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The article focuses on a study regarding somatic hypoxia-inducible factor 2 alpha (HIF2A) mutations. It mentions that syndrome of multiple paragangliomas (PGL) and duodenal somatostatinomas are associated with polycythemia found in females. It also mentions that gain-of-function somatic HIF2A mutations are located in oxygen-dependent degradation domain (ODD) of HIF2 alpha.

: Hypoxia-inducible factors are transcription factors controlling energy, iron metabolism, erythropoiesis, and development. When these proteins are dysregulated, they contribute to tumorigenesis and cancer progression. However, mutations in genes encoding α subunits of hypoxia-inducible factors (HIF-α) have not previously been identified in any cancer. Here we report two novel somatic gain-of-function mutations in the gene encoding hypoxia-inducible factor 2α (HIF2A) in two patients, one presenting with paraganglioma and the other with paraganglioma and somatostatinoma, both of whom had polycythemia. The two mutations were associated with increased HIF-2α activity and increased protein half-life. [ABSTRACT FROM PUBLISHER]

Background To determine the origin of the neoplastic cell in central nervous system (CNS) hemangioblastomas in von Hippel-Lindau disease (VHL) and its role in tumor formation and distribution, we characterized and differentiated neoplastic cells from hemangioblastomas removed from VHL patients. Methods and Findings A total of 31 CNS hemangioblastomas from 25 VHL patients were resected and analyzed. Tumor cells from the hemangioblastomas were characterized, grown, and differentiated into multiple lineages. Resected hemangioblastomas were located in the cerebellum (11 tumors), brainstem (five tumors), and spinal cord (15 tumors). Consistent with an embryologically derived hemangioblast, the neoplastic cells demonstrated coexpression of the mesodermal markers brachyury, Flk-1 (vascular endothelial growth factor-2), and stem cell leukemia (Scl). The neoplastic cells also expressed hematopoietic stem cell antigens and receptors including CD133, CD34, c-kit, Scl, erythropoietin, and erythropoietin receptor. Under specific microenvironments, neoplastic cells (hemangioblasts) were expanded and differentiated into erythrocytic, granulocytic, and endothelial progenitors. Deletion of the wild-type VHL allele in the hematopoietic and endothelial progeny confirmed their neoplastic origin. Conclusions The neoplastic cell of origin for CNS hemangioblastomas in VHL patients is the mesoderm-derived, embryologically arrested hemangioblast. The hematopoietic and endothelial differentiation potential of these cells can be reactivated under suitable conditions. These findings may also explain the unique tissue distribution of tumor involvement. [ABSTRACT FROM AUTHOR]

A 13-year-old male presented with a 10-day history of left eye swelling and pain. These symptoms prompted presentation to the emergency department. He had no significant past medical history and no preceding fevers or chills. He was found on examination of the eyes and the orbit to have left supraorbital erythema, edema, and pain with upward and medial gaze. Examination of the globe, fundus, and visual fields were normal. His white blood cell count was 6.2 (x1000/mm3) with an erythrocyte sedimentation rate of 4 (mm/hr). Diagnostic endoscopic biopsy was performed. Here we present this case alongside clinical reasoning and diagnostic evaluation with relevant input from respective experts. This case discussion reviews the final diagnosis, as well as the corresponding evaluation and management. Diagnostic algorithms based on literature review and clinical experience are also included. [ABSTRACT FROM AUTHOR]

Protein phosphatases play essential roles as negative regulators of kinases and signaling cascades involved in cytoskeletal organization. Protein phosphatase 2A (PP2A) is highly conserved and is the predominant serine/threonine phosphatase in the nervous system, constituting more than 70% of all neuronal phosphatases. PP2A is involved in diverse regulatory functions, including cell cycle progression, apoptosis, and DNA repair. Although PP2A has historically been identified as a tumor suppressor, inhibition of PP2A has paradoxically demonstrated potential as a therapeutic target for various cancers. LB100, a water-soluble, small-molecule competitive inhibitor of PP2A, has shown particular promise as a chemo- and radio-sensitizing agent. Preclinical success has led to a profusion of clinical trials on LB100 adjuvant therapies, including a phase I trial in extensive-stage small-cell lung cancer, a phase I/II trial in myelodysplastic syndrome, a phase II trial in recurrent glioblastoma, and a completed phase I trial assessing the safety of LB100 and docetaxel in various relapsed solid tumors. Herein, we review the development of LB100, the role of PP2A in cancer biology, and recent advances in targeting PP2A inhibition in immunotherapy. [ABSTRACT FROM AUTHOR]

