Caspase-dependent Cdk activity is a requisite effector of apoptotic death events

The caspase-dependent activation of cyclin-dependent kinases (Cdks) in varied cell types in response to disparate suicidal stimuli has prompted our examination of the role of Cdks in cell death. We have tested the functional role of Cdk activity in cell death genetically, with the expression of dominant negative Cdk mutants (DN-Cdks) and Cdk inhibitory genes. Here we demonstrate that Cdk2 activity is necessary for death-associated chromatin condensation and other manifestations of apoptotic death, including cell shrinkage and the loss of adhesion to substrate. Susceptibility to the induction of the cell death pathway, including the activation of the caspase cascade, is unimpaired in cells in which Cdk2 activity is inhibited. The direct visualization of active caspase activity in these cells confirms that death-associated Cdk2 acts downstream of the caspase cascade. Cdk inhibition also does not prevent the loss of mitochondrial membrane potential and membrane phospholipid asymmetry, which may be direct consequences of caspase activity, and dissociates these events from apoptotic condensation. Our data suggest that caspase activity is necessary, but not sufficient, for the full physiological cell death program and that a requisite function of the proteolytic caspase cascade is the activation of effector Cdks.     

The molecular control of DNA damage-induced cell death

Because of the singular importance of DNA for genetic inheritance, all organisms have evolved mechanisms to recognize and respond to DNA damage. In metazoans, cells can respond to DNA damage either by undergoing cell cycle arrest, to facilitate DNA repair, or by undergoing cell suicide. Cell death can either occur by activation of the apoptotic machinery or simply be a consequence of irreparable damage that prevents further cell division. In germ cells, mechanisms for limiting alterations to the genome are required for faithful propagation of the species whereas in somatic cells, responses to DNA damage prevent the accumulation of mutations that might lead to aberrant cell proliferation or behavior. Several of the genes that regulate cellular responses to DNA damage function as tumor suppressors. The clinical use of DNA damaging agents in the treatment of cancer can activate these tumor suppressors and exploits the cellular suicide and growth arrest mechanisms that they regulate. It appears that in some but not all types of tumors the propensity to undergo apoptosis is a critical determinant of their sensitivity to anti-cancer therapy. This review describes current understanding of the molecular control of DNA damage-induced apoptosis with particular attention to its role in tumor suppression and cancer therapy.     

Caspase-like protease involvement in the control of plant cell death

Cell death as a highly regulated process has now been recognized to be an important, if not essential, pathway that is ubiquitous in all multicellular eukaryotes. In addition to playing key roles in the morphogenesis and sculpting of the organs to give rise to highly specialized forms and shapes, cell death also participates in the programmed creation of specialized cell types for essential functions such as the selection of B cells in the immune system of mammals and the formation of tracheids in the xylem of vascular plants. Studies of apoptosis, the most well-characterized form of animal programmed cell death, have culminated in the identification of a central tripartite death switch the enzymatic component of which is a conserved family of cysteine proteases called caspases. Studies in invertebrates and other animal models suggest that caspases are conserved regulators of apoptotic cell death in all metazoans. In plant systems, the identities of the main executioners that orchestrate cell death remain elusive. Recent evidence from inhibitor studies and biochemical approaches suggests that caspase-like proteases may also be involved in cell death control in higher plants. Furthermore, the mitochondrion and reactive oxygen species may well constitute a common pathway for cell death activation in both animal and plant cells. Cloning of plant caspase-like proteases and elucidation of the mechanisms through which mitochondria may regulate cell death in both systems should shed light on the evolution of cell death control in eukaryotes and may help to identify essential components that are highly conserved in eukaryotes.     

Programmed cell death in neurotumour cells involves the generation of ceramide

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 µM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.Abbreviations PCD programmed cell death - PKC protein kinase C - HPTLC high-performance thin-layer chromatography - DETAPAC diethylenetriaminepentaacetic acid - DMEM Dubelco's modified Eagle's medium - FCS fetal calf serum - PBS phosphate-buffered saline - DAG diacylglycerol - DDI distilled-deionized - Cer ceramide - SM sphingomyelin Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Molecular genetics of cell death in the nematode Caenorhabditis elegans

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insightinto the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells. © 1992 John Wiley & Sons, Inc.     

