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GROWTH, DIFFERENTIATION, AND APOPTOSIS
1Department of Biology, University of Maryland, College Park, Maryland; 2Department of Biomedical Engineering, Columbia University, New York, New York; 3Johns Hopkins University, School of Public Health, Baltimore, Maryland; and 4Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, Bronx, New York
Submitted 15 September 2006 ; accepted in final form 25 November 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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respiration; voltage-dependent anion-selective channel; apoptosis; cell death
Further research has demonstrated direct binding of G3139 to the mitochondrial surface and has correlated its potency to induce cytochrome c release with the level of voltage-dependent anion-selective channel (VDAC) expression in a cell-line dependent manner. G3139 has also been shown to inhibit the conductance of VDAC reconstituted into planar phospholipid membranes (21). As VDAC has been widely recognized as a participant in cell apoptosis, these findings suggest a new proapoptotic role for G3139.
VDAC is the major permeability pathway by which metabolites cross the mitochondrial outer membrane (9, 18, 33). There are claims that proapoptotic signals stimulate VDAC (alone or together with proapoptotic Bcl-2 family proteins) to form a larger channel by which cytochrome c is released into the cytosol (38, 39, 46). These proposals are in conflict with the physico-chemical properties of VDAC and, where tested, have been found to be incorrect (34, 35). Indeed, VDAC activity seems to be antiapoptotic. VDAC2 inhibits the activation of the proapoptotic protein Bak, suppressing its ability to induce apoptosis (5). An alternative proposal consistent with this view is that VDAC closure favors mitochondria-initiated apoptosis. VDAC, in its closed states, allows small ion flow but virtually excludes large highly charged metabolic anions, such as ADP and ATP (32). VDAC closure results in failure to exchange metabolites between the cytosol and mitochondria. This process, in a still undefined manner, favors the permeabilization of the mitochondrial outer membrane (MOM) and the release of cytochrome c and other proteins into the cytosol. Some of these proteins become components of the apoptosome (25), activating caspase-9 and initiating the apoptotic cascade.
The addition of G3139 to pure VDAC channels formed in phospholipid membranes results in channel closure (21). This demonstrates a direct effect of G3139 on VDAC in the absence of other proteins. Thus, G3139 is able to induce VDAC closure, in agreement with the hypothesis that VDAC closure favors apoptosis (44).
In this paper, we report a phosphorothioate-oligonucleotide-specific strong reduction of MOM permeability to ADP by G3139. This is consistent with the inferred in situ reduction of VDAC permeability and supports the hypothesis that G3139 induces cytochrome c release by closing VDAC.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To measure the respiration of mitoplasts, mildly shocked mitochondria were employed (23). These were generated by the addition to the mitochondrial suspension, 2 volumes of cold double-distilled water. The shocked mitochondria were incubated for 10 min on ice followed by the addition of 5 volumes of respiration buffer. Finally, 2 volumes of double-concentrated respiration buffer were added to restore normal osmotic pressure. These steps were used to minimize damage to the inner membrane.
Measurement of MOM intactness by adenylate kinase release assay. Mildly shocked mitochondria containing 1 mg protein were pelleted at 14,000 g for 5 min at 4°C, and the supernatant was kept on ice until assayed. Thirty microliters of supernatant was added to 700 µl of adenylate kinase reaction mixture (50 mM Tris·HCl, pH 7.5, 5 mM MgSO4, 10 mM glucose, 5 mM ADP, 0.2 mM NADP, 10 units of hexokinase, and 10 units of glucose-6-phosphate dehydrogenase) (40). The activity of adenylate kinase was detected as an increase in absorbance at 340 nm. Intact mitochondria and mitochondria with lysed outer membranes served as negative and positive controls. The mitochondria with lysed outer membranes were hypotonically shocked by adding 50 volumes of cold double-distilled water to the mitochondria.
The assay of protein content. Mitochondrial protein was measured using the BCA method (Pierce, Rockford, IL) following the addition of Triton X-100 to the mitochondrial suspension (1% vol/vol final). BSA was the standard.
Oligonucleotides. G3139 was kindly donated by Dr. R. Brown, Genta (Berkeley Heights, NJ). The N-mers, the oligonucleotides of random sequence, were a generous gift of Trilink Biotechnologies (San Diego, CA).
Planar phospholipid membrane studies. The planar phospholipid membranes were generated according to standard methods (8, 27). The membranes were formed from phospholipid monolayers consisting of diphytanoyl phosphatidylcholine, asolectin (soybean phospholipid polar extract), and cholesterol (1:1:0.1 mass ratios).
