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. 2004 Jan 19;199(2):199-207.
doi: 10.1084/jem.20031791. Epub 2004 Jan 12.

Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics

Nathalie Méthot  1 , JingQi Huang, Nathalie Coulombe, John P Vaillancourt, Dita Rasper, John Tam, Yongxin Han, John Colucci, Robert Zamboni, Steven Xanthoudakis, Sylvie Toulmond, Donald W Nicholson, Sophie Roy
Affiliations

Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics

Nathalie Méthot et al. J Exp Med. .

Abstract

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

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Figures

Figure. 1
Figure. 1
Effect of Bcl-2 on apoptotic manifestations in the thymus of CLP-induced septic mice. Transgenic (T cell–specific Bcl-2 overexpression) and WT mice siblings underwent CLP or sham surgery, for a total of four groups (WT CLP n = 4; Bcl-2 CLP n = 7; WT sham n = 4; Bcl-2 sham n = 3). (A) Western blot from two representative animals in all four experimental groups. Detection of caspase-3, PARP, and αII-spectrin cleavage products in thymi of CLP or sham-operated mice. +, animals that carried the bcl-2 transgene. Average percentage of annexin V–positive thymocytes (B) and PI-permeable thymocytes (C) by flow cytometry. Average DNA fragmentation assessed by a DNA-histone sandwich ELISA (D) or subdiploid DNA content (E). In this figure and all subsequent figures, the error bars represent one SD. One-way analysis of variance (ANOVA) was performed with p-values < 0.001 between CLP WT and CLP Bcl-2 groups (B–E).
Figure 2.
Figure 2.
Effect of caspase-3 inhibitors on DNA fragmentation and αII-spectrin cleavage in the thymus of CLP-induced septic rats. (A) Structure of the selective caspase-3 inhibitor M867 ((3S)-3-({(2S)-2-[5-tert-butyl-3-{[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino}-2-oxopyrazin-1(2H)-yl]butanoyl}amino)-5-[methyl(pentyl)amino]-4-oxopentanoic acid). Structures and potencies for M826, M920, and M791 can be found in references and . (B) Schematic representation of the experimental design. Average αII-spectrin cleavage index (C; as described in Materials and Methods) and DNA fragmentation (D; percentage of subdiploid thymocytes) in CLP rats treated with M867 (n = 10) or vehicle (veh; n = 10) relative to vehicle-treated sham animals (n = 3). Average αII-spectrin cleavage index (D) and DNA fragmentation (E; percentage of subdiploid thymocytes) in CLP rats treated with vehicle (n = 4), M867 (n = 5), or inactive enantiomer compound (M761; n = 4) relative to vehicle-treated sham animals (n = 3). Statistical analysis (one-way ANOVA) indicates a significant difference (P < 0.01) between vehicle- and M867-treated CLP rats. M867 had no effect on basal apoptotic parameters in sham-operated animals (unpublished data).
Figure 3.
Figure 3.
Potencies of caspase-3 inhibitor M867 and ICADdm on apoptotic manifestations in cultured thymocytes. (A) Effect of increasing concentrations of M867 on DNA fragmentation (squares), spectrin cleavage (circles), and annexin V labeling (triangles) in cultured rat thymocytes. IC50s were determined by 4-parameter curve fits and are as follows: DNA fragmentation, 0.23 μM; αII-spectrin cleavage, 0.064 μM; annexin V, 0.08 μM. Annexin V–PI labeling (B and D) and DNA fragmentation (C and E) in WT (B and C) and ICADdm (D and E) mouse thymocytes cultured for 24 h. The experiment was performed with thymocytes from three WT and three ICADdm mice. Shown here are representative results from one animal of each genotype.
Figure 4.
Figure 4.
Effect of M867 dose titration on caspase-3 cleavage, αII-spectrin cleavage, and DNA fragmentation in CLP-induced septic rats. Rats that underwent CLP surgery were dosed by continuous i.v. infusion of either vehicle (n = 6) or M867 (n = 1 for each dose) for 24 h. (A) Percentage of residual αII-spectrin cleavage (circles) and DNA fragmentation (triangles) in each animal after administration of various doses of M867. Percentages were calculated from the average αII-spectrin and DNA fragmentation values obtained with the vehicle-treated animals. (B) Caspase-3 Western blotting on thymus extracts of animals dosed with M867.
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