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Takahiro Ochiya

Publications and source records attributed to Takahiro Ochiya.

At least 19 recordsLinked to original sources
PubMed2006

A photon counting technique for quantitatively evaluating progression of peritoneal tumor dissemination.

We recently established a mouse model of peritoneal dissemination of human gastric carcinoma, including the formation of ascites, by orthotopic transplantation of cultured gastric carcinoma cells. To clarify the processes of expansion of the tumors in this model, nude mice were sacrificed and autopsied at different points of time after the orthotopic transplantation of the cancer cells for macroscopic and histopathologic examination of the tumors. The cancer cells grew actively in the gastric submucosa and invaded the deeper layers to reach the serosal plane. The tumor cells then underwent exfoliation and became free followed by the formation of metastatic lesions initially in the greater omentum and subsequent colonization and proliferation of the tumors on the peritoneum. Although this model allowed the detection of even minute metastases, it was not satisfactory from the viewpoint of quantitative and objective evaluation. To resolve these problems, we introduced a luciferase gene into this tumor cell line with a high metastasizing potential and carried out in vivo photon counting analysis. This photon counting technique was found to allow objective and quantitative evaluation of the progression of peritoneal dissemination on a real-time basis. This animal metastatic model is useful for monitoring the responses of tumors to anticancer agents.

Animals↗
PubMed2006

Ductal origin of pancreatic adenocarcinomas induced by conditional activation of a human Ha-ras oncogene in rat pancreas.

Pancreatic ductal adenocarcinoma is one of the most debilitating malignancies in humans. Currently, radiation and chemotherapy are ineffective, with median survival times after treatment of <12 months. Animal models that reflect the human condition and can be used to explore screening and therapeutic approaches are clearly desirable. One feature of human pancreatic adenocarcinoma is an exceedingly high frequency of K-ras mutation. The present study was conducted to determine if targeted activation of a human oncogenic-ras transgene in rat pancreas would induce carcinomas correspondent to human pancreatic ductal adenocarcinomas. We established transgenic (Hras250) rats in which expression of a human Ha-rasG12V oncogene is regulated by the Cre/lox system. Targeted pancreatic activation of the transgene was accomplished by injection of Cre-carrying adenovirus into the pancreatic ducts and acini through the common bile duct. Adenoviral infection of injected animals was exclusive to the pancreas; infected cells could be identified in duct, intercalated duct, centroacinar and, less frequently, acinar cells, but not in endocrine islet cells. Four weeks after injection, proliferative lesions in the duct epithelium, intercalated ducts and centroacinar cells, but not acinar cells, were widespread. Tumorigenesis in other tissues was not observed. Most lesions, including atypical duct proliferative lesions, PanIN-like lesions and carcinomas, were positive for cytokeratins 19 and 7, cyclooxygenase 2 and MMP-7 but negative for amylase and chymotrypsin. Many adenocarcinoma lesions were positive for EGF and EGFR. Duct epithelial and atypical duct proliferative lesions and carcinoma lesions were all positive for transduced Ha-rasG12V oncogene expression. The cytogenesis of pancreatic ductal type carcinoma was depicted. This model exhibits important similarities to the human disease and promises to advance our understanding of the behavior of pancreas adenocarcinomas and expedite screening and therapy.

Adenocarcinoma↗
PubMed2006

FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation.

The FGF-4 (fibroblast growth factor 4, known as HST-1) protein is an important mitogen for a variety of cell types. However, only limited information is available concerning tissue distribution and the biological role of FGF-4 in the brain. In situ hybridization analysis revealed localization of mouse Fgf-4 mRNA in the normal postnatal mouse hippocampus, subventricular zone (SVZ), and the rostral migratory stream where new neurons generate, migrate, and become incorporated into the functional circuitry of the brain. We also investigated whether FGF-4 could promote both proliferation and differentiation of the neural progenitor cells by using an in vitro neurosphere assay. The addition of recombinant FGF-4 generated large proliferative spheres that have a multipotent differentiation ability. Furthermore, recombinant FGF-4 significantly promotes neuronal differentiation in attached clonal neurosphere culture. These findings suggest that FGF-4 has an ability to promote neural stem cell proliferation and neuronal differentiation in the postnatal brain.

