Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H₂O₂) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO₄. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H₂O₂ were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.     

Inactivation of Ca2(+)-, Na+K(+)-, and H+K(+)-ATPases with a carbodiimide derivative of ATP

The gamma-P adduct of ATP with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ATP-EDC) was synthesized and incubated with the Ca-ATPase of sarcoplasmic reticulum with the result that time-dependent complete loss of the enzyme's activity occurred. The inactivation required calcium and magnesium while ATP had a protective effect. ATP-EDC incubation with the NaK-ATPase and HK-ATPase produced partial (greater than 50%) inactivation, but had no effect on myosin S1, pyruvate kinase and hexokinase, suggesting that this ATP analog is a specific inactivator of the so-called 'P-type' ATPases.     

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.     

在自然衰老和诱导条件下棉花悬浮细胞程序性死亡的发生

Cotton suspension cells grew well in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT. Senescence occurred when the cells were unsubcultured. The cells began to lose their viabilities on the 17th day, and on the 21th day oligonucleosomal sized DNA fragments ( DNA ladder) could be detected. Oligonucleosomal sized DNA fragments ( DNA ladder) was the hallmark of the programmed cell death. Programmed cell death of cotton suspension cells could be induced respectively by some stress factors which included heatshock (42+/-3 degrees C for 8 hours), 10 micromol/L camptothecin, 20 micromol/L fumonisin B1 and 50 mmol/L cycloheximide. The cotton suspension cells which grew in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT differred physiologically from the cells in the MS medium supplemented with 0.1 mg/L IBA and 0.1 mg/L KT, and they responded differentially to the heatshock, 10 micromol/L camptothecin and 20 micromol/L fumonisin B1, while the same to 50 mmol/L cycloheximide.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis

Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.     

The mechanism by which the mitochondrial ATP-sensitive K+ channel opening and H2O2 inhibit the mitochondrial permeability transition

Myocardial infarction is a manifestation of necrotic cell death as a result of opening of the mitochondrial permeability transition (MPT). Receptor-mediated cardioprotection is triggered by an intracellular signaling pathway that includes phosphatidylinositol 3-kinase, endothelial nitric-oxide synthase, guanylyl cyclase, protein kinase G (PKG), and the mitochondrial K(ATP) channel (mitoK(ATP)). In this study, we explored the pathway that links mitoK(ATP) with the MPT. We confirmed previous findings that diazoxide and activators of PKG or protein kinase C (PKC) inhibited MPT opening. We extended these results and showed that other K(+) channel openers as well as the K(+) ionophore valinomycin also inhibited MPT opening and that this inhibition required reactive oxygen species. By using isoform-specific peptides, we found that the effects of K(ATP) channel openers, PKG, or valinomycin were mediated by a PKCepsilon. Activation of PKCepsilon by phorbol 12-myristate 13-acetate or H(2)O(2) resulted in mitoK(ATP)-independent inhibition of MPT opening, whereas activation of PKCepsilon by PKG or the specific PKCepsilon agonist psiepsilon receptor for activated C kinase caused mitoK(ATP)-dependent inhibition of MPT opening. Exogenous H(2)O(2) inhibited MPT, because of its activation of PKCepsilon, with an IC(50) of 0.4 (+/-0.1) microm. On the basis of these results, we propose that two different PKCepsilon pools regulate this signaling pathway, one in association with mitoK(ATP) and the other in association with MPT.     

Critical O2 and NO concentrations in NO-induced cell death in a rat liver sinusoidal endothelial cell line

Nitric oxide (NO) plus oxygen (O2) are known to cause cell damage via formation of reactive nitrogen species. NO itself directly inhibits cytochrome oxidase of the mitochondrial respiratory chain in competition with O2, thus inducing a hypoxic-like injury. To assess the critical NO and O2 concentrations for both mechanisms of NO-induced cell injury, cells of a rat liver sinusoidal endothelial cell line were incubated in the presence of the NO donor spermineNONOate at different O2 concentrations, and their loss of viability was determined by the release of lactate dehydrogenase. Protection by ascorbic acid was used as indication for the involvement of reactive nitrogen species, whereas a hypoxic-like injury was indicated by the protective effects of glycine and glucose and the increase in NAD(P)H fluorescence. High concentrations of NO (approx. 10 microM NO) and O2 (21% O2) were required to induce endothelial cell death mediated by formation of reactive nitrogen species. On the other hand, pathophysiologically relevant NO concentrations at low but physiological O2 concentrations (ca. 2 microM NO at 5% O2 and about 1 microM NO at 2% O2) induced hypoxic-like cell death in the endothelial cells that was prevented by the presence of glucose.     

Involvement of 5-lipoxygenase in programmed cell death of cancer cells

We investigated the involvement of 5-lipoxygenase activity in the early phases of programmed cell death (PCD) induced by H2O2 or retinoids in different human tumour cells (erythroleukaemia, neuroblastoma, melanoma). Apoptotic cells showed enhanced 5-lipoxygenase activity which was paralleled by decreased superoxide dismutase activity and increased light emission. Ultraweak luminescence, mainly due to membrane lipid peroxidation by lipoxygenase activation, increased in all cell lines tested within 10-15 min after induction of PCD, in a concentration and time-dependent manner. At the same time, we observed a significant increase in the intracellular steady state level of the 5-lipoxygenase metabolite leukotriene B4. Furthermore, 5-lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid was able to induce PCD in all cell lines tested. Conversely, the general lipoxygenase inhibitor nordihydroguaiaretic acid and the selective 5-lipoxygenase inhibitor caffeic acid protected the different tumour cells from H2O2-induced PCD to a similar extent. These results show the activation of the 5-lipoxygenase pathway in PCD of three different cancer cell lines.     

Elicitor-induced defence reactions in cell suspension cultures of soybean cultivars

Suspension cultured soybean (Glycine max [L.] Merr.) cells of four cultivars (Wilis, Lumut, Kalmit, Doko RC) were compared for their response to different fungal and bacterial elicitors. Cells were treated either with crude cell wall extracts of the fungal pathogens Phytophthora sojae (Pmg-elicitor) and Rhizoctonia solani (Riso-elicitor) or with two isolates of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg01/02) and a broad spectrum of antimicrobial defence reactions was measured. Cells of all four cultivars showed the same elicitor-induced rapid (H2O2 accumulation, alkalinization of the culture medium, peroxidative cross-linking of cell wall proteins) and slow (activation of phenylpropanoid metabolism, accumulation of phenolic compounds, induction of PR-proteins) defence responses. However, the reactivity of the cultivars was not identical in terms of time courses and intensities. Furthermore, the ability of the various elicitors to induce defence responses varied markedly. These differences indicate that (1) cells of the same species but of different cultivars are equipped with the same array of perception systems to recognise various stimuli but (2) the sensitivity of these perception systems or later steps in the signal transduction seem to be stimulated to a different extent in the analysed cultivars.     

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Proposed mechanism for regulation of H2O2-induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H₂O₂-induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H₂O₂ treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.     

Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. We find that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induced an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Using genetic mutants and RNAi-mediated knockdown of drp-1, we find that Drp-1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila.     

Taxol-induced apoptotic cell death in suspension cultures of Taxus cuspidata

Taxol caused apoptotic cell death of Taxus cuspidata in suspension cultures. Typical morphological and biochemical changes of apoptosis were observed by microscopy and total DNA agarose gel electrophoresis. Taxus cuspidata responded to the added Taxol by increasing the biosynthesis of Taxol. The percentage of apoptotic cells in total cells increased with the concentration of added Taxol. With Taxol added at 10 mg l^–1, the maximum concentration of Taxol produced was 23 mg l^–1, 3 times higher than that of the control culture.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary

The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis.     

Influence of b-cyclodextrin in bio-transformation of (+)-3-carene by cell suspension cultures of Acetobacter acetii

Acetobacter acetii, a local isolate transformed (+)-3-carene. b-Cyclodextrin enhanced the stability of the products and also aided the generation of step-wise transformation giving rise to oxygented derivatives at positions 2,3,4 and 5. Both the control and b-cyclodextrin mediated transformations gave rise to 17 transformation products which included 8-hydroxy-m-cymene, carane-3,4-diol-2,5-dione, 3-carene-2-ol-5-one, 3,4-epoxy-carane, 3-carene-2,5-diol and carane-carboxylic acid. A plausible orientation of (+)-3-carene inside b-cyclodextrin cavity is also suggested.     

Involvement of TatD nuclease during programmed cell death in the protozoan parasite Trypanosoma brucei

In this report, we describe the involvement of TatD nuclease during programmed cell death (PCD) in the human protozoan parasite Trypanosoma brucei. T. brucei TatD nuclease showed intrinsic DNase activity, was localized in the cytoplasm and translocated to the nucleus when cells were treated with inducers previously demonstrated to cause PCD in T. brucei. Overexpression of TatD nuclease resulted in elevated PCD and conversely, loss of TatD expression by RNAi conferred significant resistance to the induction of PCD in T. brucei. Co‐immunoprecipitation studies revealed that TatD nuclease interacts with endonucleaseG suggesting that these two nucleases could form a DNA degradation complex in the nucleus. Together, biochemical activity, RNAi and subcellular localization results demonstrate the role of TatD nuclease activity in DNA degradation during PCD in these evolutionarily ancient eukaryotic organisms. Further, in conjunction with endonucleaseG, TatD may represent a critical nuclease in a caspase‐independent PCD pathway in trypanosomatid parasites since caspases have not been identified in these organisms.