ARC, an apoptosis suppressor limited to terminally differentiated cells, is induced in human breast cancer and confers chemo- and radiation-resistance.
Explore the source record for details and available documents.
Apoptosis Regulatory Proteins↗
Biomedical subjects
R C Armstrong
Publications and source records attributed to R C Armstrong.
Cardiomyocyte apoptosis induced by Galphaq signaling is mediated by permeability transition pore formation and activation of the mitochondrial death pathway.
Expression of the wild-type alpha subunit of Gq stimulates phospholipase C and induces hypertrophy in cardiomyocytes. Addition of Gq-coupled receptor agonists additionally activates phospholipase C, as does expression of a constitutively active mutant form of Galphaq. Under these conditions, hypertrophy is rapidly succeeded by apoptotic cellular and molecular changes, including myofilament disorganization, loss of mitochondrial membrane potential, alterations in Bcl-2 family protein levels, DNA fragmentation, increased caspase activity ( approximately 4-fold), cytochrome c redistribution, and nuclear chromatin condensation in approximately 12% of the cells. We used various interventions to define the molecular relationships between these events and identify potential sites at which these features of apoptosis could be rescued. Treatment with caspase inhibitors prevented DNA fragmentation and promoted myocyte survival; however, cytochrome c release and loss of mitochondrial membrane potential still occurred. In contrast, treatment with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore, not only prevented DNA fragmentation and reduced nuclear chromatin condensation but also preserved mitochondrial membrane potential and limited cytochrome c redistribution to only approximately 2% of cells. These data demonstrate the central role of mitochondrial membrane potential in initiation of caspase activation and downstream apoptotic events and suggest that preservation of mitochondrial integrity is crucial for prolonging the life and function of cardiomyocytes exposed to pathological levels of stress.
Adenoviridae↗
Fibroblast growth factor 2 (FGF2) and FGF receptor expression in an experimental demyelinating disease with extensive remyelination.
Fibroblast growth factor 2 (FGF2) is an excellent candidate to regulate remyelination based on its proposed actions in oligodendrocyte lineage cell development in conjunction with its involvement in CNS regeneration. To assess the potential for FGF2 to play a role in remyelination, we examined the expression pattern of FGF2 and FGF receptors (FGFRs) in an experimental demyelinating disease with extensive remyelination. Adult mice were intracranially injected with murine hepatitis virus strain A-59 (MHV-A59) to induce focally demyelinated spinal cord lesions that spontaneously remyelinate, with corresponding recovery of motor function. Using kinetic RT-PCR analysis of spinal cord RNA, we found significantly increased levels of FGF2 mRNA transcripts, which peaked during the initial stage of remyelination. Analysis of tissue sections demonstrated that increased levels of FGF2 mRNA and protein were localized within demyelinated regions of white matter, including high FGF2 expression associated with astrocytes. The expression of corresponding FGF receptors was significantly increased in lesion areas during the initial stage of remyelination. In normal and lesioned white matter, oligodendrocyte lineage cells, including progenitors and mature cells, were found to express multiple FGFR types (FGFR1, FGFR2, and/or FGFR3). In addition, in lesion areas, astrocytes expressed FGFR1, FGFR2, and FGFR3. These findings indicate that, during remyelination, FGF2 may play a role in directly regulating oligodendrocyte lineage cell responses and may also act through paracrine or autocrine effects on astrocytes, which are known to synthesize other growth factors and immunoregulatory molecules that influence oligodendrocyte lineage cells.
Animals↗
Foamy cells with oligodendroglial phenotype in childhood ataxia with diffuse central nervous system hypomyelination syndrome.