Many solid tumors, including pheochromocytoma (PHEO) and paraganglioma (PGL), are characterized by a (pseudo)hypoxic signature. (Pseudo)hypoxia has been shown to promote both tumor progression and resistance to therapy.The major mediators of the transcriptional hypoxic response are hypoxia-inducible factors (HIFs). High levels of HlFs lead to transcription of hypoxia-responsive genes, which are involved in tumorigenesis. PHEOs and PGLs are catecholamine-producing tumors arising from sympathetic- or parasympathetic-derived chromaffin tissue. In recent years, substantial progress has been made in understanding the metabolic disturbances present in PHEO and PGL, especially because of the identification of some disease-susceptibility genes.To date, fifteen PHEO and PGL susceptibility genes have been identified. Based on the main transcription signatures of the mutated genes, PHEOs and PGLs have been divided into two clusters, pseudohypoxic cluster 1 and cluster 2, rich in kinase receptor signaling and protein translation pathways. Although these two clusters seem to show distinct signaling pathways, recent data suggest that both clusters are interconnected by HIF signaling as the important driver in their tumorigenesis, and mutations in most PHEO and PGL susceptibility genes seem to affect HIF-a regulation and its downstream signaling pathways. HIF signaling appears to play an important role in the development and growth of PHEOs and PGLs, which could suggest new therapeutic approaches for the treatment of these tumors. [ABSTRACT FROM AUTHOR]

Presents a study that examined the possible mechanisms of tumor formation in patients with multiple endocrine neoplasia type 2 (MEN 2)-related pheochromocytoma. Descriptions of von Hippel-Lindau disease; Background on MEN 2; Development of pheochromocytoma.

Background/Aims: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that mediates cell cycle regulation and metabolism. Mounting evidence has indicated that PP2A inhibition exhibits considerable anticancer potency in multiple types of human cancers. However, the efficacy of PP2A inhibition remains unexplored in mucoepidermoid carcinoma (MEC), especially in locally advanced and metastatic cases with limited systemic treatment. In this study, we demonstrated the therapeutic potency of LB100 in mucoepidermoid carcinoma. Methods: In this study, the expression of PP2A was evaluated using immunohistochemical (IHC) staining. The effects associated with LB100 alone and in combination with cisplatin for the treatment of mucoepidermoid carcinoma were investigated both in vitro, regarding metabolism, proliferation, and migration, and in vivo in a mucoepidermoid carcinoma xenograft model. In addition, with LB100 treatment and in response to an insulin stimulus, the expression levels and phosphorylation levels of targets in the PI3K-AKT pathway were determined using western blot analysis and immunoblotting. Results: The expression of protein phosphatase 2A was significantly upregulated in the clinical specimens of high-grade MECs compared with those of low-/medium-grade MECs and normal controls. In this article, we report that a small molecule PP2A inhibitor, LB100, decreased cellular viability and glycolytic activity and induced G2/M cell cycle arrest. Importantly, LB100 enhanced the efficacy of cisplatin in mucoepidermoid carcinoma cells both in vitro and in vivo. PP2A inhibition by LB100 increased the phosphorylation of insulin receptor substrate 1(IRS- 1) on serine residues, downregulated the expression of phosphatidylinositol 3-kinase (PI3K) p110 alpha subunit and dephosphorylated AKT at Ser473 and Thr308 in mucoepidermoid carcinoma cells in response to insulin stimulus. Conclusion: These results highlight the translational potential of PP2A inhibition to synergize with cisplatin in mucoepidermoid carcinoma treatment. [ABSTRACT FROM AUTHOR]

