Ionophore-induced apoptosis: role of DNA fragmentation and calcium fluxes

Two ionophores specific for K+, valinomycin and beauvericin, induce a type of cell death very similar to apoptosis due to tumor necrosis factor (TNF alpha). Both ionophores cause cytolysis accompanied by internucleosomal DNA fragmentation of the dying cell into units of 200 base pairs. Morphologically, the cell death appears to consist of a mixture of nuclear apoptotic changes and cytoplasmic necrotic changes. As in the case for TNF alpha-mediated death, metabolic inhibitors have no effect on the course of cell death, but DNA fragmentation and cytolysis are decreased by the endonuclease inhibitor, zinc. Beauvericin and valinomycin trigger an increase in the cytoplasmic calcium concentration, most likely due to release of calcium from intracellular stores, and chelation of cytoplasmic calcium with quin-2 inhibits DNA fragmentation. Thus, these ionophores set off apoptosis through a calcium-activatable endonuclease, suggesting that other nonphysiological toxins might also cause apoptosis through their ability to indirectly elevate the cytoplasmic calcium concentration, without the need to invoke specific surface receptors.     

Induction of Apoptosis in Leukemia U937 Cells by 5′-Deoxy-5′-methylthioadenosine, a Potent Inhibitor of Protein Carboxylmethyltransferase

We found dramatic changes in leukemia U937 cells treated with 5′-deoxy-5′-methylthioadenosine (MTA), a potent inhibitor of protein carboxylmethyltransferase (protein methylase II). Initiation of cell death was observed by 1 day after MTA treatment, and it was induced in a dose- and time-dependent manner. However, cell viability measured by trypan blue exclusion was not consistent with the actual percentage of cell death. These results indirectly indicated that the type of cell death is apoptosis rather than necrosis. Nuclear fragmentation and DNA condensation of MTA-treated U937 cells were analyzed by both fluorescent and electron microscopy. MTA-treated cells first began to arrest in the M phase of the cell cycle, and they then exhibited a mitotic-like nuclear fragmentation process with partially membraneless chromatin. Furthermore, agarose gel electrophoresis of DNA extracted from cells treated with MTA showed DNA laddering with production of fragments of approximately 200 bp multiples. These studies indicated that cell death induced by MTA has the characteristics of apoptosis, although nuclear fragmentation is atypical. It seems likely that the process of apoptosis in U937 cells induced by MTA correlates with incomplete assembly of the nuclear envelope, since MTA itself could inhibit the carboxylmethylation of nuclear lamin B and delayed incorporation of lamin B into the nuclear envelope.     

Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death

Dexamethasone and corticosterone kill mouse thymocytes, as measured by eosin uptake, after several hours of in vitro incubation. This killing requires RNA and protein synthesis, because it is inhibited by cycloheximide, emetine, or actinomycin D. An earlier event than cell death is the extensive fragmentation of nuclear DNA into oligonucleosomal subunits; this fragmentation also requires RNA and protein synthesis. The DNA cleavage results from the action of an endonuclease that preferentially cleaves DNA in the linker regions between nucleosomes. This endonuclease is found constitutively in the nuclei of thymocytes and some other cells, and requires calcium and magnesium ions for its activation; if isolated fresh thymocyte nuclei are incubated with these ions, as much as 77% of their DNA is cleaved within 90 min. Thus, the protein for which synthesis is necessary for glucocorticoid-induced thymocyte death is not the endonuclease itself, but is in some way involved in its activation; we suggest that it may be part of a system for transporting calcium into the nucleus. The endonuclease is inhibited by zinc, which also prevents thymocyte death. It appears that glucocorticoids cause thymocyte death by activating an enzyme that rapidly and extensively degrades DNA. This "death from within" is biochemically and morphologically different from toxic or accidental cell death, such as that induced by azide, heat, or antibody and complement treatment. Although mature T cells also contain the endogenous endonuclease, they lack the glucocorticoid-inducible mechanism for activating it, and are thus glucocorticoid-resistant.     