Childhood ataxia with diffuse central nervous system hypomyelination syndrome (CACH) is a recently described leukodystrophy of unknown etiology. To characterize the neuropathological features and gain insight as to the pathogenesis of this disorder, we studied cerebral tissue from six patients with the CACH syndrome. Evaluation of toluidine blue-stained, semithin sections of white matter from CACH patients disclosed unusual cells with "foamy" cytoplasm, small round nuclei and fine chromatin. Electron microscopy (EM) revealed cells in the white matter with abundant cytoplasm containing many mitochondria and loosely clustered, membranous structures, but lacking the lysosomal structures seen in macrophages. Further analysis of tissue sections with antibodies and special stains demonstrated that the abnormal cells with abundant cytoplasm labeled with oligodendroglial markers, but did not react with macrophage or astrocytic markers. Double immunolabeling with macrophage and oligodendroglial markers clearly distinguished macrophages from the "foamy" oligodendroglial cells (FODCs). Proteolipid protein (PLP) mRNA in situ hybridization demonstrated PLP mRNA transcripts in a high proportion of oligodendrocytes in CACH patients compared to control patients, and PLP mRNA transcript signal in cells, morphologically consistent with FODCs. Normal and pathological brain control tissues did not contain FODCs. These neuropathological findings will be useful pathological identifiers of CACH, and may provide clues to the pathogenesis of this disorder.
Ataxia↗
Dysembryoplastic neuroepithelial tumor: an immunohistochemical study with myelin oligodendrocyte glycoprotein.
CONTEXT: The dysembryoplastic neuroepithelial tumor (DNT) is an uncommon lesion characterized by a heterogeneous population of neurons, astrocytes, and oligodendroglia-like cells (OLCs). The basic nature of the DNT and its constituent cells, particularly the OLCs, remains unresolved; some authors favor a neuronal origin, and others propose a glial or mixed origin for these cells. DESIGN: We examined 11 DNTs with antibodies to myelin oligodendrocyte glycoprotein, a marker of mature oligodendrocytes. RESULTS: All DNTs studied (7 from males, 4 from females; age range of patients, 2-37 years) were composed of varying proportions of neurons, astrocytes, and OLCs. Membrane or cytoplasmic immunoreactivity for myelin oligodendrocyte glycoprotein was found in many OLCs in 9 of 11 cases. The number of myelin oligodendrocyte glycoprotein-positive OLCs was variable: >75% of the OLCs were positive in 5 cases, 25% to 75% of the OLCs were positive in 2 cases, and <25% of the OLCs were positive in 2 cases. CONCLUSION: These findings suggest that many of the OLCs represent mature oligodendrocytes and support the notion that DNTs are heterogenous lesions composed of multiple, mature cell types.
Adolescent↗
Activation of membrane-associated procaspase-3 is regulated by Bcl-2.
The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.
Apoptosis↗
Intracellular signals and cytoskeletal elements involved in oligodendrocyte progenitor migration.
We have examined the potential roles of intracellular Ca2+ regulation and of multiple cytoskeletal elements in control of the directed migration of cultured oligodendrocyte progenitor cells (OPs). OPs were found to migrate in response to platelet-derived growth factor (PDGF) or to a lesser extent to basic fibroblast growth factor (FGF) in a non-additive manner. This response was inhibited by chelation of intracellular Ca2+ by using BAPTA-AM. OP migration was not evoked by the neurotransmitter agonists phenylephrine or methacholine, which elevate OP Ca2+ levels. Inhibition of the MAP kinase pathway with PD 098059 did not affect OP migration to PDGF. Within growth cone-like leading edges of migratory OP processes, monomeric and filamentous actin were found to be colocalized with myosin and filamentous actin was prominent in filopodia extending beyond the leading edge. Tubulin was distributed throughout OP processes and cell bodies. Inhibition of actin or tubulin polymerization, by using cytochalasin B or nocodazole, respectively, altered OP morphology and markedly impaired migration. Inhibition of the myosin ATPase by BDM, which prevents force-generating actin/myosin interactions, greatly inhibited the chemotaxic response at concentrations that did not disrupt cell morphology. These results indicate that growth factors stimulate OP migration by activating pathways which include intracellular Ca2+ regulation, and characterize the distribution of multiple cytoskeletal elements involved in the generation of directed OP movement.
Animals↗
Peptidomimetic fluoromethylketone rescues mice from lethal endotoxic shock.
