BimS-ER activation induced the mitochondrial pathway of apoptosis. Parental 293T and 293T cells stably expressing BimS-ER/GFP or BimSG68E-ER/GFP were treated with 200 nM of 4-hydroxytamoxifen (OHT). (A) Schematic representation of BimS-ER fusion protein. Shown are the BH3 domain, the C-terminal region of BimS (Cter), and the tamoxifen-binding region of the estrogen receptor (ER-tam). (B) Expression of WT but not G68E mutant BimS-ER confers the ability of OHT to induce Bax activation in 293T cells. Cells with cultured with or without OHT for the indicated times, and Bax activation was monitored by flow cytometry using an antibody (N20) specific for activated Bax. (C) WT, but not G68E, BimS-ER induces cytochrome c release. Cells were cultured for 6 h with or without OHT, then the cytosolic and mitochondrial fractions were analyzed for cytochrome c by immunoblot. (D and E) WT but not mutant BimS-ER induces hallmarks of apoptosis: PS externalization and mitochondrial depolarization. Parental 293T and derivatives stably expressing BimS-ER/GFP or the BH3-domain mutant BimS(G68E)-ER/GFP were treated with OHT or vehicle for the times indicated. Flow cytometry was used to assess the percentage of cells either positive for binding of annexinV (D) or negative for staining with TMRE (E). (F) WT but not mutant BimS-ER induces apoptotic DNA morphology and loss of mitochondrial ΔΨ_m (TMRE staining) as seen by laser confocal microscopy. Parental, BimS-ER/GFP or BimS(G68E)-ER/GFP 293T cells were incubated in the presence of OHT for 6 h and stained with both Hoechst 33258 (1 mg/ml) and TMRE (200 nM) for 10 and 20 min, respectively, at 37°C. Images were acquired using Zeiss Axiovert 200 imaging station. (G) Time course of caspase-dependent mitochondrial depolarization. 293T cells stably expressing BimS-ER or BimS(G68E)-ER were cultured with or without 200 nM of OHT and with or without caspase inhibitor zVAD-fmk (100 μM) for 3 or 6 h. TMRE retention (ΔΨ_m) was analyzed by flow cytometry. Error bars, SD from three independent experiments.

BimS-ER-induced MOMP did not trigger the release of AIF and endoG from mitochondria concomitantly with release of cytochrome c. BimS-ER/GFP–expressing cells were cultured with or without OHT in the presence or absence of zVAD-fmk for 24 h. (A) Lack of concomitant release of AIF with cytochrome c. In overlay images, green, red, and blue colors represent, respectively, the distributions of GFP, cytochrome c, and AIF. (B) Lack of concomitant release of endoG with cytochrome c. In overlay images, green, red, and blue colors represent, respectively, the distributions of GFP, cytochrome c, and endoG.

