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PubMed2003

Strategic Research Institute--first international siRNA conference. Prospect for new therapeutics and commercial opportunities for pharma and biotech. 24-25 March 2003, LaJolla, CA, USA.

Small interfering RNAs (siRNAs), with their power to selectively silence genes, have gained much attention from biotech and pharmaceutical companies and investors. Key players in the field, from innovative biotech start-ups to big pharmaceutical companies, gathered at Strategic Research Institute's First International siRNA conference in the scenic Hilton La Jolla Torrey Pines. Topics addressed ranged from the latest technology advances and applications of RNA interference (RNAi) in drug discovery, to critical business issues such as intellectual property portfolio strategy and market prospects. While RNAi is indisputably accepted as a powerful tool in target validation and functional genomics, the concept of an siRNA drug is still viewed by big pharma companies as next-generation therapeutics. Yet challenges seem tractable. Several companies, such as Ribozyme Pharmaceuticals Inc, Alnylam Pharmaceuticals and Intradigm Corp, are working hard to prove that the bigger companies are being too conservative. The conference provided a clear vision of RNAi in drug discovery today, its potential and remaining challenges.

Animals↗
PubMed2004

The effect of stress on genome regulation and structure.

BACKGROUND: Stresses exert evolutionary pressures on all organisms, which have developed sophisticated responses to cope and survive. These responses involve cellular physiology, gene regulation and genome remodelling. SCOPE: In this review, the effects of stress on genomes and the connected responses are considered. Recent developments in our understanding of epigenetic genome regulation, including the role of RNA interference (RNAi), suggest a function for this in stress initiation and response. We review our knowledge of how different stresses, tissue culture, pathogen attack, abiotic stress, and hybridization, affect genomes. Using allopolyploid hybridization as an example, we examine mechanisms that may mediate genomic responses, focusing on RNAi-mediated perturbations. CONCLUSIONS: A common response to stresses may be the relaxation of epigenetic regulation, leading to activation of suppressed sequences and secondary effects as regulatory systems attempt to re-establish genomic order.

Climate↗
PubMed2005

High-throughput RNA interference strategies for target discovery and validation by using synthetic short interfering RNAs: functional genomics investigations of biological pathways.

During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.

Apoptosis↗
PubMed2005

TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function.

UNLABELLED: Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation. INTRODUCTION: TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor gamma chain (FcRgamma) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption. MATERIALS AND METHODS: We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts. RESULTS: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF. CONCLUSIONS: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.

Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.

Adenosine Triphosphatases↗
PubMed2003

An siRNA ribonucleoprotein is found associated with polyribosomes in Trypanosoma brucei.

Genetic interference by double-stranded RNA (dsRNA) or RNA interference (RNAi) triggers the sequence-specific degradation of cellular transcripts. The mediators of mRNA degradation are small interfering RNAs (siRNAs). Here, we report that in Trypanosoma brucei, 10%-20% of siRNAs cosediment with polyribosomes. Preventing the assembly of ribosomes on mRNAs results in a concomitant decrease of siRNAs sedimenting as high-molecular-weight complexes. We further provide evidence that siRNAs are associated with translating ribosomes and that this association is mediated by a 70-kD ribonucleoprotein complex.

Animals↗
PubMed2006

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis.

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Amino Acid Sequence↗
PubMed2005

Towards mutation-independent silencing of genes involved in retinal degeneration by RNA interference.

More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation-independent therapies for these diseases. We describe use of RNA interference (RNAi) and specifically short hairpin RNAs (shRNAs) expressed from DNA templates to silence both normal and mutant (P23H) human rhodopsin alleles by 94.34+/-2.17 and 94.9+/-1.9%, respectively, in human embryonic retinoblasts. Degeneracy of the genetic code was used to engineer a codon-exchanged mRNA (cmRNA) that demonstrated complete resistance to silencing by the shRNA. Simulation of autosomal dominant retinitis pigmentosa in cell culture through triple transfection of DNAs expressing a cmRNA, a P23H mRNA and an shRNA revealed shRNA-mediated silencing, specifically of P23H rhodopsin by 90.64+/-5.19% and no loss of rhodopsin translation from the cmRNA in those cells. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin. Our results have implications for the treatment of a very large variety of retinal degenerations in a mutation-independent manner.

Base Sequence↗
PubMed2006

Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance.

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.

Acetylation↗
PubMed2003

The adenosine analog tubercidin inhibits glycolysis in Trypanosoma brucei as revealed by an RNA interference library.

We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAi-based genomic library, we used 5 microm tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidin-resistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC50 of 7.5 microm. We conclude that 5 microm tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.

Adaptation, Physiological↗
PubMed2005

Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference.

Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by approximately 90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.

Cell Line↗
PubMed2006

Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and BCL2A1 as critical target genes.

Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPbeta and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.

Anaplastic Lymphoma Kinase↗
PubMed2005

Isoform-specific silencing of the Livin gene by RNA interference defines Livin beta as key mediator of apoptosis inhibition in HeLa cells.

Livin (alternatively called ML-IAP or KIAP) is a cancer-associated member of the antiapoptotic inhibitor of apoptosis protein family. Two splicing variants of Livin, designated Livin alpha and Livin beta, have been identified. The significance of these isoforms for Livin-mediated apoptosis inhibition is largely unclear. Using an isoform-specific RNA interference (RNAi) strategy, we silenced endogenous Livin expression in HeLa cells. We found that the targeted inhibition of Livin beta, but not of Livin alpha, blocked the growth of HeLa cells in clonogenic survival assays. In addition, silencing of Livin beta, but not of Livin alpha, sensitized HeLa cells to different proapoptotic stimuli such as UV irradiation, tumor necrosis factor alpha, or etoposide. These events were linked to activation of caspase-3 and increased poly(ADP-ribose) polymerase cleavage, specifically upon silencing of Livin beta. The proapoptotic sensitization of HeLa cells upon RNAi-mediated silencing of the endogenous livin gene was specifically reverted by ectopic expression of Livin beta but not of Livin alpha. We conclude that the Livin beta isoform plays the key role for the antiapoptotic protection of HeLa cells by the livin gene. Our results show that the Livin isoforms can strongly differ in their functional significance for the antiapoptotic resistance of tumor cells. Studies evaluating Livin as a novel diagnostic and prognostic tumor marker should benefit from isoform-specific expression analyses.

Adaptor Proteins, Signal Transducing↗
PubMed2004

siRNA technology.

RNA interference (RNAi) represents a mechanism invented by nature to protect the genome. In the past few years the field has emerged at a surprisingly high pace. The underlying molecular mechanism of gene silencing provides us with short interfering RNAs (siRNAs) which allows to target any gene with high specificity and efficiency. siRNAs can now be obtained in various ways allowing for numerous in vitro and in vivo applications. Successful knock-downs of disease-related genes indicate that siRNAs open the door for novel therapeutic procedures.

Animals↗
PubMed2006

Destabilization of the VCP-Ufd1-Npl4 complex is associated with decreased levels of ERAD substrates.

p97/VCP associated with Ufd1-Npl4 is considered a key player in ER-associated degradation (ERAD). RNA interference (RNAi) of one component of the Ufd1-Npl4 heterodimer destabilizes the VCP-Ufd1-Npl4 complex inducing proteasome-dependent degradation of the other component and releasing free VCP. In contrast to RNAi of VCP, RNAi of Ufd1 or Npl4 depleting approximately 90% of the VCP-Ufd1-Npl4 complexes does not induce unfolded protein response, indicating that the Ufd1-Npl4 dimer is not involved in the regulation of ER function by VCP. RNAi of Ufd1 or Npl4 is associated with a 2-fold increase in the levels of polyubiquitinated proteins, which form dispersed aggregates often associated with calnexin-positive structures. However, contrary to the effects of proteasome inhibition, RNAi of Ufd1 or Npl4 does not induce an accumulation of alpha-TCR and delta-CD3, two ERAD substrates overexpressed in HeLa cells. Instead, a 60-70% decrease in their levels is observed. The decrease in alpha-TCR levels is associated with a 50% decrease of its half-life. Upregulation of the putative channel forming protein, derlin-1, may contribute to the increased degradation of ERAD substrates. To explain our findings, we propose a model, where association of emerging ERAD substrates with VCP-Ufd1-Npl4 is not required for their degradation but has a regulatory role.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

siRNA--getting the message out.

The recent observation that potent and sequence-specific gene silencing by injection of double-stranded RNA (dsRNA) has sparked the phenomenon known as "RNA interference" (RNAi) and has enabled the gene-specific knockdown of drug transport proteins and metabolizing enzymes. The application of small interfering RNAs (siRNAs) is broad and the potential for use as research tools is now well established in vitro. In vivo use is still a challenge that is primarily focused on the difficulty of delivering siRNAs to target cells. The potential use of siRNAs as therapeutic agents is also exciting and holds great promise for future. For the study of drug transporter function in absorption, distribution, metabolism and excretion (ADME) and in the treatment of diseases, siRNA offers a way to gather interpretable mechanistic data-a distinct advantage over the use of "specific" chemical inhibitors. This mini review provides background information on siRNA as well as examples of the use of siRNA as applied to drug transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Cancer-derived VEGF plays no role in malignant ascites formation in the mouse.

AIM: Vascular endothelial growth factor (VEGF) is a potent mediator of peritoneal fluid accumulation following tumor progression. This study investigated the role of VEGF secreted by cancerous cells in the formation of malignant ascites. METHODS: VEGF expression was eliminated by knockdown in the pancreas cancer cell-line PancO2 using vector-based short-hairpin type RNA interference (RNAi). Malignant ascites formation in the mouse was analyzed by intraperitoneal injection of PancO2 cells expressing VEGF or with expression knockdown. RESULTS: The VEGF knockdown PancO2 cell was successfully established. Knockdown of VEGF did not affect cancer cell proliferation in vitro or in vivo. The volume of ascites following peritoneal expansion of the tumor in VEGF knockdown cells and control cells did not differ statistically in this in vivo study. Moreover, the VEGF concentration in the ascites did not differ statistically. CONCLUSION: Malignant ascites formation might be mediated by VEGF production in noncancerous tissues, such as stromal compartments. An anti-VEGF strategy against malignant ascites could be applied to various tumors regardless of whether they secrete VEGF.

Animals↗
PubMed2005

SH2-containing 5'-inositol phosphatase, SHIP2, regulates cytoskeleton organization and ligand-dependent down-regulation of the epidermal growth factor receptor.

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3'-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors.

Actins↗