Eukaryotic Cell Death Research Articles

Identification of a transcriptionally active hVH-5 pseudogene on 10q22.2

Mitogen-activated protein kinases (MAPKs) are important regulators of a vast number of biological functions that affect life and death of eukaryotic cells and are tightly regulated by the concerted action of several phosphatases. Among these is the human homologue of vaccinia virus H1 phosphatase gene clone 5 ( hVH-5) product, which dephosphorylates and thus inhibits members of the MAPK family. Here, we analyzed hVH-5 transcripts in mammary carcinoma cell lines and discovered a sequence with 88% similarity to hVH-5 transcripts. Because this variant of hVH-5 lacked intronic sequences in its genomic structure, we suggest it might be a processed pseudogene of hVH-5. ψhVH-5 transcripts were detected in human peripheral tissues as well as in 11 of 14 breast cancer cell lines. In respect to the normal hVH-5 gene, the pseudogene contains several point mutations and a frame shift due to the deletion of 2 bases that would lead to the truncation of the putative ψhVH-5 product.

Apoptosis, Pyroptosis, and Necrosis: Mechanistic Description of Dead and Dying Eukaryotic Cells

A wide variety of pathogenic microorganisms have been demonstrated to cause eukaryotic cell death, either as a consequence of infecting host cells or by producing toxic products. Pathogen-induced host cell death has been characterized as apoptosis in many of these systems. It is increasingly being

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus

When the opportunistic pathogen Aspergillus fumigatus entered the stationary phase, there was a rapid loss in cell viability which was associated with the appearance of markers characteristic of apoptosis, namely annexin V-FITC binding to the cytoplasmic membrane, demonstrating exposure of phosphatidylserine to the outer leaflet of the membrane; and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining of the nuclei, indicating DNA fragmentation. This was followed later by a loss of membrane integrity as revealed by propidium iodide staining. The development of the apoptotic phenotype was blocked when the protein synthesis inhibitor cycloheximide was added to the culture 1 h prior to the onset of the stationary phase, demonstrating active participation of the cell. In addition, intracellular activity against substrates specific for caspase-1 and -8 also increased on stationary phase entry and the development of the apoptotic phenotype was blocked when the cell permeant caspase inhibitor Z-FAD-fmk was present in the medium. Cell death in A. fumigatus during the stationary phase therefore appears to share similarities to apoptotic cell death in higher eukaryotes and to be dependent on a caspase-like activity.

The Escherichia coli mazEF suicide module mediates thymineless death.

In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its molecular mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions. MazF is a stable toxin, and MazE is an unstable antitoxin. Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P(2) is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.

Ceramides modulate programmed cell death in plants.

The balance between the bioactive sphingolipid ceramide and its phosphorylated derivative has been proposed to modulate the amount of programmed cell death (PCD) in eukaryotes. We characterized the first ceramide kinase (CERK) mutant in any organism. The Arabidopsis CERK mutant, called accelerated cell death 5, accumulates CERK substrates and shows enhanced disease symptoms during pathogen attack and apoptotic-like cell death dependent on defense signaling late in development. ACD5 protein shows high specificity for ceramides in vitro. Strikingly, C2 ceramide induces, whereas its phosphorylated derivative partially blocks, plant PCD, supporting a role for ceramide phosphorylation in modulating cell death in plants.

Biochemical Characterization of Two Analogues of the Apoptosis-linked Gene 2 Protein in Dictyostelium discoideumand Interaction with a Physiological Partner in Mammals, Murine Alix