Programmed cell death is induced by hydrogen peroxide but not by excessive ionic stress of sodium chloride in the unicellular green alga Chlamydomonas reinhardtii

Eukaryotic microalgae serve as indicators of environmental change when exposed to severe seasonal fluctuations. Several environmental stress conditions are known to produce reactive oxygen species in cellular compartments, resulting in oxidative damage and apoptosis. The study of cell death in higher plants and animals has revealed the existence of an active ‘programmed cell death’ (PCD) process and similarities between such processes suggest an evolutionary origin. A study was undertaken to examine the morphological, biochemical and molecular responses of the unicellular green alga Chlamydomonas reinhardtii after exposure to oxidative (10 mM H₂O₂) and osmotic (200 mM NaCl and 360 mM sorbitol) stress. Concentrations of H₂O₂ (2–50 mM), NaCl and sorbitol (100–800 mM) were negatively correlated with growth. Biochemical analyses showed an increase in intracellular H₂O₂ production (2.2-fold with H₂O₂ and ~1.2–1.4-fold with NaCl and sorbitol) and activities of some antioxidant enzymes [super oxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)]. Alteration of mitochondrial membrane potential (MMP) was observed upon treatment with H₂O₂ and NaCl, but not with sorbitol, indicating that the ionic stress component of NaCl altered the MMP. In addition, H₂O₂ led to the activation of a caspase-3-like protein, increase in the cleavage of a poly(ADP) ribose polymerase-1 (PARP-1)-like enzyme and formation of DNA nicks and laddering. With NaCl and sorbitol, no caspase activation, nor oligonucleosomal DNA laddering was observed, indicating non-apoptotic death. However, genomic DNA of NaCl (800 mM)-stressed cells, but not those of sorbitol-treated cells showed complete shearing. We conclude that the ionic rather than the osmotic component of NaCl leads to necrosis. These results unequivocally suggest that the vegetative cells of C. reinhardtii respond differentially to various stress agents, leading to different death types in the same organism. Moreover, unlike most other organisms, when exposed to NaCl this alga does not undergo PCD.     

Differential expression of putative cell death regulator genes in near-isogenic,resistant and susceptible barley lines during interaction with the powdery mildew fungus

We analysed pathogenesis-related expression of genes, that are assumed to be involved in ubiquitous plant defence mechanisms like the oxidative burst, the hypersensitive cell death reaction (HR) and formation of localized cell wall appositions (papillae). We carried out comparative northern blot and RT-PCR studies with near-isogenic barley (Hordeum vulgareL. cv. Pallas) lines (NILs) resistant or susceptible to the powdery mildew fungus race A6 (Blumeria graminis f.sp. hordei, BghA6). The NILs carrying one of the R-genes Mla12, Mlg or the mlo mutant allele mlo5 arrest fungal development by cell wall appositions (mlo5) or a HR (Mla12) or both (Mlg). Expression of an aspartate protease gene, an ascorbate peroxidase gene and a newly identified cysteine protease gene was up-regulated after inoculation with BghA6, whereas the constitutive expression-level of a BAS gene, that encodes an alkyl hydroperoxide reductase, was reduced. Expression of a newly identified barley homologue of a mammalian cell death regulator, Bax inhibitor 1, was enhanced after powdery mildew inoculation. An oxalate oxidase-like protein was stronger expressed in NILS expressing penetration resistance. A so far unknown gene that putatively encodes the large subunit of a superoxide generating NADPH oxidases was constitutively expressed in barley leaves and its expression pattern did not change after inoculation. A newly identified barley Rac1 homologue was expressed constitutively, such as the functionally linked NADPH oxidase gene. Gene expression patterns are discussed with regard to defence mechanisms and signal transduction.     

