Adenine nucleotide (ADP/ATP) translocase 3 participates in the tumor necrosis factor induced apoptosis of MCF-7 cells - PubMed (original) (raw)

Adenine nucleotide (ADP/ATP) translocase 3 participates in the tumor necrosis factor induced apoptosis of MCF-7 cells

Ziqiang Yang et al. Mol Biol Cell. 2007 Nov.

Abstract

Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion-mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-alpha-induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress-induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-alpha-induced apoptosis observed in ANT3 mutant (ANT3(mut)) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type- and stimulus-dependent.

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Figures

Figure 1.

ANT3 mutation in MCF-7 cells led to a resistance to TNF-α–induced cell death. (A) Parental wild-type (WT) and ANT3mut MCF-7 cells were treated with TNF-α (50 ng/ml) for different periods of time, and cell survival rates were measured by PI exclusion. (B) WT and ANT3mut cells were treated with TNF-α (50 ng/ml) + CHX (1 μg/ml) for different periods of time, and cell survival rates were measured by PI exclusion. (C) Southern blot analysis of genomic DNA digested with EcoRI and XmnI. A probe containing the exon 4 sequence of ANT3 was used. (D) Semiquantitative RT-PCR was performed using the total RNA from WT and ANT3mut cells to determine the mRNA levels of ANT1, ANT2, and ANT3. GAPDH was used as control. An ethidium bromide stain is shown. (E) siRNA of ANT2 or ANT3 was used to treat WT and ANT3mut cells for 3 d, and the ANT3 or ANT2 protein levels were analyzed by WB using anti-ANT antibody. (F) ANT3mut cells were infected with a lentivirus encoding GFP or ANT3, and the resulting cells were designated as ANT3mut+GFP or ANT3mut+ANT3, respectively. Thirty-six hours after infection, these cells were treated with TNF-α (50 ng/ml) for different periods of time, and cell survival rates were measured by PI exclusion. ANT3 mRNA levels in these cells were determined by semiquantitative RT-PCR. The ectopic expression of ANT3 was confirmed by Western blotting with anti-ANT antibody. (G) WT cells were infected with lentiviruses encoding siRNA targeting ANT3 or a control lentivirus. The resulting cells were designated as siANT3 or siVector, respectively. Forty-eight hours after infection, these cells were treated with TNF-α (50 ng/ml) for different periods of time, and the cell survival rates were measured. ANT3 mRNA levels in these cells were determined by semiquantitative RT-PCR. (H) The same as E except siANT2 was used. Comparable results were obtained in 2–5 independent experiments. The representative data are shown.

Figure 2.

ANT3mut MCF-7 cells are resistant to oxidative stress–induced cell death. WT and ANT3mut cells were either treated with different concentrations of H2O2 (A), DQ (B), vincristine (D), mitomycin (E), 5-fluorouracil (F), or valinomycin (H) for 18, 24, 96, 96, 96, and 72 h, respectively, or they were incubated with glucose-free medium (C) or 20 μM oligomycin (G) for different periods of time. Cell survival rates were measured by PI exclusion. (I) ANT3mut cells were infected with a lentivirus encoding GFP or ANT3. Thirty-six hours after infection, these cells were treated with TNF-α (50 ng/ml), H2O2 (1 mM), or glucose starvation for 24 h, and cell survival rates were measured by PI exclusion. *p < 0.005, ** p < 0.05 (Student's t test). Data are the mean ± SD of triplicate samples.

Figure 3.

ANT3 mutation–mediated death resistance is not related to cellular ATP levels or the NF-κB pathway. (A) WT and ANT3mut cells were left untreated (Ctrl) or were treated with TNF-α (50 ng/ml), DQ (0.5 mM), glucose starvation, or oligomycin (10 μM) for 12, 12, 9, and 9 h, respectively. Cells were collected and relative ATP levels were determined using the Enliten ATP assay kit (Promega). (B) WT and ANT3mut cells were treated with TNF-α (50 ng/ml) for different periods of time. NF-κB activity was measured by EMSA. Comparable results were obtained in three independent experiments. The representative data are shown.

Figure 4.

ANT3mut cells have impaired cyt c release in apoptosis. (A) WT and ANT3mut cells were treated with TNF-α alone (50 ng/ml) or TNF-α (50 ng/ml) + zVAD (1 μM) for different periods of time. Cell survival rates were measured by PI exclusion. (B) WT and ANTmut cells were treated with DQ (0.5 mM) alone or DQ (0.5 mM) + zVAD (1 μM) for different periods of time. Cell survival rates were measured by PI exclusion. (C) WT and ANT3mut cells were treated with TNF-α (50 ng/ml) for different periods of time. Cells were collected and subjected to fractionation. cyt c levels in the cytosol fraction were analyzed by Western blotting. β-actin levels were measured as a loading control. (D) WT and ANT3mut cells were treated with DQ (0.5 mM) for different periods of time. Cells were subjected to analysis of cyt c release as in C. Comparable results were obtained in 2–3 independent experiments. The representative data are shown.

Figure 5.

Comparison of Δψm changes and CsA's effect in WT and ANT3mut cells. WT and ANT3mut cells were treated with TNF-α (50 ng/ml; A) or DQ (0.5 mM; B) or were subjected to glucose starvation (C) for different periods of time. Cells were collected, and the mitochondria inner membrane potential (Δψm) in live cells (PI-negative cells) was analyzed. J-aggregate–positive cells are those with normal Δψm values. Cell death was measured by PI exclusion (D) or annexin staining (E) in WT and ANT3mut cells that were treated with or without TNF-α for 24 h in the presence or absence of 5 μM CsA. *p < 0.01 (Student's t test). Comparable results were obtained in two independent experiments. The representative data are shown.

Figure 6.

Disrupting Δψm by valinomycin overcomes the difference between WT and ANT3mut cells with respect to TNF-α–induced cell death. (A) WT and ANT3mut cells were treated with valinomycin (5 μg/ml) for different periods of time. Cell survival rates were measured by PI exclusion. (B) Δψm in cells described in A were analyzed as described in Figure 5. (C) WT and ANT3mut cells were treated with TNF-α (50 ng/ml) together with valinomycin (5 μg/ml) for different periods of time. Cell survival rates were measured. (D) Percentages of J-aggregate–positive cells in cells described in C. Comparable results were obtained in two independent experiments. The representative data are shown.

Figure 7.

ANT3 mutation in MCF-7 cells alters TNF-α and glucose starvation–induced ROS production. (A) WT and ANT3mut cells were treated with TNF-α (50 ng/ml) for different periods of time. Cells were collected, and live cells were gated for ROS analysis using HE or DCFH-DA. (B) WT and ANT3mut cells were changed to glucose-free medium for different periods of time. ROS levels were analyzed as in A. (C) siANT3- or control vector-treated MCF-7 cells were stimulated with TNF-α for time periods as indicated. ROS levels in live cells were analyzed using DCFH-DA. (D) The same as C except ANT3 was ectopically expressed in MCF-7 cells before the analysis. Comparable results were obtained in 2–3 independent experiments. The representative data are shown.

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Adenine nucleotide (ADP/ATP) translocase 3 participates in the tumor necrosis factor induced apoptosis of MCF-7 cells - PubMed (original) (raw)