Staurosporine Induces Programmed Cell Death in Embryonic Neurons and Activation of the Ceramide Pathway

Abstract: We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1–1.0 µ M ) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N -Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [³H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 µ M ), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death.     

Over‐expression of HSP70 attenuates caspase‐dependent and caspase‐independent pathways and inhibits neuronal apoptosis

HSP70 is a member of the family of heat‐shock proteins that are known to be up‐regulated in neurons following injury and/or stress. HSP70 over‐expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over‐expression by transfection with HSP70‐expression plasmids in primary cortical neurons and the SH‐SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2‐ceramide, and β‐Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease‐activating factor 1, as well as apoptosis‐inducing factor, key molecules involved in development of caspase‐dependent and caspase‐independent PCD, respectively. Markers of caspase‐dependent PCD, including active caspase‐3, caspase‐9, and cleaved PARP were attenuated in neurons over‐expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase‐dependent and caspase‐independent PCD pathways.     

The kiss of death

The programmed cell death (PCD) of neurons is generally thought to be cell autonomous and not to require a death signal from other cells. A recent study by Marín-Teva et al., in this issue of Neuron, brings this theory into question and suggests that neighboring microglia actively participate in the PCD of Purkinje cells in the cerebellum.     

Cell cycle phase-specific death response of tobacco BY-2 cell line to cadmium treatment

The character of programmed cell death (PCD) in plants differs in connection with the context, triggering factors and differentiation state of the target cells. To study the interconnections between cell cycle progression and cell death induction, we treated synchronized tobacco BY-2 cells with cadmium ions that represent a general abiotic stressor influencing both dividing and differentiated cells in planta. Cadmium induced massive cell death after application in all stages of the cell cycle; however, both the progression and the forms of the cell death differed pronouncedly. Apoptosis-like PCD induced by cadmium application in the S and G2 was characterized by pronounced internucleosomal DNA fragmentation. In contrast, application of cadmium in M and G1 phases was not accompanied by DNA cleavage, indicating suppression of autolysis and non-programmed character of the death. We interpret these results in the context of the situation in planta, where the induction of apoptosis-like PCD in the S and G2 phase might be connected with a need to preserve genetic integrity of dividing meristematic cells, whereas suppression of PCD response in differentiated cells (situated in G1/G0 phase) might help to avoid death of the whole plant, and thus enable initiation of the recovery and adaptation processes.     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.     

木质部细胞分化的程序

本文主要对近十几年来有关木质部细胞分化研究中使用的实验系统及用这些系统所取得的重要进展作了评述.并以作者实验室的研究成果为基础,结合国内外研究进展,提出木质部细胞分化程序由参与细胞编程死亡(PCD)和次生壁构建的全部基因综合编制而成.以PCD过程各阶段的划分标准来看,木质部细胞分化中从IAA诱导形成层细胞平周分裂到细胞扩大前为PCD的起始阶段,其间包括死亡信号的发生、接受和传导,以及启始caspase(半胱氨酰基天门冬氨酸蛋白酶)类似物(例如caspase-8类似物)的活化;木质部母细胞的径向扩大为PCD的效应阶段,而效应caspase类似物(例如caspase-3类似物)活化DNase、DNA的片段化及次生细胞壁的构建和各种细胞器的解体则为PCD的清除降解阶段.至今还无法将DNase活化及其引起的DNA断裂过程与次生细胞壁构建过程分开.     

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three- dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.     

