Apoptosis by leukemia cell-targeted diphtheria toxin occurs via receptor-independent activation of Fas-associated death domain protein.
OBJECTIVE
We examined the mechanism of action of a targeted fusion toxin consisting of diphtheria toxin fused to granulocyte macrophage colony stimulating factor (GMCSF) (DT(388)-GMCSF), which was designed to selectively kill acute myeloid leukemia cells.


RESULTS
U937 cells treated with DT(388)-GMCSF underwent apoptosis as shown by chromatin degradation and cellular and nuclear fragmentation. This apoptosis was prevented by a general caspase inhibitor. DT(388)-GMCSF treatment resulted in activation of the initiator caspases 8 and 9 and effector caspases. A selective caspase 8 inhibitor prevented activation of caspase 9, whereas a selective caspase 9 inhibitor did not prevent activation of caspase 8, indicating that caspase 8 activation is the proximal event in DT(388)-GMCSF-induced apoptosis. Caspase 8 was activated through a Fas-associated death domain protein (FADD)-dependent mechanism as demonstrated by inhibition of DT(388)-GMCSF-induced apoptosis on expression of a dominant negative FADD molecule. However, unlike most FADD-dependent apoptosis, this pathway may not involve death receptors, including Fas, tumor necrosis factor receptor 1, or tumor necrosis factor-related apoptosis-inducing ligand receptors, because inhibitors of the receptors did not prevent DT(388)-GMCSF-induced apoptosis.


CONCLUSIONS
These data indicate that targeted toxins induce apoptosis by activating components of the death receptor pathway in a receptor-independent manner.
Thorburn
Jacqueline
J
Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.
Frankel
Arthur E
AE
Thorburn
Andrew
A
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000187:drug effects / Q000502:physiology*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D004167:Diphtheria Toxin / Q000633:toxicity*; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D020298:U937 Cells
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004167:Diphtheria Toxin; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases