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

Méthot N, Huang J, Coulombe N, Vaillancourt JP, Rasper D, Tam J, Han Y, Colucci J, Zamboni R, Xanthoudakis S, Toulmond S, Nicholson DW, Roy S - J. Exp. Med. (2004)

Bottom Line: 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.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.Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

View Article: PubMed Central - PubMed

Affiliation: Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories, Montreal, Quebec, Canada H9H 3L1. nathalie_methot@merck.com

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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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).
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fig1: 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).

Mentions: We first determined the contribution of necrosis and the relevance of various apoptotic markers during sepsis by examining several indicators of cell viability in transgenic mice that overexpress Bcl-2 in T cells. Bcl-2 is a potent antiapoptotic protein that works in part by blocking cytochrome-c release from mitochondria and the ensuing caspase activation (17, 18). CLP was used to induce sepsis and resulted in the appearance of the processed form of caspase-3 (p17 fragment) in thymocytes from WT but not of Bcl-2 transgenic animals. The p17 product was not detected in sham animals regardless of their genotype (Fig. 1 A). Hence, caspase-3 processing is induced during sepsis but can be blocked by Bcl-2 overexpression. Next, we examined whether caspase substrates were proteolytically processed and whether thymocytes exhibited characteristic signs of apoptosis. The markers chosen reflect either direct substrate cleavage (αII-spectrin and PARP) or the consequence of caspase activation on cell function (membrane permeability, PS exposure, and DNA fragmentation). PARP is a nuclear enzyme that participates in DNA repair and has been one of the first proteins identified as a caspase substrate (19). The 24-kD NH2-terminal PARP cleavage fragment was abundant in all WT mice that had undergone CLP but was absent in most Bcl-2–overexpressing animals. Some of the CLP-operated Bcl-2 transgenic and sham-operated WT animals showed slight PARP 24-kD cleavage product (Fig. 1 A). Another caspase substrate, αII-spectrin, is a major component of the cortical cytoskeleton and is proteolytically cleaved during lymphocyte apoptosis (20). Both calpain and caspase-3 cleave αII-spectrin at multiple sites, with the p120 fragment specifically generated upon caspase-3 cleavage (21–23). The p120 αII-spectrin product was prominent in WT mice but was absent in thymi from septic Bcl-2 animals. No p120 fragment was observed in sham-operated animals (Fig. 1 A). The 150-kD αII-spectrin cleavage product, which results from both calpain and caspase activity, was present in all animals but was more abundant in thymi from CLP-operated mice. Thus, both PARP and αII-spectrin cleavage at the p120 site are protected from caspase cleavage in Bcl-2–overexpressing thymocytes during sepsis. Bcl-2 overexpression did not affect significantly the αII-spectrin p150 cleavage site. Loss of membrane phospholipid asymmetry and PS externalization are detected by FITC-labeled annexin V (24). During sepsis, a higher proportion of thymocytes were annexin V positive in CLP relative to sham-operated WT animals (49 versus 18%, respectively, P < 0.001). In contrast, the number of annexin V–positive thymocytes was virtually identical in CLP or sham animals overexpressing Bcl-2 (11.8 versus 9.3%, respectively; Fig. 1 B) but was lower than in sham-treated WT animals. Similar results were obtained with membrane permeability and PI uptake (Fig. 1 C). We conclude that the loss of membrane integrity and phospholipid asymmetry is largely dependent on the mitochondrial apoptotic pathway during sepsis. Cleavage of DNA at internucleosomal spaces is a well-known apoptotic marker and is mediated by DFF40/caspase-activated DNase (CAD) (25–27). CAD activation requires cleavage of DFF45/inhibitor of caspase-activated DNase (ICAD) by caspases (25–27), principally caspase-3 (23, 28–30). We measured DNA fragmentation by the Cell Death ELISA (CDE) method and by flow cytometry using PI staining (as described in Materials and Methods). Thymocytes from mice that have undergone CLP exhibited high levels of DNA fragmentation. Overexpression of Bcl-2 in T cells abrogated DNA cleavage and brought the signal level to or near values found in sham-operated animals (Fig. 1, D and E). We conclude that DNA fragmentation, annexin V binding and αII-spectrin cleavage are dependent on the mitochondrial apoptotic pathway during sepsis (Table I).


