Cell-permeable Dye Research Articles

Dark Hydrazone Fluorescence Labeling Agents Enable Imaging of Cellular Aldehydic Load.

Aldehydes are key intermediates in many cellular processes, from endogenous metabolic pathways like glycolysis to undesired exogenously induced processes such as lipid peroxidation and DNA interstrand cross-linking. Alkyl aldehydes are well documented to be cytotoxic, affecting the functions of DNA and protein, and their levels are tightly regulated by the oxidative enzyme ALDH2. Mutations in this enzyme are associated with cardiac damage, diseases such as Fanconi anemia (FA), and cancer. Many attempts have been made to identify and quantify the overall level of these alkyl aldehydes inside cells, yet there are few practical methods available to detect and monitor these volatile aldehydes in real time. Here, we describe a multicolor fluorogenic hydrazone transfer ("DarkZone") system to label alkyl aldehydes, yielding up to 30-fold light-up response in vitro. A cell-permeant DarkZone dye design was applied to detect small-molecule aldehydes in the cellular environment. The new dye design also enabled the monitoring of cellular acetaldehyde production from ethanol over time by flow cytometry, demonstrating the utility of the DarkZone dyes for measuring and imaging the aldehydic load related to human disease.

Two-photon excited photoconversion of cyanine-based dyes.

The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue.

Boron nitride nanotubes as vehicles for intracellular delivery of fluorescent drugs and probes.

To evaluate the response of cells to boron nitride nanotubes (BNNTs) carrying fluorescent probes or drugs in their inner channel by assessment of the cellular localization of the fluorescent cargo, evaluation of the in vitro release and biological activity of a drug (curcumin) loaded in BNNTs. Cells treated with curcumin-loaded BNNTs and stimulated with lipopolysaccharide were assessed for nitric oxide release and stimulation of IL-6 and TNF-α. The cellular trafficking of two cell-permeant dyes and a non-cell-permeant dye loaded within BNNTs was imaged. BNNTs loaded with up to 13 wt% fluorophores were internalized by cells and controlled release of curcumin triggered cellular pathways associated with the known anti-inflammatory effects of the drug. The overall findings indicate that BNNTs can function as nanocarriers of biologically relevant probes/drugs allowing one to examine/control their local intracellular localization and biochemical effects, leading the way to applications as intracellular nanosensors.

Abstract B55: Suppression of CD8 T-cell activation by tryptophan catabolism in triple-negative breast cancer

