Norio Arita

Integrin α3β1‐mediated interaction with laminin‐5 stimulates adhesion, migration and invasion of malignant glioma cells

Gliomas, characterized by their progressively invasive phenotype, express integrin α3β1 as a major receptor for the extracellular matrix both in vivo and in vitro. Since the integrin α3β1 has been shown to be a specific receptor for laminin‐5 (α3β3γ2), we examined the effects of purified human laminin‐5 on adhesion, migration and invasion of human glioma cells. Among different types of laminin variants and other matrix proteins including fibronectin and vitronectin, laminin‐5 was most potent in promoting adhesion and migration of different kinds of glioma cells. Laminin‐5‐mediated adhesion and migration were specifically inhibited by monoclonal antibodies against integrin α3 and β1 chains, confirming the role of integrin α3β1 as the major laminin‐5 receptor. Invasion of the reconstituted basement membrane (i.e., Matrigel) by glioma cells was also selectively stimulated by laminin‐5. Out results show that laminin‐5 is the major extracellular stimulant for glioma cell adhesion, migration and invasion. The immunohistochemical distribution of laminin γ2 chain, a laminin subunit unique to laminin‐5, showed that it was expressed in the tumor parenchyma of human glioma tissues. Expression of laminin α3, β3 and γ2 chains in glioma tissues and in glioma cell lines was also demonstrated at the messenger RNA level by reverse transcription polymerase chain reaction. Our results, taken together, show that laminin‐5 may be involved in the invasive phenotype of malignant gliomas both in vitro and in vivo. Int. J. Cancer76:63–72, 1998.© 1998 Wiley‐Liss, Inc.

Microsatellite instability and mutated type II transforming growth factor-β receptor gene in gliomas

Microsatellite instability has been reported in familial cancer syndrome and in various kinds of human sporadic tumors. We investigated the replication error (RER) and mutation rate of the transforming growth factor-beta type II receptor (TGF-beta RII) gene to determine the frequency of the RER+ phenotype and elucidate the relation between the mutation of the TGF-beta RII gene and RER in the tumorigenesis of glioma. We screened genomic DNA from 40 gliomas, comprised from 24 glioblastomas (GB), 11 anaplastic astrocytomas (AA) and five astrocytomas (AS) and compared the results with DNA from corresponding leukocytes. Seven of the 40 (18%) gliomas had the RER+ phenotype: five (21%) of 24 GB and two (18%) of 11 AA. In six gliomas we detected mutation of the TGF-beta RII gene. Five (71%) of seven RER+ and one (3%) of 33 RER-tumors had one A deletion in the (A)10 repeat of the TGF-beta RII gene. No mutations were detected in the (GT)3 repeat area of the TGF-beta RII gene. As the normal cells of these glioma patients had no mutations, we concluded that the mutations were somatic. We posit that the observed mutations inactivate the receptor through a frameshift mutation resulting in protein truncation. Our data suggest that the TGF-beta RII (A)10 repeat may be one area of genomic instability in the early stages of malignant glioma tumorigenesis.

Isolation and expression analysis of a novel human homologue of the Drosophila glial cells missing (gcm) gene

A novel human homologue (GCMB) of the Drosophila glial cells missing gene (dGCM) was isolated using RACE. GCMB contained a gcm motif sequence and a nuclear targeting sequence similar to that of dGCM and mouse GCMb. Homology searches indicated that GCMB was located within chromosome 6p24.2. Transcripts of GCMB were detected by means of RT‐PCR in fetal brain, normal adult kidney, 3/3 medulloblastomas, 1/3 gliomas and 4/8 non‐neuroepithelial tumor cell lines. Our data suggest that humans have two homologues of gcm like mice and that human gcm genes form a novel family which may function not only during fetal development but also in the postnatal or pathological stage.

Fibronectin-mediated cell migration promotes glioma cell invasion through chemokinetic activity

In order to investigate the biological role of fibronectin in glioma cell invasion, we studied the relation between migratory responses or adhesiveness of glioma cells to fibronectin and the in vitro invasion in three human malignant glioma cell lines, A172, T98G and U373MG. All these cell lines chemotactically migrated in a dose-dependent manner to fibronectin in concentrations ranging from 0.5 to 10 µg/ml, with A172 cells showing the strongest migration and U373 cells the weakest. Checkerboard analyses demonstrated that A172 and T98G cells showed much stronger chemokinetic responses to fibronectin than U373MG cells. In contrast to the migratory responses, A172 and U373MG cells showed an almost equally high adhesion to fibronectin and T98G cells a low adhesion. The degree of expression of the integrin α5 subunit correlated well with the strength of glioma cell adhesion to fibronectin rather than that of migration to the molecule. Furthermore, the cell adhesion to fibronectin was almost completely inhibited by arginine-glycine-aspartic acid (RGD)-containing peptides, but the fibronectin-stimulated cell migration was only partially inhibited. An in vitro invasion assay disclosed that U373MG cells invaded the artificial basement membrane barrier the most and A172 cells the least. However, addition of fibronectin to the glioma cells markedly enhanced the invasive activity of A172 and T98G cells but had little effect on that of U373MG cells. These results indicate that fibronectin-stimulated migration can be one of the factors promoting invasiveness of glioma cells and that the chemokinetic activity of fibronectin may play a crucial role in glioma invasion through conferring motor-driving force on the glioma cells.

