Granulocytes do not express but acquire monocyte-derived tissue factor in whole blood: evidence for a direct transfer

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1208-1216 ◽  
Author(s):  
Elena M. Egorina ◽  
Mikhail A. Sovershaev ◽  
Jan O. Olsen ◽  
Bjarne Østerud
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
No Reduction ◽  
Yellow Fluorescent Protein ◽  
Direct Transfer ◽  
Unanimous Opinion ◽  
Protein Levels ◽  
Interfering Rna

AbstractUnlike unanimous opinion on tissue factor (TF) expression in monocytes, the quest for TF presence in granulocytes has been going on for decades. To study the cell origin and track the blood-borne TF, we assessed TF activity and protein levels, knocked-down endogenous TF expression with small interfering RNA (siRNA), and overexpressed TF–yellow fluorescent protein (TF-YFP) fusion in immunologically isolated human monocytes and granulocytes. Monocytes and, to a much lesser extent, granulocytes isolated from lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)–stimulated whole blood contained active TF antigen. However, only monocytes possessed significant TF activity and protein levels when stimulated with LPS/PMA in suspension. Reintroduction of TF-silenced monocytes to whole blood led to a profound reduction of LPS/PMA-stimulated TF activity in both mononuclear cell (MNC) and granulocyte fractions. No reduction in TF activity in MNC and granulocyte fractions was observed when TF-silenced granulocytes were reintroduced to whole blood. As shown by immunoblotting, flow cytometry, and confocal microscopy, granulocytes became positive for TF-YFP when isolated from whole blood reconstituted with TF-YFP–expressing monocytes. Together, we pinpoint monocytes as a major source of TF and provide solid experimental evidence for a direct transfer of TF protein from the monocytes to granulocytes in the blood.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

scholarly journals The FDA-Approved Anti-Asthma Medicine Ciclesonide Inhibits Lung Cancer Stem Cells through Hedgehog Signaling-Mediated SOX2 Regulation

2020 ◽  
Vol 21 (3) ◽  
pp. 1014 ◽  
Author(s):  
Hack Sun Choi ◽  
Su-Lim Kim ◽  
Ji-Hyang Kim ◽  
Dong-Sun Lee
Keyword(s):  
Lung Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Small Interfering Rna ◽  
Hedgehog Signaling ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Protein Levels ◽  
Lung Cancer Stem Cells ◽  
Interfering Rna

Ciclesonide is an FDA-approved glucocorticoid (GC) used to treat asthma and allergic rhinitis. However, its effects on cancer and cancer stem cells (CSCs) are unknown. Our study focuses on investigating the inhibitory effect of ciclesonide on lung cancer and CSCs and its underlying mechanism. In this study, we showed that ciclesonide inhibits the proliferation of lung cancer cells and the growth of CSCs. Similar glucocorticoids, such as dexamethasone and prednisone, do not inhibit CSC formation. We show that ciclesonide is important for CSC formation through the Hedgehog signaling pathway. Ciclesonide reduces the protein levels of GL1, GL2, and Smoothened (SMO), and a small interfering RNA (siRNA) targeting SMO inhibits tumorsphere formation. Additionally, ciclesonide reduces the transcript and protein levels of SOX2, and an siRNA targeting SOX2 inhibits tumorsphere formation. To regulate breast CSC formation, ciclesonide regulates GL1, GL2, SMO, and SOX2. Our results unveil a novel mechanism involving Hedgehog signaling and SOX2 regulated by ciclesonide in lung CSCs, and also open up the possibility of targeting Hedgehog signaling and SOX2 to prevent lung CSC formation.

scholarly journals Use of Degradation Tags To Control Protein Levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803

2013 ◽  
Vol 79 (8) ◽  
pp. 2833-2835 ◽  
Author(s):  
Brian P. Landry ◽  
Jana Stöckel ◽  
Himadri B. Pakrasi
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Terminal Protein ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Protein Levels ◽  
Steady State Fluorescence ◽  
Tunable Range ◽  
Control Protein ◽  
Synechocystis Sp

ABSTRACTWe generated a collection ofssrA-based C-terminal protein degradation tags with different degradation strengths. The steady-state fluorescence levels of different enhanced yellow fluorescent protein (eYFP) tag variants in aSynechocystissp. indicated a tunable range from 1% to 50% of untagged eYFP.

scholarly journals Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5255-5266 ◽  
Author(s):  
Angelo Cignarelli ◽  
Mariangela Melchiorre ◽  
Alessandro Peschechera ◽  
Antonella Conserva ◽  
Lucia Adelaide Renna ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Interfering Rna ◽  
Protein Modification ◽  
Covalent Attachment ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Induction ◽  
Induction Of Differentiation ◽  
Interfering Rna

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2–6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.

scholarly journals Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes

2008 ◽  
Vol 28 (19) ◽  
pp. 5829-5836 ◽  
Author(s):  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Proteolytic Processing ◽  
Tunel Staining ◽  
Interfering Rna ◽  
Capping Enzymes

ABSTRACT Addition of a 5′ cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK−/− BAX−/− double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

Evidence for direct transfer of tissue factor from monocytes to platelets in whole blood

2012 ◽  
Vol 23 (4) ◽  
pp. 345-350 ◽  
Author(s):  
Mikhail A. Sovershaev ◽  
Elena M. Egorina ◽  
Bjarne Østerud ◽  
John-Bjarne Hansen
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Direct Transfer

scholarly journals The Protective Role of a Small GTPase RhoE against UVB-induced DNA Damage in Keratinocytes

2006 ◽  
Vol 282 (7) ◽  
pp. 4850-4858 ◽  
Author(s):  
Sarah A. Boswell ◽  
Pat P. Ongusaha ◽  
Paul Nghiem ◽  
Sam W. Lee
Keyword(s):  
Cyclin D1 ◽  
Small Interfering Rna ◽  
Critical Factor ◽  
Human Keratinocytes ◽  
Protective Role ◽  
Small Gtpase ◽  
Uvb Irradiation ◽  
Protein Levels ◽  
Rho Family ◽  
Interfering Rna

RhoE, a p53 target gene, was identified as a critical factor for the survival of human keratinocytes in response to UVB. The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton, acting as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho family proteins, RhoE (also known as Rnd3) is GTPase-deficient and thus expected to be constitutively active. In this study, we investigated the response of cultured human keratinocyte cells to UVB irradiation. RhoE protein levels increase upon exposure to UVB, and ablation of RhoE induction through small interfering RNA resulted in a significant increase in apoptosis and a reduction in the levels of the pro-survival targets p21, Cox-2, and cyclin D1, as well as an increase of reactive oxygen species levels when compared with control cells. These data indicate that RhoE is a pro-survival factor acting upstream of p38, JNK, p21, and cyclin D1. HaCat cells expressing small interfering RNA to p53 indicate that RhoE functions independently of its known associates, p53 and Rho-associated kinase I (ROCK I). Targeted expression of RhoE in epidermis using skin-specific transgenic mouse model resulted in a significant reduction in the number of apoptotic cells following UVB irradiation. Thus, RhoE induction counteracts UVB-induced apoptosis and may serve as a novel target for the prevention of UVB-induced photodamage regardless of p53 status.

scholarly journals Twist Modulates Human Trophoblastic Cell Invasion via Regulation of N-Cadherin

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 925-936 ◽  
Author(s):  
York Hunt Ng ◽  
Hua Zhu ◽  
Peter C. K. Leung
Keyword(s):  
Cell Invasion ◽  
Small Interfering Rna ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Trophoblastic Cell ◽  
Protein Levels ◽  
Bewo Cells ◽  
Interfering Rna ◽  
Tgf Β1

The invasion of extravillous cytotrophoblasts (EVT) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms involved in the development of the invasive phenotype of EVT include many that were first discovered for their role in cancer cell metastasis. Previous studies have demonstrated that N-cadherin and its regulatory transcription factor Twist play important roles in the onset and progression of cancers, but their roles in human trophoblastic cell invasion is not clear. The goal of the study was to examine the role of Twist and N-cadherin in human trophoblastic cell invasion. Twist and N-cadherin mRNA and protein levels were determined by RT-PCR and Western blotting in human placental tissues, highly invasive EVT, and poorly invasive JEG-3 and BeWo cells. Whether IL-1β and TGF-β1 regulate Twist mRNA and protein levels in the EVT was also examined. A small interfering RNA strategy was employed to determine the role of Twist and N-cadherin in HTR-8/SVneo cell invasion. Matrigel assays were used to assess cell invasion. Twist and N-cadherin were highly expressed in EVT but were poorly expressed in JEG-3 and BeWo cells. IL-1β and TGF-β1 differentially regulated Twist expression in EVT in a time- and concentration-dependent manner. Small interfering RNA specific for Twist decreased N-cadherin and reduced invasion of HTR-8/SVneo cells. Similarly, a reduction in N-cadherin decreased the invasive capacity of HTR-8/SVneo cells. Twist is an upstream regulator of N-cadherin-mediated invasion of human trophoblastic cells.

scholarly journals Hibiscus chlorotic ringspot virus coat protein inhibits trans-acting small interfering RNA biogenesis in Arabidopsis

2008 ◽  
Vol 89 (9) ◽  
pp. 2349-2358 ◽  
Author(s):  
Chunying Meng ◽  
Jun Chen ◽  
Shou-wei Ding ◽  
Jinrong Peng ◽  
Sek-Man Wong
Keyword(s):  
Coat Protein ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Ringspot Virus ◽  
Pcr Analysis ◽  
Transcriptional Gene Silencing ◽  
Interfering Rna

Many plant and animal viruses have evolved suppressor proteins to block host RNA silencing at various stages of the RNA silencing pathways. Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is capable of suppressing the transiently expressed sense-RNA-induced post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana. Here, constitutively expressed HCRSV CP from transgenic Arabidopsis was found to be able to rescue expression of the silenced GUS transgene. The HCRSV CP-transgenic Arabidopsis (line CP6) displayed several developmental abnormalities: elongated, downwardly curled leaves and a lack of coordination between stamen and carpel, resulting in reduced seed set. These abnormalities are similar to those observed in mutations of the genes of Arabidopsis RNA-dependent polymerase 6 (rdr6), suppressor of gene silencing 3 (sgs3), ZIPPY (zip) and dicer-like 4 (dcl4). The accumulation of microRNA (miRNA) miR173 remained stable; however, the downstream trans-acting small interfering RNA (ta-siRNA) siR255 was greatly reduced. Real-time PCR analysis showed that expression of the ta-siRNA-targeted At4g29770, At5g18040, PPR and ARF3 genes increased significantly, especially in the inflorescences. Genetic crossing of CP6 with an amplicon-silenced line (containing a potato virus X–green fluorescent protein transgene under the control of the 35S cauliflower mosaic virus promoter) suggested that HCRSV CP probably interfered with gene silencing at a step after RDR6. The reduced accumulation of ta-siRNA might result from the interference of HCRSV CP with Dicer-like protein(s), responsible for the generation of dsRNA in ta-siRNA biogenesis.

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