Elena M. Akhmametyeva

cDNA microarray analysis of vestibular schwannomas.

Background Vestibular schwannomas are known to harbor mutations in the neurofibromatosis type 2 tumor suppressor gene, but the mechanism of the neurofibromatosis type 2 tumor suppressor gene action is not well understood. Identification of genes differentially expressed in normal and diseased tissues through the use of a large-scale, cDNA microarray approach may lead to increased understanding of pathways that lead to tumor formation. Objective The objectives of this study were to evaluate the gene expression profiles in vestibular schwannomas in comparison with normal vestibular nerve tissues and to identify pathways that may be altered in schwannomas. Methods Total RNA was extracted from one normal vestibular nerve and seven vestibular schwannomas. The normal vestibular nerve was from one of the seven patients with small vestibular schwannomas. Radiolabeled cDNA was synthesized and hybridized to cDNA microarray filters that contained 25,920 known genes or expressed sequence tags. Expression profiles were imaged and analyzed. Selected genes that showed three-fold or greater difference in the intensity between the normal nerve and the schwannomas were further examined by real-time polymerase chain reaction and by immunohistochemical staining. Results Forty-two genes (0.2%) were upregulated 3-fold or more in at least 5 of the 7 tumors when the filter images were compared with a normal adjacent vestibular nerve. Among them, osteonectin, an angiogenesis mediator, and RhoB GTPase, which is important in cell signaling, were significantly upregulated in 5 of 7 tumors. Among genes that were downregulated, an apoptosis-related LUCA-15 gene was highly underexpressed in 6 of 7 schwannomas when compared with the normal nerve. Also, ezrin, a relative of the NF2 protein, was significantly downregulated in 5 of 7 tumors. Real-time PCR and immunohistochemistry data support the cDNA microarray findings. Conclusion Our cDNA microarray analysis of schwannomas suggested several interesting and potentially important tumorigenesis pathways associated with vestibular schwannoma formation. Further in vivo study is necessary to define the roles of these identified genes and their potential relationships with the neurofibromatosis type 2 tumor suppressor gene.

The role of Drosophila Merlin in spermatogenesis

BackgroundDrosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer3 allele, a missense mutation (Met177→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.ResultsTestis examination reveals that hemizygous Mer3 mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer3, Mer4 (Gln170→stop), and control testes at various stages of spermatogenesis, we show that Merlin mutations affect meiotic cytokinesis of spermatocytes, cyst polarization and nuclear shaping during spermatid elongation, and spermatid individualization. We also demonstrate that the lethality and sterility phenotype of the Mer4 mutant is rescued by the introduction of a wild-type Merlin gene. Immunostaining demonstrates that the Merlin protein is redistributed to the area associated with the microtubules of the central spindle in telophase and its staining is less in the region of the contractile ring during meiotic cytokinesis. At the onion stage, Merlin is concentrated in the Nebenkern of spermatids, and this mitochondrial localization is maintained throughout sperm formation. Also, Merlin exhibits punctate staining in the acrosomal region of mature sperm.ConclusionMerlin mutations affect spermatogenesis at multiple stages. The Merlin protein is dynamically redistributed during meiosis of spermatocytes and is concentrated in the Nebenkern of spermatids. Our results demonstrated for the first time the mitochondrial localization of Merlin and suggest that Merlin may play a role in mitochondria formation and function during spermatogenesis.

Growth Inhibitory and Anti-Tumour Activities of OSU-03012, a Novel PDK-1 Inhibitor, on Vestibular Schwannoma and Malignant Schwannoma Cells

BACKGROUND Vestibular schwannomas (VS) frequently express high levels of activated AKT. Small-molecule inhibitors of AKT signalling may have therapeutic potential in suppressing the growth of benign VS and malignant schwannomas. METHOD Primary VS and Schwann cells, human malignant schwannoma HMS-97 cells and mouse Nf2(-/-) Schwann cells and schwannoma cells were prepared to investigate the growth inhibitory and anti-tumour activities of OSU-03012, a celecoxib-derived small-molecule inhibitor of phosphoinositide-dependent kinase-1. Cell proliferation assays, apoptosis, Western blot, in vivo xenograft analysis using SCID mice and immunohistochemistry were performed. RESULTS OSU-03012 inhibited cell proliferation more effectively in both VS and HMS-97 cells than in normal human Schwann cells. The IC5) of OSU-03012 at 48h was approximately 3.1 microM for VS cells and 2.6 microM for HMS-97 cells, compared with the IC(50) of greater than 12 microM for human Schwann cells. Similarly, mouse Nf2(-/-) schwannoma and Nf2(-/-) Schwann cells were more sensitive to growth inhibition by OSU-03012 than wild-type mouse Schwann cells and mouse schwannoma cells established from transgenic mice carrying the NF2 promoter-driven SV40 T-antigen gene. Like VS cells, malignant schwannoma HMS-97 cells expressed high levels of activated AKT. OSU-03012 induced apoptosis in both VS and HMS-97 cells and caused a marked reduction of AKT phosphorylation at both the Ser-308 and Thr-473 sites in a dose-dependent manner. In vivo xenograft analysis showed that OSU-03012 was well tolerated and inhibited the growth of HMS-97 schwannoma xenografts by 55% after 9 weeks of oral treatment. The anti-tumour activity correlated with reduced AKT phosphorylation. CONCLUSION OSU-03012 is a potential chemotherapeutic agent for VS and malignant schwannomas.

