265–1272PubMedCrossRefGoogle Scholar
  13. Hers HG (1976) Control of glycogen metabolism in the liver. Ann Rev Biochem 45:167–189PubMedCrossRefGoogle Scholar
  14. Hwang P, Stern M, Fletterick RJ (1984) Purification and crys- tallization of bovine liver phosphorylase. Biochim Biophys Acta 791:252–258PubMedGoogle Scholar
  15. Klimek F, Mayer D, Bannasch P (1984) Biochemical micro- analysis of glycogen content and glucose-6-phosphate dehy- drogenase activity in focal lesions of the rat liver induced by N-nitrosomorpholine. Carcinogenesis 5:265–268PubMedCrossRefGoogle Scholar
  16. Livanova NB, Eronina TB, Silonova GV, Ramensky EV (1976) ω-Aminohexyl-Sepharose in purification of liver glycogen phosphorylase b. FEBS-Lett 69:95–98PubMedCrossRefGoogle Scholar
  17. Moore MA, Kitagawa T (1986) Hepatocarcinogenesis in the rat; the effect of the promoters and carcinogens in vivo and in vitro. Int Rev Cytol 101:125–173PubMedCrossRefGoogle Scholar
  18. Polak JM, Van Noorden S (1986) Immunocytochemistry, mod- ern methods and applications. 2nd edition. John Wright & Sons Ltd, BristolGoogle Scholar
  19. Sato K, Morris HP, Weinhouse S (1973) Characterization of glycogen synthetases and phosphorylases in transplantable rat hepatomas. Cancer Res 33:724–733PubMedGoogle Scholar
  20. Sato K, Weinhouse S (1973) Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relation to a fetal form. Arch Biochem Biophys 159:151–159PubMedCrossRefGoogle Scholar
  21. Sato K, Satoh K, Sato T, Imai F, Morris HP (1976) Isoenzyme patterns of glycogen phosphorylase in rat tissues and trans- plantable hepatomas. Cancer Res 36:487–495PubMedGoogle Scholar
  22. Stalmans W (1976) The role of liver in the homeostasis of blood glucose. Curr Top Cell Regul 11:51–97PubMedGoogle Scholar
  23. Stalmans W, Gevers G (1981) The catalytic activity of phospho- rylase b in the liver with a note on the assay in the glycoge- nolytic direction. Biochem J 200:327–336PubMedGoogle Scholar
  24. Vandenheede JR, De Wulf H, Merlevede W (1979) Liver phos- phorylase b kinase. Cyclic AMP-mediated activation and properties of the partially purified rat-liver enzyme. Eur J Biochem 101:51–58PubMedCrossRefGoogle Scholar
  25. Vesselinovitch SD, Hacker HJ, Bannasch P (1985) Histochemi- cal characterization of focal hepatic lesions induced by sin- gle diethylnitrosamine treatment in infant mice. Cancer Res 45:2774–2780PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • Gabriele Seelmann-Eggebert
    • 1
    • 2
  • Doris Mayer
    • 1
    • 2
  • Dieter Mecke
    • 3
  • Peter Bannasch
    • 1
    • 2
  1. 1.Institut für Experimentelle PathologieDeutsches KrebsforschungszentrumHeidelberg
  2. 2.Heidelberg
  3. 3.Physiologisch-Chemisches Institut der UniversitätTübingenFederal Republic of Germany

Personalised recommendations