BACKGROUND: Septic shock is a leading cause of mortality in intensive care units. No new interventions in the last 20 years have made a substantial impact on the outcome of patients with septic shock. Identification of inhibitable pathways that mediate death in shock is an important goal. MATERIALS AND METHODS: Two novel caspase inhibitors, (2-indolyl)-carbonyl-Ala-Asp-fluoromethylketone (IDN 1529) and (1-methyl-3-methyl-2-indolyl)-carbonyl-Val-Asp-fluoromethylketone (IDN 1965), were studied in a murine model of endotoxic shock. RESULTS: IDN 1529 prolonged survival when given before or up to 3 hr after high-dose LPS (p < 0.01) and increased by 2.2-fold the number of animals surviving longterm after a lower dose of LPS (p < 0.01). Despite its similar chemical structure, IDN 1965 lacked these protective effects. Both compounds inhibited caspases 1, 2, 3, 6, 8, and 9, and both afforded comparable reduction in Fas- and LPS-induced caspase 3-like activity and apoptosis. Paradoxically, administration of IDN 1529 but not IDN 1965 led to an increase in the LPS-induced elevation of serum cytokines related directly (IL-1beta, IL-18) or indirectly (IL-1alpha, IL-1Ra) to the action of caspase 1. CONCLUSIONS: A process that appears to be distinct from both apoptosis and the release of inflammatory cytokines is a late-acting requirement for lethality in endotoxic shock. Inhibition of this process can rescue mice even when therapy is initiated after LPS has made the mice severely ill.
Animals↗
In vivo proliferation of oligodendrocyte progenitors expressing PDGFalphaR during early remyelination.
Endogenous oligodendrocyte lineage cells spontaneously remyelinate focal areas of demyelination induced by murine hepatitis virus A59 infection of C57Bl/6 mice. We used this model to examine the potential for platelet-derived growth factor (PDGF) to have a role in repopulating demyelinated lesions, and in doing so we also further characterized the in vivo responses of oligodendrocyte lineage cells following demyelination. Very early in the progress of remyelination, we administered a 4-h in vivo pulse of bromodeoxyuridine (BrdU) and subsequently performed in situ hybridization for PDGF-alpha receptor (PDGFalphaR), an established marker for oligodendrocyte progenitors in vivo, or for proteolipid protein (PLP), to identify oligodendrocytes. Sections of lesioned spinal cords had a 14.5-fold increase in the number of BrdU-labeled oligodendrocyte progenitor cells (PDGFalphaR+), while BrdU-labeled oligodendrocytes (PLP+) were extremely rare. Immunocytochemistry of similar sections demonstrated that immunoreactivity for both PDGFalphaR and NG2, another marker of oligodendrocyte progenitors, was locally increased in areas of white-matter lesions. High-resolution immunofluorescence imaging was used to detect oligodendrocyte progenitor cells expressing receptors for both PDGF and fibroblast growth factor. In addition, expression of PDGF-A mRNA transcripts was increased in sections of lesioned spinal cords and reactive astrocytes in lesions exhibited immunoreactivity for PDGF ligand. Our findings indicate that during the initial stages of remyelination, oligodendrocyte progenitors proliferate locally, and that this response may potentially involve PDGF.
Animals↗
Proteolytic cleavage of HsRad51 during apoptosis.
The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.
Antibodies, Monoclonal↗
A secreted DNA-binding protein that is translated through an internal ribosome entry site (IRES) and distributed in a discrete pattern in the central nervous system.
Internal initiation of translation, a mechanism infrequently used by cellular messages, avoids the requirement of a methyl cap structure for translation of messenger RNAs. The mRNA transcript encoding the DNA-binding protein MYT2 represents one of the exceptional cellular messages that contains an internal ribosome entry site (IRES). The RNA pseudoknot structure located in the 5' untranslated region of MYT2 functions to promote translation in vivo. MYT2 was cloned by its specific binding to a TTCCA motif in the promoter region of a glial-specific gene, myelin proteolipid protein. MYT2 also recognizes single-stranded nucleic acids. In the central nervous system, MYT2 protein is found in oligodendrocyte progenitor cells, subsets of neurons, and cells of the choroid plexus together with ciliated ependymal cells. MYT2 protein can also be secreted from cells, an atypical event for a DNA-binding protein. The presence of an internal ribosome entry site in MYT2, together with the unusual localization of MYT2, suggests that this nucleic acid-binding protein may be in the class of proteins involved in cellular growth control and survival in the nervous system.
Amino Acid Sequence↗
Isolation and characterization of immature oligodendrocyte lineage cells.
