Friend Leukemia Virus Integration Research Articles

Constitutive activation of c-Abl/protein kinase C-δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma

A noncanonical pathway of transforming growth factor-β signalling, the c-Abl/protein kinase C-δ (PKC-δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. To investigate the significance of the c-Abl/PKC-δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). The activation status of the c-Abl/PKC-δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet-derived growth factor receptor inhibitor AG1296 and gene silencing of c-Abl on the expression levels of type I collagen were evaluated by immunoblotting. The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c-Abl were elevated compared with cultured normal fibroblasts and PKC-δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c-Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. Constitutive activation of the c-Abl/PKC-δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.

Transcription factors Fli1 and EKLF in the differentiation of megakaryocytic and erythroid progenitor in 5q- syndrome and in Diamond–Blackfan anemia

Friend leukemia virus integration 1 (Fli1) and erythroid Krüppel-like factor (EKLF) participate under experimental conditions in the differentiation of megakaryocytic and erythroid progenitor in cooperation with other transcription factors, cytokines, cytokine receptors, and microRNAs. Defective erythropoiesis with refractory anemia and effective megakaryopoiesis with normal or increased platelet count is typical for 5q- syndrome. We decided to evaluate the roles of EKLF and Fli1 in the pathogenesis of this syndrome and of another ribosomopathy, Diamond-Blackfan anemia (DBA). Fli1 and EKLF mRNA levels were examined in mononuclear blood and bone marrow cells from patients with 5q- syndrome, low-risk MDS patients with normal chromosome 5, DBA patients, and healthy controls. In 5q- syndrome, high Fli1 mRNA levels in the blood and bone marrow mononuclear cells were found. In DBA, Fli1 expression did not differ from the controls. EKLF mRNA level was significantly decreased in the blood and bone marrow of 5q- syndrome and in all DBA patients. We propose that the elevated Fli1 in 5q- syndrome protects megakaryocytic cells from ribosomal stress contrary to erythroid cells and contributes to effective though dysplastic megakaryopoiesis.

Transcriptional regulatory networks in human lung adenocarcinoma

Lung adenocarcinoma (AC) is the most common histological subtype of lung cancer worldwide and its absolute incidence is increasing markedly. Transcriptional regulation is one of the most fundamental processes in lung AC development. However, high-throughput functional analyses of multiple transcription factors and their target genes in lung AC are rare. Thus, the objective of our study was to interpret the mechanisms of human AC through the regulatory network using the GSE2514 microarray data. Our results identified the genes peroxisome proliferator activated receptor-γ (PPARG), CCAAT/enhancer binding proteinβ (CEBPB), ets variant 4 (ETV4), Friend leukemia virus integration 1 (FLI1), T-cell acute lymphocytic leukemia 1 (TAL1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1) as hub nodes in the transcriptome network. Among these genes, it appears that: PPARG promotes the PPAR signaling pathway via the upregulation of lipoprotein lipase (LPL) expression, but suppresses the cell cycle pathway via downregulation of growth arrest and DNA-damage-inducible, γ (GADD45G) expression; ETV4 stimulates matrix metallopeptidase 9 (MMP9) expression to induce the bladder cancer pathway; FLI upregulates transforming growth factor, β receptorII (TGFBR2) expression to activate TGF-β signaling and upregulates cyclin D3 (CCND3) expression to promote the cell cycle pathway; NFKB1 upregulates interleukin 1, β (IL-1B) expression and initiates the prostate cancer pathway; CEBPB upregulates IL-6 expression and promotes pathways in cancer; and TAL1 promotes kinase insert domain receptor (KDR) expression to promote the TGF-β signaling pathway. This transcriptional regulation analysis may provide an improved understanding of the molecular mechanisms and potential therapeutic targets in the treatment of lung AC.

Molecular investigation of Ewing sarcoma: about detecting translocations.

Molecular investigation of Ewing sarcoma: about detecting translocations.

FLI1 (Friend leukemia virus integration 1)

Review on FLI1 (Friend leukemia virus integration 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Important Genes in the Pathogenesis of 5q- Syndrome and Their Connection with Ribosomal Stress and the Innate Immune System Pathway

Myelodysplastic syndrome (MDS) with interstitial deletion of a segment of the long arm of chromosome 5q [del(5q)] is characterized by bone marrow erythroid hyperplasia, atypical megakaryocytes, thrombocythemia, refractory anemia, and low risk of progression to acute myeloid leukemia (AML) compared with other types of MDS. The long arm of chromosome 5 contains two distinct commonly deleted regions (CDRs). The more distal CDR lies in 5q33.1 and contains 40 protein-coding genes and genes coding microRNAs (miR-143, miR-145). In 5q-syndrome one allele is deleted that accounts for haploinsufficiency of these genes. The mechanism of erythroid failure appears to involve the decreased expression of the ribosomal protein S14 (RPS14) gene and the upregulation of the p53 pathway by ribosomal stress. Friend leukemia virus integration 1 (Fli1) is one of the target genes of miR145. Increased Fli1 expression enables effective megakaryopoiesis in 5q-syndrome.

The Significance of Megakaryocytic Transcription Factor Fli1 and Erythroid Transcription Factor EKLF in the Ribosomopathies: 5q Minus Syndrome and Diamond-Blackfan Anemia. the Role of Fli1 in p53 Regulation and in 5q Minus Syndrome Megakaryopoiesis,

Abstract 3825 Introduction:5q- syndrome and DBA belong to ribosomopathies. 5q- syndrome patients (pts) have haploinsufficient RPS14 gene and approximately 50% DBA pts have mutated some of genes for ribosomal proteins. These two disorders share similar erythroid and megakaryocytic characteristics: macrocytic anemia, decreased erythropoiesis, normal platelet count and they differ in: frequent neutropenia in 5q- syndrome, rare thrombocytemia and absence of hypolobulated megakaryocytes in DBA. We recently studied two transcription factors Fli1(Friend leukemia virus integration 1) and EKLF (Erythroid Krüppel like factor, also named KLF1) involved in MEP (common megakaryocytic and erythroid progenitor) differentiation in MDS 5q-syndrome pts. Now we present the examination of these factors in DBA pts.There exists cross-antagonism between Fli1 and EKLF with probable dominance of EKLF. Fli1 mRNA is target for microRNA-l45 (miR-145) localized in common deleted region of 5q. Haploinsuffciency of miR-145 in 5q- syndrome stabilizes Fli1 mRNA and increases Fli1 (Kumar et al. Blood 2009;114 abstr.947). Fli1 is also increased by interkleukin 6. IL-6 is induced by haploinsufficiency of miR-145 and miR-146a. Fli1 gene promoter is upregulated by Fli1 itself. Transcription factor PU.1 is positive regulator of Fli1 gene expression. In mice Fli1 regulates p53 via MDM2 (mouse double minute 2 or HDM2 in humans), an E3 ubiquitin ligase, which promotes p53 degradation in proteasomes (Truong et al. Oncogene 2005;24:962–69). Patients and Methods:Mononuclear cells were isolated from blood of 31 pts with 5q- syndrome, 26 MDS low risk pts with normal chromosome 5, 16 healthy controls and 10 DBA pts (7 with mutation of RP and 3 without proved mutation). Further, mononuclear cells were obtained from bone marrow of 17 pts with 5q-syndrome, 15 MDS low risk pts with normal chromosome 5, and 8 healthy controls. In total RNA of blood and bone marrow mononucklear cells Fli1, EKLF, p53, HDM2 and PU.1 mRNA levels were determined by quantitative real-time PCR. Related levels of mRNAs were calculated to the level of housekeeping GAPDH mRNA. We examined methylation status of EKLF promoter region in 5q- syndrome pts by bisulfite genomic DNA sequencing. All MDS pts involved in this study were examined by FISH and in all 5q- syndrome pts the deletion of 5q31 was confirmed. Results:In 5q- syndrome pts íncreased levels of Fli1 mRNA and decreased EKLF mRNA were detected both in blood and bone marrow mononuclear cells in comparison with healthy controls. In MDS low risk pts with normal chromosome 5 the increased Fli1 mRNA was found only in peripheral blood. There was no difference between EKLF mRNA levels in pts and in controls.In 8 of 10 DBA pts EKLF mRNA was decreased in blood in comparison to healthy controls, while Fli1 mRNA levels did not differ from controls with one pt exception. Methylation of EKLF gene promoter region is not responsible for the decrease EKLF mRNA in 5q- syndrome. Significantly increased level of p53 mRNA was found only in bone marrow of 5q- pts but not in blood of 5q- or DBA pts. There was no significant difference in HDM2 and PU.1 mRNA levels in blood and bone marrow of both groups of MDS pts and also in blood of DBA pts. Discussion:Significant EKLF decrease in all pts with 5q- syndrome and in 8 of 10 DBA pts corresponds to anemia and decreased erythropoiesis in both groups, but the cause of the EKLF decrease remains unknown. In 5q- syndrome low EKLF might be explained by the cross-antagonism between Fli1 and EKLF.Most significant is high Fli1 in all 5q- pts. This finding is important for the explanation of effective megakaryopoiesis contrary to defective erythropoiesis. The situation is different in DBA pts, where Fli1 mRNA is not increased with one exception (pt with atypical RPL5 mutation).Increased Fli1 might be the clue for the maintenance of effective megakaryopoiesis in 5q- syndrome. In erythroid cells ribosomal proteins inhibit HDM2, p53 is not degraded and increased p53 enhances apoptosis of erythroid cells. High Fli1 in megakaryocytic cells transcriptionally stimulates HDM2. In addition, IL-6 stimulates not only Fli1 but also DNA methylase 1, which methylates TP53 promoter and silences TP53 expression. These processes prevent apoptosis of megakaryocytic cells. For effective megakaryopoiesis in DBA pts we have not explanation so far. Disclosures:No relevant conflicts of interest to declare.

Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening

Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable." We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided. Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P < .001). Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.

Abstract 4018: The role of FLI1 in breast cancer development and progression

Abstract Breast cancer is the second most common cancer-related death among women in the US. The majority of the breast cancer-related deaths are due to tumor progression. A multitude of changes in gene expression are required for the cancer cell to acquire the ability to invade and migrate. The transcriptional activation or repression of these cancer-associated genes is not clearly understood, however many ETS family members have been considered good candidates. Friend leukemia virus integration 1 (FLI1) is an ETS protein that is aberrantly expressed in retrovirus-induced hematological tumors, and is found to be rearranged in Ewing's sarcoma and related primitive neuroectodermal tumors characterized by a t(11;22)(q24;q12) translocation. Limited attention has been directed towards elucidating the potential role of FLI1 in epithelial-derived cancers, including breast cancer. Our preliminary immunohistochemical analyses show that FLI1 protein is decreased in human invasive breast tumors compared to normal breast tissue. A decrease of FLI1 mRNA and protein was also demonstrated in breast cell cancer cell lines through Real Time RT-PCR and western blot, respectively. We used adenovirus to examine the effects of FLI1 expression in two invasive breast cancer cell lines (MDA-MB-231 and MDA-MB-157). Re-expression of FLI1 in both MDA-MB-231 and MDA-MB-157 inhibited cell growth, mainly due to a decrease in cellular proliferation. FLI1 expression also inhibited the motility and invasiveness of breast cancer cell lines, determined by the use of Transwell inserts coated with fibronectin or Matrigel, respectively. We hypothesize that the loss of FLI1 is a critical step for breast cancer progression. Loss-of-function studies using shRNA are being performed to complement the above gain-of-function studies. Future studies will investigate potential downstream targets of FLI1. Inhibiting the reduction of FLI1 or regulating its downstream targets may be to be unique targets of breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2011-4018

Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.

