Gene expression of neural cell adhesion molecule L1 in malignant gliomas and biological significance of L1 in glioma invasion.

Neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily that is expressed in the nervous system. Its functions have been mainly studied in vitro using premature neuronal cells. We show that all glioma cells tested, as well as normal glia cells, express a short type of L1, L1cs mRNA. The expression of L1 protein in glioma cells was confirmed by Western blotting and flow cytometric analysis. Migration assay showed that C6 glioma cells were stimulated to migrate to soluble L1 and L1cs released from L1- or L1cs-transfected fibroblast cells. The L1-stimulated migration was significantly inhibited by antibody that recognizes the immunoglobulin C2-like domain of L1. However, antibodies that recognize the fibronectin type III-like domain and the cytoplasmic (IC) domain of L1 had no effect on migration. Our in vivo migration study demonstrated the migration of L1 on C6 glioma cells that had been transfected in rat brains. These results suggest that L1cs expressed on glioma cells may play an important role in the adhesion and migration of glioma cells by homophilic binding (probably through the extracellular immunoglobulin C2 domain of L1) and that L1cs participates in tumor invasion along neuronal fibers.

A novel model of glioma cell invasion using organotypic brain slice culture.

A novel model of glioma cell invasion was established by using an organotypic culture of rat brains. Brain slices prepared from 2-day-old rat neonates were maintained in a culture at the interface between air and the culture medium. The slices were placed on double-layered membranes consisting of a polycarbonate membrane with 8-microm pores and a membrane with 0.4-microm pores. The organotypic cytoarchitecture of the cultured brain slices remained well preserved, and the neuronal viability was kept intact for over 2 months. When C6 glioma spheroids were cocultured with the brain slices, the tumor cells migrated in a scattered fashion around the spheroids. Exogenous L1 or glioma motility factor I strongly stimulated the cell migration, whereas fibronectin, tenascin, and glioma motility factor II had little or no effect. When C6 glioma cells placed on the brain slices were incubated while being stimulated by L1-transfected fibroblast cells for 2 days, many more tumor cells invaded and reached the bottom of the upper membrane. This L1-stimulated glioma cell invasion into brain slices was significantly inhibited by an anti-L1 antibody. Our novel invasion model, which mimics the in vivo conditions of the central nervous system, may make it possible to analyze actual events of glioma cell invasion in normal brains in situ.

Telomerase activity and alterations in telomere length in human brain tumors.

Telomerase activity was examined in 170 human brain tumor tissues, and terminal restriction fragment (TRF) length was examined in 152 of the 170. Telomerase activity was detected in 61.7% (66 of 107) of the neuroepithelial tumors. However, the detection rates of telomerase activity were widely different for different histopathological entities. In the case of astrocytic tumors, the detection rate was 20.0% (3 of 15) for grade II astrocytomas, 40.0% (6 of 15) for anaplastic astrocytomas, and 72.3% (34 of 47) for glioblastomas. The mean TRF length of the tumors with telomerase activity was significantly shorter than that of the tumors with undetectable telomerase activity for each tumor entity. In grade II and anaplastic astrocytomas, telomerase activity was an indicator of early histological progression and reduced survival of the patients, although there was no difference in MIB-1 staining indices between the tumors with and without telomerase activity at onset. In three astrocytic tumors, concurrence of telomere shortening and telomerase reactivation was observed at recurrence; in these cases, tumors progressed to a higher grade. Ten glioblastomas that progressed from lower-grade tumors exhibited telomerase activity, and their TRF lengths were reduced in 80% (8 of 10). In contrast, telomerase activity was detected in only 63.3% (19 of 30; P < 0.05) and the TRF length remained compatible with normal values in 56.7% (17 of 30; P < 0.01) of de novo glioblastomas. Thus, telomerase activity strongly correlated with potential tumor progression in the short term as well as with progression itself of the astrocytic tumors, whereas telomeres may still have been in the process of shortening in some of the de novo glioblastomas. High telomerase activity was exhibited in all primitive neuroectodermal tumors, anaplastic oligoastrocytomas, neuroblastomas, and oligodendrogliomas. TRF length was reduced in the majority (14 of 15) of three previously high-grade tumors, whereas it was compatible with that of normal brain tissues in the oligodendrogliomas, suggesting that telomerase activity with shortened telomeres correlates with the aggressive growth of high-grade neuroepithelial tumors. Tumor cell lines could be established from 17.2% (5 of 29) of neuroepithelial tumors with telomerase activity but not from tumors without this activity (P < 0.05), suggesting that telomerase reactivation is an essential event in the neuroepithelial cell immortalization in vitro. In nonneuroepithelial tumors, telomerase activity was detected in malignant tumors, such as germ cell tumors, lymphomas, metastatic adenocarcinomas, hemangiopericytomas, and an anaplastic meningioma. In contrast, such activity was not detected in benign tumors, including meningiomas, pituitary adenomas, hemangioblastomas and schwannomas, except for one hemangioblastoma that recurred four times and displayed malignant features at the fourth recurrence. These findings suggest that telomerase activity can be an index of malignant potential or malignancy itself in nonneuroepithelial brain tumors.

