Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Microvascular proliferation of brain metastases mimics glioblastomas in squash cytology.

OBJECTIVE: Although microvascular proliferation is a key feature in the diagnosis of high-grade glioma, the characteristics of metastatic tumour vessels in smear preparations have not been documented. In this study, the vascular changes in metastatic brain tumours, using squash cytology to examine the vascular patterns in brain metastases, were reviewed. METHODS: One hundred and forty-three squash smears of brain tissue, including 25 normal or reactive tissue, 23 malignant lymphomas, 8 grade I glioma (pilocytic astrocytoma), 23 grade II glioma (diffuse astrocytoma and oligodendroglioma), 42 grade IV glioma (glioblastoma), and 22 metastasis, were assessed. Two vascular patterns were assessed: thick and branching, and glomeruloid. The vessel density, nuclear layer and the number of vessel branches were compared. Furthermore, tumour vessels of brain metastases were analysed by histology and for immunohistochemical expression of CD34, α-smooth muscle actin (SMA) and high-molecular-weight caldesmon (h-CD). RESULTS: Among 22 metastatic tumours, thick and branching vessels were found in 17 (77%) and glomeruloid vessels in 13 (59%). These incidences of microvascular proliferation patterns were similar to those of glioblastomas or pilocytic astrocytomas. Vessel density, nuclear layer and vessel wall branches were significantly higher in metastatic tumours than malignant lymphomas, grade II gliomas or normal brain tissues. Glomeruloid vessels consisted of CD34-positive cells and α-SMA-positive cells, and α-SMA-positive cells had a low h-CD expression. These immunohistochemical patterns were similar to those of high-grade gliomas. CONCLUSIONS: The vascular features of metastatic brain tumours are similar to those of glioblastomas, suggesting that these microvascular proliferations contribute to the progression of metastatic tumours.

The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas.

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

Characteristics of tumour vessels in cytological squash smears of astrocytic tumours.

OBJECTIVE: Smear preparations are useful tools from which to diagnose brain tumours intraoperatively. Although vascular proliferation is histologically a key feature of high-grade astrocytoma, the characteristics of tumour vessels in smear preparations have not been determined. METHODS: We examined the density and morphological parameters (area, width, nuclear layer and branches of vessel wall) of tumour vessels in squash smears of 43 primary astrocytomas (grade II diffuse astrocytomas, n=9; grade III anaplastic astrocytomas, n=13; grade IV glioblastomas, n=21) and normal brain tissues (n=11). RESULTS: Vessel density and all morphological parameters were significantly higher in grade IV than in the other grades of tumours and in normal brain tissue. Vessel area, width and nuclear layer were greater in grade III than in normal brain tissue. The sensitivity and specificity of these vessel parameters for astrocytomas were 75-100% and 82-100%, respectively. CONCLUSIONS: Tumour vessel evaluations from squash smears provide useful information for the intraoperative diagnosis and grading of astrocytic tumours.

Correlation with larval body size of mRNA levels of growth hormone, growth hormone receptor I and insulin-like growth factor I in larval torafugu Takifugu rubripes.

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

BACE inhibitor treatment of mice induces hyperactivity in a Seizure-related gene 6 family dependent manner without altering learning and memory.