VDAC was purified from mitochondria isolated from rat liver (2, 13). A 1- to 3-µl aliquot of the VDAC-containing solution (2.5% Triton X100, 50 mM KCl, 10 mM Tris, 1 mM EDTA, 15% DMSO, pH 7.0) was stirred into 4–6 ml of aqueous solution containing 1.0 M KCl, 5 mM CaCl2, 1 mM EDTA, and 5 mM HEPES (pH 7.2) on the cis side of the chamber. The trans side, containing the same aqueous solution, was held at virtual ground by the voltage clamp. All of the experiments were performed at 
23°C.
Cell culture. The mycoplasma-free human melanoma cell line 518A2 was a kind gift of Dr. Volker Wacheck (University of Vienna, Vienna, Austria). Cells were grown in DMEM supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin G (sodium salt) and 100 µg/ml streptomycin sulfate. Stock cultures of all cells were maintained at 37°C in a humidified 5% CO2 incubator.
Subcellular fractionation and oligonucleotide treatment of mitochondria from melanoma cells.
Cells were harvested by trypsinization and were washed with cold PBS. Cell pellets were resuspended in 300 µl of buffer A (250 mM sucrose, 10 mM Tris·HCl, pH 7.4, 1 mM EGTA, 50 µg/ml Pefabloc, and 15 µg/ml leupeptin, aprotinin, and pepstatin). Cells were then homogenized on ice in a dounce homogenizer until 90% of cells were disrupted, as judged by Trypan blue staining. Crude lysates were centrifuged at 1,000 g for 10 min at 4°C twice to remove nuclei and unbroken cells. The supernatant was collected and subjected to a 10,000 g centrifugation for 30 min at 4°C. The pelleted mitochondria were resuspended in 20 µl of energizing buffer B (250 mM sucrose, 10 mM Tris·HCl, pH 7.4, 1 mM EGTA, 50 µg/ml Pefabloc, 10 mM KCl, 3 mM KH2PO4, 5 mM succinate, 100 µM ADP, and 15 µg/ml leupeptin, aprotinin, and pepstatin), which contained increasing concentrations of oligonucleotides (10 to 40 µM). After 2 h of incubation at 10°C water bath, samples were centrifuged to pellet the mitochondria, and the supernatant was colleted and subjected to Western blot analysis for cytochrome c release.
Western blot analysis. Aliquots of protein samples, containing 25–40 µg of protein, were resolved by SDS-PAGE, and then transferred to Hybond ECL filter paper (Amersham, Arlington Heights, IL). The filters were incubated at room temperature for 1–2 h in 5% milk in TBS containing 0.5% Tween 20. The filters were then probed with 1:200 dilutions of the anti cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 5% milk in TBS containing 0.5% Tween 20 at 4°C overnight. After being washed in TBS containing 0.5% Tween 20, the filters were incubated for 1 h at room temperature in 5% milk in TBS containing 0.5% Tween with a 1:3,000 dilution of a peroxidase-conjugated secondary antibody (Amersham). After the washing (3 x 10 min), ECL was performed according to the manufacturer's instructions.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 1, A and B, when 1 µM G3139 was added to isolated mitochondria, it reduced the rate of respiration in the transition between state III and state IV. The calculated rates of ADP consumption are shown in Fig. 1C. In the presence of G3139, the rate of ADP consumption saturates at more than 100 µM external ADP. Without G3139, the rate of ADP consumption essentially saturates at about 50 µM external ADP. At the lower ADP levels, which are more physiological, 1 µM G3139 reduced the rate of state III respiration by as much as 50% (Fig. 1C). The presence of adenylate kinase inhibitor, P1,P5-di(adenosine-5')pentaphosphate, does not change G3139 inhibition of mitochondrial respiration, showing that adenylate kinase is not involved (data not shown).
As VDAC is the major pathway for metabolite flux across the MOM (7, 9, 33), the reduced permeability should be due to VDAC closure. This was also observed in planar membrane experiments, which assess the direct interaction between G3139 and VDAC. Forty micromolar of G3139 were able to fully close a VDAC channel reconstituted into a planar membrane (Fig. 2). Channels closed in this way remain closed irrespective of the applied voltage. This effect can be reversed by removing G3139 from the system (Fig. 2), demonstrating a reversible interaction. The reversibility was also observed in mitochondrial respiration experiments (data not shown). The direct effect of G3139 on VDAC indicates that VDAC closure is responsible for the lowered respiration rate observed at low ADP levels. Closure of some of the VDAC channels slows down the permeation of ADP and decreases the intermembrane space and matrix ADP levels.
