Application of Flow Cytometry to Determine Differential Redistribution of Cytochrome c and Smac/DIABLO from Mitochondria during Cell Death Signaling

Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK(-) cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK(-) cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.     

Relative timing of redistribution of cytochrome c and Smac/DIABLO from mitochondria during apoptosis assessed by double immunocytochemistry on mammalian cells

Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (< or = 15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.     

Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.     

Differentiation-induced HL-60 cell apoptosis: A mechanism independent of mitochondrial disruption?

HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells

Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.     

Cytochrome c is released in a single step during apoptosis

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).     

Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.     

Smac/DIABLO and cytochrome c are released from mitochondria through a similar mechanism during UV-induced apoptosis

During apoptosis, a key event is the release of Smac/DIABLO (an inhibitor of XIAP) and cytochrome c (Cyt-c, an activator of caspase-9) from mitochondria to cytosol. It was not clear, however, whether the releasing mechanisms of these two proteins are the same. Using a combination of single living-cell analysis and immunostaining techniques, we investigated the dynamic process of Smac and Cyt-c release during UV-induced apoptosis in HeLa cells. We found that YFP-labeled Smac and GFP-labeled Cyt-c were released from mitochondria in the same time window, which coincided with the mitochondrial membrane potential depolarization. Furthermore, using immunostaining, we found that the endogenous Smac and Cyt-c were always released together within an individual cell. Finally, when cells were pre-treated with caspase inhibitor (z-VAD-fmk) to block caspase activation, the process of Smac release, like that of Cyt-c, was not affected. This was true for both YFP-labeled Smac and endogenous Smac. These results suggest that in HeLa cells, both Smac and Cyt-c are released from mitochondria during UV-induced apoptosis through the same permeability transition mechanism, which we believe is triggered by the aggregation of Bax in the outer mitochondrial membrane to form lipid-protein complex.     

MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells

The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 microM), PD98059 (40 microM), or PD184352 (10 microM) with 10-100 microM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (DeltaPsi(m)), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and pro-caspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of DeltaPsi(m), implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells

Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.     

Tip30-induced apoptosis requires translocation of Bax and involves mitochondrial release of cytochrome c and Smac/DIABLO in hepatocellular carcinoma cells

TIP30 (Tat-interacting protein 30), a newly found proapoptotic factor, appears to be involved in multiple functions including metabolic suppression, apoptosis induction, and diminishing angiogenic properties. In the present study, we reported that mitochondrial events were required for apoptosis induced by TIP30 in hepatocellular carcinoma cells (HCC cells). Translocation of Bax was essential for TIP30-induced apoptosis, whereas overexpression of the anti-apoptotic protein Bcl-xL delayed both second mitochondria-derived activator of caspases (Smac/DIABLO) release and onset of apoptosis. Furthermore, TIP30-induced apoptosis was dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (z-VAD-fmk) blocked DNA fragmentation. Release of Smac/DIABLO from the mitochondria through the TIP30-P53-Bax cascade was required to remove the inhibitory effect of XIAP (X-linked Inhibitor of Apoptosis) and allowed apoptosis to proceed. Our results showed for the first time that Bax-dependent release of Smac/DIABLO, cytochrome c and AIF from the mitochondria mediated the contribution of the mitochondrial pathway to TIP30-mediated apoptosis. Our data suggested that adenovirus-mediated overexpression of TIP30 was capable of inducing therapeutic programmed cell death in vitro by activating the mitochondrial pathway of apoptosis. On the basis of these studies, elucidating the mechanism by which TIP30 induces cell death might establish it as an anticancer approach.     

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.     

Constitutive presence of cytochrome c in the cytosol of a chemoresistant leukemic cell line

The release of holocytochrome c (cyt c) from mitochondria into the cytosol is reportedly a landmark of the execution phase of apoptosis. As shown here, the P-glycoprotein- (P-gp) expressing K562/ADR cell line (but not the parental K562 cell line) exhibits both cytosolic and mitochondrial cyt c in the absence of any signs of apoptosis. K562/ADR cells were found to be relatively resistant to a variety of different inducers of apoptosis, and blocking the P-gp did not reverse this resistance. The release of cyt c in non-apoptotic K562/ADR cells was not accompanied by that of any other mitochondrial apoptogenic protein, such as AIF or Smac/DIABLO, and was inhibited by Bcl-2 over expression. In addition, using a cell-free system, we show that mitochondria isolated from K562/ADR cells spontaneously released cyt c. These data suggest that cyt c release may be compatible with the preservation of mitochondrial integrity and function, as well as cell proliferation.     