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

Méthot N, Huang J, Coulombe N, Vaillancourt JP, Rasper D, Tam J, Han Y, Colucci J, Zamboni R, Xanthoudakis S, Toulmond S, Nicholson DW, Roy S - J. Exp. Med. (2004)

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).
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Related In: Results  -  Collection

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fig1: 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).
Mentions: We first determined the contribution of necrosis and the relevance of various apoptotic markers during sepsis by examining several indicators of cell viability in transgenic mice that overexpress Bcl-2 in T cells. Bcl-2 is a potent antiapoptotic protein that works in part by blocking cytochrome-c release from mitochondria and the ensuing caspase activation (17, 18). CLP was used to induce sepsis and resulted in the appearance of the processed form of caspase-3 (p17 fragment) in thymocytes from WT but not of Bcl-2 transgenic animals. The p17 product was not detected in sham animals regardless of their genotype (Fig. 1 A). Hence, caspase-3 processing is induced during sepsis but can be blocked by Bcl-2 overexpression. Next, we examined whether caspase substrates were proteolytically processed and whether thymocytes exhibited characteristic signs of apoptosis. The markers chosen reflect either direct substrate cleavage (αII-spectrin and PARP) or the consequence of caspase activation on cell function (membrane permeability, PS exposure, and DNA fragmentation). PARP is a nuclear enzyme that participates in DNA repair and has been one of the first proteins identified as a caspase substrate (19). The 24-kD NH2-terminal PARP cleavage fragment was abundant in all WT mice that had undergone CLP but was absent in most Bcl-2–overexpressing animals. Some of the CLP-operated Bcl-2 transgenic and sham-operated WT animals showed slight PARP 24-kD cleavage product (Fig. 1 A). Another caspase substrate, αII-spectrin, is a major component of the cortical cytoskeleton and is proteolytically cleaved during lymphocyte apoptosis (20). Both calpain and caspase-3 cleave αII-spectrin at multiple sites, with the p120 fragment specifically generated upon caspase-3 cleavage (21–23). The p120 αII-spectrin product was prominent in WT mice but was absent in thymi from septic Bcl-2 animals. No p120 fragment was observed in sham-operated animals (Fig. 1 A). The 150-kD αII-spectrin cleavage product, which results from both calpain and caspase activity, was present in all animals but was more abundant in thymi from CLP-operated mice. Thus, both PARP and αII-spectrin cleavage at the p120 site are protected from caspase cleavage in Bcl-2–overexpressing thymocytes during sepsis. Bcl-2 overexpression did not affect significantly the αII-spectrin p150 cleavage site. Loss of membrane phospholipid asymmetry and PS externalization are detected by FITC-labeled annexin V (24). During sepsis, a higher proportion of thymocytes were annexin V positive in CLP relative to sham-operated WT animals (49 versus 18%, respectively, P < 0.001). In contrast, the number of annexin V–positive thymocytes was virtually identical in CLP or sham animals overexpressing Bcl-2 (11.8 versus 9.3%, respectively; Fig. 1 B) but was lower than in sham-treated WT animals. Similar results were obtained with membrane permeability and PI uptake (Fig. 1 C). We conclude that the loss of membrane integrity and phospholipid asymmetry is largely dependent on the mitochondrial apoptotic pathway during sepsis. Cleavage of DNA at internucleosomal spaces is a well-known apoptotic marker and is mediated by DFF40/caspase-activated DNase (CAD) (25–27). CAD activation requires cleavage of DFF45/inhibitor of caspase-activated DNase (ICAD) by caspases (25–27), principally caspase-3 (23, 28–30). We measured DNA fragmentation by the Cell Death ELISA (CDE) method and by flow cytometry using PI staining (as described in Materials and Methods). Thymocytes from mice that have undergone CLP exhibited high levels of DNA fragmentation. Overexpression of Bcl-2 in T cells abrogated DNA cleavage and brought the signal level to or near values found in sham-operated animals (Fig. 1, D and E). We conclude that DNA fragmentation, annexin V binding and αII-spectrin cleavage are dependent on the mitochondrial apoptotic pathway during sepsis (Table I).

Bottom Line: 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.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.Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

View Article: PubMed Central - PubMed

Affiliation: Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories, Montreal, Quebec, Canada H9H 3L1. nathalie_methot@merck.com

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.

Show MeSH
Related in: MedlinePlus