Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.     

Prostaglandin A2 activates intrinsic apoptotic pathway by direct interaction with mitochondria in HL-60 cells

HL-60 cells treated by prostaglandin (PG) A₂ showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA₂-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA₂-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA₂, mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA₂. Finally, it was shown that PGA₂-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA₂ activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA₂ plays a pivotal role.     

Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.     

Stabilization and translocation of p53 to mitochondria is linked to Bax translocation to mitochondria in simvastatin-induced apoptosis

Statins are cholesterol-lowing drugs with pleiotropic effects including cytotoxicity to cancer cells. In this study, we investigated the signaling pathways leading to apoptosis by simvastatin. Simvastatin induced cardinal features of apoptosis including increased DNA fragmentation, disruption of mitochondrial membrane potential (MMP), and increased caspase-3 activity by depleting isoprenoids in MethA fibrosarcoma cells. Interestingly, the simvastatin-induced apoptosis was accompanied by p53 stabilization involving Mdm2 degradation. The apoptosis was ameliorated in p53 knockdown clones of MethA cells as well as p53^−/− HCT116 cells. The stabilized p53 protein translocated to mitochondria with Bax, and cytochrome c was released into cytosol. Moreover, knockdown or deficiency of p53 expression reduced both Bax translocation to mitochondria and MMP disruption in simvastatin-induced apoptosis. Taken together, these all indicate that stabilization and translocation of p53 to mitochondria is involved in Bax translocation to mitochondria in simvastatin-induced apoptosis.     

SARS-CoV-2 membrane protein causes the mitochondrial apoptosis and pulmonary edema via targeting BOK

Deaths caused by coronavirus disease 2019 (COVID-19) are largely due to the lungs edema resulting from the disruption of the lung alveolo-capillary barrier, induced by SARS-CoV-2-triggered pulmonary cell apoptosis. However, the molecular mechanism underlying the proapoptotic role of SARS-CoV-2 is still unclear. Here, we revealed that SARS-CoV-2 membrane (M) protein could induce lung epithelial cells mitochondrial apoptosis. Notably, M protein stabilized B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) via inhibiting its ubiquitination and promoted BOK mitochondria translocation. The endodomain of M protein was required for its interaction with BOK. Knockout of BOK by CRISPR/Cas9 increased cellular resistance to M protein-induced apoptosis. BOK was rescued in the BOK-knockout cells, which led to apoptosis induced by M protein. M protein induced BOK to trigger apoptosis in the absence of BAX and BAK. Furthermore, the BH2 domain of BOK was required for interaction with M protein and proapoptosis. In vivo M protein recombinant lentivirus infection induced caspase-associated apoptosis and increased alveolar-capillary permeability in the mouse lungs. BOK knockdown improved the lung edema due to lentivirus-M protein infection. Overall, M protein activated the BOK-dependent apoptotic pathway and thus exacerbated SARS-CoV-2 associated lung injury in vivo. These findings proposed a proapoptotic role for M protein in SARS-CoV-2 pathogenesis, which may provide potential targets for COVID-19 treatments.Subject terms: Infectious diseases, Immunopathogenesis

In SARS-CoV-2-infected lung epithelial cells, endodomain of M protein binds to the BH2 domain of BOK and inhibits ubiquitination. BOK is stabilized and translocate to the mitochondrial outer membrane, promoting Cyt c release. Cyt c released outside the mitochondria activates CASP 9 mediated apoptosis, thereby inducing pulmonary edema.     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

Glucosamine sulfate–induced apoptosis in chronic myelogenous leukemia K562 cells is associated with translocation of cathepsin D and downregulation of Bcl-xL