Recently, activating mutations of the hypoxia-inducible factor 2α gene (HIF2A/EPAS1) have been recognized to predispose to multiple paragangliomas (PGLs) and duodenal somatostatinomas associated with polycythemia, and ocular abnormalities. Previously, mutations in the SDHA/B/C/D, SDHAF2, VHL, FH, PHD1, and PHD2 genes have been associated with HIF activation and the development of pseudohypoxic (cluster-1) PGLs. These tumors overlap in terms of tumor location, syndromic presentation, and noradrenergic phenotype to a certain extent. However, they also differ especially by clinical outcome and by presence of other tumors or abnormalities. In the present study, we aimed to establish additional molecular differences between HIF2A and non-HIF2A pseudohypoxic PGLs. RNA expression patterns of HIF2A PGLs (n = 6) from 2 patients were compared with normal adrenal medullas (n = 8) and other hereditary pseudohypoxic PGLs (VHL: n = 13, SDHB: n = 15, and SDHD: n = 14). Unsupervised hierarchical clustering showed that HIF2A PGLs made up a separate cluster from other pseudohypoxic PGLs. Significance analysis of microarray yielded 875 differentially expressed genes between HIF2A and other pseudohypoxic PGLs after normalization to adrenal medulla (false discovery rate 0.01). Prediction analysis of microarray allowed correct classification of all HIF2A samples based on as little as three genes (TRHDE, LRRC63, IGSF10; error rate: 0.02). Genes with the highest expression difference between normal medulla and HIF2A PGLs were selected for confirmatory quantitative reverse transcriptase polymerase chain reaction. In conclusion, HIF2A PGLs show a characteristic expression signature that separates them from non-HIF2A pseudohypoxic PGLs. Unexpectedly, the most significantly differentially expressed genes have not been previously described as HIF target genes. [ABSTRACT FROM AUTHOR]

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation. [ABSTRACT FROM AUTHOR]

Background: With the rapid advancement of cell biology, the evaluation of a given protein's synthesis and release in cells becomes critical. However, up to now there has been no technique available to morphologically visualize and measure a newly synthesized protein in cells, nor can we measure the protein's release from the cells. Results: In this study, we developed a set of assays combining pulse chase amino acid substitution, non-radioactive labeling, and immunofluorescence co-localization to visualize newly synthesized proteins in individual cells and then to detect their release using modified ELISA. We demonstrated the synthesis and release of Bcl-2, MMP-9, and immunoglobulin G (IgG) in a human trophoblast cell line, of which the last finding has not been reported previously. Conclusions: This new technique offers a powerful tool to evaluate the dynamics of the synthesis and release of target proteins in individual cultured cells with wide applications in genetic and protein analysis. [ABSTRACT FROM AUTHOR]

Previous studies on the association between alcohol intake and the risk of myelodysplastic syndromes (MDS) have been inconclusive. We conducted a meta-analysis to evaluate alcohol intake as a risk factor for MDS. We performed a systematic literature search of articles published before March, 2014 using Web of Science, PubMed and the Cochrane Library. Two evaluators independently selected and reviewed studies based on predetermined selection criteria. The fixed- or random-effects models were used to summarize the estimates of odds ratios (ORs) with 95% confidence intervals (CIs). A total of 9 studies (8 case-control and 1 cohort) met the inclusion criteria of this meta-analysis. No significant association was observed between alcohol consumption and MDS when comparing drinkers to non-drinkers (OR=1.16, 95% CI: 0.88-1.53) or between subgroups stratified by alcohol history, gender, ethnicity, study design, source of patients or MDS subtypes. However, the data indicated a stronger association of alcohol with MDS in individuals who consumed ≥10 g/day (OR=1.55, 95% CI: 1.08-2.21) vs. those who consumed <10 g/day (OR=1.09, 95% CI: 0.78-1.53). This meta-analysis suggests that alcohol intake may increase the risk of MDS in a dose-dependent manner. However, additional well-designed, prospective cohort studies are required to verify these findings and identify other risk factors associated with MDS. [ABSTRACT FROM AUTHOR]

