<Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium infection in the porcine intestine: evidence for caspase-3-dependent and -independent programmed cell death

The normal intestinal epithelium is renewed with a turnover rate of 3–5 days. During Salmonella infection increased cell loss is observed, possibly as a result of programmed cell death (PCD). We have, therefore, studied the effects of Salmonella Typhimurium infection on three elements involved in PCD: caspase-3 activation, c-Jun phosphorylation on serine 63 (both detected by immunocytochemistry), and DNA fragmentation (detected by TUNEL reaction), using a pig jejunal loop model. Additionally, we used nuclear staining for detecting signs of classical apoptosis. Activated caspase-3 was detected in scattered epithelial cells and the number of positive cells increased with increasing times of exposure to Salmonella (P<0.0001). An increase in phospho-c-Jun in epithelial cells was already detectable 5 min after infection and often occurred in cells that appeared not to be invaded by the organism. Changes in caspase-3 activation and c-Jun phosphorylation were most marked in the proximal region of the jejunum. Although rarely observed in the epithelium, proper TUNEL-positive cells were frequently found in the intestinal lumen. Some, but not all, TUNEL-positive cells were also positive for caspase-3, indicating that both caspase-3-dependent and -independent pathways of PCD increased upon infection.     

Caspase-3-dependent and -independent apoptosis in focal brain ischemia

BACKGROUND: Although extensive caspase-3 activation has been demonstrated in experimental brain ischemia produced in neonatal rat, the role this caspase plays in the focal ischemia of adult brain is not clear, as the levels of caspase-3 in adult rat brain are extremely low. This raises the question whether caspase-3 synthesis and activation are essential for execution of the apoptotic program and DNA fragmentation in permanent brain ischemia, a condition that impairs cellular protein synthesis. MATERIALS AND METHODS: Rat middle cerebral artery was permanently occluded and histochemical detection of procaspase-3, active caspase-3 and DFF 40/CAD and apoptotic morphology analysis were performed at 6, 24, 48, and 72 hours after occlusion. RESULTS: Necrosis and two types of programmed cell death (PCD) are identified in this study of permanent focal brain ischemia. The first type of PCD is represented by active caspase-3 and DFF 40/CAD-positive cells. The second type of PCD is represented by caspase-3 and DFF40/CAD negative cells, which display morphological signs of apoptosis-like PCD: namely, nuclear chromatin condensation in lump masses and apoptotic body formation. The cells of the first type have a maximum number noted after 24 hours of ischemia. The cells of the second type are primarily seen after 48 and 72 hours of ischemia. Necrotic cells, which are also detected in the stroke, are caspase-3 negative, and have swollen nuclei, without chromatin condensation and apoptotic body formation. CONCLUSIONS: Our results indicate that in permanent brain ischemia in adult rats, PCD processes occur differently in various parts of ischemic zone. In conditions of severe energy depletion, the reactions of cellular disassembly and packaging into apoptotic bodies are accomplished without either caspase-3 expression or the activation of caspase-3-dependent deoxyribonuclease.     

Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to approximately 31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H(2)O(2)- and alpha-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H(2)O(2)/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.     

Distribution of E-cadherin and β-catenin in relation to cell maturation and cell extrusion in rat and mouse small intestines

In the small intestines, cell renewal from stem cells present in the crypts is balanced by cell extrusion from the tips of the villi. The mechanism by which extrusion occurs is unknown. Recent in vitro data suggested that loss of E-cadherin could contribute to cell extrusion and induction of programmed cell death (PCD) in mouse small intestinal epithelium. We have studied if this also occurs in the intact rodent small intestine. Our results confirm that extruded cells are negative for E-cadherin. However, loss of the E-cadherin-interacting protein β-catenin preceded both extrusion and loss of E-cadherin. Thus, all extruded cells as well as all cells in the process of extrusion lacked staining for β-catenin. Moreover, almost 80% of all cells undergoing programmed cell death, as detected by the TUNEL reaction, lacked β-catenin whereas over 70% of such cells were positive for E-cadherin. However, most cells lacking β-catenin did not display signs of PCD as detected by the TUNEL method or by staining for active caspase-3. Therefore, these results suggest that loss of β-catenin precedes the onset of programmed cell death, loss of E-cadherin and extrusion from the villi.     

