A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells

A simple, “mix-and-measure” microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z’ values of 0.6-0.7.     

Distinct localization of lipid rafts and externalized phosphatidylserine at the surface of apoptotic cells

Externalization of phosphatidylserine (PS) takes place in apoptotic cells as well as in viable cells under certain circumstances. Recent studies showed that externalized PS is localized at the lipid raft in viable activated immune cells. We found that lipid rafts and PS existed in a mutually exclusive manner in apoptotic cells. The number of PS-exposing apoptotic cells decreased when lipid rafts were disrupted. BCtheta;, which binds selectively to cholesterol in a cholesterol-rich region, did not effectively recognize lipid rafts of apoptotic cells. Lipid rafts rich in GM1 were successfully prepared from apoptotic cells, but the lipid raft protein LAT was not enriched in the preparation. Furthermore, the amount of PS and phosphatidylethanolamine but not of cholesterol in lipid rafts appeared to change after induction of apoptosis. These results suggest that lipid rafts are structurally modified during apoptosis and, despite being localized differently from PS, are involved in the externalization of PS.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.     

Programmed cell clearance: Molecular regulation of the elimination of apoptotic cell corpses and its role in the resolution of inflammation

Programmed cell clearance is a physiological process of elimination of apoptotic cell corpses. Recent studies have disclosed several ligand-receptor interactions that dictate the recognition or non-recognition of cells by macrophages and other phagocytes. The externalization of the anionic phospholipid, phosphatidylserine is effectively recognized by specific receptors on professional phagocytes and facilitates the clearance of apoptotic cells. Macrophage disposal of cells at sites of inflammation is believed to play an important role in the resolution of the inflammatory process, and recent studies have suggested a role for the NADPH oxidase in the process of macrophage elimination of activated neutrophils. The present review will focus on the molecular regulation of programmed cell clearance, and discuss the role of cell elimination in the resolution of inflammation.     

High tolerance to apoptotic stimuli induced by serum depletion and ceramide in side-population cells: high expression of CD55 as a novel character for side-population

Cancer stem cells are supposed to be resistant to apoptosis, but information for this is quite limited. Cancer stem cells are usually isolated as dye-effluxing cells with Hoechst 33342 staining, called side-population (SP) cells. Because Hoechst 33342 dye itself induces apoptosis, the SP cells isolated by such method are not suitable for evaluation of apoptosis. For accurate assessment, SP cells must be isolated without Hoechst 33342. Here, we found that CD55 was highly expressed in SP cells of two mammary gland carcinoma cell lines. Then, the high expression of CD55 was used for isolation of cancer stem cells among mammary carcinoma cell lines as a surrogate character. Cells expressing high level of CD55 (CD55(hi)) were resistant to apoptosis induced by serum depletion as in the case of SP cells. In ceramide-inducing apoptosis, CD55(hi) cells showed high tolerance. Anti-apoptotic molecules such as Bcl-2 were abundantly expressed in both SP cells and CD55(hi) cells. These findings indicated that SP cells as revealed to be CD55(hi) cells were tolerant to apoptosis. The high expression of CD55 may be a useful character for SP cells in evaluating their functions.     

Syntaxin 4 is required for acid sphingomyelinase activity and apoptotic function

Acid sphingomyelinase (A-SMase) is an important enzyme in sphingolipid metabolism and plays key roles in apoptosis, immunity, development, and cancer. In addition, it mediates cytotoxicity of cisplatin and some other chemotherapeutic drugs. The mechanism of A-SMase activation is still undefined. We now demonstrate that, upon CD95 stimulation, A-SMase is activated through translocation from intracellular compartments to the plasma membrane in an exocytic pathway requiring the t-SNARE protein syntaxin 4. Indeed, down-regulation of syntaxin 4 inhibits A-SMase translocation and activation induced by CD95 stimulation. This leads to inhibition of the CD95-triggered signaling events, including caspase 3 and 9 activation and apoptosis, activation of the survival pathway involving the protein kinase Akt, and important changes in cell cycle and proliferation. The molecular interaction between A-SMase and syntaxin 4 was not known and clarifies the mechanism of A-SMase activation. The novel actions of syntaxin 4 in sphingolipid metabolism and exocytosis we describe here define signaling mechanisms of broad relevance in cell pathophysiology.     