BACE inhibitors, which decrease BACE1 (ß-secretase 1) cleavage of the amyloid precursor protein, are a potential treatment for Alzheimer's disease. Clinical trials using BACE inhibitors have reported a lack of positive effect on patient symptoms and, in some cases, have led to increased adverse events, cognitive worsening and hippocampal atrophy. A potential drawback of this strategy is the effect of BACE inhibition on other BACE1 substrates such as Seizure-related gene 6 (Sez6) family proteins which are known to have a role in neuronal function. Mice were treated with an in-diet BACE inhibitor for 4-8 weeks to achieve a clinically-relevant level of amyloid-ß40 reduction in the brain. Mice underwent behavioural testing and postmortem analysis of dendritic spine number and morphology with Golgi-Cox staining. Sez6 family triple knockout mice were tested alongside wild-type mice to identify whether any effects of the treatment were due to altered cleavage of Sez6 family proteins. Wild-type mice treated with BACE inhibitor displayed hyperactivity on the elevated open field, as indicated by greater distance travelled, but this effect was not observed in treated Sez6 triple knockout mice. BACE inhibitor treatment did not lead to significant changes in spatial or fear learning, reference memory, cognitive flexibility or anxiety in mice as assessed by the Morris water maze, context fear conditioning, or light-dark box tests. Chronic BACE inhibitor treatment reduced the density of mushroom-type spines in the somatosensory cortex, regardless of genotype, but did not affect steady-state dendritic spine density or morphology in the CA1 region of the hippocampus. Chronic BACE inhibition for 1-2 months in mice led to increased locomotor output but did not alter memory or cognitive flexibility. While the mechanism underlying the treatment-induced hyperactivity is unknown, the absence of this response in Sez6 triple knockout mice indicates that blocking ectodomain shedding of Sez6 family proteins is a contributing factor. In contrast, the decrease in mature spine density in cortical neurons was not attributable to lack of shed Sez6 family protein ectodomains. Therefore, other BACE1 substrates are implicated in this effect and, potentially, in the cognitive decline in longer-term chronically treated patients.

Characterization of Carotid Plaque Components by Quantitative Susceptibility Mapping.

BACKGROUND AND PURPOSE: Intraplaque hemorrhage in the carotid artery is related to an increased risk of cerebrovascular ischemic events. We aimed to investigate whether quantitative susceptibility mapping can characterize carotid artery plaque components and quantify the severity of intraplaque hemorrhage. MATERIALS AND METHODS: For this ex vivo quantitative susceptibility mapping study, 9 carotid endarterectomy specimens were imaged on a 3T MR imaging scanner using a 3D multi-echo gradient-echo sequence and a microscopy coil. The samples were examined histologically using immunostains, including glycophorin A and Prussian blue. The areas of erythrocytes, iron deposits, calcification, and fibrous matrices observed on stained sections were compared with quantitative susceptibility mapping findings and their mean susceptibility values. RESULTS: Intraplaque hemorrhage and iron deposits were observed only in areas hyperintense on quantitative susceptibility mapping; calcifications and fibrous matrices were prevalent in hypointense areas. The mean susceptibility values for necrotic cores with intraplaque hemorrhage but no iron deposits, cores with iron deposits but no intraplaque hemorrhage, cores without either intraplaque hemorrhage or iron deposits, and cores with calcification were 188 ± 51, 129 ± 49, -11 ± 17, and -158 ± 78 parts per billion, respectively. There was a significant difference in the mean susceptibility values among the 4 histologic components (P < .01). The mean susceptibility values of the whole plaque positively correlated with the percentage area positive for glycophorin A (r = 0.65, P < .001) and Prussian blue (r = 0.47, P < .001). CONCLUSIONS: Our findings suggest that quantitative susceptibility mapping can characterize the composition of carotid plaques and quantify the degree of intraplaque hemorrhage and iron deposits.

Usefulness of Contrast-Enhanced 3D-FLAIR MR Imaging for Differentiating Rathke Cleft Cyst from Cystic Craniopharyngioma.

