Alterations of protein 4.1 family members in ependymomas: a study of 84 cases.

Ependymomas are common pediatric and adult CNS malignancies with a wide biologic spectrum that is often hard to predict using classic prognostic variables. The molecular pathogenesis is also poorly understood and few reproducible genetic alterations have been identified. The most common genetic alteration has been the loss of the Protein 4.1 family member, NF2, predominantly in spinal ependymomas. In contrast, a pilot study suggested that 4.1B deletions might be more common in intracranial ependymomas. These findings prompted us to study Protein 4.1 family members (NF2, 4.1B, 4.1R, 4.1G) in a larger cohort of 84 ependymomas (51 intracranial and 33 spinal; 11 WHO grade I, 43 grade II, 30 grade III). Fluorescence in situ hybridization was performed using NF2, 4.1B, 4.1R and 4.1G probes and immunohistochemical staining was performed in a subset using merlin, Protein 4.1B and Protein 4.1R antibodies. Additionally, frozen tissue from nine ependymomas (four intracranial and five spinal) was obtained for Western blot analysis for merlin, 4.1B and 4.1R expression. The majority of cases harbored one or more detectable genetic alterations, but we found that 4.1B gene deletions and 4.1R loss of expression were statistically more common in the pediatric vs adult, intracranial vs spinal, and grade III vs grade I/II subsets (P-values of 0.038 to <0.001). Also, 4.1G deletions were seen in 11/27 (41%) patients who either died of disease or had residual/recurrent tumor vs 5/41 patients with no evidence of disease at last follow-up (P=0.009). We conclude that alterations of Protein 4.1 family members are common in ependymal tumors and that specific alterations are associated with distinct clinicopathologic subsets.     

Inactivation patterns of NF2 and DAL-1/4.1B (EPB41L3) in sporadic meningioma

The molecular basis of tumorigenesis and tumor progression in meningiomas is not fully understood. The neurofibromatosis 2 (NF2) locus is inactivated in 50-60% of sporadic meningiomas, but the genetic basis of sporadic meningiomas not inactivated at the NF2 locus remains unclear. Specifically, there is conflicting data regarding the role of the tumor suppressor gene DAL-1/4.1B. Using microsatellite markers, we studied 63 sporadic meningiomas to determine loss of heterozygosity (LOH) at the NF2 and DAL-1/4.1B loci. Array comparative genomic hybridization analysis of 52 of these tumors was performed to determine copy number changes on chromosomes 18 and 22. Forty-one of 62 informative tumors showed LOH at the NF2 locus (66%) while only 12 of 62 informative tumors (19%) showed LOH of DAL-1/4.1B. Eleven of 12 (92%) tumors with DAL-1/4.1B LOH also had NF2 LOH. Monosomy or large deletions of chromosomes 18 and 22 were the main mechanism for LOH in these tumors. These studies implicate the DAL-1/4.1B locus in sporadic meningiomas less commonly than reported previously, and suggest that it is a progression rather than an initiation locus. Furthermore, we found the majority of meningiomas developed monosomy rather than isodisomy at the NF2 and DAL-1/4.1B loci as the mechanism for LOH.     

Analysis of the NF2 gene in oligodendrogliomas and ependymomas

Allelic losses of chromosome 22 are commonly found in ependymomas and oligodendrogliomas, suggesting that at least one tumor suppressor gene on chromosome 22 must be inactivated during the multistep process of tumorigenesis in these glial tumors. The neurofibromatosis 2 gene (NF2) located at 22q12, is a candidate tumor suppressor gene potentially involved in the pathogenesis of gliomas. Because there have been only a few studies of the NF2 gene in glial tumors other than astrocytoma, we screened the entire 17 NF2 exons for mutations in a series of 47 nonastrocytic tumors, including 40 oligodendrogliomas and 7 ependymomas. Only one mutation was detected, a 59-base pair insertion in exon 3 from a spinal anaplastic ependymoma. These results concur with previous findings proposing preferential inactivation of the NF2 gene in a subgroup of ependymomas, and suggest that the NF2 gene is not the target of chromosome 22 aberrations in oligodendrogliomas.     