Osmostress-Induced Apoptosis in Xenopus Oocytes: Role of Stress Protein Kinases,Calpains and Smac/DIABLO

Hyperosmotic shock induces cytochrome c release and capase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.     

Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels

Many viruses belonging to diverse viral families with differing structure and replication strategies induce apoptosis both in cultured cells in vitro and in tissues in vivo. Despite this fact, little is known about the specific cellular apoptotic pathways induced during viral infection. We have previously shown that reovirus-induced apoptosis of HEK cells is initiated by death receptor activation but requires augmentation by mitochondrial apoptotic pathways for its maximal expression. We now show that reovirus infection of HEK cells is associated with selective cytosolic release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, but not the release of apoptosis-inducing factor. Release of these factors is not associated with loss of mitochondrial transmembrane potential and is blocked by overexpression of Bcl-2. Stable expression of caspase-9b, a dominant-negative form of caspase-9, blocks reovirus-induced caspase-9 activation but fails to significantly reduce activation of the key effector caspase, caspase-3. Smac/DIABLO enhances apoptosis through its action on cellular inhibitor of apoptosis proteins (IAPs). Reovirus infection is associated with selective down-regulation of cellular IAPs, including c-IAP1, XIAP, and survivin, effects that are blocked by Bcl-2 expression, establishing the dependence of IAP down-regulation on mitochondrial events. Taken together, these results are consistent with a model in which Smac/DIABLO-mediated inhibition of IAPs, rather than cytochrome c-mediated activation of caspase-9, is the key event responsible for mitochondrial augmentation of reovirus-induced apoptosis. These studies provide the first evidence for the association of Smac/DIABLO with virus-induced apoptosis.     

Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization.

Cleavage of Bid has been shown to promote apoptosis by inducing mitochondrial membrane permeabilization with the resultant release of apoptosis-inducing proteins from the intermembrane space into the cytosol. However, direct visualization of the Bid-induced release of various proteins from the highly compartmentalized intermembrane space and the changes in the mitochondrial metabolic machinery remain elusive. Using green fluorescent protein fusion proteins and immunostaining in individual permeabilized HepG2 cells, first we demonstrated that truncated Bid (15.5-kDa C-terminal fragment, tBid) evoked a rapid and essentially complete release of cytochrome c and Smac/DIABLO from every mitochondrion. To establish at a resolution of seconds the kinetics of tBid-induced cytochrome c and Smac/DIABLO release and depolarization, we monitored the mitochondrial membrane potential (DeltaPsi(m)) fluorimetrically in permeabilized cells and applied a rapid filtration method to obtain cytosolic fractions for Western blotting. We found that subnanomolar doses of tBid were sufficient to evoke cytochrome c release and mitochondrial depolarization, whereas full-length Bid was 100-fold less effective. Bcl-x(L) prevented tBid-induced cytochrome c release and depolarization. In response to 2.5 nm tBid, cytochrome c release started after a 10 s delay, displayed rapid progression, and was complete at 50-70 s. Release of Smac/DIABLO was synchronized with cytochrome c release, whereas the loss of DeltaPsi(m) lagged slightly behind cytochrome c release. Furthermore, tBid-induced cytochrome c release was insensitive to changes in substrate composition, but tBid-induced depolarization did not occur in the presence of extramitochondrial ATP supply. Thus, tBid-induced permeabilization of the outer membrane permits rapid release of cytochrome c and Smac/DIABLO from all domains of the intermembrane space. The tBid-induced loss of DeltaPsi(m) occurs after cytochrome c release and reflects impairment of oxidative metabolism.     

Novel anti-apoptotic effect of Bcl-2: prevention of polyamine depletion-induced cell death

The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the polyamine pools in the breast cancer cell line L56Br-C1 and induces apoptotic cell death via the mitochondrial pathway. In this study, we have over-expressed the anti-apoptotic protein Bcl-2 in L56Br-C1 cells and investigated the effect of DENSPM treatment. DENSPM-induced cell death was significantly reduced in Bcl-2 over-expressing cells. Bcl-2 over-expression reduced DENSPM-induced release of the pro-apoptotic proteins AIF, cytochrome c, and Smac/DIABLO from the mitochondria. Bcl-2 over-expression reduced the DENSPM-induced activation of caspase-3. Bcl-2 over-expression also prevented DENSPM-induced Bax cleavage and reduction of Bcl-X(L) and survivin levels. The DENSPM-induced activation of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase was reduced by Bcl-2 over-expression, partly preventing polyamine depletion. Thus, Bcl-2 over-expression prevented a number of DENSPM-induced apoptotic effects.