title: Modulation of the apoptotic response of human myeloid leukemia cells to a diphtheria toxin granulocyte-macrophage colony-stimulating factor fusion protein.
abstract: It has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor-bearing cells. We now report that DT388-GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF-receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I-GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388-GM-CSF at 4 x 10(-9) mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 x 10(-9) mol/L to 3 x 10(-13) mol/L. GM-CSF-receptor occupancy at 0.7 nmol/L GM-CSF-ligand concentration correlated with the protein synthesis IC50. Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL, in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388-GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.
authors.affiliation: Department of Medicine, Medical University of South Carolina, Charleston, SC 29401, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Frankel
authors.forename: P D
authors.initials: PD
authors.lastname: Hall
authors.forename: C
authors.initials: C
authors.lastname: Burbage
authors.forename: J
authors.initials: J
authors.lastname: Vesely
authors.forename: M
authors.initials: M
authors.lastname: Willingham
authors.forename: K
authors.initials: K
authors.lastname: Bhalla
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D004167:Diphtheria Toxin / Q000235:genetics / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000473:pathology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D004167:Diphtheria Toxin; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor