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GEA 3162, a peroxynitrite donor, induces Bcl-2-sensitive, p53-independent apoptosis in murine bone marrow cells.

Taylor EL, Li JT, Tupper JC, Rossi AG, Winn RK, Harlan JM - Biochem. Pharmacol. (2007)

Bottom Line: Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential.Thus, we have demonstrated that the ONOO(-) donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes.This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical and Clinical Science, Peninsula Medical School, Universities of Exeter and Plymouth, St Luke's Campus, Heavitree Rd, Exeter, Devon EX1 2LU, UK. emma.taylor@pms.ac.uk

ABSTRACT
Apoptosis may be regulated by oxidants such as peroxynitrite (ONOO(-)). The tumour suppressor, p53, has been reported to play a crucial role in apoptosis induced by oxidants, therefore we assessed the ability of a ONOO(-) donor, GEA 3162, to activate caspases and induce mitochondrial permeability in a p53-deficient murine bone marrow cell line, Jaws II. Furthermore, these cells were stably transfected with Bcl-2, in order to investigate the impact of this survival protein on ONOO(-)-induced apoptosis. GEA 3162 activated caspases and induced loss of mitochondrial membrane potential in Jaws II cells. In particular, caspases 3 and 2 were activated, alongside minor activation of caspases 8 and 9, and apoptosis was partially dependent upon p38 MAP kinase activation, with little or no role for JNK. Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential. Thus, we have demonstrated that the ONOO(-) donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes. This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane.

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Specific caspases activated by GEA 3162. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of the cleavage of specific fluorogenic substrates for (A) caspase 3, (B) caspase 8, (C) caspase 9 and (D) caspase 2. Cells were harvested, centrifuged and lysed, then lysates incubated with specific caspase substrates immobilised on a plate, with fluorescence indicating the extent of caspase activation. Data represents mean ± S.E.M. following n = 4 experiments performed in duplicate. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type, and hashes represent significant differences between fluorescence in GFP and Bcl-2 cells for a given treatment (repeated measures ANOVA with Student–Newman–Keuls post-test).
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Figure 6: Specific caspases activated by GEA 3162. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of the cleavage of specific fluorogenic substrates for (A) caspase 3, (B) caspase 8, (C) caspase 9 and (D) caspase 2. Cells were harvested, centrifuged and lysed, then lysates incubated with specific caspase substrates immobilised on a plate, with fluorescence indicating the extent of caspase activation. Data represents mean ± S.E.M. following n = 4 experiments performed in duplicate. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type, and hashes represent significant differences between fluorescence in GFP and Bcl-2 cells for a given treatment (repeated measures ANOVA with Student–Newman–Keuls post-test).

Mentions: Analysis of specific caspase activation showed that exposure of Jaws II-GFP cells to GEA 3162 leads to a large increase in the activity of caspases 2 and 3, and a smaller increase in caspases 8 and 9 (Fig. 6). Treatment with 30 or 100 μM GEA 3162 produced a concentration-dependent increase in caspase 3 substrate cleavage in cells transfected with the GFP vector alone, with fluorescence levels reaching approximately 230% (p > 0.05) and 950% (p < 0.05) of control levels respectively. Similarly, relatively high levels of activation were seen with caspase 2, with 30 μM GEA 3162 producing around 160% (p > 0.05) and 100 μM producing 650 % (p < 0.05) of control fluorescence. However, caspase 8 activation was less pronounced, with fluorescence in response to 30 μM GEA 3162 being virtually identical to control (p > 0.05), and 100 μM measured at 200% (p < 0.05) of control. Caspase 9 levels were virtually identical in control and 30 μM GEA 3162-treated cells (p > 0.05) while 100 μM-treated cells showed 185% (p > 0.05) of control fluorescence. In contrast, overexpression of Bcl-2 greatly reduced or abolished all GEA 3162-induced increases in fluorescence, and there was no significant difference between any treatments in these cells for any of the caspases measured, indicating that Bcl-2 overexpression prevents up-regulation of the activity of multiple caspases in response to ONOO−.