A form of cell death with some features resembling apoptosis in the amitochondrial unicellular organism Trichomonas vaginalis

One of hallmarks of apoptosis is the degradation and concomitant compaction of chromatin. It is assumed that caspases and caspase-independent pathways are rate limiting for the development of nuclear apoptosis. The caspase-independent pathway involves apoptosis-inducing factor (AIF) and leads to DNA fragmentation and peripheral chromatin condensation. Both pathways are the result of activation of death signals that the mitochondrion receives, integrates, and responds to with the release of various molecules (e.g., cytochrome c and AIF). In fact, both pathways have in common the final point of the DNA fragmentation and the mitochondrial origin of molecules that initiate the apoptotic events. Here, we examine the question of whether apoptosis or apoptotic-like processes exist in a unicellular organism that lacks mitochondria. We herein show that a form of cell death with some features resembling apoptosis is indeed present in Trichomonas vaginalis. Characterization of morphological aspects implicated in this event together with the preliminary biochemical data provided may lead to new insight about the evolutionary relationships between the different forms of programmed cell death identified so far.     

The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway

Cells with aberrations in chromosomal ploidy are normally removed by apoptosis. However, aneuploid neurons have been shown to remain functional and active both in the cortex and in the retina. Lim1 horizontal progenitor cells in the chicken retina have a heterogenic final cell cycle, producing some cells that enter S-phase without proceeding into M-phase. The cells become heteroploid but do not undergo developmental cell death. This prompted us to investigate if the final cell cycle of these cells is under the regulation of an active DNA damage response. Our results show that the DNA damage response pathway, including γ-H2AX and Rad51 foci, is not triggered during any phase of the different final cell cycles of horizontal progenitor cells. However, chemically inducing DNA adducts or double-strand breaks in Lim1 horizontal progenitor cells activated the DNA damage response pathway, showing that the cells are capable of a functional response to DNA damage. Moreover, manipulation of the DNA damage response pathway during the final cell cycle using inhibitors of ATM/ATR, Chk1/2, and p38MAPK, neither induced apoptosis nor mitosis in the Lim1 horizontal progenitor cells. We conclude that the DNA damage response pathway is functional in the Lim1 horizontal progenitor cells, but that it is not directly involved in the regulation of the final cell cycle that gives rise to the heteroploid horizontal cell population.     

Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.     

Modulation of G(2) arrest enhances cell death induced by the antitumor 1-nitroacridine derivative,Nitracrine

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC₉₉ concentration delayed progression through the S phase and transiently arrested cells in G₂/M as found by flow cytometry. Higher drug concentration (2 × IC₉₉) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC₉₉ concentration and irreversible arrest in early S phase at the higher dose (2 × IC₉₉). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G₂ arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G₂ arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.     

Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells

Summary. Programmed cell death occurs in plants during several developmental processes and during the expression of resistance to pathogen attack (i.e., the hypersensitive response). An unsolved question of plant programmed cell death is whether a unique signaling pathway or different, possibly convergent pathways exist. This problem was addressed in cultured sycamore (Acer pseudoplatanus L.) cells by comparing the effects of fusicoccin, Tunicamycin and Brefeldin A, inducers of programmed cell death with well-defined molecular and cellular targets, on some of the parameters involved in the regulation of this process. In addition to cell death, the inducers are able to stimulate the production of H₂O₂, the leakage of cytochrome c from mitochondria, the accumulation of cytosolic 14-3-3 proteins, and changes at the endoplasmic reticulum level, such as accumulation of the molecular chaperone binding protein and modifications in the organelle architecture. Interestingly, no additive effect on any of these parameters is observed when fusicoccin is administered in combination with Tunicamycin or Brefeldin A. Thus, these inducers seem to utilize the same or largely coincident pathways to induce programmed cell death and involvement of the endoplasmic reticulum, in addition to that of mitochondria, appears to be a common step.Correspondence and reprints: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.