Biomedical subjects

M Miyagishi

Publications and source records attributed to M Miyagishi.

18 recordsLinked to original sources

PubMed2006

Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers.

Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.

Animals↗

PubMed2006

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Base Sequence↗

PubMed2005

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

Adenoviridae↗

PubMed2002

Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries.

A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo : that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.

Gene Expression Profiling↗

PubMed2001

Transport of intracellularly active ribozymes to the cytoplasm.

Ribozymes are RNA molecules with enzymatic activity which selectively bind and cleave specific target RNAs. To date, numerous studies directed toward the application of ribozymes in vivo have been performed and many successful experiments have been reported. However, to induce high-level activities of ribozymes in vivo, several factors must be considered. Here we report that the cytoplasmic localization of ribozymes is important for their intracellular activity in mammalian cells. Northern blot analysis revealed that a tRNA(Val) ribozyme, which can assume a cloverleaf structure similar to that of a native tRNA, is efficiently transported to the cytoplasm. In contrast, the tRNAiMet-driven ribozyme, which does not maintain the cloverleaf structure, remained predominantly in the nucleus. In correlation with the localization, the activity of the exported ribozyme was higher than that of the ribozyme retained in the nucleus. These results should provide insight into the design of ribozymes that have high-level activity in mammalian cells.

Base Sequence↗

PubMed2001

Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription.

BACKGROUND: Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes. The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors. To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2. RESULT: We found that Nrf2 contains two transcription activation domains, Neh4 and Neh5, which act synergistically to attain maximum a activation of reporter gene expression. Neh4 and Neh5 both individually and cooperatively bind to CBP (CREB (cAMP Responsive Element Binding protein) Binding Protein). In fact, the specific inhibitor of CBP, adenovirus E1A protein, significantly reduced Nrf2 activity. Importantly, the CBP-binding activity of Nrf2 deletion mutants positively correlated with their transactivation activity. Neh5 contains a motif which is commonly conserved among the CNC factors, whereas Neh4 contains the novel CBP-interacting motif recently identified in p53 and E2F. CONCLUSIONS: Our results indicate that Nrf2 exploits the cooperative binding of two independent transactivation domains to CBP in the acquisition of a potent transactivation activity.

Amino Acid Sequence↗

PubMed2001

Suppression of gene expression by RNA interference in cultured plant cells.

Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

Animals↗

PubMed2001

A simple and rapid system for the quantitation of RNA interference in plant cultured cells.

The phenomenon known as RNA interference (RNAi) by double-stranded RNA (dsRNA) that was reported recently in the nematode Caenorhabditis elegans has been shown to operate by a mechanism that is widely conserved among species including plant cells. No quantitative analysis of the effects of RNAi on the expression of specific genes in plant cultured cells has been reported. An RNAi effect was observed 24 h after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

Cell Line↗

PubMed2000

Regulation of Lef-mediated transcription and p53-dependent pathway by associating beta-catenin with CBP/p300.

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that beta-catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of beta-catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable beta-catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of beta-catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.

Apoptosis↗

PubMed2000

Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes. Cross-talk via trans-activation of the tyrosine kinase c-kit receptor.

We previously reported that activation of mitogen-activated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-1 (ET-1). In the present study, we determined signaling mechanisms upstream of MAPK activation that are involved in ET-1 stimulation and their synergism with stem cell factor (SCF). Pretreatment of cultured NHMC with ET(B) receptor antagonists, pertussis toxin, a specific phospholipase C inhibitor (), or a protein kinase C inhibitor (calphostine) blocked a transient tyrosine phosphorylation of MAPK induced by ET-1, whereas the addition of a calcium chelator (BAPTA) failed to inhibit that tyrosine phosphorylation of MAPK. Treatment with ET-1 and SCF together synergistically increased DNA synthesis, which was accompanied by synergism for MAPK phosphorylation. The time course of inositol 1,4,5-trisphosphate formation revealed that there is no difference in the level of inositol 1,4,5-trisphosphate stimulated by ET-1 + SCF or by ET-1 alone. Evaluations of the serine phosphorylation of MEK and Raf-1 activity showed a synergistic effect in SCF + ET-1-treated NHMC. Stimulation with SCF + ET-1 induced a more rapid and stronger tyrosyl phosphorylation of proteins corresponding to p52 and p66 Shc than did stimulation with SCF only, and this was accompanied by a stronger association of tyrosine-phosphorylated Shc with Grb2. Interestingly, a more rapid and marked tyrosine phosphorylation of c-kit was also detected in NHMC-treated with SCF + ET-1 than NHMC treated with SCF only. These data indicate that the synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of tyrosine phosphorylation of c-kit, which results in the enhanced formation of the Shc-Grb(2) complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines. The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells. Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities. Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.