DNA degradation and nuclear degeneration during programmed cell death in petals of Antirrhinum, Argyranthemum, and Petunia

Programmed cell death (PCD) was studied in the petals of Antirrhinum majus, Argyranthemum frutescens, and Petunia hybrida, using DNA degradation and changes in nuclear morphology as parameters. The petals exhibit loss of turgor (wilting) as a visible symptom of PCD. DNA degradation, as shown on agarose gels, occurred in all species studied, prior to visible wilting. The number of DNA masses in all the petals of a flower, determined by flow cytometry, markedly increased in Argyranthemum and Petunia, but decreased in Antirrhinum. Many small DNA masses were observed in Argyranthemum and Petunia. The surface of each small DNA mass stained with the lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide (DiOC6), indicating that these masses were surrounded by a membrane. In Antirrhinum, in contrast, the chromatin fragmented into several small spherical clumps that remained inside a large membranous structure. Nuclear fragmentation, therefore, did not occur in Antirrhinum, whereas nuclear fragmentation possibly was a cause of the small DNA masses in Argyranthemum and Petunia. It is concluded that at least two contrasting nuclear morphologies exist during PCD. In the first, the chromatin fragments inside the nucleus, not accompanied--or followed--by nuclear fragmentation. In the second, a large number of DNA masses were observed each enveloped by a membrane. The second type was probably due, at least partially, to nuclear fragmentation.     

Ectopic expression of mitochondria endonuclease Pnu1p from Schizosaccharomyces pombe induces cell death of the yeast

Endonuclease G (EndoG) is a mitochondrial non-specific nuclease that is highly conserved among the eukaryotes. Although the precise role of EndoG in mitochondria is not yet known, the enzyme is released from the mitochondria and digests nuclear DNA during apoptosis in mammalian cells. Schizosaccharomyces pombe has an EndoG homolog Pnu1p (previously named SpNuc1) that is produced as a precursor protein with a mitochondrial targeting sequence. During the sorting into mitochondria the signal sequence is cleaved to yield the functionally active endonuclease. From the analogy to EndoG, active extramitochondrial Pnu1p may trigger cell killing by degrading nuclear DNA. Here, we tested this possibility by expressing a truncated Pnu1p lacking the signal sequence in the extramitochondrial region of pnu1-deleted cells. The truncated Pnu1p was localized in the cytosol and nuclei of yeast cells. And ectopic expression of active Pnu1p led to cell death with fragmentation of nuclear DNA. This suggests that the Pnu1p is possibly involved in a certain type of yeast cell death via DNA fragmentation. Although expression of human Bak in S. pombe was lethal, Pnu1p nuclease is not necessary for hBak-induced cell death.     

Nuclear DNA fragmentation during cell death of short-lived ray tracheids in the conifer <Emphasis Type="Italic">Pinus densiflora</Emphasis>

One key event in the programmed cell death is nuclear DNA fragmentation. We investigated the timing of nuclear DNA fragmentation during the cell death of short-lived ray tracheids in Pinus densiflora using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fluorescence due to TUNEL was detected only in deformed nuclei that lacked obvious chromatin in ray tracheids that were adjacent to ray tracheids that no longer contained nuclei. Our observations revealed that nuclear DNA fragmentation occurred only at the final stage of cell death in ray tracheids in situ.     

The level of free intracellular zinc mediates programmed cell death/cell survival decisions in plant embryos

Zinc is a potent regulator of programmed cell death (PCD) in animals. While certain, cell-type-specific concentrations of intracellular free zinc are required to protect cells from death, zinc depletion commits cells to death in diverse systems. As in animals, PCD has a fundamental role in plant biology, but its molecular regulation is poorly understood. In particular, the involvement of zinc in the control of plant PCD remains unknown. Here, we used somatic embryos of Norway spruce (Picea abies) to investigate the role of zinc in developmental PCD, which is crucial for correct embryonic patterning. Staining of the early embryos with zinc-specific molecular probes (Zinquin-ethyl-ester and Dansylaminoethyl-cyclen) has revealed high accumulation of zinc in the proliferating cells of the embryonal masses and abrupt decrease of zinc content in the dying terminally differentiated suspensor cells. Exposure of early embryos to a membrane-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine led to embryonic lethality, as it induced ectopic cell death affecting embryonal masses. This cell death involved the loss of plasma membrane integrity, metacaspase-like proteolytic activity, and nuclear DNA fragmentation. To verify the anti-cell death effect of zinc, we incubated early embryos with increased concentrations of zinc sulfate. Zinc supplementation inhibited developmental PCD and led to suppression of terminal differentiation and elimination of the embryo suspensors, causing inhibition of embryo maturation. Our data demonstrate that perturbation of zinc homeostasis disrupts the balance between cell proliferation and PCD required for plant embryogenesis. This establishes zinc as an important cue governing cell fate decisions in plants.     