Background The methylation inhibitor 5-Aza-2'-deoxycytidine (decitabine, DAC) has a great therapeutic value for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). But decitabine monotherapy was associated with a relatively low rate of complete remission in AML and MDS. We aimed to investigate the effect of several anti-leukemia drugs in combination with decitabine on the proliferation of myeloid leukemia cells, to select the most efficient combination group and explore the associated mechanisms of these combination therapies. Methods Cell proliferation was tested by MTT assay and CFU-GM assay. Cell apoptosis was evaluated by Annexin V and PI staining in cell culture, TUNEL assay and transmission electron microscopy in animal study. MicroPET was used to imaging the tumor in mouse model. Molecular studies were conducted using microarray expression analysis, which was used to explore associated pathways, and real-time quantitative reverse transcription-PCR, western blot and immunohistochemistry, used to assess regulation of Wnt/β-catenin pathway. Statistical significance among groups was determined by one-way ANOVA analysis followed by post hoc Bonferroni's multiple comparison test. Results Among five anti-leukemia agents in combining with decitabine, the sequential combination of decitabine and idarubicin induced synergistic cell death in U937 cells, and this effect was verified in HEL, SKM-1 cells and AML cells isolated from AML patients. Importantly, tumor growth inhibition in this sequential combination was found to be higher than in single agent or controls in vivo. Moreover, sequential combination of the two agents induced apoptosis and depression of the Wnt/β-catenin pathway in both AML cell culture and animal studies. Conclusions The findings demonstrated that sequentially combination of decitabine and idarubicin had synergistic anti-leukemia effects. These effects were mainly attributed to demethylation of Wnt/β-catenin pathway inhibitors and downregulation of Wnt/β-catenin pathway nuclear targets. [ABSTRACT FROM AUTHOR]

Gaucher disease is caused by mutations in the glucosidase, beta, acid gene that encodes glucocerebrosidase (GCase). Glucosidase, beta, acid mutations often cause protein misfolding and quantitative loss of GCase. In the present study, we found that celastrol, an herb derivative with known anticancer, anti-inflammatory, and antioxidant activity, significantly increased the quantity and catalytic activity of GCase. Celastrol interfered with the establishment of the heat-shock protein 90/Hsp90 cochaperone Cdc37/Hsp90-Hsp70-organizing protein chaperone complex with mutant GCase and reduced heat-shock protein 90-associated protein degradation. In addition, celastrol modulated the expression of molecular chaperones. Bcl2-associated athanogene 3 and heat shock 70kDa proteins 1A and 1B were significantly increased by celastrol. Furthermore, BAG family molecular chaperone regulator 3 assisted protein folding and maturation of mutant GCase. These findings provide insight into a therapeutic strategy for Gaucher disease and other human disorders that are associated with protein misfolding. [ABSTRACT FROM AUTHOR]

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in transformed and tumor cells but not in normal cells, making it a promising agent for cancer therapy. However, many cancer cells are resistant to TRAIL, and the underlying mechanisms are not fully understood. Here, we show that the regulation of the PP2A and Src interaction plays a critical role in TRAIL resistance. Specifically, we show that TRAIL treatment activates the tyrosine kinase Src, which subsequently phosphorylates caspase-8 at tyrosine 380, leading to the inhibition of caspase-8 activation. We also show that upon TRAIL treatment, Src, caspase-8, and PP2A/C (a catalytic subunit of the PP2A phosphatase) are redistributed into lipid rafts, a microdomain of the plasma membrane enriched with cholesterol, where PP2A dephosphorylates Src at tyrosine 418 and in turn inhibits caspase-8 phosphorylation. Furthermore, we find that TRAIL treatment causes PP2A/C degradation. These data suggest that the balance between Src-mediated caspase-8 phosphorylation and the inactivation of Src-mediated caspase-8 phosphorylation by PP2A determines the outcome of TRAIL treatment in breast cancer cells. Therefore, this work identifies a novel mechanism by which the interaction between PP2A and Src in the context of caspase-8 activation modulates TRAIL sensitivity in cancer cells. [ABSTRACT FROM AUTHOR]

Acute traumatic brain injury (TBI) is associated with long-term cognitive and behavioral dysfunction. In vivo studies have shown histone deacetylase inhibitors (HDACis) to be neuroprotective following TBI in rodent models. HDACis are intriguing candidates because they are capable of provoking widespread genetic changes and modulation of protein function. By using known HDACis and a unique small-molecule pan-HDACi (LB-205), we investigated the effects and mechanisms associated with HDACi-induced neuroprotection following CNS injury in an astrocyte scratch assay in vitro and a rat TBI model in vivo. We demonstrate the preservation of sufficient expression of nerve growth factor (NGF) and activation of the neurotrophic tyrosine kinase receptor type 1 (TrkA) pathway following HDACi treatment to be crucial in stimulating the survival of CNS cells after TBI. HDACi treatment up-regulated the expression of NGF, phospho-TrkA, phospho-protein kinase B (p-AKT), NF-?B, and B-cell lymphoma 2 (Bcl-2) cell survival factors while down-regulating the expression of p75 neurotrophin receptor (NTR), phospho-JNK, and Bcl-2-associated X protein apoptosis factors. HDACi treatment also increased the expression of the stem cell biomarker nestin, and decreased the expression of reactive astrocyte biomarker GFAP within damaged tissue following TBI. These findings provide further insight into the mechanisms by which HDACi treatment after TBI is neuroprotective and support the continued study of HDACis following acute TBI. [ABSTRACT FROM AUTHOR]