The Evaluation of CD99 Immunoreactivity and EWS/FLI1 Translocation by Fluorescence in situ Hybridization in Central PNETs and Ewing’s Sarcoma Family of Tumors

Ewing's sarcoma family of tumors (ESFTs) are indicated by malignant, small, round and blue cell tumors of the bone and soft tissue. Gene rearrangements between EWS gene on chromosome 22q12 and members of the ETS gene family are common in and specific to ESFTs. Another defining characteristic of ESFTs is their membranous expression of the CD99. In contrast, such translocations and immunoreactivity are not found in central primitive neuroectodermal tumors (cPNETs). The aim of this study was to investigate the detection of EWS/FLI1 translocations and CD99 immunoreactivity in order to evaluate their clinicopathological features and their roles in the differential diagnosis of these tumors. In this study, we investigated CD99 immunoreactivity using immunohistochemistry and Ewing's sarcoma / Friend leukaemia virus integration 1 (EWS/FLI1) translocation using the fluorescence in situ hybridization (FISH) method in 23 cases. CD99 expression was detected in 10/11 (90%) ESFT cases and 2/7 cPNET cases. In 18 cases EWS/FLI1 translocation was examined using the FISH method. The EWS/FLI1 translocations were detected in 7/8 (87.5%) ESFTs cases, whereas non of 8 cPNET cases were detected with this translocation. One case could not be classified as either central or peripheral, showed EWS/FLI1 translocation. There was a statistically significant difference in CD99 expression (p = 0.0013) and EWS/FLI1 translocation (p = 0,002) between cPNETs and ESFTs cases. In conclusion, CD99 expression and EWS/FLI1 translocation are specific and sensitive markers in the diagnosis of ESFTs. However, these were often not found in cases of cPNET. Therefore, in the diagnosis of ESFTs, clinical, radiological, histopathological and immunohistochemical parameters should always be evaluated together.

Downregulation of Friend Leukemia Virus Integration 1 as a Feedback Mechanism That Restrains Lipopolysaccharide Induction of Matrix Metalloproteases and Interleukin-10 in Human Macrophages

The E26 transformation-specific (Ets) proteins are a family of transcription factors with important roles in a variety of cellular processes ranging from proliferation and differentiation to transformation and metastasis. Tissue-specific expression of Ets proteins and their ability to interact with other families of transcription factors contribute to their versatility. In this study, we investigated the regulation of Ets factors in primary human monocytes and macrophages, and their role in matrix metalloprotease (MMP) and cytokine production. The macrophage-activating Toll-like receptor ligand, lipopolysaccharide (LPS), induced the expression of Ets family members epithelium-specific Ets factor 3 (ESE-3) and TEL-2 but rapidly suppressed Friend leukemia virus integration 1 (FLI-1) expression. Modulation of FLI-1 expression using either RNA interference or forced expression identified a positive role for FLI-1 in contributing to LPS-induced expression of MMP-1, MMP-3, MMP-10, and interleukin-10 (IL-10). Thus, the rapid downregulation of FLI-1 expression after LPS stimulation attenuates the induction of various MMPs and IL-10 under inflammatory conditions. In contrast, the expression of IL-6 and TNFα and the effects of interferon (IFN)γ on LPS responses were not dependent on FLI-1. Our results define a novel FLI-1-mediated self-regulatory feedback loop that limits MMP expression and thus may attenuate extent of tissue destruction associated with inflammatory responses.

Putative tumor suppressor miR‐145 inhibits colon cancer cell growth by targeting oncogene friend leukemia virus integration 1 gene

Tumor suppressor microRNA miR-145 is commonly down-regulated in colon carcinoma tissues, but its specific role in tumors remains unknown. In this study, the authors identified the Friend leukemia virus integration 1 gene (FLI1) as a novel target of miR-145. FLI1 is involved in t(11;22)(q24:q12) reciprocal chromosomal translocation in Ewing sarcoma, and its expression appears to be associated with biologically more aggressive tumors. The authors demonstrated that miR-145 targets a putative microRNA regulatory element in the 3'-untranslated region (UTR) of FLI1, and its abundance is reversely associated with FLI1 expression in colon cancer tissues and cell lines. By using a luciferase/FLI1 3'-UTR reporter system, they found that miR-145 down-regulated the reporter activity, and this down-regulation was reversed by anti-miR-145. Mutation of the miR-145 microRNA regulatory element sequence in the FLI1 3'-UTR abolished the activity of miR-145. miR-145 decreased FLI1 protein but not FLI1 mRNA, suggesting a mechanism of translational regulation. Furthermore, the authors demonstrated that miR-145 inhibited cell proliferation and sensitized LS174T cells to 5-fluorouracil-induced apoptosis. Taken together, these results suggest that miR-145 functions as a tumor suppressor by down-regulating oncogenic FLI1 in colon cancer.

