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. 1991 Feb 1;88(3):770–774. doi: 10.1073/pnas.88.3.770

Mutated recombinant human heavy-chain ferritins and myelosuppression in vitro and in vivo: a link between ferritin ferroxidase activity and biological function.

H E Broxmeyer 1, S Cooper 1, S Levi 1, P Arosio 1
PMCID: PMC50895  PMID: 1992468

Abstract

Human heavy-chain (H-) ferritin muteins obtained by oligonucleotide site-directed mutagenesis, together with wild-type recombinant human H- and light-chain (L-) ferritins, were evaluated for in vitro effects on the suppression of human bone marrow myeloid progenitor cells and for in vivo effects on marrow and splenic myelopoiesis in C3H/HeJ mice. The 10 H-ferritin muteins exhibited alterations of various regions of the molecule, including ones exposed on the outer surface, on the inner cavity, and on the hydrophilic and hydrophobic channels and of the four-alpha-helix bundle forming the subunit structure. They were stable and were electrophoretically analogous to wild-type H-ferritin. The muteins showed in vitro and in vivo myelosuppressive activity analogous to wild type, except for mutein 222, which was totally inactive and which lacked ferroxidase activity. Recombinant human L-ferritin, devoid of ferroxidase activity, was also inactive as a suppressor. The results demonstrate that H-ferritin myelosuppressive and ferroxidase activities are linked. One possibility is that ferroxidase activity may interfere with the cellular uptake of transferrin iron that is needed for cell proliferation, an interpretation consistent with the presently described ability of hemin to overcome H-ferritin suppressive effects.

