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 《-》

Effects of caspase inhibition on camptothecin-induced apoptosis of HL-60 cells.

During camptothecin (CAM)-induced apoptosis of HL-60 cells, the external exposure of phosphatidylserine (PS) can either precede or follow DNA cleavage. The evidence suggests that cells in S-phase when CAM is added undergo rapid DNA, nuclear, and cellular disintegration before exposing PS on the outside of the plasma membrane, whereas cells moving from G1 into S-phase after CAM is added expose PS before they manifest the other phenomena. This study describes further investigations using the broad spectrum caspase inhibitor Z-VAD-FMK. The cells were cultured for a period long enough to ascertain whether a particular phenomenon was only delayed or was blocked completely. Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC) to PS, uptake of propidium iodide (PI) as a measure of plasma membrane integrity, and DNA content after membrane fixation/permeabilization were monitored by flow cytometry during 24-h cultures. Fluorescence microscopy was used to examine cell morphology. Caspase inhibition blocked DNA cleavage, breakdown of the nuclear membrane, and formation of apoptotic bodies. It also revealed the existence of a CAM-activated early S-phase checkpoint. Cells arrested in early S-phase preceded the appearance of PS-positive cells. Caspase inhibition delayed both PS exposure and loss of plasma membrane integrity but did not prevent either. The results support the hypothesis that the sequence of apoptotic phenomena in an individual CAM-treated HL-60 cell depends on the stage of proliferation of that cell when it encounters the CAM. They are also consistent with the hypothesis that caspases are not required for PS exposure or the loss of plasma membrane integrity, but they are involved indirectly in promoting these phenomena.

King MA ，Radicchi-Mastroianni MA 《-》

There is substantial nuclear and cellular disintegration before detectable phosphatidylserine exposure during the camptothecin-induced apoptosis of HL-60 cells.

An early sign of apoptosis in many cells is the appearance of phosphatidylserine (PS) on the outside of the plasma membrane, whilst the cells still retain the ability to exclude DNA-binding molecules such as propidium iodide and 7-aminoactinomycin D (7-AAD). The protein annexin V binds preferentially to PS and has often been used to monitor the early phase of apoptosis. There have been some conflicting results concerning whether annexin V binds to camptothecin (CAM)-treated HL-60 cells, a commonly used model for apoptosis. We investigated the effects of culturing HL-60 cells for up to 8 h with a range of CAM concentrations. We used flow cytometry to measure cellular light scatter, annexin V-FITC binding, and 7-AAD uptake, and DNA content after fixation and permeabilization. We also used microscopy to examine the morphology of cells (both unsorted and sorted according to their light scatter) after cytocentrifugation. We found that CAM caused the rapid appearance of low light scatter apoptotic bodies. Even among cells with "normal" light scatter, there was widespread DNA cleavage and nuclear fragmentation by 3 h. The percentage of apoptotic bodies peaked at about 4 h and it was only afterward that annexin V binding could be detected to both intact cells and to apoptotic bodies. When they first appeared, the intact annexin V+ cells had S-phase DNA content. During CAM-induced apoptosis of HL-60 cells, the external exposure of PS can either precede or follow DNA cleavage, which suggests that PS exposure is not always an indicator of early apoptosis.

King MA ，Radicchi-Mastroianni MA ，Wells JV 《-》

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》

Intracellular determination of activated caspases (IDAC) by flow cytometry using a pancaspase inhibitor labeled with FITC.

Flow cytometry-based methods of simultaneous detection of multiple activated caspases in single cells are of diagnostic value and remain to be fully explored. Genomic DNA fragmentation was determined by agarose gel electrophoresis and flow cytometry. Whole cells were incubated with the cell-permeable pancaspase inhibitor, FITC-Val-Ala-Asp-fluoro-methyl-Rerone (FITC-VAD-FMK), and propidium iodide (PI) and analyzed for intracellular activated caspases and plasma membrane integrity, respectively, by flow cytometry. Two distinct populations of apoptotic cells were identified by flow cytometry: an early apoptotic population indicated by FITC-VAD-FMK binding and the late apoptotic cells characterized by FITC-VAD-FMK staining and permeability to PI. The binding of FITC-VAD-FMK to apoptotic cells was time dependent and concurred with DNA fragmentation. Pretreatment with the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-methyl-fluoro-methyl-kerone (Z-VAD (OMe)-FMK), coordinately attenuated apoptosis induction and activation of caspases. The pancaspase inhibitor also competitively blocked the binding of FITC-VAD-FMK to early apoptotic cells in vitro. Significantly, the use of FITC-VAD-FMK permitted apoptosis determination in a number of cell lines including the caspase-3 gene-deleted MCF-7 responding to various apoptotic stimuli. Apoptosis detection based on FITC-VAD-FMK binding is specific and easily performed by flow cytometry in a variety of cells. This novel technique has implications for detecting apoptosis in pathophysiologic conditions.

Jayaraman S 《CYTOMETRY PART A》