Release Of Cyt Research Articles

Vanadium compounds induced mitochondria permeability transition pore (PTP) opening related to oxidative stress

Vanadium compounds have been regarded as promising in therapeutic treatment of diabetes and in cancer prevention. In the present work, we studied the effects of vanadium compounds on mitochondria to investigate the mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with a variety of vanadium compounds, i.e. VOSO(4), NaVO(3), and vanadyl complexes with organic ligands. Our studies indicated that VO(2+), VO(3)(-), VO(acac)(2) and VOcit (1-100microM) could induce mitochondrial swelling in a concentration dependent manner and disrupt mitochondrial membrane potential (Deltapsi(m)) in a time dependent manner, which is quite different from the rapid Deltapsi(m) collapse caused by Ca(2+) or CCCP (carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupling reagent). Release of cytochrome c (Cyt c) was observed and could be inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (PTP). Interestingly, VOdipic caused release of Cyt c without mitochondrial swelling and Deltapsi(m) disruption, an action previously only observed on the Bax protein, suggesting a potentially role of VOdipic in regulating PTP opening. In addition, all the vanadium compounds tested stimulated mitochondrial production of reactive oxygen species (ROS). Antioxidants, i.e. vitamin C and E, significantly delayed the Deltapsi(m) disruption. Overall, our experimental evidence indicated vanadium compounds exhibited multiple actions on mitochondria. Vanadium compounds did induce oxidative stress on mitochondrial and thus caused PTP opening, which led to collapse of Deltapsi(m) and Cyt c release as the initiation of cell apoptosis.

Morin fosters apoptosis in experimental hepatocellular carcinogenesis model

Here we investigated the in vivo effect of morin (500 ppm in diet) in fostering apoptosis in diethylnitrosamine (DEN) (200 mg/kg bodyweight) mediated experimental hepatocellular carcinogenesis model. We analyzed the expression of cytosolic protein Akt and their important apoptotic downstream targets like caspase-9, Bcl-2, Bax, GSK-3β in vivo, by immunoblot analysis. In silico docking studies indicated that morin could serve as a better inhibitor than the classical PI3K inhibitor LY294002. The results obtained from in vivo studies confirm this. We also demonstrate here that morin's interaction with a defined set of amino acids of PI3K p110γ catalytic subunit resulted in the down-regulation of p-Akt Ser473, p-Akt Thr308 and total Akt causing the attenuation of its downstream targets in DEN-induced hepatocellular carcinoma. Further, morin caused the up-regulation of tumor suppressor PTEN, an important negative regulator of Akt, thus initiating apoptosis. Supplementation of morin to experimental animals modulated Bcl-2/Bax ratio causing the release of cyt C and up-regulation of caspase-3 and -9. Morin was also found to prevent the Akt-mediated suppression of GSK-3β possibly causing cell cycle arrest at the G1/S phase. These observations were supported by the DNA fragmentation and transmission electron microscopy results, which showed the occurrence of apoptosis. In conclusion, our findings demonstrate that morin begets apoptosis in DEN-induced hepatocellular carcinoma.

Dexamethasone-induced apoptosis and up-regulation of Bim is dependent on glycogen synthase kinase-3

Glucocorticoids are commonly used in the treatment of lymphoid malignancies. In this study, we show that apoptosis induced by dexamethasone (Dex), a synthetic glucocorticoid, was dependent on mitochondria, since overexpression of Bcl-XL prevented Dex-induced apoptotic changes. Dominant negative (DN) caspase-9 also prevented Dex-induced apoptotic changes including the loss of mitochondrial membrane potential indicating that caspase-9 controls mitochondrial changes. In addition, we evaluated the role of glycogen synthase kinase (GSK3) in Dex-induced apoptosis. Inhibition of GSK3 attenuated Dex-induced up-regulation of Bim, loss of mitochondrial membrane potential, release of cyt c and DNA fragmentation. These results indicate that GSK3 contributes to Dex-induced apoptosis by controlling up-regulation of Bim.

Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration

Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc1 complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s−1 whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s−1. Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.

Endomorphins and morphine limit anoxia–reoxygenation-induced brain mitochondrial dysfunction in the mouse

The protection of brain mitochondria from oxidative stress is an important therapeutic strategy against ischemia–reperfusion injury and neurodegenerative disorders. Isolated brain mitochondria subjected to a 5 min period of anoxia followed by 5 min reoxygenation mirrored the effect of oxidative stress in the brain. The present study attempts to evaluate the protective effects of endomorphin 1 (EM1), endomorphin 2 (EM2), and morphine (Mor) in an in vitro mouse brain mitochondria anoxia–reoxygenation model. Endomorphins (EM1/2) and Mor were added to mitochondria prior to anoxia or reoxygenation. EM1/2 and Mor markedly improved mitochondrial respiratory activity with a decrease in state 4 and increases in state 3, respiratory control ratio (RCR) and the oxidative phosphorylation efficiency (ADP/O ratio), suggesting that they may play a protective role in mitochondria. These drugs inhibited alterations in mitochondrial membrane fluidity, lipoperoxidation, and cardiolipin (CL) release, which indicates protection of the mitochondrial membranes from oxidative damage. The protective effects of these drugs were concentration-dependent. Furthermore, these drugs blocked the enhanced release of cytochrome c (Cyt c), and consequently inhibited the cell apoptosis induced by the release of Cyt c. Our results suggest that EM1/2 and Mor effectively protect brain mitochondria against oxidative stresses induced by in vitro anoxia–reoxygenation and may play an important role in the prevention of deleterious effects during brain ischemia–reperfusion and neurodegenerative diseases.

Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C(2)-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 micromol/L C(2)-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (DeltaPhi(m)). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C(2)-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that DeltaPhi(m) began to decrease from 6 h after 25 and 50 micromol/L C(2)-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the collapse of DeltaPhi(m) through regulating mitochondrial membrane permeability transition pore. There was no effect of C(2)-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 micromol/L C(2)-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C(2)-ceramide treatment. After the treatment with caspase inhibitor, C(2)-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C(2)-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.

Apoptotic inaccessibility with maturity

Mature neurons wrap up chromatin around death-sensitizing genes to prevent unwanted apoptosis, say Wright et al. Figure 1 Apaf-1 is acetylated and open in young neurons (P5) but methylated and silent in mature neurons (P28). Its derepression allows older neurons to apoptose (bottom, left to right) upon release of cyt c (white). Neurons die off by the handful during development, when their proliferating brethren can easily replace them. But once neurons mature, they shut off proliferation pathways, and survival becomes precious. One way older neurons become less sensitive to apoptosis is by preventing a protein called Bax from reaching mitochondria, where it pokes holes that let out cytochrome c (cyt c). The new findings identify an additional protective step downstream of cyt c release. Older sympathetic neurons survived injections of cyt c because they had less Apaf-1, a protein that recruits cyt c to the death-inducing apoptosome complex. Apoptosis, however, still killed mature neurons in response to DNA damage, the authors found. This death resulted from the return of Apaf-1. In young neurons, Apaf-1 expression is induced by a cell cycle–regulated transcription factor called E2F1. When neurons stop proliferating, E2F1 is shut down along with other cell cycle proteins. DNA damage restored E2F production. But the team found that giving cells E2F1 alone was not enough to force older, undamaged neurons to express Apaf-1. The cells also had to be prodded to open up the chromatin around the Apaf-1 promoter. In mature neurons, the histones at the Apaf-1 promoter were decorated with methyl groups that signify inaccessible, silent chromatin. The same promoter in young neurons was instead tagged with acetyl groups, which indicate accessibility. When mature neurons were given both E2F1 and drugs that open chromatin by inhibiting histone deacetylases, they again made Apaf-1 and underwent apoptosis in response to cyt c. Since similar drugs are used in chemotherapy, oncologists might want to keep an eye out for neuronal side effects. It is currently unclear how DNA-damaged neurons initiate pathways that unwrap chromatin around Apaf-1 (and possibly other apoptotic genes). And whether the same thing happens under pathological conditions such as a stroke or neurodegenerative disorders is unknown. Reference: Wright, K.M., et al. 2007. J. Cell Biol. 179:825–832. [PMC free article] [PubMed]

Mechanism of Cell Death Caused by Complex I Defects in a Rat Dopaminergic Cell Line

Defects in the proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria are linked to neurodegenerative disorders. The mechanism leading to cell death elicited by complex I deficiency remains elusive. We have shown that expression of a rotenone-insensitive yeast NADH-quinone oxidoreductase (Ndi1) can rescue mammalian cells from complex I dysfunction. By using the Ndi1 enzyme, we have investigated the key events in the process of cell death using a rat dopaminergic cell line, PC12. We found that complex I inhibition provokes the following events: 1) activation of specific kinase pathways; 2) release of mitochondrial proapoptotic factors, apoptosis inducing factor, and endonuclease G. AS601245, a kinase inhibitor, exhibited significant protection against these apoptotic events. The traditional caspase pathway does not seems to be involved because caspase 3 activation was not observed. Our data suggest that overproduction of reactive oxygen species (ROS) caused by complex I inhibition is responsible for triggering the kinase activation, for the release of the proapoptotic factors, and then for cell death. Nearly perfect prevention of apoptotic cell death by Ndi1 agrees with our earlier observation that the presence of Ndi1 diminishes rotenone-induced ROS generation from complex I. In fact, this study demonstrated that Ndi1 keeps the redox potential high even in the presence of rotenone. Under these conditions, ROS formation by complex I is known to be minimal. Possible use of our cellular model is discussed with regard to development of therapeutic strategies for neurodegenerative diseases caused by complex I defects.

Avicins, a novel plant-derived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane

Avicins are pro-apoptotic, anti-inflammatory molecules with antioxidant effects both in vitro and in vivo. Based on their ability to perturb mitochondrial functions and initiate apoptosis in tumor cells, we chose to study the bioenergetic effects of avicins on tumor cell mitochondria. Avicin-treated Jurkat cells, showed a decrease in the levels of cellular ATP as well as the rate of oxygen consumption. These effects on cellular metabolism appear to be a result of avicin’s actions on the outer mitochondrial membrane (OMM). We speculate that avicins might initially inhibit the exchange of metabolites across the OMM leading to its subsequent permeabilization to cytochrome c. This speculation is supported by biophysical studies using lipid bilayers, which suggest that upstream of these effects, avicins target and close the voltage dependent anion channel (VDAC). Closure of VDAC would lead to an overall lowering of the cell energy metabolism, subsequently pushing these cells towards the apoptotic pathway by permeabilization of the OMM and release of cyt- c. Avicins therefore not only represent a novel pharmacological tool for treatment of cancers, but also highlight the influence ancient plant metabolites could have on human health.

Cardiolipin deficiency releases cytochrome c from the inner mitochondrial membrane and accelerates stimuli-elicited apoptosis

Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.

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.

Formation of FGF-2-Apatite Composite Layer on Hydroxyapatite Ceramic

A FGF-2-apatite composite layer was formed on hydroxyapatite (HAP) ceramics using supersaturated calcium phosphate solutions containing various amounts of potassium chloride. The optimum conditions for the formation of the composite layer were determined by using cytochrome C (cyt C), one of the dummy proteins of FGF-2, and an immersion solution with a Ca/P molar ratio of 0.19 and a potassium chloride concentration of 8 mM. The amounts of cyt C and FGF-2 immobilized in the composite layer were 1.28±0.30 and 3.18±0.01 µg cm-2, respectively. The release of FGF-2 from the composite layer continued for at least 3 days. It was considered that the release period of FGF-2 could be extended to day 10 because the release of cyt C continued for at least 10 days. When the composite layer is formed on commercial bone substitutes, it is expected that the promotive effect on bone formation around the bone substitutes will continue for at least 10 days.

Mitochondrial permeability as a target for neurodegenerative disorders

Mitochondria-dependent cell death is a primary mechanism of cell death in both acute and chronic neurodegenerative disorders, e.g., stroke and Alzheimer's disease. Ischemia, glutamate excitotoxicity and reactive oxygen species (ROS) all target the mitochondria. In response to these lethal stimuli, the mitochondria release death factors, primarily cytochrome c (Cyt c), initiating the apoptotic cascade. Cyt c release occurs by at least two mechanisms, i.e., mitochondrial permeability transition (mPT) and mitochondrial outer membrane permeabilization (MOMP) through a Bax channel. Blocking the release of Cyt c may have therapeutic potential in neurodegenerative disorders. Roche has described compounds that target the voltage-dependent anion channel component of the mPT. 2-Aminoethoxydiphenyl borate is able to inhibit Ca 2+ -induced Ca 2+ release, and tricyclic antidepressants can inhibit mPT. Substituted carbazoles from Serono are capable of blocking Bax channel formation and the release of Cyt c from isolated mitochondria. At MGI PHARMA, GPI-19410 was discovered to have a complex effect in blocking mitochondria-dependent cell death: it prevented mitochondrial depolarization in response to Ca 2+ , blocked Ca 2+ -induced mitochondrial swelling and Cyt c release, and blocked t-Bid-induced Cyt c release in a manner distinct from cyclosporin.

Coprecipitation of cytochrome C with calcium phosphate on hydroxyapatite ceramic

Cytochrome C (cyt C), which has a molecular weight and an isoelectric point similar to those of fibroblast growth factor (FGF-2), was coprecipitated with calcium phosphates in the presence or absence of a hydroxyapatite ceramic (HAP) using supersaturated calcium phosphate solutions that can be prepared by mixing infusion fluids. The precipitates formed in the solution consisted of amorphous calcium phosphate in the absence of a HAP. In the presence of a HAP, precipitates morphologically identical to bone-like apatite formed a calcium phosphate layer on the surface of HAP. The optimum conditions for coprecipitation of cyt C with calcium phosphate on HAP were a Ca/P molar ratio of 1.5 and a NaHCO 3 concentration of 7.90 mM. The release of cyt C from the calcium phosphate layer continued for at least 10 days in the physiological salt solution. The results suggest that FGF-2 is also expected to be coprecipitated with calcium phosphates onto HAPs using infusion fluids.

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.

Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS‐dependent pathway

1. Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primary target, the mitochondrion. 2. Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome c (Cyt c) release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H) stores in brain-isolated mitochondria. 3. Although, mitochondrial potential collapse was almost immediate after malonate addition, mitochondrial swelling was not evident before 15 min of drug presence. This latter effect was blocked by cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. 4. Malonate added to SH-SY5Y cell cultures produced a marked loss of cell viability together with the release of Cyt c and depletion of GSH and NAD(P)H concentrations. All these effects were not apparent in SH-SY5Y cells overexpressing Bcl-xL. 5. When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection afforded by Bcl-xL overexpression was not evident anymore. 6. Taken together, all these data suggest that malonate causes a rapid mitochondrial potential collapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacity and leads to mitochondrial swelling. Further permeability transition pore opening and the subsequent release of proapoptotic factors such as Cyt c could therefore be, at least in part, responsible for malonate-induced toxicity.

Death receptor signaling

Apoptosis or programmed cell death is a common property of multicellular organisms ([Danial and Korsmeyer, 2004][1]; [Krammer, 2000][2]). It can be triggered by a number of factors, including UV- or γ-irradiation, chemotherapeutic drugs or signaling by death receptors (DR). The DR family is part of

Cytochrome c release is required for phosphatidylserine peroxidation during fas‐triggered apoptosis in lung epithelial A549 cells

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.

Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas.

Non-small-cell lung carcinomas (NSCLCs) are resistant to the induction of apoptosis by conventional anticancer treatment. However, NSCLC cell lines are sensitive to the action of the broad protein kinase inhibitor, staurosporine (STS). In the NSCLC cell line U1810, STS induced the mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) followed by activation of caspases, nuclear condensation, DNA fragmentation and finally cell death. Although preincubation of U1810 cells with the broad-spectrum caspase inhibitor z-VAD.fmk delayed the occurrence of nuclear apoptosis induced by STS, it did not impede mitochondrial alterations (such as the release of Cyt c and AIF) and cell death to occur. Moreover, the microinjection of neither Cyt c nor recombinant active caspase-3 into the cytoplasm promoted nuclear apoptosis-related changes in U1810 cells. Evaluation of the role of the caspase-independent factor AIF in STS-mediated death revealed that, upon immunodepletion of AIF, cytosols from STS-treated U1810 lost their capacity to induce nuclear condensation when incubated with isolated nuclei. In addition, microinjection of an anti-AIF antibody prevented AIF from translocating to the nuclei of STS-treated U1810 cells and reduced STS-induced cell death. Finally, although the transfection-enforced overexpression of AIF was not sufficient to induce cell death, it did enhance STS-mediated cell killing. Altogether, these results indicate that activation of caspases is not sufficient to kill U1810 cells and rather suggests an important role for the AIF-mediated mitochondrial-mediated death pathway.

Cytochrome c and IP 3 R: A Deadly Handshake

The mitochondria and endoplasmic reticulum (ER) are both involved in the apoptosis pathway, and release of calcium from the ER contributes to this process of cell death (see Mattson and Chan). ER calcium release is also involved in many nonpathological signaling processes, and Boehning et al. show that the interaction of cytochrome c (cyt c) released from mitochondria with the inositol trisphosphate receptors (IP 3 Rs) of the ER may be how the ER calcium release can reach pathologic levels. An interaction between cyt c and the IP 3 R was identified in a yeast two-hybrid screen, and these two proteins coimmunoprecipitated from cells treated with the broad-spectrum kinase inhibitor STS, which induces apoptosis. Furthermore, cell fractionation studies of cells treated with STS showed that cyt c accumulated in the ER fraction and was depleted from the mitochondrial fraction. Movement of cyt c to the ER fraction was not inhibited by exposure of the cells to caspase inhibitors, which suggests that translocation of cyt c occurred before caspase activation during apoptosis. Fluorescence resonance energy transfer assays indicated that cyt c and IP 3 Rs come into close association after induction of apoptosis with STS. IP 3 Rs are sensitive to calcium such that at concentrations in the nanomolar range (resting levels in a cell), calcium stimulates IP 3 R calcium release. In contrast, at higher micromolar concentrations of calcium, IP 3 R-induced calcium release is inhibited by calcium. Microsomal preparations exposed to cyt c and micromolar concentrations of calcium did not exhibit calcium-induced inhibition of IP 3 R calcium release. In vivo calcium imaging suggested that a spike in calcium precedes cyt c release from the mitochondria and then calcium levels remain elevated. Expression of the cyt c-binding domain of the IP 3 R acted as a dominant negative, preventing calcium oscillations and cyt c release in response to STS. Thus, release of small amounts of cyt c leads to an interaction with the IP 3 R on the ER, preventing calcium inhibition of ER calcium release. The resulting global increase in calcium triggers massive release of cyt c, which then acts in the positive feedback loop to maintain ER calcium release through the IP 3 Rs. M. P. Mattson, S. L. Chan, Calcium orchestrates apoptosis. Nat. Cell Biol. 5 , 1041-1043 (2003). [Online Journal] D. Boehning, L. Patterson, L. Sadaghat, N. O. Blebova, T. Kurosaki, S. H. Snyder, Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat. Cell Biol. 5 , 1051-1061 (2003). [Online Journal]