Hypoxia-Inducible factors (HIFs) control the cellular response to hypoxia and, when dysregulated, contribute to tumorigenesis. Previously, we identified 2 gain-of-function somatic mutations in patients presenting with multiple paragangliomas or somatos-tatinomas, and polycythemia. Here, we report 2 additional unique HIF2A mutations, which disrupt the hydroxylation domain recognized by prolyl hydroxylase domain-2 containing protein, leading to stabilization of HIF-2A and increased expression of hypoxia-related genes. [ABSTRACT FROM AUTHOR]

Gaucher disease is caused by mutations of the GBA gene that encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA mutations often result in protein misfolding and premature degradation, but usually exert less effect on catalytic activity. In this study, we identified the molecular mechanism by which histone deacetylase inhibitors increase the quantity and activity of GCase. Specifically, these inhibitors limit the deacetylation of heat shock protein 90, resulting in less recognition of the mutant peptide and GCase degradation. These findings provide insight into a possible therapeutic strategy for Gaucher disease and other genetic disorders by modifying molecular chaperone and protein degradation pathways. [ABSTRACT FROM AUTHOR]

Mutations of succinate dehydrogenase subunit B (SDHB) play a crucial role in the pathogenesis of the most aggressive and metastatic pheochromocyto-mas (PHEOs) and paragangliomas (PGLs). Although a variety of missense mutations in the coding sequence of the SDHB gene have been found in PHEOs and PGLs, it has been unclear whether these mutations impair mRNA expression, protein stability, subcellular localization, or intrinsic protein function. RT-PCR and Western blot analysis of SDHB mRNA and protein expression from SDHB-related PHEOs and PGLs demonstrated intact mRNA expression but significantly reduced protein expression compared to non-SDHB PHEOs and PGLs. A pulse-chase assay of common SDHB missense mutations in transfected HeLa cell lines demonstrated that the loss of SDHB function was due to a reduction in mutant protein half-life, whereas colocalization of SDHB with mitochondria and immunoprecipitation with SDHA demonstrated intact subcellular localization and complex formation. The half-life of the SDHB protein increased after treatment with histone deacetylase inhibitors (HDACis), implicating the protein quality control machinery in the degradation of mutant SDHB protein. These findings provide the first direct mechanism of functional loss resulting from SDHB mutations and suggest that reducing protein degradation with HDACis may serve as a novel therapeutic paradigm for preventing the development of SDHB-related tumors. [ABSTRACT FROM AUTHOR]

Astrocytes are the most abundant cell of the CNS and demonstrate contact inhibition in which a nonproliferative. nonmotile cellular state is achieved once stable intercellular contacts are formed between mature cells. Cellular injury disrupts these intercellular contacts, causing a loss of contact inhibition and the rapid initiation of healing. Dysregulation of the molecular pathways involved in this process is thought to lead to an aggressive cellular state associated with neoplasia. We investigated whether a comparable correlation exists between the response of astrocytes to injury and the malignant phenotype of astrocytomas. We discovered that the loss of contact inhibition plays a critical role in the initiation and regulation of reactive astrocytes in the healing of wounds. In particular, injury of the astrocytes interrupts and destabilizes the cadherin-catenin complexes at the cell membrane leading to nuclear translocation of β-catenin and characteristic changes associated with the activation of astrocytes. Similar signaling pathways are found to be active-but dysregulased-in astrocytomas. Inhibition of β-catenin signaling diminished both the response of assrocytes to injury and induction of the malignant phenotype of astrocytomas. The findings shed light on a unique mechanism associated with the pathogenesis of astrocytomas and provide a model for the loss of contact inhibition that may broadly apply to understanding the mechanisms of tissue repair and tumorigenesis in the brain. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new "multidimensional" capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional in vitro studies utilizing the U87 glioma cell line, we found that expression of NNAT is associated with significant increases in cellular proliferation. Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P = 0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD) is caused by a spectrum of genetic mutations within the gene encoding the lysosomal enzyme glucocerebrosidase (GCase). These mutations often lead to misfolded proteins that are recognized by the unfolded protein response system and are degraded through the ubiquitin—proteasome pathway. Modulating this pathway with histone deacetylase inhibitors (HDACis) has been shown to improve protein stability in other disease settings. To identify the mechanisms involved in the regulation of GCase and determine the effects of HDACis on protein stability, we investigated the most prevalent mutations for nonneuronopathic (N370S) and neuronopathic (L444P) GD in cultured fibroblasts derived from GD patients and HeLa cells transfected with these mutations. The half-lives of mutant GCase proteins correspond to decreases in protein levels and enzymatic activity. GCase was found to bind to Hsp70, which directed the protein to TCP1 for proper folding, and to Hsp90, which directed the protein to the ubiquitin—proteasome pathway. Using a known HDACi (SAHA) and a unique small-molecule HDACi (LB-205), GCase levels increased rescuing enzymatic activity in mutant cells. The increase in the quantity of protein can be attributed to increases in protein half-life that correspond primarily with a decrease in degradation rather than an increase in chaperoned folding. HDACis reduce binding to Hsp90 and prevent subsequent ubiquitination and proteasomal degradation without affecting binding to Hsp70 or TCP1. These findings provide insight into the pathogenesis of GD and indicate a potent therapeutic potential of HDAC inhibitors for the treatment of GD and other human protein misfolding disorders. [ABSTRACT FROM AUTHOR]

