Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT388-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF

DT388-GM-CSF, a targeted fusion toxin constructed by conjugation of human g ranulocyte-macrophagecolony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I tria ls for patients with resistant acute myelold leukemia. HL-60/VCR, a multidr ug-resistant human myelold leukemia cell line, and wild-type HL-60 cells we re used to study the impact of DT388-GM-CSF on metabolism of ceramide, a mo dulator of apoptosis. After 48 hours with DT388-GM-CSF (10 nM), ceramide le vels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-C SF alone was without influence. Similar results were obtained in HL-60 cell s. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT388-GMCSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetin e failed to elevate ceramide levels or induce apoptosis at concentrations t hat inhibited protein synthesis by 50%. Exposure to C-6-ceramide inhibited protein synthesis (EC50 similar to5 muM) and decreased viability (EC50 simi lar to6 muM). Sphingomyelinase treatment depleted sphingomyelin by about 10 %, while increasing ceramide levels and inhibiting protein synthesis. Dipht heria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT388-GM-C SF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate t rigger ceramide formation that contributes to apoptosis in human leukemia c ells through caspase activation and inhibition of protein synthesis.