Inhibition of virus-induced neuronal apoptosis by Bax.

The Bax protein is widely known as a pro-apoptotic Bcl-2 family member that when overexpressed can trigger apoptosis in multiple cell types and is important for the developmental cell death of neurons. However, Bax was found here to be a potent inhibitor of neuronal cell death in mice infected with Sindbis virus. Newborn mice, which are highly susceptible to a fatal infection with neurotropic Sindbis virus, were significantly protected from neuronal apoptosis and fatal disease when infected with a recombinant Sindbis virus encoding Bax. Deletion of the N terminus of Bax, which mimics cleaved Bax, converted Bax into a pro-apoptotic factor in vivo. As mice mature during the first week after birth, they acquire resistance to a fatal Sindbis virus infection. However, Bax-deficient mice remained very sensitive to fatal disease compared with their control littermates, indicating that endogenous Bax functions as a survival factor and contributes to age-dependent resistance to Sindbis virus-induced mortality. The protective effects of Bax were reproduced in cultured hippocampal neurons but not in cultured dorsal root ganglia neurons. These findings indicate that cell-specific factors determine the anti-apoptotic versus pro-apoptotic function of Bax.

Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription.

Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

Mitochondrial fission and fusion dynamics: the long and short of it.

Maintenance of functional mitochondria requires fusion and fission of these dynamic organelles. The proteins that regulate mitochondrial dynamics are now associated with a broad range of cellular functions. Mitochondrial fission and fusion are often viewed as a finely tuned balance within cells, yet an integrated and quantitative understanding of how these processes interact with each other and with other mitochondrial and cellular processes is not well formulated. Direct visual observation of mitochondrial fission and fusion events, together with computational approaches promise to provide new insight.

Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport.

Protein sequence requirements for cleavage of the signal peptide from the Rous sarcoma virus glycoprotein have been investigated through the use of deletion mutagenesis. The phenotypes of these mutants have been characterized by expression of the cloned, mutated env genes in CV-1 cells using a late replacement SV40 vector. The deletion mutations were generated by Ba131 digestion at the XhoI site located near the 5' end of the coding sequence for the structural protein gp85, which is found at the amino terminus of the precursor glycoprotein, Pr95. The results of experiments with three mutants (X1, X2, and X3) are presented. Mutant X1 has a 14 amino acid deletion encompassing amino acids 4-17 of gp85, which results in the loss of one potential glycosylation site. In mutants X2 and X3 the amino terminal nine and six amino acids, respectively, of gp85 are deleted. During the biosynthesis of all three mutant polypeptides, the signal peptide is efficiently and accurately cleaved from the nascent protein, even though in mutants X2 and X3 the cleavage site itself has been altered. In these mutants the alanine/aspartic acid cleavage site has been mutated to alanine/asparagine and alanine/glutamine, respectively. These results are consistent with the concept that sequences C-terminal to the signal peptidase site are unimportant in defining the site of cleavage in eucaryotes. Mutants X2 and X3 behave like wild-type with respect to protein glycosylation, palmitic acid addition, cleavage to gp85 and gp37, and expression on the cell surface. Mutant X1, on the other hand, is defective in intracellular transport. Although it is translocated across the rough endoplasmic reticulum and core-glycosylated, its transport appears to be blocked at an early Golgi compartment. No terminal glycosylation of the protein, cleavage of the precursor protein to the mature products, or expression on the cell surface is observed. The deletion in X1 thus appears to destroy signals required for export to the cell surface.

Suppression of steady-state, but not stimulus-induced NF-kappaB activity inhibits alphavirus-induced apoptosis.

Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus.

Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B.

Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SV-induced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 microM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Over-expression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SV-induced NF-kappa B activation. To determine if NF-kappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.

Antibody-mediated clearance of alphavirus infection from neurons.

Humoral immunity is important for protection against viral infection and neutralization of extracellular virus, but clearance of virus from infected tissues is thought to be mediated solely by cellular immunity. However, in a SCID mouse model of persistent alphavirus encephalomyelitis, adoptive transfer of hyperimmune serum resulted in clearance of infectious virus and viral RNA from the nervous system, whereas adoptive transfer of sensitized T lymphocytes had no effect on viral replication. Three monoclonal antibodies to two different epitopes on the E2 envelope glycoprotein mediated viral clearance. Treatment of alphavirus-infected primary cultured rat neurons with these monoclonal antibodies to E2 resulted in decreased viral protein synthesis, followed by gradual termination of mature infectious virion production. Thus, antibody can mediate clearance of alphavirus infection from neurons by restricting viral gene expression.

Conversion of Bcl-2 to a Bax-like death effector by caspases.

Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.

Mitochondrial factors with dual roles in death and survival.

At least in mammals, we have some understanding of how caspases facilitate mitochondria-mediated cell death, but the biochemical mechanisms by which other factors promote or inhibit programmed cell death are not understood. Moreover, most of these factors are only studied after treating cells with a death stimulus. A growing body of new evidence suggests that cell death regulators also have 'day jobs' in healthy cells. Even caspases, mitochondrial fission proteins and pro-death Bcl-2 family proteins appear to have normal cellular functions that promote cell survival. Here, we review some of the supporting evidence and stretch beyond the evidence to seek an understanding of the remaining questions.

Pro-B-cell-specific transcription and proapoptotic function of protein kinase Ceta.

Using a subtractive cloning scheme on cDNA prepared from primary pro-B and pre-B cells, we identified several genes whose products regulate apoptosis. We further characterized one of these genes, encoding protein kinase Ceta (PKCeta). PKCeta transcripts were readily detected in pro-B cells but were absent in pre-B cells. Although both a full-length and a truncated form of PKCeta were detectable in bone marrow pro-B cells, transition to the pre-B-cell stage was associated with increased relative levels of truncated PKCeta. We found that PKCeta is proteolyzed in apoptotic lymphocytes, generating a kinase-active fragment identical to the truncated form which is capable of inducing apoptosis when expressed in a pro-B cell line. Caspase-3 can generate an identical PKCeta cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKCeta results in cell cycle arrest at the G(1)/S transition. These results suggest that the expression and proteolytic activation of PKCeta play an important role in the regulation of cell division and cell death during early B-cell development.

Perspective: virus infections and the death of neurons.

Neuronotropic viruses induce apoptosis in neurons, and Bcl-2-related anti-apoptotic proteins and caspase inhibitors decrease mortality from acute viral encephalitis. Infected neurons develop cytoplasmic blebbing characteristic of apoptosis, but a paucity of apoptotic nuclear changes potentially indicates unique aspects of virus-induced neuronal apoptosis that remain to be discovered.

Persistence of alphaviruses in vertebrate hosts.

Alphaviruses are a group of arthropod-borne, positive-strand RNA viruses that cause acute encephalitis or arthritis. These viruses were previously thought to cause only acute infections in vertebrates, but recent evidence suggests that host immunological and tissue-specific factors may act together to promote the persistence of alphavirus genomes in vivo.

Viral versus cellular BCL-2 proteins.

All gamma herpesviruses and a few other viruses encode at least one homologue of the mammalian cell death inhibitor BCL-2. Gamma herpesviruses are associated with human and animal lymphoid and epithelial tumours. However, the role of these viral BCL-2 homologues in the virus replication cycle or in human disease is not known, though recent developments show progress in this area. The structure of viral BCL-2 family protein, KSBcl-2, is similar to that of cellular family members, but viral BCL-2 proteins differ functionally from the cellular proteins, apparently escaping the regulatory mechanisms to which their cellular counterparts are subjected. Thus, exploring the biochemical and biological functions of the viral BCL-2 family proteins will increase our understanding of their role in virus infections and will undoubtedly teach us something about their cellular kin.

Anti-apoptotic genes of baculoviruses.

Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Age-dependent susceptibility to fatal encephalitis: alphavirus infection of neurons.

Sindbis virus encephalitis in mice provides a model for studying age-dependent susceptibility to acute viral encephalitis. The AR339 strain of SV causes fatal encephalitis in newborn mice, but weanling mice recover uneventfully. Increased virulence for older mice is associated with a single amino acid change from Gln to His at position 55 of the E2 glycoprotein. Weanling mice with normal immune systems clear infectious virus from neurons through an antibody-mediated mechanism. This does not happen in newborn mice because the infected neurons die soon after they are infected. Death in immature neurons, as well as most other mammalian cells infected with Sindbis virus, occurs by induction of apoptosis. This can be prevented by cellular expression of bcl-2, an inhibitor of apoptosis, which is expressed by mature neurons in culture. We conclude that mature neurons are resistant to induction of apoptosis after infection with SV through expression of cellular inhibitors of apoptosis. This provides the opportunity for antibody to clear virus by a noncytolytic mechanism.

Regulation of apoptosis by viruses that infect insects.

Orthobunyviruses and alphaviruses cause encephalitis, neuronal apoptosis and mortality in mammals, but fail to kill the mosquitoes that transmit these viruses. Therefore, host cell factors, as well as viral factors, regulate the outcome of infection. Drosophila Reaper is a pro-death factor in the Drosophila nervous system during development but homologues were not previously known to exist outside flies. Recent discovery of a Reaper protein encoded by orthobunyaviruses provides interesting insight into the mechanisms by which viruses modulate the death pathway in both vertebrate and invertebrate hosts.

Role of bilateral adrenalectomy (and oophorectomy) in the management of patients with metastatic breast cancer.

One hundred consecutive patients who underwent bilateral adrenalectomy for metastatic breast cancer with at least a 2 year "tumor-free" interval and with symptomatic bony, local-regional, pleural, or discrete pulmonary or mediastinal metastases had objective remission rates of 65 per cent at 6 months, 48 per cent at 12 months, 28 per cent at 18 months, and 16 per cent at 24 months.

Genome evolution in yeast reveals connections between rare mutations in human cancers.

Cancer cells are riddled with mutations. Less than one percent of these are thought to be mutations that drive cancer phenotypes. However, a recent study conducted on the yeast knockout collections by Teng et al. [Mol. Cell (2013) 52: 485-494] provides hard evidence that single gene deletions/mutations in most non-essential genes can drive the selection for cancer-like mutations.