ABCA1-dependent but apoA-I-independent cholesterol efflux mediated by fatty acid-bile acid conjugates (FABACs)

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.     

Activation of DNA-dependent protein kinase may play a role in apoptosis of human neuroblastoma cells

Treating SH-SY5Y human neuroblastoma cells with 1 microM staurosporine resulted in a three- to fourfold higher DNA-dependent protein kinase (DNA-PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA-PK activity: an initial increase that peaked by 4 h and a rapid decline that reached approximately 5-10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA-PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase-1 activity but stimulated caspase-3 activity by 10-15-fold in a time-dependent manner, similar to morphological changes. Similar time-dependent changes in DNA-PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 microM ceramide or UV radiation. In all these cases the increase in DNA-PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell-permeable inhibitor of DNA-PK, OK-1035, significantly reduced staurosporine-induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA-PK may be involved with the induction of apoptotic cell death.     

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.     

The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique.

BACKGROUND: To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death. METHODS: Apoptosis was induced in Jurkat cells by gamma-irradiation, incubation with camptothecin (CPT), or cytosine beta-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI- CD34+ fractions were cultured in a single-cell single-well (SCSW) assay. RESULTS: Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI- cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80-90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI- fractions (overall range 23-62%). Within total AnV+ and AnV+/PI- populations, only a minority of CD34+ cells showed ISEL positivity (range 4-8% and 0.8-6%, respectively). Different fractions of AnV+/PI- CD34+ cells did have clonogenic capacity. CONCLUSIONS: PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI- CD34+ cells in the SCSW assay.     

Cellular features of an apoptotic form of programmed cell death during the development of the ascidian, Boltenia villosa

The phylogenetic position of ascidians near the base of the chordate tree makes them ideal organisms for evolutionary developmental studies of programmed cell death (PCD). In the present study, the following key features of an apoptotic form of PCD are described in Boltenia villosa: fragmentation of DNA, increases in plasma membrane permeability, decreases in mitochondrial activity, production of reactive oxygen species (ROS), and caspase activation. First, evidence is presented for apoptosis of cells within the ovary. Later in development, during the early phase of larval tail resorption at the beginning of metamorphosis, some notochord nuclei showed DNA fragmentation and their cell corpses were rapidly eliminated from the larval body. In striking contrast to the rapid demise of notochord cells, larval muscle cells persisted for more than a week within developing juveniles. Rhodamine 123 and MTT experiments suggest that mitochondria within some of the resorbed larval tail muscle cells were metabolically active for more than a week. Furthermore, resorbed tail muscle cells contained a muscle-specific intermediate filament, termed p58, despite relatively high levels of ROS activity and the ubiquitination of their plasma membranes at day two. Corpses of larval tail muscle cells containing aggregated pigment granules survived within juveniles for more than a month, in contrast to the rapid elimination of notochord cells. Evidence consistent with the formation of larval muscle cell apoptotic bodies is presented. The most surprising result of the present study was that caspase-8, usually associated with apoptotic signaling, was activated in larval endoderm cells that develop into adult structures. When the present results were compared to features of PCD previously reported in other ascidians, significant species differences in PCD were revealed.     

Correlates of developmental cell death in Dictyostelium discoideum

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

Ultraviolet-C overexposure induces programmed cell death in Arabidopsis, which is mediated by caspase-like activities and which can be suppressed by caspase inhibitors, p35 and Defender against Apoptotic Death

Plants, animals, and several branches of unicellular eukaryotes use programmed cell death (PCD) for defense or developmental mechanisms. This argues for a common ancestral apoptotic system in eukaryotes. However, at the molecular level, very few regulatory proteins or protein domains have been identified as conserved across all eukaryotic PCD forms. A very important goal is to determine which molecular components may be used in the execution of PCD in plants, which have been conserved during evolution, and which are plant-specific. Using Arabidopsis thaliana, we have shown that UV radiation can induce apoptosis-like changes at the cellular level and that a UV experimental system is relevant to the study of PCD in plants. We report here that UV induction of PCD required light and that a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) was induced within 30 min and peaked at 1 h. This DEVDase appears to be related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase-1 and caspase-3 inhibitors and the pan-caspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that a YVADase activity and an inducible DEVDase activity possibly mediate DNA fragmentation during plant PCD induced by UV overexposure. We also report that At-DAD1 and At-DAD2, the two A. thaliana homologs of Defender against Apoptotic Death-1, could suppress the onset of DNA fragmentation in A. thaliana, supporting an involvement of the endoplasmic reticulum in this form of the plant PCD pathway.     

