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Molecular and Cellular Biology, February 2007, p. 1222-1235, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01535-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Loss of Mcl-1 Protein and Inhibition of Electron Transport Chain Together Induce Anoxic Cell Death{triangledown} ,{dagger}

Joslyn K. Brunelle,1 Emelyn H. Shroff,1 Harris Perlman,2 Andreas Strasser,3 Carlos T. Moraes,4 Richard A. Flavell,5 Nika N. Danial,6 Brian Keith,7 Craig B. Thompson,7 and Navdeep S. Chandel1*

Department of Medicine and Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611,1 Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, St. Louis, Missouri 63104,2 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia,3 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136,4 Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520,5 Department of Cancer Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115,6 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 191047

Received 17 August 2006/ Returned for modification 11 October 2006/ Accepted 27 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
How cells die in the absence of oxygen (anoxia) is not understood. Here we report that cells deficient in Bax and Bak or caspase-9 do not undergo anoxia-induced cell death. However, the caspase-9 null cells do not survive reoxygenation due to the generation of mitochondrial reactive oxygen species. The individual loss of Bim, Bid, Puma, Noxa, Bad, caspase-2, or hypoxia-inducible factor 1ß, which are potential upstream regulators of Bax or Bak, did not prevent anoxia-induced cell death. Anoxia triggered the loss of the Mcl-1 protein upstream of Bax/Bak activation. Cells containing a mitochondrial DNA cytochrome b 4-base-pair deletion ([rho–] cells) and cells depleted of their entire mitochondrial DNA ([rho0] cells) are oxidative phosphorylation incompetent and displayed loss of the Mcl-1 protein under anoxia. [rho0] cells, in contrast to [rho–] cells, did not die under anoxia. However, [rho0] cells did undergo cell death in the presence of the Bad BH3 peptide, an inhibitor of Bcl-XL/Bcl-2 proteins. These results indicate that [rho0] cells survive under anoxia despite the loss of Mcl-1 protein due to residual prosurvival activity of the Bcl-XL/Bcl-2 proteins. Collectively, these results demonstrate that anoxia-induced cell death requires the loss of Mcl-1 protein and inhibition of the electron transport chain to negate Bcl-XL/Bcl-2 proteins.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxygen deprivation (anoxia) occurs in a variety of disease states, including cardiovascular disease and cancer. Anoxia (0 to 0.5% O2), unlike hypoxia (0.5 to 3% O2), elicits cells to undergo an apoptotic cell death (37, 47). If cells are deprived of both oxygen and glucose, then a necrotic type of death occurs due to a complete lack of ATP (32). The intracellular signaling pathways regulating anoxia-induced cell death are not fully understood. Anoxic cell death occurs through the intrinsic apoptotic pathway (32). The intrinsic apoptotic pathway is initiated within the cell by a loss of the outer mitochondrial membrane integrity, leading to the redistribution of cytochrome c and other apoptotic regulatory proteins into the cytosol (55). Subsequently, cytochrome c in the cytoplasm interacts directly with Apaf-1, leading to the ATP-dependent formation of a macromolecular complex known as the apoptosome. This complex recruits and activates the aspartyl-directed protease caspase-9 (26, 60). Activated caspase-9 can activate caspase-3 and -7, resulting in the morphological features of apoptosis (45). Previous studies have shown that caspase-9 null cells survive anoxia-induced cell death (52). However, whether caspase-9 null cells can survive upon reoxygenation from anoxia is not known.

Bcl-2 family members are key regulators of the outer mitochondrial membrane integrity during cell death and can be divided into anti- or proapoptotic members (53). Antiapoptotic proteins include Bcl-2, Bcl-XL, and Mcl-1, which contain three or four conserved Bcl-2 homology (BH) domains. Overexpression of antiapoptotic Bcl-2 family members prevents death under conditions of oxygen deprivation (32, 50). Proapoptotic proteins can be separated into multidomain (BH1 to -3) and BH3-only categories. Multidomain proapoptotic proteins, including Bax and Bak, are sufficient to initiate the loss of outer mitochondrial membrane integrity, resulting in apoptosis. Bax translocates from the cytosol to the mitochondria during oxygen deprivation (46) In addition, bax–/–/bak–/– mouse embryonic fibroblasts (MEFs) are resistant to a variety of death stimuli, including oxygen deprivation (5, 32, 56). The upstream regulators of Bax and Bak are the BH3-only proteins (25). Recent studies have shown that BH3-only proteins bind pro- and antiapoptotic Bcl-2 family members with various specificities and selectivities (8, 22). The exact mechanism of direct activation of Bax and Bak by the BH3-only proteins remains unclear. One direct mechanism involves the factthat certain BH3-only proteins can interact with Bax and Bak as well as negate the antiapoptotic Bcl-2 family members to induce permeabilization of outer mitochondrial membrane (7, 22). Alternatively, the indirect mechanism involves BH3-only proteins binding to the antiapoptotic proteins, thereby derepressing Bax and Bak (8, 57). Activity of BH3-only proteins can be regulated by transcriptional and/or posttranslational mechanisms (41). It is presently unknown which BH3-only proteins act upstream of Bax or Bak in oxygen deprivation-induced cell death. Moreover, the mechanisms by which cells sense a loss of oxygen and activate BH3-only proteins, resulting in a Bax/Bak-dependent cell death, also remain unknown.