Abstract Background: The ability of cancer cells to evade and suppress an antitumor immune response is increasingly recognized as a critical feature of aggressive, metastatic disease. Conversion of the amino acid tryptophan (trp) into the catabolite kynurenine (kyn) and the subsequent secretion of kyn is a current area of interest in the field of antitumor immunity, given that trp depletion is a trigger for T-cell death, and that kyn binds to and activates the aryl-hydrocarbon receptor (AhR) in immune cells, often with immunosuppressive consequences. In a screen to identify mechanisms promoting survival of triple-negative breast cancer (TNBC) cells in transit during the metastatic cascade, our lab discovered that trp-to-kyn catabolism is significantly increased by TNBC cells in forced-suspension culture due to upregulation of the gene encoding the rate-limiting trp-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO2). Hypothesis: In addition to promoting survival of TNBC cells during metastasis, TDO2-dependent trp-to-kyn catabolism may facilitate tumor-mediated immune suppression, further contributing to the aggressive nature of this breast cancer subtype. Methods: TNBC cell lines were plated in adherent or forced-suspension culture for 24 hours, using poly (2-hydroxyethyl methacrylate)-coated plates to prevent adhesion. TDO2 mRNA (using quantitative PCR) and protein (immunoblotting) were measured in adherent versus suspended cells. Secreted kyn was measured using liquid chromatography-mass spectrometry and inhibited with a selective TDO2 inhibitor, 680C91. To test the effect of kyn on CD8 T-cell function, primary human CD8 T-cells were isolated from the blood of healthy human donors using a positive selection kit. T-cell proliferation was measured by dilution of the cell-permeable dye carboxyfluorescein succinimidyl ester and T-cell death was measured by incorporation of a fixable viability dye. Flow cytometry was used to measure both surface and intracellular T-cell proteins. Results: The rate limiting enzyme TDO2 was upregulated in BT549 cells in suspension by 20.1-fold at the mRNA level and 5.4-fold at the protein level. Secreted kyn increased by 2.4-fold in suspension culture (0.46 μM secreted kyn by adherent cells, 1.09 μM kyn secreted by suspended cells, n=3 replicates), and this increase was reversed by addition of the TDO2 inhibitor 680C91 (p<0.0001). Over the course of a five day CD3/CD28 activation, purified kyn reduced CD8 T-cell proliferation and increased CD8 T-cell death in a dose-dependent manner (n=6-9 donors, p<0.05 at 25, 50, and 100 μM kyn), and these effects were partially reversed by the addition of an AhR antagonist (n=6 donors, p<0.05 at 25 μM kyn). Kyn also reduced expression of granzyme B by live CD8 T-cells at high doses (n=4 donors, p=0.05 at 100 μM kyn), but did not alter expression of perforin or tumor necrosis factor alpha. Consistent with these results, culture of human CD8 T-cells in conditioned media from suspended BT549 cells over the course of a five day CD3/CD28 activation resulted in reduced proliferation and increased cell death in CD8 T-cells (n=6 donors, p<0.05) compared to culture in conditioned media from adherent BT549 cells. Expression of the pro-inflammatory cytokine interferon gamma by CD8 T-cells cultured in conditioned media from suspended BT549 cells may also be reduced (n=4 donors, p=0.08). Conclusions: Together, these results suggest a role for TNBC-mediated trp-to-kyn catabolism in suppressing cytotoxic T-cells. Further in vivo studies will indicate whether TDO2 activity might be a rational therapeutic target for improving antitumor immunity in patients with this aggressive subtype of breast cancer. Citation Format: Lisa I. Greene, Tullia C. Bruno, Thomas J. Rogers, Jill E. Slansky, Virginia F. Borges, Jennifer K. Richer. Suppression of CD8 T-cell activation by tryptophan catabolism in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B55.

Hemin-Induced Mast Cell-Extracellular Traps Impart Resistance to Therapy in a Sickle Microenvironment

Mast cell activation in the skin contributes to inflammation, neurogenic inflammation and hyperalgesia in sickle mice (Vincent et al., Blood 2013). However, the underlying cause of mast cell activation and challenges in inhibiting mast cell activation remain unclear. Hemolysis in sickle cell disease leads to excess free heme, which contributes to endothelial activation and neutrophil extracellular trap (NET) formation. We therefore hypothesized that free heme activates mast cells and extracellular trap formation in a sickle microenvironment. We examined hemin-induced activation in mast cells from the skin of HbAA-BERK control and HbSS-BERK sickle mice. Mast cells in culture were incubated with vehicle, or I ng/ml TNF-α for 4 hours, or 40 μM hemin for 2 hours, or with hemin for 2 hours after 2 hours of priming with TNF-α to create an inflammatory sickle microenvironment. Cells were stained with cell impermeable DNA dye SYTOX orange and cell permeable dye SYTO13 to visualize the extracellular DNA fibers. Incubation with TNF-α or hemin did not show significant eruption of DNA from the cell body. However, sickle mast cells incubated with both TNF-α and hemin showed distinct DNA containing fibers exploding from the cell body with the appearance of spider web like formation similar to NETs. The length of majority of individual fibers was more than 50 μm stretching to more than 500 μM, indicative of TRAP formation. This response was blunted in mast cells from control mice. Thus, mast cells in a sickle microenvironment are primed and activated, and further priming with cytokines such as TNF-α leads to hemin-induced mast cell extracellular trap (MET) formation. Sickle mast cells express significantly higher TLR4 and FcεRI as compared to control mice (Vincent et al., Blood 2013). Therefore, we examined if silencing of FcεRI and/or inhibition of TLR4 attenuated TNF-α primed/hemin-induced MET formation. TAK242 at a dose of 1 μM but not at 0.5 μM inhibited TNF-α primed/hemin-induced MET formation, but silencing of FcεRI had no effect. Since cannabinoids, imatinib, palmitoylethanolamide (PEA) and cromolyn are known mast cell inhibitors, we examined their effect on TNF-α/hemin-induced METs. Imatinib (30 - 100 μM), a known mast cell inhibitor, showed no significant effect, while cromolyn (100 μM) led to a modest decrease in MET formation. Cannabinoid CP 55,940 and PEA at a relatively low dose of 30 μM completely blocked MET formation. Functionally, imatinib, PEA and CP 55,940 reduced the release of cytokines TNF-α and RANTES from mast cells incubated with TNF-α/hemin for 24 hours. Hemin in the presence of TNF-α stimulated sustained activation of NLRP3 inflammasome signaling. Thus, mast cells in a sickle microenvironment are activated due to a rich inflammatory cytokine milieu, via activation of the inflammasome signaling upon stimulation with hemin. The unique phenomenon of MET formation is orchestrated by a combined action of inflammatory milieu and hemin, imparting resistance to common mast cell inhibitors such as cromolyn and imatinib. This new phenomenon of MET formation identified by us may in part contribute to challenges of treating sickle cell disease and other mast cell associated pathological conditions. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye.