3T3 Cells↗

PubMed2000

Molecular characterization of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

Members of the basic helix-loop-helix (bHLH) family of transcription factors have been implicated in the regulation of cell proliferation and differentiation. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Northern blot analysis showed that POD-1/Capsulin are preferentially expressed in human vascular smooth muscle cells, and are enriched in mouse heart, kidney, testis, spleen, and lung. Ectopic expression of the HA-tagged POD-1/Capsulin in 293T cells revealed that this protein has an apparent molecular mass of 26 kDa and localized in the nucleus. Furthermore, in order to identify possible interactants of the bHLH protein, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified alternative-splicing form of HEB, as potential heterodimeric partner of POD-1/Capsulin. Our results provide the molecular basis for cell type-specific transcriptional regulation by POD-1/Capsulin.

3T3 Cells↗

PubMed1997

Functional association between CBP and HNF4 in trans-activation.

CBP functions as a key transcriptional coactivator for a variety of transcription factors. We show here that the hepatocyte nuclear factor 4 (HNF4), a transcription factor in the nuclear receptor superfamily with no defined ligand, is cloned by yeast two-hybrid system using CBP as a bait. GST-pull down assay with nuclear extracts or in vitro translation products revealed that CBP and HNF4 interact with each other at the middle portion (aa 119-375) of HNF4 and two distinct regions (aa 271-451 and 1626-2259) of CBP, respectively, in the ligand-independent manner. Co-transfection experiments indicated that CBP is capable of activating HNF4 site-mediated transcription. These results suggested a functional association between CBP and HNF4 in trans-activation.

Animals↗

PubMed1997

The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis.

The paracrine linkage of endothelins (ET) between keratinocytes and melanocytes suggested that ETs are intrinsic mediators for human melanocytes in UVB-induced pigmentation. In this study, the role of ET-1 in the epidermal hyperpigmentation was investigated in vivo and in vitro. The addition of 10 nM ET-1 induced a H-7 (10 microM) suppressible-increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase-related protein-1 mRNA expression as shown by Northern blotting. Analysis of signaling mechanisms leading to tyrosinase activation demonstrated the involvements of quick translocation of PKC, the H-7 (10 microM) suppressible-phosphorylation of the threonine residue of several proteins, and highly elevated level of cyclic AMP (4-fold over control). Reverse transcription polymerase chain reaction (RT-PCR) of RNA isolated from the epidermis of human skin exposed to UVB revealed that UVB irradiation with a dose of 2 MED caused a significant increase in the expressions of ET-1, IL-1 alpha, and tyrosinase mRNA signals 5 days after irradiation. The involvement of ET-1 in UVB-pigmentation was also corroborated by the experiments that the extracts of M. Chamomilla, which can act as an antagonist for ET-receptor binding-mediated signaling but has no inhibitory effect on tyrosinase activity in culture, had a significant inhibitory effect on UVB-induced pigmentation in vivo when daily applied immediately after UVB exposure to human skin. These findings suggest that ET-1 is an important mediator in the epidermis for UVB-induced pigmentation in vivo.

Cell Division↗

PubMed1996

Role of endothelin-1 in hyperpigmentation in seborrhoeic keratosis.

Seborrhoeic keratosis (SK) is a benign epidermal tumour with a varying degree of pigmentation. We have recently demonstrated that endothelin-1 (ET-1) is a strong keratinocyte-derived mitogen and melanogen for human melanocytes in UVB-induced melanosis. To clarify the role of ET-1 in hyperpigmentation in SK, we have used immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to see whether the production of ET-1 is accentuated in SK. Immunohistochemical analysis in SK (n = 7; acanthotic and deeply pigmented types) revealed marked immunostaining with anti-ET-1 in almost all basaloid and basal cells as compared with definite staining confined to basal cells in the perilesional normal controls. In parallel, RT-PCR of ET-1 mRNA demonstrated accentuated expression of ET-1 transcript in SK (n = 4) in comparison with that in the perilesional normal controls, accompanied by a similarly accentuated expression of tyrosinase mRNA. These findings suggest that ET-1 plays a part in the hyperpigmentation seen in SK.

