Cyanide sensitive and insensitive bioenergetics in a clonal neuroblastoma x glioma hybrid cell line

The primary mechanism of cyanide (CN) intoxication is the inhibition of metabolism in the central nervous system. We determined the effects of CN on several biochemical processes in neuroblastoma x glioma hybrid NG108-15 cells, which possess numerous neuronal properties. These cells were not sensitive to a high concentration (1 mM) of NaCN, but became sensitive in the presence of the anaerobic glycolysis inhibitors sodium iodoacetate (IA) and 2-deoxyglucose (2-DG): cellular metabolic processes (e.g., DNA, RNA and protein synthesis) decreased, to about 40% of control due to treatment with 0.5 mM NaCN+0.05 mM IA and 0.1 mM NaCN+20 mM 2-DG. ATP in cells exposed to 0.01 or 0.1 mM NaCN+20 mM 2-DG was reduced 75% and 100%, respectively within one min. Pretreatment of cells with the CN antidote cobalt (II) chloride (CoCl₂) (0.06–0.18 mM) for 5 min prevented the depression of both [³H]leucine incorporation and ATP synthesis due to 1 mM NaCN+20 mM 2-DG in a concentration-dependent manner. A proposed CN antidote alpha-ketoglutaric acid (disodium salt) also prevented the depression of cellular metabolism due to NaCN plus 2-DG. These results indicate that blocking anaerobic glycolysis makes NG108-15 cells sensitive to a low concentration of CN. Thus NG108-15 cells should be useful to study the mechanisms of neurotoxicity of CN and to test antidotes.     

Chemically induced mutation of L5178Y mouse leukemia cells from asparagine-dependence to asparagine-independence

L₅₁₇8Y mouse lymphoblastic leukemia cells are auxotrophic for l-asparagine (ASN) and have been widely used as a model system for studies on l-asparagine independence, were treated with known chemical mutagens to investigate the molecular basis of this mutation. Mutagens which primarily induce base pair substitutions—ethyl methanesullfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)—as well as those which induce frame-shift mutations (the acridine half-mustards ICR-₃₇₂ and ICR-191) each increased the frequency of ASN⁺ cells in treated cultures to at least ten times the usual background frequency of 1 to 2 ASN⁺ cells per 10⁶ cells. The effectiveness of both classes of mutagens indicates that the change to asparagine prototrophy might occur by a mechanism other than, or in addition to, reversion of a specific base pair, point mutation. The mutability of this easily assayed nutritional genetic marker in a cell line that can be grown either in vitro or in vivo may provide a useful system for assay of other agents of unknown mutagenic potential.     

Toll‐like receptor 9 protects non‐immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca²⁺ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.     

Evidence of lysosomal membrane permeabilization in mucopolysaccharidosis type I: Rupture of calcium and proton homeostasis

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α‐iduronidase (IDUA), which leads to intralysosomal accumulation of glysosaminoglycans. Patients with MPS I present a wide range of clinical manifestations, but the mechanisms by which these alterations occur are still not fully understood. Genotype–phenotype correlations have not been well established for MPS I; hence, it is likely that secondary and tertiary alterations in cellular metabolism and signaling may contribute to the physiopathology of the disease. The aim of this study was to analyze Ca²⁺ and H⁺ homeostasis, lysosomal leakage of cysteine proteases, and apoptosis in a murine model of MPS I. After exposition to specific drugs, cells from Idua?/? mice were shown to release more Ca²⁺ from the lysosomes and endoplasmic reticulum than Idua+/+ control mice, suggesting a higher intraorganelle store of this ion. A lower content of H⁺ in the lysosomes and in the cytosol was found in cells from Idua?/? mice, suggesting an alteration of pH homeostasis. In addition, Idua?/? cells presented a higher activity of cysteine proteases in the cytosol and an increased rate of apoptotic cells when compared to the control group, indicating that lysosomal membrane permeabilization might occur in this model. Altogether, our results suggest that secondary alterations—as changes in Ca²⁺ and H⁺ homeostasis and lysosomal membrane permeabilization—may contribute for cellular damage and death in the physiopathology of MPS I. J. Cell. Physiol. 223: 335–342, 2010. © 2010 Wiley‐Liss, Inc.     

Fat mass and obesity-associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

As the first identified N⁶-methyladenosine (m⁶A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m⁶A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m⁶A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m⁶A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m⁶A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m⁶A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m⁶A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m⁶A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.     