Neurofibromatosis type 2 (NF2) is a multiple neoplasia syndrome and is caused by a mutation of the NF2 tumor suppressor gene that encodes for the tumor suppressor protein merlin. Biallelic NF2 gene inactivation results in the development of central nervous system tumors, including schwannomas, meningiomas, ependymomas, and astrocytomas. Although a wide variety of missense germline mutations in the coding sequences of the NF2 gene can cause loss of merlin function, the mechanism of this functional loss is unknown. To gain insight into the mechanisms underlying loss of merlin function in NF2, we investigated mutated merlin homeostasis and function in NF2-associated tumors and cell lines. Quantitative protein and RT-PCR analysis revealed that whereas merlin protein expression was significantly reduced in NF2-associated tumors, mRNA expression levels were unchanged. Transfection of genetic constructs of common NF2 missense mutations into NF2 gene-deficient meningioma cell lines revealed that merlin loss of function is due to a reduction in mutant protein half-life and increased protein degradation. Transfection analysis also demonstrated that recovery of tumor suppressor protein function is possible, indicating that these mutants maintain intrinsic functional capacity. Further, increased expression of mutant protein is possible after treatment with specific proteostasis regulators, implicating protein quality control systems in the degradative fate of mutant tumor suppressor proteins. These findings provide direct insight into protein function and tumorigenesis in NF2 and indicate a unique treatment paradigm for this disorder. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD), the most common lysosomal storage disorder of humans, iscaused by mutations in the gene coding for the enzyme glucocerebrosidase (GCase). Clinical manifestations vary among patients with the three types of GD, and phenotypic heterogeneity occurs even among patients with identical mutations. To gain insight into why phenotypic heterogeneity occurs in GD. we investigated mechanisms underlying the net loss of GCase catalytic activity in cultured skin fibroblasts derived from patients with the three types of GD. The findings indicate that the loss of catalytic activity of GCase correlates with its quantitative reduction, rather than a decrease in functional capacity of mutant enzyme. Use of a proteasome inhibitor, lactacystin, resulted in increased expression of GCase, suggesting a mechanism of protein degradation in GD. Furthermore, reduced binding of GCase to TCP1 ring complex (TRiC), a regulator of correct protein folding. may result in defective maturation of nascent GCase in GD cells. Additionally, increased interaction between GCase and c-Cbl, an E3 ubiquitin ligase, may be involved in the degradation and loss of GCase in GD. The findings suggest that specific molecular mediators involved in GCase maturation and degradation could be responsible for phenotypic variation among patients with the same genotypes and that these mediators could be therapeutically targeted to increase GCase activity in patients with GD. [ABSTRACT FROM AUTHOR]