Clinicopathological study of cellular proliferation and invasion in gliomatosis cerebri: important role of neural cell adhesion molecule L1 in tumour invasion.

BACKGROUND/AIMS: In patients with gliomatosis cerebri (GC), glial fibrillary acidic protein (GFAP) positive cells invade the entire brain, particularly the white matter. Because the nosological definition and histogenesis of GC remain controversial, the morphology and immunohistochemical staining patterns of neoplastic GC cells were compared with those of other gliomas. METHODS: An immunohistochemical analysis of neoplastic cells from four patients with GC and 20 with astrocytic tumours using antibodies against Ki-67, GFAP, and L1, the last of which is a neural cell adhesion molecule putatively related to glioma invasion. RESULTS: GC tumour cells can be divided into two types, those mainly composed of strongly GFAP and L1 positive gemistocytic cells, the other composed of small, GFAP and L1 negative spindle shaped cells. The two types did not differ with respect to Ki-67 positivity. Cells from patients with other gliomas were positive for GFAP but concurrent L1 expression was negative or weakly positive. CONCLUSION: The strong expression of L1 in patients with GC and its poor expression in the 20 patients with other types of glioma, including those with GFAP positive gemistocytic astrocytomas, suggest that L1 expression may play a role in the histogenesis of GC.

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 microg/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 alpha5 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.

Role of fibronectin-stimulated tumor cell migration in glioma invasion in vivo: clinical significance of fibronectin and fibronectin receptor expressed in human glioma tissues.

In order to clarify the role of fibronectin in glioma invasion in vivo, we analyzed the relationship between fibronectin-stimulated cell migration and adhesion in 14 primary glioma cells and the expression of fibronectin and the fibronectin receptor in the corresponding tumor tissues. The tumors comprised nine glioblastomas (GB) and five anaplastic gliomas (AG) consisting of two astrocytomas, two oligoastrocytomas and one ependymoma. All glioma cells tested in the primary cell culture were found to migrate to fibronectin in a dose-dependent manner. The extent of cell migration to fibronectin was not significantly different for the GB and AG groups. On the other hand, cell adhesion to fibronectin in the AG was much stronger than that in the GB group. Immunohistochemistry demonstrated that fibronectin positively stained in the extracellular matrix (ECM) in eight cases and that the fibronectin receptor was positive in tumor cell membranes in 10 cases. In addition, cellular fibronectin isoforms containing ED-A and ED-B sequences were found to be immunolocalized in the tumor cells and the ECM of GB. These isoforms were also specifically expressed in tumor vessels within tumor tissues, but not in those within normal brain tissues. Cell migration tended to be expressed more strongly by glioma cells derived from tumor tissues in which fibronectin was positively immunolocalized in the ECM than from tissues with negative fibronectin in the ECM. Four glioma cells derived from GB whose tumor cells did not positively stain for fibronectin receptors migrated much less extensively to fibronectin than other glioma cells whose tissues showed positive staining for the fibronectin receptor. Of these four GB, two had loss of heterozygosity in the locus of fibronectin receptor beta1 gene. These results suggest that fibronectin deposited in the extracellular matrix of tumors, which can be derived from both plasma and the tumor cell itself, strongly promotes the migration of glioma cells, and that expression of the fibronectin receptor may play a critical role in the biological behavior of the tumor cells, particularly in fibronectin-stimulated cell migration in vivo.