Reduced shear stress: A major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity

BHK-21 cells were cultured under various shear stress conditions in an Integrated Rotating-Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm² (simulated microgravity) to 0.92 dyn/cm². Under simulated microgravity conditions, BHK-21 cells complexed into three-dimensional cellular aggregates attaining 6 × 10⁶ cells/ml as compared to growth under 0.92 dyn cm² conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells. © 1993 Wiley-Liss, Inc.     

Stable nuclear RNA returns to post-division nuclei following release to the cytoplasm during mitosis

When nuclei from ³H-RNA-containing amebae (A. proteus), chased for as many as 8 cell generations, are implanted into unlabeled enucleate cells, the nuclei retain 30% or more of the cellular ³H-RNA (or at least 15 times the cytoplasmic concentration of ³H-RNA). After such cells divide, the daughter nuclei retain approximately the same proportion of total cellular ³H-RNA—although all (or almost all) of the nuclear RNA is liberated to the cytoplasm during mitosis. Thus, we conclude that RNA stably associated with the interphase nucleus has a particular affinity for the nucleus despite the fact it is in the cytoplasm when the chromosomes are condensed and the nuclear envelope is not intact.     

Autoradiographic studies on glucose utilization in individual zone of the rat thymus

Summary Wistar rats were injected intraperitoneally with 2-(¹⁴C)deoxyglucose and their thymuses were processed for thaw-mount autoradiography after 5, 10 or 35 min. Highest levels of radioactivity were demonstrated in the thymic medulla (5-fold higher than in the cortex). Scanning of autoradiograms for regional differences in grain densities indicated particularly intense glucose utilization in the cortico-medullary zone. Differences in glucose utilization between individual thymic zones seem to reflect differences in cellular composition, i. e., ratio of stroma cells to thymocytes.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Characterization of Odorous Compounds in Rotten Blue-green Algae

An ice-nucleating bacterium, strain KUIN-1, was isolated from the leaves of field beans (Phaseolus vulgaris L.). Strain KUIN-1 was identified as Pseudomonas fluorescens from its taxonomical characteristics. Ice-nucleating activity was obtained when strain KUIN-1 was cultured aerobically in a medium containing Koser citrate broth (pH 7.0) for 24 hr at 18°C. The ice- nucleating activity did not appear until the bacterial cell concentration reached 10⁷ to 10⁸/ml. Nucleation at — 3.0°C was detected in suspensions (1.8 × 10⁹ cells/ml) of cells that had been grown on the medium containing Koser citrate broth. Strain KUIN-1 produced a lower nucleation frequency (i.e. the number of ice nuclei/cell) than did ice-nucleating Pseudomonas syringae No. 31 suspensions, particularly at temperatures above — 5°C. The nucleation frequency of strain KUIN- 1-suspensions was similar to that obtained for an ice-nucleating Erwinia herbicola No. 26 at — 5°C.     

pH effect on cellular uptake of Sn(IV) chlorine e6 dichloride trisodium salt by cancer cells in vitro

The effects of pH value and presence of serum in an incubation medium on photosensitizer drug cellular uptake in MCF7 cancer cells have been investigated. The results showed that the presence of serum in an incubation medium reduced the drug cellular uptake at all pH values. It has been found that decreasing on pH values of the incubation medium increased the cellular uptake of the drug, demonstrating selective uptake of the sensitizer. The HepG2 liver cancer cells exhibited more drug cellular uptake than CCD-18CO normal colon cells, which assessed the selectivity uptake of photosensitizer on cancerous cells. The concentration of photosensitizer measured in 10⁶ cells showed a good correlation to the incubation time. Fluorescence and absorption spectroscopy been have used to examine the cells.     

Aberrant Ca2+ oscillations in smooth muscle cells from overactive human bladders

Overactive bladder (OAB) syndrome is highly prevalent and costly, but its pathogenesis remains unclear; in particular, the origin of involuntary detrusor muscle activity. To identify the functional substrate for detrusor muscle overactivity, we examined intracellular Ca²⁺ oscillations in smooth muscle cells from pathologically overactive human bladders. Basal cytoplasmic Ca²⁺ concentration was elevated in smooth muscle cells from overactive bladders. Unprovoked, spontaneous rises of Ca²⁺ were also identified. These spontaneous Ca²⁺ oscillations were Ca²⁺-dependent, sensitive to L-type Ca²⁺ channel antagonist verapamil and also attenuated by blocking SR Ca²⁺ reuptake. The fraction of spontaneously active cells was higher in cells from overactive bladders and the magnitude of spontaneous Ca²⁺ oscillations also greater. Spontaneous action potentials or depolarising oscillations were also observed, associated with Ca²⁺ rise; with a higher percentage of cells from idiopathic OAB, but not in neurogenic OAB. Low concentrations of NiCl₂ attenuated both spontaneous electrical and Ca²⁺ activation. This study provides the first evidence that spontaneous, autonomous cellular activity—Ca²⁺ and membrane potential oscillations, originates from detrusor smooth muscle in human bladders, mediated by extracellular Ca²⁺ influx and intracellular release. Such cellular activity underlies spontaneous muscle contraction and defective Ca²⁺ activation contributes to up-regulated contractile activity in overactive bladders.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Effect of peroxynitrite on production of superoxide anion radicals and calcium homeostasis in cells of astroglial origin

We studied the effect of a donor of peroxynitrite, SIN-1, on the morphological characteristics of interweaved rat C6 glioma cells, on menadione-induced production of superoxide anion radicals, and on the concentration of Ca²⁺ in these cells. In concentrations of 1.25·10⁻⁴ to 2.5·10⁻⁷ M, SIN-1 demonstrated cytotoxic and antimitogenic effects. This donor of peroxynitrite caused abnormal modifications of the size of C6 cells and the structure of cellular organelles, intensified in a dose-dependent manner the release of Ca²⁺ from cellular stores into the cytoplasm, and suppressed menadione-induced production of superoxide anion radicals. Therefore, it should be believed that peroxynitrite exerts a modifying effect on the processes of mitotic division and induces apoptosis; it is also involved in the processes of intracellular signalling providing an increase in the concentration of cytosolic Ca²⁺ and a decrease in the redox activity of cells. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 401–406, September–December, 2006.     

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD⁺-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.     

Synthesis and biological evaluation of new C-12(α/β)-(N-) sulfamoyl-phenylamino-14-deoxy-andrographolide derivatives as potent anti-cancer agents

Andrographolide, the major diterpenoidal constituent of Andrographis paniculata (Acanthaceae) and its derivatives have been reported to possess plethora of biological properties including potent anti-cancer activity. In this work, synthesis and in-vitro anti-cancer evaluation of new C-12-substituted aryl amino 14-deoxy-andrographolide derivatives (III a–f) are reported. The substitutions include various sulfonamide moieties –SO₂-NH-R¹. The new derivatives (III a–e) exhibited improved cytotoxicity (GI₅₀, TGI and LC₅₀) compared to andrographolide (I) and the corresponding 3,14,19-O-triacetyl andrographolide (II) when evaluated against 60 NCI cell line panel. Compounds III c and III e are found to be non-toxic to normal human dermal fibroblasts (NHDF) cells compared to reference drug THZ-1.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

Cellular magnesium homeostasis

Magnesium, the second most abundant cellular cation after potassium, is essential to regulate numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signaling pathways, as attested by more than 1000 entries in the literature. Despite significant recent progress, however, our understanding of how cells regulate Mg²⁺ homeostasis and transport still remains incomplete. For example, the occurrence of major fluxes of Mg²⁺ in either direction across the plasma membrane of mammalian cells following metabolic or hormonal stimuli has been extensively documented. Yet, the mechanisms ultimately responsible for magnesium extrusion across the cell membrane have not been cloned. Even less is known about the regulation in cellular organelles. The present review is aimed at providing the reader with a comprehensive and up-to-date understanding of the mechanisms enacted by eukaryotic cells to regulate cellular Mg²⁺ homeostasis and how these mechanisms are altered under specific pathological conditions.     

Probing Novel Roles of the Mitochondrial Uniporter in Ovarian Cancer Cells Using Nanoparticles

Nanoparticles provide a potent tool for targeting and understanding disease mechanisms. In this regard, cancer cells are surprisingly resistant to the expected toxic effects of positively charged gold nanoparticles (⁺AuNPs). Our investigations led to the identification of MICU1, regulator of mitochondrial calcium uniporter, as a key molecule conferring cancer cells with resistance to ⁺AuNPs. The increase in cytosolic [Ca²⁺]_cyto in malignant cells induced by ⁺AuNPs is counteracted by MICU1, preventing cell death. Pharmacological or siRNA-mediated inhibition of mitochondrial Ca⁺² entry leads to endoplasmic reticulum stress and sensitizes cancer cells to ⁺AuNP-induced cytotoxicity. Silencing MICU1 decreases Bcl-2 expression and increases caspase-3 activity and cytosolic cytochrome c levels, thus initiating the mitochondrial pathway for apoptosis: effects further enhanced by ⁺AuNPs. This study highlights the potential of nanomaterials as a tool to broaden our understanding of cellular processes, establishes MICU1 as a novel regulator of the machinery in cancer cells that prevents apoptosis, and emphasizes the need to synergize nanoparticle design with understanding of mitochondrial machinery for enhancing targeted cellular toxicity.