BACKGROUND AND PURPOSE: Because it can be difficult to discriminate between a Rathke cleft cyst and cystic craniopharyngioma by conventional MR imaging alone, we investigated whether contrast-enhanced 3D T2-FLAIR MR imaging at 3T helps to distinguish a Rathke cleft cyst from a cystic craniopharyngioma. MATERIALS AND METHODS: We evaluated pre- and postcontrast T1-weighted and 3D T2-FLAIR images of 17 patients with pathologically confirmed Rathke cleft cyst (n = 10) or cystic craniopharyngioma (n = 7). All underwent 3T MR imaging studies before surgery. Two neuroradiologists independently recorded the enhancement grade of the lesion wall as grade 2 (most of the wall enhanced), grade 1 (some of the wall enhanced), and grade 0 (none of the wall enhanced). One neuroradiologist performed a blinded reading study of conventional MR images with/without 3D T2-FLAIR images. Interobserver agreement was determined by calculating the κ coefficient. Statistical analyses, including receiver operating characteristic curve analysis were performed. RESULTS: Interobserver agreement for postcontrast T1WI and 3D T2-FLAIR images was excellent (κ = 0.824 and κ = 0.867, respectively). Although the difference in the mean enhancement grade of Rathke cleft cysts and cystic craniopharyngiomas was not significant on postcontrast T1WIs, it was significant on postcontrast 3D T2-FLAIR images (P = .0011). The area under the receiver operating characteristic curve of the conventional MR alone and conventional MR with 3D T2-FLAIR readings was 0.879 and 1.0, respectively, though there was no significant difference in the area under the curve between the 2 readings. CONCLUSIONS: Contrast-enhanced 3D T2-FLAIR imaging at 3T helps to distinguish a Rathke cleft cyst from cystic craniopharyngioma.

Type 3 and type 1 ryanodine receptors are localized in triads of the same mammalian skeletal muscle fibers.

The type 3 ryanodine receptor (RyR3) is a ubiquitous calcium release channel that has recently been found in mammalian skeletal muscles. However, in contrast to the skeletal muscle isoform (RyR1), neither the subcellular distribution nor the physiological role of RyR3 are known. Here, we used isoform-specific antibodies to localize RyR3 in muscles of normal and RyR knockout mice. In normal hind limb and diaphragm muscles of young mice, RyR3 was expressed in all fibers where it was codistributed with RyR1 and with the skeletal muscle dihydropyridine receptor. This distribution pattern indicates that RyR3 is localized in the triadic junctions between the transverse tubules and the sarcoplasmic reticulum. During development, RyR3 expression declined rapidly in some fibers whereas other fibers maintained expression of RyR3 into adulthood. Comparing the distribution of RyR3-containing fibers with that of known fiber types did not show a direct correlation. Targeted deletion of the RyR1 or RyR3 gene resulted in the expected loss of the targeted isoform, but had no adverse effects on the expression and localization of the respective other RyR isoform. The localization of RyR3 in skeletal muscle triads, together with RyR1, is consistent with an accessory function of RyR3 in skeletal muscle excitation-contraction coupling.

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1.

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

Abnormal features in skeletal muscle from mice lacking mitsugumin29.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

Molecular architecture of membranes involved in excitation-contraction coupling of cardiac muscle.

Peripheral couplings are junctions between the sarcoplasmic reticulum (SR) and the surface membrane (SM). Feet occupy the SR/SM junctional gap and are identified as the SR calcium release channels, or ryanodine receptors (RyRs). In cardiac muscle, the activation of RyRs during excitation-contraction (e-c) coupling is initiated by surface membrane depolarization, followed by the opening of surface membrane calcium channels, the dihydropyridine receptors (DHPRs). We have studied the disposition of DHPRs and RyRs, and the structure of peripheral couplings in chick myocardium, a muscle that has no transverse tubules. Immunolabeling shows colocalization of RyRs and DHPRs in clusters at the fiber's periphery. The positions of DHPR and RyR clusters change coincidentally during development. Freeze-fracture of the surface membrane reveals the presence of domains (junctional domains) occupied by clusters of large particles. Junctional domains in the surface membrane and arrays of feet in the junctional gap have similar sizes and corresponding positions during development, suggesting that both are components of peripheral couplings. As opposed to skeletal muscle, membrane particles in junctional domains of cardiac muscle do not form tetrads. Thus, despite their proximity to the feet, they do not appear to be specifically associated with them. Two observations establish the identify of the structurally identified feet arrays/junctional domain complexes with the immunocytochemically defined RyRs/DHPRs coclusters: the concomitant changes during development and the identification of feet as the cytoplasmic domains of RyRs. We suggest that the large particles in junctional domains of the surface membrane represent DHPRs. These observations have two important functional consequences. First, the apposition of DHPRs and RyRs indicates that most of the inward calcium current flows into the restricted space where feet are located. Secondly, contrary to skeletal muscle, presumptive DHPRs do not show a specific association with the feet, which is consistent with a less direct role of charge movement in cardiac than in skeletal e-c coupling.

Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns.

Inositol 1,4,5-trisphosphate (IP3) is a second messenger that elicits complex spatiotemporal patterns of calcium ion (Ca2+) mobilization and has essential roles in the regulation of many cellular functions. In Madin-Darby canine kidney epithelial cells, green fluorescent protein-tagged pleckstrin homology domain translocated from the plasma membrane to the cytoplasm in response to increased concentration of IP3. The detection of translocation enabled monitoring of IP3 concentration changes within single cells and revealed spatiotemporal dynamics in the concentration of IP3 synchronous with Ca2+ oscillations and intracellular and intercellular IP3 waves that accompanied Ca2+ waves. Such changes in IP3 concentration may be fundamental to Ca2+ signaling.

Multiple prominent dilated perivascular spaces do not induce Wallerian degeneration as evaluated by diffusion tensor imaging.

It is unknown whether dilated perivascular spaces can affect the adjacent neuronal fibers. We describe conventional MR and diffusion tensor imaging findings of a case with multiple, prominent dilated perivascular spaces in the left cerebral hemisphere. Diffusion tensor imaging showed no alterations in the fractional anisotropy and apparent diffusion coefficient values for the corona radiata, posterior rim of the internal capsule, and the cerebral peduncle, indicating no wallerian degeneration associated with dilated perivascular spaces.

Clinical evaluation of cellulose porous beads for the therapeutic embolization of meningiomas.

BACKGROUND AND PURPOSE: Cellulose porous beads (CPBs) are a new, exceptionally uniformly sized, nonabsorbable embolic agent. We evaluated their efficacy in the preoperative embolization of meningiomas. METHODS: In 141 consecutive patients, we used CPBs (200-microm diameter) for the preoperative embolization of meningiomas. We selected patients whose tumors were > or =4 cm with 50% of blood to the tumor supplied by the external carotid artery (ECA). All patients underwent a provocation test before embolization. The percentage of blood supplied to the tumor by the internal carotid artery and ECA was determined angiographically. Nonenhanced areas on postembolization MR imaging were calculated. Intraoperative blood loss, units of blood transfusion, and hemostasis at the time of surgery were recorded for each patient. The interval between embolization and surgery was intentionally longer than 7 days. RESULTS: Of the 141 patients, 128 underwent CBP embolization. Eleven patients had positive provocation test results, and 2 had vasospasm; they were not CBP embolized. In 72% of the patients CBP embolization achieved reduction in the flow of the feeding artery by more than 50%. The nonenhanced area on MR imaging was not significantly correlated with the degree of ECA supply or devascularization. The interval between embolization and surgery was 8-26 days (mean, 9.9 days). The longer this interval, the greater was the tumor-softening effect and the rate of tumor removal. CONCLUSIONS: CPBs may be useful for the preoperative embolization of meningiomas. To increase the efficacy of CPB embolization, the interval to surgery should be at least 7 days.

