An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1.

An open reading frame, LMW5-HL, in the African swine fever virus genome displays a high degree of similarity to the proto-oncogene bcl-2 and, to a lesser degree, the Epstein-Barr virus gene BHRF1. A highly conserved central region is found in all three proteins. LMW5-HL encodes a protein of 18 kDa that is present in infected porcine macrophages at both early and late times postinfection. The similarity of LMW5-HL to bcl-2 and BHRF1 suggests a role for it in cell maintenance during productive or persistent viral infection.     

HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL,induces a caspase-independent autophagic cell death

The Drosophila spinster (spin) gene product is required for programmed cell death in the nervous and reproductive systems. We have identified a human homologue of the Drosophila spin gene product (HSpin1). HSpin1 bound to Bcl-2 and apoptosis regulator Bcl-X (Bcl-xL), but not to proapoptotic members such as Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, in cells treated with TNF-alpha. Exogenous expression of HSpin1 resulted in the cell death without inducing a release of cytochrome c from mitochondria. Overexpression of Bcl-xL inhibited the HSpin1-induced cell death. Interestingly, a necrosis inhibitor, pyrrolidine dithiocarbomate, but not the pancaspase inhibitors, carbobenzoxy-VAD-fluoromethyl ketone and p35, blocked the HSpin1-induced cell death. HSpin1-induced cell death increases autophagic vacuole and mature form of cathepsin D, suggesting a novel caspase-independent cell death, which is link to autophagy.     

The cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death

The matrix (M) protein of vesicular stomatitis virus (VSV) expressed in the absence of other viral components causes many of the cytopathic effects of VSV, including an inhibition of host gene expression and the induction of cell rounding. It was recently shown that M protein also induces apoptosis in the absence of other viral components. This raises the possibility that the activation of apoptotic pathways causes the inhibition of host gene expression and cell rounding by M protein. To test this hypothesis, host gene expression and cell rounding were analyzed after the transfection of M mRNA into HeLa cells stably overexpressing Bcl-2 (HeLa-Bcl-2 cells). We have shown previously that Bcl-2 inhibits M-protein-induced apoptosis. Here, we show that activation of the apoptotic pathways downstream of Bcl-2 is not required for the inhibition of host gene expression by M protein. In contrast, overexpression of Bcl-2 inhibited cell rounding induced by M protein, indicating that apoptotic pathways downstream of Bcl-2 are required for the cell-rounding activities of M protein.     

BCL-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy

The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases. © 1996 Wiley-Liss, Inc.     

harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L).

Programmed cell death is essential in organ development and tissue homeostasis and its deregulation is associated with the development of several diseases in mice and humans. The precise mechanisms that control cell death have not been elucidated fully, but it is well established that this form of cellular demise is regulated by a genetic program which is activated in the dying cell. Here we report the identification, cloning and characterization of harakiri, a novel gene that regulates apoptosis. The product of harakiri, Hrk, physically interacts with the death-repressor proteins Bcl-2 and Bcl-X(L), but not with death-promoting homologs, Bax or Bak. Hrk lacks conserved BH1 and BH2 regions and significant homology to Bcl-2 family members or any other protein, except for a stretch of eight amino acids that exhibits high homology with BH3 regions. Expression of Hrk induces cell death which is inhibited by Bcl-2 and Bcl-X(L). Deletion of 16 amino acids including the conserved BH3 region abolished the ability of Hrk to interact with Bcl-2 and Bcl-X(L) in mammalian cells. Moreover, the killing activity of this mutant form of Hrk (Hrk deltaBH3) was eliminated or dramatically reduced, suggesting that Hrk activates cell death at least in part by interacting with and inhibiting the protection afforded by Bcl-2 and Bcl-X(L). Because Hrk lacks conserved BH1 and BH2 domains that define Bcl-2 family members, we propose that Hrk and Bik/Nbk, another BH3-containing protein that activates apoptosis, represent a novel class of proteins that regulate apoptosis by interacting selectively with survival-promoting Bcl-2 and Bcl-X(L).     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

Anti-Bcl-2 family members,zfBcl-x<Subscript>L</Subscript> and zfMcl-1a,prevent cytochrome <Emphasis Type="Italic">c</Emphasis> release from cells undergoing betanodavirus-induced secondary necrotic cell death

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x_L and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members. J.-L. Wu and J.-R. Hong contributed equally to the research.     

