Hiroshi Isomura

Development of the blood-retinal barrier in vitro: formation of tight junctions as revealed by occludin and ZO-1 correlates with the barrier function of chick retinal pigment epithelial cells.

To elucidate the molecular mechanisms of the blood-retinal barrier (BRB), we examined chick retinal tissues histochemically using antibodies against tight junction proteins such as ZO-1, 7H6 antigen, and occludin. Retinal pigment epithelial (RPE) cells in situ in chickens and late chick embryos expressed all of the tight junctional proteins examined, showing that tight junctions seal the cell borders of chick RPE cells in vivo. On the other hand, RPE cells isolated from late chick embryos and transferred in vitro did not express occludin, ZO-1 and 7H6 antigen. The effects of differentiation-inducing agents, such as retinoic acid, dexamethasone and dimethyl sulfoxide (DMSO) were tested. Only DMSO induced an increase in transepithelial electrical resistance (TER) in a time-dependent manner. Under supplementation with DMSO, immunofluorescently demonstrable occludin and ZO-1 were induced progressively at cell borders in parallel with the increase in TER that occurred with decreases in inulin and dextran permeability. Electron microscopically tight junction-like junctional apparatus were induced in RPE cells. These results indicated that tight junctions of RPE cells play an important role in the formation of the BRB.

Polarized distribution of carcinoembryonic antigen is associated with a tight junction molecule in human colorectal adenocarcinoma.

This study we presents a novel anti‐occludin monoclonal antibody that can be used for formalin‐fixed, paraffin‐embedded tissue sections. The relationships between aberrant localization of carcinoembryonic antigen (CEA) and abnormalities of tight junctions were studied in human colorectal cancers by this antibody. Abnormalities in the cell surface expression of CEA have been shown to be characteristic of human colorectal cancer cells. Cancer cells that participated in the formation of glandular structures expressed occludin at the apical cell border and CEA was expressed more apically than occludin. Where cancer cells showed solid nests without glandular structures, occludin was completely lost and CEA was demonstrated in a diffuse pattern throughout the cells. These findings suggest that the polarized apical expression of CEA in neoplastic glandular structures depends on the expression of occludin and the fence function of tight junctions. During tumour progression, loss of occludin may lead to the loss of membrane polarity and the non‐polarized expression of CEA. The antibody described provides a powerful tool for the study of tight junctions in surgically resected human tissue. Copyright

Bacterial lipopolysaccharide reduced intestinal barrier function and altered localization of 7H6 antigen in IEC-6 rat intestinal crypt cells.

The intestinal epithelial barrier restricts the passage of potentially toxic substances into the systemic circulation and is considered to be mostly mediated by tight junctions, though the mechanisms involved in the regulation of intestinal tight junctions are not yet fully understood. In the present study, we examined whether bacterial lipopolysaccharide (LPS) altered the barrier function of tight junction and localization of tight junctional proteins, ZO‐1 and 7H6 antigen, in IEC‐6 intestinal cells. Administration of LPS to the basolateral surface of IEC‐6 cells disrupted the barrier function and caused the disappearance of 7H6 antigen from the cell border, whereas LPS administered to the apical surface altered neither the barrier function nor the localization of 7H6 antigen in IEC‐6 cells. On the other hand, the localization of ZO‐1 was not influenced by these treatments of LPS. These results suggest that the interaction of LPS with the basolateral surface of intestinal epithelial cells disrupts the barrier function and 7H6 antigen take part in the maintenance of the barrier function in IEC‐6 cells. J. Cell. Physiol. 171:284–290, 1997.

Expression of occludin, a tight-junction-associated protein, in human lung carcinomas.

Occludin is a tight-junction-associated transmembrane protein, and previous observations suggested that occludin might play a crucial role in the formation and maintenance of organized tubular structures. Based on these observations, we explored the possible role of occludin immunostaining in the diagnosis of lung carcinomas. A total of 68 lung carcinomas and surrounding normal lung tissues were studied. A formalin-fixed, paraffin-embedded section from each tumor was stained with a new anti-occludin monoclonal antibody raised in our laboratory. In normal lung tissues, the anti-occludin antibody strongly stained the apicoluminal borders of the bronchial/bronchiolar epithelia and bronchial glands as a dot or short line. The antibody also stained the intercellular borders of alveolar epithelia. In cancer cells that faced lumina of all adenocarcinomas, regardless of grade, including bronchioloalveolar carcinomas, occludin showed an expression pattern identical to that of the normal bronchial and alveolar epithelia. Occludin reactivity was not noted in any cases of squamous cell carcinoma, large cell carcinoma, small cell carcinoma, or large cell neuroendocrine carcinoma. The results of the present study suggest that occludin can serve as an immunohistochemical indicator of the “true” glandular differentiation that forms tubulo-papillary structures in human lung carcinoma tissues.

A Novel Experimental Mouse Model of Peritoneal Dissemination of Human Gastric Cancer Cells: Different Mechanisms in Peritoneal Dissemination and Hematogenous Metastasis

We established a new cell line, AZ‐P7a, with high peritoneal‐metastatic potential in nude mice. AZ‐P7a cells were derived from the human gastric carcinoma line AZ‐521, which has low capacity for peritoneal dissemination. AZ‐P7a cells developed peritoneal metastasis in 11/14 (78.6%) mice, whereas the parental AZ‐521 cells developed metastasis in 2/6 (33.3%) mice. The metastatic foci in the peritoneum showed essentially the same histological appearance as those induced by parental cells. The tumorigenicity and the motile activity of AZ‐P7a cells were stronger than those of the parental AZ‐521 cells; in contrast, adhesion to the extracellular matrix and the production of vascular endothelial growth factor by AZ‐P7a cells were decreased. In fluorescence‐activated cell sorter (FACS) analysis, AZ‐P7a cells expressed significantly greater levels of integrins α2, α3, α5, α6 and αvβ5, as compared with AZ‐521 cells. However, α1, α4, αvβ3, hCD44H, hCD44v3, hCD44v6 and hCD44v10 were not expressed in either cell line. AZ‐P7a cells developed no liver metastasis when administered by the intrasplenic injection method, though the highly liver metastatic cell line AZ‐H5c showed the same rate of peritoneal dissemination as that exhibited by AZ‐P7a cells after intraabdominal injection. These findings suggested that the mechanism of peritoneal dissemination differed from that of hematogenous metastasis. Moreover, the latter appears to be controlled by more complex mechanisms than the former. Thus, this cell line might be useful for investigating the mechanism of peritoneal dissemination of human gastric cancer.

A Novel Experimental Mouse Model of Peritoneal Dissemination of Human Gastric Cancer Cells: Analysis of the Mechanism of Peritoneal Dissemination Using cDNA Macroarrays

We established a new cell line, NUGC‐3P4T, with high peritoneal metastatic disseminating potential in nude mice. NUGC‐3P4T cells were derived from the human gastric carcinoma line NUGC‐3, which has low capacity for peritoneal dissemination. NUGC‐3P4T cells developed peritoneal dissemination in 10/10 (100%) mice, whereas the parental NUGC‐3 cells developed dissemination in 1/5 (20.0%) mice. The metastatic foci in the peritoneum showed essentially the same histological appearance as those induced by parental cells. The tumorigenicity, the motile activity and the adhesive activity to the laminin of NUGC‐3P4T cells were stronger than those of NUGC‐3 cells. Production of IL‐8 was significantly higher in NUGC‐3P4T than in NUGC‐3. cDNA macroarrays analysis showed that a variety of cytokines, interleukins, and other immunomodulators and their receptors were up‐ or down‐regulated at the mRNA level in NUGC‐3P4T cells, compared with NUGC‐3 cells. Thus, this unique cell line and in vivo model might be useful to study the biology of peritoneal dissemination of human gastric cancer.

Phase-dependent effects of transforming growth factor β1 on osteoblastic markers of human osteoblastic cell line SV-HFO during mineralization

A human osteoblastic cell line (SV-HFO) established in our laboratory expresses osteoblastic markers, including mineralization in vitro, in response to differentiation-inducing agents such as dexamethasone. In this study, we examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the mineralization of SV-HFO cells and show that TGF-beta 1 inhibited the mineralization of the cells via down regulation of tetranectin and alkaline phosphatase without influencing other osteoblastic markers. To examine precisely the effects of TGF-beta 1 on the process of mineralization, we tentatively divided the whole process of mineralization into four phases: induced ALP activity (days 0-5), maximal ALP activity (days 5-10), early mineralization (days 10-15), and progressive mineralization (days 15-20). These inhibitory effects of TGF-beta 1 on the expression of tetranectin and alkaline phosphatase, like that on mineralization, were observed only when TGF-beta 1 was applied in the early phase of the process of mineralization. On the other hand, the other osteoblastic markers were not influenced by treatment with TGF-beta 1. These results suggest that TGF-beta 1 may inhibit mineralization of osteoblasts by the downregulation of tetranectin and alkaline phosphatase expression in the early phase. Thus, TGF-beta 1 has phase-dependent effects on a human osteoblastic cell line during the process of mineralization.

Expression of occludin in human rectal carcinoid tumours as a possible marker for glandular differentiation

Aims:  To examine whether or not the tight junction‐associated transmembrane protein occludin is expressed in rosette or gland‐like structures in human rectal carcinoid tumours. The tight junction is crucial for the formation and maintenance of organized tubular structures in glandular epithelia. Previous studies have reported the presence of glandular structures in carcinoid tumours, though they are not believed to arise from glandular epithelium.

A new peritoneal dissemination model established from the human pancreatic cancer cell line.

We established a new cell line, HPC-3P4a, with high peritoneal disseminated potential in nude mice. HPC-3P4a was derived from a human pancreatic carcinoma cell line (HPC-3) that had low capacity for peritoneal dissemination. HPC-3P4a developed peritoneal dissemination in 10 of 11 (90.9%) cases, whereas parental HPC-3 developed peritoneal dissemination in one of six (16.7%) cases. The metastatic foci in the peritoneum showed essentially the same histologic appearance of parental involvement. The tumorigenicity, motility, and adhesive activity of HPC-3P4a to the extracellular matrix were stronger than were those of the HPC-3. In FACS analysis, HPC-3P4a significantly increased the expression of &agr;6 and &agr;v&bgr;5 integrins, while it decreased &agr;2 integrin, hCD44H, and hCD44v10, as compared with HPC-3. The VEGF production of HPC-3P4a was significantly lower than that of HPC-3. Analysis of gene macroarrays showed a variety of cytokines, interleukin, and other immunomodulatory, and their receptors were up-regulated and down-regulated on an mRNA level in HPC-3P4a cells, compared with HPC-3 cells. Intrasplenic injection of HPC-3P4a produced no liver metastasis. We named our original highly liver metastatic cell line HPC-3H4 (previously reported). This HPC-3H4 cell was established by repeated intrasplenic injection from parental cell HPC-3; thus, it developed high liver metastasis. Moreover, HPC-3H4 developed peritoneal dissemination by intra-abdominal injection. In contrast, HPC-3P4a did not develop liver metastasis by intrasplenic injection. These findings are very interesting and might suggest that the process of hematogenous metastasis differed from that of peritoneal dissemination. Thus, this cell line may be useful for investigating the mechanism of peritoneal dissemination in human pancreatic cancer.

Hemangioma of the Prostatic Urethra: Hematospermia and Massive Postejaculation Hematuria with Clot Retention

The case of a 53‐year‐old man with hematospermia and massive postejaculation hematuria that caused urinary retention is described. This is the sixth case in the English and Japanese language literature. Cystourethroscopic examination revealed that a solitary raised tumor was present just distal to the vermontanum, and that bleeding was from its apex. Histologic examination of an excisional biopsy sample showed features compatible with hemangioma.