The molecular biology of vestibular schwannomas: dissecting the pathogenic process at the molecular level.

Objective: The goal of this article was to review concisely what is currently known about the tumorigenesis of vestibular schwannomas. Background: Recent advances in molecular biology have led to a better understanding of the cause of vestibular schwannomas. Mutations in the neurofibromatosis type 2 tumor suppressor gene (NF2) have been identified in these tumors. In addition, the interactions of merlin, the protein product of the NF2 gene, and other cellular proteins are beginning to give us a better idea of NF2 function and the pathogenesis of vestibular schwannomas. Methods: Review of the relevant basic science studies at our institution as well as the basic science and clinical literature. Results: The clinical characteristics of vestibular schwannomas and neurofibromatosis type 2 syndromes are reviewed and related to alterations in the NF2 gene. Studies demonstrating our current understanding of tumor developmental pathways are highlighted. In addition, methods of clinical and genetic screening for neurofibromatosis type 2 disease are outlined. Avenues for the development of potential future research and therapies are discussed. Conclusion: Great strides have been made to identify why vestibular schwannomas develop at the molecular level. Continued research is needed to find targeted therapies with which to treat these tumors.

AR42, a novel histone deacetylase inhibitor, as a potential therapy for vestibular schwannomas and meningiomas

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disease that results in the formation of bilateral vestibular schwannomas (VSs) and multiple meningiomas. Treatment options for NF2-associated tumors are limited, and to date, no medical therapies are FDA approved. The ideal chemotherapeutic agent would inhibit both VS and meningiomas simultaneously. The objectives of this study are (1) to test the efficacy of AR42, a novel histone deacetylase inhibitor, to inhibit VS and meningioma growth and (2) to investigate this drugs mechanisms of action. Primary cultures of human VS and meningioma cells were established. Nf2-deficient mouse schwannoma and benign human meningioma Ben-Men-1 cells were also cultured. Cells were treated with AR42, and the drugs effects on proliferation and the cell cycle were analyzed using a methanethiosulfonate assay and flow cytometry, respectively. Human phospho-kinase arrays and Western blots were used to evaluate the effects of AR42 on intracellular signaling. The in vivo efficacy of AR42 was investigated using schwannoma xenografts. Tumor volumes were quantified using high-field, volumetric MRI, and molecular target analysis was performed using immunohistochemistry. AR42 inhibited the growth of primary human VS and Nf2-deficient mouse schwannoma cells with a half maximal inhibitory concentration (IC(50)) of 500 nM and 250-350 nM, respectively. AR42 also inhibited primary meningioma cells and the benign meningioma cell line, Ben-Men-1, with IC(50) values of 1.5 µM and 1.0 µM, respectively. AR42 treatment induced cell-cycle arrest at G(2) and apoptosis in both VS and meningioma cells. Also, AR42 exposure decreased phosphorylated Akt in schwannoma and meningioma cells. In vivo treatment with AR42 inhibited the growth of schwannoma xenografts, induced apoptosis, and decreased Akt activation. The potent growth inhibitory activity of AR42 in schwannoma and meningioma cells suggests that AR42 should be further evaluated as a potential treatment for NF2-associated tumors.

Retinoblastoma-cyclin-dependent kinase pathway deregulation in vestibular schwannomas.

Objectives The purpose of the study was to identify genes of the retinoblastoma protein (pRb)–cyclin‐dependent kinase (CDK) pathway that are deregulated in vestibular schwannomas when compared with normal vestibular nerve tissues.

