Figure 1.
Stable BCL-2 overexpression in MCF10A cells induces an ABT-737-sensitive primed for death state.
(A) Representative data from a flow cytometry experiment to determine the percentage of control-transfected (MCF10A CON) or BCL-2 overexpressing (MCF10A BCL-2) cells stained positively with Annexin V (AnxV), propidium iodide (PI), or both following a 4 h treatment with vehicle (con) or ABT-737 (10 µM). Numbers indicate the percentage of cells in the gated populations. Cells to the right of the vertical gate were considered AnxV positive and cells above the horizontal gate were considered PI positive. (B) Early apoptosis, expressed as the percentage of PI negative cells (bottom two quadrants in A) that were AnxV-positive (bottom right quadrant) after 4 h of treatment with vehicle control (con), ABT-737, Q-VD (Q, 20 µM), or ABT-737+ QVD. (C) Cell death, expressed as percentage of PI positive cells after 4 h of the treatments in B. Results in B and C are mean ± SE of three experiments performed in triplicate. *p<0.05 for ABT-737-treated relative to control-treated; †p<0.05 for MCF10A BCL-2 relative to control-transfected cells. (D) Expression levels of BCL-2 and MCL-1 relative to β-actin loading control in MCF10A CON and BCL-2 cells. Numbers below protein bands are optical density values. Protein normalized to β-actin and BCL-2:MCL-1 ratios are plotted in the right panel for lanes 1–4.
Figure 2.
MCF10A BCL-2 mitochondria are selectively sensitive to ABT-737-induced cytochrome c release.
Cyt c was quantified by ELISA in the medium and cell lysate after a 28 min incubation of permeabilized cells with vehicle, ABT-737 (as indicated), or alamethicin (alm, 80 µg/ml). Succinate (5 mM, in the presence of rotenone, 0.5 µM), ADP (1 mM), and K2HPO4 (3.6 mM) were present to support mitochondrial respiration during the treatment. Results are expressed as % cyt c released into the medium compared to the total cyt c that was quantified (mean ± SD, n = 3). Cyt c was not detectable in the medium of permeabilized cells treated with vehicle.
Figure 3.
ABT-737 induces a BAX/BAK-dependent impairment of maximal O2 consumption rate in sensitive cells.
(A) Representative bioenergetic profiles of MCF10A control-transfected (CON) and BCL-2 overexpressing (BCL-2) cells treated with vehicle (con) or ABT-737 (ABT, 10 µM), oligomycin (oligo, 0.5 µg/ml), FCCP (1 µM), and antimycin A (AA, 10 µM) as indicated. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal O2 consumption. The dotted line indicates the time point used to calculate maximal OCR. The solid line with arrows illustrates the reduction in MCF10A BCL-2 maximal OCR with ABT-737. Representative traces are means from one experiment performed in triplicate. OCR is baseline-normalized to the point prior to vehicle or ABT-737 addition. (B) Maximal OCR following addition of ABT-737 as % vehicle control (see A). Results are mean ± SE of 3–4 experiments with 2–5 replicates per experiment. *p<0.05 for ABT-737-treated relative to control-treated; †p<0.05 for MCF10A BCL-2 relative to control-transfected cells. (C) Representative bioenergetic profiles of immortalized wild type (WT) and BAX/BAK knockout (KO) mouse embryonic fibroblasts (MEF) treated with vehicle (con) or ABT-737 (ABT, 10 µM), oligo (0.2 µg/ml), FCCP (1 µM, WT, 2 µM KO), and AA (1 µM) as indicated. Traces are means from one experiment with 4–5 replicates. OCR is baseline-normalized as in A. (D) Maximal OCR following addition of ABT-737 as % vehicle control. Results are mean ± SE of 3 experiments with 2–5 replicates per experiment. Maximal OCR was determined as in A and B. Optimal oligomycin and FCCP concentrations were determined by titration for each cell type. *p<0.05 for ABT-737-treated relative to control-treated; †p<0.05 for BAX/BAK KO MEF relative to WT.
Figure 4.
ABT-737-impaired maximal O2 consumption is rescued by exogenous cytochrome c.
MCF10A BCL-2 cells were exposed to ABT-737 (10 µM) or vehicle (con) for 30 min, followed by acute plasma membrane permeabilization by saponin (sap, 10 µg/ml) in the presence of the complex II substrate succinate (5 mM), the complex I inhibitor rotenone (0.5 µM), the uncoupler FCCP (1 µM), and the presence or absence of cytochrome c (cyt c, 100 µM). The caspase inhibitor Q-VD (20 µM), when present, was added 30 min prior to ABT-737. (A) A representative experiment with treatments performed in triplicate. OCR is baseline-normalized to the point prior to vehicle or ABT-737 addition. (B) Quantification of the maximal OCR at the initial measurement point after permeabilization as a percentage of control (no ABT-737, Q-VD, or cyt c). Mean ± SE of 3–4 experiments with 2–3 replicates per experiment. *p<0.05 relative to the control treatment.
Figure 5.
A respiration-based microplate assay detects cell death priming at the level of the mitochondrion.
