Long-Sheng Chang

Transcriptional repression by YY1, a human GLI-Krüippel-related protein, and relief of repression by adenovirus E1A protein

A sequence within the transcription control region of the adeno-associated virus P5 promoter has been shown to mediate transcriptional activation by the adenovirus E1A protein. We report here that this same element mediates transcriptional repression in the absence of E1A. Two cellular proteins have been found to bind to overlapping regions within this sequence element. One of these proteins, YY1, is responsible for the repression. E1A relieves repression exerted by YY1 and further activates transcription through its binding site. A YY1-specific cDNA has been isolated. Its sequence reveals YY1 to be a zinc finger protein that belongs to the GLI-Krüppel gene family. The product of the cDNA binds to YY1 sites. When fused to the GAL4 DNA-binding domain, it is capable of repressing transcription directed by a promoter that contains GAL4-binding sites, and E1A proteins can relieve the repression and activate transcription through the fusion protein.

Identification of a TAL1 target gene reveals a positive role for the LIM domain-binding protein Ldb1 in erythroid gene expression and differentiation.

ABSTRACT The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2) gene through two E box GATA elements in its proximal promoter. Binding of this complex to DNA was dependent on the integrity of both E box and GATA sites and was demonstrated to occur on the P4.2 promoter in cells. Maximal transcription in transiently transfected cells required both E box GATA elements and expression of all five components of the complex. This complex was shown, in addition, to be capable of linking in solution double-stranded oligonucleotides corresponding to the two P4.2 E box GATA elements. This DNA-linking activity required Ldb1 and increased with dimethyl sulfoxide-induced differentiation of murine erythroleukemia (MEL) cells. In contrast, enforced expression in MEL cells of dimerization-defective mutant Ldb1, as well as wild-type Ldb1, significantly decreased E box GATA DNA-binding activities, P4.2 promoter activity, and accumulation of P4.2 and β-globin mRNAs. These studies define a physiologic target for a TAL1- and GATA-1-containing ternary complex and reveal a positive role for Ldb1 in erythroid gene expression and differentiation.

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 Human POLD1 Gene IDENTIFICATION OF AN UPSTREAM ACTIVATOR SEQUENCE, ACTIVATION BY Sp1 AND Sp3, AND CELL CYCLE REGULATION

The promoter of the human POLD1 gene encoding the catalytic subunit of DNA polymerase δ is G/C-rich and does not contain a TATA box. Transient transfection analysis in HeLa cells employing POLD1-luciferase chimeric plasmids revealed a core promoter region extending 328 base pairs (bp) from the major transcription initiation site. Multiple elements in this region including two 11-bp direct repeats located between nucleotide positions −92 and −22, play an important role in POLD1 promoter activity. Deletion or linker-replacement mutations of the repeats drastically reduced the promoter activity. A 70-bp DNA fragment containing the two repeats could stimulate the expression of the POLD1 or a heterologous promoter in an orientation-independent manner. DNase I footprinting and band-shift assays showed that HeLa nuclear extracts contained proteins specifically binding to the repeat sequences. Southwestern blot and UV cross-linking analyses identified Sp1 and two 85-kDa proteins that bound to the repeats. Additionally, screening of HeLa cDNA expression libraries for the sequence-specific DNA-binding protein using the 11-bp repeat sequences as the probe, identified a cDNA that corresponds to Sp3, a member of the Sp1 family. Cotransfection studies in Drosophila SL2 cells showed that both Sp1 and Sp3, but not Sp2, could activate the POLD1 promoter through the repeat sequences. The POLD1 promoter activity was induced about 4-fold at the late G1/S boundary in serum-stimulated cells. The 11-bp repeats together with an E2F-like sequence, located adjacent to the major transcription initiation site, were important for the stimulation. Taken together, this study provides a direct evidence for transcriptional regulation of the human POLD1 gene.

MicroRNA-10b regulates tumorigenesis in neurofibromatosis type 1

MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR‐10b was up‐regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR‐10b correlated with low neurofibromin expression was found in a neuroectodermal cell line: Ewing’s sarcoma SK‐ES‐1 cells. Antisense inhibiting miR‐10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR‐10b. Overexpression of miR‐10b in 293T cells suppressed neurofibromin expression and activated RAS signaling. Antisense inhibition of miR‐10b restored neurofibromin expression in SK‐ES‐1 cells, and decreased RAS signaling independent of neurofibromin in NF1 MPNST cells. These results suggest that miR‐10b may play an important role in NF1 tumorigenesis through targeting neurofibromin and RAS signaling. (Cancer Sci 2010)

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.

Phosphatidylinositol 3-kinase/AKT pathway activation in human vestibular schwannoma.

Hypothesis: The neurofibromatosis 2 gene, which encodes the tumor suppressor protein merlin, is frequently mutated in vestibular schwannomas (VS). Merlin can inhibit phosphatidylinositol 3 kinase (PI3 kinase) by binding to PI3 kinase enhancer long isoform. Therefore, we hypothesized that the PI3 kinase/AKT pathway is activated in VS. Background: Despite advances in diagnosis and treatment, VS continue to cause patient morbidity. A more thorough understanding of the signaling pathways deregulated in VS will aid in the development of novel medical therapeutics. Activation of the PI3 kinase/AKT pathway increases cell survival and cell proliferation and has been observed in a variety of human cancers. However, whether the PI3 kinase/AKT pathway is activated in human VS has not been reported. Methods: Complementary deoxyribonucleic acid microarrays were performed using cultured Schwann cells, 4 VS specimens, and 2 paired normal vestibular nerves. Immunohistochemical analysis using antibodies to activated phosphorylated-AKT was performed on 14 VS tissue sections. Western blots using various antibodies to components of the PI3 kinase/AKT pathways were conducted. Results: Microarray analysis demonstrated that total AKT gene expression was upregulated in VS, compared with normal vestibular nerves. Immunohistochemical analysis of 14 VS tissue sections detected positive staining for activated AKT phosphorylated at both serine-473 and threonine-308 in all tumors. Western blots comparing VS specimens with normal vestibular nerves showed that the AKT pathway was activated in VS but not in normal nerve. Total AKT, phosphorylated-AKT, PI3-kinase, phosphorylated-phosphatase and tensin homologue deleted on chromosome 10, phosphorylated-phosphoinositide-dependent protein kinase 1, phosphorylated-forkhead box O, phosphorylated-glycogen synthase kinase 3&bgr;, and phosphorylated-mammalian target of rapamycin were upregulated in VS. Conclusion: The PI3 kinase/AKT pathway is activated in VS. Using our recently reported quantifiable VS xenograft model, novel inhibitors of the PI3 kinase/AKT pathway may be tested for VS growth inhibition in vivo.

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.