Using in vitro systems, the proliferation, migration, differentiation, and survival of immature oligodendrocyte lineage cells can be examined to elucidate the cellular and molecular interactions that regulate this lineage. The ability to monitor progressive stages of differentiation within the lineage by immunophenotyping and to manipulate the cellular responses with growth factors makes these cultures advantageous as both a method for studying the cell biology of myelination and as a model system for lineage analysis in the mammalian central nervous system. In addition, cultured oligodendrocytes carry out the normal in vivo sequence of expression of a set of cell type-specific genes, some of which are extremely highly expressed, and so provide advantages for analysis of gene regulation. This paper describes commonly used methods for the preparation of mixed glial cell cultures from perinatal rodent brain. Although these cultures are most commonly derived from perinatal rat brain, a protocol for preparation from mouse brain is also provided because of the increasing number of studies that use mice to facilitate molecular biological techniques. Methods to prepare secondary cultures of different stages of oligodendrocyte lineage cells are detailed. As examples of methods to use for the characterization of these cells, immunophenotypes of each stage of the oligodendrocyte lineage are illustrated, incorporation of [3H]thymidine for analysis of cell proliferation is illustrated, and detailed methods are provided for analysis of migration in a microchemotaxis chamber.
Adult↗
Human IAP-like protein regulates programmed cell death downstream of Bcl-xL and cytochrome c.
The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-xL were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-xL. In contrast, hILP, but not Bcl-xL, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-xL may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.
Apoptosis↗
Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release.
Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
Apoptosis↗
In situ expression of fibroblast growth factor receptors by oligodendrocyte progenitors and oligodendrocytes in adult mouse central nervous system.
Basic fibroblast growth factor (bFGF) induces proliferation and alters differentiation of cultured oligodendrocyte lineage cells. In situ, bFGF is present in normal adult central nervous system (CNS) and upregulated during an early stage of various pathological conditions. We examined the expression of receptors for bFGF (FGFRs) by oligodendrocyte progenitors and oligodendrocytes in situ in normal adult mouse CNS to further understand the potential in situ response to bFGF. We found FGFR immunoreactivity in oligodendrocyte progenitors, identified by expression of NG2 or platelet-derived growth factor alpha receptor (PDGFalphaR), and in oligodendrocytes expressing 2',3'-cyclic nucleotide 3' phosphodiesterase. Particularly interesting is the demonstration that oligodendrocyte progenitors simultaneously expressing receptors for both bFGF and PDGF-AA are present in normal adult CNS. Since in vitro bFGF and PDGF-AA in combination induce oligodendrocyte progenitors from normal adult CNS to undergo rapid proliferation and migration, the in situ coexpression of FGFRs and PDGFalphaR supports the hypothesis that oligodendrocyte progenitors can respond to bFGF and PDGF-AA in situ, and that both growth factors may be critical for repopulation of demyelinated lesions during remyelination.
Animals↗
Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system.
The establishment and operation of the nervous system requires genetic regulation by a network of DNA-binding proteins, among which is the zinc finger superfamily of transcription factors. We have cloned and characterized a member of the unusual Cys-Cys-His-Cys (also referred to as Cys2HisCys, CCHC, or C2HC) class of zinc finger proteins in the developing nervous system. The novel gene, Myt1-like (Myt1l), is highly homologous to the original representative of this class, Myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992). The MYT1 gene maps to human chromosome 20, while MYT1L maps to a region of human chromosome 2. Both zinc finger proteins are found in neurons at early stages of differentiation, with germinal zone cells displaying intense staining for MyT1. Unlike Myt1, Myt1l has not been detected in the glial lineage. Neurons that express Myt1l also express TuJ1, which marks neurons around the period of terminal mitosis. The Myt1l protein resides in distinct domains within the neuronal nucleus, analogous to the discrete pattern previously noted for Myt1 (Armstrong et al.: 14:303-321, 1995). The developmental expression and localization of these two multifingered CCHC proteins suggests that each may play a role in the development of neurons and oligodendroglia in the mammalian central nervous system.
Amino Acid Sequence↗
FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis.
We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.
Adaptor Proteins, Signal Transducing↗
Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis.
To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.
Adaptor Proteins, Signal Transducing↗