Programmed cell death of tobacco BY‐2 cells induced by still culture conditions is affected by the age of the culture under agitation

Evans Blue staining indicated that actively growing tobacco BY‐2 cells in the exponential phase died more rapidly than quiescent cells in the stationary phase when the cells cultured under agitation were placed under still conditions. Fifty percent cell death was induced at about 18, 26, 80 and 140 h for early, mid, late exponential‐ and stationary‐phase cells, respectively. Actively growing cells became TUNEL (transferase‐mediated dUTP nick end labelling)‐positive more rapidly than quiescent cells, suggesting that the cell death evaluated by Evans Blue is accompanied by DNA cleavages. Electrophoresis of genomic DNA showed a typical ‘DNA laddering' pattern formed by multiples of about 200 bp internucleosomal units. Chromatin condensation was first detected at least within 24 h by light microscopy, and then cell shrinkage followed. These findings suggest that the death of BY‐2 cells induced by still conditions is PCD (programmed cell death).     

H2O2 Involvement in Polyamine-Induced Cell Death in Tobacco Leaf Discs

The response of tobacco (Nicotiana tabacum L.) wild-type SR1 leaf discs in terms of reactive oxygen species (ROS) formation and cell death occurrence was evaluated after exposure to the polyamines (PAs) putrescine (Put), spermidine (Spd), and spermine (Spm). Although NADPH oxidase-like enzyme activity was inhibited by all PAs at 3 or 21 h of treatment, H₂O₂ content increased significantly in a time- and concentration-dependent manner, suggesting that H₂O₂ accumulation was linked to the activity of other ROS-generating enzymes. Polyamine oxidase (PAO) activity, which increased markedly upon application of Spd or Spm, is a prime candidate for the increased H₂O₂ accumulation. Except for 0.1 mM Put, which maintained guaiacol peroxidase (GPOX) and catalase (CAT) activities at the same level as the control, the other PA treatments decreased CAT, ascorbate peroxidase, and GPOX activities at 21 h, contributing to the H₂O₂ increase. Esterase activity and Evans blue staining, two cell death parameters, were negatively affected at 3 h of treatment with 1 mM Spd and with both concentrations of Spm, whereas at 21 h there was an increase in cell death with both concentrations of the three PAs, except for 0.1 mM Put, which did not alter those parameters. The expression of the senescence-associated cysteine protease gene CP1 was measured to monitor senescence, a physiological cell death process. Application of all PAs increased the expression of the gene, except for 0.1 mM Put, which decreased its expression at 21 h. This result was in agreement with the prevention of cell death exerted by Put and evidenced by Evans blue staining, esterase activity, and electrolyte release.     

Abscisic acid is involved in chitosan-induced resistance to tobacco necrosis virus (TNV)

Chitosan (CHT) antiviral activity has been further investigated in the pathosystem Phaseolus vulgaris - tobacco necrosis virus (TNV). CHT application elicited both callose apposition and ABA accumulation in leaf tissues, at 12 and 24h after treatment, respectively, and induced a high level of resistance against TNV. Besides, treatment with the ABA inhibitor nordihydroguaiaretic acid (NDGA), before CHT application, reduced both callose deposition and plant resistance to the virus, thus indicating the involvement of ABA in these processes. Exogenous application of ABA also induced a significant resistance to TNV, though this resistance was abolished by NDGA pre-treatment. These results, overall, indicate that the rise of ABA synthesis induced by chitosan plays an important role in enhancing callose deposition but the latter has only a partial effect on virus spreading, which must be constraint by other resistance mechanisms.     

Morphological changes in the apex of pea roots during and after recovery from aluminium treatment

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. The growing region of the root during and after treatment with Al was examined by marking the root at intervals with India ink. Al-induced cell death was detected by staining with Evans blue. Root growth in 40 μM Al solution relative to that in Al-free solution (RRG) was approximately 45% from 6 h to12 h after the start of the treatment. However, values of RRG from 12 h to 24 h in Al-free solution for recovery or in the same Al solution were about 75% and 35%, respectively, indicating recovery from Al-induced growth inhibition. Images of the root characterized by zonal staining with Evans blue were observed in the sub-apical region (more than 1 mm from the tip) in Al-stressed roots. However, the interval of the stained zone was widened in the root after recovery from Al-induced growth inhibition, though it was narrower and more densely stained with time in the Al-stressed roots. During the recovery, the root apex may resume elongation in a specified region without Al-induced death or injury in cells detected by Evans blue.     