BimS-ER-induced MOMP leads to an early, but not immediate, loss of respiratory function. (A) Cellular amounts of mitochondrial proteins VDAC and Hsp60 remained constant for 24 h. Mitochondria were isolated from 293T cells stably expressing BimS-ER, untreated, or treated with OHT in the presence or absence of Q-VD for 24 h, and then immunoblots were probed with antibodies to VDAC or mitochondrial Hsp60. An amount of mitochondrial fraction corresponding to the same equivalent number of cells was loaded in each lane. (B) BimS-ER–expressing cell populations displayed a gradual loss of respiration after OHT addition. Respiratory rates were determined by polarography either in intact or digitonin-permeabilized cells, as indicated. Right panel, basal respiration (intact cells) or ADP-stimulated respiration (permeabilized cells); left panel, the maximal respiration after FCCP addition. Error bars, SD from three independent experiments. Note that these measurements were made with mixed live/dead cell populations, and thus these values represent an overestimate of the respiratory capacity of cells that underwent MOMP. (C) A strong and rapid decline of respiration rate follows MOMP. The percentages obtained in B were used to calculate the percentage of respiration rate remaining in cells that had undergone MOMP, based on the assumption that the cells that failed to release cytochrome c maintain their respiratory functions intact. For comparison, ATP levels from the same experiment (Figure 4B), corrected in a similar way for the percentage of cells undergoing MOMP, were included. Note that loss of respiration preceded ATP depletion. (D) A separate experiment evaluated the loss of activity of individual respiratory complexes (RCs). Shown is the percentage of apoptotic cells in this experiment, as measured by annexin V staining 8 and 24 h after OHT addition in presence or absence of Q-VD. (E) For the same experiment as shown in D, activities of Complex I, CI (top left), Complex II, CII (top right), and cytochrome c oxidase, COX (bottom left) activity on mitochondria isolated from 293T cells stably expressing BimS-ER treated or not with OHT in presence or absence of Q-VD for 8 and 24 h. Values were normalized to citrate synthase activity (CS), whose activity is not affected by the different treatments. Rotenone and potassium cyanide (KCN), specific inhibitors of CI and COX, respectively, were used as controls. The percentage of CI and COX activity inhibition was calculated compared with untreated samples and is presented in the bottom right panel. Note that MOMP in the presence of caspase inhibitor caused the loss of nearly all Complex I even as early as 8 h; ∼50% of COX activity was lost by 8 h, and this was unchanged at 24h, and Complex II activity was unaffected.

Directly-induced MOMP resulted in a delayed loss of mitochondrial proteins at different rates. (A) MOMP results in a loss of cytochrome c oxidase subunit II. At the indicated times after OHT addition, cells were fixed and coimmunostained for HtrA2/Omi and mitochondria-encoded cytochrome c oxidase subunit II. (B) Cells undergoing MOMP maintained some content of the mitochondrial matrix protein Hsp60 even at 72 h. Cells were treated as in A but coimmunostained for cytochrome c and Hsp60. The percentage of cells that had released cytochrome c but contained Hsp60 is shown at the bottom right of the right-most column.

Removing glucose after MOMP reduced cell accumulation, but did not alter the timing of cell cycle arrest. CFSE-stained BimS-ER/DsRed2–expressing cells were cultured with or without OHT in the presence of Q-VD-fmk for 8, 24, 48, and 72 h. (A) Eight hours after OHT addition, regular DMEM (25 mM glucose) was replaced by DMEM containing either 0, 0.1, 10 or 25 mM glucose. Cell proliferation was monitored by CFSE fluorescence intensity using flow cytometry. Similar results were obtained with the addition of 2-deoxyglucose to regular DMEM (not shown).

DNA synthesis and cell division continued during the first 48 h after BimS-ER activation, but then slowed. (A) DNA synthesis rate and cell cycle distribution were unaffected for the first 24 h. 293T cells stably expressing BimS-ER were cultured for the indicated times with or without 1 μM of OHT in presence or absence of 100 μM of zVAD-fmk. BrdU (10 μM) was added to the cells for the last hour of death induction. The percentage of cells in S phase in the whole population was then determined using an APC-conjugated anti-BrdU antibody by flow cytometry. (B) A subpopulation of cells exhibited gradually declining rates of DNA synthesis from 48 to 72 h after BimS-ER activation. Cells were treated as in A. Flow cytometry was used to measure BrdU incorporation versus total DNA content (cell cycle phase) in individual cells following OHT addition. The arrows mark a portion of the cells displaying a gradually declining rate of BrdU incorporation (DNA synthesis) throughout S phase. (C) Laser confocal microscopy confirmed that cells in which Htra2/Omi had been released from mitochondria (i.e., cells having undergone MOMP) were the same cells that displayed reduced BrdU incorporation at later time points. The assay was performed as in B, but after fixation, cells were costained for BrdU and HtrA2/Omi. (D) A subpopulation of cells underwent a delayed proliferative arrest ∼48 h after BimS-ER activation. 293T cells stably expressing BimS-ER/mtDsRed2 were stained with the stable plasma-membrane–associating dye CFSE and treated with vehicle or 1 μM of OHT in the presence or absence of zVAD-fmk or Q-VD for the times indicated. At each successive time point, CFSE fluorescence intensity was reduced, consistent with cell division, except in a portion of the cells indicated by red arrows, whose cytokinesis was evidently arrested or delayed.