Animals↗
PubMed2006

MOZ is essential for maintenance of hematopoietic stem cells.

Monocytic leukemia zinc-finger protein (MOZ), a MYST family histone acetyltransferase, is involved in the chromosome translocations associated with acute myeloid leukemia. MOZ acts as a transcriptional coactivator for AML1, which is essential for establishment of definitive hematopoiesis. To investigate the roles of MOZ in normal hematopoiesis, we generated MOZ-null mice. MOZ-/- mice died around embryonic day 15 (E15). In MOZ-/- E14.5 embryos, hematopoietic stem cells, lineage-committed progenitors, and B lineage cells were severely reduced. On the other hand, arrest of erythroid maturation and elevated myeloid lineage populations were observed. MOZ-deficient fetal liver cells could not reconstitute hematopoiesis of recipients after transplantation. Analysis using microarray and flow cytometry revealed that expression of thrombopoietin receptor (c-Mpl), HoxA9, and c-Kit was down-regulated. These results show that MOZ is required for maintenance of hematopoietic stem cells, and that it plays a role in differentiation of erythroid and myeloid cells. Some aspects of the MOZ-/- phenotype are similar to that observed in PU.1-deficient mice. MOZ was able to interact with PU.1 and activate PU.1-dependent transcription, thus suggesting a physical and functional link between PU.1 and MOZ.

Animals↗
PubMed2006

Hedgehog signal activation in gastric pit cell and in diffuse-type gastric cancer.

BACKGROUND & AIMS: Sonic hedgehog (SHH) and Indian hedgehog (IHH) have cell-specific actions in some organs. Only SHH has been shown to regulate parietal cell differentiation. This study examined whether SHH, 2 other ligands IHH and Desert hedgehog, and receptors or downstream targets are expressed in normal gastric epithelium or in intestinal and diffuse-type gastric cancers. The effects of a Hedgehog (Hh) inhibitor, cyclopamine, were assessed in primary gastric epithelium cultures and gastric cancer cell lines. METHODS: Reverse-transcription polymerase chain reaction and immunostaining compared expression and localization of Hh signaling molecules and phenotypic markers of pit, neck, and gland cells in situ and in cultured cells treated with cyclopamine. Bromodeoxyuridine staining assessed the effects of cyclopamine on proliferation. RESULTS: Hh signaling molecules were expressed differentially in pit, neck, and gland cells. IHH co-expressed with most downstream targets in the pit. IHH, SHH, Patched (PTCH), Smoothened (SMO), and downstream targets were expressed more frequently and highly in the diffuse as compared with intestinal type cancers. In diffuse cancers, IHH was expressed in cells with an epithelial phenotype and SHH in cells with a mesenchymal phenotype. Cyclopamine reduced the number of cells with a pit phenotype but not a gland phenotype in primary cultures. Cyclopamine had particularly potent effects of inhibiting the growth of cell lines that expressed high levels of SMO. CONCLUSIONS: Expression of IHH and downstream targets correlates with pit cells. IHH and SMO may be useful biomarkers of diffuse cancers that may show growth inhibition with Hh antagonists such as cyclopamine.

Animals↗
PubMed2006

Therapeutic potential of RNA interference against cancer.

One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment.

Animals↗
PubMed2006

Atelocollagen-mediated systemic DDS for nucleic acid medicines.

The goal of our research is to provide a practical platform for drug delivery in oligonucleotide therapy. We report here the efficacy of an atelocollagen-mediated oligonucleotide delivery system applied to systemic siRNA and antisense oligonucleotide treatments in animal disease models. Atelocollagen and oligonucleotides formed a complex of nanosized particles, which was highly stable against nucleases. The complex allowed oligonucleotides to be delivered efficiently into several organs and tissues via intravenous administration. In a tumor metastasis model, the complex successfully delivered siRNA to metastasized tumors in bone tissue and inhibited their growth. We also demonstrated that a single intravenous treatment of the antisense oligodeoxynucleotide complex suppressed ear dermatitis in a contact hypersensitivity model. These results indicate the strong potential of the atelocollagen-mediated drug delivery system for practical therapeutic technology.