Mutational analysis of the DAL-1/4.1B tumour-suppressor gene locus in meningiomas

The DAL-1/41B gene (differentially expressed in adenocarcinoma of the lung), located in the chromosome 18p11.3 region, belongs to the protein family 4.1 (membrane-associated proteins), which includes the product of the NF2 gene (merlin), and the proteins, ezrin, radixin, and moesin. DAL-1/4.1B is normally expressed at high levels in the brain, with lower levels in the kidney, intestine, and testis. DAL-1/4.1B is known to suppress growth in meningiomas and can be lost in about 60% of sporadic meningiomas as an early event in tumorigenesis; it is a critical growth regulator in the pathogenesis of neoplastic transformation. The similarity between the DAL-1/4.1B protein and merlin, with their high levels of expression in the brain and their recurrent loss in meningiomas, and the lack of previous DAL-1/4.1B mutational analysis reports initiated this mutational study of DAL-1/4.1B in a series of 83 meningiomas. We found the following sequence variations; Ala555Thr (G1663A in exon 13) and Thr950Lys (C2849A in exon 19) in two cases each, and one case with a 5pb deletion (del taaaa) in intron 18. A polymorphism in exon 14 (C2112T/Thr704Thr, also known as C2166T) was also identified; the tumoral allelic constitutions were heterozygous C/T in 15, homo- or hemizygous C in 67 and hemizygous T in one tumour. The low mutational frequency in our study discounts sequence variations in DAL-1/4.1B as the main mechanism underlying participation of this gene in the neoplastic transformation of meningiomas, and suggests that other inactivating mechanisms, such as epigenetic changes, may participate in DAL1/4.1B silencing.     

Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas

Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in approximately 60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT-PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types.     

原癌基因Wip1在室管膜瘤中表达增加并与P53相关

目的 探讨野生型P53-诱导的蛋白磷酸酶1(Wip1)在室管膜瘤中的表达及其与P53相关性.方法 用免疫组织化学、反转录聚合酶链反应(RT-PCa)和Western blot 方法检测31例室管膜瘤及10例正常脑组织中Wip1mRNA、蛋白表达.并用免疫组化检测P53.同时,回顾性分析临床病理因素与Wipl表达之间的相...     

Differential increase in the expression of heat shock protein family members during sleep deprivation and during sleep

Although sleep is thought to be restorative from prior wakeful activities, it is not clear what is being restored. To determine whether the synthesis of macromolecules is increased in the cerebral cortex during sleep, we subjected C57BL/6 mice to 6 hours of sleep deprivation and then screened the expression of 1176 genes of known function by using cDNA arrays. The expression of the heat shock proteins (HSP), endoplasmic reticulum protein (ERp72) and glucose-regulated protein (GRp78), was among the genes whose expression was significantly elevated in the cortex during sleep deprivation, whereas GRp78 and GRp94 mRNAs were elevated in the cortex during recovery sleep after sleep deprivation, as confirmed by conventional and quantitative real-time polymerase chain reaction and/or Northern analyses. A systematic evaluation of the expression of six heat shock protein family members (ERP72, GRp78, GRp94, HSP27, HSP70-1, and HSP84) in seven brain regions revealed increased mRNA levels in cortex, basal forebrain, hypothalamus, cerebellum and medulla during sleep deprivation, whereas increased mRNA levels during recovery sleep were limited to the cortex and medulla. Immunohistochemical studies identified increased numbers of GRp78-, GRp94-, and ERp72-immunoreactive cells in the dorsal and lateral cortex during sleep deprivation but, during recovery sleep, elevated numbers of these cells were found only in the lateral cortex. In the medulla, increased numbers of GRp94-immunoreactive cells were observed in nucleus tractus solitarius, dorsal motor nucleus of the vagus and the rostroventrolateral medulla during recovery sleep. The widespread increase of heat shock protein family mRNAs in brain during sleep deprivation may be a neuroprotective response to prolonged wakefulness. In contrast, the relatively limited heat shock protein family mRNA expression during recovery sleep may be related to the role of heat shock proteins in protein biogenesis and thus to the restorative function of sleep.     