BimS-ER-induced MOMP led to a gradual loss of mitochondrial polarization and cellular ATP content. 293T cells stably expressing BimS-ER/GFP were cultured with or without OHT in the presence of zVAD-fmk for the times indicated. (A) An increasing number of cells lost mitochondrial membrane potential (ΔΨ_m) over the course of 72 h after BimS-ER activation. Of the cells that had released cytochrome c, the percentage that retained TMRE staining, i.e., that were ΔΨ_m⁺, is indicated at the bottom right corners of the lower panels. (B). A steady decline of cellular ATP content after MOMP culminated in near depletion of ATP at 72 h. Left panel, ATP content in the bulk cell population, including cells that had not undergone MOMP. ATP content was determined in each sample by bioluminescence. Error bars, SD from three independent experiments each done in triplicate. Middle panel, percentage of cells that had undergone MOMP (cytochrome c release) at various times. At the indicated times after OHT addition, cells were fixed and immunostained for cytochrome c. More than 500 hundred cells were counted in each case (n = 2). Right panel, the percentages obtained in the middle panel were used to calculate the ATP content of cells that had undergone MOMP, based on the assumption that the cells that failed to release cytochrome c did not lose any ATP content.

Exogenous cytochrome c failed to reverse inhibition of respiratory function caused by MOMP. 293T BimS-ER/GFP cells were collected at various time points after treatment with vehicle (control), zVAD-fmk, or OHT plus zVAD-fmk. Cells were then treated with a low concentration of digitonin to permeabilize the plasma membrane, while the mitochondrial membranes remained intact (see Materials and Methods). (A) Representative oxygen consumption curves. Maximal respiration (in the presence of glutamate/malate and FCCP) was followed before and after the addition of exogenous cytochrome c in control, zVAD, or zVAD/OHT-treated digitonin-permeabilized cells. As a positive control, N/C-Bid (14 nM) was added to a separate sample of zVAD-treated cells 30 min after digitonin incubation. Numbers under the curves indicate respiratory rates (nmol O₂/min/10⁶ cells). As expected, respiration in the control cells was not stimulated by exogenous cytochrome c, as MOMs were intact. In contrast, N/C-Bid caused permeabilization of the MOM, and exogenous cytochrome c was therefore able to gain access to respiratory complexes to stimulate respiration. (B) Summary of effects of cytochrome c on respiration, expressed as a percentage of the maximal increase expected, based on the measured percentage of cells having undergone MOMP. Exogenous cytochrome c partially restored respiration 4 h after OHT addition, but not at later times. Data show the average change in the rate of maximal respiration after addition of exogenous cytochrome c, measured 4, 8, 24, and 72 h after zVAD/OHT treatment. Error bars, SD from three to five independent experiments.

Blocking mitochondrial, but not glycolytic, function resulted in an immediate arrest of cellular proliferation. (A) CCCP, rotenone, and oligomycin halt proliferation immediately. Twenty-four hours after CFSE staining, 293T cells were incubated with vehicle or CCCP (1 or 5 μM), rotenone (1 μM), oligomycin (1 μg/ml), or both rotenone (1 μM) and oligomycin (1 μg/ml). Cell proliferation and ΔΨ_m loss were monitored by CFSE fluorescence intensity (left panel) and TMRE staining (right panel), respectively, using flow cytometry. Note that oligomycin by itself did not dissipate ΔΨ_m but nevertheless blocked cellular proliferation. (B) Inhibition of glycolysis had no effect on cell proliferation over 72 h. CFSE-stained cells were cultured either in DMEM deprived of glucose, in the presence or absence of additional sodium pyruvate (1 mM) or in regular DMEM with or without addition of 1 or 5 mM of 2-deoxyglucose. Cell proliferation was monitored by CFSE fluorescence intensity using flow cytometry.