We have previously proposed that the mitochondrial electron transport chain is required for anoxic cell death. Cells depleted of mitochondrial DNA ([rho0] cells) are resistant to oxygen deprivation-induced cell death (32). The [rho0] cells lack a functional electron transport chain and rely on glycolysis for ATP generation. The [rho0] cells also fail to undergo cell death in response to classical electron transport chain inhibitors, such as cyanide (30). Thus, electron transport inhibition during anoxia could initiate cell death. An alternative explanation is that [rho0] cells rely primarily on glycolysis for energy and that this adaptation to glycolysis might prevent anoxia-induced cell death. Furthermore, it is not known whether anoxia fails to activate Bax or Bak in [rho0] cells. In the present study, we address three questions. (i) Do cells survive upon reoxygenation from anoxia in the presence of caspase inhibition? (ii) What are the BH3-only proteins involved in anoxia-induced cell death? (iii) Do [rho0] cells survive under anoxia due to an inability to inhibit the electron transport chain or due to adaptation to glycolysis?


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. MEFs, human lung epithelial A549 cells, and human 143B osteosarcoma cells were cultured in Dulbecco's modified essential medium (DMEM) with 4.5g/liter glucose, L-glutamine, and sodium pyruvate (Invitrogen). This was supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM HEPES. Cytochrome b null cells were cytoplasmic hybrids between [rho0] 143B cells and mitochondria containing a 4-base-pair deletion in the cytochrome b gene within mitochondrial DNA (42). These cells were cultured in DMEM as described above with 100 µg/ml uridine. [rho0] 143B cells were generously provided by Douglas Wallace (University of California, Irvine) and cultured in DMEM as described above with 100 µg/ml uridine (Sigma) and 50 ng/ml ethidium bromide (Sigma). Bid null MEFs were a generous gift from Stanley J. Korsmeyer (58). Wild-type and HIF-1B–/– MEFs were immortalized with large T antigen (28). Wild-type and caspase-2–/– MEFs were 3T3 immortalized (36). Wild-type, bax–/–/bak–/–, caspase-9–/–, bad–/–, and bid–/– MEFs were simian virus 40 transformed (27, 21). Wild-type and bim–/– lung fibroblasts were also simian virus 40 transformed (3). Wild-type, puma–/–, and noxa–/– MEFs were E1A and Ras transformed (54).

Oxygen conditions. Anoxic conditions (0% oxygen, 85% nitrogen, 10% hydrogen, and 5% carbon dioxide) were achieved in a humidified anaerobic workstation at 37°C. An anaerobic color indicator (Oxoid) confirmed anaerobicity of the chamber. Hypoxic conditions (1.5% oxygen, 93.5% nitrogen, and 5% carbon dioxide) were achieved in a humidified variable aerobic workstation (INVIVO O2; BioTrace), which contains an oxygen sensor that continuously monitors the chamber oxygen tension.

Measurement of cell death. Cell death was measured using a cytotoxicity detection kit (Roche Applied Science) according to the manufacturer's protocol. This kit is based on the measurement of lactate dehydrogenase (LDH) that is released into the medium by damaged cells. Cell death is presented as amount of LDH measured in the medium divided by the total LDH released after treatment with 1% Triton X-100. Apoptosis was detected by determining the percentage of cells that had condensed and hadfragmented nuclei by staining with Hoechst 33258 stain (1 µg/ml; Sigma) as previously described (32). All cell death results are from four independent experiments and are represented as the mean value ± standard error of the mean (SEM).

Colony survival assay. MEFs were seeded at a density of 100 cells/60-mm dish in DMEM. After 24 h, the cells were exposed to anoxia for 24 h and 48 h. Cells were reoxygenated to normal oxygen conditions at 37°C for 10 days before fixation in methanol and staining with 0.5% crystal violet. The percentage of survival is the ratio of cells exposed to anoxia to cells under normoxia times 100. Data are represented as means ± SEMs from three independent experiments.

Measurement of Bax activation. Bax activation was measured as previously published (29). Briefly, both adherent and nonadherent cells were collected. Adherent cells were removed using Cell Dissociation Solution Non-enzymatic (Sigma). Cells were fixed in 0.25% paraformaldehyde for 1 min and washed three times in phosphate-buffered saline (PBS). MEFs only were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD PharMingen clone 2.4G2) at a concentration of 1:100 for 15 min. All cells were incubated for 30 min with Bax antibody (BD PharMingen clone 6A7) at a concentration of 1:50 in PBS containing 100 µg/ml digitonin (Sigma) and then washed three times. Next, cells were incubated for 30 min with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse immunoglobulin G (BD PharMingen clone A85-1) at a concentration of 1:50 in PBS containing 100 µg/ml digitonin. Cells were washed two times in PBS and analyzed by flow cytometry.

Peptides. BH3 peptides were synthesized by the Tufts University Core Facility and purified by high-pressure liquid chromatography. The Bid BH3 amino acid sequence was EDIIRNIARHLAQVGDSMDR, and the mutant Bid BH3 sequence was EDIIRNIARHAAQVGASMDR. The Noxa BH3 peptide sequence was AELPPEFAAQLRKIGDKVYC. Eight D-arginine residues and a glycine linker residue were added to the amino termini of the peptides (25). The cell-permeative Bad BH3 peptide was purchased from Calbiochem.