Adult↗

PubMed1995

Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.

We previously demonstrated that human keratinocytes produce and secrete endothelins (ET), which can be strong mitogens for human melanocytes. Ultraviolet B (UVB) exposure highly stimulated the paracrine linkage of endothelins between keratinocytes and melanocytes, indicating that they are keratinocyte-derived intrinsic mitogens in UVB-induced pigmentation. In this study, the role of ET-1 as a melanogen in UVB melanogenesis was investigated in vitro and in vivo. In the conditioned medium of keratinocytes exposed to UVB, melanin synthesis by human melanocytes, as measured by 14C-thiouracil incorporation, was significantly accentuated. This stimulatory effect was reduced by anti-ET-1 to the level of that in the non-UVB-exposed control, suggesting an essential role of ET-1 as an intrinsic melanogen in UVB-induced melanogenesis. In a parallel study, the addition of 10 nM ET-1 induced an increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase-related protein-1 mRNA expression as shown by Northern blotting. Reverse transcription-polymerase chain reaction of RNA isolated from the epidermis of human skin exposed to UVB revealed that, whereas in non-exposed sites ET-1, IL-1 alpha, and tyrosinase mRNA signals were scarcely detected, UVB-irradiation, with a dose of twice the minimal erythema dose, caused a significant increase in the expressions of the three genes 5 d after irradiation. These findings suggest that ET-1 is an important mediator for UVB-induced pigmentation in the epidermis in vivo.

Base Sequence↗

PubMed1992

Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.

We recently demonstrated that human melanocyte proliferation and differentiation could be stimulated by endothelin (ET) derivatives via a receptor-mediated signal transduction pathway (Yada, Y., Higuchi, K., and Imokawa, G. (1991) J. Biol. Chem. 266, 18352-18357). We show here that the growth factors for human melanocytes are produced and secreted by the surrounding cells, namely human keratinocytes for ET-1 and Big-ET-1. Northern blots have revealed the presence of ET-1 gene transcripts in proliferating human keratinocytes. The ET-1 production by human keratinocytes increased after irradiation with ultraviolet B (UVB) in a dose-dependent manner, accompanied by the significant secretion of interleukin 1 alpha (IL-1 alpha). Among the cytokines related to UVB-induced cellular reactions and keratinocyte growth, only IL-1 alpha and -1 beta stimulated the secretion of ET-1 and Big-ET-1 but not of ET-3 and Big-ET-3 in a time-dependent manner. Northern blots for IL-1 alpha-stimulated or UVB-exposed human keratinocytes revealed that production of ET-1 gene transcripts markedly increased (by about 300 or 1,200%) with constant levels of beta-actin gene transcripts. In a parallel study, the medium conditioned by UVB-exposed human keratinocytes elicited a significant anti-ET-1 antibody-suppressible increase in DNA synthesis by cultured human melanocytes in a UV dose-dependent manner, which was associated with a marked and rapid (80 s) increase in the intracellular calcium level upon incubation with human melanocytes. These studies suggest that ETs produced and secreted by keratinocytes play an essential role in the maintenance of melanocyte proliferation and UV hyperpigmentation in the epidermis.

Actins↗

PubMed1990

Cloning of cDNA and amino acid sequence of one of chicken cone visual pigments.

The chicken has four kinds of color visual pigments, in addition to rhodopsin. A chicken genomic DNA library was screened with cDNA of human red-sensitive pigment and a chicken genomic DNA fragment including rhodopsin exons 2, 3 and 4, and then a genomic DNA fragment encoding a visual pigment, possibly an iodopsin, was cloned. A cDNA library, constructed from chicken retina mRNA, was screened with the genomic DNA fragment and the cDNA of human red-sensitive pigment, and the cDNA encoding the pigment was cloned. The nucleotide sequence of this cDNA was similar to that of the human red-sensitive pigment, with identities of 78% for the nucleotide sequence and 84% for the amino acid sequence with human red-sensitive pigment.

Amino Acid Sequence↗