Cell death caused by a combination of aluminum and iron in cultured tobacco cells

The inhibition of growth of tobacco cells ( Nicotiana tabacum L. cv. Samsun) after treatment with A1 in medium containing high concentrations of cations requires the presence of Fe (II or III) during the treatment. We examined whether the inhibition of the post‐treatment growth is due to cell death occurring during the treatment with A1 and Fe. In cells at the end of A1 treatment, the integrity of the plasma membrane and the integrity of the mitochondrial inner membrane were monitored by use of Evans blue staining and the cleavage of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT), respectively. Time‐course and dose‐response experiments indicate that the inhibition of post‐treatment growth is strongly related to Evans blue uptake, but not to MTT cleavage. These results suggest that the loss of integrity of the plasma membrane caused by a combination of Al and Fe directly contributes to cell death and the inhibition of post‐treatment growth.     

Tomato phospholipid hydroperoxide glutathione peroxidase inhibits cell death induced by Bax and oxidative stresses in yeast and plants

Using a conditional life or death screen in yeast, we have isolated a tomato (Lycopersicon esculentum) gene encoding a phospholipid hydroperoxide glutathione peroxidase (LePHGPx). The protein displayed reduced glutathione-dependent phospholipid hydroperoxide peroxidase activity, but differs from counterpart mammalian enzymes that instead contain an active seleno-Cys. LePHGPx functioned as a cytoprotector in yeast (Saccharomyces cerevisiae), preventing Bax, hydrogen peroxide, and heat stress induced cell death, while also delaying yeast senescence. When tobacco (Nicotiana tabacum) leaves were exposed to lethal levels of salt and heat stress, features associated with mammalian apoptosis were observed. Importantly, transient expression of LePHGPx protected tobacco leaves from salt and heat stress and suppressed the apoptotic-like features. As has been reported, conditional expression of Bax was lethal in tobacco, resulting in tissue collapse and membrane permeability to Evans blue. When LePHGPx was coexpressed with Bax, little cell death and no vital staining were observed. Moreover, stable expression of LePHGPx in tobacco conferred protection against the fungal phytopathogen Botrytis cinerea. Taken together, our data indicated that LePHGPx can protect plant tissue from a variety of stresses. Moreover, functional screens in yeast are a viable tool for the identification of plant genes that regulate cell death.     

Heat treatment and viability assessment by Evans blue in cultured Symbiodinium kawagutii cells

Evans blue was used to determine viability in Symbiodinium kawagutii cultures but heat treatments at up to 80°C for 1 h were required for detecting blue-stained dead cells. About 3 h at this temperature was necessary to obtain ~50% blue-stained dead cells, and more than 6 h for 100%. In comparison, Trypan blue showed unusual elevated numbers of viable cells at times when more than 50% cells were dead by Evans blue parameters. Respiration decreased to 17 and 13.2% after 1 and 2 h at 80°C, respectively, and a marginal but still statistically significant 7.5% was scored after 3 h. Thus, cultured S. kawagutii cells were more resistant to heat-induced death than expected, and Evans blue was reliable for assessment of their viability.     

Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner

Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY-2 tobacco cells exposed to H(2)O(2). It was shown that H(2)O(2) is able to induce various morphological cell death features in cultured tobacco BY-2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H(2)O(2) added to the cell culture. The appearance of nuclear fragmentation similar to 'apoptotic bodies' associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H(2)O(2). The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H(2)O(2) induces high necrotic cell death. These data suggest that H(2)O(2)-induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.     

Cytosine Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture

Abstract: Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC₅₀ of ∼60 µ M when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC₅₀ = 1 µ M ), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.     

Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress

Salt stress causes oxidative damage and cell death in plants. Plants accumulate proline and glycinebetaine (betaine) to mitigate detrimental effects of salt stress. The aim of this study was to investigate the protective effects of proline and betaine on cell death in NaCl-unadapted tobacco (Nicotiana tabacum) Bright Yellow-2 suspension-cultured cells subjected to salt stress. Salt stress increased reactive oxygen species (ROS) accumulation, lipid peroxidation, nuclear deformation and degradation, chromatin condensation, apoptosis-like cell death and ATP contents. Neither proline nor betaine affected apoptosis-like cell death and G(1) phase population, and increased ATP contents in the 200mM NaCl-stressed cells. However, both of them effectively decreased ROS accumulation and lipid peroxidation, and suppressed nuclear deformation and chromatin condensation induced by severe salt stress. Evans Blue staining experiment showed that both proline and betaine significantly suppressed increment of membrane permeability induced by 200mM NaCl. Furthermore, among the ROS scavenging antioxidant defense genes studied here, mRNA levels of salicylic acid-binding (SAbind) catalase (CAT) and lignin-forming peroxidase (POX) were found to be increased by proline and betaine under salt stress. It is concluded that both proline and betaine provide a protection against NaCl-induced cell death via decreasing level of ROS accumulation and lipid peroxidation as well as improvement of membrane integrity.     

Analysis of aclarubicin-induced cell death in human fibroblasts

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.     

PCD hallmarks in cell suspension cultures of white poplar (Populus alba L., cv. “Villafranca”) challenged with cadmium

Abstract

The response to oxidative stress was investigated in suspension cultures of Populus alba L. “Villafranca” exposed to cadmium. Cell death was demonstrated by Evans Blue staining. Although DNA laddering was not detected, the nuclear morphology evaluated by DAPI revealed irregularly stained granular nuclei derived from chromatin condensation, a programmed cell death hallmark.     

Nitroprusside induces cardiomyocyte death: interaction with hydrogen peroxide

We examined the hypothesis that sodium nitroprusside (SNP) produces cell death in cardiomyocytes through generation of H(2)O(2). Embryonic chick cardiomyocytes in culture were treated with SNP, and cell viability was assessed by trypan blue, MTT assay, and fluorescent activated cell sorting (FACS) analysis. SNP for 24 h induced a significant (P < 0.001) dose-dependent loss of cell viability. On MTT assay, the half-maximal effective concentration was 0.53 mM (confidence interval 0.45-0.59 mM). SNP-treated cardiomyocytes displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated SNP-induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by propidium iodide (PI) staining of permeabilized cells, increased significantly (P < 0.05) after 0.5 mM SNP for 24 h. The proportion of apoptotic cells, characterized by dual staining of intact cardiomyocytes with fluorescein diacetate and PI, was significantly (P < 0.05) increased after treatment with 0.5 mM SNP for 24 h. SNP metabolism and NO production was suggested by the significant (P < 0.05) increase in nitrite generation in the media with 0.5 mM SNP compared with control. SNP-mediated H(2)O(2) production was implicated in the mechanism of SNP-induced cell death. First, SNP produced a significant (P < 0.05) increase in H(2)O(2) detected in the media after 6 or 24 h of SNP treatment. Second, catalase completely blocked the reduction of cell viability induced by 0.1 mM SNP and significantly (P < 0.05) blunted the effect of 0.5 mM SNP. In contrast, the iron chelator deferoxamine did not alter SNP-induced loss of cell viability. FACS analysis showed that the combination of low concentrations of H(2)O(2) (10(-8) M) that did not alter cell viability augmented SNP-induced apoptosis. In contrast, the amount of necrotic cell death was unchanged by the combination of H(2)O(2) and SNP. H(2)O(2) plus SNP produced a dramatic alteration in cell structure with greater membrane bleb formation, shrunken cells, and more intense cytosolic acridine orange staining and nuclear fragmentation than either agent alone. These data indicate the vulnerability of cardiomyocytes to SNP and suggest the involvement of H(2)O(2) in the pathogenesis of SNP-induced cardiomyocyte cell death. Establishing apoptosis as a component of the type of cell death induced by SNP permitted the recognition that SNP-induced apoptosis was increased by chronic treatment with low (subtoxic) concentrations of H(2)O(2).     

