Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Intracellular acidification induces apoptosis by stimulating ICE-like protease activity.

ICE-like protease activation and DNA fragmentation are preceded by a decrease in intracellular pH (pHi) during apoptosis in the IL-3 dependent cell line BAF3. Acidification occurs after 7 hours in cells deprived of IL-3 and after 4 hours when cells are treated with etoposide, close to the time of detection of ICE-like protease activity. Increasing extracellular pH reduces ICE-like protease activation and DNA fragmentation. Bcl-2 over-expression both delays acidification and inhibits ICE-like protease activation. Generation of a rapid intracellular pH decrease, using the ionophore nigericin, induces ICE-like protease activation and apoptosis. ZVAD, a cell permeable inhibitor of ICE-like proteases, does not affect acidification but inhibits apoptosis induced by IL-3 removal or nigericin treatment. These data suggest that intracellular acidification triggers apoptosis by directly or indirectly activating ICE-like proteases.

An apoptotic endonuclease activated either by decreasing pH or by increasing calcium.

DNA fragmentation in isolated nuclei from the murine IL3-dependent bone marrow cell line BAF3 could be stimulated either by decreasing pH below 6.5 or by adding microM calcium at neutral pH. An endonuclease which could also be stimulated either by a decrease in pH, to 6.5, or by the presence of microM calcium at neutral pH, was purified 10(4)-fold from nuclei of BAF3 cells. Digestion of DNA with the purified enzyme resulted in 5'-terminal hydroxyl and 3'-terminal phosphate ends. These characteristics are distinct from those described for other mammalian endonucleases. The possible role of this enzyme in genome digestion during apoptosis is discussed.

Interleukin-3 and Bcl-2 cooperatively inhibit etoposide-induced apoptosis in a murine pre-B cell line.

Murine bone marrow-derived hemopoietic cells, dependent on interleukin (IL)-3 for their growth in culture, undergo programmed cell death, or apoptosis, upon cytokine withdrawal. The topoisomerase II inhibitor etoposide causes a more rapid onset of apoptosis in the IL-3-dependent cell line BAF3, deprived of IL-3. This acceleration of apoptosis by etoposide is prevented by inhibitors of RNA and protein synthesis and by the nucleases inhibitor aurintricarboxylic acid. The presence of IL-3 or overexpression of the oncogene bcl-2 caused a marked delay in the induction of apoptosis by etoposide, acting in a cooperative manner. The time at which the apoptotic program is irreversible is close to the induction of endonuclease activity as indicated by the effect of the delayed addition of either IL-3 or aurintricarboxylic acid on the onset of apoptosis, suggesting the importance of endonuclease activation in the development of apoptosis in hemopoietic cells.