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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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Effect of GEA 3162 on Jaws II cell viability. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of cell viability by MTT assay. Data represents mean ± S.D. following n = 2 experiments. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type (repeated measures ANOVA with Student–Newman–Keuls post-test).
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Figure 3: Effect of GEA 3162 on Jaws II cell viability. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of cell viability by MTT assay. Data represents mean ± S.D. following n = 2 experiments. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type (repeated measures ANOVA with Student–Newman–Keuls post-test).

Mentions: Assessment of Jaws II cell viability by MTT assay following exposure to GEA 3162 (30 or 100 μM) for 4 h showed a decrease in viability in Jaws II-GFP cells (Fig. 3), with a significant (p < 0.05) effect seen with the higher concentration. Jaws II-Bcl-2 cells also showed a slight decrease in viability, although this effect was reduced compared to the control cells and was associated with more variability in absorbance measured. Both sets of cells continued to exclude Trypan Blue following treatment with GEA 3162 (data not shown).


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)

Effect of GEA 3162 on Jaws II cell viability. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of cell viability by MTT assay. Data represents mean ± S.D. following n = 2 experiments. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type (repeated measures ANOVA with Student–Newman–Keuls post-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1991334&req=5

Figure 3: Effect of GEA 3162 on Jaws II cell viability. GFP or Bcl-2-overexpressing Jaws II cells were exposed to GEA 3162 (30–100 μM) for 4 h before assessment of cell viability by MTT assay. Data represents mean ± S.D. following n = 2 experiments. Asterisks represent significant (p < 0.05) difference from control (untreated) cells for each cell type (repeated measures ANOVA with Student–Newman–Keuls post-test).
Mentions: Assessment of Jaws II cell viability by MTT assay following exposure to GEA 3162 (30 or 100 μM) for 4 h showed a decrease in viability in Jaws II-GFP cells (Fig. 3), with a significant (p < 0.05) effect seen with the higher concentration. Jaws II-Bcl-2 cells also showed a slight decrease in viability, although this effect was reduced compared to the control cells and was associated with more variability in absorbance measured. Both sets of cells continued to exclude Trypan Blue following treatment with GEA 3162 (data not shown).

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.

Show MeSH
Related in: MedlinePlus