Expression of the Autographa californica nuclear polyhedrosis virus apoptotic suppressor gene p35 in nonpermissive Spodoptera littoralis cells.

Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519-525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system.     

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.     

Investigating apoptosis: Characterization and analysis of Trichoplusia ni-caspase-1 through overexpression and RNAi mediated silencing

In both mammals and invertebrates, caspases play a critical role in apoptosis. Although Lepidopteron caspases have been widely studied in Spodoptera frugiperda cells, this is not the case for Trichoplusia ni cells, despite their widespread use for the production of recombinant protein and differences in baculovirus infectivity between the two species. We have cloned, expressed, purified and characterized Tn-caspase-1 in several situations: in its overexpression, in silencing via RNA interference (RNAi), during baculovirus infection, and in interactions with baculovirus protein p35. Overexpression can transiently increase caspase activity in T. ni (High Five?) cells, while silencing results in a greater than 6-fold decrease. The reduction in caspase activity resulted in a reduction in the level of apoptosis, demonstrating the ability to affect apoptosis by modulating Tn-caspase-1. During baculovirus infection, caspase activity remains low until approximately 5 days post infection, at which point it increases dramatically, though not in those cells treated with dsRNA. Our results demonstrate that Tn-caspase-1 is presumably the principal effector caspase present in High Five cells, and that it is inhibited by baculovirus protein p35. Finally, our results indicate differences between RNAi and p35 as effector molecules for modulating caspase activity and apoptosis during cell growth and baculovirus infection.     

Functional analysis of inhibitor of apoptosis 1 of the silkworm Bombyx mori

Recent advances in genome-wide surveys have revealed a number of lepidopteran insect homologs of mammalian and Drosophila genes that are responsible for apoptosis regulation. However, the underlying molecular mechanisms for apoptosis regulation in lepidopteran insect cells remain poorly understood. In the present study, we demonstrated that the transfection of Bombyx mori BM-N cells with dsRNA against the B. mori cellular iap1 gene (cbm-iap1) induces severe apoptosis that is accompanied by an increase of caspase-3-like protease activity. In these apoptotic cells, the cleaved form of the endogenous initiator caspase Dronc (Bm-Dronc) was detected, indicating that cBm-IAP1 protein depletion by RNAi silencing resulted in the activation of Bm-Dronc. In transient expression assays in BM-N cells, cBm-IAP1 suppressed the apoptosis triggered by Bm-Dronc overexpression and depressed the elevation of caspase-3-like protease activity, but also increased the cleaved form of Bm-Dronc protein. cBm-IAP1 also suppressed the caspase-3-like protease activity stimulated by Bm-caspase-1 overexpression. Co-immunoprecipitation experiments demonstrated that cBm-IAP1 strongly interacts with Bm-Dronc, but only has weak affinity for Bm-caspase-1. Transient expression analyses showed that truncated cBm-IAP1 proteins defective in the BIR1, BIR2 or RING domain were unable to suppress Bm-Dronc-induced apoptosis. In addition, BM-N cells expressing truncated cBm-IAP1 proteins underwent apoptosis, suggesting that intact cBm-IAP1, which has anti-apoptotic activity, was replaced or displaced by the overexpressed truncated cBm-IAP1 proteins, which are incapable of interfering with the apoptotic caspase cascade. Taken together, the present results demonstrate that cBm-IAP1 is a vital negative regulator of apoptosis in BM-N cells and functions by preventing the activation and/or activity of Bm-Dronc and Bm-caspase-1.     

A novel anti-proliferative role of HMGA2?in induction of apoptosis through caspase 2?in primary human fibroblast cells

The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that γH2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.     

Superinfection-induced apoptosis and its correlation with the reduction of viral progeny in cells persistently infected with Hz-1 baculovirus.

Differential induction of necrosis or apoptosis was found upon challenge of cells of the insect Spodoptera frugiperda productively or persistently infected with Hz-1 baculovirus, respectively. Unlike parental SF9 cells, which were essentially all killed by virally induced necrosis, persistently infected cells underwent a process of massive cell death by apoptosis; cells which were not killed by apoptosis then reestablished a cell monolayer. Upon viral challenge, the yield of viral progeny was reduced greatly in persistently virus-infected cells but not in parental cells. Immunolabelling of individual cells revealed that upon viral challenge, production of viral progeny was detectable only in necrotic cells and not in apoptotic cells. These results indicated that induction of apoptosis greatly reduces the yield of viral progeny in cells persistently infected with Hz-1 baculovirus. This is the first report of apoptosis induction in persistently infected cells upon viral superinfection.     