Two homologues, Dd-ALG-2a and Dd-ALG-2b, of the mammalian calcium-binding protein ALG-2 (apoptosis-linked gene 2) have been characterized in the cellular slime mold Dictyostelium discoideum. Fluorescence titrations showed that both proteins bind calcium ions with affinities (Ca2+)(0.5) of 30 and 450 microm, respectively, at sites specific to calcium. Calcium ion binding resulted in changes of conformation associated with the unmasking of hydrophobic regions of the proteins. Surface plasmon resonance analysis showed that Dd-ALG-2a homodimers formed (K(D) of 1 microm) at calcium ion concentrations similar to those necessary for Ca2+-induced conformational changes. Deletion of the hydrophobic N-terminal sequence or EF-hand 5 of Dd-ALG-2a prevented dimerization. The Dd-ALG-2b homodimer was not detected, and the Dd-ALG-2a/2b heterodimer formed only when Dd-ALG-2b was the immobilized partner. Murine Alix formed a heterodimer (K(D) = 0.6 microm) with Dd-ALG-2a but not with Dd-ALG-2b, and the interaction strictly depended upon calcium ions. The DeltaNter construct of Dd-ALG-2a lost its interaction capacity with mouse Alix. The genes encoding both proteins, Dd-alg-2a and -2b, were expressed in growing cells. The levels of mRNA were at a maximum during aggregation (4-8 h) and decreased rapidly thereafter. In contrast, the levels of proteins remained fairly stable. Dd-ALG-2a and Dd-ALG-2b were found to be dispensable for growth and development, based on the finding that single Dd-alg2a- or Dd-alg-2b- and double Dd-alg2a-/Dd-alg-2b- mutant cell lines showed normal growth in axenic medium or on bacterial lawns and exhibited unaltered development.

Rapid cell death in Xanthomonas campestris pv. glycines.

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the etiological agent of bacterial pustule disease of soybean, exhibited post-exponential rapid cell death (RCD) in LB medium. X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961 also displayed RCD, though less pronouncedly than XcgAM2. RCD was not observed in Pseudomonas syringae pv. glycines, or Escherichia coli DH5alpha. Incubation of the post-exponential LB-grown XcgAM2 cultures at 4 degrees C arrested the RCD. RCD was also inhibited by the addition of starch during the exponential phase of LB-growing XcgAM2. Protease negative mutants of XcgAM2 were found to be devoid of RCD behavior observed in the wild type XcgAM2. While undergoing RCD, the organism was found to transform to spherical membrane bodies. The presence of membrane bodies was confirmed by using a membrane specific fluorescent label, 1,6-diphenyl 1,3,5-hexatriene (DPH), and also by visualizing these structures under microscope. The membrane bodies of XcgAM2 were found to contain DNA, which was devoid of the indigenous plasmids of the organism. The membrane bodies were found to bind annexin V indicative of the externalization of membrane phosphatidyl serine. Nicking of DNA in XcgAM2 cultures undergoing RCD in LB medium was also detected using a TUNEL assay. The RCD in XcgAM2 appeared to have features similar to the programmed cell death in eukaryotes.

Melanosynthesis, differentiation, and apoptosis in Kupffer cells from Rana esculenta.

It is well known that amphibian Kupffer cells (KCs) contain eumelanins. In this paper, we demonstrate through a molecular analysis that Rana esculenta KCs synthesize high levels of mRNA for tyrosinase and through cytochemistry that they possess dopa oxidase activity: both these data prove that frog KCs are capable of autonomously synthesizing eumelanins. On the other hand, by using a highly sensitive reverse transcription-polymerase chain reaction assay we clearly show that in mammalian KCs the tyrosinase gene is not expressed. Quite unexpectedly, we have detected tyrosinase mRNA in Rana esculenta spleen, lung, and heart; to explain this finding, we suggest that it could be due to the presence of pigmented macrophages within the spleen, that probably behave as KCs, and of melanophores in lung and heart. It also may be hypothesized that the Rana esculenta tyrosinase gene, as opposed to its mammalian counterpart, is expressed in many cell types because its promoter contains sequences that are recognized by widely synthesized transcription factors. Our experiments also demonstrate that there is an inverse correlation between the amount of tyrosinase mRNA and melanin content, and that populations of terminally differentiated KCs are characterized by a high degree of apoptosis. Based on these data, we propose that differentiating KCs start accumulating eumelanins, as a result of previous expression of high levels of tyrosinase and of dopa oxidase activity, acquire the full KC phenotype (characterized by both phagocytic and melanosynthetic ability), and then undergo apoptosis. Accordingly, we propose that these cells could represent an interesting model to study, at the molecular level, the relationship between differentiation, specific gene expression, and programmed cell death in higher eukaryotes.

Nuclear changes in necrotic HL-60 cells.

Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptosis is characterized by well-defined nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On the other hand, the nuclear modifications that occur during necrosis are largely less known. Here, we have investigated whether or not nuclear modifications occur during ethanol-induced necrotic cell death of HL-60 cells. By means of immunofluorescence staining, we demonstrate that the patterns given by antibodies directed against some nuclear proteins (lamin B1, NuMA, topoisomerase IIalpha, SC-35, B23/nucleophosmin) changed in necrotic cells. The changes in the spatial distribution of NuMA strongly resembled those described to occur during apoptosis. On the contrary, the fluorescent pattern characteristic for other nuclear proteins (C23/nucleolin, UBF, fibrillarin, RNA polymerase I) did not change during necrosis. By immunoblotting analysis, we observed that some nuclear proteins (SAF-A, SATB1, NuMA) were cleaved during necrosis, and in the case of SATB1, the apoptotic signature fragment of 70 kDa was also present to the same extent in necrotic samples. Caspase inhibitors did not prevent proteolytic cleavage of the aforementioned polypeptides during necrosis, while they were effective if apoptosis was induced. In contrast, lamin B1 and topoisomerase IIalpha were uncleaved in necrotic cells, whereas they were proteolyzed during apoptosis. Transmission electron microscopy analysis revealed that slight morphological changes were present in the nuclear matrix fraction prepared from necrotic cells. However, these modifications (mainly consisting of a rarefaction of the inner fibrogranular network) were not as striking as those we have previously described in apoptotic HL-60 cells. Taken together, our results indicate that during necrosis marked biochemical and morphological changes do occur at the nuclear level. These alterations are quite distinct from those known to take place during apoptosis. Our results identify additional biochemical and morphological criteria that could be used to discriminate between the two types of cell death. J. Cell. Biochem. Suppl. 36: 19-31, 2001.

Role of programmed cell death in development

Programmed cell death (PCD) is an integral part of both animal and plant development. In animals, model systems such as Caenorhabditis elegans, Drosophila melanogaster, and mice have shown a general cell death profile of induction, caspase mediation, cell death, and phagocytosis. Tremendous strides have been made in cell death research in animals in the past decade. The ordering of the C. elegans genes Ced-3, 4 and 9, identification of caspase-activated DNase that degrades nuclear DNA during PCD, identification of signal transduction modules involving caspases as well as the caspase-independent pathway, and the involvement of mitochondria are some of the findings of immense value in understanding animal PCDs. Similarly, the caspase inactivation mechanisms of infecting viruses to stall host cell death give a new dimension to the viral infection process. However, plant cell death profiles provide an entirely different scenario. The presence of a cell wall that cannot be phagocytosed, absence of the hallmarks of animal PCDs such as DNA laddering, formation of apoptotic bodies, a cell-death-specific nuclease, a biochemical machinery of killer enzymes such as caspases all point to novel ways of cell elimination. Large gaps in our understanding of plant cell death have prompted speculative inferences and comparisons with animal cell death mechanisms. This paper deals with both animals and plants for a holistic view on cell death in eukaryotes.

Bordetella bronchiseptica-mediated cytotoxicity to macrophages is dependent on bvg-regulated factors, including pertactin.

The effect of Bordetella bronchiseptica infection on the viability of murine macrophage-like cells and on primary porcine alveolar macrophages was investigated. The bacterium was shown to be cytotoxic for both cell types, particularly where tight cell-to-cell contacts were established. In addition, bvg mutants were poorly cytotoxic for the eukaryotic cells, while a prn mutant was significantly less toxic than wild-type bacteria. B. bronchiseptica-mediated cytotoxicity was inhibited in the presence of cytochalasin D or cycloheximide, an inhibitor of microfilament-dependent phagocytosis or de novo eukaryotic protein synthesis, respectively. The mechanism of eukaryotic cell death was examined, and cell death was found to occur primarily through a necrotic pathway, although a small proportion of the population underwent apoptosis.

Addiction modules and programmed cell death and antideath in bacterial cultures.

