Retraction: Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.
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Retraction Notice↗
Biomedical subjects
Eigo Suyama
Publications and source records attributed to Eigo Suyama.
Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo.
As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.
Animals↗
Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation.
We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.
Animals↗
A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro.
Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.
Capsid Proteins↗
Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes.
Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.
Animals↗
An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease.
Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.
Aged↗
LIM kinase-2 targeting as a possible anti-metastasis therapy.
BACKGROUND: Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. METHODS: We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. RESULTS: Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. CONCLUSIONS: Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells.
Base Sequence↗
Cell migration and metastasis as targets of small RNA-based molecular genetic analyses.
Metastatic tumor cells can migrate from one place to another in the body. This involves their adherence to host cell layers and subsequent transcellular movements by a complex process, molecular basis of which are yet to be clarified. Elucidation of genes functionally involved in metastasis may lead to deeper understanding of the mechanism of cell migration, and identification and designing of metastasis-modulating strategies for cancer therapeutics. We review here cell migration in tumor metastasis and the use of small RNA-based approaches to identify functional genes. We then describe our promising novel approach that uses randomized ribozyme libraries for identification of genes involved in cell migration, a consistent feature of metastatic cells.
Animals↗
Identification of genes involved in cell invasion by using a library of randomized hybrid ribozymes.
Libraries of RNA helicase-coupled randomized ribozymes are a powerful tool for the identification of functional genes. We have demonstrated the usefulness of this functional gene-discovery system by identifying genes involved in tumor invasion, a process that is an essential feature of tumor metastasis: the spread of cancer cells from the original tumor to other sites in the body that imposes serious problems in the prognosis and treatment of cancer. Using a filter-based invasion assay in vitro, we isolated ribozymes that enhanced the invasive properties of NIH 3T3 fibroblasts. Sequence analysis of selected clones and a database search revealed that genes such as the gene for Gem GTPase and uncharacterized genes that resemble genes for myosin phosphatase and protein-tyrosine-phosphatase are involved in cell invasion. Our system for gene identification by using ribozymes and the functional analysis of target genes should help to clarify the complex mechanisms of invasion and metastasis and might provide information that is relevant to cancer therapy.
3T3 Cells↗
siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells.
RNA interference has emerged as a powerful tool for the silencing of gene expression in animals and plants. It was reported recently that 21 nt synthetic small interfering RNAs (siRNAs) specifically suppressed the expression of endogenous genes in several lines of mammalian cells. However, the efficacy of siRNAs is dependent on the presence of a specific target site within the target mRNA and it remains very difficult to predict the best or most effective target site. In this study, we demonstrate that siRNAs that have been generated in vitro by recombinant human Dicer (re-hDicer) significantly suppress not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras, c-jun and c-fos. In our system, selection of a target site is not necessary in the design of siRNAs. However, it is important to avoid homologous sequences within a target mRNA in a given protein family. Our diced siRNA system should be a powerful tool for the inactivation of genes in mammalian cells.
Cloning, Molecular↗
Identification of genes responsible for cell migration by a library of randomized ribozymes.
Several genes appear to be associated with metastasis, but the underlying mechanisms of metastasis still remain unclear. In this study, we used a library of randomized ribozymes to identify, by inactivation of transcripts, genes involved in cell migration that is an essential aspect of metastasis. Using a chemotaxis assay, the ribozymes that inhibited cell migration were selected from the library. Among such ribozymes, we found two ribozymes that targeted and cleaved ROCK1 mRNA at independent sites. ROCK1 and ROCK2 are Rho kinases, and it has been demonstrated that they regulate the organization of the actin cytoskeleton and are responsible for cell motility and cytokinesis. The two ribozymes that specifically cleaved ROCK1 mRNA inhibited both the migration and invasion of invasive HT1080 fibrosarcoma, but neither had any effect on cell proliferation. Our analysis indicates that the ribozymes toward ROCK1 can block invasive activity but not the proliferation of HT1080 cells without having any effect on expression of ROCK2. Ribozymes identified in this study, including the ribozymes against ROCK1, might be useful in understanding the mechanisms of cell migration and metastasis.
Animals↗
Cancer gene validation using ribozymes and macroarray.
Transcription factor AP-2 seems to play an important role in the malignancy of melanoma. In this study, we constructed ribozyme expression vectors to suppress the expression of AP-2 (AP-2-ribozymes) and then examined gene expression in human A375P melanoma cells that stably expressed ribozymes targeted to the AP-2 transcript. A comparison of the gene-expression profiles of A375P cells that expressed AP-2-ribozymes and those transfected with the empty vector revealed changes in levels of expression of several genes. Here we described that the combination of gene suppression by ribozymes and the analysis of gene expression using a macro-array provides a good approach for elucidating signal transduction pathways. These results provide further insight into the role of AP-2 in human melanoma cells.
DNA-Binding Proteins↗
Use of a ribozyme library for validation of gene functions and cellular pathways.
An effective and simple technology is required to identify genes that function in a particular phenotype of interest. A system for identification of genes with libraries of randomized ribozymes has a great potential to investigate gene functions and cellular pathways. This system has been used to identify genes that are relevant to several phenotypes. In vivo application of the library of ribozymes in mice model was also performed to identify genes involved in metastasis. Libraries of ribozymes might be used in vitro and in vivo to study various aspects of basic cell biology and disease processes including metastasis.
Gene Library↗
[RNAi-mediated specific gene silencing and its application to medical treatments of viral infectious disease].
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Animals↗
[Gene discovery system with randomized ribozyme libraries].
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Apoptosis↗
[In vivo gene discovery system with libraries of randomized ribozymes].
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Animals↗
Identification of a caspase 3-independent role of pro-apoptotic factor Bak in TNF-alpha-induced apoptosis.
By using our recently developed gene discovery system, we have identified Bak, a member of the Bcl-2 family, as a pro-apoptotic factor in the tumor necrosis factor (TNF)-alpha-induced apoptotic pathway in caspase 3-deficient cells. Unlike Bcl-2, Bak stimulates several apoptotic pathways, however the molecular mechanism(s) of its action remains unclear. For example, it is unclear whether Bak induces apoptosis in caspase 3-deficient cells. In this study, we examined the effects of overexpression of Bak in MCF-7 cells that lack caspase 3. We found that despite the absence of caspase 3 in MCF-7 cells, they were more sensitive to the cell death effects of Bak as compared to caspase 3-expressing HeLa S3 cells. The targeting of Bak function by ribozymes suggests that Bak is required for the TNF-alpha-induced apoptotic pathway in caspase 3-deficient cells. This study demonstrates the caspase 3-independent function of Bak in the TNF-alpha-induced apoptotic pathway.
Apoptosis↗
Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.
Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.
Apoptosis↗