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures

Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.     

In vivo effects of thyroid hormone, corticosteroids and prolactin on cell proliferation and apoptosis in the anterior intestine of the euryhaline mudskipper (Periophthalmus modestus)

We have previously shown that anterior intestinal epithelium of the euryhaline mudskipper (Periophthalmus modestus) undergoes apoptosis during seawater (SW) acclimation, whereas elevated cell proliferation was observed in freshwater (FW)-acclimated fish. To understand the possible endocrine regulation of the gastrointestinal cell turnover during salinity acclimation, we examined the ratios of apoptotic and proliferating cells in the anterior intestine of one-third SW-acclimated mudskipper treated with triiodothyronine (T3), cortisol, 11-deoxycorticosterone (DOC, the putative teleostean mineralocorticoid), or prolactin (PRL). In situ nick end labeling of genomic DNA (TUNEL) and immunohistochemistry of proliferating cells nuclear antigen (PCNA) were used as indicators of apoptosis and cell proliferations, respectively. Cortisol significantly elevated apoptosis (P<0.05) in the epithelia and connective tissues and also stimulated the epithelial cell proliferation (P<0.05). PRL induced epithelial cell proliferation (P<0.05), but did not affect apoptotic status of the intestinal epithelium. Neither T3 nor DOC had any impact on cell proliferation or apoptosis. Together, our results suggest a role for cortisol and PRL in the regulation of anterior intestinal epithelial turnover during salinity acclimation in this species.     

Region-Specific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and hippocampus respectively were also in a good agreement with apoptotic proteins expression in these structures. In the cortex, hippocampus and cerebellum DNA fragmentation intensity was proportional to the active caspase-3 level. In contrast to these structures, in the brainstem, the lowest level of this protease was accompanied by the highest intensity of DNA fragmentation among the brain regions studied. The data suggest that cell death normally occurring during early postnatal life could be realized in the developing brainstem via caspase-3-independent pathways in animals that express this protease.     

Seasonal study of apoptotic markers in lizard oviduct

Lizard oviduct is a very dynamic organ that undergoes high tissue remodelling as a function of the cyclic reproductive activity. Until today there are no studies of molecular actors involved in cell death in the lizard oviduct. Therefore, this report is focused on some of apoptotic markers responsible of programmed cell death in this organ during the main significant phases of reproductive cycle. Apoptotic cell recognition was based on the estimation of known following markers: cleaved caspase-9 and-3; tissue transglutaminase (tTG) and DNA fragmentation. By Western blotting, expected band sizes of 29 and 17?kDa, recognizing the anti-caspase-9 and caspase-3, respectively, showed a stronger expression during the ovulation and postovulation. Enzymatic activity of caspase-9 shows the highest value at ovulation, whereas that of caspase-3 is recorded at postovulation. The expression and the activation of tTG protein are in line with the fragmentation of DNA. No tTG positive cells are detected at quiescence, when either no TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive nuclei or DNA fragmentation is observed. At this time tTG activity is at a minimum. Indeed, a consistent DNA smear is observed from the DNA extracted at postovulation, when tTG activity reached its maximum and several transglutaminase immunoreactivity cells and TUNEL positive nuclei are observed. The temporal and dynamic outlines of apoptotic parameters match with seasonal modifications of the oviduct. Taken together, our results demonstrate that the seasonal apoptotic activity of the oviduct represents a key process in the remodelling of this tissue during the reproductive cycle.     

Caspase-3 activity and carbonyl cyanide m-chlorophenylhydrazone-induced apoptosis in HL-60

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being "truly" necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as "truly" necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.