Histone Deacetylase Inhibition Suppresses the Transforming Growth Factor β1-Induced Epithelial-to-Mesenchymal Transition in Hepatocytes†,‡

Transforming growth factor beta1 (TGFbeta1) plays a crucial role in the induction of the epithelial-to-mesenchymal transition (EMT) in hepatocytes, which contributes to the pathogenesis of liver fibrosis. The inhibition of the TGFbeta1 cascade suppresses EMT and the resultant fibrosis. In this study, we focus on EMT-induced fibrosis in hepatocytes and the epigenetic regulation of the type I collagen gene. Histone acetylation is an important, major epigenetic mechanism that modulates gene transcription. We evaluated the epigenetic regulation of type I collagen in alpha mouse liver 12 hepatocytes (an untransformed mouse cell line) that had undergone EMT after treatment with TGFbeta1. The histone deacetylase inhibitor trichostatin A (TSA) inhibited EMT; this was reflected by the preservation of epithelial markers and function (E-cadherin and albumin). Fibrosis, the ultimate outcome of EMT, was abolished by TSA; this was indicated by the inhibition of type I collagen deposition. TSA exerted its anti-EMT effects by deactivating the mothers against decapentaplegic homolog 3 (Smad3)/Smad4 transcription complex and by interfering with p300, a coactivator of the type I collagen promoter, and preventing its binding to Smad3. TSA also restored Friend leukemia virus integration 1, an inhibitor of the type I collagen gene. TGFbeta1-induced EMT and its inhibition by TSA were replicated in human primary hepatocytes. Histone deacetylase inhibition abrogates TGFbeta1-induced EMT in hepatocytes and reverses EMT-induced fibrosis by epigenetic modulation of type I collagen.

Fli-1 and EKLF Gene Expression in Patients with MDS 5q- Syndrome.