Quantitative study of monocyte chemoattractant protein-1 (MCP-1) in cerebrospinal fluid and cyst fluid from patients with malignant glioma.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino acid protein that attracts monocytes. In vitro studies have reported high levels of MCP-1 messenger RNA expression, as well as the presence of MCP-1, in malignant glioma cells. PURPOSE: Our purpose was to determine whether an MCP-1 assay could be used in a clinical setting 1) to differentiate malignant from benign gliomas and from nontumor disorders of the central nervous system and 2) to detect subarachnoid dissemination of glioma cells. METHODS: MCP-1 levels in cerebrospinal fluid (CSF) and cyst fluid were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that we had previously developed. We measured MCP-1 levels in CSF samples from 19 patients with malignant glioma (glioblastoma, 10; anaplastic astrocytoma, six; anaplastic oligodendroglioma, two; and ependymoblastoma, one), nine patients with benign glioma, and seven patients with nontumor disorders of the central nervous system. Cyst fluids from four patients with malignant glioma (anaplastic astrocytoma) were also tested. The correlation between MCP-1 concentration in the CSF and subarachnoid dissemination of malignant glioma cells was also studied. RESULTS: The MCP-1 concentration (mean +/- SE) in CSF samples from patients with malignant glioma (2.3 +/- 0.4 ng/mL) was significantly higher than that from patients with benign glioma (0.6 +/- 0.1 ng/mL) (P < .01) or from patients with no tumor (0.5 +/- 0.1 ng/mL) (P < .01). Furthermore, CSF samples from patients with subarachnoid dissemination of malignant glioma contained significantly higher amounts of MCP-1 than those from patients without dissemination (P < .05). Cyst fluids from four of the patients with malignant glioma contained high concentrations of MCP-1. CONCLUSIONS: These results indicate that MCP-1 is produced by malignant glioma in vivo as well as in vitro and suggest that testing for MCP-1 in CSF may be useful in the clinic to differentiate malignant glioma from benign glioma and to detect subarachnoid dissemination of the tumor cells. IMPLICATIONS: The MCP-1 ELISA in CSF may lead to more accurate diagnosis of malignant glioma and detection of subarachnoid dissemination of tumor cells, facilitating selection of patients with these conditions for appropriate therapy.

High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.

A novel method for high-efficiency and region- controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection. A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes. The lacZ gene was efficiently transferred and expressed in the tumor cell 3 days after plasmid injection. However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes. The plasmid was not transferred without electroporation. Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system. The expressed monocyte chemoattractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in tumor tissue. This method, electrogene therapy, which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy.

Detection of cellular proteins that interact with the NF2 tumor suppressor gene product.

The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.

Impairment of cell adhesion by expression of the mutant neurofibromatosis type 2 (NF2) genes which lack exons in the ERM-homology domain.

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins which link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations were nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, we performed protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, we expressed the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) in a fibroblast cell line by using both liposome-mediated transfection and nuclear microinjection of the expression plasmids. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in detachment from the substratum. These findings suggested that the exon-missing mutations in the ERM-homology domain may affect cell membrane-cytoskeleton signaling and consequently disrupt cell-to-cell or cell-to-matrix interaction.

Mutations of the p16 gene in gliomas.

In the present study we investigated the frequency of p16 gene exon 2 mutations in 35 malignant gliomas, using either direct sequencing of the PCR products or cloning into the pCRII vector and sequencing of the cloned PCR products. No mutations were detected during direct sequencing of the PCR products. However, after sequencing of individual clones, we found multiple mutations in 5 tumors involving codons 73(GCC to ACC, Ala to Thr), 76 (GCC to GTC, Ala to Val), 85(GCT to ACT, Ala to Thr), 98(CAC to TAC, His to Tyr), 102 (GCG to GTG, Ala to Val), 106 (GTG to ATG, Val to Met), 107 (CGC to TGC, Arg to Cys), 127 (GCA to GTA, Ala to Val), 128 (CGG to TGG, Arg to Trp) and 136 (GGC to GAC, Gly to Asp). Mutations were found only in glioblastomas and were either C to T or G to A transitions. Each mutation was detected in a small percentage of tumor cells (1.3-22%) using individual colony sequencing and southern hybridization with mutant oligonucleotides, consistent with the heterogenous cell population of glioblastomas. The presence of p16 gene mutations only in glioblastomas suggests that they are late events in glioma development.