Johbu Itoh

Methodological Development of a Clonogenic Assay to Determine Endothelial Progenitor Cell Potential

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133+ cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133+ cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.

Hepatocyte Growth Factor Suppresses Profibrogenic Signal Transduction via Nuclear Export of Smad3 With Galectin-7

BACKGROUND & AIMS Hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-beta is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-beta-stimulated profibrogenic signal transduction. METHODS Effects of HGF on TGF-beta-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring alpha2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. RESULTS Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-beta-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-beta-stimulated expression of those target genes. CONCLUSIONS These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

Prohormone convertases (PC1/3 and PC2) in rat and human pancreas and islet cell tumors: Subcellular immunohistochemical analysis

Prohormons convertase 1/3 (PC1/3; also termed PC1 or PC3) and PC2 are enzymes that activate prohormones by cleaving the pairs of basic amlno acids. This mechanlsm was inltlally Interred lrom the series of several endocrine and neuroendocrine precursor protoh, inciudlng proinsulin and prolusion. To determine the cellular and sub cellular distribution of PC1/3 and PC2 in the rat snd human pancreas, Immunohlstochemistry was performed using polyclonal antlers against mouse PC1/3 (ST‐28) and mouse PC2 (ST‐29). These studles showed light and dsctron mlcroacoplc co‐locailzation of Insulln, PC1/3 and PC2, and the coexistence of glucagons and PC2 In the pancreatic islets. This tendency of colocalizstion was also depicted In one case of human insulin and three cam of human glucagonomas, as well as In rat Insullnomas. in two cases of human Insullnomas, Incomplete processing of proinsulin was suggested by the absence of PC2. At the sub cellular level in the rat pancreatic lslet, the colocalizstion of PC1/3 and insulin, and that of PC2 and glucagons, were observed in the same secretor granules by immunoelectron, microscopy and Image analysis. These studles suggest that PC1/3 and PC2 can functlon with the specifictties In the processing of proinsulin and proglucagon Into their active forms, respectively, in the normal and neoplastic pancreatic islets.

Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor

BACKGROUND Metastin, the final peptide of the KiSS-1 gene, has been proposed to suppress cell motility. OBJECTIVE This study investigated whether renal cell carcinoma (RCC) tissue expresses metastin or its receptor, and clarified whether metastin can suppress migration and/or invasion and/or proliferation of RCC cells in vitro. DESIGN, SETTING, AND PARTICIPANTS Twenty-five RCC samples were submitted. Fresh RCC tissues were prepared for real-time RT-PCR, and formalin-fixed and paraffin-embedded tissues blocks were examined by immunohistochemistry. RCC cell lines Caki-1 and ACHN were supplied for cell migration, invasion, and proliferation assays. MEASUREMENTS Real-time RT-PCR was performed by using Taq Man gene expression system. ENVISION system was used in immunohistochemistry. Wound-healing assay and matrigel assays were used to identify migration and invasion abilities of RCC cell lines. Cell Counting Kit-8 was applied to measure the cell proliferation. Cell morphology was examined under a META system. Statistical analysis was performed with SPSS15.0J. RESULTS AND LIMITATIONS In twenty-five RCC samples, the mRNA level of metastin receptor was identified to be significantly higher than non-neoplastic renal cortex by real-time RT-PCR (p=0.011). Immunohistochemical study also detected metastin receptor protein in all RCC tumors. In vitro, this study showed that metastin inhibited migration and invasion of Caki-1 and ACHN cells. In contrast, it had no effects on cell proliferation. Metastin (10 micromol/l) induced excessive formation of focal adhesions and stress fibers in Caki-1 and ACHN cells; this phenomenon was inhibited by pretreating pharmacological Rho-kinase inhibitor (Y-27632) to those cells. CONCLUSION This is the first report regarding overexpression of the metastin receptor hOT7T175 in human RCC. We demonstrate that metastin can inhibit migration and invasion of the RCC cell line, which is regulated by a Rho-kinase inhibitor. Metastin and its receptor are therefore probable targets for suppressing RCC.

Three-dimensional Imaging of the Intracellular Localization of Growth Hormone and Prolactin and Their mRNA Using Nanocrystal (Quantum Dot) and Confocal Laser Scanning Microscopy Techniques:

Semiconductor nanocrystals (Quantum dots, Qdots) have recently been used in biological research, because they do not fade on exposure to light, and they enable us to obtain multicolor imaging because of a narrow emission peak that can be excited via a single wavelength of light. There have been no reports of simultaneous localization of mRNA and protein using Qdots. We successfully applied these advantages of Qdot and confocal laser scanning microscopy (CLSM) to three-dimensional images of the intracellular localization of growth hormone and prolactin and to their mRNA. In situ hybridization and immunohistochemistry using Qdots combined with CLSM can optimally illustrate the relationship between protein and mRNA simultaneously in three dimensions. Such an approach enables us to visualize functional images of proteins in relation with mRNA synthesis and localization.

