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Activation of the intrinsic and extrinsic pathways in high pressure-induced apoptosis of murine erythroleukemia cells

Abstract

We previously demonstrated that caspase-3, an executioner of apoptosis, is activated in the pressure-induced apoptosis of murine erythroleukemia (MEL) cells (at 100 MPa). Here, we examined the pathway of caspase-3 activation using peptide substrates and caspase inhibitors. Using the substrates of caspases-8 and -9, it was found that both are activated in cells under high pressure. The production of nuclei with sub-G1 DNA content in 100 MPa-treated MEL cells was suppressed by inhibitors of caspases-8 and -9, and pan-caspase. In 100 MPa-treated cells, pan-caspase inhibitor partially prevented the cytochrome c release from the mitochondria and the breakdown of mitochondrial membrane potential. These results suggest that the intrinsic and extrinsic pathways are activated in apoptotic signaling during the high pressure-induced death of MEL cells.

Abbreviations

Ac-IETD-MCA:

acetyl-IIe-Glu-Thr-Asp-4-methylcoumaryl-7-amide

Ac-LEHD-CHO:

acetyl-Leu-Glu-His-Asp-aldehyde

Ac-LEHD-MCA:

acetyl-Leu-Glu-His-Asp-4-methylcoumaryl-7-amide

CAD:

caspase-activated deoxyribo-nuclease

DTT:

dithiothreitol

HEPES:

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

MEL:

murine erythroleukemia

NP-40:

nonidet P-40

PBS:

phosphate-buffered saline

PI:

propidium iodide

PMSF:

phenylmethanesulfonyl fluoride

RNase A:

ribonuclease A

ROS:

reactive oxygen species

UV:

ultraviolet

z-IETD-fmk:

benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone

z-VAD-fmk:

benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone

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Correspondence to Takeo Yamaguchi.

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Yamaguchi, T., Hashiguchi, K., Katsuki, S. et al. Activation of the intrinsic and extrinsic pathways in high pressure-induced apoptosis of murine erythroleukemia cells. Cell Mol Biol Lett 13, 49–57 (2008). https://doi.org/10.2478/s11658-007-0034-x

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Key words

  • Apoptosis
  • Caspases
  • Cytochrome c
  • Flow cytometry
  • Membrane potential
  • High pressure