Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K(+) regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K(+) during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1beta and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.     

Functional genomic analysis of apoptotic DNA degradation in C. elegans

Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Using an RNAi-based functional genomic approach, we have identified seven additional cell death-related nucleases (crn genes), which along with two known nucleases (CPS-6 and NUC-1) comprise at least two independent pathways that contribute to cell killing, and likely signaling for phagocytosis, by degrading chromosomal DNA. Several crn genes have human homologs that are important for RNA processing, protein folding, DNA replication, and DNA damage repair, suggesting dual roles for CRN nucleases in cell survival and cell death. It should now be possible to systematically decipher the mechanisms of apoptotic DNA degradation.     

Cuts can kill: the roles of apoptotic nucleases in cell death and animal development

Chromosome fragmentation is one of the major biochemical hallmarks of apoptosis. However, until recently, its roles in apoptosis and mechanisms of action remained elusive. Recent biochemical and genetic studies have shown that chromosome fragmentation is a complex biochemical process that involves a plethora of conserved nucleases with distinct nuclease activities and substrate specificities. These apoptotic nucleases act cooperatively among themselves and with other nonnuclease cofactors to promote stepwise chromosome fragmentation and DNA degradation. Importantly, in addition to its direct contribution to the dismantling of the dying cell, apoptotic DNA degradation can facilitate cell killing and other apoptotic events such as clearance of apoptotic cells. Furthermore, some apoptotic nucleases apparently affect other aspects of animal development, including immune responses. The identification of new apoptotic nucleases and analysis of their functions in apoptosis and animal development should pave the way for future studies to uncover new functions for apoptotic nucleases and shed light on the hidden links between apoptotic DNA degradation and human diseases.     

PARP-1 and its associated nucleases in DNA damage response

Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) acts as a DNA damage sensor. It recognizes DNA damage and facilitates DNA repair by recruiting DNA repair machinery to damage sites. Recent studies reported that PARP-1 also plays an important role in DNA replication by recognizing the unligated Okazaki fragments and controlling the speed of fork elongation. On the other hand, emerging evidence reveals that excessive activation of PARP-1 causes chromatin DNA fragmentation and triggers an intrinsic PARP-1-dependent cell death program designated parthanatos, which can be blocked by genetic deletion or pharmacological inhibition of PARP-1. Therefore, PARP-1 plays an essential role in maintaining genomic stability by either facilitating DNA repair/replication or triggering DNA fragmentation to kill cells. A group of structure-specific nucleases is crucial for executing DNA incision and fragmentation following PARP-1 activation. In this review, we will discuss how PARP-1 coordinates with its associated nucleases to maintain genomic integrity and control the decision of cell life and death.     

Oligonucleosomal DNA Fragmentation in MCF-7 Cells Undergoing Palmitate-Induced Apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.     

Structural and functional insight into sugar-nonspecific nucleases in host defense

In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way.     

Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase

The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.     

The Roles and Acting Mechanism of Caenorhabditis elegans DNase II Genes in Apoptotic DNA Degradation and Development

DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3′ OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1''s ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3′ OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation.     

The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage

The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.     

Steroidogenesis and apoptosis in the mammalian ovary

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis in granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated up to 24h following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or TNF-alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one not involving mitochondrial Cyt C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and affymetrix DNA microarray technology we discovered that granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells. Thus, the apoptotic signals could bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures cyclicity of estradiol and progesterone release in the estrous/menstruous cycle even during the initial stages of apoptosis.     

Camptothecin-induced imbalance in intracellular cation homeostasis regulates programmed cell death in unicellular hemoflagellate Leishmania donovani

Leishmania, a unicellular trypanosomatid protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. However, it undergoes a process of programmed cell death during treatment with the topoisomerase I poison camptothecin (CPT). The present study shows that CPT-induced formation of reactive oxygen species increases the level of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (DeltaPsim), which is followed by a significant decrease in intracellular pH levels. CPT-induced oxidative stress also causes impairment of the Na+ - K+ -ATPase pump and subsequently decreases the intracellular K+ level in leishmanial cells. A decrease in both intracellular pH and K+ levels propagates the apoptotic process through activation of caspase 3-like proteases by rapid formation of cytochrome c-mediated apoptotic complex. In addition to caspase-like protease activation, a lower level of intracellular K+ also enhances the activation of apoptotic nucleases at the late stage of apoptosis. This suggests that the physiological level of pH and K+ are inhibitory for apoptotic DNA fragmentation and caspase-like protease activation in leishmanial cells. Moreover, unlike mammalian cells, the intracellular ATP level gradually decreases with an increase in the number of apoptotic cells after the loss of DeltaPsim. Taken together, the elucidation of biochemical events, which tightly regulate the process of growth arrest and death of Leishmania donovani promastigotes, allows us to define a more comprehensive view of cell death during treatment with CPT.     

Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin-treated oats

Histological and cytological evidence of where and when apoptotic cells occur in Pc-2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM-TUNEL was positive in the non-collapsed cells but not in the collapsed cells in the treated leaves. However, the EM-TUNEL assay was positive in the nuclei of the non-collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin-treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin-treated non-collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM-TUNEL assay indicated that EGTA co-incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.     

28S ribosome degradation in lymphoid cell apoptosis: evidence for caspase and Bcl-2-dependent and -independent pathways

Apoptosis, a physiological form of cell death, is characterized by the activation of a program that kills cells and recycles their constituents. We have used thymoma cell lines to examine the role of Bcl-2 and caspases in ribosomal destruction during apoptosis. Glucocorticoid- and calcium ionophore (A23187)-induced apoptosis of S49 Neo cells resulted in both 28S rRNA and DNA degradation. Interestingly, anisomycin, a potent protein synthesis inhibitor, also induced 28S rRNA and DNA fragmentation suggesting that the responsible nucleases are present in the viable cells and become activated during apoptosis. The anti-apoptotic protein, Bcl-2, inhibited both glucocorticoid- and anisomycin-induced DNA and 28S rRNA degradation but could not protect against A23187-induced nucleic acid degradation. We next examined the role of caspase activation in the generation of 28S rRNA degradation through the use of ZVAD, a general caspase inhibitor. Under conditions where ZVAD substantially decreased 28S rRNA degradation induced by glucocorticoid or anisomycin, no decrease was observed when A23187 was used to induce apoptosis. Surprisingly, RNA degradation, like DNA degradation, occurs exclusively in shrunken lymphocytes but not those with normal cell volume despite equivalent exposure of the cells to the apoptotic signals. Together, these findings indicate the ribosome is a specific target for death effectors during apoptosis and that a caspase/Bcl-2-independent pathway exists to activate its destruction.     

Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast

Protracted starvation of auxotrophic Saccharomyces cerevisiae strains for an essential amino acid is commonly used to allow investigation of adaptive mutation mechanisms during starvation-induced cell cycle arrest. Under these conditions, the majority of cells dies during the first 6 days. We investigated starving cells for markers of programmed cell death and for the production of reactive oxygen species (ROS). We observed that protracted starvation for lysine or histidine resulted in an increasing number of cells exhibiting DNA fragmentation and chromatin condensation, thus an apoptotic phenotype. Not only respiration-competent cells but also respiratory deficient rho0 cells were able to undergo programmed cell death. In addition the starving cells rapidly exhibited indicators of oxidative stress, independently of their respiratory competence. These results indicate that starvation for an essential amino acid results in severe cell stress, which may finally be the trigger of programmed cell death.     

Is There a Molecular Logic That Sustains Neuronal Functional Integrity and Survival? Lipid Signaling Is Necessary for Neuroprotective Neuronal Transcriptional Programs

A challenge to civilization is the growing incidence in the loss of sight and cognition due to increased life expectancy. Therefore, we are confronted with a rise in the occurrence of photoreceptor- and neuronal-survival failure, as reflected mainly by age-related macular degeneration (AMD) and Alzheimer’s disease (AD). Nervous system development is driven by neuronal apoptotic cell death, and thereafter, for the entire lifespan of an organism, neurons are postmitotic cells. In neurodegenerative diseases, apoptosis and other forms of cells death lead to selective neuronal loss. Although age is the main risk factor, not everyone develops these diseases during aging. Despite decades of important findings about neuronal cell death, the specific mechanisms that regulate neuronal survival remain incompletely understood.     

Activation of K+ channels: an essential pathway in programmed cell death

Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+ efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+ transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+ channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.     

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.