Synthetic activation of caspases: Artificial death switches

Abstract

The routine acceptance of gene therapy as a therapeutic option will require that it is proven to be safe. To guarantee this safety, a mechanism must be used to eliminate transferred gene products or genetically modified cells in the event that they become deleterious to the host. For this reason, “suicide genes,” such as herpes simplex virus thymidine kinase (tk), are often incorporated into gene therapy vectors (1, 2). The tk gene kills proliferating cells by converting the dideoxynucleoside analog ganciclovir into a form that can be incorporated into elongating DNA, leading to chain termination (3). In contrast, regulating apoptosis by controlling the multimerization of Fas with anti-Fas antibodies, Fas ligand, or synthetically with the dimerizing drug FK1012 has been shown to be possible in nonproliferating cells, including CD4 ^{CD8 thymocytes (}4, 5), differentiated neutrophils and monocytes (6), and hepatocytes (7). Further, unlike the viral tk gene, conditional Fas alleles can be made from human proteins, minimizing potential immunogenicity (5, 8).

Fas is a member of the tumor necrosis factor receptor superfamily whose members can induce pleiotropic responses, including proliferation, activation, differentiation, and apoptosis, depending primarily on their cytoplasmic signaling domains (for review, see ref. 9). The molecular details of Fas-mediated apoptosis are rapidly emerging and frequently reviewed (10). The interaction of Fas with the Fas ligand FasL leads to the aggregation of Fas cytoplasmic death domains (DD) and increases the affinity of the Fas DD for the DD of the adapter molecule FADD (Fas-associated with death domain) (MORT1). FADD, in turn, interacts with the cysteine protease caspase-8 [FADD-like interleukin 1β converting enzyme (ICE), FLICE/MACH], via a conserved death effector domains (DED) found in both proteins. Thus, Fas cross-linking leads to caspase-8 cross-linking. Like all caspases, caspase-8 is an aspartic acid-directed protease that is activated by the proteolytic removal of its amino-terminal prodomain and by an additional internal cleavage, producing a fully active molecule composed of two p17 and two p12 subunits. Probably by transproteolysis, the aggregation of caspase-8 contributes to this activation and the initiation of a protease cascade that includes caspase-1 (ICE)-related and caspase-3 (YAMA/CPP32)-related enzymes, ultimately leading to the irreversible cleavage of multiple proapoptotic targets. Mitochondrial-derived factors and proteins, including cytochrome c and the protease AIF (apoptosis inducing factor) seem to be essential for the activation of the most downstream members of this protease cascade, including caspases 3, 6, and 7 (for review, see ref. 11). Members of the Bcl-2 family, such as Bax or Bcl-x_L, are primarily localized in the mitochondria and help to modulate the release of these factors that occurs concomitant with an apoptosis-associated increase in the permeability of the mitochondrial membrane (12). Because many of the events in Fas signaling are regulated by protein–protein interactions, Fas signaling intermediates, such as caspases, are ideal candidates for designing conditional alleles based on chemically induced dimerization (CID) (13–15).

In this report we describe conditional alleles of the zymogens caspase-1 and caspase-3. The CID-binding domain (CBD), FKBP12 (where FKBP is FK506-binding protein), has been placed at the amino terminus of these proteins adjacent to the prodomains of the inactive proteases. Upon administration of a lipid-permeable dimerizing drug, aggregation of caspases occurs, leading to autoproteolysis and activation. We show that this chemical activation of either caspase-1 or caspase-3 is sufficient to trigger apoptosis in target cells, and we call this technique chemically induced apoptosis (CIA) and term these molecules artificial death switches (ADS). Although conditional caspase-1 alleles are somewhat “autotoxic,” like previously described conditional Fas alleles (5), the conditional caspase-3 alleles are completely nontoxic in the absence of CID, even at high levels of expression. Interestingly, a truncated caspase-3 lacking its prodomain is somewhat autotoxic, consistent with other reports that the prodomains of caspases contribute to maintaining their quiescence in unstimulated cells (16). Further, the conditional caspase-1 allele appears to be completely insensitive to excess Bcl-x_L, whereas the conditional caspase-3 allele can be blocked by an excess of Bcl-x_L levels. Finally, both conditional caspase-1 and caspase-3 alleles can trigger apoptosis in a broad range of tissues. These results confirm that cross-linking caspases can lead to their activation in intact cells and demonstrate an expanded repertoire of proteins that can be activated by CID.

Acknowledgments

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