Post-ischemic delayed expression of hepatocyte growth factor and c-Met in mouse brain following focal cerebral ischemia.

We investigated long-term changes in the expression of protein and mRNA of hepatocyte growth factor (HGF) and its receptor c-Met in mouse brain after permanent occlusion of the middle cerebral artery, by using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. HGF-immunopositive cells were observed in the periinfarct region from 4 days after occlusion, peaking at 14-28 days. The area containing HGF-immunopositive cells continued to expand until 28 days after occlusion. c-Met-immunopositive cells were observed exclusively at the periinfarct region at 7 and 14 days after occlusion. At 28 days after occlusion, there were many c-Met-immunopositive cells in the widespread periinfarct region. Triple immunohistochemical staining by using confocal laser scanning microscopy (CLSM) demonstrated that most of the HGF-immunopositive cells were localized to reactive astrocytes. The c-Met-immunopositive cells were also localized to reactive astrocytes. HGF mRNA was upregulated exclusively in the periinfarct region at 14 days. c-Met mRNA was upregulated in the periinfarct region from as late as 28 days after occlusion. Thus, HGF and c-Met show delayed expression in the periinfarct region at both protein and mRNA levels after induction of ischemia. Because HGF was recently shown to play critical roles in angiogenesis and neurotrophic activities, the temporal profiles of their expression may imply the involvement of HGF in the process of post-ischemic brain tissue repair.

Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo.

We have identified a novel human septin family gene Bradeion, which is specifically expressed in human colorectal cancer and malignant melanoma. In order to analyze the implications of tumor-specific gene expression, ribozymes and its derivatives were specifically designed and transfected into various colorectal adenocarcinoma cell lines for Bradeion inactivation. We constructed ribozyme expression plasmids controlled by a human tRNAVal promoter, and both hammerhead ribozyme and its allosteric derivative maxizyme were used for two different forms of Bradeion mRNA. The sequence-specific cleavage of Bradeion mRNA resulted in significant growth inhibition and G2 arrest in human cancer cell lines, detected by flow cytometry analysis. In addition, in vivo mice studies demonstrated marked tumor growth suppression by the Bradeion-specific ribozymes. Thus, the tumor-specific and selective marker Bradeion also provides valuable tools as a potential target for colorectal cancer therapy.

In situ hybridization analysis of Pit-1 mRNA and hormonal production in human pituitary adenomas

Abstract The pituitary-specific transcriptional factor, Pit-1, is a member of the POU-domain family which has a role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Recent investigations have suggested the involvement of specific regulation of Pit-1 transcripts in human pituitary adenomas. In this study, we analyzed the expression of Pit-1 gene and Pit-1 product in various human pituitary adenomas using in situ hybridization (ISH) and immunohistochemistry (IHC). Northern hybridization analysis revealed 2.4- and 4.1-kb Pit-1 transcripts in normal pituitary, growth hormone (GH)-, prolactin (PRL)- and thyrotropin (TSH)-secreting adenomas. By ISH analysis, Pit-1 mRNA was detected in 42 (84%) of 50 adenomas. The highest incidence was observed in 15 GH-secreting adenomas and 8 TSH-secreting adenomas, in which Pit-1 mRNA was detected in all cases. Pit-1 mRNA expression was detected in 11 (85%) of 13 PRL-secreting adenomas. In 12 clinically non-functioning adenomas, Pit-1 mRNA was also present in 8 cases, and 5 of these were associated with immunohistochemical expression of Pit-1 product. By combined ISH and IHC, Pit-1 mRNA was often colocalized with GH, PRL or TSHβ immunoreactivities and sometimes colocalized with α-subunit of glycoprotein (αSU) immunoreactivity. The expression of Pit-1 mRNA in various cell types of human pituitary adenomas in addition to GH, TSHβ and PRL immunoreactivities suggests that Pit-1 may play a role in functional development of pituitary adenomas, including clinically non-functioning adenomas. However, some additional transcriptional factors or enhancers may be required.

Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome

The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(−), ER(+)/HER2(+), ER(−)/HER2(+), and ER(−)/HER2(−) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(−)/progesterone receptor (PgR)(−)/HER2(−) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(−)/PgR(−)/HER2(−) status, a basal phenotype, and a worse clinical outcome.

Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.