Ryouichi Hamasuna

Regulation of matrix metalloproteinase-2 (MMP-2) by hepatocyte growth factor/scatter factor (HGF/SF) in human glioma cells : HGF/SF enhances MMP-2 expression and activation accompanying up-regulation of membrane type-1 MMP

Hepatocyte growth factor/scatter factor (HGF/SF) contributes to the malignant progression of human gliomas. We investigated the effect of HGF/SF on matrix metalloproteinase‐2 (MMP‐2), membrane type 1 matrix metalloproteinase (MT1‐MMP) and tissue inhibitors of metalloproteinases (TIMPs), expressions of c‐Met/HGF receptor‐positive human glioblastoma cells. Treatment of U251 human glioblastoma cells with HGF/SF resulted in enhanced secretion of MMP‐2 with an increased level of the active form. This was accompanied by enhanced expression (2.5‐fold) of mRNA specific for MMP‐2. The stimulatory effect of HGF/SF on MMP‐2 expression did not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. MT1‐MMP, a cell‐surface activator of proMMP‐2, was also up‐regulated by HGF/SF in a dose‐dependent manner. By contrast, the level of TIMP‐1 mRNAs was not altered significantly and that of TIMP‐2 was reduced mildly by the HGF/SF treatment, suggesting that HGF/SF may eventually modulate a balance between MMP‐2 and TIMPs in favor of the proteinase activity in the glioma cell microenvironment. HGF/SF also stimulated MMP‐2 expression of other glioblastoma cell lines. Since glioblastomas frequently co‐express HGF/SF and its receptor, our results suggest that HGF/SF might contribute to the invasiveness of glioblastoma cells through autocrine induction of MMP‐2 expression and activation. Int. J. Cancer 82:274–281, 1999.

Reduced expression of hepatocyte growth factor activator inhibitor type‐2/placental bikunin (HAI‐2/PB) in human glioblastomas: Implication for anti‐invasive role of HAI‐2/PB in glioblastoma cells

Hepatocyte growth factor activator inhibitor type‐2/placental bikunin (HAI‐2/PB) is a serine proteinase inhibitor that contains 2 Kunitz‐domains and a presumed transmembrane domain. It has broad inhibitory spectra against various serine proteinases showing potent inhibitory activities not only to hepatocyte growth factor activator but also to plasmin, trypsin and kallikreins. In this study, we investigated the expression of HAI‐2/PB in human gliomas in vivo and the effects of HAI‐2/PB on the fibrinolytic and invasive capabilities of human glioblastoma cells in vitro. With RNA blot analysis, HAI‐2/PB mRNA was expressed in normal brain and in low‐grade astrocytomas, but was hardly detectable in anaplastic astrocytomas and glioblastomas, indicating that its expression levels were inversely correlated with the histological grade of human gliomas. To further explore the possible role of HAI‐2/PB in glioma progression, cultured human glioblastoma cell lines (U251 and YKG‐1) were transiently transfected with an expression vector harboring human HAI‐2/PB cDNA. Subsequent analysis indicated that the expression of HAI‐2/PB suppressed the fibrinolytic activities of both glioblastoma cell lines. Moreover, HAI‐2/PB inhibited Matrigel invasion of U251 and YKG‐1 cells by 30% and 64%, respectively. This anti‐invasive effect appeared to be mediated primarily by the inhibitory activity of HAI‐2/PB against the serine proteinase‐dependent matrix degradation. These findings suggest that the reduced expression of HAI‐2/PB is possibly involved in the progression of human gliomas.

Overexpression of intestinal trefoil factor in human colon carcinoma cells reduces cellular growth in vitro and in vivo

BACKGROUND & AIMS Intestinal trefoil factor (ITF) has a role in gastrointestinal mucosal integrity and the repair of damaged mucosa. However, little is known about its role in tumors. To analyze the role of ITF in colon carcinomas, overexpression of the ITF gene in colon carcinoma cells was used. METHODS Human colon carcinoma cell lines LoVo and SW837, expressing no endogenous ITF, and WiDr expressing a low level of ITF were stably transfected with an expression vector harboring human ITF complementary DNA. The effects of ITF overexpression on in vitro growth, morphology in collagen gel, response to epidermal growth factor (EGF), mitogen-activated protein kinase (MAPK) activity, and growth in nude mice were assessed. RESULTS Overexpression of ITF in LoVo and SW837 resulted in significantly reduced growth in vitro and in vivo. In collagen gels, the ITF-expressing LoVo clones formed smaller, more dispersed colonies. EGF-induced phosphorylation of MAPKs was modestly reduced in the ITF-expressing clones. The growth of WiDr was modestly suppressed only in vivo by ITF overexpression. CONCLUSIONS Overexpression of ITF suppressed the growth of colon carcinoma cells. ITF may function as an inhibitory factor for the growth of colonic neoplasm.

Solitary fibrous tumor of the spinal nerve rootlet : Case report and literature survey

Solitary fibrous tumor (SFT) is a rare tumor that arises most commonly in the pleura. Recent evidence indicated that it is a tumor that originates from mesenchymal, probably fibroblastic, cells and is not restricted to the pleura. This report presents a case of primary SFT occurring as a dumbbell‐shaped tumor of the cervical spine (C4/5) in a 46‐year‐old Japanese female, probably originating from the spinal rootlet. The tumor was predominantly extradural, loosely attached to the dura mater, with a small intradural extramedullary part attached to the C5 anterior and posterior rootlets. Histologically, the tumor was predominantly composed of a haphazard proliferation of spindle cells separated by abundant collagen. Immunohistochemically, the cells were strongly positive for CD34, bcl‐2 and vimentin, but were negative for S‐100 protein, neuron specific enolase, cytokeratin and epithelial membrane antigen. The present case and review of the literature strongly suggest that SFT is an entity that should be considered in the differential diagnosis of tumors of the cerebrospinal region.

Mouse hepatocyte growth factor activator gene: its expression not only in the liver but also in the gastrointestinal tract.

A cDNA encoding mouse hepatocyte growth factor activator (HGFA) has been cloned by RT-PCR, based on the screening result from the database of expressed sequence tags. Subsequently, its gene was cloned from a mouse genomic bacterial artificial chromosome library using the cDNA as a probe. Sequencing analysis revealed that mouse HGFA protein deduced from the cDNA, similar to its human and rat counterparts, has two epidermal growth factor-like domains, type 1 and 2 fibronectin homology domains, a single kringle domain and a catalytic domain of serine proteinase, and the gene consists of 14 exon spanning approximately 7.5 kb. Interestingly, mouse HGFA mRNA was detected not only in the liver but also in the gastrointestinal tract by RNA blot analysis. Since hepatocyte growth factor (HGF) is up-regulated in the damaged gastrointestinal mucosa, our present data suggest that HGFA might activate proHGF directly in the gastrointestinal mucosa and play an important role in wound repair throughout the gastrointestinal tract.

Simultaneous up-regulation of urokinase-type plasminogen activator (uPA) and uPA receptor by hepatocyte growth factor/scatter factor in human glioma cells.

Several lines of evidence indicate that hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, c-Met, may play an important role in progression of human glioma. In this study, effects of HGF/SF on urokinase- type plasminogen activator (uPA)-mediated proteolysis network were examined in c-Met-positive human glioma cell lines. Treatment of the glioma cells with various concentrations of HGF/SF resulted in an enhanced secretion of uPA proteins accompanying increased transcription of uPA mRNA in a dose dependent fashion. The levels of uPA receptor (uPAR) mRNAs were also elevated simultaneously upon HGF/SF stimulation, and the cell-surface associated uPA activity was also elevated by the treatment. Since concomitant expression of HGF and its receptor c-Met are frequently observed in malignant gliomas, these results suggest that HGF/SF participates in invasive process of malignant glioma cells not only by its motility-stimulating activity but also through enhanced degradation of the extracellular matrix induced by autocrine activation of uPA proteolysis network.

Conserved expression of hepatocyte growth factor activator inhibitor type-2/placental bikunin in human colorectal carcinomas

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) was recently identified as a potent inhibitor of hepatocyte growth factor activator. It was also independently reported as placental bikunin (PB) and as a protein over-expressed in pancreatic cancer. The expression of HAI-2/PB was analyzed in human normal colon mucosa, adenomas, and carcinomas. HAI-2/PB mRNA was consistently expressed in the colorectal mucosa. The expression was conserved in the neoplastic colorectal mucosa, and no relationship was found between HAI-2/PB mRNA levels and tumor stages. Moreover, 13 out of 14 colorectal carcinoma cell lines expressed HAI-2/PB mRNA. Immunohistochemically, HAI-2/PB proteins were predominantly stained beneath the apical surface of normal enterocytes. In tumor tissues, rather disarranged intracytoplasmic granular staining was observed. The HAI-2/PB immunoreactivity was well conserved in the colonic adenoma-carcinoma sequence, and this protein may have important unknown function in the intestinal mucosa.

Establishment and characterization of a new human glioblastoma cells line, NYGM

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF advation of GBM cells.

TI-201 uptake in an intracranial primary malignant melanoma

A 32-year-old man had a giant pigmented nevus in general at birth in the head, face, chest, and back, and mental retardation and right hemiparesls (unknown cause) developed. A malignant melanoma was suspected on clinical evidence plus the images. At cranlotomy, an intracranial primary malignant melanoma was diagnosed that originated in the pia matter with neurocutaneous melanosis.

Enhanced expression of hepatocyte growth factor activator inhibitor type 2‐related small peptide at the invasive front of colon cancers

Background: Hepatocyte growth factor activator inhibitor type 2-related small peptide (H2RSP) is a small nuclear protein abundantly expressed in the gastrointestinal epithelium. However, its functions remain unknown. Aims: To investigate the expression and localisation of H2RSP in normal, injured and neoplastic human intestinal tissue. Methods: Immunohistochemical examination and in situ hybridisation for H2RSP were performed using normal and diseased intestinal specimens. Its subcellular localisation and effects on the cellular proliferation and invasiveness were examined using cultured cells. Results: In the normal intestine, H2RSP was observed in the nuclei of surface epithelial cells and this nuclear localisation was impaired in regenerating epithelium. In vitro, the nuclear translocation of H2RSP was observed along with increasing cellular density, and an overexpression of H2RSP resulted in a reduced growth rate and enhanced invasiveness. H2RSP expression was down regulated in well-differentiated colorectal adenocarcinomas. However, a marked up regulation of the cytoplasmic H2RSP immunoreactivity was observed in cancer cells at the invasive front. These cells showed low MIB-1 labelling, an enhanced p16 expression and nuclear β-catenin. The number of H2RSP-positive cells in the invasive front of well-differentiated adenocarcinomas was considerably higher in the cases with lymph node metastases than in node-negative ones. Conclusion: In the normal intestine, the nuclear accumulation of H2RSP is a marker of differentiated epithelial cells. Although H2RSP was down regulated in colorectal adenocarcinomas, a paradoxical up regulation was observed in actively invading carcinoma cells. H2RSP immunoreactivity at the invasive front may serve as a marker of invasive phenotype of well-differentiated colon cancers.