Familial amyloidotic polyneuropathy presenting with carpal tunnel syndrome and a new transthyretin mutation, asparagine 70.

We report familial amyloidotic polyneuropathy in a pedigree of German ancestry residing in New Jersey. Eight affected subjects presented in the third to seventh decade with carpal tunnel syndrome (CTS) and one subject presented with vitreous opacification. Transmission was autosomal dominant and survival was prolonged. Affected subjects were heterozygous for a novel mutation in serum transthyretin (TTR), resulting in an asparagine for lysine substitution at residue 70 of the TTR monomer. We report two methods for rapid identification of the mutation based on the polymerase chain reaction. This pedigree further emphasizes the evolving phenotypic and genotypic heterogeneity of the transthyretinopathies. Familial or sporadic CTS or unexplained vitreous opacification suggest the possibility of TTR amyloidosis and should prompt a search for TTR mutations.

Microsatellite instability and mutated type II transforming growth factor-beta 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.

Homozygous deletions of p16INK4A/MTS1 and p15INK4B/MTS2 genes in glioma cells and primary glioma tissues.

The p16INK4A/MTS1 (p16) and p15INK4B/MTS2 (p15) genes map to 9p21 where genetic alterations have been frequently reported in various human tumors. Using the polymerase chain reaction (PCR), we investigated the loss of these genes on primary glioma samples and cultured glioma cells. All or any of three exons of the p16 gene were homozygously delted in 11 (35.5%) of 31 glioblastomas, none of 9 anaplastic astrocytomas and 5 astrocytomas, and in all 6 human glioma cell lines. Exon 2 of the p15 gene was homozygously deleted in 4 (12.9%) of 31 glioblastomas, but not in lower grade gliomas. It was homozygously deleted in 5 (83.3%) of 6 glioma cell lines. In 12 short-term cultures of cells derived from primary glioma samples, 5 (41.7%) and 2 (16.7%) glioblastoma-derived cells had homozygous deletion of all or any of the three exons of the p16 gene and exon 2 of the p15 gene, respectively. The deletion pattern of these genes in cultured cells was completely consistent with that seen in the primary tumors. Furthermore, two long-term cultures retained both genes that were identical to those in the original tumor tissues. Our results indicate that loss of the p16 and p15 genes may be involved in tumor progression in human gliomas, especially in the development of glioblastoma, that this loss may give growth advantage to the cells in culture, and that it is not the result of culture artifacts.

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.

Epidermal growth factor receptor in human glioma.

Distribution of the epidermal growth factor (EGF) receptor in the surgical specimen of the human glioma was studied by immunohistochemical techniques using a monoclonal anti-EGF receptor antibody. Of 11 gliomas examined, EGF receptors were detected in nine glioblastomas and in one fibrillary astrocytoma. In the majority of cells, staining was observed over the cell membrane. Nuclear and cytoplasmic staining was also seen. In four glioblastomas, EGF receptor-positive cells were diffusely distributed in the tumor tissue. In one glioblastoma and one fibrillary astrocytoma, only a few positive cells were observed. These results imply the possible role of EGF receptors in the cellular proliferation of the human glioma.

Motility factor produced by malignant glioma cells: role in tumor invasion.

