Cell Biology and Toxicology

, Volume 22, Issue 3, pp 213–219 | Cite as

Selective cytotoxicity of recombinant STXA1-GM-CSF protein in hematopoetic cancer cells

  • M. Habibi Roudkenar
  • A. Jafari
  • M. Oloomi
  • S. Bouzari


Chimeric proteins are composed of a cell-targeting moiety and a cell-killing moiety. In this study, a chimeric protein, STXA1-GM-CSF, composed of catalytic domain of Shiga toxin (A1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed and expressed in E. coli. Cytotoxicity, receptor blocking, and neutralization experiments revealed that the chimeric protein induced cytotoxic effect on different cell lines. This effect was found to be specific, due to the presence of the killing moiety (A1), which exerts its effect through a specific GM-CSF-targeting domain, by binding to its receptor present on those cell lines. These initial investigations indicate that the chimeric protein was functional; further analyses are required for its application.


shiga-toxin hGM-CSF cancer cytotoxicity chimeric protein 



acute myeloid leukemia


(human) granulocyte-macrophage colony-stimulating factor


3-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyletrazolium bromide


ribosome-inactivating protein


sodium docecyl sulfate–polyacrylamide gel electrophoresis


Shiga-like toxin


Shiga toxin


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Al-Jaufy AY, Haddad JA, King SR, McPhee RA, Jackson MP. Cytotoxicity of a Shiga toxin A subunit-CD4 fusion protein to human immunodeficiency virus-infected cells. Infect Immun. 1994;62:956–60.PubMedGoogle Scholar
  2. Bendle AE, Shao Y, Davies SM, et al. A recombinant fusion toxin targeted to the granulocyte-macrophage colony-stimulating factor receptor. Leuk Lymphoma. 1997;25:257–70.Google Scholar
  3. Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. Functional isolation and characterisation of human hemopoietic stem cells. Science. 1995;267:104–8.PubMedGoogle Scholar
  4. Budel LM, Touw IP, Delwel R, Clark SC, Lowenberg B. Interlekin-3 and granulocyte-monocyte colony-stimulating factor receptors on human acute myelocytic leukemia cells and relationship to the proliferative response. Blood. 1989;74:565–71.PubMedGoogle Scholar
  5. Chiba S, Shibuya K, Piao Y, et al. Identification and cellular distribution of distinct proteins forming human GM-CSF receptor. Cell Regul. 1990;1:327–35.PubMedGoogle Scholar
  6. Frankel AE, Hall PD, Burbage C, et al. Modulation of the apoptotic response of human myeloid leukemia cells of a diphtheria toxin granulocyte-macrophage colony-stimulating factor fusion protein. Blood. 1997;90:3654–61.PubMedGoogle Scholar
  7. Frankel AE, Powell BL, Hall PD, Case LD, Kreitman JR. Phase I trial of a novel diphtheria toxin/GMCSF fusion protein (DT388GCSF) for refractory or relapsed acute myeloid leukaemia (AML). Clin Cancer Res. 2002a;8:1004–13.Google Scholar
  8. Frankel AE. Receptor upregulation enhances cell surface receptor targeted therapies. Leuk Res. 2002b;26:1069–71.CrossRefGoogle Scholar
  9. Habibi Roudkenar M, Bouzari S, Oloomi M, Jafari A, Shahrokhi N, Shokrgozar MA. Expression of a chimeric protein containing the catalytic domain of Shiga-like toxin and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in Escherichia coli and its recognition by reciprocal antibodies. Iranian Biomed J. 2005;9:143–8.Google Scholar
  10. Kojio S, Zhang HM, Ohumara M, Gondaira F, Kobayashi N, Yamamoto T. Caspase-3 inactivation and apoptosis induction coupled with the retrograde transport of Shiga toxin: inhibition by brefeldin A. FEMS Immunol Med Microbiol. 2000;29;275–81.PubMedGoogle Scholar
  11. Lyne PD, Bamborough TL, Duncan D, Richards WG. Molecular modelling of the GM-CSF and IL-3 receptor complexes. Protein Sci. 1995;4:2223–33.PubMedCrossRefGoogle Scholar
  12. Narayanan S, Suredranath K, Bora N, Surolia A, Karande AA. Ribosome inactivating protein and apoptosis. FEBS Lett. 2005;579:1324–31.CrossRefPubMedGoogle Scholar
  13. Sakamaki K, Miyajima I, Kitamura T, Miyajima A. Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosporylation. EMBO J. 1992;11:3541–9.PubMedGoogle Scholar
  14. Skinner LM, Jackson MP. Inhibition of prokaryotic translation by the Shiga toxin enzymatic subunit. Microb Pathog. 1998;24:117–22.CrossRefPubMedGoogle Scholar
  15. Stirpe F. Ribosome-inactivating proteins. Toxicon. 2004;44:371–83.CrossRefPubMedGoogle Scholar
  16. Tada H, Shiho O, Kuroshima K, Koyama M, Tsukamoto K. An improved colorimetric assay for interleukin 2. J Immunol Methods. 1986;9:157–65.CrossRefGoogle Scholar
  17. Terpstra W, Rozemuller H, Breems DA, et al. Diphtheria toxin fused to granulocyte-macrophage stimulating-factor eliminates acute myeloid leukaemia cells with the potential to initiate leukaemia in immunodeficient mice, but spares normal hemopoietic stem cells. Blood. 1997;90:3735–42.PubMedGoogle Scholar
  18. Williams MD, Rostovtsev A, Narla RK, Uckun FM. Production of recombinant DTct GMSF in a Baculovirus expression vector system for biotherapy of GMCSF-receptor sensitive hematologic malignancies. Protein Expr Purif. 1998;13:210–21.CrossRefPubMedGoogle Scholar
  19. Wognum AW, Westerman Y, Visser TP, Wagemaker G. Distribution of receptors for granulocyte-macrophage colony-simulating factor on immature CD34+ bone marrow cells, differentiating monomyeloid progenitors, and mature blood cell subsets. Blood. 1994;84:764–74.PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • M. Habibi Roudkenar
    • 1
  • A. Jafari
    • 1
  • M. Oloomi
    • 1
  • S. Bouzari
    • 1
    • 2
  1. 1.Molecular Biology UnitPasteur Institute of IranTehranIran
  2. 2.Molecular Biology UnitPasteur Institute of IranTehranIran

Personalised recommendations