ABSTRACT
The majority of single nucleotide variants (SNVs) identified in Genome Wide Association Studies (GWAS) fall within non-protein coding DNA and have the potential to alter gene expression. Non-protein coding DNA can control gene expression by acting as transcription factor (TF) binding sites or by regulating the organization of DNA into chromatin. SNVs in non-coding DNA sequences can disrupt TF binding and chromatin structure and this can result in pathology. Further, environmental health studies have shown that exposure to xenobiotics can disrupt the ability of TFs to regulate entire gene networks and result in pathology. However, there is a large amount of interindividual variability in exposure-linked health outcomes. One explanation for this heterogeneity is that genetic variation and exposure combine to disrupt gene regulation, and this eventually manifests in disease. Many resources exist that annotate common variants from GWAS and combine them with conservation, functional genomics, and TF binding data. These annotation tools provide clues regarding the biological implications of an SNV, as well as lead to the generation of hypotheses regarding potentially disrupted target genes, epigenetic markers, pathways, and cell types. Collectively this information can be used to predict how SNVs can alter an individual's response to exposure and disease risk. A basic understanding of the regulatory information contained within non-protein coding DNA is needed to predict the biological consequences of SNVs, and to determine how these SNVs impact exposure-related disease. We hope that this review will aid in the characterization of disease-associated genetic variation in the non-protein coding genome.
Subject(s)
Gene-Environment Interaction , Genetic Variation , Genomics/methods , Untranslated Regions , Animals , Gene Expression , Genetic Variation/genetics , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide/genetics , Untranslated Regions/drug effectsABSTRACT
Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal "homeostatic" state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5' and 3' untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Antineoplastic Agents/administration & dosage , High-Throughput Screening Assays/methods , Neoplasms/drug therapy , Untranslated Regions/drug effects , Vascular Endothelial Growth Factor A/genetics , Administration, Oral , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Mice , Neoplasms/genetics , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND/AIMS: Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system. METHODS: For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively. RESULTS: A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct. CONCLUSIONS: This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.