Buffy,a Drosophila Bcl-2 protein,has anti-apoptotic and cell cycle inhibitory functions

Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila: Here we report that Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Rpr, Grim and Hid, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th(5)) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G(1)/early-S phase arrest. Our data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins.     

Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1

The molecular pathways responsible for apoptosis in response to granzyme B have remained unresolved. Here we present data supporting the notion that granzyme B-mediated cell death is largely dependent on a pathway that is inhibitable by Bcl-2 or its viral analog BHRF1. We used a panel of stably transfected FDC-P1 mouse myeloid cell lines to show that overexpression of functional, wild-type Bcl-2 or BHRF1 rescued cells from granzyme B-mediated apoptosis, whereas mutated (Gly145-->Glu) Bcl-2, or wild-type Bcl-2 directed to the plasma membrane conferred no protection. Overexpression of Bcl-2 resulted in inhibition of multiple parameters of apoptosis in response to purified perforin and granzyme B, including DNA fragmentation, changes in light scatter profile indicating cell shrinkage and increased refractivity, loss of mitochondrial membrane potential and inhibited colony formation in clonogenic assays. Nevertheless, when exposed to cytotoxic lymphocytes, FDC-P1 and YAC-1 cells overexpressing Bcl-2 remained susceptible to death imparted by cytolytic granules, irrespective of whether the granules contained granzyme B. Thus, alternative granzyme B-independent pathways can be activated by intact lymphocytes to overcome Bcl-2-like inhibitors of apoptosis, enabling CTLs to overcome potential viral blocks to granzyme B-mediated cell death.     

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Whether viral pathogens that induce ER stress responses benefit the host or the virus remains controversial. In this study we show that betanodavirus induced ER stress responses up-regulate GRP78, which regulates the viral replication and host cellular mitochondrial-mediated cell death. Betanodavirus (redspotted grouper nervous necrosis virus, RGNNV) infection resulted in the following increased ER stress responses in fish GF-1 grouper fin cells: (1) IRE-1 and ATF-6 sensors at 48 h post-infection (p.i.) that up-regulated chaperone protein GRP78; (2) activation of caspase-12; and (3) PERK phosphorylation and down-regulation of Bcl-2. Analyses of GRP78 functions during viral replication using either loss-of-function or gain-of-function approaches showed that GRP78 over-expression also enhanced viral replication and induced cell death. Then, we found that zfGRP78 localization gradually increased in mitochondria after RGNNV infection by EGFP tagging approach. Furthermore, zfGRP78 can interact with viral RNA-dependent RNA polymerase (RdRp) by using immunofluorescent and immunoprecipitation assays. Finally, we found that blocking GRP78-mediated ER signals can reduce the viral death factors protein α and protein B2 expression and decrease the Bcl-2 down-regulation mediated mitochondria-dependent cell death, which also enhances host cellular viability. Taken together, our results suggest that RGNNV infection and expression can trigger ER stress responses, which up-regulate the chaperone GRP78 at early replication stage. Then, GRP78 can interact with RdRp that may enhance the viral replication for increasing viral death factors’ expressions at middle-late replication stage, which can enhance mitochondrial-mediated cell death pathway and viral spreading. These results may provide new insights into the mechanism of ER stress-mediated cell death in RNA viruses.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

Toll-like receptor 3 (TLR3) induces apoptosis via death receptors and mitochondria by up-regulating the transactivating p63 isoform alpha (TAP63alpha)