Other reagents. MG132 (10 µM; Sigma), MitoQ (18), Eukarion-134 (EUK-134) (Eukarion, Inc.), Q-VD-OPH (Enzyme System Products at MP Biomedicals) were used. The human Mcl-1 cDNA cloned in the retrovirus LNCX was kindly provided by Aly Karsan (British Columbia Cancer Research Centre, Canada).

Western blotting. Cells were scraped and lysed using 1x cell lysis buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride, and the Bio-Rad protein assay was used to measure the protein concentration. Whole-cell lysates (50 µg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad) and transferred to a Hybond-ECL nitrocellulose membrane (Amersham). Membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 buffer. Primary antibodies used were Mcl-1 antibody 1 (Santa Cruz S-19) at 1:500, Mcl-1 antibody 2 (Rockland) at 1:10,000, and alpha-tubulin antibody (Sigma clone B-5-1-2) at 1:2,000. Secondary antibodies used were horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signaling) 1:1000. SuperSignal chemiluminescent substrate (Pierce) was used to develop the blot.

Real-time PCR. Total RNA was isolated from wild-type MEFs by using the Ambion RNAqueous-4PCR kit and the Bio-Rad Aurum total RNA minikit. cDNA was made using the Ambion RETROscript kit. The expression of Mcl-1 and housekeeping gene L19 was determined using SYBR green supermix (Bio-Rad) and Bio-Rad's iCycler iQ system. The mouse Mcl-1 primers used were 5'-CGA ACC ATT AGC AGA AAC TAT CAC (sense) and 5'-AAA CCC ATC CCA GCC TCT TT (antisense). The mouse L19 primers were 5'-CAT CAA GCG ATC AGG GAA TG (sense) and 5'-GAG GAT TAT ACA GTT CAA AGC AAA T (antisense). Data were analyzed by the Pfaffl method (38).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anoxia-induced cell death occurs through the intrinsic apoptotic pathway. To test whether the intrinsic apoptotic pathway regulates anoxia-induced cell death, wild-type, bax–/–/bak–/–, and caspase-9–/– MEFs were exposed to 0% oxygen for 24, 48, and 72 h. Wild-type MEFs underwent apoptosis in response to anoxia, while both bax–/–/bak–/– and caspase-9–/– MEFs were resistant (Fig. 1 A to D). Bax was activated in both wild-type and caspase-9–/– MEFs, indicating that Bax activation is upstream of caspase-9 (Fig. 1E). Bax activation was assessed by use of an antibody recognizing the proapoptotic competent form of Bax (29). These data indicate that anoxia-induced cell death occurs through the intrinsic apoptotic pathway.


Figure 1
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  		FIG. 1. Anoxia-induced cell death requires Bax or Bak and caspase-9. (A and B) Cell death of wild-type, bax–/–/bak–/–, and caspase-9–/– MEFs exposed to either 21% or 0% oxygen for 24, 48, and 72 h was assessed by LDH release. Mean values ± SEMs from four independent experiments are shown. (C and D) Percentage of apoptotic cells scored by Hoechst staining among wild-type, bax–/–/bak–/–, and caspase-9–/– MEFs exposed to 21% or 0% oxygen for 24 h or 72 h. (E) Bax activation in wild-type and caspase-9–/– MEFs exposed to 21% or 0% oxygen for 24 h.

 
Reoxygenation induces cell death in caspase-9–/– cells but not bax–/–/bak–/– cells. Cells exposed to sustained oxygen deprivation, such as tumor cells, eventually become exposed to oxygen again. To determine the effects of reoxygenation, bax–/–/bak–/– and caspase-9–/– MEFs were exposed to 0% oxygen for 48 h and subsequently exposed to 21% oxygen for 24, 48, and 72 h. While bax–/–/bak–/– MEFs survived reoxygenation for up to 72 h (Fig. 2A), caspase-9–/– MEFs began to die upon reoxygenation (Fig. 2B). We confirmed the ability of bax–/–/bak–/– MEFs to survive reoxygenation by demonstrating clonal survival of these cells under normoxia (Fig. 2C). The broad-spectrum caspase inhibitor Q-VD-OPH was used to test whether the activation of additional caspases contributed to the death of caspase-9–/– MEFs upon reoxygenation. Q-VD-OPH has been shown to be more effective in preventing caspase-dependent cell death than the commonly used inhibitor ZVAD-fmk, due to higher specificity and less toxicity to cells (6). While both 10 µM and 50 µM concentrations of Q-VD-OPH effectively inhibited the anoxia-induced cell death of wild-type MEFs (see Fig. S1A in the supplemental material), addition of Q-VD-OPH was not able to protect caspase-9–/– MEFs from death after reoxygenation (Fig. 2D). Thus, death of caspase-9–/– MEFs upon reoxygenation is independent of caspases.


Figure 2
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  		FIG. 2. Caspase-9 null cells die upon reoxygenation independent of caspases. (A and B) Cell death of bax–/–/bak–/– or caspase-9–/– MEFs exposed to 0% oxygen for 48 h and subsequently exposed to 21% oxygen for 24, 48, and 72 h was assessed by LDH release. Mean values ± SEMs from four independent experiments are shown. (C) Wild-type, bax–/–/bak–/–, or caspase-9–/– were exposed to 0% oxygen for 24 or 48 h and subsequently incubated under normoxia for 10 days. Mean values ± SEMs from three independent experiments are shown. (D) Cell death of caspase-9–/– MEFs exposed to 0% oxygen for 48 h and subsequently exposed to 21% oxygen for 24, 48, and 72 h was assessed by LDH release. Upon reoxygenation, fresh medium containing 10 µM and 50 µM of Q-VD-OPH was added to the cells.

