Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death

Abstract

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-X(L) prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-X(L) to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F(1)F(0)-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-X(L), cells devoid of mitochondrial DNA (rho degrees cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax(-/-) bak(-/-) mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.

Figures

FIG. 1.

Bcl-XL prevents cell death in response to oxygen deprivation but not oxygen and glucose deprivation. (A) Rat1a fibroblasts stably transfected with a control vector (Neo) or Bcl-XL were exposed to 0% O2 for 24 and 48 h and assayed for LDH release, as described in Materials and Methods. (B) Neo control and Bcl-XL cells were exposed to 0% O2 for 24 h and assayed for cytoplasmic histone-associated-DNA fragments. (C) Neo control and Bcl-XL cells in glucose-free media supplemented with 12 mM 2-DOG were exposed to 0% O2 for 8 and 12 h and assayed for LDH release. (D) Neo control and Bcl-XL cells in glucose-free media supplemented with 2-DOG (12 mM) were exposed to 0% O2 for 12 h and assayed for cytoplasmic histone-associated-DNA fragments. (E) Percentage of apoptotic cells scored by Hoechst staining of Neo control and Bcl-XL cells exposed to 0% O2 with or without 2-DOG (12 mM) for 24 h. ∗, P < 0.05 compared with Neo control cells exposed to 21% O2.

FIG. 2.

Cells exposed to oxygen deprivation commit to death at the point of cytochrome c release. (A) Rat1a fibroblasts were exposed to 0% O2 for 8, 16, 24, and 48 h and assayed for LDH release. (B) Rat1a cells were exposed to 0% O2 for 8 and 16 h and then reintroduced into 21% O2 for 16 and 8 h, respectively, and assayed for LDH release. ∗, P < 0.05 compared with cells exposed to 21% O2. (C) Cytosolic fractions probed with either cytochrome c or cytochrome c oxidase subunit IV following 8 and 16 h of oxygen deprivation (0% O2) or 8 h of oxygen and glucose deprivation (0% O2 + 2-DOG) in Neo and Bcl-XL cells. The mitochondrial fraction was included as a control.

FIG. 3.

Caspase-9 is activated during oxygen deprivation. (A) Neo control and Bcl-XL-transfected cells were exposed to 0% O2 for 8 and 16 h or 0% O2 plus 2-DOG for 8 h and assayed for caspase-9 activity. Data are normalized to the Neo control cells at 21% O2. (B) Neo control and Bcl-XL cells were exposed to 0% O2 for 0, 8, and 16 h, and the levels of ATP were measured and standardized for ATP content at 0 h.

FIG. 4.

Akt does not prevent oxygen deprivation-induced cell death. (A) Rat1a cells stably transfected with a control vector (Puro) or Src myristoylated Akt (Myr-Akt) were exposed to 0% O2 for 24 and 48 h and assayed for LDH release. ∗, P < 0.05 compared with Puro control cells exposed to 21% O2. (B) Control, Myr-Akt, and Bcl-XL-transfected cells were exposed to 21 and 1.5% O2 for 24 h and assayed for VEGF release into the culture supernatant. ∗, P < 0.05 compared with Puro or Neo control cells exposed to 21% O2.

FIG. 5.

Oxygen deprivation-induced cell death requires functional electron transport and is dependent upon Bax or Bak. (A) Wild-type and ρ° HT1080 human fibrosarcoma cells were exposed to 0% O2 or doxorubicin (DOXO, 0.25 μg/ml) for 48 h and assayed for LDH release. (B) Wild-type, bax−/− bak−/−, and p53−/− MEFs were exposed to 0% O2 for 48 and 72 h and assayed for LDH release. The data are the means and standard errors of the means of six independent experiments. ∗, P < 0.05 compared with wild-type cells exposed to 21% O2.

FIG. 6.

Oxygen deprivation-induced cell death occurs independently of the mitochondrial PTP or ROS. Cell death was assayed by LDH release. (A) Rat1a fibroblasts were pretreated with the PTP inhibitors cyclosporine (CyA, 0.5 or 5 μM) and TPZ (5 μM) for 1 h, followed by exposure to 0% O2 for 48 h. (B) Rat1a fibroblasts were pretreated with cyclosporine (CyA, 0.5 μM). Subsequently, cells were exposed to thapsigargin (TG, 1.5 μM) for 1 h followed by a 6-h exposure to hydrogen peroxide (H2O2, 100 μM). (C) Rat1a fibroblasts were pretreated with ROS inhibitors MnTBAP (50 μM) and NAC (1 mM) and exposed to 0% O2 for 48 h. (D) HT1080 fibrosarcoma cells transfected with a control vector or with MnSOD and catalase targeted to the mitochondria (HT1080-MnSOD/mCAT) for 0% O2 for 48 h. ∗, P < 0.05 compared with cells exposed to 21% O2.

FIG. 7.

Bcl-XL prevents cell death by allowing glycolytic ATP to maintain a partial mitochondrial membrane potential via the F1F0-ATP synthase. (A) Neo and Bcl-XL-transfected cells were exposed to 0% O2 for 8, 16, 24, and 48 h, and the mitochondrial membrane potential (Ψ) was measured as described in Materials and Methods. A large fraction of Neo cells at 48 h were dead; therefore, Ψ was not measured. ∗, P < 0.05 for Bcl-XL cells compared with Neo cells. (B) Bcl-XL cells were exposed to 0% O2 for 22 h and subsequently incubated with the fluorescence probes TMRE and MITO for 2 h at 0% O2 in the presence of antimycin a (Anti, 1 μg/ml), 2-DOG (12 mM), aurovertin B (Auro, 30 μM), oligomycin (Oligo, 1 μg/ml), and atractyloside (Atract, 1 mM). ∗, P < 0.05 compared with Bcl-XL cells exposed to 0% O2. (C) Bcl-XL cells were exposed to 0% O2 for 48 h in the presence of antimycin a (Anti, 1 μg/ml), atractyloside (Atract, 1 mM), aurovertin B (Auro, 30 μM), and oligomycin (Oligo, 1 μg/ml) and assayed for LDH release. ∗, P < 0.05 compared with Neo control cells exposed to 21% O2.