Differential role of 14-3-3 family members in Xenopus development.

The 14-3-3 proteins are intracellular dimeric phosphoserine/threonine binding molecules that participate in signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. Previous work established that 14-3-3 proteins are required in early Xenopus laevis development by modulating fibroblast growth factor signaling. Although this general requirement for 14-3-3 proteins in Xenopus early embryogenesis is established, there is no information about the specific role of individual 14-3-3 genes. Botanical studies previously demonstrated functional specificity among 14-3-3 genes during plant development. In this study, an antisense morpholino oligo microinjection approach was used to characterize the requirement for six specific 14-3-3 family members in Xenopus embryogenesis. Microinjection experiments followed by Western blot analysis showed that morpholinos reduced specific 14-3-3 protein levels. Embryos lacking specific 14-3-3 isoforms displayed unique phenotypic defects. In particular, reduction of 14-3-3 tau (tau) protein, and to a lesser extent, 14-3-3 epsilon (epsilon), resulted in embryos with prominent gastrulation and axial patterning defects and reduced mesodermal marker gene expression. In contrast, reduction of 14-3-3 zeta (zeta) protein caused no obvious phenotypic abnormalities. Reduction of 14-3-3 gamma (gamma) protein resulted in eye defects without gastrulation abnormalities. Therefore, individual 14-3-3 genes have separable functions in vertebrate embryonic development.     

Calcium pyrophosphate dihydrate crystal-induced inhibition of neutrophil apoptosis: involvement of Bcl-2 family members

Introduction  

The inflammation associated with calcium pyrophosphate dihydrate (CPPD) crystal-induced arthritis arises from the activation of neutrophils with crystals in the synovial joint. Furthermore, constitutive neutrophil apoptosis is inhibited by this interaction with CPPD so that the lifetime of the cells and the duration of the inflammatory response are extended. The objective of this study was to investigate the role of bcl-2 protein family members in the CPPD-induced prosurvival response.     

Differential expression of slitrk family members in the mouse nervous system

The Slitrk family of transmembrane proteins is composed of six members that are highly expressed in the nervous system. To date, the function of Slitrks during development of the nervous system has yet to be defined. The high homology between the extracellular region of Slitrks and the repulsive axon guidance molecules Slits suggests that Slitrks may regulate axon outgrowth during development. To begin to evaluate their role during development, we have examined the expression of the Slitrk genes in the developing murine nervous system using in situ hybridization. Here, we show that despite some overlap in expression, the Slitrks display distinct patterns of expression in the olfactory system, the eye, forebrain structures, the cerebellum, the spinal cord, and dorsal root ganglia. These diverse patterns of expression suggest that Slitrk family members may have different functions during development of the nervous system. Developmental Dynamics 238:3285–3296, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of inhibitor-of-apoptosis protein family members in malignant mesothelioma

Inhibitor-of-apoptosis proteins (IAPs) mediate cancer cell survival and chemoresistance. We analyzed the expressions of X-linked IAP (XIAP), survivin, and livin in malignant mesothelioma. Ten effusions were analyzed for XIAP, survivin, and livin expression using immunoblotting. Based on the immunoblotting results, 112 mesotheliomas from 94 patients (pleural, n = 77; peritoneal, n = 35; solid, n = 68; effusions, n = 44) were immunostained for XIAP and survivin expression. Results were analyzed for associations with anatomic site (pleura versus peritoneum), specimen type (solid versus effusion), proliferation (Ki-67 score), and survival. Immunoblotting showed expression of XIAP in 9 of 10 effusions and that of survivin in 4 of 10 effusions, but no expression of livin. Immunohistochemistry showed cytoplasmic XIAP expression in 71 of the 112 (63%) tumors. XIAP expression was significantly higher in peritoneal mesotheliomas than in pleural mesotheliomas (P = .001) and in effusions than in solid lesions (P = .017). Cytoplasmic survivin was found in 75 of the 112 (67%) tumors and showed no site-related difference. Nuclear survivin was expressed in 37 of the 112 (33%) tumors, with a trend for positive association with the Ki-67 score (P = .051). Nuclear survivin (P = .003) and Ki-67 (P = .013) were downregulated in effusions as compared with solid tumors. Higher XIAP expression and Ki-67 score were associated with a trend for poor overall survival (P = .064 for both) in the univariate analysis. XIAP and survivin, but not livin, are frequently expressed in malignant mesotheliomas. Nuclear survivin expression is reduced in effusions as compared with solid lesions concomitantly with reduced proliferation. XIAP is upregulated in mesothelioma effusions and peritoneal mesotheliomas, suggesting a prosurvival role in malignant mesothelioma cells, particularly at these anatomic sites.     

