Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium that is conserved in higher metazoans.

Species belonging to the lowest metazoan phylum, the sponges (Porifera), exhibit a surprisingly complex and multifaceted Bauplan (body plan). Recently, key molecules have been isolated from sponges which demonstrate that the cells of these animals are provided with characteristic metazoan adhesion and signal transduction molecules, allowing tissue formation. In order to understand which factors control the spatial organization of these cells in the sponge body plan, we screened for a cDNA encoding a soluble modulator of the behaviour of endothelial cells. A cDNA encoding a putative protein, which is highly similar to the human and mouse endothelial monocyte-activating polypeptide (EMAP) II has been isolated from a library of the marine sponge Geodia cydonium. The sponge EMAP-related polypeptide (EMAPR) has been termed EMAPR1_GC. The full-length cDNA clone, GCEMAPR1, has a size of 592 nucleotides (nt) and contains a 447 nt-long potential open reading frame; the molecular weight (MW) of the deduced amino acid sequence, 16,499 Da, is close to that of mature mammalian EMAP II (ca. 18 kDa). The sponge polypeptide is also closely related to a deduced polypeptide from the cosmid clone F58B3 isolated from Caenorhabditis elegans. A phylogenetic analysis revealed that the sponge and the nematode EMAPR molecules form a cluster which is significantly separated from the corresponding mammalian EMAP molecules. The function of the first cloned putative soluble modulator of endothelial cells in sponges remains to be determined.     

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.     

Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

BACKGROUND: Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. METHODS: Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. RESULTS: AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. CONCLUSIONS: The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.     

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.     

Spontaneous human monocyte apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin and is independent of caspase-1.

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.     

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.     

Apoptosis in the malaria protozoan,Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito

Death by apoptosis regulates cell numbers in metazoan tissues and it is mediated by activation of caspases and results in characteristic morphological and biochemical changes. We report here that the malaria protozoan, Plasmodium berghei, exhibits features typical of metazoan apoptotic cells including condensation of chromatin, fragmentation of the nuclear DNA and movement of phosphatidylserine from the inner to the outer lamellae of the cell membrane. In addition, proteins with caspase-like activity were identified in the cytoplasm of the ookinete suggesting that the cellular mechanism of cell death may be similar to that of multicellular eukaryotes. Our data show that more than 50% of the mosquito midgut stages of the parasite die naturally by apoptosis before gut invasion. Cell death was prevented by a caspase inhibitor, treatment resulting in a doubling of parasite intensity. All these features also occur in vitro. Cell suicide thus plays a major and hitherto unrecognised role in controlling parasite populations and could be a novel target for malaria control strategies.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.     

Identification of caspases and apoptosis in the simple metazoan Hydra.

Apoptosis is a normal process by which cells die and are eliminated from tissue by phagocytosis [1]. It is involved in regulating cell numbers in adult tissues and in eliminating 'excess' cells during embryogenesis and development. Apoptosis is mediated by activation of caspases, which then cleave a variety of cellular substrates and thereby cause the characteristic morphology of apoptotic cells (rounded cells, condensed chromatin, susceptibility to phagocytosis) [2]. Although apoptosis has been well documented in nematodes, insects and mammals, it is not yet clear how early in evolution apoptosis or its component enzymes arose. In the simple metazoan Hydra vulgaris, cell death regulates cell numbers [3] [4] [5]. In starved animals, for example, epithelial cell proliferation continues at a nearly normal rate although the tissue does not increase in size; the excess cells produced are eliminated by phagocytosis. Cell death can also be induced in wild-type hydra by treatment with colchicine [6] or in a mutant strain (sf-1) by temperature shock [7]. Here, we show that cell death in hydra is morphologically indistinguishable from apoptosis in higher animals, that hydra polyps express two genes with strong homology to members of the caspase 3 family, and that caspase-3-specific enzyme activity accompanies apoptosis in hydra. The occurrence of apoptosis and caspases in a member of the ancient metazoan phylum Cnidaria supports the idea that the invention of apoptosis was an essential feature of the evolution of multicellular animals.     

Caspase inhibition and N6-benzyladenosine-induced apoptosis in HL-60 cells.