 
Reoxygenation leads to ROS-dependent cell death in caspase-9–/– MEFs. We next examined whether reoxygenation-induced reactive oxygen species (ROS) were responsible for the cell death in caspase-9–/– MEFs. EUK-134, a synthetic superoxide dismutase and catalase mimetic, was utilized as a broad-based antioxidant to scavenge ROS. Indeed, the addition of 20 µM and 50 µM EUK-134 was able to decrease cell death in caspase-9–/– MEFs upon reoxygenation (Fig. 3A). To test whether mitochondrially derived ROS are responsible for the cell death in caspase-9–/– MEFs upon reoxygenation, the mitochondrion-targeted antioxidant MitoQ was utilized. The addition of 2 µM and 4 µM MitoQ to caspase-9–/– MEFs upon reoxygenation was able to prevent death (Fig. 3B). Furthermore, the addition of MitoQ improved caspase-9–/– MEF clonogeneic survival upon reoxygenation (Fig. 3C). Therefore, the death of caspase-9–/– MEFs upon reoxygenation is dependent on ROS production. By contrast, the cell death of wild-type MEFs under anoxia is not prevented by MitoQ (see Fig. S1B in the supplemental material), indicating that ROS are not required for anoxia-induced cell death. These data indicate that ROS are required only for the execution of cell death upon reoxygenation in caspase-9-deficient cells.


Figure 3
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  		FIG. 3. Caspase-9 null cells die upon reoxygenation due to mitochondrial ROS. (A and B) Cell death of caspase-9–/– MEFs exposed to 0% oxygen for 48 h and subsequently exposed to 21% oxygen for 24, 48, and 72 h was assessed by LDH release. Upon reoxygenation, fresh medium containing 5 µM and 20 µM of the synthetic superoxide dismutase and catalase mimetic EUK-134 or containing 2 µM and 4 µM of the mitochondrial targeted antioxidant MitoQ was added to the cells. Mean values ± SEMs from four independent experiments are shown. (C) caspase-9–/– MEFs were exposed to 0% oxygen for 24 or 48 h and subsequently incubated under normoxia for 10 days. MitoQ (2 µM) was added 2 h prior to reoxygenation. Mean values ± SEMs from three independent experiments are shown.

 
Individual loss of BH3-only proteins is not sufficient to prevent anoxia-induced cell death. The upstream signaling molecules necessary for Bax and Bak activation and release of cytochrome c from the mitochondria are the BH3-only proteins (52). Bim and Bid are BH3-only proteins that can bind prosurvival Bcl-2 family members and activate Bax or Bak (22, 25). Both bid–/– and bim–/– fibroblasts died at rates similar to those for wild-type cells under anoxia, indicating that the loss of Bim or Bid was not sufficient to prevent cell death (Fig. 4A and B). The loss of the BH3-only protein Puma, which binds to all the prosurvival Bcl-2 family members, was not sufficient to prevent anoxia-induced cell death (Fig. 4C). The BH3-only proteins Noxa and Bad negate prosurvival functions of Mcl-1 and Bcl-2/Bcl-XL, respectively. Neither noxa–/– nor bad–/– MEFs were protected from anoxia-induced cell death (Fig. 4D and E). The failure of individual BH3 proteins to prevent anoxia-induced cell death led us to examine whether a caspase-2 might be involved anoxic cell death. Caspase-2 has been implicated as an initiator caspase activating Bax in certain cell types (23). However, caspase-2–/– MEFs also displayed significant cell death under anoxia (see Fig. S2 in the supplemental material).


Figure 4
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  		FIG. 4. Individual loss of Bid, Bim, Puma, Noxa, Bad, and HIF-1 does not protect against anoxia-induced cell death. Cell death of wild-type, bid–/–, bim–/–, puma–/–, noxa–/–, bad–/–, and HIF-1ß–/– fibroblasts exposed to 21% or 0% oxygen for 24 and 48 h was assessed by LDH release. Mean values ± SEMs from four independent experiments are shown.

 
The transcription factor hypoxia-inducible factor 1 (HIF-1) has been suggested to mediate anoxia-induced cell death through transcriptionally increasing potential death factors, such as BNIP3, HGTD-P, RTP801, and Noxa (4, 14, 19, 24, 51). HIF-1 is a heterodimer consisting of {alpha} and ß subunits (49). To investigate whether HIF-1 was necessary for anoxia-induced cell death, we exposed HIF-1ß–/– MEFs to anoxia for 24 and 48 h. HIF-1ß–/– MEFs failed to activate HIF-1 transcriptional activity as assessed by a luciferase reporter assay (see Fig. S3 in the supplemental material). At 24 h there was a slight decrease in cell death in HIF-1ß–/– MEFs compared to wild-type cells. However, significant cell death was observed at 48 h in both wild-type and HIF-1ß–/– MEFs (Fig. 4F). Collectively, these results indicate that the individual loss of upstream regulators of Bax or Bak is not sufficient to prevent anoxia-induced cell death.

