15 research outputs found

    Human Kidney Disease-causing INF2 Mutations Perturb Rho/Dia Signaling in the Glomerulus

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    Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans

    Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

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    <p>Abstract</p> <p>Background</p> <p>Methylation-mediated silencing of genes is one epigenetic mechanism implicated in cancer. Studies regarding the role of modulation of gene expression utilizing inhibitors of DNA methylation, such as decitabine, in osteosarcoma (OS) have been limited. A biological understanding of the overall effects of decitabine in OS is important because this particular agent is currently undergoing clinical trials. The objective of this study was to measure the response of the OS cell line, U2OS, to decitabine treatment both <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p>Microarray expression profiling was used to distinguish decitabine-dependent changes in gene expression in U2OS cells, and to identify responsive loci with demethylated CpG promoter regions. U2OS xenografts were established under the sub-renal capsule of immune-deficient mice to study the effect of decitabine <it>in vivo </it>on tumor growth and differentiation. Reduced nuclear methylation levels could be detected in xenografts derived from treated mice by immunohistochemistry utilizing a 5-methylcytidine antibody. Decitabine treatment reduced tumor xenograft size significantly (p < 0.05). Histological analysis of treated U2OS xenograft sections revealed a lower mitotic activity (p < 0.0001), increased bone matrix production (p < 0.0001), and a higher number of apoptotic cells (p = 0.0329). Microarray expression profiling of U2OS cultured cells showed that decitabine treatment caused a significant induction (p < 0.0025) in the expression of 88 genes. Thirteen had a ≥2-fold change, 11 of which had CpG-island-associated promoters. Interestingly, 6 of these 11 were pro-apoptotic genes and decitabine resulted in a significant induction of cell death in U2OS cells <it>in vitro </it>(p < 0.05). The 6 pro-apoptotic genes (<it>GADD45A</it>, <it>HSPA9B</it>, <it>PAWR</it>, <it>PDCD5</it>, <it>NFKBIA</it>, and <it>TNFAIP3</it>) were also induced to ≥2-fold <it>in vivo</it>. Quantitative methylation pyrosequencing confirmed that the tested pro-apoptotic genes had CpG-island DNA demethylationas a result of U2OS decitabine treatment both <it>in vitro </it>and in xenografts</p> <p>Conclusion</p> <p>These data provide new insights regarding the use of epigenetic modifiers in OS, and have important implications for therapeutic trials involving demethylation drugs. Collectively, these data have provided biological evidence that one mode of action of decitabine may be the induction of apoptosis utilizing promoter-CpG demethylation of specific effectors in cell death pathways in OS.</p

    In Vitro Analysis of Integrated Global High-Resolution DNA Methylation Profiling with Genomic Imbalance and Gene Expression in Osteosarcoma

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    Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks

    Decitabine-Induced Demethylation of 5′ CpG Island in GADD45A Leads to Apoptosis in Osteosarcoma Cells1

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    GADD45 genes are epigenetically inactivated in various types of cancer and tumor cell lines. To date, defects of the GADD45 gene family have not been implicated in osteosarcoma (OS) oncogenesis, and the role of this pathway in regulating apoptosis in this tumor is unknown. The therapeutic potential of Gadd45 in OS emerged when our previous studies showed that GADD45A was reexpressed by treatment with the demethylation drug decitabine. In this study, we analyze the OS cell lines MG63 and U2OS and show that on treatment with decitabine, a significant loss of DNA methylation of GADD45A was associated with elevated expression and induction of apoptosis. In vivo affects of decitabine treatment in mice showed that untreated control xenografts exhibited low nuclear staining for Gadd45a protein, whereas the nuclei from xenografts in decitabine-treated mice exhibited increased amounts of protein and elevated apoptosis. To show the specificity of this gene for decitabine-induced apoptosis in OS, GADD45A mRNAs were disrupted using short interference RNA, and the ability of the drug to induce apoptosis was reduced. Understanding the role of demethylation of GADD45A in reexpression of this pathway and restoration of apoptotic control is important for understanding OS oncogenesis and for more targeted therapeutic approaches

    Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-0

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    <p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>e y-axis indicates cell number in millions and the x-axis indicates time points in days. The results are expressed as cell counts at each corresponding time point. Each data point is the Mean of cell counts from 2 experiments (5 passages apart) each consist of 2 independent cultures and the error bars indicate the standard deviation. The findings indicate a slight increase of U2OS cells' doubling time and a decrease of 18% (p = 0.045) in the viability of treated cells compared to untreated control , Cell death in U2OS cells caused by decitabine treatment at 1 μM concentration (light column) compared to no-treatment (dark column). The results are expressed as percentage of cell death (fraction of cells with positive PI stain). The y-axis indicates the percentage of cells with PI staining (dead cells). Each column is the Mean of 3 experiments with error bars indicating standard deviation. The asterisk indicates significant increase in cell death (p < 0.05) as a result of decitabine treatment

    Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation-2

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    <p><b>Copyright information:</b></p><p>Taken from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells and in xenografts: Identification of apoptotic genes as targets for demethylation"</p><p>http://www.cancerci.com/content/7/1/14</p><p>Cancer Cell International 2007;7():14-14.</p><p>Published online 10 Sep 2007</p><p>PMCID:PMC2034371.</p><p></p>n is the Mean of tumor volumes measured in 12 xenograft tumors. There was a significant decrease (p < 0.05) in tumor volumes as a result of decitabine treatment. , Osteoid assessment . Each column is the Mean of osteoid evaluation of 9 sections. There was significant increase (p < 0.0001) in osteoid formation as a result of decitabine treatment. , Representation images of H&E sections. Control xenograft tumors (left) show solid sheets of poorly differentiated cells with minimal osteoid (image magnification × 100). Decitabine treated tumors shows less dense cell population and increased areas of osteoid seen as light pink and lacy matrix with the nuclei of osteoblasts sitting closer to the produced matrix (image magnification × 100). The arrows in the enlargement show the osteoid matrix surrounding osteoblasts (defined as eosinophilic osteoid-like material). And , Mitotic count as identified in the same sections used to asses for osteoid evaluation. Each column is the Mean mitotic count in 9 sections (a minimum of 1100 nuclei were scanned per section). Decitabine treatment resulted in a significantly lower mitotic count (p < 0.0001). The arrows in the enlargement image in (left) indicate mitotic nuclei. , Results for apoptosis analysis by TUNEL assay. Each column is the Mean count of TUNEL positive nuclei seen in 9 images representing 9 sections (≥1000 nuclei were scanned per section). There was a significant increase (p < 0.05) of apoptotic cells as a result of decitabine treatment. , Representation images from TUNEL assay of control tumors (left) and decitabine treated tumors [50] (image magnification × 200). The arrows in the enlargement image show the TUNEL-positive nuclei (apoptotic nuclei). Error bars indicate standard deviation from the Mean values and p-values are based on comparison between control and decitabine treated tumors using student t-test
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