Hisashi Hashimoto

Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression

In order to verify whether tenascin‐C (TN‐C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375‐GFP), implanted subcutaneously into BALB/cA nude (WT) and TN‐C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375‐GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell‐cell interactions. Both the content of TN‐C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375‐GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN‐C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN‐C expression of A375‐GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN‐C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN‐C. These results clearly indicated that the expressions of both TN‐C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN‐C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development.

Experimental IgA Nephropathy Induced by a Low-Dose Environmental Mycotoxin, Nivalenol

Based on the hypothesis that IgA nephropathy (IgAN) is triggered by some exogenous antigen(s) which induces dysregulation of the mucosal immune system, we developed an experimental model of orally induced IgAN by an environmental mycotoxin, nivalenol (NIV), which often contaminates agricultural products in Southeast Asia and Japan. In the present study, low doses of oral NIV reproducibly induced significant IgA deposits in the glomerular mesangium and elevated serum IgA levels in mice irrespective of the strain; the degree of immunopathological changes analogous to human IgAN was associated with the dose and duration of NIV treatment. Furthermore, a competitive enzyme-linked immunosorbent assay with an NIV analogue-protein conjugate disclosed that the IgA antibody in the sera from the NIV model mice had a higher affinity to the mycotoxin. Conclusively, these findings suggest that NIV induces some pathological changes in mice which resemble those in human IgAN, and that this mycotoxin is associated with pathogenesis in some types of glomerulonephritis.

Establishment and characterization of a human malignant choroids plexus papilloma cell line (HIBCPP)

A cell line designated “HIBSPP” was established from a human malignant choroids plexus papilloma of 29-year-old Japanese woman. This line grew well without interruption for 3 years and was subcultivated over 70 times. The cells were spindle, oval, and polygonal in shape, and neoplastic and pleomorphic features, a jigsaw puzzle-like arrangement, multilayering and forming papillary shuctures without contact inhibition. The cells proliferated slowly, and the population doubling time was about 69 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the hypo-tetraploid range, and many marker chromosomes were observed. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

Three-dimensional analysis of the developing pituitary gland in the mouse†

Prenatal development of the mouse pituitary gland was analyzed in three‐dimensions by using confocal laser‐scanning microscopy. In any wholemount preparation of the fetal pituitary gland from day 10 to day 18, immunofluorescence of laminin was observed in the deeper regions of the organ and demarcated the boundary of the epithelial tissue from the surrounding mesenchyme. Three‐dimensional capillary networks in the fetal pituitary gland at day 13, day 15, and day 18 were visualized clearly by perfusing the blood vessels with an fluorescein isothiocyanate‐labelled gelatin solution.

PREPARATION OF WHOLE MOUNTS AND THICK SECTIONS FOR CONFOCAL MICROSCOPY

Publisher Summary The chapter presents a study related to the preparation of whole mounts and thick sections for confocal microscopy. The chapter presents a method that was developed primarily to observe the three-dimensional (3-D) distribution of the extracellular matrixes—laminin and tenascin. Laminin is a noncollagenous glycoprotein and is one of the main constituents of basement membranes in both fetal and adult organs. 3-D visualization reveals branching in the fetal lung and the development of the pituitary gland. The expression of tenascin is restricted spatiotemporally and its heterogeneous distribution is well indicated by 3-D images. The method is composed simply of fixation, sectioning if necessary, pretreatment, and immunostaining as usual, but each step was re-examined. The method is now applicable to other tissue components, such as neurotransmitters, including vasoactive intestinal peptide, somatostatin, and substance P, peptide hormones, neurofilaments, and interleukins. In addition, the chapter also introduces a novel method for the 3-D investigation of vascular nets. One of the most important and intrinsic issues found on a confocal image is that the fluorescence intensity from a structure oriented in parallel with the optical axis becomes high and that from a structure perpendicular to the optical axis becomes relatively low. 3-D images reconstructed with a confocal laser scanning microscope (CLSM) will afford meritorious information on the biological system to researchers.