Anoxia diminishes levels of the Mcl-1 protein. Recent studies have indicated that a critical step in the activation of Bak is the loss of the antiapoptotic Bcl-2 family member Mcl-1 (10, 34, 57). Protein levels of Mcl-1 decreased upon exposure to anoxic conditions (0% oxygen) in wild-type MEFs (Fig. 5A). The decrease in Mcl-1 protein levels was detected at around 16 h, and levels were further decreased by 24 h of oxygen deprivation. In contrast, Bcl-XL levels did not change under anoxia (Fig. 5A). Mcl-1 levels did not change during normoxic (21% O2) or hypoxic (1.5% O2) conditions or under serum deprivation (see Fig. S4 in the supplemental material). The decrease in Mcl-1 protein levels under anoxia occurred in bax–/–/bak–/– MEFs, indicating that loss of the Mcl-1 protein is upstream of Bax or Bak activation (Fig. 5B). The decrease in Mcl-1 protein levels during anoxia was partially prevented by 10 µM MG132, a proteasome inhibitor (Fig. 5B). Furthermore, mRNA levels of Mcl-1 slightly increased under 0% or 1.5% O2 (Fig. 5C). To determine whether Mcl-1 protein was degraded faster under anoxia, we examined Mcl-1 protein levels in the presence of an inhibitor of protein synthesis, cycloheximide, after 16 h of anoxia. Mcl-1 protein levels diminished faster under anoxia than under normoxia (Fig. 5D).


Figure 5
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  		FIG. 5. Mcl-1 levels decrease upon exposure to anoxia. (A) Mcl-1 and Bcl-XL protein levels in wild-type MEFs exposed to either 21% O2 or 0% O2 were determined. (B) Protein levels of Mcl-1 in wild-type and bax–/–/bak–/– MEFs that were exposed to 0% oxygen for 8, 16, and 24 h in the presence and absence of 10 µM MG132, a proteasome inhibitor, were determined. (C) Real-time PCR analysis of Mcl-1 mRNA levels from wild-type MEFs that were exposed to 21%, 1.5%, or 0% oxygen for 8 and 16 h. (D) Wild-type MEFs were incubated for 16 h under anoxia, at which point they were treated with cycloheximide (5 µg/ml), and Mcl-1 protein levels were assessed by densitometry for the next 2 hours.

 
Mcl-1 mRNA has been reported to increase through HIF-1 under low-oxygen conditions (39). Nevertheless, Mcl-1 protein levels did decrease in HIF-1ß–/– MEFs under anoxia (Fig. 6A). Recent evidence suggests that Noxa is required for Mcl-1 degradation during UV radiation (57). However, Mcl-1 protein levels were still diminished in noxa–/– MEFs under anoxia (Fig. 6B). We attempted to test whether the overexpression of Mcl-1 would be sufficient to prevent anoxia-induced cell death. However, the exogenous addition of Mcl-1 protein also decreased under anoxia (Fig. 6C), and consequently the Mcl-1-overexpressing cells died under anoxia (data not shown). By contrast, the overexpression of the prosurvival Bcl-2 family member Bcl-XL can protect cells from anoxia, since it was not degraded in the absence of oxygen (see Fig. S5 in the supplemental material). Together these results indicate that anoxia triggers the loss of the Mcl-1 protein upstream of Bax or Bak activation, which does not require Noxa or HIF-1.


Figure 6
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  		FIG. 6. Noxa or HIF-1ß is not required for the anoxia-induced decrease in Mcl-1 protein. (A and B) Mcl-1 protein levels in noxa–/– or HIF-1ß–/– MEFs exposed to 21% or 0% oxygen for 8, 16, and 24 h were determined. (C) Wild-type MEFs were retrovirally infected with pLNCX vector containing human Mcl-1 or with the control LNCX vector and exposed to 21% or 0% oxygen for 8, 16, and 24 h. Endogenous (Endo.) mouse Mcl-1 is shown as well.

 
The electron transport chain is required for anoxia-induced cell death. The initiation of cell death under anoxia is ultimately triggered by an oxygen-sensing machinery that is able to detect changes in oxygen levels and trigger the activation of Bax or Bak. The mitochondrial electron transport chain would be a likely candidate for the oxygen-sensing machinery, since cytochrome c oxidase utilizes the majority of oxygen in cells. In the absence of oxygen, cytochrome c oxidase ceases to function and mitochondria become depolarized and do not generate ATP. Indeed cells undergo a Bax/Bak-dependent cell death due to inhibition of cytochrome c oxidase by azide (1 mM) under normoxia (Fig. 7A). To investigate whether a functional electron transport chain was required for anoxia-induced cell death, A549 cells were depleted of their mitochondrial DNA ([rho0]-A549 cells). The [rho0]-A549 cells lack a functional electron transport chain and rely on glycolysis for survival. Wild-type A549 cells died upon exposure to anoxia after 48 h, while [rho0] cells were markedly protected (Fig. 7B). Bax was activated in A549 cells but not in [rho0]-A549 cells exposed to anoxia (Fig. 7C). Treatment of A549 and [rho0]-A549 cells under normoxia with 50 µM of a peptide encoding the BH3 domain of Bid resulted in death, while a peptide with a mutated Bid BH3 domain was not able to induce cell death (Fig. 7D). These data indicate that the Bax/Bak apoptotic pathway was intact in [rho0]-A549 cells.


