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.

Subcellular distribution of the anticancer drug mitoxantrone in human and drug-resistant murine cells analyzed by flow cytometry and confocal microscopy and its relationship to the induction of DNA damage.

Flow cytometry and laser scanning confocal imaging have been used to analyze the uptake of the anticancer topoisomerase II poison mitoxantrone by intact mammalian cells and the results correlated with the induction of DNA damage. Unlike Adriamycin, mitoxantrone displays only minimal levels of red fluorescence when excited at 514 wavelength. However, using these excitation and emission conditions, flow cytometry could detect low levels of fluorescence in human transformed fibroblasts exposed to high concentrations (5-20 microM) of mitoxantrone for 1 h. Over this dose range whole cell fluorescence was a function of cell size and increased with drug concentration while drug-induced DNA-protein cross-linking showed saturation. Confocal microscopy revealed the time- and dose-dependent appearance of fluorescence, interpreted here as reflecting the disposition of drug molecules, preferentially within the cytoplasm, nuclear membrane, and nucleoli. This pattern contrasted with the intense intranuclear fluorescence observed in Adriamycin-treated human cells. Loss of the nuclear membrane during mitosis resulted in an apparent increase in chromatin-associated fluorescence. Photon counting procedures revealed a predominantly cytoplasmic, possibly lysosomal, location for fluorescence from human cells exposed for 1 h to a low but cytotoxic concentration (0.1 microM, yielding approximately 90% cell kill) of mitoxantrone. At this low concentration, human cells displayed minimal levels of DNA strand cleavage or DNA-protein cross-linking. Murine cells, displaying mitoxantrone resistance as part of the P-glycoprotein-mediated multidrug resistance phenotype, showed specific extinction of mitoxantrone-associated fluorescence from inside nuclei but not from within extranuclear compartments. The study demonstrates the feasibility of high resolution studies on the intracellular distribution of mitoxantrone in intact living cells. We suggest a mechanism by which cytoplasmic sequestration of mitoxantrone may be important in determining the response of normal and multidrug-resistant cells as they attempt to progress through mitosis.