Animals↗
PubMed2006

Central genetic alterations common to all HCV-positive, HBV-positive and non-B, non-C hepatocellular carcinoma: a new approach to identify novel tumor markers.

Hepatocellular carcinoma (HCC) is a common malignancy, but the prognosis remains poor due to the lack of sensitive diagnostic markers. To gain insight into the central molecular features common to all types of HCC, and to identify novel diagnostic markers or therapeutic targets for HCC, we performed a gene expression profiling analysis using a high throughput RT-PCR system. After examining the mRNA expression of 3,072 genes in 204 (119 tumor and 85 non-tumor) liver samples, we identified differential gene expression between the HCV group (n=80), HBV group (n=19) and non-B, non-C group (n=20) with a principal component analysis and a correlation spectrum analysis. After selection of genes differentially expressed between tumor and non-tumor tissues (p<0.01) within each HCC group, a total of 51 differentially expressed genes (23 upregulated and 28 downregulated genes) were found to be common to the three HCC groups. Gene Ontology grouping analysis revealed that genes with functions related to cell proliferation or differentiation and genes encoding extracellular proteins were found to be significantly enriched in these 51 common genes. Using an atelocollagen-based cell transfection array for functional analysis of eight upregulated genes, five (CANX, FAM34A, PVRL2, LAMR1, and GBA) significantly inhibited cellular apoptosis by two independent assays. In conclusion, we identified 51 differentially expressed genes, common to all HCC types. Among these genes, there was a high incidence of anti-apoptotic activity. This combination approach with the advanced statistical methods and the bioinformatical analysis may be useful for finding novel molecular targets for diagnosis and therapy.

Apoptosis↗
PubMed2006

Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer.

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.

Animals↗
PubMed2005

Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and/or legs. We also show that the siRNA/atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3'-hydroxykinase p110-alpha-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-alpha levels was not associated with the in vivo administration of the siRNA/atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA.

Animals↗
PubMed2005

Gene expression profiling of cerebellar development with high-throughput functional analysis.

We measured the expression levels of 450 genes during mouse postnatal cerebellar development by quantitative PCR using RNA purified from layers of the cerebellar cortex. Principal component analysis of the data matrix demonstrated that the first and second components corresponded to general levels of gene expression and gene expression patterns, respectively. We introduced 288 of the 450 genes into PC12 cells using a high-throughput transfection assay based on atelocollagen and determined the ability of each gene to promote neurite outgrowth or cell proliferation. Five genes induced neurite outgrowth, and seven genes enhanced proliferation. Evaluation of the functional data and gene expression patterns showed that none of these genes exhibited elevated expression at maturation, suggesting that genes characteristic of mature neurons are not likely to participate in neuronal development. These results demonstrate that functional data can facilitate interpretation of expression profiles and identification of new molecules that participate in biological processes.

Animals↗
PubMed2005

Direct hepatic fate specification from mouse embryonic stem cells.

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fate-specification from ES cells by using simple adherent monolayer culture conditions. ES cell-derived hepatocytes showed liver-specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell-derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. Supplementary material for this article can be found on the HEPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Animals↗
PubMed2005

Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells.

Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3beta/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se.

Animals↗
PubMed2005

Development and biological analysis of peritoneal metastasis mouse models for human scirrhous stomach cancer.