Figure 7
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  		FIG. 7. A549 cells depleted of mitochondrial DNA ([rho0] cells) fail to activate Bax and do not die during anoxia. (A) Cell death of wild-type and bax–/–/bak–/– MEFs exposed to 21% in the presence of 1 mM azide, a cytochrome c oxidase inhibitor, for 24 and 48 h was assessed by LDH release. (B) Wild-type A549 and A549-[rho0] cells were exposed to 21% or 0% oxygen for 48 h, and cell death was measured by LDH release. Mean values ± SEMs from four independent experiments are shown. (C) Wild-type A549 and A549-[rho0] cells were exposed to 21% or 0% oxygen for 24 h and analyzed for activation of Bax. (D) Wild-type A549 and A549-[rho0] cells were treated with 50 µM of Bid BH3 peptide or mutant (Mut) Bid BH3 peptide for 16 h, and cell death was measured by LDH release. Mean values ± SEMs from four independent experiments are shown.

 
Adaptation to glycolysis does not protect against anoxia-induced cell death. The adaptation to glycolysis could also explain the inability of [rho0] cells to undergo oxygen deprivation-induced cell death. We utilized 143B cytochrome b null cells to determine whether adaptation to glycolysis was responsible for the protection against oxygen deprivation-induced cell death. Cytochrome b null cells contain a 4-base-pair deletion within the gene carried by mitochondrial DNA and are termed [rho–] cells (42). Cytochrome b null cells are incompetent at oxidative phosphorylation and rely solely on glycolysis for ATP production. Both wild-type and cytochrome b null cells died at similar rates after 72 h of oxygen deprivation (Fig. 8A). Bax was activated in wild-type and cytochrome b null cells after 48 h of oxygen deprivation (Fig. 8C). Interestingly, [rho0] cells in the 143B cell background were protected from anoxia-induced cell death, indicating that the ability of electron transfer through the respiratory chain and not the ability to conduct oxidative phosphorylation was the major requirement for anoxia-induced cell death (Fig. 8B). Overexpression of Bcl-XL was able to protect both wild-type and cytochrome b null cells from oxygen deprivation-induced cell death after 72 h (see Fig. S6 in the supplemental material), indicating that these cells still died through an intrinsic apoptotic pathway. In addition, Mcl-1 protein levels significantly decreased after 16 and 24 h of oxygen deprivation in wild-type and cytochrome b null cells (Fig. 8D). These data are further supported by results indicating that cells deficient in mitochondrial complex I activity also died under anoxia (see Fig. S7 in the supplemental material). Similar to the case for cytochrome b null cells, a deficiency in mitochondrial complex I activity would render cells incompetent at oxidative phosphorylation. Therefore, adaptation to glycolysis does not protect against oxygen deprivation-induced cell death.


Figure 8
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  		FIG. 8. Cells containing a deletion in the cytochrome b gene ([rho–] cells) die normally during anoxia. (A) Wild-type and cytochrome (cyto) b null cells were exposed to 21% or 0% oxygen for 24, 48, and 72 h, and cell death was assessed by LDH release. Mean values ± SEMs from four independent experiments are shown. (B) Cell death of wild-type and [rho0] 143B cells exposed to 21% or 0% oxygen for 72 h was measured by LDH release. Mean values ± SEMs from four independent experiments are shown. (C) Wild-type and cytochrome b null cells were exposed to 21% or 0% oxygen for 24 h and analyzed for activation of Bax. (D) Mcl-1 protein levels in wild-type and cytochrome b null cells exposed to 0% oxygen for 8, 16, and 24 h were determined.

 
Loss of Mcl-1 protein coupled with negation of Bcl-2/Bcl-XL triggers anoxia-induced cell death. Previous studies have demonstrated that loss of Mcl-1 protein coupled with neutralization of other antiapoptotic Bcl-2 family members, such as Bcl-2, Bcl-XL, and Bcl-w, is sufficient to result in rapid cell death (57). Indeed, [rho0] cells exposed to the Noxa BH3 peptide do not undergo cell death (Fig. 9A). However, [rho0] cells exposed to the Bad BH3 peptide in combination with the Noxa BH3 peptide undergo cell death (Fig. 9A). The Noxa BH3 peptide binds to the Mcl-1 protein, and the Bad BH3 peptide binds to Bcl-2, Bcl-XL, and Bcl-w. [rho0] cells survived despite their decrease in the Mcl-1 protein levels (Fig. 9B). This indicates that these cells have sufficient prosurvival activity from the remaining antiapoptotic proteins, Bcl-2, and Bcl-XL, to prevent cell death. To test whether negating the residual prosurvival functions of Bcl-2/Bcl-XL would induce these [rho0] cells to die under anoxia, we treated these cells with the Bad BH3 peptide. The Bad BH3 peptide did not induce cell death in [rho0] cells under normoxia because Mcl-1 protein is present. However, the Bad BH3 peptide did induce cell death in [rho0] cells under anoxia, where Mcl-1 protein is not abundant (Fig. 9C). These data indicate that the loss of the Mcl-1 protein coupled with negation of Bcl-2/Bcl-XL prosurvival function is sufficient to induce cell death during anoxia.