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A linear plot of Pclosed vs. Pclosed/[G] was generated to determine K and Pmax (Fig. 3, A and B). Ki is equal to 1/K. The inset of Fig. 3A shows the linearized results that yield a Ki of 0.18 µM and maximum permeability reduction of 85%.
G3139 also closes VDAC channels in a concentration-dependent manner (Fig. 3B). However, a higher concentration is needed for G3139 to induce the same extent of closure as observed in the mitochondrial experiments. The Ki of 9.6 µM is about 50 times greater, but the maximum conductance drop of 86% is indistinguishable in the two types of experiments (Fig. 3B, inset). The difference in sensitivity could be due to endogenous mitochondrial factors that influence the closure of VDAC, factors eliminated by VDAC purification and reconstitution into phospholipids membranes. One example of such a factor is the presence of modulating proteins in the mitochondrial intermembrane space that induce the VDAC closure by favoring the closed states (9). Despite the quantitative difference, both results are strikingly similar, thus providing evidence that G3139 is actually closing VDAC in the MOM.
G3139-induced mitochondrial respiration reduction is not due to release of cytochrome c. G3139 causes the release of cytochrome c from mitochondria, but this release takes time and is evident after 2 h (21). The permeability changes reported here are early and immediate effects. However, we observed that elevated levels of G3139 induced small decreases in light scattering at 600 nm, consistent with some mitochondrial swelling (Fig. 4A). Large-scale mitochondrial swelling is usually due to the formation of a permeability transition, and this could lead to outer membrane tearing and cytochrome c release2 . Loss of cytochrome c impairs electron transport and therefore respiration. However, at concentrations between 0.5 and 1 µM, G3139 does not induce significant swelling (n = 3, 95% confidence). Even at concentrations of 2 or 5 µM of G3139, the apparent swelling is very small compared with swelling induced by calcium ions, associated with the permeability transition. This slight change in light scattering is unlikely to reflect a matrix swelling that would tear the outer membrane and release cytochrome c (Fig. 4A).
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The effects of G3139 on the vital respiratory functions of the inner membrane were tested by measuring state III, state IV, and uncoupled respiration rates as a function of [G3139] (Fig. 5). Please note that true state III is only achieved, in the presence of G3139, at high levels of added ADP. We rationalize that the high levels overcome the reduced outer membrane permeability, allowing maximal ADP-dependent stimulation of respiration. Thus at 80 µM ADP, there is an apparent reduction in ADP-dependent respiration that probably arises from the reduced permeability of the outer membrane. Even at the high ADP levels used (as in Fig. 1A), G3139 reduces state III and increases state IV respiration, resulting in a reduced respiration control ratio. However, at 0.5 µM and 1 µM, these effects are not significant. The increase in state IV at higher concentrations is consistent with some uncoupling. The decrease of state III respiration rate was mainly caused by inhibition of the electron transport chain, confirmed by the carbonyl cyanide m-chlorophenylhydrazone (CCCP) uncoupled mitochondrial respiration rate (Fig. 5B). CCCP eliminates the protomotive force and should result in maximal rates of respiration. At 5 µM G3139 there was a significant drop in the rate of respiration in the presence of CCCP. However, even at a concentration of 5 µM, G3139 only influenced state III/state IV respiration by 20% compared with more than 80% reduction in MOM permeability (Fig. 5). Thus, effects observed at higher concentrations of G3139 cannot account for the permeability change (Fig. 5). Moreover, the ratio of state III to uncoupled rate does not change with increased amounts of G3139, indicating that ANT can fully function at least at high ADP concentrations (Fig. 5B).
