Improved Artificial Death Switches Based on Caspases and FADD

Abstract

A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperproliferative disorders, such as cancer or restenosis. However, most of these approaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial death switches based on chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules function efficiently in both dividing and nondividing cells. In this approach, lipid-permeable, nontoxic CIDs are used to conditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previous reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. Since the maximum efficacy of these artificial death switches requires low basal and high specific activity, we have optimized these death switches for three parameters: (1) extent of oligomerization, (2) spacing between CBDs and target proteins, and (3) intracellular localization. We describe improved conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subnanomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonstrate for the first time that oligomerization of the death effector domain of the Fas-associated protein, FADD, is sufficient to trigger apoptosis, suggesting that the primary function of FADD, like that of Apaf-1, is oligomerization of associated caspases. Finally, we demonstrate that nuclear-targeted caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that the cleavage of nuclear targets is sufficient for apoptosis.

Ablation of microvessels in vivo upon dimerization of iCaspase-9

“…28,29 In contrast, approaches that directly target the cell death machinery might be more effective in inducing endothelial cell apoptosis and disrupting microvessels in vivo. Because the caspase-9 allele used in the present studies acts as a cellular artificial death switch (ADS) which is activated with dimerizer drugs, the construct used here or related caspase-based suicide switches 18,21 could be activated in a controlled manner in tumors.…”

“…Because apoptotic stimuli often engage multiple intracellular pathways, it is unknown whether activation of apical caspases is sufficient to induce endothelial cell apoptosis and disruption of microvessels in vivo. In this report, we expressed iCaspase-9, a conditional caspase-9 molecule that is activated upon drug-induced dimerization, [18][19][20][21] in primary endothelial cells and evaluated the effect of active iCaspase-9 in functional human microvessels engineered in immunodeficient mice.…”

“…3 Briefly, a 2-kb cassette containing HAtagged human F v 2-caspase-9, 21 now called iCaspase-9, was inserted into the HpaI cloning site of the retroviral vector LXSN (gift from AD Miller, Fred Hutchinson Cancer Research Center, Seattle, WA, USA). The iCaspase-9 construct, reverse orientation control, or vector alone was transfected into PA317 amphotropic packaging cells with Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN, USA) to generate recombinant retroviruses.…”

Double‐inducible gene activation system for caspase 3 and 9 in epidermis

“…With the application of RU486, the Glp65 fusion protein was exclusively expressed in skin keratinocytes, which subsequently initiated caspase precursor induction. In the presence of lipid-permeable dimeric ligand CID (AP20187, ARIAD Pharmaceuticals), which was applied intraperitoneally to adult mice or daubed topically to the back skin of neonates, the inactive caspase was forced to dimerize, leading to an autoproteolysis and self-activation (Fan et al, 1999;Mallet et al, 2002;Shariat et al, 2001).…”

“…1b). The precursor will remain biologically inactive until its exposure to CID (Fan et al, 1999) (Shariat et al, 2001). Both inducible systems are specific, reversible, nontoxic, and have a high induction potential.…”