Figure 9
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  		FIG. 9. Loss of Mcl-1 protein coupled with negation of Bcl-2/Bcl-XL triggers anoxia-induced cell death. (A) A549-[rho0] cells were treated with Noxa BH3 peptide (50 µM) and Bad BH3 peptide (50 µM) at 21% oxygen for 24 h, and cell death was assessed by LDH release. (B) Mcl-1 protein levels in wild-type A549 and A549-[rho0] cells exposed to 0% oxygen for 8, 16, and 24 h were determined. (C) A549-[rho0] cells were treated with the Bad BH3 peptide (50 µM) and exposed to 21% or 0% oxygen for 24 h, and cell death was assessed by LDH release. Mean values ± SEMs from four independent experiments are shown.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The molecular details underlying how cells decide to commit to die under anoxia is not fully understood. In the present study we demonstrate that cells under anoxia commit to the intrinsic apoptotic pathway. Anoxia activates Bax, and bax–/–/bak–/– MEFs are protected against anoxia-induced cell death. Interestingly, caspase-9–/– MEFs are also resistant to anoxia-induced cell death, despite their activation of Bax. However, caspase-9–/– MEFs, unlike bax–/–/bak–/– MEFs, did undergo cell death upon reoxygenation. The death upon reoxygenation in caspase-9–/– MEFs was independent of caspases but dependent on mitochondrial ROS. These results are consistent with growing evidence that indicates that apoptotic inducers can also initiate death that does not rely on caspase activation (9). An implication of these findings is that caspase inhibition downstream of Bax or Bak activation is not likely to be effective, since cells deprived of oxygen are ultimately replenished with oxygen. An example of this occurrence is in the development of a solid tumor (1, 33). Oxygen deprivation develops when the rate of cell growth and metabolism exceeds the supply of oxygen in solid tumors. The low oxygen level in the center of the tumor triggers angiogenesis. Therefore, tumor cells deficient in caspase-9 or Apaf-1 might be initially resistant to anoxia-induced death in the developing tumor, but the restoration of oxygen by angiogenesis is likely to induce death in these cells. The caspase-9 null cells are sensitive to reoxygenation after anoxia because these cells have their outer mitochondrial membrane disrupted. This would allow leakage of ROS during reoxygenation, resulting in ROS-dependent cell death. Indeed, the administration of mitochondrion-targeted antioxidants to caspase-9 prevents cell death upon reoxygenation. In contrast, Bax/Bak null cells are resistant to reoxygenation injury because they have an intact outer mitochondrial membrane which would buffer the release of damaging ROS during reoxygenation. Although caspase-9 has been implicated as a tumor suppressor, our data suggest that Apaf-1 or caspase-9 might not be a tumor suppressor, whereas Bax/Bak would be a tumor suppressor (11, 52). The loss of caspase-9 would not prevent reoxygenation injury, thus limiting tumor growth. This concept is consistent with observations that Apaf-1 and caspase-9 do not determine growth factor withdrawal or drug treatment-induced cell death, and they do not have an essential suppressive role in myc-induced lymphomagenesis (12, 48).

The best-described upstream regulators of Bax or Bak activation are BH3-only proteins or caspase-2 and p53. We have previously demonstrated that p53 is not required for anoxia-induced cell death (5, 32). In the present study, we found that loss of capsase-2 was not sufficient to prevent anoxia-induced cell death. Surprisingly, the individual loss of BH3-only protein Bim, Bid, Puma, or Noxa was also not sufficient to inhibit anoxia-induced cell death. Previous studies have indicated that the individual loss of these BH3-only proteins attenuates death against a variety of stimuli. For example, loss of Bim or Puma reduces cytokine deprivation-induced death (3, 16). Puma and Noxa are required for DNA damage-induced cell death (54). Puma also reduces endoplasmic reticulum(ER) stress-induced cell death (44). The observation that Bid is not required for anoxia-induced cell death indicates that death receptor-dependent activation of Bax and/or Bak is not likely an initiating mechanism of cell death. We also examined whether the transcription factor HIF-1 could also regulate anoxic cell death. HIF-1 has been shown to regulate numerous prodeath factors, such as BNIP3, HGTD-P, RTP801, and Noxa (4, 14, 19, 24, 51). However, the loss of HIF-1 transcriptional activity did not reduce anoxia-induced cell death. Based on these observations, we conclude that either multiple BH3-only proteins or a novel protein that either contains a BH3 domain or functions analogously to BH3-only proteins is required for anoxia-induced cell death.

Although cell death depends on the proapoptotic proteins Bax and Bak under anoxia, this study indicates that antiapoptotic proteins are also likely to be key regulators of cell death during anoxia. Among the antiapoptotic members of the Bcl-2 family proteins, the loss of Mcl-1 protein has been shown to be a critical regulator of cell death primarily due to DNA damage early in the signaling cascade (10, 34). Loss of Mcl-1 protein upon activation of the DNA damage response is thought to be mediated by the inhibition of synthesis and continuous degradation of Mcl-1 through its E3 ligase Mule (59). Furthermore, the BH3-only protein Noxa has been shown to be required for the loss of the Mcl-1 protein upon UV radiation-induced DNA damage (57). Glycogen synthase kinase-3 can also control MCL-1 stability by phosphorylation of serine 159 (31). Here, we found that anoxia decreases the Mcl-1 protein levels without affecting the mRNA levels. The loss of Mcl-1 protein is independent of Bax or Bak, as well as Noxa. The decrease in the Mcl-1 protein levels is not observed under hypoxia (1.5% O2), which does not trigger cell death (37, 47). The degradation of Mcl-1 is faster under anoxia. The mechanisms by which Mcl-1 protein levels decrease under anoxia is dependent on the proteasome. The cellular signals linking anoxia to proteasomal degradation of Mcl-1 protein remains unknown. A possibility is that anoxia could induce DNA damage which would result in proteasomal degradation of Mcl-1 protein. However, this is unlikely, since previous reports have indicated that anoxia does not induce detectable levels of DNA damage (15). Anoxia induces a replication arrest that results in phosphorylation of ATR/ATM targets such as p53 and H2AX (15). Another possibility for the loss of Mcl-1 protein is ER stress caused by anoxia (20). Although caspase-12 null MEFs are not protected from anoxia-induced cell death, the loss of PERK does impair cell survival under anoxia (2). Future studies will have to examine the relationship between Mcl-1 and ER stress.