DNA Digestion and Chromatin Condensation during Nuclear Death inTetrahymena

DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organismTetrahymena.We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agree- ment with a previous study [17], indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn²⁺and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion inTetrahymena,suggesting a requirement here for an endonuclease which is Ca²⁺-independent and Zn²⁺-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.     

Caspase-activated DNase Is Necessary and Sufficient for Oligonucleosomal DNA Breakdown,but Not for Chromatin Disassembly during Caspase-dependent Apoptosis of LN-18 Glioblastoma Cells

Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.     

Extracellular ATP as a trigger for apoptosis or programmed cell death

Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.     

Alterations in cell nuclei during apoptosis

Apoptosis is a genetically programmed phenomenon that aids in maintaining homeostasis in multicellular organisms. The characteristic morphological features of apoptosis are highly conservative and are dependent on the cell type and the apoptotic inducer. The nuclear events occurring during apoptosis include changes at the molecular level (i.e. DNA cleavage, modifications of nuclear polypeptides, and proteolysis of several proteins important for cell maintenance), and, consequently, alterations at the morphological level (i.e. chromatin condensation, nuclear shrinkage, DNA fragmentation and apoptotic body formation). These events are still not fully understood. It is very probable that a progressive decrease in pH could also be an essential factor for the induction of nuclease and protease activities, and an important element of the optimal conditions for their function. This review details the current state of knowledge on apoptotic nuclear events, with particular focus on the proteins involved in the execution of apoptosis in cell nuclei, and on the differences in substrate cleavage profiles for different types of cell undergoing cell death.     

Sulindac activates nuclear translocation of AIF, DFF40 and endonuclease G but not induces oligonucleosomal DNA fragmentation in HT-29 cells

Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100-300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells.     

Overexpression of Helicard,a CARD-containing helicase cleaved during apoptosis,accelerates DNA degradation

Apoptotic cell death is characterized by several morphological nuclear changes, such as chromatin condensation and extensive fragmentation of chromosomal DNA. These alterations are primarily triggered through the activation of caspases, which subsequently cleave nuclear substrates. Caspase-3 induces processing of Acinus, which leads to chromatin condensation. DNA fragmentation is dependent on the DNase CAD, which is released from its inhibitor, ICAD, upon cleavage by caspase-3. DNA degradation is also induced by AIF and endonuclease G, which are both released from mitochondria upon death stimuli but do not require prior processing by caspases for their DNase activity. Here we report the identification of a widely expressed helicase designated Helicard, which contains two N-terminal CARD domains and a C-terminal helicase domain. Upon apoptotic stimuli, Helicard is cleaved by caspases, thereby separating the CARD domains from the helicase domain. While Helicard localizes in the cytoplasm, the helicase-containing fragment is found in the nucleus. Helicard accelerates Fas ligand-mediated DNA degradation, whereas a noncleavable or a helicase-dead Helicard mutant does not, implicating Helicard in the nuclear remodeling occurring during apoptosis.     

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3 end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN₃, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3end labelling of DNA breaks.     

Cell nucleus and DNA fragmentation are not required for apoptosis

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.     

Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells

Zinc ions have both essential and toxic effects on mammalian cells. Here we report the ability of zinc to act as an inducer of apoptosis in C6 rat glioma cells. Incubation with 150 to 300 microM ZnCl2 caused cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, nuclear fragmentation and breakdown of the mitochondrial membrane potential. On the other hand, zinc deprivation by the membrane permeable chelator TPEN [N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine] also induced programmed death in this cell line, indicating the existence of intracellular zinc levels below and above which apoptosis is induced. Zinc-induced apoptosis in C6 cells was independent of major signaling pathways (protein kinase C, mitogen activated protein kinase and guanylate cyclase) and protein synthesis, but was increased by facilitating zinc uptake with the ionophore pyrithione. Lanthanum(III)chloride was also able to increase the net zinc uptake, but nevertheless apoptotic features and zinc toxicity were reduced. Remarkably, lanthanum suppressed the zinc-induced breakdown of the mitochondrial membrane potential. We conclude that in C6 cells lanthanum acts in two different ways, as a promoter of net zinc uptake and as a suppressor of zinc-induced apoptosis.     

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.     

Loss of Acinus inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis

Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.