Cathepsin D (cat D) reportedly plays an important role in certain apoptotic processes, the downstream pathways of which involve release of cytochrome c (cyt c) from mitochondria and activation of the caspase cascade. Previous studies revealed that the B-cell lymphoma 2 (Bcl-2) family members Bax or Bid play important roles in apoptotic signal transduction between cat D and mitochondria. Here, we show that glucosamine sulfate (GS) inhibits the proliferation and induces apoptosis of human chronic myelogenous leukemia K562 cells in vitro. GS interfered with the maturation of cat D. Activation of caspase-3, cleavage of poly-(ADP-ribose)-polymerase, release of cyt c, and downregulation of Bcl-xL accompanied GS-induced apoptosis, and these processes were inhibited by the cat D inhibitor pepstatin A. However, we did not detect any altered gene expression of Bcl-2, Bax, or Bid during apoptosis. Translocation of cat D from the lysosome to the cytosol was observed in GS-treated K562 cells. These findings suggest that GS-induced K562 cell apoptosis involves the translocation of cat D from the lysosome to the cytosol. Furthermore, our findings suggest that downregulation of Bcl-xL (but not Bcl-2, Bax, or Bid) connects cat D and the mitochondrial pathway, which causes the release of cyt c and activation of the caspase cascade during GS-induced apoptosis of K562 cells.     

Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-x_L markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-x_L co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.     

Calcium-sensing receptor activating phosphorylation of PKC�� translocation on mitochondria to induce cardiomyocyte apoptosis during ischemia/reperfusion

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that activates intracellular effectors; for example, it causes inositol phosphate (IP) and 1,2 diacylglycerol (DAG) accumulation, stimulating the release of intracellular calcium and the activation of the protein kinase Cs (PKCs). The activation of CaSR by ischemia/reperfusion (I/R) induces cardiomyocyte apoptosis through the mitochondrial apoptotic pathway; however, the underlying mechanisms remain unclear. In this study, rat hearts were subjected to 30 min of ischemia followed by 2 h of reperfusion in the presence of a CaSR activator, GdCl₃. Our results revealed that, under these conditions, the expression of CaSR was increased, the number of apoptotic cardiomyocytes was significantly increased (as shown by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay) and the cells with a typical apoptotic morphology were observed using transmission electron microscopy. Our data further showed that mitochondria isolated from hearts that had undergone I/R combined with GdCl₃ exhibited a significant increase in the translocation of phosphorylated PKC?? to the mitochondria, an increase in cytochrome c (cyt c) release from the mitochondria and a marked decrease in mitochondrial potential. The administration of rottlerin, an inhibitor of PKC??, significantly reduced reperfusion-induced apoptosis, phospho-PKC?? translocation to the mitochondria and the release of cyt c from the mitochondria. Thus, the involvement of CaSR in cardiac apoptosis through the mitochondrial pathway during I/R with GdCl₃ is related to phospho-PKC?? translocation to the mitochondria.     

Dissimilar mechanisms of cytochrome c release induced by octyl glucoside-activated BAX and by BAX activated with truncated BID

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAX_oligo) and by a combination of monomeric BAX (BAX_mono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, t^cBID). We also examined whether the effects induced by BAX_oligo or by BAX_mono activated with t^cBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that t^cBID plus BAX_mono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAX_oligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAX_oligo could be at least partially suppressed by mitochondrial depolarization. The effects of t^cBID plus BAX_mono were insensitive to depolarization. BAX_oligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K⁺ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAX_oligo, but failed to influence the effects produced by t^cBID plus BAX_mono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAX_oligo and by BAX activated with t^cBID.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

Swainsonine promotes apoptosis in human oesophageal squamous cell carcinoma cells <Emphasis Type="BoldItalic">in vitro</Emphasis> and <Emphasis Type="BoldItalic">in vivo</Emphasis> through activation of mitochondrial pathway

Swainsonine, a natural indolizidine alkaloid, has been reported to have antitumour effects, and can induce apoptosis in human gastric and lung cancer cells. In the present study, we evaluated the antitumour effects of swainsonine on several oesophageal squamous cell carcinoma cells and investigated relative molecular mechanisms. Swainsonine treatment inhibited the growth of Eca-109, TE-1 and TE-10 cells in a concentration-dependent manner as measured by MTT assay. Morphological observation, DNA laddering detection and flow cytometry analysis demonstrated that swainsonine treatment induced Eca-109 cell apoptosis in vitro. Further results showed that swainsonine treatment up-regulated Bax, down-regulated Bcl-2 expression, triggered Bax translocation to mitochondria, destructed mitochondria integrity and activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c, which in turn activated caspase-9 and caspase-3, promoted the cleavage of PARP, resulting in Eca-109 cell apoptosis. Moreover, swainsonine treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activation in xenograft tumour cells, resulting in a significant decrease of tumour volume and tumour weight in the swainsonine-treated xenograft mice groups compared with that in the control group. Taken together, this study demonstrated that swainsonine inhibited Eca-109 cells growth through activation of mitochondria-mediated caspase-dependent pathway.     