Despite resistance of most gliomas to chemotherapy, approximately 2/3 of oligodendrogliomas show sensitivity to such agents. This sensitivity has been associated with deletions on chromosome 1p alone or in combination with 19q. Higher expression of the enzyme glyoxalase I has been found in oligodendrogliomas with chromosome 1p intact compared to those with a deletion. Higher expression of this enzyme is also associated with tumor chemoresistance in other cancers. The present study tested whether the drug troglitazone would make a glioma cell line more sensitive to chemotherapeutic agents. This drug was chosen because it has been shown to decrease glyoxalase I enzyme activity in cells. Treatment with troglitazone decreased expression of glyoxalase I, and potentiated cell death when used in combination with chemotherapeutic agents. This decrease in glyoxalase I protein may be one mechanism by which this potentiation occurs, and troglitazone may be a candidate for use in glioma therapy. [ABSTRACT FROM AUTHOR]

A variety of mechanisms maintain the integrity of the genome in the face of cell stress. Cancer cell response to chemotherapeutic and radiation-induced DNA damage is mediated by multiple defense mechanisms including polo-like kinase 1 (P1k-1), protein kinase B (Akt-1), and/or p53 pathways leading to either apoptosis or cell cycle arrest. Subsequently, a subpopulation of arrested viable cancer cells may remain and recur despite aggressive and repetitive therapy. Here, we show that modulation (activation of Akt-1 and P1k-1 and repression of p53) of these pathways simultaneously results in paradoxical enhancement of the effectiveness of cytotoxic chemotherapy. We demonstrate that a small molecule inhibitor. LB-1.2, of protein phosphatase 2A (PP2A) activates P1k-1 and Akt-1 and decreases p53 abundance in tumor cells. Combined with temozolomide (TMZ; a DNA-methylating chemotherapeutic drug), LB-1.2 causes complete regression of glioblastoma multiforme (GBM) xenografts without recurrence in 50% of animals (up to 28 weeks) and complete inhibition of growth of neuroblastoma (NB) xenografts. Treatment with either drug alone results in only short-term inhibition/regression with all xenografts resuming rapid growth. Combined with another widely used anticancer drug, Doxorubicin (DOX, a DNA intercalating agent), LB-1.2 also causes marked GBM xenograft regression, whereas DOX alone only slows growth. Inhibition of PP2A by LB-1.2 blocks cell-cycle arrest and increases progression of cell cycle in the presence of TMZ or DOX. Pharmacologic inhibition of PP2A may be a general method for enhancing the effectiveness of cancer treatments that damage DNA or disrupt components of cell replication. [ABSTRACT FROM AUTHOR]

The article examines the molecular pathogenesis, genetics, clinical findings and management strategies for an autosomal-dominant multiple neoplasia syndrome neurofibromatosis type 2. Patients with the syndrome are reportedly predisposed to the development of lesions of the nervous system, eyes and skin which are characterised with bilateral vestibular nerve schwannomas. Its diagnosis is based on clinical criteria, including the Manchester diagnostic criteria that consists of patients without a family history of the disorder or bilateral vestibular schwannoma but who have multiple other related lesions. Data on the frequency of lesions associated with the syndrome is also provided.

Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 ± 0.094 vs. 0.018 ± 0.009 day-1), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease. [ABSTRACT FROM AUTHOR]

Inactivation of the von Hippel--Lindau (VHL) gene and activation of the hypoxia-inducible factor (HIF) in susceptible cells precedes formation of tumorlets and frank tumor in the epididymis of male VHL patients. We performed detailed histologic and molecular pathologic analysis of tumor-free epididymal tissues from VHL patients to obtain further insight into early epididymal tumorigenesis. Four epididymides from two VHL patients were serially sectioned to allow for three-dimensional visualization of morphologic changes. Areas of interest were genetically analyzed by tissue microdissection, immunohistochemistry for HIF and markers for mesonephric differentiation, and in situ hybridization for HIF downstream target vascular endothelial growth factor. Structural analysis of the epididymides revealed marked deviations from the regular anatomic structure resulting from impaired organogenesis. Selected efferent ductules were represented by disorganized mesonephric cells, and the maldeveloped mesonephric material was VHL-deficient by allelic deletion analysis. Furthermore, we observed maldeveloped mesonephric material near cystic structures, which were also VHL-deficient and were apparent derivatives of maldeveloped material. Finally, a subset of VHL-deficient cells was structurally integrated in regular efferent ductules; proliferation of intraductular VHL-deficient cells manifests itself as papillary growth into the ductular lumen. Furthermore, we clarify that that there is a pathogenetic continuum between microscopic tumorlets and formation of tumor. In multiple locations, three-dimensional reconstruction revealed papillary growth to extend deeply into ductular lumina, indicative of progression into early hamartoma-like neoplasia. We conclude epididymal tumorigenesis in VHL disease to occur in two distinct sequential steps: maldevelopment of VHL-deficient mesonephric cells, followed by neoplastic papillary proliferation. [ABSTRACT FROM AUTHOR]

