Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling

Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling

Dynamics of outer mitochondrial membrane permeabilization during apoptosis.

Individual cells within a population undergo apoptosis at distinct, apparently random time points. By analyzing cellular mitotic history, we identified that sibling HeLa cell pairs, in contrast to random cell pairs, underwent apoptosis synchronously. This allowed us to use high-speed cellular imaging to investigate mitochondrial outer membrane permeabilization (MOMP), a highly coordinated, rapid process during apoptosis, at a temporal resolution approximately 100 times higher than possible previously. We obtained new functional and mechanistic insight into the process of MOMP: We were able to determine the kinetics of pore formation in the outer mitochondrial membrane from the initiation phase of cytochrome-c-GFP redistribution, and showed differential pore formation kinetics in response to intrinsic or extrinsic apoptotic stimuli (staurosporine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)). We also detected that the onset of mitochondrial permeabilization frequently proceeded as a wave through the cytosol, and that the frequency of wave occurrence in response to TRAIL was reduced by inhibition of protein kinase CK2. Computational analysis by a partial differential equation model suggested that the spread of permeabilization signals could sufficiently be explained by diffusion-adsorption velocities of locally generated permeabilization inducers. Taken together, our study yielded the first comprehensive analysis of clonal cell-to-cell variability in apoptosis execution and allowed to visualize and explain the dynamics of MOMP in cells undergoing apoptosis

Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles

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Improving quality in nanoparticle-induced cytotoxicity testing by a tiered inter-laboratory comparison study

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.Web of Science108art. no. 143

Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif

PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-b family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a nove

High Content Analysis Provides Mechanistic Insights on the Pathways of Toxicity Induced by Amine-Modified Polystyrene Nanoparticles

<div><p>The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH₂) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH₂ NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH₂ NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for <i>in vitro</i> screening of nanomaterials in highthroughput format.</p></div

Revisiting the paradigm of silica pathogenicity with synthetic quartz crystals: the role of crystallinity and surface disorder

Background: Exposure to some - but not all - quartz particles is associated to silicosis, lung cancer and autoimmune diseases. What imparts pathogenicity to any single quartz source is however still unclear. Crystallinity and various surface features are implied in toxicity. Quartz dusts used so far in particle toxicology have been obtained by grinding rocks containing natural quartz, a process which affects crystallinity and yields dusts with variable surface states. To clarify the role of crystallinity in quartz pathogenicity we have grown intact quartz crystals in respirable size. Methods: Quartz crystals were grown and compared with a fractured specimen obtained by grinding the largest synthetic crystals and a mineral quartz (positive control). The key physico-chemical features relevant to particle toxicity - particle size distribution, micromorphology, crystallinity, surface charge, cell-free oxidative potential - were evaluated. Membranolysis was assessed on biological and artificial membranes. Endpoints of cellular stress were evaluated on RAW 264.7 murine macrophages by High Content Analysis after ascertaining cellular uptake by bio-TEM imaging of quartz-exposed cells. Results: Quartz crystals were grown in the submicron (n-Qz-syn) or micron (μ-Qz-syn) range by modulating the synthetic procedure. Independently from size as-grown quartz crystals with regular intact faces did not elicit cellular toxicity and lysosomal stress on RAW 264.7 macrophages, and were non-membranolytic on liposome and red blood cells. When fractured, synthetic quartz (μ-Qz-syn-f) attained particle morphology and size close to the mineral quartz dust (Qz-f, positive control) and similarly induced cellular toxicity and membranolysis. Fracturing imparted a higher heterogeneity of silanol acidic sites and radical species at the quartz surface. Conclusions: Our data support the hypothesis that the biological activity of quartz dust is not due to crystallinity but to crystal fragmentation, when conchoidal fractures are formed. Besides radical generation, fracturing upsets the expected long-range order of non-radical surface moieties - silanols, silanolates, siloxanes - which disrupt membranes and induce cellular toxicity, both outcomes associated to the inflammatory response to quartz

PS-NH₂ NPs cause dose-dependent changes in lysosomal properties.

<p>I321N1 and HepG2 cells were exposed to vehicle (ctrl) or increasing concentrations of PS-COOH or PS-NH₂ NPs for 24 hours and analysed by HCA. The Spot Detection Bioapplication was used to analyse the Lysotracker green positive vesicles inside the exposed cells. A. Representative images of cells exposed to vehicle or 25 µg/ml PS-NH₂ NPs showing the increased Lysotracker green fluorescence and the analysis performed using the Spot Detector Bioapplication. Scale bar = 20 µm. B. Dose-dependent changes parameters associated with Lysotracker green positive vesicles: counts, area and intensity of Lysotracker green positive vescicles for each cell were calculated; dose dependent changes in individual spots were also recorded. C. Comparison among spot counts, spot total area and spot total intensity/cell within each cell line. The results suggest that increasing concentrations of PS-NH₂ NPs cause swelling of lysosomes, as indicated by increased Lysotracker green intensity, followed by a reduction in spot counts and reduciton in the spot area/cell and their intensity, indicative of lysosomal rupture. Data are shown as average +/− SD of object numbers, fluorescence intensity, area per cell, or average fluorescence intensity per identified object respectively from a representative experiment. Data were fitted with a gaussian curve for visual purposes only.</p

Sequences of EC50 thresholds suggest that all cell lines tested except Raw264.7 execute apoptotic cell death, while Raw264.7 undergo a non-regulated form of cell death.

<p>EC₅₀/IC₅₀ for each parameter measured for the selected cell lines exposed to vehicle (ctrl) or increasing doses of PS-Plain, PS-COOH or PS-NH₂ NPs, for 24 hours or 72 hours were calculated as described in the Methods. EC50/IC50 measured values after exposure for 24 hours (A). C. Schematic representation showing EC50/IC50 threshold sequences as predictive of time sequences. The graphs and sequences of EC50 shown were derived from the measurements obtained for 1321n1 cells. After 24 hours exposure the sequence of thresholds suggests that all cell lines except RAW264.7 underwent apoptosis following initial lysosomal damage; the EC50 thresholds observed for RAW264.7 suggest a sudden/non regulated mechanism of cell death compatible with necrosis. The EC50/IC50 values for all the parameters analysed shifted to lower concentrations after exposure for 72 hours, suggesting that longer exposure can lead to potential toxicity for doses that resulted non toxic after 24 hour exposure.</p