Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.

King MA 《CYTOMETRY PART A》

Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fa

Perchellet EM ，Wang Y ，Weber RL ，Sperfslage BJ ，Lou K ，Crossland J ，Hua DH ，Perchellet JP ... -  《BIOCHEMICAL PHARMACOLOGY》

Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes deltapsim loss, caspase-9 activity, and apoptosis in Jurkat cells.

Gamen S ，Anel A ，Pérez-Galán P ，Lasierra P ，Johnson D ，Piñeiro A ，Naval J ... -  《EXPERIMENTAL CELL RESEARCH》

Antimycin A-induced apoptosis of HL-60 cells.

Previous experiments in our laboratory investigating apoptosis induced in HL-60 cells by camptothecin (CAM) have revealed that the sequence and rapidity of the apoptotic phenomena in an individual cell depend on the proliferative state of that cell when it encounters CAM. The role of mitochondria in HL-60 apoptosis was explored using an inhibitor of oxidative phosphorylation, antimycin A (AMA). Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC), uptake of propidium iodide (PI), and DNA content after membrane fixation/permeabilization were monitored by flow cytometry. Z-VAD-FMK was used to inhibit caspases. Fluorescence microscopy was used to examine cell morphology. Cells in the G1 phase of the cell cycle were the first to exhibit signs of apoptosis in response to 100 microM AMA and some of these cells disintegrated without exposing to phosphatidylserine (PS). Caspase inhibition prevented fragmentation of DNA, the nucleus, and the cell, but only delayed PS exposure and loss of plasma membrane integrity. The highly active mitochondria of G1-phase HL-60 cells make them particularly sensitive to AMA. PS exposure and plasma membrane damage are mediated by noncaspase molecules released from mitochondria. We hypothesize that if mitochondria are subjected to a sufficiently severe insult, whether indirectly as a result of extensive CAM-induced DNA damage or directly by the effect of AMA on electron transport, the nature and quantities of the proapoptotic molecules released are such that apoptosis proceeds to the point of cell disintegration before the PS exposure pathway is complete.

King MA ，Radicchi-Mastroianni MA 《-》

Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells.

Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL-induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 microg/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL-induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL-induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.

Chen J ，Mehta JL ，Haider N ，Zhang X ，Narula J ，Li D ... -  《-》