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Selected References

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  1. Arosio P., Cozzi A., Ingrassia R., Levi S., Luzzago A., Ruggeri G., Iacobello C., Santambrogio P., Albertini A. A mutational analysis of the epitopes of recombinant human H-ferritin. Biochim Biophys Acta. 1990 Jun 19;1039(2):197–203. doi: 10.1016/0167-4838(90)90186-j. [DOI] [PubMed] [Google Scholar]
  2. Bhalla K., Cole J., MacLaughlin W., Baker M., Arlin Z., Graham G., Grant S. Deoxycytidine stimulates the in vitro growth of normal CFU-GM and reverses the negative regulatory effects of acidic isoferritin and prostaglandin E1. Blood. 1986 Nov;68(5):1136–1141. [PubMed] [Google Scholar]
  3. Broxmeyer H. E., Bognacki J., Dorner M. H., de Sousa M. Identification of leukemia-associated inhibitory activity as acidic isoferritins. A regulatory role for acidic isoferritins in the production of granulocytes and macrophages. J Exp Med. 1981 Jun 1;153(6):1426–1444. doi: 10.1084/jem.153.6.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broxmeyer H. E., Bognacki J., Ralph P., Dörner M. H., Lu L., Castro-Malaspina H. Monocyte-macrophage-derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells in vitro. Blood. 1982 Sep;60(3):595–607. [PubMed] [Google Scholar]
  5. Broxmeyer H. E., Cooper S., Lu L., Miller M. E., Langefeld C. D., Ralph P. Enhanced stimulation of human bone marrow macrophage colony formation in vitro by recombinant human macrophage colony-stimulating factor in agarose medium and at low oxygen tension. Blood. 1990 Jul 15;76(2):323–329. [PubMed] [Google Scholar]
  6. Broxmeyer H. E., Dupont B. A role for class-II major histocompatibility complex antigens in the regulation of myelopoiesis. Prog Allergy. 1985;36:203–221. [PubMed] [Google Scholar]
  7. Broxmeyer H. E., Gentile P., Cooper S., Lu L., Juliano L., Piacibello W., Meyers P. A., Cavanna F. Functional activities of acidic isoferritins and lactoferrin in vitro and in vivo. Blood Cells. 1984;10(2-3):397–426. [PubMed] [Google Scholar]
  8. Broxmeyer H. E., Lu L., Bicknell D. C., Williams D. E., Cooper S., Levi S., Salfeld J., Arosio P. The influence of purified recombinant human heavy-subunit and light-subunit ferritins on colony formation in vitro by granulocyte-macrophage and erythroid progenitor cells. Blood. 1986 Dec;68(6):1257–1263. [PubMed] [Google Scholar]
  9. Broxmeyer H. E., Piacibello W., Juliano L., Platzer E., Berman E., Rubin B. Y. Gamma interferon induces colony-forming cells of the human monoblast cell line U937 to respond to inhibition by lactoferrin, transferrin, and acidic isoferritins. Exp Hematol. 1986 Jan;14(1):35–43. [PubMed] [Google Scholar]
  10. Broxmeyer H. E., Ralph P., Gilbertson S., Margolis V. B. Induction of leukemia-associated inhibitory activity and bone marrow granulocyte-macrophage progenitor cell alterations during infection with Abelson virus. Cancer Res. 1980 Nov;40(11):3928–3933. [PubMed] [Google Scholar]
  11. Broxmeyer H. E. Relationship of cell-cycle expression of Ia-like antigenic determinants on normal and leukemia human granulocyte-macrophage progenitor cells to regulation in vitro by acidic isoferritins. J Clin Invest. 1982 Mar;69(3):632–642. doi: 10.1172/JCI110490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Broxmeyer H. E., Williams D. E., Geissler K., Hangoc G., Cooper S., Bicknell D. C., Levi S., Arosio P. Suppressive effects in vivo of purified recombinant human H-subunit (acidic) ferritin on murine myelopoiesis. Blood. 1989 Jan;73(1):74–79. [PubMed] [Google Scholar]
  13. Broxmeyer H. E., Williams D. E. The production of myeloid blood cells and their regulation during health and disease. Crit Rev Oncol Hematol. 1988;8(3):173–226. doi: 10.1016/s1040-8428(88)80016-7. [DOI] [PubMed] [Google Scholar]
  14. Covell A. M., Cook J. D. Interaction of acidic isoferritins with human promyelocytic HL60 cells. Br J Haematol. 1988 Aug;69(4):559–563. doi: 10.1111/j.1365-2141.1988.tb02415.x. [DOI] [PubMed] [Google Scholar]
  15. Covell A. M., Einspahr D. E., Skikne B. S., Cook J. D. Specific binding of acidic isoferritins to erythroleukemia K562 cells. J Lab Clin Med. 1987 Dec;110(6):784–790. [PubMed] [Google Scholar]
