DNA Fragmentation Induced by Cytotoxic T Lymphocytes Can Result in Target Cell Death

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.     

Autophagic-Like Cell Death in Neutrophils Induced by Autoantibodies

Human neutrophils undergo autophagic-like cell death following Sialic acid binding immunoglobulin-like lectin-9 (Siglec-9) ligation and concurrent stimulation with certain, but not all, neutrophil survival cytokines. Caspase inhibition by these cytokines is required, but is not sufficient, to trigger this particular form of cell death. Additional mechanisms may involve reactive oxygen species (ROS), and blocking of ROS or prevention of ROS production prevents autophagic-like neutrophil death. Interestingly, human intravenous immunoglobulin (IVIg) preparations contain natural anti-Siglec-9 autoantibodies, which are able to ligate Siglec-9 on neutrophils and induce autophagic-like cell death in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and some other survival cytokines. Here, we discuss the pathophysiological and therapeutic implications of these recent findings.

Addendum to:

Immunological and Functional Evidence for Anti-Siglec-9 Autoantibodies in Intravenous Immunoglobulin (IVIg) Preparations

S. von Gunten, A. Schaub, M. Vogel, B.M. Stadler, S. Miescher and H.-U. Simon

Blood 2006; In press     

镉胁迫引起烟草悬浮细胞程序性死亡

镉胁迫会造成烟草悬浮细胞大规模死亡。通过TUNEL技术和琼脂糖凝胶电泳技术的检测发现,这种细胞死亡伴随有典型的DNA“梯形带”出现,表明这种由Cd胁迫引起的细胞死亡是一种程序性死亡。受胁迫细胞氧化性增强及细胞中丙二醛(MDA)水平升高,说明Cd胁迫时会在细胞中造成大量活性氧(ROS),暗示烟草细胞的程序性死亡可能与ROS有关。     

渗透胁迫诱导的小麦叶片细胞程序性死亡

用20%PEG6000（-0.63MPa）溶液对小麦（Triticumaestivum）根系进行渗透胁迫,在DNA琼脂糖凝胶电泳图谱上观察到明显的梯状DNA条带,表明PEG处理诱发了DNA核小体间的断裂,从而表现出典型的细胞程序性死亡的生化特征;末端脱氧核糖核酸转移酶介导的3′-OH末端标记法（terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end     

Vero Cell Proteomic Changes Induced by Zika Virus Infection

The re‐emergence and the recent spread of the Zika virus (ZIKV) has raised significant global concerns due to lack of information in patient diagnosis and management. Thus, in addition to gaining more basic information about ZIKV biology, appropriate interventions and management strategies are being sought to control ZIKV‐associated diseases and its spread. This study's objective is to identify host cell proteins that are significantly dysregulated during ZIKV infection. SOMAScan, a novel aptamer‐based assay, is used to simultaneously screen >1300 host proteins to detect ZIKV‐induced host protein dysregulation at multiple time points during infection. A total of 125 Vero cell host proteins, including cytokines such as CXCL11 and CCL5, interferon stimulated gene 15, and translation initiation factors EIF5A and EIF4G2, are significantly dysregulated after ZIKV infection. Bioinformatic analyses of 77 host proteins, that are significantly dysregulated ≥1.25‐fold, identify several activated biological processes, including the JAK/STAT, Tec kinase, and complement cascade pathways.     

The Cleavage of Semaphorin 3C Induced by ADAMTS1 Promotes Cell Migration

Metastasis is a sequential process that allows cells to move from the primary tumor and grow elsewhere. Because of their ability to cleave a variety of extracellular signaling and adhesion molecules, metalloproteases have been long considered key components of the metastatic program. However, the function of certain metalloproteases, such as ADAMTS1, is not clear and seems to depend on the cellular environment and/or the stage of tumor progression. To characterize the function of ADAMTS1, we performed two alternative proteomic approaches, difference gel electrophoresis and stable isotope labeling by amino acids in cell culture, to identify novel substrates of the metalloprotease. Both techniques showed that overexpression of ADAMTS1 leads to the release of semaphorin 3C from the extracellular matrix. Although semaphorins are well known regulators of axon guidance, accumulating evidence shows that they may also participate in tumor progression. Here, we show that the cleavage of semaphorin 3C induced by ADAMTS1 promotes the migration of breast cancer cells, indicating that the co-expression of these molecules in tumors may contribute to the metastatic program.     

Factors that Affect the Type of Cell Death Induced by Chemicals

Surveying about 1000 compounds, we found that several low molecular weight α,β-unsaturated ketones induced non-apoptotic cell death characterized by the formation of autophagosomes, occasionally accompanied by mitochondrial shrinkage. The cytotoxic activity of these compounds was significantly reduced by the addition of N-acetyl-L-cysteine, suggesting their interaction with SH groups of intracellular targeted molecules (the so-called “non-sterically hindered Michael acceptor”). This suggests that the nature of the chemical structure as well as the type of target cells is another factor that determines the type of cell death induced by chemicals.

Addendum to:

Tumor-Specificity and Type of Cell Death Induced by Trihaloacetylazulenes in Human Tumor Cell Lines

T. Sekine, J. Takahashi, M. Nishishiro, A. Arai, H. Wakabayashi, T. Kurihara, M. Kobayashi, K. Hashimoto, H. Kikuchi, T. Katayama, Y. Kanda, S. Kunii, N. Motohashi and H. Sakagami

Anticancer Res 2007; 27:133-44     

IgY—Ricin A诱导癌细胞程序化死亡的研究

研究了抗癌导向药物IgY-Ricin A杀伤癌细胞的作用机理。人胃低分化粘液腺癌MGC-803细胞经IgY-Ricin A处理,细胞的增殖明显受到抑制,而同样处理的人胚正常肺细胞2BS,其生长不受这种药物的影响。FCM实验结果表明,IgY-Ricin A处理的MGC-803细胞,在8h开始出现细胞程序化死亡峰Apo,同样处理的2BS细胞则无Apo峰出现。经lgY-Ricin A处理的MGC-803细胞核由均一状态变为浓缩凝集状,经激光共聚焦显微镜进行光切片和三维重组,发现MGC-803细胞核由原来的球形变成高度浓缩凝集的点状结构。DNA凝胶电泳分析显示,IgY-Ricin A处理的MGC-803细胞DNA被降解,呈现梯状电泳条带这一典型的细胞程序化死亡指标。研究结果表明,IgY-Ricin A通过诱导细胞程序化死亡来控制MGC-803细胞的增殖,最终杀死癌细胞。     

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.     

Inhibition of Ced-3/ICE-related Proteases Does Not Prevent Cell Death Induced by Oncogenes, DNA Damage, or the Bcl-2 Homologue Bak

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1β– converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode “death” gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage–induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing.     

Biochemical and Morphological Features of Rice Cell Death Induced by Pseudomonas avenae

Pseudomonas avenae is a Gram-negative phytopathogenie bacteriumthat causes the symptom of a brown stripe in infected susceptibleplants. The host range of P. avenae is wide among the monocotyledonousplants, however, individual strains can infect only one or afew host species. A rice-incompatible strain, N1141, causedrapid cell death in sheath sections and in cultured rice cells.A rice-compatible strain, H8301, also induced cell death, however,this cell death in a compatible interaction was delayed comparedto the cell death induced by the N1141 incompatible strain.Inoculation of N1141 strain induced expression of EL2 gene whichis thought to be one of the defense-related gene. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of culturedrice cells showed that DNA cleavage occurred only in N1141-inoculatedrice cells. N1141 strain caused cytoplasmic condensation, shrinkage,and plasma membrane blebbing, all of which are important morphologicalcharacteristics of programmed cell death (PCD). In contrast,H8301 strain inoculated rice cells appeared to show weakeningof the cell wall instead of cytoplasm condensation, shrinkageand membrane blebbing. These results suggest that the rapidcell death of rice induced by the incompatible strain is characterizedas PCD. (Received May 22, 1999; Accepted July 22, 1999)     

Oxidative Cleavage of Diverse Ethers by an Extracellular Fungal Peroxygenase

Many litter-decay fungi secrete heme-thiolate peroxygenases that oxidize various organic chemicals, but little is known about the role or mechanism of these enzymes. We found that the extracellular peroxygenase of Agrocybe aegerita catalyzed the H₂O₂-dependent cleavage of environmentally significant ethers, including methyl t-butyl ether, tetrahydrofuran, and 1,4-dioxane. Experiments with tetrahydrofuran showed the reaction was a two-electron oxidation that generated one aldehyde group and one alcohol group, yielding the ring-opened product 4-hydroxybutanal. Investigations with several model substrates provided information about the route for ether cleavage: (a) steady-state kinetics results with methyl 3,4-dimethoxybenzyl ether, which was oxidized to 3,4-dimethoxybenzaldehyde, gave parallel double reciprocal plots suggestive of a ping-pong mechanism (K_m(peroxide), 1.99 ± 0.25 mm; K_m(ether), 1.43 ± 0.23 mm; k_cat, 720 ± 87 s⁻¹), (b) the cleavage of methyl 4-nitrobenzyl ether in the presence of H₂¹⁸O₂ resulted in incorporation of ¹⁸O into the carbonyl group of the resulting 4-nitrobenzaldehyde, and (c) the demethylation of 1-methoxy-4-trideuteromethoxybenzene showed an observed intramolecular deuterium isotope effect [(k_H/k_D)_obs] of 11.9 ± 0.4. These results suggest a hydrogen abstraction and oxygen rebound mechanism that oxidizes ethers to hemiacetals, which subsequently hydrolyze. The peroxygenase appeared to lack activity on macromolecular ethers, but otherwise exhibited a broad substrate range. It may accordingly have a role in the biodegradation of natural and anthropogenic low molecular weight ethers in soils and plant litter.Recently, a new group of extracellular peroxygenases was described in agaric fungi that are ubiquitous biodegraders of lignocellulose in soils and plant litter. These heme-thiolate enzymes catalyze H₂O₂-dependent halogenations and hydroxylations of numerous aromatic substrates, and thus show some functional similarity to heme chloroperoxidase and to cytochromes P450 (P450s),³ which are also heme-thiolate proteins (1–4). However, the best-characterized fungal peroxygenase, from Agrocybe aegerita, exhibits low sequence identity (∼25%) with heme chloroperoxidase and no significant sequence identity with the P450s (5). On the other hand, the absorption spectrum of the native peroxygenase and of its carbon monoxide adduct closely resemble those of P450s (6). So far, little is known about the catalytic cycle of the A. aegerita peroxygenase.The physiological function of these peroxygenases is also unclear, but their extracellular location suggests a role in the biodegradation or detoxification of organic chemicals encountered by the fungi. Ethers stand out as potential substrates for several reasons. First, ether linkages are widespread in soils and litter, not only in abundant natural substances such as lignin, flavonoids, and lignans, but also in anthropogenic compounds that include many solvents, biocides, and surfactants (7–11). Second, an oxidative mechanism is required for the biodegradation of ethers, which are relatively recalcitrant because they do not hydrolyze at physiological pH values (7). Finally, it is already known that functionally similar monooxygenases, including P450s, are capable of ether scission and have a role in the intracellular metabolism of these compounds by some organisms (7, 12–15).Here we show that the extracellular peroxygenase from A. aegerita cleaves many ethers, including some significant environmental pollutants, and we evaluate some limitations on the etherolytic reactions that the enzyme can accomplish. In addition, we report data from stoichiometrical analyses, steady-state kinetics experiments, an H₂¹⁸O₂-labeling study, and intramolecular deuterium isotope effect determinations. These results provide insights into the enzymatic mechanism for ether cleavage.     

GPx对活性氧诱发细胞程序性死亡的影响

谷胱甘肽过氧化物酶（GPx）是细胞内清除活性氧的主要抗氧化酶之一.以稳定表达GPx的CHO细胞系为模型,研究GPx对百草枯（paraquat）和叔丁基脂氢过氧化物（tbOOH）细胞毒性的影响,发现paraquat和tbOOH都能够诱导CHO细胞产生典型的细胞程序性死亡的形态学改变和特征性的DNA“梯子状”断裂,而稳定表达GPx的细胞系能明显抵抗tbOOH诱发的细胞程序性死亡,但不能抵抗paraquat诱发的细胞程序性死亡.该结果揭示,GPx能选择性抑制活性氧诱发的细胞凋亡.     

Cell Death Induced on Cell Cultures and Nude Mouse Skin by Non-Thermal,Nanosecond-Pulsed Generated Plasma

Non-thermal plasmas are gaseous mixtures of molecules, radicals, and excited species with a small proportion of ions and energetic electrons. Non-thermal plasmas can be generated with any high electro-magnetic field. We studied here the pathological effects, and in particular cell death, induced by nanosecond-pulsed high voltage generated plasmas homogeneously applied on cell cultures and nude mouse skin. In vitro, Jurkat cells and HMEC exhibited apoptosis and necrosis, in dose-dependent manner. In vivo, on nude mouse skin, cell death occurred for doses above 113 J/cm² for the epidermis, 281 J/cm² for the dermis, and 394 J/cm² for the hypodermis. Using electron microscopy, we characterized apoptosis for low doses and necrosis for high doses. We demonstrated that these effects were not related to thermal, photonic or pH variations, and were due to the production of free radicals. The ability of cold plasmas to generate apoptosis on cells in suspension and, without any sensitizer, on precise skin areas, opens new fields of application in dermatology for extracorporeal blood cell treatment and the eradication of superficial skin lesions.     

Patterns of Genomic Integration of Nuclear Chloroplast DNA Fragments in Plant Species

The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts.     

Cleavage of DNA by viologen related compounds and their incorporation into oligodeoxyribonucleotides.

The DNA cleavage reaction by viologen and related compound such as 2,7-diazapyrenium salt was investigated. These viologen analogues were successfully incorporated into the oligothymidylate in the form of covalent bonding at the site of the phosphorous backbone through the linker arm.     

Cu2+对拟南芥根的局部毒性及诱导DNA损伤和细胞死亡

该文探讨了不同浓度的Cu²⁺胁迫对拟南芥(Arabidopsis thaliana)根生长、活性氧(ROS)积累、抗氧化酶活性、质膜完整性和细胞活性的影响, 通过分根实验初步分析了Cu²⁺毒性效应的影响范围。结果表明, Cu²⁺胁迫可显著抑制拟南芥主根伸长, 诱导ROS积累及DNA损伤, 促发抗氧化酶活性升高, 破坏质膜完整性, 且Cu²⁺浓度越高, 毒性效应越明显, 在高浓度Cu²⁺胁迫下细胞活性显著降低。分析各参数之间的关系, 表明ROS的积累与超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)及抗坏血酸过氧化物酶(APX)的活性呈显著正相关; ROS积累与DNA损伤、质膜完整性、细胞活性之间具有显著的近线性关系。分根实验结果表明, 只有在添加重金属Cu²⁺(75 μmol·L^–1)一侧培养基中的根生长受抑制, 并出现ROS积累、细胞死亡, 暗示Cu²⁺对拟南芥根系的局部毒性效应可能是由于ROS的局部性积累导致受胁迫根系一侧的细胞死亡所引起的。     

Apoptosis: A Functional Paradigm for Programmed Plant Cell Death Induced by a Host-Selective Phytotoxin and Invoked during Development

The host-selective AAL toxins secreted by Alternaria alternata f sp lycopersici are primary chemical determinants in the Alternaria stem canker disease of tomato. The AAL toxins are members of a new class of sphinganine analog mycotoxins that cause cell death in both animals and plants. Here, we report detection of stereotypic hallmarks of apoptosis during cell death induced by these toxins in tomato. DNA ladders were observed during cell death in toxin-treated tomato protoplasts and leaflets. The intensity of the DNA ladders was enhanced by Ca2+ and inhibited by Zn2+. The progressive delineation of fragmented DNA into distinct bodies, coincident with the appearance of DNA ladders, also was observed during death of toxin-treated tomato protoplasts. In situ analysis of cells dying during development in both onion root caps and tomato leaf tracheary elements revealed DNA fragmentation localized to the dying cells as well as the additional formation of apoptotic-like bodies in sloughing root cap cells. We conclude that the fundamental elements of apoptosis, as characterized in animals, are conserved in plants. The apoptotic process may be expressed during some developmental transitions and is the functional process by which symptomatic lesions are formed in the Alternaria stem canker disease of tomato. Sphinganine analog mycotoxins may be used to characterize further signaling pathways leading to apoptosis in plants.     

Sensing Cytosolic RpsL by Macrophages Induces Lysosomal Cell Death and Termination of Bacterial Infection

The intracellular bacterial pathogen Legionella pneumophila provokes strong host responses and has proven to be a valuable model for the discovery of novel immunosurveillance pathways. Our previous work revealed that an environmental isolate of L. pneumophila induces a noncanonical form of cell death, leading to restriction of bacterial replication in primary mouse macrophages. Here we show that such restriction also occurs in infections with wild type clinical isolates. Importantly, we found that a lysine to arginine mutation at residue 88 (K88R) in the ribosome protein RpsL that not only confers bacterial resistance to streptomycin, but more importantly, severely attenuated the induction of host cell death and enabled L. pneumophila to replicate in primary mouse macrophages. Although conferring similar resistance to streptomycin, a K43N mutation in RpsL does not allow productive intracellular bacterial replication. Further analysis indicated that RpsL is capable of effectively inducing macrophage death via a pathway involved in lysosomal membrane permeabilization; the K88R mutant elicits similar responses but is less potent. Moreover, cathepsin B, a lysosomal protease that causes cell death after being released into the cytosol upon the loss of membrane integrity, is required for efficient RpsL-induced macrophage death. Furthermore, despite the critical role of cathepsin B in delaying RpsL-induced cell death, macrophages lacking cathepsin B do not support productive intracellular replication of L. pneumophila harboring wild type RpsL. This suggests the involvement of other yet unidentified components in the restriction of bacterial replication. Our results identified RpsL as a regulator in the interactions between bacteria such as L. pneumophila and primary mouse macrophages by triggering unique cellular pathways that restrict intracellular bacterial replication.