We examined whether the loss of Mcl-1 protein under anoxia is related to inhibition of the mitochondrial electron transport chain. However, our data indicate that loss of Mcl-1 protein is independent of the mitochondrial electron transport chain, since the decrease in Mcl-1 protein levels is observed in the respiration-incompetent [rho0] cells. Interestingly, the [rho0] cells do not activate Bax or undergo cell death under anoxia despite the decrease in Mcl-1 protein levels. In the absence of a functional electron transport chain, such as in [rho0] cells, the loss of Mcl-1 protein does not trigger cell death due to other antiapoptotic Bcl-2 family members preventing Bax and Bak activation. This is consistent with previous reports demonstrating that the loss of Mcl-1 is not sufficient to initiate cell death due to the prevention of Bax and Bak activation by Bcl-XL (57). Indeed, the [rho0] cells underwent rapid cell death under anoxia only in the presence of a Bad BH3 peptide that binds to Bcl-2, Bcl-XL, and Bcl-w. Based on these observations, we propose a model for anoxic cell death involving two critical events: the loss of the Mcl-1 protein and inhibition of the electron transport chain that negates the function of additional antiapoptotic Bcl-2 family members (e.g., Bcl-XL) (Fig. 10).


Figure 10
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  		FIG. 10. Based on our current observations, we propose that cells exposed to anoxia activate Bax/Bak-dependent cell death through two critical steps: (i) decrease of the Mcl-1 protein and (ii) inhibition of the electron transport chain to diminish the prosurvival activity of Bcl-XL/Bcl-2.

 
How the electron transport chain would negate Bcl-2, Bcl-XL, and Bcl-w function remains unknown. One explanation could be that the respiration-incompetent [rho0] cells are resistant to anoxia-induced death due to adaptation to glycolysis. Glycolytic metabolism has been shown to regulate apoptosis through the regulation of Bcl-2 family members (13, 40, 43). However, the requirement of the electron transport chain in the initiation of cell death under anoxia does not require adaptation to glycolysis. This is supported by the observation that cytochrome b null cells underwent anoxia-induced cell death. These cells are adapted to glycolysis due to a lack of complex III activity. Thus, they are respiration deficient and oxidative phosphorylation incompetent. These cells have some residual electron transfer capacity through the respiratory chain (42). We speculate that during anoxia the residual electron transfer capacity ceases, which results in depolarization of mitochondrial membrane potential. This would release mitochondrial calcium in to the cytosol to negate Bcl-2/Bcl-XL proteins.

Our current findings have implications for cancer. Tumor cells that are distal from a blood vessel can develop regions of hypoxia and anoxia. Mcl-1 is known to be overexpressed in some types of human cancers (17). The high levels of the Mcl-1 protein in tumor cells would provide an advantage for these cells by allowing them to maintain viability under anoxic conditions as pending angiogenesis replenishes oxygen levels. Furthermore, the decrease in Mcl-1 levels in anoxic regions would make these tumor cells sensitive to small molecules capable of binding the other antiapoptotic Bcl-2 family members, such as Bcl-2, Bcl-XL, and Bcl-w. A recent study did demonstrate that the small molecule ABT-737 is capable of binding Bcl-2, Bcl-XL, and Bcl-w and displays antitumor activity as a single agent in certain cancer cells (35). It is conceivable that the low-oxygen environment of the tumor cells lowered Mcl-1 levels and cooperated with this small molecule to trigger tumor regression. In summary, our data indicate a mechanism by which loss of the Mcl-1 protein cooperates with electron transport inhibition to initiate cell death under anoxia.


   ACKNOWLEDGMENTS
 
This work was supported in part by National Institutes of Health grants (GM60472-07 and P01HL071643-03004) and an American Heart Association grant (0350054N) to N.S.C. J.K.B. and E.H.S. are supported by National Institutes of Health predoctoral training grants and grants CA009560 and HL076139, respectively. A.S. is supported by NHMRC (Canberra), NIH, the Leukemia and Lymphoma Society of America, and JDRF/NHMRC.

We thank I. de Coo (University Hospital Rotterdam) for the original cytochrome b mutant fibroblasts. We thank Aly Karsan for the LNCX containing the human Mcl-1 cDNA. We thank Michael Murphy for providing MitoQ.


   FOOTNOTES
 
* Corresponding author. Mailing address: McGaw Pavilion, 240 East Huron Avenue, M-334, Northwestern University Medical School, Chicago, IL 60611. Phone: (312) 503-2549. Fax: (312) 908-4650. E-mail: nav{at}northwestern.edu. Back

{triangledown} Published ahead of print on 4 December 2006. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, February 2007, p. 1222-1235, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01535-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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