Down?Regulation of Human Deoxyuridine Triphosphate Nucleotidohydrolase (dUTPase) Using Small Interfering RNA (siRNA)

ChemInform ◽  
2005 ◽  
Vol 36 (12) ◽  
Author(s):  
M. V. Williams ◽  
A. W. Studebaker
Keyword(s):  
Small Interfering Rna ◽  
Down Regulation ◽  
Deoxyuridine Triphosphate ◽  
Interfering Rna

scholarly journals Functional Analysis of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Differentially Expressed by Variants of Human HT-1080 Fibrosarcoma Exhibiting High and Low Levels of Intravasation and Metastasis

2007 ◽  
Vol 282 (49) ◽  
pp. 35964-35977 ◽  
Author(s):  
Juneth J. Partridge ◽  
Mark A. Madsen ◽  
Veronica C. Ardi ◽  
Thales Papagiannakopoulos ◽  
Tatyana A. Kupriyanova ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Cancer Cell ◽  
Small Interfering Rna ◽  
Cultured Cells ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Down Regulation ◽  
Primary Tumors ◽  
Tissue Inhibitors ◽  
Interfering Rna

The role of tumor-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in cancer cell dissemination was analyzed by employing two variants of human HT-1080 fibrosarcoma, HT-hi/diss and HT-lo/diss, which differ by 50-100-fold in their ability to intravasate and metastasize in the chick embryo. HT-hi/diss and HT-lo/diss were compared by quantitative reverse transcription-PCR and Western blot analyses for mRNA and protein expression of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3) in cultured cells in vitro and in primary tumors in vivo. MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, both in cultures and in tumors, whereas the HT-lo/diss variant consistently expressed higher levels of MMP-2, TIMP-1, and TIMP-2. Small interfering RNA-mediated down-regulation of MMP-2 and TIMP-2 increased intravasation of HT-lo/diss cells. Coordinately, treatment of the developing HT-hi/diss tumors with recombinant TIMP-1 and TIMP-2 significantly reduced HT-hi/diss cell intravasation. However, a substantial increase of HT-hi/diss dissemination was observed upon small interfering RNA-mediated down-regulation of three secreted MMPs, including the interstitial collagenase MMP-1 and the two gelatinases, MMP-2 and MMP-9, but not the membrane-tethered MMP-14. The addition of recombinant pro-MMP-9 protein to the HT-hi/diss tumors reversed the increased intravasation of HT-hi/diss cells, in which MMP-9 was stably down-regulated by short hairpin RNA interference. This rescue did not occur if the pro-MMP-9 was stoichiometrically complexed with TIMP-1, pointing to a direct role of the MMP-9 enzyme in regulation of HT-hi/diss intravasation. Collectively, these findings demonstrate that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. not promoting but rather catalytically interfering with the early stages of cancer dissemination.

Flt3 Mutations in Proximity to an Ubiquitin Dependent Endocytosis Motif Suspend Its Hdm2 Modulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4320-4320
Author(s):  
Line Wergeland ◽  
Eystein Oveland ◽  
Gry Sjoholt ◽  
Siv Lise Bedringaas ◽  
Randi T. Hovland ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Inhibitory Effect ◽  
Down Regulation ◽  
Reciprocal Regulation ◽  
Surface Receptors ◽  
Flt3 Mutations ◽  
Elevated Expression ◽  
Interfering Rna ◽  
Small Molecular Inhibitors ◽  
Tandem Duplications

Abstract Acute myeloid leukemia (AML) frequently features mutations in the receptor tyrosine kinase Flt3 and elevated expression of the oncogenic E3 ubiquitin ligase Hdm2. Additional to the p53 inhibitory effect of Hdm2, Hdm2 appears involved in endocytosis of cell surface receptors. In this study we explore the possibility of Flt3 modulation by Hdm2 in primary AML cells and cell lines (NB4 and MV4–11) with wild type Flt3 (Flt3-wt) or mutated Flt3 (Flt3-ITD). Flt3 ligand (FL), small molecular inhibitors and small interfering RNA (siRNA) were used to elucidate the relation between Flt3 and Hdm2 on protein level, mRNA expression and modulation of apoptosis. The basal level of Flt3 is higher in AML patients with Flt3-ITD than in patients with Flt3-wt. Flt3-ITD affects a ubiquitin endocytosis motif that in some patients are duplicated, possibly resulting in enhanced receptor cycling. Down-regulation of Flt3-wt by FL, small interfering RNA or PKC412 resulted in elevated level of Hdm2. Similarly, Hdm2 attenuation resulted in increased Flt3 protein expression. Flt3-ITD responded less to Flt3 down-regulation, and was only weakly responding to Hdm2 modulation. We demonstrate that modulation of Flt3 or Hdm2 results in reciprocal regulation, and that Flt3 with internal tandem duplications may suspend its Hdm2 modulation. Together, Flt3-ITD results in dysregulated receptor turnover and elevated Hdm2 thus interconnecting the two pathways of Flt3 and p53, both related to chemoresistance in AML.

Calcitriol Prevents In Vitro Vascular Smooth Muscle Cell Mineralization by Regulating Calcium-Sensing Receptor Expression

Endocrinology ◽  
2015 ◽  
Vol 156 (6) ◽  
pp. 1965-1974 ◽  
Author(s):  
Aurélien Mary ◽  
Lucie Hénaut ◽  
Cédric Boudot ◽  
Isabelle Six ◽  
Michel Brazier ◽  
...  
Keyword(s):  
Vitamin D ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Small Interfering Rna ◽  
Receptor Expression ◽  
Calcium Sensing Receptor ◽  
Down Regulation ◽  
Calcium Sensing ◽  
Cardiovascular Morbidity And Mortality ◽  
Interfering Rna

Abstract Vascular calcification (VC) is a degenerative disease that contributes to cardiovascular morbidity and mortality. A negative relationship has been demonstrated between VC and calcium sensing receptor (CaSR) expression in the vasculature. Of interest, vitamin D response elements, which allow responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], are present in the promoters of the CaSR gene. We hypothesized that 1,25(OH)2D3, by modulating CaSR expression in vascular smooth muscle cells (VSMCs), might protect against VC. Human VSMCs were exposed to increasing concentrations of 1,25(OH)2D3 (0.01–10 nmol/L) in noncalcifying (1.8 mmol/L) or procalcifying Ca2+0 condition (5.0 mmol/L). Using quantitative RT-PCR and Western blotting we observed a significant increase in both CaSR mRNA and protein levels after exposure to 1.0 nmol/L 1,25(OH)2D3. This effect was associated with a maximal increase in CaSR expression at the cell surface after 48 hours of 1,25(OH)2D3 treatment, as assessed by flow cytometry. Down-regulation of the vitamin D receptor by small interfering RNA abolished these effects. In the procalcifying condition, 1.0 nmol/L 1,25(OH)2D3 blocked the Ca2+0-induced decrease in total and surface CaSR expression and protected against mineralization. Down-regulation of CaSR expression by CaSR small interfering RNA abolished this protective effect. 1,25(OH)2D3 concentrations of 0.5 and 5.0 nmol/L were also effective, but other (0.01, 0.1, and 10 nmol/L) concentrations did not modify CaSR expression and human VSMC mineralization. In conclusion, these findings suggest that nanomolar concentrations of 1,25(OH)2D3 induce a CaSR-dependent protection against VC. Both lower and higher concentrations are either ineffective or may even promote VC. Whether this also holds true in the clinical setting requires further study.

Snail1 Down-Regulation using Small Interfering RNA Complexes Delivered through Collagen Scaffolds

2009 ◽  
Vol 20 (12) ◽  
pp. 2262-2269 ◽  
Author(s):  
Rosa Viñas-Castells ◽  
Carolyn Holladay ◽  
Andrea di Luca ◽  
Victor Manuel Díaz ◽  
Abhay Pandit
Keyword(s):  
Small Interfering Rna ◽  
Collagen Scaffolds ◽  
Down Regulation ◽  
Interfering Rna ◽  
Rna Complexes

scholarly journals Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase IIIα

2008 ◽  
Vol 19 (2) ◽  
pp. 711-721 ◽  
Author(s):  
Andras Balla ◽  
Yeun Ju Kim ◽  
Peter Varnai ◽  
Zsofia Szentpetery ◽  
Zachary Knight ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Small Interfering Rna ◽  
Type Ii ◽  
Measurable Effect ◽  
Down Regulation ◽  
Type Iii ◽  
Hormone Sensitive ◽  
Phosphatidylinositol 4 ◽  
Interfering Rna

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

scholarly journals Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone Deacetylase in Prostate Cancer

2005 ◽  
Vol 280 (23) ◽  
pp. 22437-22444 ◽  
Author(s):  
Hong Chen ◽  
Szu-wei Tu ◽  
Jer-Tsong Hsieh
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Epithelial Cells ◽  
Histone Deacetylase ◽  
Promoter Region ◽  
Small Interfering Rna ◽  
Gene Promoter ◽  
Down Regulation ◽  
Ezh2 Expression ◽  
Interfering Rna

Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.

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