Nuclear-Cycle Dependence of the Development of the Preprophase Band in Tobacco BY-2 Cells

When tobacco BY-2 cells that had been treated with aphidicolinfor 24 h were cultured in the absence of aphidicolin, DNA synthesiswas initiated within 1 h. DNA synthesis was completed within4 h and then the preprophase band of microtubules (PPB) developed.However, when cells that had been treated with aphidicolin werecultured in the absence of aphidicolin for 1 h and then againin its presence, DNA synthesis, which was initiated during thehour in the absence of aphidicolin, was not completed withina further 10 h in the presence of aphidicolin. Moreover, thePPB did not develop even after the PPB had appeared and disappearedin cells that were cultured contemporaneously in the continuedabsence of aphidicolin. The development of the PPB seems to be causally associated withthe nuclear cycle of cell division in tobacco BY-2 cells. Thisprocess seems to require the completion of the replication ofall, or almost all, of the nuclear DNA. (Received July 25, 1992; Accepted November 24, 1992)     

The role of changes in the cytoplasmic concentration of Ca2+ in dexamethasone-induced thymocyte death]

The putative role of changes in cytosolic Ca2+ concentration ([Ca2+]i) in the dexamethasone (DM) induced thymocyte death was investigated. Incubation of rat thymocytes with 10(-7) M DM for different time intervals from 0.1 to 8 h did not change the basal [Ca2+]i level ca 100 nM as measured with Ca(2+)-fluorescent probe Quin-2. Ca2+ influx measured by the rate of 45Ca2+ uptake was also just the same in DM treated and control cells. At the same time a 6-8 h incubation of cell suspension with 10(-7) M DM results in significant increase in DNA fragmentation and pyknosis, and a 24 h incubation is associated with the decrease in the percentage of cells not staining with Trypan blue. Thus, the results obtained indicate that 10(-7) M DM induces thymocyte death without any significant and constant [Ca2+]i rise during the first 8 h after hormone application.     

Human Bcl-2 protects against AMPA receptor-mediated apoptosis

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.     

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers. 1. The Possible Role of Cell Death

The ability of a cell-free secretion from germinating conidia(germination fluid) and heat-killed conidia of Botrytis cinereaPers. ex Pers. to induce resistance in exposed carrot root tissueswas unaffected during freezing to –25 °C and rapidthawing, and by heating to 50 °C for 10 min. Activity waslost by boiling for 10 min or at room temperature after 24 h. There was less cell death (Evans blue and neutral red staining)in the surface tissue of slices pretreated with heat-killedconidia and subsequently exposed to infection than in similartissue of untreated, inoculated slices. Both inducing preparationsalone caused maceration and cell death in one or two cell layersof the slice surface. Healthy tissue killed by freezing to –25°Cfor 2 h and rapid thawing, released a factor which, when similarlytested on slices, resulted in germ-tube inhibition and reducedvisual symptoms following inoculation with live spores. Thepossibility that cell death triggered the release of a hostelicitor which caused enhanced accumulation of phytoalexinsand suberin deposition is discussed. Botrytis cinerea, carrot, induced resistance, cell death, vital staining     

Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619-626). After culture for 72 h, cells were further cultured for 24-72 h in medium without FBS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The number of wild-type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (0.1 or 1 ng/ml) or thapsigargin (10(-7) or 10(-6) M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF-alpha (10 ng/ml) or thapsigargin (10(-5) M) caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity in wild-type cells was significantly increased by culture with TNF-alpha (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10(-5) M) caused a significant increase in Ca(2+)/calmodulin-dependent NO synthase activity in wild-type cells or transfectants. TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-nitro-L-arginine (10(-4) M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with thapsigargin (10(-6) M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin-induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF-alpha or thapsigargin.