Protection of ginsenoside Rg1 on chondrocyte from IL-1β-induced mitochondria-activated apoptosis through PI3K/Akt signaling

Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Ginsenoside Rg1 protects cells by antagonizing apoptosis. This study aimed to investigate the protective effect of Rg1 on interleukin 1β (IL-1β)-induced chondrocyte apoptosis and the underlying molecular mechanisms. Chondrocytes were harvested from the joints of 1-week-old Sprague–Dawley rats. After treated with 10 μg/mL Rg1 for 2 h, the chondrocytes were cultured with 10 ng/mL IL-1β to induce cytotoxicity. Cell viability was assessed with MTT assays. Annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling were used to detect chondrocyte apoptosis. The contents of total Akt, phosphorylated Akt (p-Akt), Bcl-2, Bax, and cytochrome C (Cyt c) were determined by Western blotting assay. A quantitative colorimetric assay was used to determine caspase-3 activity. Our present findings have shown that pre-treatment of chondrocytes with Rg1 reduces IL-1β induced cytotoxicity/apoptosis. Rg1 pretreatment also decreases the activity of IL-1β that reduces expression of Bcl-2 and level of p-Akt, and increases Bax activity, Cyt c release, and caspase-3 activation. It also reverses the activity of IL-1β that reduces the expression of tissue inhibitor of metalloproteinase-1 expression and increased the synthesis of matrix metalloproteinase-13, with the net effect of inhibiting extracellular matrix degradation. These results indicate that Rg1 may protect chondrocytes from IL-1β-induced apoptosis via the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, through preventing caspase-3 release.     

Haplophytin-A induces caspase-8-mediated apoptosis via the formation of death-inducing signaling complex in human promyelocytic leukemia HL-60 cells

Haplophytin-A (10-methoxy-2,2-dimethyl-2,6-dihydro-pyrano[3,2-c]quinolin-5-one), a novel quinoline alkaloid, was isolated from the Haplophyllum acutifolium. In this study, we investigated the effect of haplophytin-A on the apoptotic activity and the molecular mechanism of action in human promyelocytic leukemia HL-60 cells. Treatment with haplophytin-A (50 μM) induced classical features of apoptosis, such as, DNA fragmentation, DNA ladder formation, and the externalization of annexin-V-targeted phosphatidylserine residues in HL-60 cells. In addition, haplophytin-A triggered the activations of caspase-8, -9, and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, haplophytin-A caused the loss of mitochondrial membrane potential (ΔΨ_m) and the release of cytochrome c and Smac/DIABLO to the cytosol, and modulated the expression levels of Bcl-2 family proteins. We further demonstrated that knockdown of caspase-8 using its siRNA inhibited the mitochondrial translocation of tBid, the activations of caspase-9 and caspase-3, and subsequent DNA fragmentation by haplophytin-A. Furthermore, haplophytin-A-induced the formation of death-inducing signaling complex (DISC) and then activated caspase-8 in HL-60 cells. During haplophytin-A-induced apoptosis, caspase-8-stimulated tBid provide a link between the death receptor-mediated extrinsic pathway and the mitochondria- mediated intrinsic pathway. Taken together, these results suggest that the novel compound haplophytin-A play therapeutical role for leukemia via the potent apoptotic activity through the extrinsic pathway, involving the intrinsic pathway.     

Inhibition of Nitric-Oxide-Mediated Apoptosis in Jurkat Leukemia Cells despite Cytochrome c Release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.     