  16. Cukrová V., Hrkal Z., Koprivová H., Neuwirt J. Identification of leukemia cell-derived inhibitory activity (LIA) in conditioned media from human myeloid leukemic cell line ML-2. Blut. 1986 Jan;52(1):51–58. doi: 10.1007/BF00320142. [DOI] [PubMed] [Google Scholar]
  17. Dezza L., Cazzola M., Bergamaschi G., Stella C. C., Pedrazzoli P., Recalde H. R. Effects of recombinant human H-subunit and L-subunit ferritins on in vitro growth of human granulocyte-monocyte progenitors. Br J Haematol. 1988 Mar;68(3):367–372. doi: 10.1111/j.1365-2141.1988.tb04216.x. [DOI] [PubMed] [Google Scholar]
  18. Dezza L., Cazzola M., Danova M., Carlo-Stella C., Bergamaschi G., Brugnatelli S., Invernizzi R., Mazzini G., Riccardi A., Ascari E. Effects of desferrioxamine on normal and leukemic human hematopoietic cell growth: in vitro and in vivo studies. Leukemia. 1989 Feb;3(2):104–107. [PubMed] [Google Scholar]
  19. Dezza L., Cazzola M., Piacibello W., Arosio P., Levi S., Aglietta M. Effect of acidic and basic isoferritins on in vitro growth of human granulocyte-monocyte progenitors. Blood. 1986 Mar;67(3):789–795. [PubMed] [Google Scholar]
  20. Fargion S., Arosio P., Fracanzani A. L., Cislaghi V., Levi S., Cozzi A., Piperno A., Fiorelli G. Characteristics and expression of binding sites specific for ferritin H-chain on human cell lines. Blood. 1988 Mar;71(3):753–757. [PubMed] [Google Scholar]
  21. Galbraith R. A., Sassa S., Kappas A. Heme binding to murine erythroleukemia cells. Evidence for a heme receptor. J Biol Chem. 1985 Oct 5;260(22):12198–12202. [PubMed] [Google Scholar]
  22. Guimaraes J. E., Berney J. J., Broxmeyer H. E., Hoffbrand A. V., Francis G. E. Acidic isoferritin stimulates differentiation of normal granulomonocytic progenitors. Leukemia. 1988 Jul;2(7):466–471. [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. Johnson C. S., Marcelletti J., Longley C., Furmanski P. Inhibition of normal erythropoiesis in mice with Friend virus induced erythroleukemia. Exp Hematol. 1982 Oct;10(9):743–753. [PubMed] [Google Scholar]
  25. Lawson D. M., Treffry A., Artymiuk P. J., Harrison P. M., Yewdall S. J., Luzzago A., Cesareni G., Levi S., Arosio P. Identification of the ferroxidase centre in ferritin. FEBS Lett. 1989 Aug 28;254(1-2):207–210. doi: 10.1016/0014-5793(89)81040-3. [DOI] [PubMed] [Google Scholar]
  26. Levi S., Luzzago A., Cesareni G., Cozzi A., Franceschinelli F., Albertini A., Arosio P. Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants. J Biol Chem. 1988 Dec 5;263(34):18086–18092. [PubMed] [Google Scholar]
  27. Levi S., Luzzago A., Franceschinelli F., Santambrogio P., Cesareni G., Arosio P. Mutational analysis of the channel and loop sequences of human ferritin H-chain. Biochem J. 1989 Dec 1;264(2):381–388. doi: 10.1042/bj2640381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Levi S., Salfeld J., Franceschinelli F., Cozzi A., Dorner M. H., Arosio P. Expression and structural and functional properties of human ferritin L-chain from Escherichia coli. Biochemistry. 1989 Jun 13;28(12):5179–5184. doi: 10.1021/bi00438a040. [DOI] [PubMed] [Google Scholar]
  29. Lu L., Broxmeyer H. E., Meyers P. A., Moore M. A., Thaler H. T. Association of cell cycle expression of Ia-like antigenic determinations on normal human multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins. Blood. 1983 Feb;61(2):250–256. [PubMed] [Google Scholar]
  30. Lu L., Broxmeyer H. E., Moore M. A., Sheridan A. P., Gentile P. Abnormalities in myelopoietic regulatory interactions with acidic isoferritins and lactoferrin in mice infected with Friend virus complex: association with altered expression of Ia antigens on effector and responding cells. Blood. 1985 Jan;65(1):91–99. [PubMed] [Google Scholar]
  31. Lu L., Welte K., Gabrilove J. L., Hangoc G., Bruno E., Hoffman R., Broxmeyer H. E. Effects of recombinant human tumor necrosis factor alpha, recombinant human gamma-interferon, and prostaglandin E on colony formation of human hematopoietic progenitor cells stimulated by natural human pluripotent colony-stimulating factor, pluripoietin alpha, and recombinant erythropoietin in serum-free cultures. Cancer Res. 1986 Sep;46(9):4357–4361. [PubMed] [Google Scholar]