Ceramide Induces Apoptosis to Immature Cerebellar Granule Cells in Culture

A recent study revealed that ceramide acts as a second messenger in the sphingomyelin pathway and thus plays an important regulatory role in programmed cell death (apoptosis) to cell the lines induced by tumor-necrosis factor (TNF)- and interleukin (IL)-1, although its effect remains controversial regarding primary neuronal culture. We investigated the effect of a cell-permeable ceramide analog (C₂-ceramide) on cultures of cerebellar granule cells, which is thought to have active sphingomyelin pathway during development. The presence of C₂-ceramide decreased the number of cerebellar granule cells (CGCs) in a concentration-dependent manner when added at DIV 1 (1 day in vitro). The ED₅₀ was 60 M. After DIV2, CGCs became less sensitive to C₂-ceramide and the ED₅₀ was 200 M at DIV 7. DNA staining with Hoechst 33258 showed the morphology of apoptotic nuclei in the degenerating neurons. Internucleosomal DNA degradation could also be observed by gel electrophoresis. Protein and RNA synthesis inhibitors prevented the death of neurons. C₂-dihydroceramide, which lacks the 4–5 trans double bond and failed to induce neuronal death. These results thus demonstrated that C₂-ceramide induces apoptosis to the CGCs at the early stage in vitro, however the CGCs were found to be less sensitive to C₂-ceramide at the later stage in vitro.     

Cytokinins: new apoptotic inducers in plants

High concentrations of cytokinins block cell proliferation and induce programmed cell death (PCD) in both carrot ( Daucus carota L.) and Arabidopsis thaliana (L.) Heynh. cell cultures [13 and 27 micro M N(6)-benzylaminopurine (BAP), respectively]. In the present work, cell death was scored by Evan's blue staining and was also demonstrated to be programmed by various parameters, including chromatin condensation, oligonucleosomal DNA degradation (laddering), and release of cytochrome c from mitochondria. In carrot cells, this induction takes approximately 24 h, with proliferating cells being more sensitive than quiescent ones. Two hormones, namely abscisic acid and 2,4-dichlorophenoxyacetic acid (2,4-D), protect cells against the cytokinin-induced death. PCD is not merely a consequence of the inability of the culture to proliferate, since high levels of 2,4-D block carrot cell proliferation without promoting PCD. Increased ethylene production was also observed in BAP-treated cultures, although this increase was not responsible for PCD because inhibitors of ethylene synthesis and action did not block PCD in BAP-treated cultures. Programmed cell death in the form of DNA laddering was also seen in plants treated with cytokinins. This process was accompanied by accelerated senescence in the form of leaf yellowing.     

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells

Background information. PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). Results. SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling)‐positive cells both in inactive and feeding animals. The DA‐induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto‐c (cytochrome c) translocation into the cytosol and methyl‐β‐cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto‐c was under the control of Bcl‐2 and Bad. DA also increased the active caspase‐3 in gland cells while D2 receptor antagonists and TEA attenuated it. Conclusion. Our results provide evidence for a type of transmitter‐mediated pathway that regulates the PCD of secretory cells in a mitochondrial‐caspase‐dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto‐c, ceramide, Bcl‐2 proteins and caspase‐3, but not caspase‐8, was demonstrated in cells involved in the DA‐induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.     

Kinetin induces cell death in root cortex cells of Vicia faba ssp. minor seedlings

The double fluorescence staining with acridine orange and ethidium bromide (AO/EB) revealed that treatment of Vicia faba ssp. minor seedlings with kinetin-induced programmed cell death (PCD) in root cortex cells. Kinetin-induced cell death reflected by the morphological changes of nuclei including their invagination, volume increase, chromatin condensation and degradation as well as formation of micronuclei showed by AO/EB and 4,6-diamidino-2-phenylindol staining was accompanied by changes including increase in conductivity of cell electrolytes secreted to culture media, decrease in the number of the G1- and G2-phase cells and appearance of fraction of hypoploid cells as the effect of DNA degradation without ladder formation. Decrease in the number of mitochondria and in the activity of cellular dehydrogenases, production of reactive oxygen species (ROS), appearance of small and then large lytic vacuoles and increase in the amount of cytosolic calcium ions were also observed. The PCD was also manifested by increased width and weight of apical fragments of roots as well as decreased length of cortex cells which led to shortening of the whole roots. The kinetin-induced PCD process was almost completely inhibited by adenine, an inhibitor of phosphoribosyl transferase, and mannitol, an inhibitor of ROS production. These cell-death hallmarks and pathway of this process suggested that the induction of kinetin-specific vacuolar type of death, expressed itself with similar intensity on both morphological and metabolic levels, was a transient protecting whole roots and whole seedlings against elimination.     

棉花体细胞增殖和胚胎发生中的细胞程序性死亡

棉花组织培养中愈伤组织褐化可能与细胞程序性死亡(PCD)有关.对棉花组培中不同时期的愈伤组织DNA进行琼脂糖电泳,观察到:仅在第一次愈伤组织继代后10 d左右和愈伤组织第一次继代到分化培养基中培养10 d左右,愈伤组织的DNA产生了180 bp左右的片断或其整数倍片断大小的DNA带,呈DNA梯(DNA ladder)状电泳,说明在这两个时期PCD达到了高峰.在这两次PCD高峰后1周均出现愈伤组织大规模的褐化死亡.显微镜观察到第一次PCD发生高峰的愈伤组织存在许多管状、内含物少的细胞,这些细胞分布在愈伤组织的边缘和内部;第二次PCD发生高峰观察到体细胞胚胎分化、PCD细胞的木栓化和水渍化.对体细胞胚胎分化时期的原生质体进行荧光染色(DAPI),荧光显微镜下观察到发生细胞程序性死亡的细胞核浓缩、染色质凝聚、结块、边缘化和形成PCD小体.     

The murine TRAIL receptor signals caspase-independent cell death through ceramide

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.     

Nitric oxide and hydrogen peroxide involvement during programmed cell death of Sechium edule nucellus

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we show that nucellus PCD is accompanied by a considerable production of both nitric oxide and hydrogen peroxide (NO and H₂O₂). Interestingly, each of the two molecules is able to induce the production of the other and to cause cell death when applied to a living nucellus. We show that the induced cell death has features of a PCD, accompanied by profound changes in the morphology of the nuclei and by a massive degradation of nuclear DNA. Moreover, we report that NO and H₂O₂ cause an induction of caspase‐like proteases previously characterized in physiological nucellar PCD.     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.     

Studies on the induction of apoptosis in WEHI 231 cells by pharmacological agents and lipid mediators. Sphingosine and ceramide induce apoptosis in WEHI 231

We report the results of a systematic study of the effects of pharmacological agents known to cause or modify physiological cell death (PCD). Using WEHI 231 cells as a model, we investigated the effects of dexamethasone, cAMP, selected growth factors/ cytokines, DNA damaging agents, metabolic inhibitors and lipid mediators. We found that WEHI 231 cells are not affected by cAMP(1-90 microM) or TGFbeta (1-50 ng/ml), both of which are known to induce PCD in other systems. We also failed to detect protection from PCD in WEHI 231 cells cultured with Zn(++), E64 and leupeptin. In contrast, dexamethasone (400 microg/ml), etoposide (10(-4)M), emetine (10(-5)M), calyculin (10(-5)M), sphingosine (8-16 microM) and ceramide (20-40 microM) all cause PCD in WEHI 231 cells. The effects of ceramide can be blocked by LPS but not by overexpression of bcl2.The role of killer lipids in PCD is discussed.     

Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

Lipid accumulation due to nitrogen depletion has been studied extensively in Chlamydomonas reinhardtii and the metabolic changes that lead to triacylglycerol biosynthesis have been of particular interest to researchers in the biodiesel industry. The induction of programmed cell death (PCD) in response to nitrogen starvation has also been documented in related chlorophytes. Here, we examined the temporal and metabolic overlap of lipid accumulation and PCD in response to nitrogen starvation in the important model organism C. reinhardtii. Nitrogen starvation induced physiological stress, measured by the progressive decline in chlorophyll a fluorescence, reduced photosynthetic efficiency and decreased growth. In keeping with previous reports, cells accumulated lipids reaching a peak after 2–3 days. At the same time, DNA nicking and caspase‐like protease activity was observed in a proportion of cells, and ultrastructural observations confirmed that death was via PCD. Our results demonstrate that DNA nicking and caspase‐like activity are observed during PCD in C. reinhardtii in response to nitrogen starvation, and that death occurs at the same time as lipid biosynthesis. Microalgal lipid production due to nitrogen depletion in C. reinhardtii is limited by the decrease in culture growth and knowing that the loss of culture density is, at least in part, due to PCD is important for the biotechnology industry.