In bacteria, programmed cell death is mediated through "addiction modules" consisting of two genes. The product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. Here we extensively review what is known about those modules that are borne by one of a number of Escherichia coli extrachromosomal elements and are responsible for the postsegregational killing effect. We focus on a recently discovered chromosomally borne regulatable addiction module in E. coli that responds to nutritional stress and also on an antideath gene of the E. coli bacteriophage lambda. We consider the relation of these two to programmed cell death and antideath in bacterial cultures. Finally, we discuss the similarities between basic features of programmed cell death and antideath in both prokaryotes and eukaryotes and the possibility that they share a common evolutionary origin.

Gene Transcription Through Myc Family Members in Eukaryotic Cells

c-Myc family proteins, encoded by c-myc family proto-oncogenes, play critical roles in mechanisms associated with proliferation, differentiation and apoptotic death in eukaryotic cells. These functions are mediated by transcriptional activity of these proteins through binding to the E-box core sequence CACGTG referred to as a Myc core element located at a promoter or enhancer region of the individual target genes in the nucleus. Recent studies have demonstrated the presence of novel nuclear proteins that specifically recognize a Myc core element, in addition to c-Myc, Max, Mad and Mxil. On the other hand, a Myc core element has alternating purine/pyrimidine repeats which could undergo a conformational transition from right-handed (B-DNA) to left-handed (Z-DNA) forms in the presence of a high concentration of salts such as Mg2+ and polyamines. Similarly, a Myc element has a homopurine-homopyrimidine site that may take a triplex configuration in particular situations. We have searched for nuclear proteins that can specifically recognize a Myc core element in different topological variations in murine brain.

The role of macrophage cell death in tuberculosis.

Studies of host responses to infection have traditionally focused on the direct antimicrobial activity of effector molecules (antibodies, complement, defensins, reactive oxygen and nitrogen intermediates) and immunocytes (macrophages, lymphocytes, and neutrophils among others). The discovery of the systems for programmed cell death of eukaryotic cells has revealed a unique role for this process in the complex interplay between microorganisms and their cellular targets or responding immunocytes. In particular, cells of the monocyte/macrophage lineage have been demonstrated to undergo apoptosis following intracellular infection with certain pathogens that are otherwise capable of surviving within the hostile environment of the phagosome or which can escape the phagosome. Mycobacterium tuberculosis is a prototypical 'intracellular parasite' of macrophages, and the direct induction of macrophage apoptosis by this organism has recently been reported from several laboratories. This paper reviews the current understanding of the mechanism and regulation of macrophage apoptosis in response to M. tuberculosis and examines the role this process plays in protective immunity and microbial virulence.

Thymine metabolism and thymineless death in prokaryotes and eukaryotes.

For many years it has been known that thymine auxotrophic microorganisms undergo cell death in response to thymine starvation [thymineless death (TLD)]. This effect is unusual in that deprivation of many other nutritional requirements has a biostatic, but not lethal, effect. Studies of numerous microbes have indicated that thymine starvation has both direct and indirect effects. The direct effects involve both single- and double-strand DNA breaks. The former may be repaired effectively, but the latter lead to cell death. DNA damaged by thymine starvation is a substrate for DNA repair processes, in particular recombinational repair. Mutations in recBCD recombinational repair genes increase sensitivity to thymineless death, whereas mutations in RecF repair protein genes enhance the recovery process. This suggests that the RecF repair pathway may be critical to cell death, perhaps because it increases the occurrence of double-strand DNA breaks with unique DNA configurations at lesion sites. Indirect effects in bacteria include elimination of plasmids, loss of transforming ability, filamentation, changes in the pool sizes of various nucleotides and nucleosides and in their excretion, and phage induction. Yeast cells show effects similar to those of bacteria upon thymine starvation, although there are some unique features. The mode of action of certain anticancer drugs and antibiotics is based on the interruption of thymidylate metabolism and provides a major impetus for further studies on TLD. There are similarities between TLD of bacteria and death of eukaryotic cells. Also, bacteria have "survival" genes other than thy (thymidylate synthetase), and this raises the question of whether there is a relationship between the two. A model is presented for a molecular basis of TLD.

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.