This work expands our tissue proteome capabilities from the analysis of soluble proteins in previous studies to the examination of membrane proteins within the pellets of enriched and selectively isolated tumor cells procured from microdissected tissue specimens. The pellets of targeted ovarian tumor cells are treated by two different membrane protein extraction methods, including the use of detergent and organic solvent. The detergent-based membrane protein preparation protocol not only extracts proteins effectively from cell pellets but also is compatible with subsequent proteome analysis using combined capillary isoelctric focusing/nano reversed-phase liquid chromatography separations coupled with nano electrospray ionization mass spectrometry. Among proteins identified from an amount of pellet equivalent to 20 000 cells, 773 proteins are predicted to contain one or more transmembrane domains, corresponding to 22% membrane proteome coverage within the SwissProt Human protein sequence entries. [ABSTRACT FROM AUTHOR]

This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high- confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 μg of total protein, an amount corresponding to 20000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of ClEF fractions without an accompanying increase in sample consumption. [ABSTRACT FROM AUTHOR]

A 4-week-old child presented with lethargy, emesis, decreased spontaneous movements, and a bulging fontanelle. Neuroimaging demonstrated a large, hemispheric, multicystic lesion with multiple enhancing nodules, which, on pathological examination, proved to be multiple, distinct hemangioblastomas. Careful molecular analysis failed to reveal alterations of the VHL gene. This represents an uncommon presentation for these tumors and suggests that genes other than VHL may be important in the genesis of these tumors. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Presents a study that investigated primary tumors for deletion of 11q13 sequences to verify whether a cervical cancer tumor suppressor gene may be localized to chromosome 11q13. Methods for the deletion of 11q13 sequences in primary tumors; Confirmation of 11q13 loss using fluorescence in situ hybridization; Mapping of chromosome 11q13 deletion in primary tumors and HeLa cell lines.

Investigates the association of male germ-line-specific protein with epigenetic reprogramming events. Involvement of the 11-zinc-finger factor in gene regulation; Formation of methylation-sensitive insulators; Identification of the methylation marks in somatic cells.

Presents a study that examined the point mutations of the wild-type allele in tumors from patients with Von Hippel-Lindau (VHL) germline deletion. Background on the presence of VHL germline mutation in patients with VHL; Characteristics of VHL disease; Percentage of VHL patients with no germline mutations of the VHL gene; Information on the deletion of the wild-type VHL.

Discusses the detection of somatic mutations of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene in sporadic angiofibromas. Characteristics of mutations; Types of proliferating cells involved in angiofibromas; Role of the Men1 gene product in the initiation and regression of a subset of sporadic angiofibromas.

We developed an Age-related macular degeneration (AMD) model, Ccl2/Cx3crl deficient mouse, which displays AMD-like lesions including drusen formation, retinal pigment epithelium (RPE) hypopigmentation and accumulation of lipofuscin, photoreceptor degeneration, and choroidal neovascularization. This study aimed to detect causal factors directly contributing to the phenotype of this model. The proteomics showed that 4 proteins were differentially expressed in the retina of Ccl2-/-/Cx3crl-/-. Mass spectrophotometer confirmed that two of them were ERp29 precursor and CBP140, proteins from endoplasmic reticulum (ER). RT-PCR, Western Blotting and immunochemistry indicated that ERp29 and CBP140 significantly down-regulated in the retina of Ccl2-/-/Cx3crl-/- in comparison with the wild type. Our data identified at least 2 ER proteins that are associated with an AMD-like phenotype in Ccl2-/-/Cx3crl-/-. ER proteins function as chaperone in protein folding and maturation. Improper function of ER leads to the accumulation of unfolded protein, which is believed to be the machismo of several age-related or neurodegenerative diseases including AMD. ERp29 is one such ER-resident chaperone. We expect that the ER and ER-related proteins might play a role in the retinal phenotype of Cel2-/-/Cx3crl-/-. [ABSTRACT FROM AUTHOR]