Evaluation of NF2 gene deletion in sporadic schwannomas, meningiomas, and ependymomas by chromogenic in situ hybridization

Fluorescence in situ hybridization, loss of heterozygosity testing, and comparative genomic hybridization have been used to detect NF2 gene alterations in both sporadic and neurofibromatosis type 2 (NF2)-associated central nervous system tumors. In this study, we performed chromogenic in situ hybridization (CISH) and immunohistochemistry to evaluate for NF2 gene deletion in a group of sporadic meningiomas, schwannomas, and ependymomas. Twenty-two sporadic tumors, including 9 ependymomas, 10 meningiomas, and 3 schwannomas, were studied. CISH and immunohistochemistry were performed using the NF2 gene deletion probe and NF2 polyclonal antibody. Deletion of the NF2 gene was identified in 11 (50%) tumors, including 60% (6/10) of meningiomas, 33% (3/9) of ependymomas, and 67% (2/3) of schwannomas. The remaining 11 (50%) cases were diploid. Overall, immunoexpression of NF2 protein was observed in 50% (11/22) tumors, and concordance between CISH and immunohistochemistry was observed in 73% of cases. Our results support previous observations that schwannomas and meningiomas, and to a lesser degree, ependymomas, express a high incidence of NF2 gene deletion, which supports the hypothesis that NF2 gene plays an important role in their tumorigenesis. In addition, we have validated CISH as an efficient, economic, and reliable method for routinely assessing NF2 gene deletion in these tumors.     

Unique features of different members of the human guanylate-binding protein family.

Guanylate-binding proteins (GBPs) are the most abundant cellular proteins expressed in response to interferon-gamma (IFN-gamma), with seven highly homologous members in humans, termed HuGBP-1 to HuGBP-7. To date, differential features that may indicate differential functions of these proteins have not been described. Here, we investigated the expression and subcellular localization of the different HuGBPs in endothelial cells (EC). IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) induced the expression of HuGBP-1, HuGBP-2, and HuGBP-3 at similar high levels. In contrast, expression of HuGBP-4 and HuGBP-5 was robustly induced only by IFN-gamma and not by TNF-alpha and IL-1beta. Expression of HuGBP-6 and HuGBP-7 was not detected in EC under the various conditions examined. Investigating subcellular localization of the EC-expressed HuGBPs, HuGBP-1, HuGBP-3, and HuGBP-5 were exclusively detected in the cytoplasm, whereas HuGBP-2 and HuGBP-4 displayed a nucleocytoplasmic distribution. Treatment of the cells with IFN-gamma and aluminum fluoride caused rapid enrichment of HuGBP-1 and HuGBP-2 in the Golgi apparatus, as demonstrated by time-lapse microscopy and fluorescence analyses of GFP-tagged HuGBPs. HuGBP-3 and HuGBP-4 were never detected in the Golgi apparatus, whereas HuGBP-5 was constitutively enriched in this cytosolic compartment, irrespective of stimulation. These results assign a characteristic pattern of expression and subcellular localization to each of the HuGBPs, indicating for the first time that these proteins may have different cellular functions.