Simultaneous observation of capillary nets and tenascin in intestinal villi

In order to reveal the biological role of capillaries in a tissue, it is desirable to study the three‐dimensional distribution of capillary nets in relation to tissue architecture. However, the simultaneous observation of the three‐dimensional distribution of these nets and other substances has been rarely performed to date. In the present study, we have developed a novel method for investigating the three‐dimensional distribution of capillary nets simultaneously with that of extracellular matrix components, such as tenascin, using the confocal laser scanning microscope.

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and lng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a biehybrid typed liver apparatus.

Three-dimensional Distribution of Extracellular Matrix in the Mouse Small Intestinal Villi. Laminin and Tenascin

To investigate the three-dimensional distribution of laminin and tenascin in the villi of the mouse small intestine, we designed a new tissue preparation procedure for confocal laser scanning microscopy. Fixed tissue was pretreated with sodium deoxycholate and immunostained. The specimens displayed intense immunofluorescence even in the deeper sites of the lamina propria. Laminin was distributed like a sheet in the basement membrane of the epithelium as well as that of the capillaries. Additionally, it was associated with some tubular structures in the lamina propria. Many pores were found in the laminin sheet. By comparison, tenascin was distributed at the epithelial basement membrane and in the underlying connective tissue exhibiting a striped pattern. Tenascin was observed wrapping around the capillary wall in a spiral but was absent at the tip of the villi except in the capillary wall. Pores in the staining pattern of tenascin were also found as was the case for laminin. This three-dimensional reconstruction made it possible to observe more clearly the localization of these two extracellular matrix molecules.

Establishment and characterization of a human ovarian small cell carcinoma, hypercalcemic type, cell line (OS-1) secreting PTH, PthrP and ACTH--special reference to the susceptibility of anti-cancer drugs.

We successfully established a novel cell line (OS-1) derived from human ovarian small cell carcinoma, hypercalcemic type secreted PTH, PTH-rP and ACTH. The OS-1 cell line was established from metastatic focus of uterus. A patient was 25-year-old Japanese woman. The first she received left ovariectomy on April 2002. The histopathological diagnosis was ovarian small cell carcinoma, pT2c, Nx, Mx. Then on June 2003, metastatic focus of uterus was ectomied. A part of the recurrent tumor of uterus was cut into small pieces with razor blades, and dissociated with 0.1% trypsin-0.02% EDTA/ PBS(-) solution at room temperature. The single cells and small cell clusters were seeded into 60mm dishes and cultured in growth medium (GM: DMEM/F12 supplemented with 20% fetal bovine serum and 0.1% non-essential amino acids solution) at 37°C, 4.7% CO2 in humidified air. Medium was exchanged twice a week. The OS-1 cells grew as floating cultures in the dishes. Radioimmunoassay of the conditioned media was revealed that the cultures secreted large amount of PTH, PTHrP and ACTH simultaneously. Susceptibilities of anti-cancer drugs to the OS-1 cells were examined using oxygen electrode meter (Daikin), and the results suggested VLB and TXL were effective, and CDDP, CPT-11, VP-16, VCR, CPA, MMC and CBDCA were not effective.In our knowledge, it is the first report that the cell line secreting PTH, PTHrP and ACTH was successfully established from ovarian small cell carcinoma, hypercalcemic type. We expect that OS-1 cell line contribute to study on the mechanism of ectopic hormone secretion and susceptibility of anti cancer drugs to the small cell carcinoma.

The Differentiation of Early Embryonic Stem Cells into Adipocytes-like Cells

An early embryonic stem cell line, EES-6 cells, was established fiom 2-cell stage embryos of ddY mice. The cells were maintained in an undifferentiated state with D-MEM/FlZ medium supplemented with 10% fetal bovine semm (FEW (GM) and lng of leukemia inhibitory factor (LIF) without any feeder cells. In this study, EES cells were cultured with a medium containing embryotrophic factors (ETFs) which promoted the differentiation of EES cells into white and brown adipocytes-like cells for a period of 5 days. fipid droplets in brown adipocyte-like cells were stained with Sudan III; however, large lipid-like droplets in white or brown adipocyte-like cells were unstained with either Sudan III or alcian blue. These findings have numerous possibilities for therapeutic use such as regeneration of skin and wound healing.