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Further evidence that G3139 acts by reducing outer membrane permeability, was obtained from mildly shocked mitochondria. The mitochondrial outer membrane was damaged to bypass any permeability barrier of this membrane. Both mild hypotonic shock and carefully titrated amounts of digitonin were used for the initial trials. Of these, the hypotonic shock did the least damage, as assessed by measuring ADP-dependent respiration. There was an unavoidable reduction in respiration following hypotonic shock, and a trade-off was achieved between MOM breakage and respiration reduction. The effects of G3139 are clear despite this limitation. The state III/state IV respiration with and without G3139 is nearly the same, especially compared with the huge difference observed with the intact mitochondria (Fig. 6A). Even more persuasive, in the presence of 1 µM G3139, disruption of the outer membrane restored state III respiration to a level close to the rate observed without G3139, indicating that the major factor inhibiting respiration is the limitation of the rate of ADP transport through the outer membrane (Fig. 6A). However, it is clear that mildly shocking only partially restores the permeability value (Fig. 6B). Is this a sign that G3139 affects ANT? To address this quantitatively, one must compensate for the mitochondria that remained intact following the hypotonic shock. The adenylate kinase activity assay showed that 37% ± 1% (n = 4) of mitochondria were still intact after a mild shock. These remaining intact mitochondria should still respond to G3139 and thus there should still be a small reduction in MOM permeability upon addition of G3139. Assuming the remaining intact mitochondria were still inhibited by G3139, we calculated predicted values of changes in relative permeability upon the addition of G3139 (Fig. 6B). It is clear that at 0.5 and 1 µM concentrations, the restoration of permeability following hypotonic shock was complete (compare experimental values to predicted values). However, at 2 and 5 µM, G3139 had a greater inhibitory effect than can be accounted for by incomplete MOM damage and therefore, at these higher levels, G3139 does have effects on inner-membrane processes.
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Important features of the oligonucleotide. The isosequential phosphodiester analog of G3139 does not induce mitochondrial swelling, and it does not inhibit the respiration of mitochondria (Fig. 7). This agrees with our previous findings that this single-stranded oligodeoxyribonucleotide does not interact with VDAC and does not induce cytochrome c release from mitochondria (21). In fact, there is a small increase of the MOM permeability, which is understandable, considering that this highly charged molecule can decrease the voltage difference across the VDAC channels in the MOM (9) and thus could favor the open state.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fact that VDAC is the major pathway for metabolites indicates that G3139 reduces the permeability of the MOM by closing VDAC. This is supported by the fact that phosphorothioate oligodeoxynucleotides induce closure of VDAC reconstituted into planar phospholipid membranes.
The major effect of G3139 is on the MOM. We have demonstrated this in a variety of ways: 1) The G3139-induced reduction of permeability to ADP is not inhibited by cyclosporin A (Fig. 4B), indicating that it is not due to a permeability transition. 2) At concentrations less than or equal to 1 µM, the effect of G3139 on the MIM is insignificant (Figs. 5 and 6B), and even at higher concentrations, the effect on MIM cannot fully explain the permeability reduction. (Figs. 5, 6B). 3) Damaging the MOM actually restores state III respiration that was inhibited by 0.5 and 1 µM G3139, indicating that the inhibition is on the outer membrane, not on the ANT (Fig. 6A). 4) The constant ratio of state III to CCCP uncoupled respiration rate confirms that ANT was not inhibited by G3139 (Fig. 5B).
Additionally, the mitochondrial permeability reductions induced by G3139 were essentially the same when 5 mM malate and glutamate were used as the substrate (Fig. 9), showing that pyridine nucleotides were not depleted from the matrix during G3139 treatment.
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The mechanism and specificity of the oligonucleotide-induced permeability decrease. Our study has shown that the length of the oligonucleotide plays a role in its ability to affect the MOM permeability and cytochrome c release from mitochondria, while the sequence may be less important (Table 2). The presence of phosphorothioate linkages in the oligomer is very important for its ability to reduce the MOM permeability (Fig. 7) and subsequently induce cytochrome c release and cell apoptosis (21).
The importance of the phosphorothioate linkage has also been observed in other oligonucleotide-protein interactions. The binding of a phosphorothioate oligodeoxyribonucleotide to albumin increased with the number of phosphorothioate linkages (16). The binding of dA36 to the g5p protein increases more than 300-fold after substitution of sulfur for oxygen in an oligonucleotide (29).
The differences between sulfur and oxygen atoms may provide insight into the requirement for the phosphorothioate linkage. Compared with oxygen, the sulfur atom has less electronegativity, and its larger van der Waal's radius decreases its surface charge density, which may allow it to carry a full negative charge in solution. (12, 14). The sulfur also lacks the ability to hydrogen bond with water, and so water dissociates more readily from the oligonucleotide. This is critical because the binding energy is the difference between the energy of interaction between the protein and the oligonucleotide and the energies of dehydration of the interacting surfaces. The low-charge density of the sulfur also reduces the enthalpy needed to strip small ions before binding to VDAC (6). This property also increases the polarizability (37) of the sulfur atoms, strengthening the interaction with lower charge density groups found in proteins.
A simple explanation for the length dependence of the permeability decrease induced by the phosphorothioate oligonucleotides with a randomized sequence is the importance of sheer size. A larger inhibitor would generate a more effective steric block or electrostatic block of the channel. However, the reality may be more complex. For example, multiple interactions may be needed to stabilize the complex.