(A) MCF10A BCL-2 cells were exposed at the arrow to saponin (sap, 10 µg/ml) plus succinate (5 mM), rotenone (0.5 mM), ADP (1 mM), K2HPO4 (3.6 mM), and ABT-737 (ABT), vehicle (con), or alamethicin (alm, 80 µg/ml). Numbers in legend correspond to ABT-737 concentration in µM. (B) MCF10A CON cells receiving the treatments described in A. (C) MCF10A BCL-2 cells were exposed to saponin (10 µg/ml) plus succinate (5 mM), rotenone (0.5 mM), ADP (1 mM), and K2HPO4 (3.6 mM), followed by ABT-737 (ABT, 10 µM) or vehicle (con), cyt c (100 µM) or con, and sodium azide (5 mM). (D) MCF10A CON cells receiving the treatments described in C. All results are means from one experiment in triplicate and are representative of at least three independent experiments. OCR is baseline-normalized to the point prior to saponin addition.
Figure 6.
The BAX inhibitor propranolol delays the ABT-737-induced decline in respiration.
(A) MCF10A BCL-2 cells were permeabilized as in Figure 5 in the presence or absence of propranolol (PL), and then exposed to ABT-737 (10 µM) or vehicle (con). Results are normalized to OCR just prior to ABT-737 or vehicle addition and are means from one experiment in triplicate. (B) Quantification of the experiment depicted in A. OCR at the second point after ABT-737 or con addition (arrowhead in A) is expressed as a percentage of control (no ABT-737 or PL). Mean ± SE of 3–4 experiments with 2–3 replicates per experiment. *p<0.05 relative to the control treatment; †p<0.05 relative to treatment with ABT-737 alone; #p<0.05 for 0.75 or 1 mM PL+ ABT-737 relative to 0.5 mM PL+ ABT-737.
Figure 7.
ABT-737 induces dose-dependent impairment of maximal O2 consumption rate in B-cell lymphoma cells.
(A–C) Representative bioenergetic profiles of SP53 (A), JeKo-1 (B), and WEHI-231 (C) cells treated with vehicle (con) or ABT-737 (ABT), oligomycin (oligo, 0.3 µg/ml), FCCP (1 µM for SP53 cells, 3 µM for JeKo-1 and WEHI-231 cells), and antimycin A (AA, 1 µM) as indicated. Optimal oligomycin and FCCP concentrations were determined by titration for each cell type. Pyruvate (10 mM) was added in combination with FCCP. Numbers in legends correspond to ABT-737 concentration in µM. Representative traces are means from one experiment performed in triplicate. OCR is baseline-normalized to the point prior to vehicle or ABT-737 addition. (D) Maximal OCR following addition of ABT-737 as % vehicle control. Maximal OCR was determined as in Fig. 3. Results are mean ± SE of 3 experiments with 2–3 replicates per experiment. *p<0.05 for ABT-737-treated relative to control-treated; †p<0.05 relative to WEHI-231 cells treated with the same concentration of ABT-737.
Figure 8.
Dose-dependent cytochrome c redistribution in SP53 cells but not WEHI-231 cells treated with ABT-737.
Saponin (10 µg/ml) was injected to liberate the contents of the cytoplasm into the assay medium after a 30 min incubation of intact cells with vehicle or ABT-737 (as indicated). Assays were conducted using the XF24 and the saponin injection corresponded to the time of oligomycin injection in Fig. 7. Cyt c was quantified by ELISA in the medium and cell lysate. Results are expressed as % cyt c released into the medium compared to the total cyt c that was quantified (mean ± SD, n = 3). (A) SP53 cells. (B) WEHI-231 cells.
Figure 9.
SP53 and JeKo-1 lymphoma cells exhibit a high BCL-2:MCL-1 ratio and a primed phenotype.
(A) Expression levels of BCL-2 and MCL-1 relative to β-actin loading control in B-cell lymphoma cell lines. Mouse MCL-1 protein (WEHI-231) is known to migrate faster than the human form of the protein (SP53 and JeKo-1). (B) BCL-2 or MCL-1 protein levels normalized to β-actin and BCL-2:MCL-1 ratios following densitometric analysis of the immunoblots in A. Only the upper MCL-1 band corresponding to the full length protein was used for quantification. Results are mean ± SD, n = 3. *p<0.05 relative to WEHI-231 cells. (C) Early apoptosis, expressed as the percentage of PI negative cells that were AnxV-positive after 4 h of treatment with vehicle control or the indicated concentration of ABT-737. Results are mean ± SD of one experiment performed in triplicate. (D) Apoptosis, as determined by the percentage of sub-diploid nuclei. Results are mean ± SE of three (SP53) or five (JeKo-1 and WEHI-231) experiments performed in triplicate. *p<0.05 for ABT-737-treated relative to control-treated; †p<0.05 relative to WEHI-231 cells treated with the same concentration of ABT-737.
Figure 10.
Bioenergetics-based profiling predicts BAX/BAK-dependent cytochrome c release.
The schematic diagram and table (inset) outline drugs and drug targets used in the study. In bioenergetic-profiling assays, electrons are donated to the electron transport chain via complex I or complex II substrate (pyruvate or succinate, respectively). Cyt c (C), normally present in excess, is required to transfer electrons from complex III to complex IV. ABT-737 (or ABT-263) disrupts the binding of BCL-2 to BH3-only proteins (BH3) such as BIM on the mitochondrial outer membrane, enabling BAX/BAK activation, pore formation, and consequent cyt c release. The depletion of mitochondrial cyt c causes respiratory inhibition under conditions of high electron transport, such as in the presence of the uncoupler FCCP (Fig. 3–4,7) or a high ADP/ATP ratio (Fig. 5–6). Saponin permeabilizes the plasma membrane by extracting cholesterol, enabling the introduction of exogenous purified cyt c, which reverses respiratory inhibition due to cyt c release (Fig. 5). Propranolol, an inhibitor of BAX-induced cyt c release, delays the ABT-737-mediated loss of respiration (Fig. 6).