To better understand the cellular mechanism of tumor invasion, the production of a cell motility-stimulating factor by malignant glioma cells was studied in vitro. Serum-free conditioned media from cultures of rat C6 and human T98G cell lines contained a factor that stimulated the locomotion of the producer cells. This factor was termed the "glioma-derived motility factor." The glioma-derived motility factor is a heat-labile protein with a molecular weight greater than 10 kD and has relative stability to acid. The factor showed not only chemotactic activity but also chemokinetic (stimulated random locomotion) activity in the two types of glioma cells studied. Although glioma-derived motility factors in conditioned media obtained from two different cell origins are likely to be the same, chemokinetic migration of T98G cells to their conditioned medium was much stronger than that of C6 cells to theirs. Coincubation of cells with cytochalasin B, which disrupts the assembly of cellular actin microfilaments, almost completely inhibited the cell migration stimulated by glioma-derived motility factor. Cytochalasin B also induced marked alterations in cell morphology, including cell retraction and arborization, while the drug did not affect cell attachment to culture dishes. These results indicate that glioma cells produce a motility factor which may play a role particularly when tumor cells are detached and migrate away from the original tumor mass, thus promoting tumor invasion. Also, glioma cell migration stimulated by the motility factor requires the normal organization of cytoskeletons such as actin microfilaments.

A clinical and neuroradiological study of X-linked hydrocephalus in Japan.

To clarify the clinicopathological features of X-linked hydrocephalus, the authors studied 30 affected males from 15 families. In utero ultrasonography, performed at 21 to 40 weeks of gestation, revealed 18 fetuses with hydrocephalus. Computerized tomography (CT) revealed bilateral enlargement of the lateral ventricle with preponderant dilation of the posterior horn. In five patients with complete magnetic resonance (MR) imaging data, the most specific finding was localized atrophy of the anterior vermian lobe. Other MR imaging findings included a large massa intermedia, flat corpora quadrigemina, a small brainstem, and diffuse hypoplasia of the cerebral white matter. In all cases, the corpus callosum was hypoplastic or aplastic. The aqueduct was patent in four of five cases. Asymmetrical reduction of the ventricular size and a rippled ventricular wall were characteristic postshunt CT findings. Progressive macrocephaly and symptoms due to increased intracranial pressure were ameliorated by the shunt; however, the neurological outcome was not improved by shunting. Of 14 patients who lived to be between 2 and 18 years of age, all are retarded. These results indicate that X-linked hydrocephalus is not a disease of simple ventriculomegaly due to aqueduct stenosis alone but involves other complicated central nervous system anomalies.

Effect of MX2, a new morpholino anthracycline, against experimental brain tumors.

A new lipophilic morpholino anthracycline derivative, MX2, has an antitumor activity against a wide spectrum of experimental tumors. We examined the effect of MX2 against experimental brain tumors. At a low dose, MX2 exhibited strong growth inhibitory activity against human and rat glioma cells. The survival time of rats with meningeal carcinomatosis induced by intracisternal inoculation of Walker 256 carcinosarcoma cells was significantly prolonged by intravenous MX2. The growth of subcutaneously implanted glioma in rats was significantly retarded by intravenous MX2. These results suggest that MX2 warrants further experimental evaluation of its efficacy against malignant brain tumors including gliomas.

Chromatography of crotamiton and its application to the determination of active ingredients in ointments.

Crotamiton, which is a mixture of cis and trans isomers, was investigated by several separation techniques. One of the HPLC modes, in which crotamiton eluted as a single peak, was selected for the determination of five active ingredients (crotamiton, prednisolone, glycyrrhetinic acid, dibucaine and chlorhexidine hydrochloride) in an ointment. The simultaneous determination was performed using isocratic reversed-phase mode within 20 min by employing an octyl (C8) column and a mobile phase containing sodium dodecyl sulfate (SDS) and 2-propanol. The method was successfully applied to quality control and stability testing of the ointment.

Abdominal cyst formation following ventriculoperitoneal shunt in a case of hydrocephalus due to cryptococcal meningitis. Case report: completely cured by surgical removal of the cyst and treatment with a newly developed anti-fungal drug (Difulcan).