Abstract 2788Poster Board II-764 Introduction.Patients with MDS-5q- syndrome have macrocytic anemia often with hypoplastic erythropoiesis and on the contrary thrombocythemia with effective though dysplastic megakaryopoiesis. Megakaryocytes and erythroid cells are thought to share a common progenitor MEP (T.P.McDonald et al., Exp. Hematology 1993). There are two key transcription factors which together with other transcription factors and relevant cytokines and receptors determine the hemopoietic differentiation of the common stem cell: erythroid Krüppel-like factor (EKLF) for erythroid lineage and Friend leukemia virus integration 1 (FLi-1) for megakaryopoiesis (Pilar Frontelo et al., Blood 2007; G.A.Blobel, Blood 2007; F.Bouilloux et al., Blood 2008). There is functional cross antagonism between FLi-1 and EKLF (J.Starck et al., Mol. Cel. Biology 2003). FLi-1 is active only if dephosphorylated (H.Huang et al., ASH Abstracts 2008). The question is whether both factors play any role in 5q- syndrome. Methods.FLi-1 and EKLF gene expressions were determined in mononuclear cells isolated from the whole blood or bone marrow using Ficoll-Paque PLUS. Expression of both factors was measured by quantitative real-time PCR. RT-PCR products were verified by electrophoresis and direct sequencing. The assays were performed for sample in duplicate. Glyceraldehyd-3-phosphate dehydrogenase (GAPDH), FLi-1 and EKLF were amplified in 25 μl reaction mixture containing 12.5 μl SYBR Green JumpStart Taq Ready Mix, 2.5 μl 2 μM FLi-1 or EKLF forward and reverse primers, 0,25 μl internal reference dye and 1 μl cDNA. Relative levels of FLi-1 and EKLF mRNAs were calculated to the level of housekeeping GAPDH mRNA. Results.FLi-1 and EKLF were measured in blood mononuclear cells of 8 patients fulfilling all criterias of 5q- syndrome. FLi1mRNA/GAPDHmRNA was higher in all samples, average value was 0.0930 (0.0242-0.4274) compared to control value 0.0194. FLi1mRNA/GAPDHmRNA in bone marrow mononuclear cells of 7 patients with 5q- syndrome was higher in all samples but one. The average value was 0.0827 (0.0070-0.2554) compared to healthy controls 0.0044.EKLF gives very low values in the majority of patients′ blood and bone marrow samples as well as in healthy controls. The evaluation is therefore less reliable then FLi-1 assessment. EKLFmRNA/GAPDHmRNA in blood was 0.0004 (0.0-0.0023) compared to the control 0.0222. The results of EKLF in 5 bone marrow samples are inconsistent. Three are lower than the control (0.0068), 1 of remaining 2 samples is extremely high (0.3491). It is interesting that this patient is the only one who responded to erythropoietin and is transfusion independent. Summary.Our preliminary results with FLi-1 and EKLF gene expression measurement are in agreement with expected findings: increased FLi-1 expression corresponds to thrombocytemia in 5q- syndrome patients and expression of EKLF, lower than in controls would correspond to anemia in these patients. However, EKLF values are less reliable because of very low values in patients as well as in controls and because of inconsistent results in bone marrow samples. We prepare to follow both factors in 5q- patients after the treatment with lenalidomide. Lenalidomide improves anemia in 5q- syndrome patients and temporarily causes decrease of thrombocytes (A.List et al., N.Engl.J.Med. 2005, 2006). Inhibition of phosphatases by lenalidomide (S.Wei et al., Proc.Natl.Acad.Sci.USA 2009) can stop FLi-1 dephosphorylation which leads to FLi-1 inactivation. Hypotetically inactive FLi-1 would enable EKLF to induce MEP into erythroid lineage.Supported by MSM 0021620808 Disclosures:No relevant conflicts of interest to declare.

Phosphorylation of Ewing's sarcoma protein (EWS) and EWS-Fli1 in response to DNA damage

In Ewing's sarcomas, chromosomal translocations cause the N-terminal domain of the EWS (Ewing's sarcoma protein) to fuse with the DNA-binding domains of the Ets (E26 transformation-specific) family of transcription factors. Here we show that EWS and EWS-Fli1 (Friend leukaemia virus integration 1), the fusion most frequently found in Ewing's sarcomas, become phosphorylated at Thr(79) in response to either mitogens or DNA-damaging agents. The much weaker mitogen-induced phosphorylation of EWS is catalysed by the MAPKs (mitogen-activated protein kinases) ERK1 (extracellular signal-regulated kinase 1) and ERK2, whereas the much stronger phosphorylation of EWS induced by the DNA alkylating agent MMS (methyl methanesulphonate) can be catalysed by JNK (c-Jun N-terminal kinase) and at least one other protein kinase distinct from ERK1/ERK2. In contrast, the phosphorylation of EWS-Fli1 induced by MMS was largely mediated by p38alpha/p38beta MAPKs. MMS induced a much stronger phosphorylation of EWS-Fli1 than EWS in heterodimers comprising both proteins.

Heavy Ion Beam Irradiation Regulates the mRNA Expression in Megakaryocytopoiesis from Human Hematopoietic Stem/Progenitor Cells