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Cellular communication via directed protrusion growth: Critical length-scales and membrane morphology

We investigated the growth of cell protrusions from adherent HEK 293 cells and their capability to bridge cytophobic Teflon® AF microgaps, establishing a critical length scale, beyond which cells cannot probe free space. For this purpose, we employed a photolithography-based surface fabrication strategy for producing micropatterned substrates composed of glass and the amorphous polymer Teflon® AF. Cell protrusions growing from HEK 293 cells on these substrates were confined to extend on 2 μm wide glass lanes, intersected by Teflon® AF microgaps of various lengths between 2 and 16 μm. After 24 hours of incubation, the frequency of cell protrusions crossing the gap was found to be strongly dependent on the gap size. Gaps which are greater than 4 μm were found to be increasingly difficult to cross. Cell extensions crossing the microgaps either appeared as nanosized connections, in approximately 30% of all observed cases, or as microsized connections. Molecular transport in the established cell-to-cell connection across the microgap was investigated by activation of TRPM8 ion channels followed by supply of Ca2+ to one of the connected cells. The diffusion of the Ca2+ ions was visualized by means of a cell-permeant pre-fluorescent dye. We observed both open- and closed-ended intercellular connections in both nano- and microsized cell protrusions.

Detection of Mitochondrial Mass, Damage, and Reactive Oxygen Species by Flow Cytometry.

The reagents and procedures highlighted here will give the investigators an indication of the health status and volume of mitochondria in primary cells and cell lines through the use of a number of cell-permeable dyes. Mitochondrial volume can be monitored by using the probe MitoTracker Green FM. This reagent labels mitochondria in a manner that is independent of the membrane potential, therefore providing a readout relating purely to the mitochondrial mass of the cell. In contrast, MitoTracker Red CMXRos, tetramethylrhodamine methyl ester, and 10-N-nonyl acridine orange label mitochondria in a manner dependent on the membrane potential, thus giving an indication of mitochondrial stress. Using MitoSOX Red, it is also possible to analyze the production of the mitochondrial superoxide anion. Like the MitoTracker probes, MitoSOX Red is taken up passively by cells. In the mitochondria, the probe is oxidized by superoxide, resulting in the emission of red fluorescence.

A facile method for simultaneously measuring neuronal cell viability and neurite outgrowth.