The JNK inhibitor SP600125 enhances dihydroartemisinin-induced apoptosis by accelerating Bax translocation into mitochondria in human lung adenocarcinoma cells

The C-Jun N-terminal Kinase (JNK) inhibitor SP600125 is widely used to inhibit the JNK-mediated Bax activation and cell apoptosis. However, this report demonstrates that SP600125 synergistically enhances the dihydroartemisinin (DHA)-induced human lung adenocarcinoma cell apoptosis by accelerating Bax translocation and subsequent intrinsic apoptotic pathway involving mitochondrial membrane depolarization, cytochrome c release, caspase-9 and caspase-3 activation. The dynamical analysis of GFP-Bax mobility inside single living cells using fluorescence recovery after photobleaching revealed that SP600125 aggravated the DHA-induced decrease of Bax mobility and Bax translocation. These results for the first time present a novel pro-apoptotic action of SP600125 in DHA-induced apoptosis.     

Elucidating cytochrome C release from mitochondria: insights from an in silico three-dimensional model

Mitochondrial regulation of apoptosis depends on the programmed release of proapoptotic proteins such as cytochrome c (Cyt c) through the outer mitochondrial membrane (OMM). Although a few key processes involved in this release have been identified, including the liberation of inner membrane-bound Cyt c and formation of diffusible pores on the OMM, other details like the transport of Cyt c within complex mitochondrial compartments, e.g., the cristae and crista junctions, are not yet fully understood (to our knowledge). In particular, a remodeling of the inner mitochondrial membrane accompanying apoptosis seen in a few studies, in which crista junctions widen, has been hypothesized to be a necessary step in the Cyt c release. Using a three-dimensional spatial modeling of mitochondrial crista and the crista junction, model simulations and analysis illustrated how the interplay among solubilization of Cyt c, fast diffusion of Cyt c, and OMM permeabilization gives rise to the observed experimental release profile. Importantly, the widening of the crista junction was found to have a negligible effect on the transport of free Cyt c from cristae. Finally, model simulations showed that increasing the fraction of free/loosely-bound Cyt c can sensitize the cell to apoptotic stimuli in a threshold manner, which may explain increased sensitivity to cell death associated with aging.     

Bcl-2 and tBid proteins counter-regulate mitochondrial potassium transport

The mechanism of cytochrome c release from mitochondria in apoptosis remains obscure, although it is known to be regulated by bcl-2 family proteins. Here we describe a set of novel apoptotic phenomena—stimulation of the mitochondrial potassium uptake preceding cytochrome c release and regulation of such potassium uptake by bcl-2 family proteins. As a result of increased potassium uptake, mitochondria undergo moderate swelling sufficient to release cytochrome c. Overexpression of bcl-2 protein prevented the mitochondrial potassium uptake as well as cytochrome c release in apoptosis. Bcl-2 was found to upregulate the mitochondrial potassium efflux mechanism—the K/H exchanger. Specific activation of the mitochondrial K-uniporter led to cytochrome c release, which was inhibited by bcl-2. tBid had an opposite effect—it stimulated mitochondrial potassium uptake resulting in cytochrome c release. The described counter-regulation of mitochondrial potassium transport by bcl-2 and Bid suggests a novel view of a mechanism of cytochrome c release from mitochondria in apoptosis.     

Activation of the intrinsic and extrinsic pathways in high pressure-induced apoptosis of murine erythroleukemia cells

We previously demonstrated that caspase-3, an executioner of apoptosis, is activated in the pressure-induced apoptosis of murine erythroleukemia (MEL) cells (at 100 MPa). Here, we examined the pathway of caspase-3 activation using peptide substrates and caspase inhibitors. Using the substrates of caspases-8 and -9, it was found that both are activated in cells under high pressure. The production of nuclei with sub-G1 DNA content in 100 MPa-treated MEL cells was suppressed by inhibitors of caspases-8 and -9, and pan-caspase. In 100 MPa-treated cells, pan-caspase inhibitor partially prevented the cytochrome c release from the mitochondria and the breakdown of mitochondrial membrane potential. These results suggest that the intrinsic and extrinsic pathways are activated in apoptotic signaling during the high pressure-induced death of MEL cells.