Regulation of the Neurofibromatosis 2 gene promoter expression during embryonic development

Mutations in the Neurofibromatosis 2 (NF2) gene are associated with predisposition to vestibular schwannomas, spinal schwannomas, meningiomas, and ependymomas. Presently, how NF2 is expressed during embryonic development and in the tissues affected by neurofibromatosis type 2 (NF2) has not been well defined. To examine NF2 expression in vivo, we generated transgenic mice carrying a 2.4‐kb NF2 promoter driving β‐galactosidase (β‐gal) with a nuclear localization signal. Whole‐mount embryo staining revealed that the NF2 promoter directed β‐gal expression as early as embryonic day E5.5. Strong expression was detected at E6.5 in the embryonic ectoderm containing many mitotic cells. β‐gal staining was also found in parts of embryonic endoderm and mesoderm. The β‐gal staining pattern in the embryonic tissues was corroborated by in situ hybridization analysis of endogenous Nf2 RNA expression. Importantly, we observed strong NF2 promoter activity in the developing brain and in sites containing migrating cells including the neural tube closure, branchial arches, dorsal aorta, and paraaortic splanchnopleura. Furthermore, we noted a transient change of NF2 promoter activity during neural crest cell migration. While little β‐gal activity was detected in premigratory neural crest cells at the dorsal ridge region of the neural fold, significant activity was seen in the neural crest cells already migrating away from the dorsal neural tube. In addition, we detected considerable NF2 promoter activity in various NF2‐affected tissues such as acoustic ganglion, trigeminal ganglion, spinal ganglia, optic chiasma, the ependymal cell‐containing tela choroidea, and the pigmented epithelium of the retina. The NF2 promoter expression pattern during embryogenesis suggests a specific regulation of the NF2 gene during neural crest cell migration and further supports the role of merlin in cell adhesion, motility, and proliferation during development. Developmental Dynamics 235:2771–2785, 2006.

Growth of benign and malignant schwannoma xenografts in severe combined immunodeficiency mice

Objectives: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice.

Analysis of the human neurofibromatosis type 2 gene promoter and its expression

OBJECTIVE: It is hypothesized that transcriptional regulation plays an important role for neurofibromatosis type 2 (NF2) expression in Schwann cells and other cell types. The objective of this study is the isolation and characterization of the transcriptional regulatory elements of the NF2 gene. STUDY DESIGN AND SETTING: A bacterial artificial chromosome library and a partial genomic DNA library were used to isolate the human NF2 gene; NF2 promoter-luciferase constructs were generated, and promoter activities were assayed. This study was carried out in a molecular biology laboratory. RESULTS: A bacterial artificial chromosome clone with an approximately 100-kilobase insert containing nearly the entire human NF2 gene has been isolated. An additional 5’ NF2 sequence has also been cloned. Transient transfection experiments demonstrate strong promoter activity from the NF2 5’ flanking DNA. CONCLUSIONS: The NF2 gene is approximately 100 kilobases long. Both positive and negative regulatory elements are present in NF2 5’ flanking regions. SIGNIFICANCE: Better understanding of the NF2 gene and its regulation will improve molecular diagnostics and ultimately treatment of patients with NF2. (Otolaryngol Head Neck Surg 2000; 123:413-8.)

Induction of apoptosis by the p53-related p73 and partial inhibition by the baculovirus-encoded p35 in neuronal cells

To better understand whether the p53-related p73 gene could induce neuronal apoptosis, we tested whether p73 induced cell killing in three neuronal cell lines and whether apoptosis could be inhibited by p35, a baculovirus-encoded protein that blocks caspase 3. Recombinant adenoviruses carrying the hemagglutinin (HA)-tagged p73beta or p35, or the green fluorescent protein gene driven by the cytomegalovirus immediate-early promoter were constructed, and used to infect human SK-N-AS and SK-N-SH neuroblastoma, and rat PC12 pheochromocytoma cells. Infection with Adp73beta virus resulted in p73beta over-expression and substantial reduction of cell viability due to apoptosis in all three neuronal cell lines as compared with the control AdGFP virus. These results indicate that p73beta over-expression in neuronal cells could induce apoptotic cell death regardless of the endogenous expression of p73. The p73 effect was partially blocked by co-expression of the wild-type p35, suggesting caspase-mediated cell killing. Insertion of a hemagglutinin (HA) tag at the N-terminus of p35 markedly reduced its ability to inhibit the p73 effect compared with the wild-type p35, while insertion of an HA tag to the C-terminus of p35 had no appreciable effect. Taken together, our results suggest that the N-terminal structure of p35 is critical for its anti-apoptotic activity on p73-induced apoptosis in neuronal cells.