Neurite outgrowth is an important morphological phenotype of neuronal cells that correlates with their function and cell health, yet there are limited methods available for measuring this phenomenon. Current approaches to measuring neurite outgrowth are laborious and time-consuming, relying largely upon immunocytochemical staining of neuronal markers (e.g., beta-III tubulin or MAP2) followed by manual or automated microscopy for image acquisition and analysis. Here we report the development of a quick and simple dual-color fluorescent dye-based staining method that allows for the simultaneous measurement of neuronal cell health and relative neurite outgrowth from the same sample. An orangered fluorescent dye that stains cell membrane surfaces is used as an indirect reporter of changes in relative neurite outgrowth due to alterations in the number or length of membrane projections emanating from neuronal cell bodies. Cell viability is assessed simultaneously via the use of a cell-permeant dye that is converted by intracellular esterase activity from a non-fluorescent substrate to a green-fluorescent product. Using Neuroscreen-1 cells (a PC-12 subclone), primary rat cortex neurons, and human induced pluripotent stem cell (iPSC)-derived neurons, we demonstrate that this multiplex assay allows for rapid visualization and unbiased, quantitative plate reader analysis of neuronal cell health and neurite outgrowth.

Rapid, Specific, No-wash, Far-red Fluorogen Activation in Subcellular Compartments by Targeted Fluorogen Activating Proteins

Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20–30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments.

Mapping Live Cell Viscosity with an Aggregation‐Induced Emission Fluorogen by Means of Two‐Photon Fluorescence Lifetime Imaging

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE-Cy, a cell-permeable dye with aggregation-induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE-Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE-Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.

Enhanced nitric oxide generation in root transition zone during the early stage of cadmium stress is required for maintaining root growth in barley

The objective of this study was to analyse the role of nitric oxide (NO) in relation to reactive oxygen species (ROS) production and root growth inhibition in the early response of barley root tip to cadmium. We used cell-permeable NO-binding dye DAF-2DA for the NO localization and cell-impermeable dye DAR-4M for the quantification of NO in barley root apex. A considerable increase in NO production was detected in the transition zone of root tips already 30 min after the immersion of barley roots into 15 μM Cd. This Cd-induced NO generation further increased during the recovery period and reached its maximum level 2 h after the short-term treatment. At this stage an enhanced Cd-induced NO generation was observed in the meristematic, transition and in the beginning of elongation zone in comparison with control roots. Inhibitor of NO synthase activity markedly reduced root growth in control seedlings and had a synergistic growth inhibitory effect with Cd. On the contrary, this Cd-induced root growth inhibition was markedly alleviated by the NO donor. Exposure of roots to Cd markedly increased the production of ROS in the proximal elongation zone of root. While NO did not affect ROS generation, inhibition of its synthesis slightly decreased ROS generation in both control and Cd-treated roots. Our results revealed that enhanced NO production is a very early response of barley root to Cd stress and it is involved in the regulation of root growth mitigating Cd-induced root growth inhibition.

Synthesis and characterization of the anticancer and metal binding properties of novel pyrimidinylhydrazone derivatives

Three novel pyrimidinylhydrazones substituted at either the aromatic moiety or at the imine carbon atom were synthesized and characterized by standard analytical methods. All compounds were found to be toxic in the micro- to submicromolar range against a diverse panel of cancer cell lines including multidrug resistant (MDR) derivatives expressing P-glycoprotein (Pgp). UV–visible spectrophotometry experiments demonstrated that the most active compound (3) forms highly stable complexes with iron(III) and copper(II) in a wide pH range with a stronger preference towards iron(III). The redox activity of the iron and copper complexes of ligand 3 was investigated using cyclic voltammetry and was tested with cellular reductants. The impact of reactive oxygen species (ROS) on the mechanism of toxicity was assessed using the ROS-sensitive cell permeable dye 2′,7′-dichlorofluorescin diacetate (DCFDA). Our results demonstrate that the studied pyrimidinylhydrazones form redox-active iron and copper complexes that are capable of producing intracellular ROS, which might lead to cellular damage and cell death in cancer cells regardless of their resistance status.

