MODULATION OF THE APOPTOTIC RESPONSE OF HUMAN MYELOID-LEUKEMIA CELLS TO A DIPHTHERIA-TOXIN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTORFUSION PROTEIN

It has previously been shown that human granulocyte-macrophage colony- stimulating factor (GM-CSF) can he fused to a truncated diphtheria tox in (DT) to produce a recombinant fusion toxin that kills GM-CSF recept or-bearing cells. We now report that DT388-GM-CSF induces apoptosis an d inhibition of colony formation in semisolid medium in receptor posit ive cells, and that the induction of apoptosis correlates with GM-CSF- receptor occupancy at low ligand concentrations. Also, the induction o f apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic protei ns (Bcl2 and Bc1X(L)). Nine myeloid leukemia cells lines and four nonm yeloid leukemia cell lines were incubated with 0.7 nmol/L of I-125-GM- CSF in the presence or absence of excess cold GM-CSF and bound label m easured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombi nant fusion toxin for 48 hours and incorporation of H-3-leucine (prote in synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. D T388-CM-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 IC(50)s 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 GMCSF-liga nd concentration correlated with the protein synthesis IC50. Similarly , the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclX(L), 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 sens itivities will depend on the presence of at least 100 high affinity GM -CSF receptors/cell and the absence of overexpressed antiapopiotic pro teins. (C) 1997 by The American Society oi Hematology.