The number of published studies on peritoneal dissemination of scirrhous gastric carcinoma is very small as a result of the unavailability of highly reproducible animal models. Orthotopic implantation of HSC-44PE and HSC-58 (scirrhous gastric carcinoma-derived cell lines) cells into nude mice led to dissemination of the tumor cells to the greater omentum, mesenterium, peritoneum and so on, and caused ascites in a small number of animals. Cycles of isolation of the ascitic tumor cells and orthotopic inoculation of these cells were repeated in turn to animals. This was to isolate highly metastatic cell lines with a strong capability of inducing the formation of ascites (44As3 from HSC-44PE; 58As1 and 58As9 from HSC-58). All three cell lines induced tumor formation at the site of orthotopic injection, and caused fatal cancerous peritonitis and bloody ascites in 90-100% of the animals approximately 3-5 weeks after the inoculation. When the parent cells were implanted, the animals became moribund in approximately 12-18 weeks, however, none of the animals developed ascites. Complementary DNA microarray and immunohistochemical analyses revealed differences in the expression levels of genes coding for the matrix proteinase, cell adhesion, motility, angiogenesis and proliferation between the highly metastatic- and parent-cell lines. The usefulness of this model for the evaluation of drugs was assessed by analyzing the stability of the metastatic potential of the cells and the reproducibility. Animals intravenously treated with CPT-11 and GEM showed suppressed tumor growth and significantly prolonged survival. The metastatic cell lines and the in vivo model established in the present study are expected to serve as a model of cancerous peritonitis developing from primary lesions, and as a useful means of clarifying the pathophysiology of peritoneal dissemination of scirrhous gastric carcinoma and the development of drugs for its treatment.

Adenocarcinoma, Scirrhous↗
PubMed2005

Long-term maintenance of liver-specific functions in cultured ES cell-derived hepatocytes with hyaluronan sponge.

This study investigated the three-dimensional culture of hepatocytes differentiated from mouse embryonic stem (ES) cells with a porous hyaluronan (HA) sponge support. Hepatocytes were immobilized within the pores of the support. Spheroids could be observed within the support, each containing between 20 and 50 hepatocytes. To examine the liver-specific functions of the hepatocytes in the culture, the levels of albumin secreted into the medium were analyzed. The secretion of albumin was stable over the course of 32 days, longer than that in both conventional monolayer and collagen sponge cultures. To elucidate further the liver-specific functions of hepatocytes embedded in the HA sponge, metabolic activities of the hepatocytes were examined for their ability to eliminate ammonia from culture media and the synthesis of urea nitrogen. While rates of ammonia removal and urea nitrogen synthesis were similar to those in both conventional monolayer and in collagen sponge cultures, these functions were maintained for longer duration in cells embedded in the HA sponge. These results demonstrate that the porous HA sponge is an effective support for the in vitro culture of ES-derived hepatocytes used for both basic and applied studies for cell transplantation.

Animals↗
PubMed2004

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo.

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

Animals↗
PubMed2004

HST-1/FGF-4 plays a critical role in crypt cell survival and facilitates epithelial cell restitution and proliferation.

The fibroblast growth factor-4 (HST-1/FGF-4) is a heparin-binding growth factor that influences on epithelial and many other cells through interaction with FGF receptors. It has been demonstrated that the HST-1/FGF-4 gene protects mice from lethal irradiation by preventing bone marrow damage and intestinal tract damage. However, the radioprotective mechanism is unknown. In this study, we have investigated the expression of Hst-1/Fgf-4 in mouse small intestine after irradiation, and determined the role of HST-1/FGF-4 in mouse intestinal crypt cell survival and epithelial cell proliferation and restitution. We found the induction of endogenous Hst-1/Fgf-4 expression in intestine when mice are exposed to 9.0 Gy irradiation. Laser-captured microdissection (LCM) coupled with RT-PCR analysis revealed that expression of Hst-1/Fgf-4 was found in epithelial cell of the villi and crypt cells. Pretreatment of HST-1/FGF-4 caused an increase in the number of surviving crypt cells, and clearly suppresses the radiation-induced apoptosis of the crypt cells. Moreover, exogenous HST-1/FGF-4 enhances epithelial cell restitution and proliferation in an in vitro model. These data suggest that HST-1/FGF-4 is induced by irradiation injury, and that HST-1/FGF-4 will find a therapeutic role in the prevention of intestinal cell toxicity following intensive chemotherapy and radiation therapy protocols, and in allogenic cell transplantation.

Animals↗