Looking beneath the Surface: The Cell Death Pathway of Fas/APO-1 (CD95)

The biochemical basis of programmed cell death is poorly understood in mammals. The cell surface receptor Fas/APO-1 (CD95) is one molecule known to be central to a number of mammalian cell death processes. Several studies in the past year have led to insights about the role of Fas/APO-1 in vivo and have also given some clues about the biochemical components of the Fas/APO-1 death pathway. This article reviews those studies and discuss models of Fas/APO-1 signaling and function. Cell death occurs as a normal process in a wide variety of developmental and homeostatic contexts in metazoan organisms (1); it represents the timely and appropriate fate for many or even the majority of cells born in certain organ systems. Despite the importance and ubiquitous nature of such physiologic, or "programmed", cell death, little is known about the molecular events that mediate this process. That a conserved biochemical pathway exists is suggested by the observation that programmed cell death is almost always accompanied by a consistent set of morphologic changes, an appearance known as apoptosis (2). The identification of the genes that control programmed cell death in higher eukaryotes has been hampered by several inherent difficulties. First, the genetic tools so useful in dissecting cell death pathways in Caenorhabditis elegans (3) and Drosophila (4) have not been available in higher eukaryotes. Second, the death-inducing properties of such genes makes genetic selection an impractical means of identification. Third, it appears that many cell death genes are constitutively expressed and present in an inactive form (5), making it unlikely that they could be discovered by techniques relying upon differential gene expression. Finally, genes identified by virtue of an ability to induce death when overexpressed must be subjected to rigorous criteria to determine whether the cell death is of physiologic importance, since it is likely that overexpression of certain proteins may lead to toxic effects that are distinct from the in vivo roles of those proteins. Two approaches to date have yielded the most information about cell death processes: (i) identification of cell death genes by classical genetic means coupled with characterization of their mammalian homologs and (ii) screening for proteins capable of inducing cell death directly in mammalian cells. The Fas antigen/APO-1 is an example of a protein discovered using the latter approach, as it was first discovered as an inducer of cell death and later shown to be necessary and sufficient for certain programmed deaths in vivo. More recent studies have connected Fas to elements of cell death pathways in other species. It has been proposed that Fas is related to the Drosophila cell death protein Reaper, and that in signaling cell death Fas relies upon a relative of the C. elegans cell death protein CED-3. Fas may therefore represent an evolutionarily conserved component of a universal cell death pathway.

Monoclonal-antibody-mediated apoptosis in adult T-cell leukaemia

The monoclonal antibody anti-APO-1 recognises a 52 kD cell membrane protein (APO-1) on some lymphoid tumour cell lines and on activated T cells. Binding of anti-APO-1 to cells expressing APO-1 results in programmed cell death, apoptosis, the most common form of death in eukaryotic cells. Expression of the antigen and sensitivity to the induction of cell death by anti-APO-1 were studied in human T-cell lines transformed by human leukaemia virus type 1 (HTLV-I) and in cultured cells from patients with adult T-cell leukaemia (ATL). APO-1 was strongly expressed on both types of cells and incubation of the cells with anti-APO-1 resulted in inhibition of proliferation and apoptosis. Induction of apoptosis may therefore be a possible therapeutic tool in HTLV-I-associated malignant disorders.

Ultrastructural Analysis of Apoptosis Induced by the Monoclonal Antibody Anti-APO-1 on a Lymphoblastoid B Cell Line

Apoptosis (programmed cell death) is the most common form of death in eukaryotic cells. We recently described a monoclonal antibody, anti-APO-1, which induces apoptosis of cells from the human B-lymphoblastoid line SKW 6.4 and of cells from a variety of other human lymphoid cell lines. This model of apoptosis was now studied ultrastructurally. SKW 6.4 cells undergoing apoptosis showed the following morphological changes: condensation of the cytoplasm and karyoplasm, formation of large electron-opaque aggregates of the chromatin lining the nuclear membrane, "blebbing" of the cell membrane at an early stage of apoptosis, and dilatation of the mitochondria. Two hours after adding anti-APO-1, the nuclear membrane was ruptured. Occasionally, large vesicular enlargement of endoplasmic reticulum or Golgi appeared in the cytoplasm. Finally, total breakdown of all cell membranes and cellular disintegration was observed.