Synthetic activation of caspases: artificial death switches - PubMed (original) (raw)

Synthetic activation of caspases: artificial death switches

R A MacCorkle et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal "suicide switch" would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1beta converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-xL-independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-xL, that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Design of conditional alleles of Fas and caspases. (A) Model of Fas ligand-mediated Fas signaling. Fas cross-linking causes association with FADD, triggering caspase-8 activation, and signaling cascades that lead to apoptosis. (B and C) In each example, administration of a CID leads to the cross-linking of chimeric proteins by their CBDs. Asterisk, active site QACRG. (B) Conditional Fas receptor showing Fas-associated proteins, FADD, and caspase-8. (C) Conditional caspases illustrating CID-mediated removal of the prodomain and conversion to fully processed form. (D) Schematic of CID-regulated proapoptotic molecules. Signaling molecules can be cloned 5′ or 3′ of FKBPs. (S/X), _Sal_I/_Xho_I composite site, destroying both; M, myristoylation targeting peptide; E, hemagglutinin epitope.

Figure 2

Figure 2

Cross-linking caspase-1 triggers apoptosis in mammalian cells. Jurkat-TAg cells were transiently transfected with 4 μg of Fpk3-casp-1 (○), 2 μg of Fpk3-casp-1 (•), 1 μg of Fpk3-casp-1 (▵), 0.5 μg of Fpk3-casp-1 (▴), 0.25 μg of Fpk3-casp-1 (□), or 4 μg of Fpk3 (▪). After 20 h, transfected cells were treated with FK1012. After an additional 24 h, SEAP activity was assayed and reported directly (A) or as a percentage of activity from untreated cells in identical aliquots from the same transfections (B). Data are representative of three experiments performed in duplicate.

Figure 3

Figure 3

Conditional caspase-3 triggers apoptosis in mammalian cells. Jurkat-TAg cells were transiently transfected as follows: (A and B) Fv2-casp-3 (2 μg; □) or Fv2-casp-3/S163 (2 μg; ▪). (C) Fpk3-casp-3/S163 (2 μg; ▴), Fpk3-casp-3 (2 μg; □), or Fpk3-Δ20casp-3 (2 μg; ▪). After 20 h, transfected cells were treated and analyzed as before. Data are representative of at least three experiments performed in duplicate. (D) Anti-hemagglutinin epitope immunoblot of extracts from Jurkat TAg cells transiently transfected with 2 μg of Fv2-casp-3 or Fv2-casp-3/S163 and treated with 100 or 500 nM AP1903 as indicated. (E) Anti-hemagglutinin epitope immunoblot of extracts from similarly transfected Jurkat-TAg cells treated for 8 h with half-logarithmic dilutions of AP1903 as indicated.

Figure 4

Figure 4

Conditional caspase-1 and caspase-3 can bypass Bcl-xL. Jurkat-TAg cells were transiently transfected with the following plasmids: (A) Fpk3-casp-1 (▵; C, bar 1), Fpk3-casp-1 + 1 μg of Bcl-xL (▴; C, bar 2), Fpk3-casp-1 + 2 μg of Bcl-xL (□; C, bar 3), Fpk3-casp-1 + 4 μg Bcl-xL (▪; C, bar 4), or Fpk3 + 4 μg Bcl-xL (•; C, bar 5). (B) Fv2-casp-3 (▵; D, bar 1), Fv2-casp-3 + 1 μg of Bcl-xL (▴; D, bar 2), Fv2-casp-3 + 2 μg of Bcl-xL (□; D, bar 3), Fv2-casp-3 + 4 μg of Bcl-xL (▪; D, bar 4), or Fv2-casp-3/S163 + 4 μg of Bcl-xL (•; D, bar 5). Cells were transfected and assayed as above. Data are representative of at least three experiments. (C and D) Aliquots of cells from A and B were treated with anti-Fas antibody CH-11 and assayed as above. Bars 1–5 are described in A and B. Data are given relative to untreated cells from the same transfection. (E) Jurkat-TAg cells were transiently transfected with a constitutive GFP reporter and the indicated plasmids. Transfected cells were enriched to greater than 60% and split into two equal cultures, one of which was treated with 500 nM AP1903 (Fv chimeras) or FK1012 (Fpk chimeras). After 24 h, cells were stained with propidium iodide and analyzed by flow cytometry to determine the percentage of viable GFP-positive/propidium iodide-negative cells. The percent survival indicated is the percentage of viable cells after treatment with drug relative to the untreated aliquots.

Figure 5

Figure 5

Sensitivity of multiple cell lines to conditional Fas, caspase-1, and caspase-3. Jurkat-Tag, 293, and HeLa cells were transiently transfected with a constitutively expressing luciferase reporter plasmid and control vector MFv2 (shaded bars), MFv2-Fas plasmid (wide cross-hatched bars), control vector Fpk3 (narrow cross-hatched bars), Fpk3-casp-1 (bricked bars), or Fv2-casp-3 (solid bars). After 24 h, transfected cells were split into duplicate cultures and 500 nM drug (AP1903 for Fv or FK1012 for Fpk) was added to one culture for an additional 24 h. The percent relative reporter activity is the percent of luciferase activity after drug addition relative to untreated cells. Error bars represent the SD of the mean activity of three transfections.

Similar articles

Cited by

References

    1. Moolten F L. Cancer Res. 1986;46:5276–5281. - PubMed
    1. Culver K W, Ram Z, Wallbridge S, Ishii H, Oldfield E H, Blaese R M. Science. 1992;256:1550–1552. - PubMed
    1. Matthews, T. & Boehme, R. (1988) Rev. Infect. Dis. 10 Suppl. 3, S490–S494. - PubMed
    1. Ogasawara J, Suda T, Nagata S. J Exp Med. 1995;181:485–491. - PMC - PubMed
    1. Spencer D M, Belshaw P J, Chen L, Ho S N, Randazzo F, Crabtree G R, Schreiber S L. Curr Biol. 1996;6:839–847. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources