Overexpression of α-synuclein in an astrocyte cell line promotes autophagy inhibition and apoptosis.

α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis.

Inhibition of cytoplasmic p53 differentially modulates Ca(2+) signaling and cellular viability in young and aged striata.

The p53 protein, a transcription factor with many gene targets, can also trigger apoptosis in the cytoplasm. The disruption of cell homeostasis, such as Ca(2+) signaling and mitochondrial respiration, contributes to the loss of viability and ultimately leads to cell death. However, the link between Ca(2+) signaling and p53 signaling remains unclear. During aging, there are alterations in cell physiology that are commonly associated with a reduced adaptive stress response, thus increasing cell vulnerability. In this work, we examined the effects of a cytoplasmic p53 inhibitor (pifithrin µ) in the striatum of young and aged rats by evaluating Ca(2+) signaling, mitochondrial respiration, apoptotic protein expression, and tissue viability. Our results showed that pifithrin µ differentially modulated cytoplasmic and mitochondrial Ca(2+) in young and aged rats. Cytoplasmic p53 inhibition appeared to reduce the mitochondrial respiration rate in both groups. In addition, p53 phosphorylation and Bax protein levels were elevated upon cytoplasmic p53 inhibition and could contribute to the reduction of tissue viability. Following glutamate challenge, pifithrin µ improved cell viability in aged tissue, reduced reactive oxygen species (ROS) generation, and reduced mitochondrial membrane potential (ΔΨm). Taken together, these results indicate that cytoplasmic p53 may have a special role in cell viability by influencing cellular Ca(2+) homeostasis and respiration and may produce differential effects in the striatum of young and aged rats.

Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase ß subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect ß subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

The matrix (M) protein of Newcastle disease virus binds to human bax through its BH3 domain.

The underlying mechanisms by which Newcastle disease virus (NDV) kills cancer cells are still unclear. Recent discoveries have shown that many viruses contain Bcl-2 homology-like domains which enabled their interaction with Bcl-2 family members, and thereby accounting for their virulence and pathogenicity. Alignment of the protein sequences of Malaysian strain of NDV, known as AF2240, with those from members of the human Bcl-2 family showed many similar regions; most notably we found that its matrix (AF2240-M) protein, large (AF2240-L) protein and fusion (AF2240-F) protein all contain BH3-like regions. In addition, there are BH1-like domains in these proteins, where AF2240-F and Mcl-1 share 55% identity within this region. To further investigate our hypothesis that the presence of the BH3-like domains in these proteins may convey cytotoxicity, AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expression of these constructs promoted cell death. As shown by flow cytometry, AF2240-M protein with deleted BH3-like region showed five-fold decrease in apoptosis. Moreover, the construct containing the N-terminal of AF2240-M showed nearly the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein participates in promoting cell death. Moreover, AF2240-M transfection promoted Bax redistribution to mitochondria. Therefore, to determine whether there is any direct interaction between NDV viral proteins with some members of the Bcl-2 family, various constructs were co-transfected into HeLa cells. Co-immunoprecipitation trials showed that the AF2240-M indeed directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. AF2240-F failed to interact with any of the tested proteins, although Bcl-XL slowed down the rate of cell death caused by this construct by nearly five-fold. In a parallel experiment, the level of expression of endogenous Bax and Bcl-2 after infection of HeLa cells with NDV was assessed by qRT-PCR, but no statistically significant change was observed. Consequently, the Bax/Bcl-2 ratio at the mRNA level did not alter. Overall, our study has shed additional light into the mechanisms by which NDV induces apoptosis.

Mitochondrially targeted ceramides preferentially promote autophagy, retard cell growth, and induce apoptosis.

C(6)-pyridinium (D-erythro-2-N-[6'-(1''-pyridinium)-hexanoyl]sphingosine bromide [LCL29]) is a cationic mitochondrion-targeting ceramide analog that promotes mitochondrial permeabilization and cancer cell death. In this study, we compared the biological effects of that compound with those of D-erythro-C(6)-ceramide, its non-mitochondrion-targeting analog. In MCF7 cells it was found that C(6)-pyridinium ceramide preferentially promoted autophagosome formation and retarded cell growth more extensively than its uncharged analog. This preferential inhibition of cell growth was also observed in breast epithelial cells and other breast cancer cells. In addition, this compound could promote Bax translocation to mitochondria. This redistribution of Bax in MCF7 cells could be blocked by the pan-caspase inhibitor zVAD-fmk but via a Bid-independent signaling pathway. Moreover, C(6)-pyridinium ceramide-induced translocation of Bax to mitochondria led to mitochondrial permeabilization and cell death. Overall, we show that mitochondrial targeting of C(6)-pyridinium ceramide significantly enhances cellular response to this compound.

Kallistatin attenuates endothelial apoptosis through inhibition of oxidative stress and activation of Akt-eNOS signaling.

Kallistatin is a regulator of vascular homeostasis capable of controlling a wide spectrum of biological actions in the cardiovascular and renal systems. We previously reported that kallistatin inhibited intracellular reactive oxygen species formation in cultured cardiac and renal cells. The present study was aimed to investigate the role and mechanisms of kallistatin in protection against oxidative stress-induced vascular injury and endothelial cell apoptosis. We found that kallistatin gene delivery significantly attenuated aortic superoxide formation and glomerular capillary loss in hypertensive DOCA-salt rats. In cultured endothelial cells, kallistatin suppressed TNF-α-induced cellular apoptosis, and the effect was blocked by the pharmacological inhibition of phosphatidylinositol 3-kinase and nitric oxide synthase (NOS) and by the knockdown of endothelial NOS (eNOS) expression. The transduction of endothelial cells with adenovirus expressing dominant-negative Akt abolished the protective effect of kallistatin on endothelial apoptosis and caspase activity. In addition, kallistatin inhibited TNF-α-induced reactive oxygen species formation and NADPH oxidase activity, and these effects were attenuated by phosphatidylinositol 3-kinase and NOS inhibition. Kallistatin also prevented the induction of Bim protein and mRNA expression by oxidative stress. Moreover, the downregulation of forkhead box O 1 (FOXO1) and Bim expression suppressed TNF-α-mediated endothelial cell death. Furthermore, the antiapoptotic actions of kallistatin were accompanied by Akt-mediated FOXO1 and eNOS phosphorylation, as well as increased NOS activity. These findings indicate a novel role of kallistatin in the protection against vascular injury and oxidative stress-induced endothelial apoptosis via the activation of Akt-dependent eNOS signaling.

Newcastle disease virus infection promotes Bax redistribution to mitochondria and cell death in HeLa cells.

BACKGROUND/AIMS: Newcastle disease virus (NDV) is an avian paramyxovirus that has gained a lot of interest in cancer viro-therapeutic applications because of its ability to selectively induce apoptosis in human cancer cells. However, the underlying mechanisms by which NDV induces apoptosis in human cancer cells are still not entirely understood. METHODS: In this study we examined the effect of a Malaysian velogenic strain of NDV, known as AF2240, on some elements of the intrinsic pathway of apoptosis. RESULTS: We show that NDV infection leads to conformational change of Bax protein. This is associated with the translocation of Bax from the cytoplasm to mitochondria and the release of cytochrome c into the cytoplasm. Interestingly, the level of Bcl-2 protein was not affected by NDV treatment. CONCLUSION: We have shown that Bax conformational change and subcellular distribution is involved in the intrinsic pathway of apoptosis induced by NDV.

Kruppel-like factor 4 is a novel mediator of Kallistatin in inhibiting endothelial inflammation via increased endothelial nitric-oxide synthase expression.

Kallistatin is a plasma protein that exhibits pleiotropic effects in vasodilation, anti-angiogenesis, and anti-inflammation. To isolate a kallistatin-binding protein that mediates the vascular actions of kallistatin, we screened and identified a positive clone from a human heart cDNA expression library by using an alkaline phosphatase-kallistatin fusion protein binding assay. Sequence analysis revealed that kallistatin-binding protein is human Kruppel-like factor 4 (KLF4). KLF4 was localized on the plasma membrane of HEK-293 cells and endothelial cells overexpressing KLF4. KLF4 and kallistatin complex formation was identified in endothelial cells by immunoprecipitation followed by immunoblotting. We showed that kallistatin inhibits tumor necrosis factor-alpha-induced NF-kappaB activation, as well as vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression in endothelial cells, whereas knockdown of KLF4 by small interfering RNA oligonucleotide abolished the effect of kallistatin. Kallistatin increased endothelial nitric-oxide synthase (eNOS) expression and nitric oxide levels, and these effects were also blocked by KLF4 small interfering RNA oligonucleotide. Moreover, inhibition of eNOS by RNA interference or by NOS inhibitor abolished the blocking effect of kallistatin on vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression. In summary, we identified KLF4 as a kallistatin-binding protein, which has a novel role in mediating the anti-inflammatory actions of kallistatin via increasing eNOS expression in endothelial cells. This study provides a new target for modulating endothelial function in vascular disease.

AMPK inhibitor Compound C stimulates ceramide production and promotes Bax redistribution and apoptosis in MCF7 breast carcinoma cells.

Compound C is commonly used as an inhibitor of AMP-activated protein kinase (AMPK), which serves as a key energy sensor in cells. In this study, we found that Compound C treatment of MCF7 cells led to Bax redistribution from the cytoplasm to mitochondria and cell death. However, this effect does not involve AMPK. In addition, we found that treatment with this compound leads to an enhanced ceramide production. Analyses by quantitative PCR and ceramide synthase activity assay suggest that ceramide synthase 5 (LASS/CerS 5) is involved in Compound C-induced ceramide upregulation. Downregulation of LASS/CerS 5 was found to attenuate Compound C-mediated ceramide production, Bax redistribution, and cell death.

Ceramide generated by sphingomyelin hydrolysis and the salvage pathway is involved in hypoxia/reoxygenation-induced Bax redistribution to mitochondria in NT-2 cells.

Ceramide functions as an important second messenger in apoptosis signaling pathways. In this report, we show that treatment of NT-2 neuronal precursor cells with hypoxia/reoxygenation (H/R) resulted in ceramide up-regulation. This elevation in ceramide was primarily due to the actions of acid sphingomyelinase and ceramide synthase LASS 5, demonstrating the action of the salvage pathway. Hypoxia/reoxygenation treatment led to Bax translocation from the cytoplasm to mitochondria and cytochrome c release from mitochondria. Down-regulation of either acid sphingomyelinase or LASS 5-attenuated ceramide accumulation and H/R-induced Bax translocation to mitochondria. Overall, we have demonstrated that ceramide up-regulation following H/R is pertinent to Bax activation to promote cell death.

Evaluation of some cell death features by real time real space microscopy.

In the past few years, the investigation of many cell death features, especially ones associated with changes in DeltaPsi(m), have gained an important insights with the development of high-resolution fluorescence microscopy. With the use of real time real space measurements, it was possible to perform dynamic studies not only to investigate the location of the organelles, but also to follow changes in transport mechanism, such as Ca(2+) concentration in different subcellular compartments. In addition, this technique has been used for the simultaneous tracking of organelle location, ion measurements, and DeltaPsi(m,) which clearly contributed to further understanding mechanisms related to the control of cell death. This chapter describes the methodology employed to study changes in DeltaPsi(m), Bax translocation, and Ca(2+) measurements upon apoptotic induction. It also details the new technique developed and employed in our laboratory to measure Ca(2+) signaling in brain slices by confocal microscopy. This method has been applied to investigate real time real space studies in different models of neurodegenerative processes, such as Huntington's disease and aging.

Differential immunoscreening identifies a glycosylation variant of the epidermal growth factor receptor in ME-180 cervical carcinoma cells.

In this report, we describe the development and characterization of an anti-ME-180 cervical cancer-specific epidermal growth factor (EGF) receptor monoclonal antibody (MAb). This MAb, 6C7, specifically binds to ME-180 cervical cancer cells and not to normal cervical epithelial cells. By immunoaffinity chromatography, we have shown that the 6C7 antibody binds to a 205-kDa protein. Subsequent mass spectrometry sequencing analysis identified this protein as an EGF receptor. In addition, treatment of the ME-180 EGF receptor with N- and O-linked glycosidases indicated that this antibody binds to the carbohydrate portion of the glycoprotein. Moreover, Western blotting analysis with an anti-EGF receptor antibody indicated that this protein is present in abundance in all cervical cancer cell lines, including ME-180, HeLa, Ca Ski, HT-3, SiHa, and Hs 588.T. However, the 6C7 antibody only binds to the EGF receptor from ME-180 cells, suggesting that this protein is differentially glycosylated in ME-180 cells, compared to other cervical cancer cell lines. Finally, we have shown that this antibody could selectively block EGF-mediated cell proliferation in ME-180 cells but not in HeLa cells. Overall, our study suggests that the differentially glycosylated EGF receptor could potentially serve as a unique target for the immunotherapeutic treatment of cervical cancer.

Distinct domains of Bcl-XL are involved in Bax and Bad antagonism and in apoptosis inhibition.

Pro-survival factor Bcl-X(L) can antagonize the pro-apoptotic functions of Bax and Bad via two distinct mechanisms. It can block Bax-mediated cell death by preventing Bax translocation from the cytosol to mitochondria. On the other hand, Bcl-X(L) can neutralize Bad by sequestering it to mitochondria. In order to map the domains of Bcl-X(L) involved in inhibiting Bax and Bad, we have carried out mutational analyses of this protein. This was done by deleting the key domains of Bcl-X(L), including its BH1-4 domains, the flexible loop, the C-terminal hydrophobic domain, and segments of the alpha5-alpha6 hairpin. The resulting Bcl-X(L) mutant constructs were then co-transfected with either GFP-Bax or GFP-Bad. We found that the BH1-4 domains and the C-terminal segment of Bcl-X(L) were essential for blocking Bax localization to mitochondria. On the other hand, only its BH1 and BH3 domains and the C-terminal hydrophobic segment were necessary for sequestering Bad to mitochondria. In addition, by immunoprecipitation analyses, we found that these deletions differentially affected the ability of the Bcl-X(L) mutant proteins to bind Bax and Bad. Finally, cell viability assays indicated that the BH1-4 domains of Bcl-X(L) were the primary domains required for inhibiting staurosporine-induced apoptosis, suggesting that distinct domains of Bcl-X(L) are involved in antagonizing Bax and Bad and in apoptosis inhibition.

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis.

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.

A mitochondrial pool of sphingomyelin is involved in TNFalpha-induced Bax translocation to mitochondria.

We recently showed that targeting bSMase (bacterial sphingomyelinase) specifically to mitochondria caused accumulation of ceramide in mitochondria, and induced cytochrome c release and cell death [Birbes, El Bawab, Hannun and Obeid (2001) FASEB J., 15, 2669-2679]. In the present study, we investigated the role of this mitochondrial pool of ceramide in response to a receptor-mediated event, namely TNFalpha (tumour necrosis factor alpha), and the involvement of this mitochondrial pool of ceramide in Bax translocation to mitochondria, an event that precedes cytochrome c release. Treatment of MCF7 cells with TNFalpha caused an increase in ceramide levels in the mitochondrial fraction which accompanied Bax translocation to mitochondria. Targeting bSMase to mitochondria specifically resulted in Bax translocation to mitochondria, suggesting that the mitochondrial ceramide pool is involved in Bax translocation. Moreover, in a reconstituted cell-free system, treatment of isolated mitochondria with bSMase enhanced Bax association with mitochondrial membranes. Collectively, these results suggest that the generation of ceramide in mitochondria in response to TNFalpha is sufficient to induce Bax translocation to mitochondria and subsequent cytochrome c release and cell death.

Generation and characterization of monoclonal antibodies directed against the surface antigens of cervical cancer cells.

In this report, we describe the development and characterization of monoclonal antibodies against the surface antigens of cervical cancer cells. Using HeLa cervical carcinoma cells as the immunogen, we have developed a number of antibodies that specifically label cervical cancer cells but not normal cervical epithelial cells. These antibodies displayed differential reactivity towards various cervical cancer cell lines as determined by immunofluorescence labeling and western blotting analyses. One of these antibodies, 13G4, which showed the strongest labeling to HeLa cells and has the widest range of reactivity to other cervical cancer cell lines, was extensively characterized. By immunoaffinity chromatography, we purified a 90-kDa protein that appears to be the principal target recognized by this antibody. This protein was subsequently identified as decay accelerating factor (DAF) or CD55 by the mass spec sequencing analysis of the tryptic peptides derived from this protein. Digestion of HeLa DAF with glycosidases that removed its N- and O-linked carbohydrates has revealed that the 13G4 antibody binds to the peptide portion of this glycoprotein. Overall, our approach of generating and characterizing monoclonal antibodies directed against the surface antigens of cervical cancer cells serves as a stepping stone towards the eventual development of a unique panel of monoclonal antibodies that could potentially be used for the detection and therapeutic treatment of cervical cancer.

Bcl-x(L) sequesters its C-terminal membrane anchor in soluble, cytosolic homodimers.

Bcl-x(L) is a potent inhibitor of apoptosis. While Bcl-x(L) can be bound to mitochondria, a substantial fraction, depending on the cell type or tissue, is found in the cytosol of healthy cells. Gel filtration and crosslinking experiments reveal that, unlike monomeric Bax, Bcl-x(L) migrates in a complex of approximately 50 kDa in the cytosol. Co-immunoprecipitation experiments indicate that Bcl-x(L) in the cytosol forms homodimers. The C-terminal hydrophobic tails of two Bcl-x(L) molecules are involved in homodimer formation, and analysis of mutants demonstrates that the C-terminal lysine residue and the G138 residue lining the BH3-binding pocket are required for homodimerization. The flexible loop preceding the C-terminal tail in Bcl-x(L) is longer than that of several monomeric Bcl-2 family members and is a requisite for the homodimer formation. Bad binding to Bcl-x(L) dissociates the homodimers and triggers Bcl-x(L) binding to mitochondrial membranes. The C-terminal tail of Bcl-x(L) is also required to mediate Bcl-x(L)/Bax heterodimer formation. Both mitochondrial import and antiapoptotic activity of different Bcl-x(L) mutants correlate with their ability to form homodimers.

Generation and characterization of species-specific anti-Bcl-X(L) monoclonal antibodies.

Bcl-X(L) is a pro-survival member of the Bcl-2 family that plays an important role in apoptosis regulation. As a first step to carry out the molecular characterization of this protein, we have generated five monoclonal antibodies (MAbs) directed against synthetic peptides corresponding to the flexible loop region of human and murine Bcl-X(L). These antibodies display species specificity and are suitable for Western blotting, immunofluorescence labeling, and immunoprecipitation. These results suggest that these antibodies can serve as important tools for the detection and purification of endogenous and ectopically expressed human and murine Bcl-X(L).