GEA 3162, a peroxynitrite donor, induces Bcl-2-sensitive, p53-independent apoptosis in murine bone marrow cells.

Taylor EL, Li JT, Tupper JC, Rossi AG, Winn RK, Harlan JM - Biochem. Pharmacol. (2007)

Specific caspases activated by GEA 3162. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of the cleavage of specific fluorogenic substrates for (A) caspase 3, (B) caspase 8, (C) caspase 9 and (D) caspase 2. Cells were harvested, centrifuged and lysed, then lysates incubated with specific caspase substrates immobilised on a plate, with fluorescence indicating the extent of caspase activation. Data represents mean ± S.E.M. following n = 4 experiments performed in duplicate. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type, and hashes represent significant differences between fluorescence in GFP and Bcl-2 cells for a given treatment (repeated measures ANOVA with Student–Newman–Keuls post-test).
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Figure 6: Specific caspases activated by GEA 3162. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of the cleavage of specific fluorogenic substrates for (A) caspase 3, (B) caspase 8, (C) caspase 9 and (D) caspase 2. Cells were harvested, centrifuged and lysed, then lysates incubated with specific caspase substrates immobilised on a plate, with fluorescence indicating the extent of caspase activation. Data represents mean ± S.E.M. following n = 4 experiments performed in duplicate. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type, and hashes represent significant differences between fluorescence in GFP and Bcl-2 cells for a given treatment (repeated measures ANOVA with Student–Newman–Keuls post-test).
Mentions: Analysis of specific caspase activation showed that exposure of Jaws II-GFP cells to GEA 3162 leads to a large increase in the activity of caspases 2 and 3, and a smaller increase in caspases 8 and 9 (Fig. 6). Treatment with 30 or 100 μM GEA 3162 produced a concentration-dependent increase in caspase 3 substrate cleavage in cells transfected with the GFP vector alone, with fluorescence levels reaching approximately 230% (p > 0.05) and 950% (p < 0.05) of control levels respectively. Similarly, relatively high levels of activation were seen with caspase 2, with 30 μM GEA 3162 producing around 160% (p > 0.05) and 100 μM producing 650 % (p < 0.05) of control fluorescence. However, caspase 8 activation was less pronounced, with fluorescence in response to 30 μM GEA 3162 being virtually identical to control (p > 0.05), and 100 μM measured at 200% (p < 0.05) of control. Caspase 9 levels were virtually identical in control and 30 μM GEA 3162-treated cells (p > 0.05) while 100 μM-treated cells showed 185% (p > 0.05) of control fluorescence. In contrast, overexpression of Bcl-2 greatly reduced or abolished all GEA 3162-induced increases in fluorescence, and there was no significant difference between any treatments in these cells for any of the caspases measured, indicating that Bcl-2 overexpression prevents up-regulation of the activity of multiple caspases in response to ONOO−.

Bottom Line: Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential.Thus, we have demonstrated that the ONOO(-) donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes.This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical and Clinical Science, Peninsula Medical School, Universities of Exeter and Plymouth, St Luke's Campus, Heavitree Rd, Exeter, Devon EX1 2LU, UK. emma.taylor@pms.ac.uk

ABSTRACT
Apoptosis may be regulated by oxidants such as peroxynitrite (ONOO(-)). The tumour suppressor, p53, has been reported to play a crucial role in apoptosis induced by oxidants, therefore we assessed the ability of a ONOO(-) donor, GEA 3162, to activate caspases and induce mitochondrial permeability in a p53-deficient murine bone marrow cell line, Jaws II. Furthermore, these cells were stably transfected with Bcl-2, in order to investigate the impact of this survival protein on ONOO(-)-induced apoptosis. GEA 3162 activated caspases and induced loss of mitochondrial membrane potential in Jaws II cells. In particular, caspases 3 and 2 were activated, alongside minor activation of caspases 8 and 9, and apoptosis was partially dependent upon p38 MAP kinase activation, with little or no role for JNK. Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential. Thus, we have demonstrated that the ONOO(-) donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes. This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane.

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Related in: MedlinePlus