  32. Luzzago A., Cesareni G. Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli. EMBO J. 1989 Feb;8(2):569–576. doi: 10.1002/j.1460-2075.1989.tb03411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marcelletti J., Furmanski P. A murine model system for the study of the human leukemia-associated inhibitory activity. Blood. 1980 Jul;56(1):134–137. [PubMed] [Google Scholar]
  34. Moore R. N., Joshi J. G., Deana D. G., Pitruzzello F. J., Horohov D. W., Rouse B. T. Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin. J Immunol. 1986 Mar 1;136(5):1605–1611. [PubMed] [Google Scholar]
  35. Nocka K. H., Pelus L. M. Cell cycle specific effects of deferoxamine on human and murine hematopoietic progenitor cells. Cancer Res. 1988 Jul 1;48(13):3571–3575. [PubMed] [Google Scholar]
  36. Núez M. T., Gaete V., Watkins J. A., Glass J. Mobilization of iron from endocytic vesicles. The effects of acidification and reduction. J Biol Chem. 1990 Apr 25;265(12):6688–6692. [PubMed] [Google Scholar]
  37. Pearse B. M. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc Natl Acad Sci U S A. 1982 Jan;79(2):451–455. doi: 10.1073/pnas.79.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pelus L. M. Association between colony forming units-granulocyte macrophage expression of Ia-like (HLA-DR) antigen and control of granulocyte and macrophage production. A new role for prostaglandin E. J Clin Invest. 1982 Sep;70(3):568–578. doi: 10.1172/JCI110649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Seligman P. A. Structure and function of the transferrin receptor. Prog Hematol. 1983;13:131–147. [PubMed] [Google Scholar]
  40. Taetle R. Acidic isoferritins (leukemia-associated inhibitory activity) fail to inhibit blast proliferation in acute myelogenous leukemia. Blood. 1981 Sep;58(3):653–657. [PubMed] [Google Scholar]
  41. Taetle R., Honeysett J. M., Trowbridge I. Effects of anti-transferrin receptor antibodies on growth of normal and malignant myeloid cells. Int J Cancer. 1983 Sep 15;32(3):343–349. doi: 10.1002/ijc.2910320314. [DOI] [PubMed] [Google Scholar]
  42. Taetle R., Rhyner K., Castagnola J., To D., Mendelsohn J. Role of transferrin, Fe, and transferrin receptors in myeloid leukemia cell growth. Studies with an antitransferrin receptor monoclonal antibody. J Clin Invest. 1985 Mar;75(3):1061–1067. doi: 10.1172/JCI111768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taetle R. The role of transferrin receptors in hemopoietic cell growth. Exp Hematol. 1990 May;18(4):360–365. [PubMed] [Google Scholar]
  44. Thorstensen K., Romslo I. Uptake of iron from transferrin by isolated hepatocytes. The effect of cellular energy metabolism on the intracellular distribution of iron and transferrin. Scand J Clin Lab Invest. 1987 Dec;47(8):837–846. [PubMed] [Google Scholar]
  45. Thorstensen K., Romslo I. Uptake of iron from transferrin by isolated rat hepatocytes. A redox-mediated plasma membrane process? J Biol Chem. 1988 Jun 25;263(18):8844–8850. [PubMed] [Google Scholar]
  46. Treffry A., Harrison P. M., Luzzago A., Cesareni G. Recombinant H-chain ferritins: effects of changes in the 3-fold channels. FEBS Lett. 1989 Apr 24;247(2):268–272. doi: 10.1016/0014-5793(89)81350-x. [DOI] [PubMed] [Google Scholar]
  47. Viola L., Biagini R., Barbieri R., Gambari R. Inhibition of hemoglobin accumulation by monoclonal antibodies to the human transferrin receptor is reversed by hemin. Exp Hematol. 1987 Dec;15(11):1145–1152. [PubMed] [Google Scholar]
  48. Williams D. E., Cooper S., Broxmeyer H. E. Effects of hematopoietic suppressor molecules on the in vitro proliferation of purified murine granulocyte-macrophage progenitor cells. Cancer Res. 1988 Mar 15;48(6):1548–1550. [PubMed] [Google Scholar]
  49. Zhan H., Pollack S., Weaver J. Hemolysates reduce iron released from transferrin. Am J Hematol. 1989 Jul;31(3):203–207. doi: 10.1002/ajh.2830310311. [DOI] [PubMed] [Google Scholar]

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