Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1991 Sep;88(3):789–797. doi: 10.1172/JCI115378

Autocrine stimulation by erythropoietin and autonomous growth of human erythroid leukemic cells in vitro.

M T Mitjavila 1, J P Le Couedic 1, N Casadevall 1, S Navarro 1, J L Villeval 1, A Dubart 1, W Vainchenker 1
PMCID: PMC295463  PMID: 1653276

Abstract

Autonomous colony formation is a frequent event in erythroleukemia. In 13 cases of early erythroid leukemias, we investigated whether erythropoietin (Epo) autocrine stimulation was responsible for the growth factor autonomy. Epo transcripts were detected by Northern blotting in cells from one patient. These cells also expressed an Epo receptor (1,000 receptors per cell) with a 420-pM affinity and Epo was detected in the supernatant of cultured cells. In 8 of the 13 cases, Epo transcripts were revealed by the polymerase chain reaction ranging from 0.5 to 500 copies per cell. In situ hybridization proved that these Epo transcripts were present in the blast cells. No Epo gene abnormalities were detected by Southern blotting. In two cases, leukemic cells were grown in the presence of Epo-neutralizing antibodies or Epo antisense oligomers. In one case, the antibody significantly reduced autonomous growth. In contrast, the antibody had no effect in the second case in which blast cells transcribed the Epo gene at a low level. However, Epo antisense oligomers partially inhibited autonomous growth. This inhibition was reversed by addition of exogenous Epo. Overall, these results suggest that an extracellular or intracellular autocrine Epo stimulation occurs in some cases of erythroid malignancies.

Full text

PDF
794

Images in this article

792Image
on p.792
792Image
on p.792
792Image
on p.792
793Image
on p.793
793Image
on p.793
793Image
on p.793
793Image
on p.793
793Image
on p.793
793Image
on p.793
794Image
on p.794