As an extension of our recently published work (Mlejnek and Kuglík [2000] J. Cell. Biochem. 77:6-17), the role of caspases in N(6)-benzylaminopurine riboside (BAPR)-induced apotosis in HL-60 cells was evaluated in this study. Here, BAPR-induced apoptosis was accompanied by activation of caspase-3 and caspase-9. However, when these caspases were selectively inhibited, the progression of BAPR-induced apoptosis was not markedly affected. Besides that, activation of caspase-3 and caspase-9 was found to be rather late event in apoptotic process. These results suggested that other caspases might be critically implicated. Indeed, pan-specific caspase inhibitor, Z-VAD-FMK, completely prevented DNA cleavage and apoptotic bodies formation. However, Z-VAD-FMK failed to prevent cell death and it was incapable to fully counteract the main apoptotic hallmark-chromatin condensation. Finally, our data indicate that cellular decision between apoptosis and necrosis is made upon the availability of both caspase proteases and intracellular ATP.     

Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.     

A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation

Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.     

Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies

Ectromelia virus (EV), which causes mousepox, is a member of the orthopoxviruses that are defined as being able to suppress apoptosis. Caspase-3 is one of the key effector proteases which regulates the apoptotic cascade and which is responsible for DNA fragmentation observed during apoptosis. It is well known that viruses, especially poxviruses, can inhibit caspase activity. Here, we report that EV can regulate apoptosis in vitro, suppressing the activity of caspases recognizing the DEVD (Asp-Glu-Val-Asp) motif (caspase-3 and -7) before successful virus replication is completed. Caspase-3 activity measurement showed that an increase in caspase-3 activity preceded the peak of DNA fragmentation demonstrated by TUNEL staining of L929 and RK-13 cells. By using specific caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO and zVAD-fmk), we showed that caspase-3 and -7 (DEVDases) are major effector caspases during EV-induced apoptosis in permissive L929 and RK-13 cell cultures. Apoptosis in vivo seems to play an important role during viraemia as well as during the clearance of EV from genetically susceptible BALB/c (H-2(d)) mice. However, as shown by measurement of caspase-3 activity, caspase-3 protein detection and M30-antibody staining, both DEVDases seem to play an important role during EV clearance from draining lymph nodes and conjunctivae at 15 days p.i. up to 20 days p.i., whereas in the liver and spleen DNA fragmentation coexisted with viral multiplication and secondary viraemia. Apoptosis was DEVDase dependent only in the liver, while spleen DNA fragmentation observed between 5 and 10 days p.i. was caspase independent. Therefore, we conclude that DEVDase- (caspase-3- and caspase-7-) dependent apoptosis is an important mechanism regulating the resolution of EV infection.     

Caspase-7 gene disruption reveals an involvement of the enzyme during the early stages of apoptosis

Caspases play a key role during apoptotic execution. In an attempt to elucidate the specific role of caspase-7 we generated a chicken DT40 cell line in which both alleles of the gene were disrupted. Viability assays showed that caspase-7-/- clones are more resistant to the common apoptosis-inducing drugs etoposide and staurosporine. Caspase-7-/- cells show a delay in phosphatidylserine externalization and DNA fragmentation as well as cleavage of the caspase substrates poly(ADP-ribose) polymerase 1 and lamins B1 and B2. Caspase affinity labeling and activity assays indicated that deficient cells exhibit a delay in caspase activation compared with wild type DT40 cells, providing an explanation for the differences in apoptotic execution between caspase-7 null and wild type DT40 cells. These results strongly suggest that caspase-7 is involved earlier than other effector caspases in the apoptotic execution process in DT40 B lymphocytes.     

Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors.     

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.     

Human, insect and nematode caspases kill Saccharomyces cerevisiae independently of YCA1 and Aif1p

This study characterised the impact of active metazoan apoptotic proteases (caspases) on Saccharomyces cerevisiae viability. Expression of active caspase-3 or caspase-8 in yeast ruptured plasma and nuclear membranes and dramatically impaired clonogenic survival, but did not damage DNA. Deletion of the proposed yeast apoptosis regulators YCA1 or Aif1p did not affect the ability of human, insect or nematode caspases to kill yeast. These data indicate that expression of active metazoan caspases causes irreversible damage to yeast membranes and organelles, in a manner independent of YCA1 and Aif1p.