Jacqueline Thorburn1, Arthur E Frankel, Andrew Thorburn. 1. Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.
Abstract
PURPOSE: 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. EXPERIMENTAL DESIGN AND 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.
PURPOSE: 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. EXPERIMENTAL DESIGN AND 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.
Authors: Adva Cohen-Fredarow; Ari Tadmor; Tal Raz; Naama Meterani; Yoseph Addadi; Nava Nevo; Inna Solomonov; Irit Sagi; Gil Mor; Michal Neeman; Nava Dekel Journal: Mol Endocrinol Date: 2014-05-13