Improved artificial death switches based on caspases and FADD

A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperprolif erative disorders, such as cancer or restenosis. However, most of these app roaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial dea th switches based on chemical inducers of dimerization (CIDs) and endogenou s proapoptotic molecules function efficiently in both dividing and nondivid ing cells. In this approach, lipid-permeable, nontoxic CIDs are used to con ditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previo us reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. S ince the maximum efficacy of these artificial death switches requires low b asal 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 improv ed conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subn anomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonst rate for the first time that oligomerization of the death effector domain o f the Fas-associated protein, FADD, is sufficient to trigger apoptosis, sug gesting that the primary function of FADD, like that of Apaf-1, is oligomer ization of associated caspases. Finally, we demonstrate that nuclear-target ed caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that th e cleavage of nuclear targets is sufficient for apoptosis.