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamson J. W., Fialkow P. J., Murphy S., Prchal J. F., Steinmann L. Polycythemia vera: stem-cell and probable clonal origin of the disease. N Engl J Med. 1976 Oct 21;295(17):913–916. doi: 10.1056/NEJM197610212951702. [DOI] [PubMed] [Google Scholar]
  2. Anderson W. F., Beckman B., Beltran G., Fisher J. W., Stuckey W. J. Erythropoietin-independent erythroid colony formation in patients with erythroleukaemia (M6) and related disorders. Br J Haematol. 1982 Oct;52(2):311–317. doi: 10.1111/j.1365-2141.1982.tb03894.x. [DOI] [PubMed] [Google Scholar]
  3. Andersson L. C., Gahmberg C. G., Teerenhovi L., Vuopio P. Glycophorin A as a cell surface marker of early erythroid differentiation in acute leukemia. Int J Cancer. 1979 Dec 15;24(6):717–720. doi: 10.1002/ijc.2910240603. [DOI] [PubMed] [Google Scholar]
  4. Andrejauskas E., Moroni C. Reversible abrogation of IL-3 dependence by an inducible H-ras oncogene. EMBO J. 1989 Sep;8(9):2575–2581. doi: 10.1002/j.1460-2075.1989.tb08396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A., Gralnick H. R., Sultan C. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976 Aug;33(4):451–458. doi: 10.1111/j.1365-2141.1976.tb03563.x. [DOI] [PubMed] [Google Scholar]
  6. Bot F. J., Schipper P., Broeders L., Delwel R., Kaushansky K., Löwenberg B. Interleukin-1 alpha also induces granulocyte-macrophage colony-stimulating factor in immature normal bone marrow cells. Blood. 1990 Jul 15;76(2):307–311. [PubMed] [Google Scholar]
  7. Breton-Gorius J., Villeval J. L., Mitjavila M. T., Vinci G., Guichard J., Rochant H., Flandrin G., Vainchenker W. Ultrastructural and cytochemical characterization of blasts from early erythroblastic leukemias. Leukemia. 1987 Mar;1(3):173–181. [PubMed] [Google Scholar]
  8. Caracciolo D., Venturelli D., Valtieri M., Peschle C., Gewirtz A. M., Calabretta B. Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis. J Clin Invest. 1990 Jan;85(1):55–61. doi: 10.1172/JCI114433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Casadevall N., Vainchenker W., Lacombe C., Vinci G., Chapman J., Breton-Gorius J., Varet B. Erythroid progenitors in polycythemia vera: demonstration of their hypersensitivity to erythropoietin using serum free cultures. Blood. 1982 Feb;59(2):447–451. [PubMed] [Google Scholar]
  10. Chang J. M., Metcalf D., Lang R. A., Gonda T. J., Johnson G. R. Nonneoplastic hematopoietic myeloproliferative syndrome induced by dysregulated multi-CSF (IL-3) expression. Blood. 1989 May 1;73(6):1487–1497. [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Choppin J., Lacombe C., Casadevall N., Muller O., Tambourin P., Varet B. Characterization of erythropoietin produced by IW32 murine erythroleukemia cells. Blood. 1984 Aug;64(2):341–347. [PubMed] [Google Scholar]
  13. Chung S. W., Wolff L., Ruscetti S. K. Transmembrane domain of the envelope gene of a polycythemia-inducing retrovirus determines erythropoietin-independent growth. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7957–7960. doi: 10.1073/pnas.86.20.7957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cox K. H., DeLeon D. V., Angerer L. M., Angerer R. C. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol. 1984 Feb;101(2):485–502. doi: 10.1016/0012-1606(84)90162-3. [DOI] [PubMed] [Google Scholar]
  15. Dunbar C. E., Browder T. M., Abrams J. S., Nienhuis A. W. COOH-terminal-modified interleukin-3 is retained intracellularly and stimulates autocrine growth. Science. 1989 Sep 29;245(4925):1493–1496. doi: 10.1126/science.2789432. [DOI] [PubMed] [Google Scholar]
  16. Eaves C. J., Eaves A. C. Erythropoietin (Ep) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood. 1978 Dec;52(6):1196–1210. [PubMed] [Google Scholar]
  17. Egrie J. C., Cotes P. M., Lane J., Gaines Das R. E., Tam R. C. Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin as tracer and immunogen. J Immunol Methods. 1987 May 20;99(2):235–241. doi: 10.1016/0022-1759(87)90133-5. [DOI] [PubMed] [Google Scholar]
  18. Eridani S., Dudley J. M., Sawyer B. M., Pearson T. C. Erythropoietic colonies in a serum-free system: results in primary proliferative polycythaemia and thrombocythaemia. Br J Haematol. 1987 Dec;67(4):387–391. doi: 10.1111/j.1365-2141.1987.tb06158.x. [DOI] [PubMed] [Google Scholar]
