Ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells.
Treatment failure of patients with acute myelogenous leukemia (AML) is frequently due to the development of multidrug resistance phenotype blasts. We have expressed a fusion protein consisting of human granulocyte-macrophage colony stimulating factor (GMCSF) fused to the N-terminus of a lectin-deficient ricin toxin B chain (RTB) in Spodoptera frugiperda insect cells. The fusion protein was purified by immunoaffinity chromatography and reassociated with chemically deglycosylated ricin toxin A chain (RTA). The resulting fusion toxin was found to react with antibodies to GMCSF, RTB and RTA and had the predicted molecular mass of 80 kDa. GMCSF-ricin bound poorly to asialofetuin (Kd = 10(6) M-1) and receptor negative cells indicating loss of lectin activity, but bound strongly to GMCSF receptor positive HL60 cells. Ligand displacement assays showed fusion toxin affinity 2.6-fold less than native GMCSF. Selective inhibition of protein synthesis was observed on receptor positive cells. Induction of apoptosis was also observed on receptor positive cells. Cells expressing multidrug resistance gene products (P-gp, Bcl2 and BclXL) were also sensitive to fusion toxin. These results suggest that GMCSF-ricin deserves further preclinical development.
Burbage
C
C
Department of Medicine, Medical University of South Carolina, Charleston, USA.
Tagge
E P
EP
Harris
B
B
Hall
P
P
Fu
T
T
Willingham
M C
MC
Frankel
A E
AE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D001212:Asialoglycoproteins / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D018432:Drug Resistance, Multiple; D060748:Fetuins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute; D008970:Molecular Weight; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000096:biosynthesis / Q000378:metabolism / Q000633:toxicity*; D018411:Spodoptera; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000378:metabolism; D051020:bcl-X Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies; D001212:Asialoglycoproteins; C494811:BCL2L1 protein, human; D060748:Fetuins; D019253:Proto-Oncogene Proteins c-bcl-2; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D051020:bcl-X Protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D012276:Ricin