Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro

The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

Heat shock proteins in leukaemia cell differentiation and cell death

When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1μM), or CB3717 (5μM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comparable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.

The importance of Na(plus)-K(plus)-Cl- cotransport in nitrogen mustard induced cell death

The incubation of murine leukaemic L1210 cells in vitro for 4 hours (hr) with 10uM nitrogen mustard (HN2), a bifunctional alkylating agent, inhibited the influx of the potassium congener, 88rubidium+ ( 86Rb+) by the selective inhibition of the Na+-K+-CI- cotransporter. The aim of this project was to investigate the importance of this lesion in HN2-induced cytotoxicity. 86Rb+ uptake in human erythrocytes was inhibited by high concentrations of HN2 (2mM) and occurred in two phases.In the first hour both the Na+/K+ ATPase pump and the Na+-K+-CI- cotransporter were equally inhibited but after 2 hrs exposure to 2mM HN2, the Na+ -K+ -CI- cotransporter was significantly more inhibited than the Na+/K+ ATPase pump. In contrast, both potassium transport systems were equally inhibited in L1210 cells incubated for 10 minutes with 1mM HN2. The selective inhibition of the Na+-K+-CI- cotransporter, after a 3 hrs exposure to 10uM HN2, was not absolved by coincubation with 5ug/ml cycloheximide (CHX), an inhibitor of protein synthesis. Incubation of L1210 cells with concentrations of diuretics which completely inhibited Na+-K+-CI- cotransport did not enhance the cytotoxicity of either HN2 or its monofunctional analogue 2-chloroethyldimethylamine (Me-HN1). The incubation of L1210 cells with a twice strength Rosewell Park Memorial Institute 1640 media did not enhance the toxicity of HN2. An L1210 cell line (L1210FR) was prepared which was able to grow in toxic concentrations of furosemide and exhibited a similiar sensitivity to HN2 as parental L1210 cells. Treatment of L1210 cells with 10uM HN2 resulted in a decrease in cell volume which was concurrent with the inhibition of the Na+-K+-CI- cotransporter. This was not observed in L1210 cells treated with either 1 or O.SuM HN2. Thus, possible differences in the cell death, in terms of necrosis and apoptosis, induced by the different concentrations of HN2 was investigated. The cell cycle of L1210 cells appeared to be blocked non-specifically by 10uM HN2 and in S and G2/M by either 1 or 0.5uM HN2. There were no significant changes in the cytosolic calcium concentrations of L1210 cells for up to 48 hrs after exposure to the three concentrations of HN2. No protection against th_ toxic effects of HN2 was observed in L1210 cells incubated with 5ug/ml CHX for up to 6 hrs. Incubation for 12 or 18 hrs with a non-toxic concentration (5mM) of L-Azetidine-2- carboxylic acid (ACA) enhanced the toxicity of low concentrations (<0.5uM) of HN2.

Dealing with cell death – an ageing problem?

The aged population have an increased susceptibility to infection, therefore function of the innate immune system may be impaired as we age. Macrophages, and their precursors monocytes, play an important role in host defence in the form of phagocytosis, and also link the innate and adaptive immune system via antigen presentation. Classically-activated ‘M1’ macrophages are pro-inflammatory, which can be induced by encountering pathogenic material or pro-inflammatory mediators. Alternatively activated ‘M2’ macrophages have a largely reparative role, including clearance of apoptotic bodies and debris from tissues. Despite some innate immune receptors being implicated in the clearance of apoptotic cells, the process has been observed to have a dominant anti-inflammatory phenotype with cytokines such as IL-10 and TGF-ß being implicated. The atherosclerotic plaque contains recruited monocytes and macrophages, and is a highly inflammatory environment despite high levels of apoptosis. At these sites, monocytes differentiate into macrophages and gorge on lipoproteins, resulting in formation of ‘foam cells’ which then undergo apoptosis, recruiting further monocytes. This project seeks to understand why, given high levels of apoptosis, the plaque is a pro-inflammatory environment. This phenomenon may be the result of the aged environment or an inability of foam cells to elicit an anti-inflammatory effect in response to dying cells. Here we demonstrate that lipoprotein treatment of macrophages in culture results in reduced capacity to clear apoptotic cells. The capability of lipoprotein treated macrophages to respond to inflammatory stimuli is also shown. Monocyte recruitment to the plaque is currently under study, as is apoptotic cell-mediated immune modulation of human monocyte-derived macrophages.

Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells:role of ERK1/2 in H2O2-induced cell death

Reactive oxygen species including H2O2 activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H2O2 in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H2(O2 on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H2O2 stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H2O2 treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H2O2-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H2O2-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H2O2 (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H2O2-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H2O2-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H2O2 stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H2O2-induced cell death.

Programmed cell death (PCD) processes begin extremely early in Alstroemeria petal senescence

-In the Liliaceous species Alstroemeria, petal senescence is characterized by wilting and inrolling, terminating in abscission 8-10 d after flower opening. -In many species, flower development and senescence involves programmed cell death (PCD). PCD in Alstroemeria petals was investigated by light (LM) and transmission electron microscopy (TEM) (to study nuclear degradation and cellular integrity), DNA laddering and the expression programme of the DAD-1 gene. -TEM showed nuclear and cellular degradation commenced before the flowers were fully open and that epidermal cells remained intact whilst the mesophyll cells degenerated completely. DNA laddering increased throughout petal development. Expression of the ALSDAD-1 partial cDNA was shown to be downregulated after flower opening. -We conclude that some PCD processes are started extremely early and proceed throughout flower opening and senescence, whereas others occur more rapidly between stages 4-6 (i.e. postanthesis). The spatial distribution of PCD across the petals is discussed. Several molecular and physiological markers of PCD are present during Alstroemeria petal senescence. © New Phytologist (2003).

Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death:a mechanism for maintaining tissue integrity

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which cross-links proteins via e(g-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca2+ homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca2+ homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca2+, but it is independent of intracellular proteases and is near maximal after only 20min post-wounding. Using labelled primary amines as an indicator of tTG activity within these 'wounded cells', we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.

Microarray analysis of expression of cell death-associated genes in spinal cord cells with cyclic tensile strain

Previous studies have described alterations in gene expression following spinal cord injury, but this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile strain on cultured spinal cord cells from E15 Sprague-Dawley rats. Microarray analysis of gene expression and categorization of identified genes were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. The application of cyclic tensile strain reduced the viability of cultured spinal cord cells significantly in a dose- and time-dependent manner. GO analysis identified candidate genes related to apoptosis (44) and to response to stimulus (17). KEGG analysis identified changes in the expression levels of 12 genes of the mitogen-activated protein kinase (MAPK) signaling pathway, which were confirmed to be upregulated and validated by RT-PCR analysis. Spinal cord cells undergo cell death in response to cyclic tensile strain, which were dose- and time-dependent, with upregulation of various genes, in particular of the MAPK pathway.

The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro:implications for degenerative disc disease

The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.

Apoptotic cell‐associated ICAM‐3 in the phagocytic removal of apoptotic cells

Apoptosis is a highly controlled cell death programme that culminates in the exposure of molecular ‘flags’ at the dying cell surface that permit recognition and removal by viable phagocytes. Failure to efficiently remove dying cells can lead to devastating inflammatory and autoimmune disorders. The molecular mechanisms underlying apoptotic cell surface changes are poorly understood. Our previous work has shown an apoptosis-associated functional change in ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) resulting in a molecular ‘flag’ to mediate corpse removal. Here we detail apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. We show ICAM-3 functions to tether apoptotic leukocytes to macrophages via an undefined receptor. Though CD14 has been suggested as a possible receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Furthermore, we demonstrate leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates reduced cell volume throughout apoptosis. This loss of ICAM-3 occurs via shedding of ICAM-3 in microparticles (‘apoptotic bodies’). Such microparticles are potent chemoattractants for macrophages. Notably, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. These data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of apoptosis.

Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes. © 2012 Macmillan Publishers Limited All rights reserved.

Development of a post-mitotic, neuronal-astrocytic cell system, for the prediction of acute neurotoxicity in humans

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.