  19. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  20. Graf T., Beug H. Role of the v-erbA and v-erbB oncogenes of avian erythroblastosis virus in erythroid cell transformation. Cell. 1983 Aug;34(1):7–9. doi: 10.1016/0092-8674(83)90130-7. [DOI] [PubMed] [Google Scholar]
  21. Greaves M. F., Sieff C., Edwards P. A. Monoclonal antiglycophorin as a probe for erythroleukemias. Blood. 1983 Apr;61(4):645–651. [PubMed] [Google Scholar]
  22. Heard J. M., Roussel M. F., Rettenmier C. W., Sherr C. J. Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene. Cell. 1987 Nov 20;51(4):663–673. doi: 10.1016/0092-8674(87)90135-8. [DOI] [PubMed] [Google Scholar]
  23. Iscove N. N., Guilbert L. J., Weyman C. Complete replacement of serum in primary cultures of erythropoietin-dependent red cell precursors (CFU-E) by albumin, transferrin, iron, unsaturated fatty acid, lecithin and cholesterol. Exp Cell Res. 1980 Mar;126(1):121–126. doi: 10.1016/0014-4827(80)90476-0. [DOI] [PubMed] [Google Scholar]
  24. Jacobs K., Shoemaker C., Rudersdorf R., Neill S. D., Kaufman R. J., Mufson A., Seehra J., Jones S. S., Hewick R., Fritsch E. F. Isolation and characterization of genomic and cDNA clones of human erythropoietin. 1985 Feb 28-Mar 6Nature. 313(6005):806–810. doi: 10.1038/313806a0. [DOI] [PubMed] [Google Scholar]
  25. Johnson G. R., Gonda T. J., Metcalf D., Hariharan I. K., Cory S. A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte-macrophage colony stimulating factor. EMBO J. 1989 Feb;8(2):441–448. doi: 10.1002/j.1460-2075.1989.tb03396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koury S. T., Bondurant M. C., Koury M. J. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood. 1988 Feb;71(2):524–527. [PubMed] [Google Scholar]
  27. Krantz S. B., Goldwasser E. Specific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7574–7578. doi: 10.1073/pnas.81.23.7574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lacombe C., Casadevall N., Varet B. Polycythaemia vera: in vitro studies of circulating erythroid progenitors. Br J Haematol. 1980 Feb;44(2):189–199. doi: 10.1111/j.1365-2141.1980.tb01201.x. [DOI] [PubMed] [Google Scholar]
  29. Lacombe C., Da Silva J. L., Bruneval P., Fournier J. G., Wendling F., Casadevall N., Camilleri J. P., Bariety J., Varet B., Tambourin P. Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest. 1988 Feb;81(2):620–623. doi: 10.1172/JCI113363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laker C., Stocking C., Bergholz U., Hess N., De Lamarter J. F., Ostertag W. Autocrine stimulation after transfer of the granulocyte/macrophage colony-stimulating factor gene and autonomous growth are distinct but interdependent steps in the oncogenic pathway. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8458–8462. doi: 10.1073/pnas.84.23.8458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lang R. A., Metcalf D., Cuthbertson R. A., Lyons I., Stanley E., Kelso A., Kannourakis G., Williamson D. J., Klintworth G. K., Gonda T. J. Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage. Cell. 1987 Nov 20;51(4):675–686. doi: 10.1016/0092-8674(87)90136-x. [DOI] [PubMed] [Google Scholar]
  32. Lang R. A., Metcalf D., Gough N. M., Dunn A. R., Gonda T. J. Expression of a hemopoietic growth factor cDNA in a factor-dependent cell line results in autonomous growth and tumorigenicity. Cell. 1985 Dec;43(2 Pt 1):531–542. doi: 10.1016/0092-8674(85)90182-5. [DOI] [PubMed] [Google Scholar]
  33. Lawrence J. B., Singer R. H. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 1985 Mar 11;13(5):1777–1799. doi: 10.1093/nar/13.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Li J. P., D'Andrea A. D., Lodish H. F., Baltimore D. Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor. Nature. 1990 Feb 22;343(6260):762–764. doi: 10.1038/343762a0. [DOI] [PubMed] [Google Scholar]
  35. Lin F. K., Suggs S., Lin C. H., Browne J. K., Smalling R., Egrie J. C., Chen K. K., Fox G. M., Martin F., Stabinsky Z. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7580–7584. doi: 10.1073/pnas.82.22.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liszka K., Majdic O., Bettelheim P., Knapp W. Glycophorin A expression in malignant hematopoiesis. Am J Hematol. 1983 Nov;15(3):219–226. doi: 10.1002/ajh.2830150303. [DOI] [PubMed] [Google Scholar]