Sex differences in the level of Bcl‐2 family proteins and caspase‐3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal rats

In developing rats, sex differences in the number of apoptotic cells are found in the central division of the medial preoptic nucleus (MPNc), which is a significant component of the sexually dimorphic nucleus of the preoptic area, and in the anteroventral periventricular nucleus (AVPV). Specifically, male rats have more apoptotic cells in the developing AVPV, whereas females have more apoptotic cells in the developing MPNc. To determine the mechanisms for the sex differences in apoptosis in these nuclei, we compared the expression of the Bcl‐2 family members and active caspase‐3 in postnatal female and male rats. Western blot analyses for the Bcl‐2 family proteins were performed using preoptic tissues isolated from the brain on postnatal day (PD) 1 (day of birth) or on PD8. In the AVPV‐containing tissues of PD1 rats, there were significant sex differences in the level of Bcl‐2 (female > male) and Bax (female < male) proteins, but not of Bcl‐xL or Bad proteins. In the MPNc‐containing tissues of PD8 rats, there were significant sex differences in the protein levels for Bcl‐2 (female < male), Bax (female > male), and Bad (female < male), but not for Bcl‐xL. Immunohistochemical analyses showed significant sex differences in the number of active caspase‐3‐immunoreactive cells in the AVPV on PD1 (female < male) and in the MPNc on PD8 (female > male). We further found that active caspase‐3‐immunoreactive cells of the AVPV and MPNc were immunoreactive for NeuN, a neuronal marker. These results suggest that there are sex differences in the induction of apoptosis via the mitochondrial pathway during development of the AVPV and MPNc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Active depletion of host cell inhibitor-of-apoptosis proteins triggers apoptosis upon baculovirus DNA replication

Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects.     

Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

喜树碱诱导的草地贪夜蛾Sf9细胞凋亡

传统植物源杀虫剂喜树碱具有优异的抑制昆虫生长发育活性, 其诱导昆虫细胞凋亡的作用方式和机制尚不明确, 极大地限制了喜树碱在植物保护领域的应用开发。本研究以1 μmol/L喜树碱诱导草地贪夜蛾Spodoptera frugiperda Sf9细胞呈现细胞皱缩、微绒毛消失和染色质边集等典型细胞凋亡早期超微结构形态特征, 中期凋亡小体逐渐出现并急剧增多, DNA电泳分析可见清晰DNA片段化凋亡特征。流式细胞术分析表明1 μmol/L喜树碱诱导Sf9细胞12 h凋亡率达到最大值39.67%, 是对照的13.13倍, 随后减小。喜树碱诱导Sf9细胞凋亡在12 h和24 h 时Sf caspase-1分别出现两个活性高峰, 表明其作为效应因子在细胞凋亡级联反应过程中具有影响作用。喜树碱显著抑制Sf9细胞拓扑异构酶Ⅰ活性, 阻断解旋负超螺旋pBR322 DNA, 导致DNA损伤进而启动细胞凋亡级联反应使Sf caspase-1活性增加, 提示其信号转导过程是细胞凋亡诱导机制之一。本研究通过分析喜树碱的诱导昆虫Sf9细胞凋亡, 对揭示喜树碱诱导昆虫细胞凋亡的作用机制具有重要启示和帮助。     

Characterization of the apoptosis suppressor protein P49 from the Spodoptera littoralis nucleopolyhedrovirus

Two antiapoptotic types of genes, iap and p35, were found in baculoviruses. P35 is a 35-kDa protein that can suppress apoptosis induced by virus infection or by diverse stimuli in vertebrates or invertebrates. iap homologues were identified in insects and mammals. Recently, we have identified sl-p49, a novel apoptosis suppressor gene and the first homologue of p35, in the genome of the Spodoptera littoralis nucleopolyhedrovirus. Here we show that sl-p49 encodes a 49-kDa protein, confirmed its primary structure that displays 48.8% identity to P35, and performed computer-assisted modeling of P49 based on the structure of P35. We demonstrated that P49 is able to inhibit insect and human effector caspases, which requires P49 cleavage at Asp(94). Finally we identified domains important for P49's antiapoptotic function that include a reactive site loop (RSL) protruding from a beta-barrel domain. RSL begins at an amphipathic alpha1 helix, traverses the beta-sheet central region, exposing Asp(94) at the apex, and rejoins the beta-barrel. Our model predicted seven alpha-helical motifs, three of them unique to P49. alpha-Helical motifs alpha(1), alpha(2), and alpha(4') were required for P49 function. The high structural homology between P49 and P35 suggests that these molecules bear a scaffold common to baculovirus "apoptotic suppressor" proteins. P49 may serve as a novel tool to analyze the contribution of different components of the caspase chain in the apoptotic response in organisms not related phylogenetically.     

Caspase‐3‐mediated cyclic stretch‐induced myoblast apoptosis via a Fas/FasL‐independent signaling pathway during myogenesis

Skeletal muscle cells are exposed to mechanical stretch during embryogenesis. Increased stretch may contribute to cell death, and the molecular regulation by stretch remains incompletely understood. The aim of this study was to investigate the effects of cyclic stretch on cell death and apoptosis in myoblast using a Flexercell Strain Unit. Apoptosis was studied by annexin V binding and PI staining, DNA size analysis, electron microphotograph, and caspase assays. Fas/FasL expression was determined by Western blot. When myoblasts were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐dependent manner. We also determined that stretch induced cleavage of caspase‐3 and increased caspase‐3 activity. Caspase‐3 inhibition reduced stretch‐induced apoptosis. Protein levels of Fas and FasL remained unchanged. Our findings implicated that stretch‐induced cell death is an apoptotic event, and that the activation of caspase cascades is required in stretch‐induced cell apoptosis. Furthermore, we had provided evidence that caspase‐3 mediated cyclic stretch‐induced myoblast apoptosis. Mechanical forces induced activation of caspase‐3 via signaling pathways independent of Fas/FasL system. J. Cell. Biochem. 107: 834–844, 2009. © 2009 Wiley‐Liss, Inc.     

Role of caspase 9 in activation of HTLV-1 LTR expression by DNA damaging agents

Adult T-cell leukemia (ATL) is caused by HTLV-I. The viral Tax oncoprotein plays a central role in initiating the process to ATL. However, after infection HTLV-1 enters into latency, during which virus gene expression is very low, so that the level of Tax is likely insufficient for exerting its oncogenic activities. Therefore only 5% of the infected individuals may develop ATL several decades after infection. It is assumed that the transition from latency to ATL development requires at least a temporary activation of the latent virus in order to elevate Tax to its oncogenic threshold. We have previously found that DNA damaging agents, which usually induce apoptosis, can also activate the viral LTR and that the anti-apoptosis Bcl-2 protein not only avoid their apoptosis induction but concomitantly prevents their LTR activation effect. Therefore, the present study was designed to identify the factor that while participating in the apoptotic cascade acts also to activate the viral LTR. For this purpose we employed ectopic vectors expressing these apoptotic factors together with potent shRNAs against each of them and anti caspase peptide inhibitors. We have found that in addition to its function as initiator of the mitochondrial apoptotic cascade, caspase 9 can acts also as an executer which among other non-apoptotic functions it forms an Sp1-p53 complex that activates the LTR by binding to an Sp1 recognition site residing in the LTR. This finding can help in designing effective preventing strategies against ATL development in clinically latent HTLV-1 carriers.     

Caspase inhibition in apoptotic T cells triggers necrotic cell death depending on the cell type and the proapoptotic stimulus

CD95 (Fas/Apo-1) triggers apoptotic cell death via a caspase-dependent pathway. Inhibition of caspase activation blocks proapoptotic signaling and thus, prevents execution of apoptosis. Besides induction of apoptotic cell death, CD95 has been reported to trigger necrotic cell death in susceptible cells. In this study, we investigated the interplay between apoptotic and necrotic cell death signaling in T cells. Using the agonistic CD95 antibody, 7C11, we found that caspase inhibition mediated by the pancaspase inhibitor, zVAD-fmk, prevented CD95-triggered cell death in Jurkat T cells but not in A3.01 T cells, although typical hallmarks of apoptosis, such as DNA fragmentation or caspase activation were blocked. Moreover, the caspase-independent cell death in A3.01 cells exhibited typical signs of necrosis as detected by a rapid loss of cell membrane integrity and could be prevented by treatment with the radical scavenger butylated hydroxyanisole (BHA). Similar to CD95-induced cell death, apoptosis triggered by the DNA topoisomerase inhibitors, camptothecin or etoposide was shifted to necrosis when capsase activation was inhibited. In contrast to this, ZVAD was fully protective when apoptosis was triggered by the serpase inhibitor, Nalpha-tosyl-phenyl-chloromethyl ketone (TPCK). TPCK was not protective when administered to anti-CD95/ZVAD-treated A3.01 cells, indicating that TPCK does not possess anti-necrotic activity but fails to activate the necrotic death pathway. Our findings show (a) that caspase inhibition does not always protect apoptotic T cells from dying but merely activates a caspase-independent mode of cell death that results in necrosis and (b) that the caspase-inhibitor-induced shift from apoptotic to necrotic cell death is dependent on the cell type and the proapoptotic stimulus.