The results are consistent with a one-to-one interaction between G3139 and VDAC. This might indicate a single binding site on VDAC or that electrostatic repulsion precludes the binding of two oligonucleotides. The analysis is complicated by an incomplete reduction in permeability. The latter finding is in harmony with the finding that some VDAC channels are resistant to closure by G3139 (21).
The physiological conditions may modify the interaction. The ability of G3139 to influence mitochondrial respiration rates depends on the free [ADP]. At high ADP levels the effects of G3139 are quite small because the lower MOM permeability is compensated by the higher [ADP]. Inside the cell, more than half the ADP is bound to proteins (3, 4, 28). The free [ADP] from various cell types is not directly known but has been estimated to be 6–90 µM (3, 36, 45). The failure to detect the ADP by 31P-NMR argues for the real concentration to be in the low end of that range (3, 36). In any event, this range is within the concentration range at which G3139-induced MOM permeability reduction limits the rate of ADP phosphorylation.
G3139 induces reductions of MOM permeability at doses that are more than one order of magnitude lower than those needed to achieve comparable reductions in the conductance of a VDAC-containing membrane. These differences in sensitivity between experiments on isolated mitochondria and those performed on pure VDAC in phospholipid membranes indicate that physiological conditions may actually augment the interaction and/or magnify the effect (Fig. 3, A and B). The presence of regulatory proteins and other local factors that favor VDAC closure (9) may act synergistically with G3139. The voltage difference across MOM may also contribute to the stronger in vivo effect.
Differences in sensitivity also appear among different mitochondria isolated on different days. The Ki values vary between 0.2 to 0.5 µM. These differences could not be attributed to differences in weight/age of the animal. Also all animals were male and were on the same diet. Hence, the difference likely arises from differences in the physiological state of the animal: different levels of VDAC regulatory proteins in the intermembrane space, different levels of electrical potential across the outer membrane, or different amounts of Bcl-2 family proteins in the MOM. For example, Bcl-xL is antiapoptotic and has been reported to open VDAC channels and to increase the MOM permeability (44). There may be competition between G3139 and Bcl-2 family proteins.
The apoptotic implication of the interaction. VDAC's role in early apoptotic events is still controversial. There is evidence that VDAC is not involved in the apoptosis of Saccharomyces cerevisiae (17), but apoptosis in yeast is very different from apoptosis in multicellular organisms. Indeed, it is generally believed that VDAC is important in the initiation of apoptosis, but different and sometimes conflicting mechanisms have been proposed. Many reports claim that VDAC is a part of the permeability transition pore (PTP) (10, 26, 41, 42). The opening of PTP causes dissipation of the mitochondrial membrane potential and matrix swelling, which leads to cytochrome c release and cell apoptosis. Some claim that VDAC can oligomerize (46) or associate with Bax (39) to form a pathway for the release of proteins from mitochondria. We favor a third mechanism, which is that VDAC closure leads to the initiation of apoptosis (43). A drastic reduction in the rate of exchange of metabolites between the mitochondria and the cytosol leads to unidentified changes that result in the release of proteins from mitochondria. This hypothesis is supported by a variety of observations (34, 44) and is consistent with the present findings. G3139 closes VDAC and induces cytochrome c release and apoptosis in cells. The perfect match of the abilities of N-mers to decrease MOM permeability and to induce cytochrome c release from isolated mitochondria (Table 2, Fig. 8) further implicate VDAC closure as the initial step leading to cytochrome c release from mitochondria.
In conclusion, phosphorothioate oligonucleotides (n > 12) are able to reduce MOM permeability through closing VDAC channels. Our findings support the hypothesis that VDAC closure leads to apoptosis and that G3139 closes VDAC channels in the MOM. In the cell the situation is more complex. G3139 is more potent than a random sequence. Among the possible factors is the presence of CpG motifs (15). Therefore, the action of G3139 is multifaceted with VDAC closure being only one of these facets.
VDAC can be viewed as an antiapoptotic protein, whose open state guarantees the exchange of metabolites through the MOM and inhibits cell apoptosis. It is also a transducer of apoptotic signals, integrating the information and communicating it in the form of a change in MOM permeability (Fig. 10).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 P/O ratio is defined as ATP produced per mole of O consumed for a particular substrate. 
2 Even at the low ionic strength used, mild hypotonic shocks that damage the outer membrane result in reductions in the rate of state III respiration that can be reversed by addition of cytochrome c.
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