Identification and developmental expression of inhibitor of caspase-activated DNase (ICAD) in Drosophila melanogaster

DNA fragmentation, a hallmark of apoptosis, is regulated by a specific nuclease called caspase-activated DNase (CAD) and its inhibitor (ICAD). When cell lysates from Drosophila S2 cells were chemically denatured and the denatured proteins were removed after dialysis, the supernatant inhibited Drosophila CAD (dCAD). To identify the inhibitor, we tested recombinant DREP-1, which was previously identified using the Drosophila EST data base and found it also inhibited dCAD DNase. An antibody against DREP-1 inhibited the ICAD activity in the S2 cell extracts, confirming the identification of DREP-1 as a Drosophila homolog of ICAD (dICAD). The recombinant DREP-1/dICAD was cleaved at a specific site by human caspase 3 as well as by extracts prepared from S2 cells undergoing apoptosis. Biochemical fractionation and immunoprecipitation of dICAD from S2 cell extracts indicated that dICAD is complexed with dCAD in proliferating cells. The expression of the caspase-resistant form of dICAD/DREP-1 in a Drosophila neuronal cell line prevented the apoptotic DNA fragmentation. Northern hybridization and the immunohistochemical analyses revealed that the expression of the dICAD gene is developmentally regulated.     

SfDronc,an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X.     

Activation of extracellular signal-regulated kinases ERK1 and ERK2 induces Bcl-xL up-regulation via inhibition of caspase activities in erythropoietin signaling

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.     

Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells

A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.     

Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction

Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.     

Protease Activation in Apoptosis Induced by MAL

The proteolytic caspase cascade plays a central role in the signaling and execution steps of apoptosis. This study investigated the activation of different caspases in apoptosis induced by MAL (a folding variant of human alpha-lactalbumin) isolated from human milk. Our results show that the caspase-3-like enzymes, and to a lesser extent the caspase-6-like enzymes, were activated in Jurkat and A549 cells exposed to MAL. Activated caspases subsequently cleaved several protein substrates, including PARP, lamin B, and alpha-fodrin. A broad-range caspase inhibitor, zVAD-fmk, blocked the caspase activation, the cleavage of proteins, and DNA fragmentation, indicating an important role for caspase activation in MAL-induced apoptosis. Since an antagonistic anti-CD95 receptor antibody, ZB4, did not influence the MAL-induced killing, we conclude that this process does not involve the CD95-mediated pathway. While MAL did not directly activate caspases in the cytosol, it colocalized with mitochondria and induced the release of cytochrome c. Thus, these results demonstrate that caspases are activated and involved in apoptosis induced by MAL and that direct interaction of MAL with mitochondria leads to the release of cytochrome c, suggesting that this release is an important step in the initiation and/or amplification of the caspase cascade in these cells.     

Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE.

Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila. We report here the identification and characterization of drICE, a D. melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr. drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.     

Identification of caspase 3-mediated cleavage and functional alteration of eukaryotic initiation factor 2alpha in apoptosis

Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.     

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.     

DNA topoisomerase IIalpha interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.     

Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP

Activation of caspases 3 and 9 is thought to commit a cell irreversibly to apoptosis. There are, however, several documented situations (e.g., during erythroblast differentiation) in which caspases are activated and caspase substrates are cleaved with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases but not in others is unclear. One possibility is that some caspase substrates generate antiapoptotic signals when cleaved. Here we show that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). Fragment C expressed alone induces apoptosis, but this effect could be totally blocked by fragment N. Fragment N could also block apoptosis induced by low levels of caspase 9. As caspase activity increases, fragment N is further cleaved into fragments N1 and N2. Apoptosis induced by high levels of caspase 9 or by cisplatin was strongly potentiated by fragment N1 or N2 but not by fragment N. The present study supports a model in which RasGAP functions as a sensor of caspase activity to determine whether or not a cell should survive. When caspases are mildly activated, the partial cleavage of RasGAP protects cells from apoptosis. When caspase activity reaches levels that allow completion of RasGAP cleavage, the resulting RasGAP fragments turn into potent proapoptotic molecules.     

Determinants of the nuclear localization of the heterodimeric DNA fragmentation factor (ICAD/CAD)

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.     

DNA topoisomerase IIα interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution 2, 3, 4 and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 4, 7, 8 (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors 6, 8. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIα (Topo IIα), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIα and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIα, and that their association enhances the decatenation activity of Topo IIα in vitro.     

Illuminating the role of caspases during Drosophila oogenesis

Cell death is a prominent feature of animal germline development. In Drosophila, the death of 15 nurse cells is linked to the development of each oocyte. In addition, females respond to poor environmental conditions by inducing egg chamber death prior to yolk uptake by the oocyte. To study these two forms of cell death, we analyzed caspase activity in the germline by expressing a transgene encoding a caspase cleavage site flanked by cyan fluorescent protein and yellow fluorescent protein. When expressed in ovaries undergoing starvation-induced apoptosis, this construct was an accurate reporter of caspase activity. However, dying nurse cells at the end of normal oogenesis showed no evidence of cytoplasmic caspase activity. Furthermore, although expression of the caspase inhibitors p35 or Drosophila inhibitor of apoptosis protein 1 blocked starvation-induced death, it did not affect normal nurse cell death or overall oogenesis in well-fed females. Our data suggest that caspases play no role in developmentally programmed nurse cell death.     

Mechanisms for the magnolol-induced cell death of CGTH W-2 thyroid carcinoma cells

Magnolol, a substance purified from the bark of Magnolia officialis, inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The aim of this study was to study the effects of magnolol on CGTH W-2 thyroid carcinoma cells. After 24 h treatment with 80 microM magnolol in serum-containing medium, about 50% of the cells exhibited apoptotic features and 20% necrotic features. Cytochrome-c staining was diffused in the cytoplasm of the apoptotic cells, but restricted to the mitochondria in control cells. Western blot analyses showed an increase in levels of activated caspases (caspase-3 and -7) and of cleaved poly (ADP-ribose) polymerase (PARP) by magnolol. Concomitantly, immunostaining for apoptosis inducing factor (AIF) showed a time-dependent translocation from the mitochondria to the nucleus. Inhibition of either PARP or caspase activity blocked magnolol-induced apoptosis, supporting the involvement of the caspases and PARP. In addition, magnolol activated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. These data suggest that magnolol promoted apoptosis probably by alleviating the inhibitory effect of Akt on caspase 9. Furthermore, inhibition of PARP activity, but not of caspase activity, completely prevented magnolol-induced necrosis, suggesting the notion that it might be caused by depletion of intracellular ATP levels due to PARP activation. These results show that magnolol initiates apoptosis via the cytochrome-c/caspase 3/PARP/AIF and PTEN/Akt/caspase 9/PARP pathways and necrosis via PARP activation.     

Post-translational modification of Bid has differential effects on its susceptibility to cleavage by caspase 8 or caspase 3

Bid is instrumental in death receptor-mediated apoptosis where it is cleaved by caspase 8 at aspartate 60 and aspartate 75 to generate truncated Bid (tBID) forms that facilitate release of mitochondrial cytochrome c. Bid is also cleaved at these sites by caspase 3 that is activated downstream of cytochrome c release after diverse apoptotic stimuli. In this context, tBid may amplify the apoptotic process. Bid is phosphorylated in vitro by casein kinases that regulate its cleavage by caspase 8 (Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L. Antonsson, A., and Martinou, J.-C. (2001) Mol. Cell 8, 601-611). Using a Bid decapeptide substrate, we observed that phosphorylation at threonine 59 inhibited cleavage by caspase 8. This was also seen when recombinant Bid (rBid) and Bid isolated from murine kidney were incubated with casein kinase II. However, there were differences in the susceptibility of rBid and isolated Bid to cleavage by caspases 3 and 8. Caspase 8 cleaved rBid to generate two C-terminal products, p15 and p13 tBid, but produced only p15 tBid from isolated Bid. Contrary to rBid, isolated Bid was resistant to cleavage by caspase 3, yet was readily cleaved within the cytosolic milieu. Our data suggest that one or more distinct cellular mechanisms regulate Bid cleavage by caspases 8 and 3 in situ.     

Unspecific activation of caspases during the induction of apoptosis by didemnin B in human cell lines

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.     

Apoptotic DNA fragmentation

Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death. It occurs in response to various apoptotic stimuli in a wide variety of cell types. Molecular characterization of this process identified a specific DNase (CAD, caspase-activated DNase) that cleaves chromosomal DNA in a caspase-dependent manner. CAD is synthesized with the help of ICAD (inhibitor of CAD), which works as a specific chaperone for CAD and is found complexed with ICAD in proliferating cells. When cells are induced to undergo apoptosis, caspases-in particular caspase 3-cleave ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. Cells that lack ICAD or that express caspase-resistant mutant ICAD thus do not show DNA fragmentation during apoptosis, although they do exhibit some other features of apoptosis and die. In this review, the molecular mechanism of and the physiological roles played by apoptotic DNA fragmentation will be discussed.