Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures

  • 31 Accesses

  • 8 Citations


The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogeneous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus non-hypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.

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


  1. Aden DP, Fogel A, Plotkin S, Damjanov I, Knowles BB (1979) Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282:615–616

  2. Bachmann S, Le Hir M, Eckardt K-U (1993) Co-localization of erythropoietin mRNA and ecto-5′-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin. J Histochem Cytochem 41:335–341

  3. Bouma ME, Rogier E, Verthier N, Labarre C, Feldmann G (1989) Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: morphology and immunocytochemical studies of hepatic-secreted proteins. In Vitro Cell Dev Biol 25:267–275

  4. Egrie JC, Cotes PM, Lane J, Gaines Das RE, Tam RC (1987) Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin as tracer and immunogen. J Immunol Methods 99:235–241

  5. Fandrey J, Bunn HF (1993) In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction. Blood 81:617–623

  6. Fandrey J, Seydel FP, Siegers C-P, Jelkmann W (1990) Role of cytochrome P450 in the control of the production of erythropoietin. Life Sci 47:127–134

  7. Fisher JW, Taylor G, Porteous DD (1965) Localization of erythropoietin in glomeruli of sheep kidney by fluorescent antibody technique. Nature 205:611–612

  8. Fukushima Y, Yanagisawa M, Nakamoto Y, Miura AB (1990) Localization of renal erythropoietin and the role of Ia antigen expression in hypoxic rat glomeruli. Tohoku J Exp Med 160:129–140

  9. Goldberg MA, Glass GA, Cunningham JM, Bunn HF (1987) The regulated expression of erythropoietin by two human hepatoma cell lines. Proc Natl Acad Sci USA 84:7972–7976

  10. Gruber DF, Zucali JR, Wleklinski J, Larussa V, Mirand EA (1977) Temporal transition in the site of rat erythropoietin production. Exp Hematol 5:399–407

  11. Hsu SM, Raine L, Fanger H (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580

  12. Imai N, Kawamura A, Higuchi M, Oh-eda M, Orita T, Kawaguchi T, Ochi N (1990) Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin. J Biochem 107:352–359

  13. Jelkmann W (1992) Erythropoietin: structure, control of production, and function. Physiol Rev 72:449–489

  14. Jelkmann W, Bauer C (1981) Demonstration of high levels of erythropoietin in rat kidneys following hypoxic hypoxia. Pflügers Arch 392:34–39

  15. Jelkmann W, Pagel H, Wolff M, Fandrey J (1992) Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys. Life Sci 50:301–308

  16. Knowles BB, Howe CC, Aden DP (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499

  17. Koury (1989) Discussion comment. Contrib Nephrol 76:22

  18. Koury ST, Bondurant MC, Koury MJ (1988) Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood 71:524–527

  19. Koury ST, Bondurant MC, Koury MJ, Semenza GL (1991) Localization of cells producing erythropoietin in murine liver by in situ hybridization. Blood 77:2497–2503

  20. Krantz SB (1991) Erythropoietin. Blood 77:419–434

  21. Kurtz A, Eckhardt K-U, Neumann R, Kaissling B, Le Hir M, Bauer C (1989) Site of erythropoietin formation. Contrib Nephrol 76:14–23

  22. Lacombe C, Da Silva J-L, Bruneval P, Fournier J-G, Wendling F, Casadevall N, Camilleri J-P, Bariety J, Varet B, Tambourin P (1988a) Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest 81:620–623

  23. Lacombe C, Da Silva J-L, Bruneval P, Camilleri J-P, Bariety J, Tambourin P, Varet B (1988b) Identification of tissues and cells producing erythropoietin in the anemic mouse. Contrib Nephrol 66:17–24

  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685

  25. Lane EB (1982) Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J Cell Biol 92:665–673

  26. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

  27. Maxwell AP, Lappin TRJ, Johnston CF, Bridges JM, McGeown MG (1990) Erythropoietin production in kidney tubular cells. Br J Haematol 74:535–539

  28. Mori S, Saito T, Morishita Y, Saito K, Urabe A, Wakabayashi T, Takaku F (1985) Glomerular epithelium as the main locus of erythropoietin in human kidney. Jpn J Exp Med 55:69–70

  29. Recny MA, Scoble HA, Kim Y (1987) Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. J Biol Chem 262:17156–17163

  30. Schooley JC, Mahlmann LJ (1972) Evidence for the de novo synthesis of erythropoietin in hypoxic rats. Blood 40:662–670

  31. Schuster SJ, Koury ST, Bohrer M, Salceda S, Caro J (1992) Cellular sites of extrarenal and renal erythropoietin production in anaemic rats. Br J Haematol 81:153–159

  32. Sternberger LA (1979) Immunocytochemistry, 2nd edn., John Wiley and Sons, New York

  33. Suzuki T, Sasaki R (1990) Immunocytochemical demonstration of erythropoietin immunoreactivity in peritubular endothelial cells of the anemic mouse kidney. Arch Histol Cytol 53:121–124

  34. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

  35. Ueno M, Seferynska I, Beckman B, Brookins J, Nakashima J, Fisher JW (1989) Enhanced erythropoietin secretion in hepatoblastoma cells in response to hypoxia. Am J Physiol 257:C743-C749

  36. Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143

  37. Wolff M, Fandrey J, Jelkmann W (1993) Microelectrode measurements of pericellular PO2 in erythropoietin-producing human hepatoma cell cultures. Am J Physiol (in press)

Download references

Author information

Correspondence to W. Jelkmann.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Herkens, C., Wolff, M., Fandrey, J. et al. Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures. Histochemistry 100, 303–309 (1993). https://doi.org/10.1007/BF00270051

Download citation


  • Erythropoietin
  • Human Hepatoma
  • Hepatoma Cell Line
  • Cell Line HepG2
  • Recombinant Human Erythropoietin