ABSTRACT
It has recently been shown that the transcription factor Erg, an Ets family member, drives constitutive expression of the intercellular adhesion molecule 2 (ICAM-2) in human umbilical vein endothelial cells (HUVECs) and that its expression is down-regulated by the pleiotropic cytokine tumor necrosis factor alpha (TNF-alpha). To identify other Erg target genes and to define its function in the endothelium, a combined approach of antisense oligonucleotides (GeneBloc) and differential gene expression was used. Treatment of HUVECs with Erg-specific GeneBloc for 24, 48, and 72 hours suppressed Erg mRNA and protein levels at all time points. Total RNA extracted from HUVECs treated with Erg-specific or control GeneBloc was analyzed for differences in gene expression using high-density, sequence-verified cDNA arrays containing 482 relevant genes. Inhibition of Erg expression resulted in decreased expression of ICAM-2, as predicted. Four more genes decreased in Erg-deficient HUVECs were the extracellular matrix proteins SPARC and thrombospondin, the adhesive glycoprotein von Willebrand factor, and the small GTPase RhoA. Each of these molecules has been directly or indirectly linked to angiogenesis because of its role in vascular remodeling, adhesion, or shape change. Therefore, the role of Erg in vascular remodeling was tested in an in vitro model, and the results showed that HUVECs treated with Erg GeneBloc had a decreased ability to form tubulelike structures when grown on Matrigel. These results suggest that Erg may be a mediator of the TNF-alpha effects on angiogenesis in vivo.
Subject(s)
Cell Differentiation , DNA-Binding Proteins , Endothelium, Vascular/cytology , Gene Expression Regulation , Oligonucleotides, Antisense/pharmacology , Oncogene Proteins/genetics , Oncogene Proteins/physiology , Trans-Activators , Transcription Factors , Antigens, CD/genetics , Blotting, Western , Cell Adhesion Molecules/genetics , Humans , Neovascularization, Physiologic , Oncogene Proteins/antagonists & inhibitors , Polymerase Chain Reaction , RNA, Messenger/analysis , Thrombospondins/genetics , Transcriptional Regulator ERG , Tumor Necrosis Factor-alpha/pharmacology , Umbilical Veins , rhoA GTP-Binding Protein/genetics , von Willebrand Factor/geneticsABSTRACT
Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/genetics , Poliovirus/genetics , RNA, Catalytic/pharmacology , RNA, Viral/genetics , Virus Replication/drug effects , Base Sequence , HeLa Cells , Humans , Molecular Sequence Data , Protein Biosynthesis , RNA, Catalytic/chemical synthesis , TransfectionABSTRACT
The nucleotide sequence of the Escherichia coli K12 beta-methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.