LPS/CD14 activation triggers SGLT-1-mediated glucose uptake and cell rescue in intestinal epithelial cells via early apoptotic signals upstream of caspase-3

Recent findings indicate that enhanced glucose uptake protects enterocytes from excessive apoptosis and barrier defects induced by LPS exposure. The aim of this study was to characterize the mechanisms responsible for increased sodium-dependent glucose cotransporter (SGLT)-1 activity in enterocytes challenged with LPS. SGLT-1-transfected Caco-2 cells were incubated with LPS in high glucose media. LPS increased SGLT-1 activity in dose- and time-dependent fashion, and is due to increased V(max) of the cotransporter. Elevated apical expression of SGLT-1 was also demonstrated. This LPS-induced effect was colchicine-inhibitable, suggesting microtubule-dependent translocation of SGLT-1 onto apical surface. Immunofluorescence staining showed expression of CD14 on the apical surface, but no TLR-4, on these cells. Neutralizing anti-CD14 decreased the LPS-induced upregulation of SGLT-1 activity, whereas anti-TLR-4 had no effect. Pharmacological studies indicated that signaling for LPS-mediated SGLT-1 glucose uptake depends on caspase-8 and -9 activation, but occurs independently of caspase-3. The findings describe a novel feedback mechanism within the apoptotic signaling pathway for SGLT-1-dependent cytoprotection. The observation suggests a new function for CD14 on enterocytes, involving the induction of the caspase-dependent SGLT-1 activity, which ultimately leads to cell rescue. The understanding of these signaling events may shed light on enterocytic cytoprotection and homeostasis mechanism upon pro-apoptotic challenges.     

Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha

Intestinal epithelial cells interact with immune cells located in the intestinal epithelium via soluble factors. An in vitro model system using coculture was constructed to analyze the effect of macrophages on intestinal epithelial cells, and human intestinal epithelial-like Caco-2 monolayers and activated macrophage-like THP-1 cells were used in this study. Coculturing with THP-1 cells resulted in an increase of lactate dehydrogenase release from Caco-2 and a decrease in the transepithelial electrical resistance of the monolayers, showing that coculturing with THP-1 induced cell damage to Caco-2 cells. This disruption was significantly suppressed by adding anti-TNF-alpha antibody and etanercept, strongly suggesting that TNF-alpha secreted from THP-1 had caused cell damage to Caco-2 monolayers. The disrupted Caco-2 monolayers showed both apoptotic and necrotic characteristics by morphological and biochemical analyses. TNFRI and NF-kappaB seem to have been involved in this regulation. It is suggested that this phenomenon is similar in some respects to that observed with IBD and that this in vitro coculture system could be a good model for searching for the drugs or food substances that can be used to treat or prevent IBD.     

Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin-treated oats

Histological and cytological evidence of where and when apoptotic cells occur in Pc-2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM-TUNEL was positive in the non-collapsed cells but not in the collapsed cells in the treated leaves. However, the EM-TUNEL assay was positive in the nuclei of the non-collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin-treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin-treated non-collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM-TUNEL assay indicated that EGTA co-incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.     

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.     

Identification and characterization of the mitochondrial membrane sorting signals in phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.     

Aberrant T cell activation and heightened apoptotic turnover in end-stage renal failure patients: a comparative evaluation between non-dialysis,haemodialysis, and peritoneal dialysis

Patients in end-stage renal disease (ESRD) have a high incidence of bacterial and viral infections. Fifteen non-dialysed (ND), 15 haemodialysed (HD), 15 patients with peritoneal dialysis (PD), and 15 healthy controls were included. T cell proliferation was measured by [3H]thymidine uptake. Apoptosis and cell phenotype were determined by FACS. sTNF-R1, sCD95, interleukin-1beta-converting enzyme (sICE), and interleukin (IL)-10 were measured by ELISA. PHA and CD3-driven T cell proliferation were significantly decreased in ESRD patients. CD3(+), CD19(+) B cells, and percentage of CD4(+) T cells were significantly reduced. Percent memory T cells (CD45RO(+)) and cells undergoing apoptosis (CD95(+)/Annexin V+) were significantly increased in ESRF. Moreover, sCD95, sTNFRI, and ICE were significantly increased. Serum level of IL-10, a Th2 cytokine, was enhanced. These findings strongly suggest that in ESRD patients Th1 T cells are selectively susceptible to undergo apoptosis. This observation provides an additional pathophysiological concept in the genesis of Th2 dominance.     

Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance

Sepsis causes lymphopenia which is inversely correlated with patient survival. The role of apoptosis-specific immune-activation and activation-induced cell-death in sepsis is incompletely understood. Fifteen septic patients and 20 healthy controls were included. T-cell proliferation was measured by [3H]thymidine uptake. Apoptosis and cell phenotype were determined by FACS. sTNFR1, sCD95, interleukin-1beta converting enzyme (sICE), and interleukin (IL)-10 were measured by ELISA. PHA and CD3-driven T-cell proliferation were significantly decreased in septic patients. The percentages of CD3(+) and CD4(+) T cells and CD19(+) B cells were significantly reduced. Percent memory T-cells (CD45RO(+)) and cells undergoing apoptosis (CD95(+)/annexin-V(+)) were significantly increased in sepsis. Moreover, sCD95, sTNFRI, and ICE were significantly increased. Anti-CD3 antibody triggering induced a 56% increase of CD4 T-cell death in septic patients vs. 7.5% in controls relative to IgG. Serum level of IL-10, a Th2 cytokine, was enhanced. These findings strongly suggest that in septic patients Th1 T-cells are selectively susceptible to undergo apoptosis. This observation provides an additional pathophysiological concept in the genesis of Th2 dominance.     

Organogallium(III) complexes as apoptosis promoting anticancer agents for head and neck squamous cell carcinoma (HNSCC) cell lines

Organogallium(III) dinuclear (1-9) and tetranuclear (10) complexes present potential therapeutic agents for the treatment of various types of cancer. The antiproliferative activity of 1-10 was evaluated with cell lines of head and neck squamous cell carcinomas, e.g. HN (soft palate), Cal27, Cal33 (tongue) and FaDu (hypopharynx) cell lines. The activity of compound 8 is comparable with that of cisplatin on cell line Cal27 (IC₅₀ 4.6 μM for both compounds). The mode of cell death induced, caspase activity and cell cycle analysis were evaluated for potential hit compounds 3, 5 and 8 Potential hit compounds 3, 5 and 8 were further evaluated for the mode of cell death, caspase activity and cell cycle analysis. Apoptosis induced by compounds 3, 5 and 8 on Cal27 and FaDu cells was confirmed by DNA laddering , as well as acridine orange (AO) and ethidium bromide (EB) double staining. These compounds (3, 5 and 8) induced caspase-independent apoptosis (within 4 h of action) in cell line Cal27. Extrinsic-mediated apoptosis associated with caspase 8 and 3 activation is the main mode of cytotoxicity induced on FaDu cells by compounds 3, 5 and 8. Cell cycle perturbations caused by these compounds are also observed. Our data suggest that compounds 3, 5 and 8 should be studied further for the treatment of head and neck cancer.     

14-3-3 proteins as signaling integration points for cell cycle control and apoptosis

14-3-3 proteins play critical roles in the regulation of cell fate through phospho-dependent binding to a large number of intracellular proteins that are targeted by various classes of protein kinases. 14-3-3 proteins play particularly important roles in coordinating progression of cells through the cell cycle, regulating their response to DNA damage, and influencing life-death decisions following internal injury or external cytokine-mediated cues. This review focuses on 14-3-3-dependent pathways that control cell cycle arrest and recovery, and the influence of 14-3-3 on the apoptotic machinery at multiple levels of regulation. Recognition of 14-3-3 proteins as signaling integrators that connect protein kinase signaling pathways to resulting cellular phenotypes, and their exquisite control through feedforward and feedback loops, identifies new drug targets for human disease, and highlights the emerging importance of using systems-based approaches to understand signal transduction events at the network biology level.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells     

Molecular basis for HEF1/NEDD9/Cas-L action as a multifunctional co-ordinator of invasion,apoptosis and cell cycle

Upregulation of the scaffolding protein HEF1, also known as NEDD9 and Cas-L, has recently been identified as a pro-metastatic stimulus in a number of different solid tumors, and has also been strongly associated with pathogenesis of BCR-Abl-dependent tumors. As the evidence mounts for HEF1/NEDD9/Cas-L as a key player in metastatic cancer, it is timely to review the molecular regulation of HEF1/NEDD9/Cas-L. Most of the mortality associated with cancer arises from uncontrolled metastases, thus a better understanding of the properties of proteins specifically associated with promotion of this process may yield insights that improve cancer diagnosis and treatment. In this review, we summarize the extensive literature regarding HEF1/NEDD9/Cas-L expression and function in signaling relevant to cell attachment, migration, invasion, cell cycle, apoptosis, and oncogenic signal transduction. The complex function of HEF1/NEDD9/Cas-L revealed by this analysis leads us to propose a model in which alleviation of cell cycle checkpoints and acquired resistance to apoptosis is permissive for a HEF1/NEDD9/Cas-L-promoted pro-metastatic phenotype. The contribution of Mahendra K. Singh and Lauren Cowell was equivalent.