Autocrine C‐peptide mechanism underlying INS1 beta cell adaptation to oxidative stress

Excessive generation of reactive oxygen species (ROS) causing oxidative stress plays a major role in the pathogenesis of diabetes by inducing beta cell secretory dysfunction and apoptosis. Recent evidence has shown that C-peptide, produced by beta cells and co-secreted with insulin in the circulation of healthy individuals, decreases ROS and prevents apoptosis in dysfunctional vascular endothelial cells. In this study, we tested the hypothesis that an autocrine activity of C-peptide similarly decreases ROS when INS1 beta cells are exposed to stressful conditions of diabetes. Reactive oxygen species and apoptosis were induced in INS1 beta cells pretreated with C-peptide by either 22 mM glucose or 100 μM hydrogen peroxide (H2 O2 ). To test C-peptide's autocrine activity, endogenous C-peptide secretion was inhibited by the KATP channel opener diazoxide and H2 O2 -induced ROS assayed after addition of either exogenous C-peptide or the secretagogue glibenclamide. In similar experiments, extracellular potassium, which depolarizes the membrane otherwise hyperpolarized by diazoxide, was used to induce endogenous C-peptide secretion. ROS was measured using the cell-permeant dye chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2 -DCFDA). Insulin secretion and apoptosis were assayed by enzyme-linked immunosorbent assay. C-peptide significantly decreased high glucose-induced and H2 O2 -induced ROS and prevented apoptosis of INS1 beta cells. Diazoxide significantly increased H2 O2 -induced ROS, which was reversed by exogenous C-peptide or glibenclamide or potassium chloride. These findings demonstrate an autocrine C-peptide mechanism in which C-peptide is bioactive on INS1 beta cells exposed to stressful conditions and might function as a natural antioxidant to limit beta cell dysfunction and loss contributing to diabetes.

Reduced Dyes Enhance Single‐Molecule Localization Density for Live Superresolution Imaging

Cell-permeable rhodamine dyes are reductively quenched by NaBH4 into a non-fluorescent leuco-rhodamine form. Quenching is reversible, and their fluorescence is recovered when the dyes are oxidized. In living cells, oxidation occurs spontaneously, and can result in up to ten-fold higher densities of single molecule localizations, and more photons per localization as compared with unmodified dyes. These two parameters directly impact the achievable resolution, and we see a significant improvement in the quality of live-cell point-localization super-resolution images taken with reduced dyes. These improvements carry over to increase the density of trajectories for single-molecule tracking experiments.

Redox Control of the Senescence Regulator Interleukin-1α and the Secretory Phenotype

Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H2O2 and intracellular Ca(2+) levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca(2+) promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca(2+) chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca(2+) chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca(2+) homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.

OP0193 Synovial Fluid Neutrophils Undergoing Netosis Contribute to Joint Inflammation by Producing Citrullinated Autoantigens

BackgroundAnti-citrullinated protein antibodies (ACPA) are characteristically detected in patients with rheumatoid arthritis (RA) and growing evidence suggests that they are involved in disease pathogenesis. During disease flares large numbers of...

Mitochondria Superoxide Is an Early Trigger of Apoptosis in an Ex Vivo Model of Human Beta Thalassemia.