Toll-like receptor 3 (TLR3), a member of the pathogen recognition receptors, is widely expressed in various cells and has been shown to activate immune signaling pathways by recognizing viral double-stranded RNA. Recently, it was reported that the activation of TLR3 induced apoptosis in some cells, but the detailed molecular mechanism is not fully understood. In this study, we found that in endothelial cells polyinosinic-polycytidylic acid (poly(I-C)) induced dose- and time-dependent cell apoptosis, which was elicited by TLR3 activation, as TLR3 neutralization and down-regulation repressed the apoptosis. Poly(I-C) induced the activation of both caspases 8 and 9, indicating that TLR3 triggered the signaling of both the extrinsic and intrinsic apoptotic pathways. Poly(I-C) up-regulated tumor necrosis factor-related apoptosis-inducing ligand and its receptors, death receptors 4/5, resulting in initiating the extrinsic pathway. Furthermore, poly(I-C) down-regulated anti-apoptotic protein, B cell lymphoma 2 (Bcl-2), and up-regulated Noxa, a key Bcl-2 homology 3-only antagonist of Bcl-2, leading to the priming of the intrinsic pathway. A p53-related protein, the transactivating p63 isoform α (TAp63α), was induced by TLR3 activation and contributed to the activation of both the intrinsic and extrinsic apoptotic pathways. Both the cells deficient in p63 gene expression by RNA interference and cells that overexpressed the N-terminally truncated p63 isoform α (ΔNp63α), a dominant-negative variant of TAp63α, by gene transfection, survived TLR3 activation. Taken together, TAp63α is a crucial regulator downstream of TLR3 to induce cell death via death receptors and mitochondria.     

Antiapoptotic oncogene bcl-2 induces a program of senescence in E1A + c-Ha-ras-transformants treated with adriamycin

Introduction of bcl-2 gene in EIA + c-Ha-ras-transformed rat embryo fibroblasts, which are unable to be arrested after damaging influences and possess high proapoptotic sensitivity, results not only in suppression of cell death but also in re-establishment of cell cycle block following DNA damage and serum starvation. Flow cytometry showed that E1A + c-Ha-ras + bcl-2-transformants treated with DNA-intercalator adriamycin are capable of being arrested at G1/S boundary for a long time (for less than 5 days). According to the growth curve data, the number of Bcl-2-overexpressing cells remanins constant for a week of cultivation with adriamycin. Clonogenic efficacy of E1A + c-Ha-ras + bcl-2-cells is brought to no already in 16 h after adriamycin addition. Apoptotic death, revealed by oligonucleosomic fragmentation of DNA, as well as cell death, occurring due to mitotic catastrophe, after adriamycin treatment are almost absent in Bcl-2-overexpressing transformants, as compared with parental E1A + c-Ha-ras-transformants. Bcl-2 introduction in E1A + c-Ha-ras-transformants is accompanied by a rise of SA beta-Gal (Senescence Associated beta-Galactosidase) activity, which is commonly considered to be a marker of cell senescence. Adriamycin treatment of E1A + c-Ha-ras + bcl-2-transformants results in a much higher rise in SA beta-Gal activity, as compared with untreated cells. Co-immunoprecipitation experiments demonstrated the introduction of Bcl-2 to result in formation of Bcl-2 complexes with early region E1A oncoproducts, which are thought to be responsible for proapoptotic susceptibility of E1A-expressing transformants. The data obtained lead to suggestion that bcl-2 transfer to E1A + c-Ha-ras-transformants may induce a switch from the cell death program on the program of senescence after DNA damage, due, presumably, to Bcl-2 interaction with the apoptosis activator the viral oncoprotein E1A.     

Viral Bcl-2 homologs and their role in virus replication and associated diseases

Cellular Bcl-2 family proteins regulate a critical step in the mammalian programmed cell death pathway by modulating mitochondrial permeability and function. Bcl-2 family proteins are also encoded by several large DNA viruses, including all known gamma herpesviruses, adenoviruses, and several other unrelated viruses. Viral Bcl-2 proteins can prevent cell death but often escape cellular regulatory mechanisms that govern their cellular counterparts. By evading the "altruistic" suicide of infected cells, viruses can ensure replication and propagation in the infected host, but sometimes in surprising ways. Many human cancers and other disorders are associated with viruses that encode Bcl-2 homologs. Here we consider the available mechanistic data for viral compared to cellular Bcl-2 protein function along with relevance to the virus life cycle and human disease states.     

A suppressive effect of the adenovirus 5 protein E1B 55K on apoptosis induced by IL-3 deprivation and gamma-irradiation

The murine IL-3-dependent myeloid cell line 32D undergoes a rapid death when deprived of interleukin-3 (IL-3), a process that is suppressed or delayed by the constitutive expression of Bcl-2 or the Bcl-2-related Bcl-xL survival protein. The adenovirus type 5 E1B region encodes an E1B 55K protein, that has been reported to bind and inactivate the p53 protein that plays an important role in the induction of apoptosis. In order to explore the potential effect of the E1B 55K protein on IL-3 deprival-induced cell death, we have established 32D cell lines overexpressing the adenovirus E1B 55K protein and compared its ability to modulate the cell death with that of the human Bcl-2 protein. We observed that E1B 55K, as Bcl-2, delays the cell death caused by either IL-3-deprivation or DNA damage induced by gamma-irradiation. Cell-cycle analysis after IL-3 deprivation indicated that surviving Bcl-2 transfectants accumulate predominantly in the G0/G1 phase of the cell cycle, while E1B 55K transfectants survive in both G0/G1 and the S and G2/M phases of the cell cycle. zVAD-fmk, a broad caspase inhibitor, prevented chromatin condensation and fragmentation, but not cell death, suggesting that IL-3 deprivation induces a cell death program in which the caspases are dispensable. In contrast, both E1B 55K and Bcl-2 allowed cell survival and prevented the typical features of programmed cell death, such as phosphatidyl-serine exposure, loss of mitochondrial membrane potential, and chromatin condensation and fragmentation. Our findings indicate that the adenovirus 5 E1B 55K protein has the capability to act as a survival factor, and suggest that E1B 55K exerts its effect upstream of the activation of effector caspases, by preventing the loss of mitochondrial membrane potential induced by IL-3 deprivation.     

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

A Bcl-2 related family member, Bad, promotes cell death, and its function is regulated by phosphorylation. In this study, we show how the IPNV elicits the induction of Bad gene expression and promotes host apoptotic death. Anti-IPNV-E1S polyclonal and anti-VP3 monoclonal antibodies are used to neutralize the virus that blocks the prime death signal via the virus receptor. In the viability assay, each antibody could also enhance cell viability during IPNV infection. We tested tyrosine kinase inhibitors on IPNV-infected cells in order to assess their effect on blocking the death signal. With 100 microg/ml genistein treatment, Bad-like gene expression was blocked, either by rescuing the IPNV-infected CHSE-214 cells or by blocking internucleosomal DNA cleavage; but the tyrphostin group did not block Bad expression. For CHSE-214 cells, treatment with the protein synthesis-inhibitor, cycloheximide (1microg/ml), blocked new protein synthesis via activated tyrosine kinase during IPNV infection. We found that Bad protein expression could be blocked, and apoptotic death prevented. Together, these results demonstrate that the IPNV exerts up-regulation of a pro-apoptotic death gene (Bad), the expression of which serves to trigger apoptotic cell death. Our data also suggests that the IPNV induces apoptotic death via a viral receptor which triggers death effector Bad gene expression, possibly through a tyrosine kinase-dependent pathway.     

Exposure to the viral by-product dsRNA or Coxsackievirus B5 triggers pancreatic beta cell apoptosis via a Bim / Mcl-1 imbalance

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.