scholarly journals Daxx Silencing Sensitizes Cells to Multiple Apoptotic Pathways

2003 ◽  
Vol 23 (20) ◽  
pp. 7108-7121 ◽  
Author(s):  
Liuh-Yow Chen ◽  
J. Don Chen
Keyword(s):  
Gene Expression ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Death Receptor ◽  
Kinase Activation ◽  
Transient Overexpression ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Caspase Gene ◽  
Interfering Rna

ABSTRACT Daxx is a nuclear protein involved in apoptosis and transcriptional repression, and it interacts with the death receptor Fas, promyelocytic leukemia protein (PML), and several transcriptional repressors. The function of Daxx in apoptosis is controversial because opposite results were obtained in transient overexpression and genetic knockout studies. Furthermore, the roles of PML and transcriptional repression in Daxx-regulated apoptosis are currently unknown. In this study, we investigated the role of Daxx in Fas- and stress-induced apoptosis by small interfering RNA-mediated Daxx silencing in mammalian cells. Daxx silencing had no apparent cytotoxic effects on mammalian cells within 72 h. Intriguingly, Daxx silencing strongly sensitized cells to Fas- and stress-induced apoptosis, which was accompanied by caspase activation, cytochrome c release, and Jun N-terminal kinase activation. Consistently, endogenous Daxx was degraded rapidly upon induction of apoptosis by stress or anti-Fas antibody. Finally, PML silencing had no effect on Daxx silencing-mediated apoptotic events, while caspase gene expression was upregulated in the absence of Daxx. These data strongly suggest that Daxx may inhibit Fas and stress-mediated apoptosis by suppressing proapoptotic gene expression outside of PML domains.

Mechanisms of mammalian zinc-regulated gene expression

2008 ◽  
Vol 36 (6) ◽  
pp. 1262-1266 ◽  
Author(s):  
Kelly A. Jackson ◽  
Ruth A. Valentine ◽  
Lisa J. Coneyworth ◽  
John C. Mathers ◽  
Dianne Ford
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Gene Promoter ◽  
Mrna Levels ◽  
Promoter Regions ◽  
Mrna Stabilization ◽  
Transcriptional Regulatory ◽  
Regulated Gene Expression

Mechanisms through which gene expression is regulated by zinc are central to cellular zinc homoeostasis. In this context, evidence for the involvement of zinc dyshomoeostasis in the aetiology of diseases, including Type 2 diabetes, Alzheimer's disease and cancer, highlights the importance of zinc-regulated gene expression. Mechanisms elucidated in bacteria and yeast provide examples of different possible modes of zinc-sensitive gene regulation, involving the zinc-regulated binding of transcriptional activators and repressors to gene promoter regions. A mammalian transcriptional regulatory mechanism that mediates zinc-induced transcriptional up-regulation, involving the transcription factor MTF1 (metal-response element-binding transcription factor 1), has been studied extensively. Gene responses in the opposite direction (reduced mRNA levels in response to increased zinc availability) have been observed in mammalian cells, but a specific transcriptional regulatory process responsible for such a response has yet to be identified. Examples of single zinc-sensitive transcription factors regulating gene expression in opposite directions are emerging. Although zinc-induced transcriptional repression by MTF1 is a possible explanation in some specific instances, such a mechanism cannot account for repression by zinc of all mammalian genes that show this mode of regulation, indicating the existence of as yet uncharacterized mechanisms of zinc-regulated transcription in mammalian cells. In addition, recent findings reveal a role for effects of zinc on mRNA stability in the regulation of specific zinc transporters. Our studies on the regulation of the human gene SLC30A5 (solute carrier 30A5), which codes for the zinc transporter ZnT5, have revealed that this gene provides a model system by which to study both zinc-induced transcriptional down-regulation and zinc-regulated mRNA stabilization.

scholarly journals Knockdown of hDaxx in normally non-permissive undifferentiated cells does not permit human cytomegalovirus immediate-early gene expression

2007 ◽  
Vol 88 (11) ◽  
pp. 2935-2940 ◽  
Author(s):  
Ian J. Groves ◽  
John H. Sinclair
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Transcriptional Repression ◽  
Cell Types ◽  
Cellular Protein ◽  
Early Gene ◽  
Immediate Early ◽  
Undifferentiated Cells ◽  
Interfering Rna

The cellular protein human Daxx (hDaxx), a component of nuclear domain 10 structures, is known to mediate transcriptional repression of human cytomegalovirus immediate-early (IE) gene expression upon infection of permissive cell types, at least in part, by regulation of chromatin structure around the major IE promoter (MIEP). As it is now clear that differentiation-dependent regulation of the MIEP also plays a pivotal role in the control of latency and reactivation, we asked whether hDaxx-mediated repression is involved in differentiation-dependent MIEP regulation. We show that downregulation of hDaxx by using small interfering RNA technology in undifferentiated NT2D1 cells does not permit expression of viral IE genes, nor does it result in changes in chromatin structure around the MIEP. Viral IE gene expression is only observed upon cellular differentiation, suggesting little involvement of hDaxx in the regulation of the viral MIEP in undifferentiated cells.

scholarly journals Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase–mediated apoptosis of HeLa cells

2006 ◽  
Vol 174 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Hyungshin Yim ◽  
In Sun Hwang ◽  
Joon-Seok Choi ◽  
Kwang-Hoon Chun ◽  
Ying Hua Jin ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Replication Initiation ◽  
Dominant Negative ◽  
Ataxia Telangiectasia Mutated ◽  
Activation Kinetics ◽  
Kinase Activation ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Atr Kinase ◽  
Interfering Rna

We show that caspase-3 cleaves Cdc6 at D290/S and D442/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-α–induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-α–induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-α– or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53–Bax-mediated apoptosis.