AbstractAbstract 2131Apoptotic cell death is a central feature of beta-thalassemia and other anemias caused by ineffective erythropoiesis. In this study, markers of apoptosis were examined using an ex vivo model of human beta-thalassemia to explore the cause(s) and timing of erythroblast cell death. Beta-globin gene and protein expression were knocked down by lentiviral transduction of beta-globin shRNA (beta-KD) in adult human CD34+ cells. Beta-globin mRNA (Control=4.0E+07 ± 1.4E+06 copies/ng, vs. beta-KD=2.5E+06 ± 1.6E+06 copies/ng, p=0.014) and protein were reduced in beta-KD cells by 90% compared to controls while alpha globin expression was maintained. The effects of imbalanced globin chain synthesis were therefore studied according to the stage of erythroblast maturation. Erythroid progenitor cell commitment and proliferation occur over the first two weeks in culture. During the third week of culture (days 14–21), the proerythroblasts undergo terminal differentiation with the characteristic loss of CD71 from the cell surface.Prior to culture day 14, phenotypic analyses demonstrated low levels of apoptosis in beta-KD and control cultures. On culture day 14, a small but significant increase in active caspase 3 was detected in the beta-KD cells compared to controls (beta-KD=4.0±1.0%, Control=0.7±0.3%, p=0.02) suggesting that apoptosis was initiated during the early stages of terminal maturation. Increases in annexin V staining did not achieve statistical significance on day 14 beta-KD cells (beta-KD=13.1±3.9%, Control=7.6±2.2%, p=0.16). By culture day 18, when orthochromic normoblasts are the most prevalent population in control cultures, a large population of apoptotic cells was detected in the beta-KD. The beta-KD erythroblasts demonstrated further increases in active caspase 3 (beta-KD=11.4±2.2% vs. Control=1.1±0.1%, p=0.014), as well as significant increases in surface annexin V (beta-KD=75.8±3.3%, vs. Control=35.9±12.7%, p=0.024). Western analysis of culture day 18 beta-KD membranes demonstrated a marked increase in alpha-chains compared with culture day 14.Since cleaved caspase 3 was increased in beta-KD cells near the beginning of their terminal maturation and prior to the accumulation of alpha chains in the cell membranes, other triggers of apoptosis were investigated. Mitochondrial superoxide is a reactive species that can be generated by iron or other mitochondrial toxins. Increased levels of mitochondrial superoxide cause apoptosis. The cultured erythroblasts were stained with MitoSOX, a cell-permeable dye that specifically detects mitochondrial superoxide. On culture day 11, mitochondrial superoxide was barely detectable in beta-KD and control cell populations (beta-KD=5.2±3.3% vs. Control=3.6±2.9%, p=0.051). Thereafter, the superoxide detection was increased significantly in beta-KD cells on culture day 14 (beta-KD=54.2±6.7% vs. Control=9.1±2.9%, p=0.003), and culture day 18 (beta-KD=81.1±3.2% vs. Control=34.6±3.8%, p=0.007).Oxidation of cellular membranes by hemichromes and free alpha chains damages thalassemic erythrocytes and their precursors. These data suggest that imbalanced globin chain synthesis also triggers apoptosis during the early stages of terminal differentiation by increasing superoxide formation in the mitochondria. Disclosures:No relevant conflicts of interest to declare.

Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death

BackgroundSimultaneous use of cell-permeant and impermeant fluorescent nuclear dyes is a common method to study cell viability and cell death progression. Although these assays are usually conducted as end-point studies, time-lapse imaging offers a powerful technique to distinguish temporal changes in cell viability at single-cell resolution. SYTO 13 and Hoechst 33342 are two commonly used cell-permeant nuclear dyes; however their suitability for live imaging has not been well characterized. We compare end-point assays with time-lapse imaging studies over a 6 h period to evaluate the compatibility of these two dyes with longitudinal imaging, using both control neurons and an apoptotic neuron model.FindingsIn longitudinal assays of untreated neurons, SYTO 13 addition caused acute necrosis within 3 h, whereas neurons imaged with Hoechst remained viable for at least 6 h. In a staurosporine-induced apoptotic model of neurotoxicity, determinations of the mode of cell death and measurements of nuclear size were identical between longitudinal studies using Hoechst and end-point assays. Alternatively, longitudinal studies using 500 nM or 5 nM SYTO 13 were not consistent with end-point assays.ConclusionsSYTO 13 is acutely neurotoxic and when used in longitudinal studies, masked end-stage morphologic evidence of apoptotic cell death. In contrast, a single application of Hoechst evoked no evidence of toxicity over a 6 h period, and was consistent with end-point characterizations of cell viability and nuclear morphology. For longitudinal characterization of acute cell death, Hoechst is a superior option.