Neural cell adhesion molecule L1 in gliomas: correlation with TGF-beta and p53.

AIMS: To assess immunohistochemically whether the neural cell adhesion molecule L1, which is a member of the immunoglobulin superfamily and has been shown recently to be a stimulating factor for glioma migration, is expressed in glioma tissues, and to investigate factors that can regulate this expression. METHODS: Twenty seven glioma tissue specimens including 13 glioblastomas, seven anaplastic astrocytomas, and seven astrocytomas were examined. Immunohistochemical analyses of L1, p53, and transforming growth cell factor beta (TGF-beta) were performed on each tumour using both polyclonal and monoclonal antibodies. RESULTS: Nine (33%) specimens (six glioblastomas and three anaplastic astrocytomas) had L1 positive immunostaining. p53 positive staining was detected in 10 (43%) of 23 glioma specimens (seven glioblastomas and three anaplastic astrocytomas). TGF-beta positive immunostaining was observed in 12 (52%) of the 23 glioma specimens (six glioblastomas, four anaplastic astrocytomas, and two astrocytomas). There was a statistical correlation between both p53 and L1 expression and TGF-beta and L1 expression. No such correlation was found between p53 and TGF-beta expression. CONCLUSIONS: These results suggest that mutation of the p53 gene or expression of TGF-beta may upregulate the expression of the L1 gene, thus resulting in high grade migration of glioma cells.

In vivo etoposide-resistant C6 glioma cell line: Significance of altered DNA topoisomerase II activity in multi-drug resistance

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase IIα isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase IIα in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase IIα isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.

A new mutation of the L1CAM gene in an X-linked hydrocephalus family

X-linked hydrocephalus is a genetic form of hydrocephalus that frequently occurs in males. It is characterized by ventricular dilatation, mental retardation, deformity of the thumb and spastic paraparesis. Recently, 23 different mutations of the gene for the neural cell adhesion molecule, L1CAM, located at chromosome region Xq28, have been reported, 16 of which were detected in families with X-linked hydrocephalus. We sequenced the coding region of the L1CAM gene of patients from two different families with X-linked hydrocephalus and found a novel mutation at nucleotide residue 1963 in one family. This mutation from adenine to guanine results in an amino acid change from lysine to glutamic acid at residue 655 of the L1CAM protein, which belongs to the fibronectin type III domain. We report another method for the rapid identification of the mutation based on the polymerase chain reaction. This mutation was not detected among 70 X chromosomes from a healthy population. Ours is the first report demonstrating this gene mutation in X-linked hydrocephalus in an Asian population. Our findings further emphasize the evolving genotypic heterogeneity in X-linked hydrocephalus.

Establishment of spontaneously immortalized rat type 1 astroglial cell lines: The role of p53 in astroglial carcinogenesis

We established five spontaneously immortalized cell lines using purified rat type 1 astroglia on a rigid transfer schedule. All the cell lines maintained their polygonal shape, regular pavement growth, low saturation density, positive glial fibrillary acidic protein expression, and serum requirements, while none were tumorigenic in nude mice. We then obtained a spontaneously transformed cell line by maintaining the cells for 6 months at a high cell density. Since alterations of the tumor suppressor p53 gene have been reported in the immortalization of some cell lines and in transformation of others, we characterized p53 in immortalized, spontaneously transformed, and 5 N‐ethyl‐N‐nitrosourea (ENU)‐transformed cell lines. While each of the ENU‐induced or the spontaneously transformed cell lines exhibited p53 gene mutations that resulted in amino acid alterations, no alterations in the p53 gene were observed in any of the immortalized cell lines. Thus, alterations of the p53 protein correlate more strongly with transformation than with immortalization of type 1 astroglia. Immortalization may be regulated by gene(s) other than p53. Spontaneously immortalized type 1 astroglial cell lines may provide a new tool to investigate an initial step of astroglial carcinogenesis.

Expression and Biological Functions of L1 Cell Adhesion Molecule in Malignant Glioma Cells

Human and rat glioma cells were screened for their expression of the L cell adhesion molecule (L1 CAM) gene and protein by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunodot analysis, respectively. All glioma cells tested expressed the L1 gene in various degrees. They also produced L1 molecules to a certain extent, while normal rat glia did not produce L1. The amount of expression of the L1 gene was related to the cell morphology and cell-cell aggregation patterns in a monolayer cell culture. That is, the more glioma cells expressed the L1 gene, the less polarity they had in cell shape and the more compactly they aggregated when growing in the early phase of cell culture. The glioma cells strongly migrated not only to a synthetic L1 polypeptide but also to L1- or L1 cs-transfected fibroblast cells. These results suggest that L1 expressed in glioma cells may play an important role in the migration of the glioma cells through homophilic or heterophilic cell adhesion, thus participating in tumor invasion, particularly along the nerve fibers.