scholarly journals Small Interfering RNA–Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

2017 ◽  
Vol 11 (5) ◽  
pp. 686-693
Author(s):  
Jong-Beom Park ◽  
Chanjoo Park
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Mrna Level ◽  
Serum Deprivation ◽  
Specific Gene ◽  
Disc Cells ◽  
Fetal Bovine ◽  
Interfering Rna

<sec><title>Study Design</title><p><italic>In vitro</italic> cell culture model.</p></sec><sec><title>Purpose</title><p>To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells.</p></sec><sec><title>Overview of Literature</title><p>Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear.</p></sec><sec><title>Methods</title><p>Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls.</p></sec><sec><title>Results</title><p>Serum deprivation increased apoptosis by 40.3% (<italic>p</italic>&lt;0.001), decreased proliferation by 45.3% (<italic>p</italic>&lt;0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (<italic>p</italic>&lt;0.001). Finally, Fas siRNA–mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (<italic>p</italic>&lt;0.05 for both).</p></sec><sec><title>Conclusions</title><p>The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.</p></sec>

scholarly journals c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis

2004 ◽  
Vol 199 (8) ◽  
pp. 1041-1052 ◽  
Author(s):  
Stephan Mathas ◽  
Andreas Lietz ◽  
Ioannis Anagnostopoulos ◽  
Franziska Hummel ◽  
Burkhard Wiesner ◽  
...  
Keyword(s):  
Death Receptor ◽  
B Cell Lymphoma ◽  
Immunohistochemical Analysis ◽  
Death Domain ◽  
Inhibitory Protein ◽  
Antiapoptotic Proteins ◽  
Signaling Complex ◽  
Induced Apoptosis ◽  
High Level ◽  
Interfering Rna

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

scholarly journals Human UDP-galactose 4′-epimerase (GALE) is required for cell-surface glycome structure and function

2019 ◽  
Vol 295 (5) ◽  
pp. 1225-1239
Author(s):  
Alex Broussard ◽  
Alyssa Florwick ◽  
Chelsea Desbiens ◽  
Nicole Nischan ◽  
Corrina Robertson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Antigen ◽  
Mammalian Cells ◽  
Death Receptor ◽  
Cell Physiology ◽  
Total Sialic Acid ◽  
Death Receptor Signaling ◽  
Induced Apoptosis ◽  
And Function

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4′-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS–based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE−/− cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

scholarly journals Extracellular Signal-Regulated Kinase Activation by Neisseria gonorrhoeae Downregulates Epithelial Cell Proapoptotic Proteins Bad and Bim

2008 ◽  
Vol 76 (6) ◽  
pp. 2715-2721 ◽  
Author(s):  
Heather L. Howie ◽  
Shelly L. Shiflett ◽  
Magdalene So
Keyword(s):  
Epithelial Cell ◽  
Neisseria Gonorrhoeae ◽  
Kinase Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Type Iv ◽  
Extracellular Signal ◽  
Domain 3 ◽  
Induced Apoptosis ◽  
Dependent Modification ◽  
Interfering Rna

ABSTRACT Neisseria gonorrhoeae expressing type IV pili (Tfp) activates extracellular signal-regulated kinase (ERK) and induces a cytoprotective state in the epithelial cell in a manner that is enhanced by pilT. As the ERK signaling pathway is well-known for its role in cytoprotection and cell survival, we tested the hypothesis that ERK is involved in producing this cytoprotective effect. Inhibiting ERK activation prior to infection attenuated the ability of these bacteria to induce cytoprotection. Activated ERK specifically targeted two proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins, Bim and Bad, for downregulation at the protein level. Bim downregulation occurred through the proteasome. ERK, in addition, inactivated Bad by triggering its phosphorylation at Ser112. Finally, reducing the level of either Bad or Bim alone by small interfering RNA was sufficient to protect uninfected cells from staurosporine-induced apoptosis. We conclude that Tfp-induced cytoprotection is due in part to ERK-dependent modification and/or downregulation of proapoptotic proteins Bad and Bim.

scholarly journals The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

2019 ◽  
Author(s):  
Alex Broussard ◽  
Alyssa Florwick ◽  
Chelsea Desbiens ◽  
Nicole Nischan ◽  
Corrina Robertson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fas Ligand ◽  
Death Receptor ◽  
Cell Physiology ◽  
Uridine Diphosphate ◽  
Death Receptor Signaling ◽  
Nucleotide Sugars ◽  
Induced Apoptosis ◽  
And Function

ABSTRACTGlycan biosynthesis relies on nucleotide-sugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)-galactose 4’-epimerase (GALE), which interconverts two pairs of essential NSs. GALE deletion in human cells triggered major imbalances in its substrate NSs and consequent dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. NS dysregulation also directly impacted cell signaling, as GALE−/− cells exhibit Fas hypoglycosylation and hypersensitivity to Fas ligand-induced apoptosis. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

scholarly journals Epithelial Cells Infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) Are Resistant to Apoptosis

2001 ◽  
Vol 69 (12) ◽  
pp. 7880-7888 ◽  
Author(s):  
Krishnaraj Rajalingam ◽  
Hesham Al-Younes ◽  
Anne Müller ◽  
Thomas F. Meyer ◽  
Agnes J. Szczepek ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chlamydia Pneumoniae ◽  
Death Receptor ◽  
Underlying Mechanism ◽  
Chlamydophila Pneumoniae ◽  
Apoptosis Resistance ◽  
Infected Cells ◽  
Obligate Intracellular Pathogen ◽  
Apoptotic Pathways ◽  
Induced Apoptosis

ABSTRACT The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.

Sign in / Sign up

Export Citation Format

Share Document