A 54-year-old woman was admitted to the hospital for evaluation of meningitis. Tuberculous meningitis was suspected initially because of general findings and a high adenosine deaminase activity (ADA) value in the cerebrospinal fluid. Administration of antituberculous drugs was not effective. Computed tomography scanning revealed progression of ventricular enlargement. A ventriculo-peritoneal shunt was placed upon diagnosis of hydrocephalus due to meningitis. The presence of a large abdominal cyst formation was demonstrated. Cryptococcus was detected in the cyst fluid, leading to a diagnosis of cryptococcal meningitis. Intravenous administration of fluconazole (400 mg/day) was begun. Excision of the cyst was performed when Cryptococcus was no longer detected in the cyst fluid. The patient recovered uneventfully.

Shifting of dural arteriovenous malformation from the cavernous sinus to the sigmoid sinus to the transverse sinus after transvenous embolization. A case of left spontaneous carotid-cavernous sinus fistula.

The angiographic features of left spontaneous carotid-cavernous sinus fistula and multiple dural arteriovenous malformations that developed after transvenous embolization are described. A dural arteriovenous malformation involving the left sigmoid sinus was demonstrated, along with a marked decrease in size of the left carotid-cavernous sinus fistula and the disappearance of venous drainage from the left cavernous to the right cavernous sinus after embolization with spring coils via the left superior ophthalmic vein. The dural arteriovenous malformation of the left sigmoid sinus subsequently extended to the transverse sinus after partial embolization of the sigmoid sinus. Finally, a dural arteriovenous malformation involving the left transverse sinus developed, with the disappearance of the arteriovenous malformation affecting the sigmoid sinus and left carotid-cavernous sinus fistula following complete embolization of the sigmoid sinus via the left transverse sinus.

Ventriculolumbar perfusion chemotherapy with methotrexate and cytosine arabinoside for meningeal carcinomatosis: a pilot study in 13 patients.

Thirteen patients with meningeal carcinomatosis were treated by ventriculolumbar perfusion using methotrexate (MTX) and cytosine arabinoside (Ara-C). MTX (10-30 mg) and Ara-C (40 mg) were infused at 8- to 12-hour intervals on six or nine occasions via an Ommaya reservoir placed in the lateral ventricle. Nine of thirteen patients had evaluable response (69% response rate with a mean survival of 8.8 months among responders) and ventriculolumbar perfusion therapy was effective in improving cerebral, cranial nerve, and spinal root signs and symptoms, especially sensorimotor disturbance in the lower limbs. Three of the six bedridden patients became ambulatory without assistance and two of the four patients who were walking with assistance became ambulatory without assistance. Urinary incontinence also markedly improved, except in one nonresponder. Lumbar cerebrospinal fluid parameters (cytological findings and tumor markers) also improved in association with the clinical improvement. Our pilot results were encouraging, especially the improvement of sensorimotor function in the lower limbs. However, the toxicity was unacceptable when compared with that of standard intrathecal chemotherapy. Thus, this therapy needs to be investigated further to establish the most appropriate drug doses and perfusate volume to reduce toxicity as well as determine its true efficacy in the treatment of meningeal carcinomatosis.

Rapid growth of a meningioma during pregnancy: relationship with estrogen and progesterone receptors--case report.

Meningiomas are often described as hormone-dependent because of their preponderance in females and their tendency to clinically manifest during or after pregnancy. We describe a case of meningioma that grew rapidly during two pregnancies over a 2-year period. The tumor's rapid growth was confirmed by high-resolution computed tomography. Its estimated doubling time was 110 days. The tumor was removed and found to be a benign meningothelial meningioma. The tumor tissue was positive for estrogen receptors (ERs) and progesterone receptors (PRs). In nine cases, including this one, in which ERs and PRs were measured, the positive rate was much higher for PRs than for ERs; only two were positive for ERs. In the case reported here, it is presumed that the growth of the meningioma was influenced by estrogen and/or progesterone binding to receptors, although it is unclear whether one or both hormones was involved.