  37. Maxwell A. P., Lappin T. R., Johnston C. F., Bridges J. M., McGeown M. G. Erythropoietin production in kidney tubular cells. Br J Haematol. 1990 Apr;74(4):535–539. doi: 10.1111/j.1365-2141.1990.tb06347.x. [DOI] [PubMed] [Google Scholar]
  38. Mayeux P., Billat C., Jacquot R. The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage. J Biol Chem. 1987 Oct 15;262(29):13985–13990. [PubMed] [Google Scholar]
  39. Mitjavila M. T., Villeval J. L., Cramer P., Henri A., Gasson J., Krystal G., Tulliez M., Berger R., Breton-Gorius J., Vainchenker W. Effects of granulocyte-macrophage colony-stimulating factor and erythropoietin on leukemic erythroid colony formation in human early erythroblastic leukemias. Blood. 1987 Oct;70(4):965–973. [PubMed] [Google Scholar]
  40. Nakazawa M., Mitjavila M. T., Debili N., Casadevall N., Mayeux P., Rouyer-Fessard P., Dubart A., Roméo P. H., Beuzard Y., Kishi K. KU 812: a pluripotent human cell line with spontaneous erythroid terminal maturation. Blood. 1989 May 15;73(7):2003–2013. [PubMed] [Google Scholar]
  41. Navarro S., Debili N., Bernaudin J. F., Vainchenker W., Doly J. Regulation of the expression of IL-6 in human monocytes. J Immunol. 1989 Jun 15;142(12):4339–4345. [PubMed] [Google Scholar]
  42. Prchal J. F., Axelrad A. A. Letter: Bone-marrow responses in polycythemia vera. N Engl J Med. 1974 Jun 13;290(24):1382–1382. doi: 10.1056/nejm197406132902419. [DOI] [PubMed] [Google Scholar]
  43. Rich I. N., Heit W., Kubanek B. Extrarenal erythropoietin production by macrophages. Blood. 1982 Oct;60(4):1007–1018. [PubMed] [Google Scholar]
  44. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  45. Semenza G. L., Traystman M. D., Gearhart J. D., Antonarakis S. E. Polycythemia in transgenic mice expressing the human erythropoietin gene. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2301–2305. doi: 10.1073/pnas.86.7.2301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tambourin P., Casadevall N., Choppin J., Lacombe C., Heard J. M., Fichelson S., Wendling F., Varet B. Production of erythropoietin-like activity by a murine erythroleukemia cell line. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6269–6273. doi: 10.1073/pnas.80.20.6269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Villeval J. L., Cramer P., Lemoine F., Henri A., Bettaieb A., Bernaudin F., Beuzard Y., Berger R., Flandrin G., Breton-Gorius J. Phenotype of early erythroblastic leukemias. Blood. 1986 Nov;68(5):1167–1174. [PubMed] [Google Scholar]
  48. Vuillet-Gaugler M. H., Breton-Gorius J., Vainchenker W., Guichard J., Leroy C., Tchernia G., Coulombel L. Loss of attachment to fibronectin with terminal human erythroid differentiation. Blood. 1990 Feb 15;75(4):865–873. [PubMed] [Google Scholar]
  49. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wong P. M., Chung S. W., Dunbar C. E., Bodine D. M., Ruscetti S., Nienhuis A. W. Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder. Mol Cell Biol. 1989 Feb;9(2):798–808. doi: 10.1128/mcb.9.2.798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ymer S., Tucker W. Q., Sanderson C. J., Hapel A. J., Campbell H. D., Young I. G. Constitutive synthesis of interleukin-3 by leukaemia cell line WEHI-3B is due to retroviral insertion near the gene. Nature. 1985 Sep 19;317(6034):255–258. doi: 10.1038/317255a0. [DOI] [PubMed] [Google Scholar]
  52. Yoshimura A., D'Andrea A. D., Lodish H. F. Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4139–4143. doi: 10.1073/pnas.87.11.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Young D. C., Griffin J. D. Autocrine secretion of GM-CSF in acute myeloblastic leukemia. Blood. 1986 Nov;68(5):1178–1181. [PubMed] [Google Scholar]
  54. Young D. C., Wagner K., Griffin J. D. Constitutive expression of the granulocyte-macrophage colony-stimulating factor gene in acute myeloblastic leukemia. J Clin Invest. 1987 Jan;79(1):100–106. doi: 10.1172/JCI112769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zanjani E. D., Lutton J. D., Hoffman R., Wasserman L. R. Erythroid colony formation by polycythemia vera bone marrow in vitro. Dependence on erythropoietin. J Clin Invest. 1977 May;59(5):841–848. doi: 10.1172/JCI108706. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES