A second signal for autophagic cell death?

Dictyostelium cells in monolayers in vitro lend themselves well to a study of autophagic cell death (ACD). There is no apoptosis machinery in the protist Dictyostelium, no caspase nor Bcl-2 family members (except a paracaspase whose inactivation does not alter cell death), thus there is no apoptosis that could interfere with, or substitute for, non-apoptotic cell death. Also, Dictyostelium, a eukaryote, has a haploid genome, which facilitates random insertional mutagenesis.     

Genetic approaches to molecular mechanisms of programmed cell death in Dictyostelium

Caspase-independent cell deaths have been observed in many species including the human. However, the molecular mechanisms which govern them are largely unknown. Our present work makes use of a model organism, the protist Dictyostelium discoideum, which displays a caspase-independent cell death during its development. In rich medium, Dictyostelium multiplies vegetatively as a unicellular organism, but in starvation conditions, Dictyostelium cells aggregate, differentiate and morphogenize into a multicellular structure, called sorocarp, containing a mass of spores supported by a stalk. Cells in the stalk are considered dead on the basis of non-regrowth in a rich medium and are vacuolized. This programmed cell death is therefore developmental and vacuolar, and in addition, caspase-independent since the Dictyostelium genome does not contain caspases genes. In order to study in detail this cell death without induction of development, an in vitro experimental protocol has been adopted, which enabled us to describe the cascade of morphological events during this cell death. An insertional mutagenesis approach, followed by appropriate selection or screening of mutants potentially resistant to death, attempted at establishing the cascade of molecular events leading to vacuolar death of Dictyostelium cells. A better understanding of alternative death pathways may allow to control different types of cell deaths in the cases of cancers or neurodegenerative diseases. In this short review, we will discuss briefly some generalities about the development of Dictyostelium in starvation conditions, and we will focus on the course of programmed cell death in Dictyostelium and on the genetic tools used to elucidate the corresponding molecular mechanisms.     

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。     

An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.     

Cell differentiation and patterning in Dictyostelium.

In Dictyostelium development, prestalk cells first differentiate at scattered positions in the aggregate and then sort out, probably by chemotaxis to cAMP. They may regulate their proportions by selective depletion of the stalk cell inducer, DIF-1. Once sorted, prestalk cells form a DIF-1 sink, which can produce gradients of DIF-1 and its metabolites in the slug. Global movements of cells in the slug may be regulated by cAMP signals, as in aggregation. Terminal differentiation of stalk and spore cells requires activation of cAMP-dependent protein kinase, possibly brought about by ammonia depletion. Finally, a technique for insertional mutagenesis promises the ready isolation of developmental genes.     

Programmed cell death

This paper reviews data on programmed cell death (apoptosis) in animals and plants. Necrosis is a pathological scenario of cell death, which entails an inflammatory response in animal tissues. Apoptosis results in the disintegration of animal/plant cells into membrane vesicles enclosing the intracellular content, which are thereupon engulfed by adjacent or specialized cells (phagocytes) in animals. Plants lack such specialized cells, and plant cell walls prevent phagocytosis. The paper considers the main molecular mechanisms of apoptosis in animals and the pathways of activation of caspases, evolutionarily conserved cysteine proteases. A self-contained section concerns itself with the process of programmed cell death (PCD) in microorganisms including: 1) cell death in the myxomycete Dictyostelium discoideum and the parasitic flagellate Trypanosoma cruzi; 2) PCD in genetically manipulated yeast expressing the proapoptotic Bax and Bak proteins; 3) the death of a part of a prokaryotic cell population upon the depletion of nutrient resources or under stress; 4) the elimination of cells after a loss of a plasmid encoding a stable cytotoxic agent in combination with an unstable antidote; and 5) PCD in phage-infected bacterial cells.     

Library-independent cloning of genomic fragments adjacent to vector integration sites: isolation of the EB4-PSV gene from a Dictyostelium gene disruption transformant.

We have designed an efficient procedure to clone genomic DNA adjacent to the integration site of transformation vectors. Using this method on a Dictyostelium gene disruption transformant, we have cloned a 5kb fragment which has previously escaped isolation by conventional library screening. Our protocol eliminates recloning of the original vectors which are often integrated in multiple tandem copies (1) but specifically recovers vectors containing genomic fragments adjacent to the integration site. The protocol is useful to isolate flanking fragments in gene disruption transformants to characterize flanking regions which may influence the expression of transformed genes by position effects and to identify sites of insertional mutagenesis.     

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.     

Isolation of spermidine synthase gene (spsA) of Dictyostelium discoideum

The gene encoding spermidine synthase (spsA) was isolated from Dictyostelium discoideum using the technique of insertional mutagenesis. Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 h of development. Sequencing of the genomic DNA and a full-length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids. The sequence is highly conserved, with amino acid identities compared to spermidine synthases of humans, 59.5%, to mouse, 61.3%, and to yeast, 58.1%. A null mutant of the spsA gene is unable to grow in the absence of exogenous spermidine. Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that have abnormally short stalks.     

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore ( Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c , FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.     

撤除外源生长素诱发棉花胚性悬浮细胞程序性死亡

棉花胚性悬浮细胞在仅含生长素的MS培养基上培养时,生长良好;但当转入到不含生长素的MS培养基上培养时,大规模死亡.通过细胞学观察发现,在转入无生长素的MS培养基培养3～4 d后可见明显的核质浓缩、胞质收缩,而高温处理引起的细胞坏死无此现象.用抽提悬浮细胞基因组DNA进行凝胶电泳发现:这种细胞死亡还伴随有典型的DNA梯度出现,而坏死的细胞和对照无DNA梯度.表明这种由生长素撤除引起的细胞死亡是一种程序性死亡.这种细胞死亡能被水解酶抑制剂和半胱氨酸蛋白酶抑制剂抑制,表明水解酶和类半胱氨酸蛋白水解酶(CLP)参与细胞程序性死亡.     

A specific pathway inducing autophagic cell death is marked by an IP3R mutation

Three main advantages make Dictyostelium a very favorable model to study the induction of autophagic cell death in vitro. First, its small, sequenced and haploid genome facilitates genetic approaches. Second, the Dictyostelium genome does not encode the two main molecular families involved in apoptosis (caspases and bcl-2 family), which therefore cannot interfere in this case with autophagic cell death. Third, induction of autophagic cell death follows in this case a two-step process, namely starvation-induced sensitization leading to autophagy but not to death, followed by a DIF-1-induced pathway leading to cell death proper. The latter, DIF-1-induced pathway is defined experimentally, through sequential additions, and most important also genetically, through random mutagenesis leading in particular to the preparation and study of an iplA mutant. The iplA gene encodes the IP3 Receptor, and its mutation leads to the absence of vacuolization and of death when autophagic cell death is triggered. Further study of the DIF-1 pathway should shed additional light on the induction of autophagic cell death (as opposed to that of just autophagy) in Dictyostelium and by extension perhaps in other organisms.     

Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

Apoptotic and non-apoptotic modes of programmed cell death in MCF-7 human breast carcinoma cells

Apoptosis is a specific mode of programmed cell death (PCD), recognized by characteristic morphological and molecular changes. Here we present evidence for a non-apoptotic type of PCD in human MCF-7 breast carcinoma cells. We used TNF-alpha and tyrphostin AG213 to induce apoptotic and non-apoptotic cell death respectively in vitro. Microscopic and immunohistochemical studies, together with DNA analysis and flow cytometric analysis of p53 and bcl-2 oncogene expression, revealed some novel characteristics of non-apoptotic cell death. We show here for the first time some of the biochemical features of an experimentally induced non-apoptotic PCD and emphasize the distinct biochemical events leading to apoptotic and non-apoptotic PCD.     

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.     

Epidermal growth factor triggers an original, caspase-independent pituitary cell death with heterogeneous phenotype

Programmed cell death (PCD) is physiologically involved in the regulation of cell division and differentiation. It encompasses caspase-dependent mitochondrial and nonmitochondrial pathways. Additional caspase-independent pathways have been characterized in mitochondrial PCDs but remain hypothetical in nonmitochondrial PCDs. Epidermal growth factor (EGF) has been shown to inhibit division of pituitary somato-lactotrope cells occurring in parallel with EGF-mediated differentiation of these precursors into lactotrope cells. We show here that in somato-lactotrope pituitary cell line GH4C1, EGF triggers a PCD characterized by an apoptosis-like DNA fragmentation, insensitivity to broad-range caspase inhibitors, and absence of either cytochrome c or apoptosis-inducing factor release from mitochondria. Dying cells display loose chromatin clustering and numerous cytoplasmic vacuoles, a fraction of which are autophagic, thus conferring a heterogeneous phenotype to this PCD. Moreover, overexpression of cell death inhibitor Bcl-2 prevented not only the EGF-induced PCD but also its prodifferentiation effects, thus pointing to a mechanistic relationship existing between these two phenomena. Overall, the characterized differentiation-linked cell death represents an original form of caspase-independent PCD. The mechanisms underlying this PCD involve combinatorial engagement of discrete death effectors leading to a heterogeneous death phenotype that might be evolutionary related to PCD seen during the differentiation of some unicellular organisms.     

The wound response in fresh-cut lettuce involves programmed cell death events

In this work, the involvement of programmed cell death (PCD) in the wound-induced postharvest browning disorder and senescence in butterhead lettuce (Lactuca sativa L.) fresh-cuts was studied. At the wounded (cut, bruised) sites, rapid browning, loss of chlorophyll and massive cell death, accompanied with accumulation of reactive oxygen species and increased electrolyte leakage occurred in a narrow strip of tissue adjacent the injury. The dead cell morphology (protoplast and nuclei shrinkage) together with the biochemical and physiological changes resembled necrotic PCD type. With a slight delay post-wounding, senescence associated with similar cell death features was initiated in distant non-wounded sites. In addition to necrotic PCD, both in wounded and senescing tissue, the appearance of empty cell corpses was observed, indicating that part of the cells might undergo vacuolar PCD (self-digestion of cellular content after vacuole collapse). The wounding-induced local cell death at the primary site of damage suggested that PCD may serve as a mechanism to seal-off the wound by building a physical barrier of dead cells. However, the cell death at sites remote from the wound suggests the distribution of long-distance senescence-inducing wound messengers. Trichomes in unwounded tissue often were the first to show H₂O₂ accumulation and dead cells; thereafter, the elevated H₂O₂ and cell death appeared in connecting cells and senescence progressed over larger areas. This suggests that trichomes may contribute to mediating the wound signalling leading to subsequent senescence. Our findings demonstrate that PCD is an integral part of the wound syndrome in fresh-cut lettuce.     

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).     

Developmental cell death in dictyostelium does not require paracaspase

Apoptotic cell death often requires caspases. Caspases are part of a family of related molecules including also paracaspases and metacaspases. Are molecules of this family generally involved in cell death? More specifically, do non-apoptotic caspase-independent types of cell death require paracaspases or metacaspases? Dictyostelium discoideum lends itself well to answering these questions because 1) it undergoes non-apoptotic developmental cell death of a vacuolar autophagic type and 2) it bears neither caspase nor metacaspase genes and apparently only one paracaspase gene. This only paracaspase gene can be inactivated by homologous recombination. Paracaspase-null clones were thus obtained in each of four distinct Dictyostelium strains. These clones were tested in two systems, developmental stalk cell death in vivo and vacuolar autophagic cell death in a monolayer system mimicking developmental cell death. Compared with parent cells, all of the paracaspase-null cells showed unaltered cell death in both test systems. In addition, paracaspase inactivation led to no alteration in development or interaction with a range of bacteria. Thus, in Dictyostelium, vacuolar programmed cell death in development and in a monolayer model in vitro would seem not to require paracaspase. To our knowledge, this is the first instance of developmental programmed cell death shown to be independent of any caspase, paracaspase or metacaspase. These results have implications as to the relationship in evolution between cell death and the caspase family.