Masashi Koono

Glioma cell extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147) stimulates production of membrane-type matrix metalloproteinases and activated gelatinase A in co-cultures with brain-derived fibroblasts

Extracellular matrix metalloproteinase inducer (EMMPRIN) also called CD147, basigin or M6 in the human is a member of the immunoglobulin superfamily that is enriched on the surface of tumor cells and stimulates adjacent stromal cells to produce several matrix metalloproteinases (MMPs). In this study, we have demonstrated that coculturing of EMMPRIN-expressing human glioblastoma multiforme cells (U251) with brain-derived human fibroblasts not only stimulates production, but also activation of pro-gelatinase A (proMMP-2), an enzyme that is enriched in malignant gliomas and most likely crucial to tumor progression. Production of membrane types 1 and 2-MMPs (MT1-MMP and MT2-MMP), which are activators of proMMP-2, was also stimulated in these cocultures. Stimulation of MMP-2, MT1-MMP and MT2-MMP production was inhibited by anti-EMMPRIN monoclonal antibody in a dose-dependent manner. Thus, we have shown, for the first time, that EMMPRIN causes increased expression of MT1-MMP and MT2-MMP, as well as increased production and activation of MMP-2.

Expression of emmprin (CD147), a cell surface inducer of matrix metalloproteinases, in normal human brain and gliomas

EMMPRIN (extracellular matrix metalloproteinase inducer), also called CD147, basigin or M6 in the human, is a member of the immunoglobulin superfamily that is present on the surface of tumor cells and stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPs). In our study, we investigated expression of EMMPRIN in human normal brain and gliomas, since mouse basigin and chicken HT7, the species homologues of human EMMPRIN, are associated with neuronal interactions and normal blood‐brain barrier function, respectively. EMMPRIN expression was detected in all samples of non‐neoplastic brain and glioma tissues examined. However, expression levels of EMMPRIN mRNA and protein were significantly higher in gliomas than in non‐neoplastic brain. Moreover, levels of mRNA expression and immunohistochemical staining correlated with tumor progression in gliomas: They were highest in the most malignant form of glioma, glioblastoma multiforme, followed by anaplastic astrocytoma and then low‐grade astrocytoma. Also, immunolocalization revealed quite different distributions in non‐neoplastic brain and glioma: EMMPRIN was demonstrated only in vascular endothelium in non‐neoplastic regions of the brain, whereas it was present in tumor cells but not in proliferating blood vessels in malignant gliomas. These data indicate that an MMP inducer molecule EMMPRIN is differently expressed in human normal brain and gliomas and could be associated with astrocytoma progression. Possible mechanisms whereby glioma cell EMMPRIN could influence tumor progression will be discussed. Int. J. Cancer 88:21–27, 2000.

Distribution of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in Human Tissues: Cellular Surface Localization of HAI-1 in Simple Columnar Epithelium and Its Modulated Expression in Injured and Regenerative Tissues

We used a specific monoclonal antibody to human hepatocyte growth factor activator inhibitor type 1 (HAI-1) in immunohistochemical procedures to determine the distribution and localization of HAI-1 in human tissues. In normal adult tissues, HAI-1 was predominantly expressed in the simple columnar epithelium of the ducts, tubules, and mucosal surface of various organs. In all cases, HAI-1 was localized predominantly on the cellular lateral (or basolateral) surface. By contrast, hepatocytes, acinar cells, endocrine cells, stromal mesenchymal cells, and inflammatory cells were hardly stainable with the antibody, and stratified squamous epithelium showed only faint immunoreactivity on the surface of cells of the basal layer. In the gastrointestinal tract, the surface epithelium was strongly stained. RNA blot analysis confirmed the presence of specific mRNA transcript in the gastrointestinal mucosa, and in situ hybridization revealed that HAI-1 mRNA showed a similar cellular distribution pattern. Although HAI-1 was not expressed in normal hepatocytes, strong immunoreactivity was observed on the epithelium of pseudo-bile ducts and on the surface of scattered hepatocytes in fulminant hepatitis. The enhanced expression was also noted in regenerating tubule epithelial cells of the kidney after infarction. We conclude that HAI-1 is preferentially expressed in the simple columnar epithelium of the mucosal surface and duct, that the predominant localization of HAI-1 is the cell surface, and that the expression of HAI-1 can be modulated by tissue injury and regeneration.

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.

Concomitant expression of hepatocyte growth factor (HGF), HGF activator and c-met genes in human glioma cells in vitro

Three new cell lines of human glioblastoma have been established. These cells co‐expressed hepatocyte growth factor (HGF) and its receptor, c‐Met, genes in vitro. Reverse‐transcriptase/polymerase‐chain reaction study revealed that the cells also expressed gene for HGF activator, a recently cloned serine proteinase, suggesting that HGF might have a role in glioma cells in vitro as an autocrine factor. The activator mRNA was also detected in other well‐established glioma cell lines, glioma tissues and normal brain. The concomitant expression of HGF, HGF activator and c‐met was also detected in one glioblastoma case in vivo out of five tested.

Expression of c‐Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study

Recent studies suggest the involvement of hepatocyte growth factor/scatter factor (HGF/SF) in glioma cell invasion and tumour progression. We investigated the distribution and rate of tumour cells that express c‐Met protein, which is the cell‐surface receptor for HGF/SF, in astrocytic tumours. The type of cells that express c‐Met in tumour tissues was also identified.

Comparative analysis of expression of hepatocyte growth factor and its receptor, c-met, in gliomas, meningiomas and schwannomas in humans

Expression of hepatocyte growth factor (HGF) and c-met, a proto-oncogene that encodes a receptor for HGF, was examined in 45 cases of human primary intracranial tumors by means of RT-PCR. In gliomas, HGF and c-met mRNAs were preferentially expressed in high-grade tumors. Co-expression of both genes was observed in glioblastomas (6/15) and in one anaplastic astrocytoma (1/5) but not in low-grade astrocytomas (0/3). By contrast, the c-met gene was consistently expressed in meningiomas (12/14) and schwannomas (8/8). The presence of c-Met protein was confirmed in the tumor cells of glioblastoma, meningioma and schwannoma by immunohistochemical staining. Moreover, all of the schwannoma cases co-expressed the HGF gene. These observations suggest that HGF/c-met expression is somehow related to the disease progression in gliomas, whereas c-Met protein might have an important fundamental biological role in meningioma and schwannoma. Moreover, since all of the schwannoma cases concomitantly expressed the ligand (HGF) and the receptor (c-met) genes, HGF may act in an autocrine fashion in schwannoma.

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: An analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of potymer‐ase chain reaction (PCR) amplification from formalin‐fixed tissues have been Investigated mostly by experiments, the feasibility of archival formalln‐flxed, paraffin‐embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin‐fixed, paraffin‐embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2–3 days and 14% (4/28) of the samples fixed for 4–6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified pS3 products from archival tissues using a non‐isotopic method, temperature gradient gel elec‐trophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding Just after 1 day or less fixation for subsequent use in PCR amplification.

Amyloid β protein precursor is involved in the growth of human colon carcinoma cell in vitro and in vivo

Amyloid β protein precursor (APP) is a membrane‐bound protein ubiquitously expressed in a variety of types of cells. However, its biological functions remain largely uncertain, particularly in non‐neural cells and tumors. Our previous studies revealed that a secreted form of APP having a Kunitz‐type inhibitor domain is a major serine proteinase inhibitor secreted by human colon carcinoma cells. In our study, we used an antisense RNA strategy to selectively inhibit the expression of APP in the human colon carcinoma cell line SW837. A vector capable of expressing an antisense mRNA complementary to 911 bases of the 5′ end of APP mRNA was transfected into SW837 cells. After selection, 2 stably transfected antisense clones were obtained in which both the APP protein and mRNA were significantly suppressed. The proliferative potential and colony‐forming efficiency of the antisense clones in vitro were markedly suppressed compared with the parent and mock‐transfected clones. The addition of the conditioned medium of parent cells or purified secretory APP enabled these antisense effects to be overcome in vitro. The suppressed growth was also observed in vivo when the cells were injected subcutaneously into nude mice. Histologically, formation of tubular structures appeared to be suppressed in the antisense clones in vivo. These observations suggest potentially important roles of APP in cellular proliferation and differentiation of colon carcinoma cells.

Lymphoblastic lymphoma expressing natural killer cell phenotype with involvement of the mediastinum and nasal cavity

Most CD56+ lymphomas display polymorphic and angiocentric/angiodestructive histologic features and are closely related to Epstein-Barr virus (EBV) infection. We report a 47-year-old Japanese man with CD56+ lymphoma that showed histologic features of lymphoblastic lymphoma with mediastinal and nasal involvement and an aggressive course. A sample specimen showed the histology of lymphoblastic lymphoma with a positive reaction for terminal deoxynucleotidyl transferase (TdT) but no angiocentric/angiodestructive features. Transmission electron microscopy revealed a few membrane-bound electron-dense granules in their cytoplasm. Immunohistochemically, lymphoma cells exhibited CD56+ cytoplasmic CD3 (cCD3)+ TdT+. A Southern blot analysis showed no integration of EBV and human T-lymphotrophic virus 1 (HTLV-1) and no rearrangement of the T-cell receptors or immunoglobulin heavy chain genes. This unusual lymphoblastic lymphoma exhibiting cCD3 + CD56 + TdT + TCR- is assumed as an immature or progenitor natural killer cell lineage.