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PubMed2001

Double-stranded RNA-mediated interference with plant virus infection.

Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not single-stranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.

Gene Silencing↗
PubMed2006

RNA interference-mediated simultaneous down-regulation of urokinase-type plasminogen activator receptor and cathepsin B induces caspase-8-mediated apoptosis in SNB19 human glioma cells.

The invasive character of gliomas depends on proteolytic cleavage of the surrounding extracellular matrix. Cathepsin B and urokinase-type plasminogen activator receptor (uPAR) together are known to be overexpressed in gliomas and, as such, are attractive targets for gene therapy. In the present study, we used plasmid constructs to induce the RNA interference (RNAi)-mediated down-regulation of uPAR and cathepsin B in SNB19 human glioma cells. We observed that the simultaneous down-regulation of uPAR and cathepsin B induces the up-regulation of proapoptotic genes and initiates a collapse in mitochondrial Deltapsi. Cathepsin B and uPAR down-regulated cells showed increases in the expression of activated caspase-8 and DFF40/caspase-activated DNase. Nuclear translocation of AIF and Fas ligand translocation to the cell membrane were also observed. Ki67 and X-linked inhibitor of apoptosis protein levels decreased, thereby indicating apoptosis. These results suggest the involvement of uPAR-cathepsin B complex on the cell surface and its role in maintaining the viability of SNB19 glioma cells. In conclusion, RNAi-mediated down-regulation of uPAR and cathepsin B initiates a partial extrinsic apoptotic cascade accompanied by the nuclear translocation of AIF. Our study shows the potential of RNAi-mediated down-regulation of uPAR and cathepsin B in developing new therapeutics for gliomas.

Apoptosis↗
PubMed2004

A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in rat-1 cells.

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.

Animals↗
PubMed2005

RNA interference-mediated silencing of the p53 tumor-suppressor protein drastically increases apoptosis after inhibition of endogenous fatty acid metabolism in breast cancer cells.

Fatty acid synthase (FAS)-dependent endogenous fatty acid synthetic activity is abnormally elevated in a biologically-aggressive subset of breast carcinomas. Remarkably, tumor-associated FAS hyperactivity represents a novel target for anti-metabolic therapy because pharmacological inhibitors of FAS are selectively cytotoxic for tumor cells, triggering their apoptotic cell death. Since the p53 tumor-suppressor protein (TP53) is thought to play a novel role in cellular responses of a variety of non-genotoxic metabolic stresses, we characterized the involvement of TP53 in the response of breast cancer cells to FAS inhibition. MCF-7 breast cancer cells were selected for study because they have an intact TP53 pathway and undergo little apoptosis following FAS blockade. Two chemically distinct inhibitors of FAS (the natural mycotoxin cerulenin and the novel small-molecule inhibitor C75) were studied in parallel to provide a broad picture of consequences suffered by the loss of FAS function on TP53 signaling. Treatment with either cerulenin or C75 induced TP53 protein accumulation at 24 h in MCF-7 cells. To determine whether the up-regulation of TP53 following exposure to cerulenin or C75 was solely due to inhibition of endogenous fatty acid metabolism, we first evaluated the cytotoxic response to chemical FAS blockers on MCF-7 cells in which FAS gene expression was previously silenced by using the highly sequence-specific mechanism of RNA interference. MCF-7 cells became insensitive to C75-induced cytotoxicity when the expression of FAS was specifically suppressed by targeted knock-down with small interfering RNA (siRNA), whereas they partially retained their sensitivity to cerulenin. These results demonstrate that C75-induced cytotoxic damage to breast cancer cells is closely dependent on its ability to inhibit FAS-catalyzed endogenous fatty acid biogenesis, thus ruling out a significant direct effect of C75 on DNA. To determine the functional role of TP53 on breast cancer cell survival after FAS blockade, we evaluated FAS inhibitor-mediated apoptosis in MCF-7 cells transiently transfected with a pool of sequence-specific double-stranded RNA oligonucleotides targeting TP53 gene. In these conditions, TP53 protein levels were unchanged during the period of FAS-inhibitor exposure. Remarkably, siRNA-induced silencing of TP53 gene expression did result in a dramatic increase (approximately 300%) in apoptotic cell death following exposure to C75. Strikingly, there was no apparent relationship between the TP53 mutational status and sensitivity to chemical FAS inhibitor in a panel of human breast cancer cell lines. However, the degree of TP53 mRNA expression was predictive of sensitivity to C75-induced cytotoxicity, with low-TP53 mRNA expressing breast cancer cells showing hypersensitivity to FAS blockade. These findings strongly suggest that: a) TP53 is a novel molecular sensor of energy imbalance after the perturbation of endogenous fatty acid metabolism in breast cancer cells; b) TP53 function closely influences the decision between apoptosis and growth arrest following FAS blockade; and c) pharmacological inhibitors of FAS activity may be clinically useful against breast carcinomas exhibiting mutation or aberrant expression of TP53.

Apoptosis↗
PubMed2002

RNA interference-mediated silencing of Sod2 in Drosophila leads to early adult-onset mortality and elevated endogenous oxidative stress.

Oxidative stress has been widely implicated as an important factor in the aging process. Because mitochondrial respiration is the principal source of reactive oxygen within cells, the mitochondrially localized superoxide dismutase (SOD) 2 is thought to play an important front-line defensive role against aging-related oxidative stress. Although genetic studies with mutants deficient in SOD1, the predominantly cytosolic isoform of SOD, have been instrumental in elucidating the role of reactive oxygen metabolism in aging in Drosophila, the lack of available mutations in the Sod2 gene has hampered an equivalent analysis of the participation of this important antioxidant enzyme in the Drosophila aging model. Here we report that ablation of mitochondrial SOD2 through expression of a GAL4-regulated, inverted-repeat Sod2 RNA-interference transgene in an otherwise normal animal causes increased endogenous oxidative stress, resulting in loss of essential enzymatic components of the mitochondrial respiratory chain and the tricarboxylic acid cycle, enhances sensitivity to applied oxidative stress, and causes early-onset mortality in young adults. In sharp contrast, ablation of SOD2 has no overt effect on the development of larvae and pupae, which may reflect a fundamental transition in oxygen utilization andor reactive oxygen metabolism that occurs during metamorphosis from larval to adult life.

Animals↗
PubMed2006

RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes.

DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes "driving" amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20-30% of tumors exhibit amplification within chromosome band 17q12, containing the ERBB2 oncogene. Analysis of array-based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon "core") at 17q12 includes two other genes, GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell-cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell-cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified with ERBB2 at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes.

Breast Neoplasms↗
PubMed2006

RNA interference-mediated prevention and therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and is on the increase worldwide. Hepatocellular carcinoma results from chronic liver disease and cirrhosis most commonly associated with chronic hepatitis B (HBV) or hepatitis C (HCV) infection. The highest incidences of HCC are found in China and Africa, where chronic HBV infection is the major risk component. In the United States, Europe and Japan, the significant increase in HCC and HCC-related deaths within the last three decades is mainly attributed to the rise in the number of HCV-infected individuals; smaller increases of HCC are associated with HBV. Given that HCV and HBV infection account for the majority of HCCs, therapeutic and prophylactic approaches to control or eliminate virus infection may prove effective in reducing the occurrence of HCC. Although anti-viral therapies exist for both HBV and HCV infections, they are ineffective for a significant number of patients. In addition, some treatments such as interferon therapy are dose limiting owing to toxic side effects. Clearly, new approaches are needed. RNA interference (RNAi)-based approaches may meet this need and have already shown promising preclinical results in cell culture and animal models. Although this paper focuses on the potential of RNAi as a prophylactic for HCC development, the potential use of RNAi-mediated approaches for HCC therapy will also be discussed.

Animals↗
PubMed2005

Assembly and function of RNA silencing complexes.

In the RNA-interference pathway, double-stranded RNA induces sequence-specific mRNA degradation through the action of the RNA-induced silencing complex (RISC). Recent work has provided our first glimpses of the RISC-assembly pathway and uncovered the biochemical roles of critical RISC components. These advances have taken our mechanistic understanding of RNA interference to a new level and promise to improve our ability to exploit this biological process for use in experimental biology and medicine.

Animals↗
PubMed2004

Preextinction viral RNA can interfere with infectivity.

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.

Animals↗
PubMed1995

Use of a polyethylene glycol-peptide conjugate in a competition gel shift assay for screening potential antagonists of HIV-1 Tat protein binding to TAR RNA.

Interference of binding of Tat protein to TAR RNA in HIV-1-infected cells may be a useful therapeutic strategy for AIDS. An electrophoretic assay to screen potential low-molecular-weight (< 2 kDa) Tat antagonists has been established. A radiolabeled TAR RNA fragment (delta TAR) is retarded in mobility when bound by a Tat peptide-polyethylene glycol conjugate (Tat-PEG), which is used in place of the Tat protein. The assay determines the ability of a potential antagonist to compete with Tat-PEG for binding to delta TAR, as measured by interference with the gel shift of delta TAR. To discriminate between specific and nonspecific interactions, the assay is done in the absence or the presence of a 250-fold molar excess of tRNA.

Amino Acid Sequence↗
PubMed2000

Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.

We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana. To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter. When introduced into the genome of A. thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference. The severity of phenotypes varied between transgenic lines. In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference. The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A. thaliana.

AGAMOUS Protein, Arabidopsis↗
PubMed2003

Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells.

Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. Clinically, MDR is one of the major causes for chemotherapeutic treatment failure in cancer patients. To explore a new approach to circumventing MDR, we adopted RNA interference to target MDR1 gene expression. RNA interference is a conserved biological response to double-stranded RNA, which results in sequence-specific gene silencing [G. J. Hannon, Nature (Lond.), 418: 244-251, 2002]. We report that introduction of an MDR1-targeted small interfering RNA duplex into drug-resistant cancer cells markedly inhibited the expression of MDR1 mRNA and P-gp, as determined by reverse transcription-PCR and Western blot. Inhibition of P-gp expression by small interfering RNA enhanced the intracellular accumulation of and selectively restored sensitivity to drugs transported by P-gp. These studies indicate that RNA interference can modulate MDR in preclinical models.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant.

BACKGROUND & AIMS: Present therapy for chronic hepatitis B attains control only in limited proportions. Small interfering RNA (siRNA) offers a new tool with potential therapeutic applications for hepatitis B virus (HBV). Given the importance of sequence identity in the effectiveness of siRNA and the heterogeneity of HBV sequences among different isolates, a short hairpin RNA (shRNA)-expressing plasmid, pSuper/HBVS1, was developed to target a region conserved among major HBV genotypes and assess its effectiveness control of HBV. METHODS: HBV replication-competent plasmid was cotransfected with pSuper/HBVS1 to HuH-7 cells or to mice. The levels of viral proteins, RNA, and DNA were examined in transfected cells and animals. The effects of pSuper/HBVS1 on clinical isolates with genotypes B and C were also determined. RESULTS: pSuper/HBVS1 significantly decreased levels of viral proteins, RNA, and DNA for HBV genotype A in cell culture and in mice. Comparable suppressive effects were observed on clinical isolates of genotypes B and C. A clone with a silent mutation in the target region was identified from a patient with genotype C. This mutant revealed diminished sensitivity to pSuper/HBVS1 and could be selected out in the presence of pSuper/HBVS1 in cell culture. CONCLUSIONS: These findings indicated that shRNA could suppress HBV expression and replication for genotypes A, B, and C, promising an advance in treatment of HBV. However, the emergence of resistant mutants in HBV quasispecies should be considered.

Animals↗
PubMed2004

RNAi: running interference for the cell.

RNA interference or RNAi is a recently characterized mechanism of eukaryotic gene regulation in which a short sequence of double-stranded RNA (dsRNA) specifically down-regulates expression of the associated gene. Preliminary characterization of this phenomenon has revealed a set of inter-related cellular pathways which appear to represent both a response to foreign RNA and a mechanism of endogenous gene regulation. Introduction of dsRNA into cells by a variety of means, including transfection of synthetic RNA duplexes, triggers the RNAi response resulting in specific suppression of target gene expression. Recent efforts on a genome wide scale have involved application of RNAi as an important new tool in cell biology to elucidate gene function in living cells.

Animals↗
PubMed2006

[A murine model of transitory optic neuropathy based on small interference RNA-induced OPA1 silencing in vivo (gene mutation associated with Kjer's disease)].

PURPOSE: Developing a murine model of OPA1 linked optic neuropathy. METHODS: Intravitreal injections (in adult C57BL/6J mice) of small interference RNA (siRNA) specific to OPA1 were performed in the left eye. The right eye served as control, injected with nonspecific siRNA (siRNA scramble). Visual evoked potentials and flash electroretinograms were performed 5 and 12 days after injection. Three months after injection, microscopy of optic nerve sections was performed. RESULTS: The electrophysiological tests showed a significant reduction in the VEP when the siRNA OPA1-injected eye was stimulated, compared with the control eye injected with siRNA scramble. The electroretinogram was normal in both eyes: no significant difference between the right and the left eye was found. Three months after injection, no measurable axonal degeneration was found in either eye. CONCLUSION: The reduced expression of OPA1 based on RNA silencing in adult mice could induce reversible dysfunction of retinal ganglion cells.

Animals↗
PubMed1982

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. II. Evidence that large N-terminal amber fragments of the beta subunit interfere with RNA polymerase function.

A collection of 95 independent, spontaneously-occurring mutants carrying amber lesions that affect expression of the beta gene, rpoB, has been isolated (see accompanying paper (Nene and Glass 1982)). Certain rpoB amber mutations act in trans, preventing a functional allele present on an F' plasmid from acting at high temperature. Two such temperature-sensitive rpoB(Am) strains are shown to produce large, N-terminal amber fragments. The possibility that these truncated polypeptides are the cause of this transdominant conditional-lethal phenotype is supported by analysis of fragment levels in thermoresistant survivors: the nonsense fragments are degraded at a significantly faster rate (half-lives 1.4- to 2.6-fold reduced) in Ts+ derivatives likely to carry second-site mutations within rpoB. We suggest that the beta fragments interfere with RNA polymerase function by interacting with one or more of the polymerase subunits.

DNA-Directed RNA Polymerases↗
PubMed2005

Growth inhibition of K562 cells by cyclin E gene-specific small interfering RNA.

OBJECTIVE: To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells. METHODS: siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via Lipofectamine2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference. RESULTS: Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G(1)-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%; as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels. CONCLUSION: RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.

Cell Proliferation↗