Heavy ion beams are a high-LET radiation that has greater biological effect than electron beams or X-rays. However, little is known about the effect of heavy ion beams on the proliferation and differentiation of human hematopoietic stem/progenitor cells (HSPCs). The present study examined the effect of heavy ion beams on gene expression in human HSPCs, especially during early stage of megakaryocytopoiesis. Human CD34+ cells were exposed to monoenergetic carbon-ion beams (290 MeV/nucleon, LET = 50 KeV/m) that were generated by an accelerator (Heavy Ion Medical Accelerator in Chiba). The expression of various genes related to early hematopoiesis, megakaryocytopoiesis/erythropoiesis, cytokine receptors and oxidative stress were analyzed by real-time RT-PCR. Friend leukemia virus integration 1, an early hematopoiesis-related gene, showed significantly higher mRNA expression than the control at 6 hr after irradiation. In contrast, no significant differences were observed in almost all of the other early hematopoiesis-related genes, cytokine receptor-coded genes and megakaryocytopoiesis/erythropoiesis-differentiation pathway-related genes, respectively. An analysis of the response of the genes to oxidative stress revealed the expression of heme oxygenase 1 to show a 1.5-fold and 11.9-fold increase from the day 0 control at 24 hr after 0.5 Gy and 2 Gy irradiation, respectively. Similarly, the NAD(P)H dehydrogenase-quinone 1 expression also showed a 22.0-fold and a 21.8-fold increase at 6 hr in comparison to the initial control. These results showed that the heavy ion beams affect megakaryocytopoiesis/ erythropoiesis differentiation of human HSPCs on the gene expression level.

Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli1

BackgroundA function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.MethodsBioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.ResultsmiR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.ConclusionsmiR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.

Gene Expression Analysis of a Porcine Native Abdominal Aortic Aneurysm Model

Introduction. We sought to characterize the gene expression patterns occurring during the development of aneurysms in the native porcine aorta. Methods. In Yorkshire swine, the infrarenal aorta was balloon dilated and infused with a solution of type I collagenase/pancreatic porcine elastase (16,000 U/1,000 U). Aneurysmal and control aortic samples were obtained at 1 (n = 3), 2 (n = 6), and 4 (n = 5) weeks following aneurysm induction. RNA was isolated, converted to biotin-modified antisense RNA and hybridized to porcine genome arrays. Aneurysmal and control gene intensities were compared using the 2-sample-for-means z-test. P < .01 was considered statistically significant. Results. Extracellular matrix remodeling genes that were upregulated in aneurysmal compared with control tissue included matrix metalloproteinase-1, -2, -3, and -9; MT-MMP; cathepsin-D, -H, -K, and -S; tissue inhibitor of metalloproteinase-1; and collagen I-a1 chain (P < .01). Elastin exhibited temporally downregulated gene expression (P < .01). Inflammatory genes that were upregulated included intercellular adhesion molecule-2, tumor necrosis factor-a, interleukin (IL)-1b, IL-10, chemokine receptor-4, and tissue plasminogen activator (P < .01). Atherosclerosis and cancer genes that were upregulated included apolipoprotein E, acyl-CoA binding protein, friend leukemia virus integration-1, and E26 transformation-specific sequence (P < .01). Conclusion. The porcine model replicates the gene expression patterns that are observed during the development of aneurysms in human studies as well as in rodent models. The porcine model thereby represents a novel method to study the impact of endovascular, cell-based, and other therapeutic interventions on AAA pathophysiology. (Surgery 2008;144:252-8.)

Clinical and molecular‐cytogenetic evaluation of a family with partial Jacobsen syndrome without thrombocytopenia caused by an ∼5 Mb deletion del(11)(q24.3)

Clinical manifestations of Jacobsen syndrome (JBS) depend on the size of the 11qter deletion, which usually varies between approximately 7 and 20 Mb. Typical JBS features include developmental delay/mental retardation, short stature, congenital heart defects, thrombocytopenia, and characteristic dysmorphic facial features. We report on a family in which a 4-year-old girl as well as her mother and maternal uncle present with subtle features of JBS. Notably, neither thrombocytopenia nor congenital anomalies were detected in this family. Cytogenetic analyses revealed normal karyotypes. Using fluorescence in situ hybridization (FISH) and whole-genome oligonucleotide array CGH analyses, we identified an approximately 5 Mb deletion of the terminal part of chromosome 11q in all the three affected family members. The deletion breakpoint was mapped between 129,511,419 and 129,519,794 bp. This is the smallest deletion reported in a JBS patient. Interestingly, the FLI1 (friend leukemia virus integration 1) hematopoiesis factor gene located approximately 6.5 Mb from 11qter and usually deleted in patients with JBS, is intact. Our data support previous hypotheses that FLI1 haploinsufficiency is responsible for thrombocytopenia in patients with JBS.