scholarly journals Overexpression of miR-210 is associated with SDH-related pheochromocytomas, paragangliomas, and gastrointestinal stromal tumours

2014 ◽  
Vol 21 (3) ◽  
pp. 415-426 ◽  
Author(s):  
V H M Tsang ◽  
T Dwight ◽  
D E Benn ◽  
G Y Meyer-Rochow ◽  
A J Gill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multivariate Analysis ◽  
Cell Lines ◽  
Fold Increase ◽  
Cellular Responses ◽  
Hek293 Cells ◽  
Gastrointestinal Stromal Tumours ◽  
Sirna Knockdown ◽  
Normal Controls

miR-210 is a key regulator of response to hypoxia. Pheochromocytomas (PCs) and paragangliomas (PGLs) with germlineSDHxorVHLmutations have pseudohypoxic gene expression signatures. We hypothesised that PC/PGLs containingSDHxorVHLmutations, and succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumours (GISTs), would overexpress miR-210 relative to non-SDHor -VHL-mutated counterparts. miR-210 was analysed by quantitative PCR in i) 39 PC/PGLs, according to genotype (oneSDHA, fiveSDHB, sevenVHL, threeNF1, sevenRET, 15 sporadic, one unknown) and pathology (18 benign, eight atypical, 11 malignant, two unknown); ii) 18 GISTs, according to SDHB immunoreactivity (nine SDH-deficient and nine SDH-proficient) and iii) two novelSDHB-mutant neurosphere cell lines. miR-210 was higher inSDHx- orVHL-mutated PC/PGLs (7.6-fold) compared with tumours withoutSDHxorVHLmutations (P=0.0016). miR-210 was higher in malignant than in unequivocally benign PC/PGLs (P=0.05), but significance was lost when benign and atypical tumours were combined (P=0.08). In multivariate analysis, elevated miR-210 was significantly associated withSDHxorVHLmutation, but not with malignancy. In GISTs, miR-210 was higher in SDH-deficient (median 2.58) compared with SDH-proficient tumours (median 0.60;P=0.0078). miR-210 was higher in patient-derived neurosphere cell lines containingSDHBmutations (6.5-fold increase) compared with normal controls, in normoxic conditions (P<0.01). Furthermore, siRNA-knockdown of SDHB in HEK293 cells increased miR-210 by 2.7-fold (P=0.001) under normoxia. Overall, our results suggest that SDH deficiency in PC, PGL and GISTs induces miR-210 expression and substantiates the role of aberrant hypoxic-type cellular responses in the development of these tumours.

scholarly journals The Role of Histone Demethylases and DNA Methyltransferases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3921-3921
Author(s):  
Katerina Rejlova ◽  
Alena Musilova ◽  
Martina Slamova ◽  
Karel Fiser ◽  
Karolina Skvarova Kramarzova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hox Genes ◽  
Fold Increase ◽  
Dna Methyltransferases ◽  
Histone Mark ◽  
Gene Promoters ◽  
Histone Demethylases ◽  
Atra Treatment ◽  
Hox Gene

Abstract Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous results showed that HOX gene expression differs among genetically characterized subtypes of pediatric acute myeloid leukemia (AML). Specifically, PML-RARa positive AML patients have overall lowest HOX gene expression which positively correlates with expression of histone 3 lysine 27 (H3K27) demethylases - JMJD3 and UTX and negatively with the expression of DNA methyltransferases - DNMT3a and DNMT3b. Interestingly, JMJD3 was already shown to be a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). From these findings we postulated a hypothesis that reduced levels of HOX genes in PML-RARa positive AML are a consequence of suppressed expression of histone demethylases resulting in increased H3K27 methylation and/or of elevated levels of DNMTs leading to de novoDNA methylation. We studied the role of histone demethylases and DNMTs in the regulation of HOX gene expression and the effect of treatment in PML-RARa positive cell lines (NB4 and ATRA-resistant clones NB4-LR2 and NB4-MR2). We treated NB4 cell line by all-trans retinoic acid (ATRA; 1uM), which was described to release the differentiation block caused by the presence of PML-RARa and to degrade the fusion protein. We observed that expression of particular HOX genes (HOXA1, HOXA3, HOXA4, HOXA5, HOXA7, HOXB4, HOXB6) measured by qPCR was significantly increased after ATRA treatment. While the level of JMJD3 was significantly increased upon ATRA treatment as well, the expression of UTX did not change. Furthermore, we detected significantly reduced expression of DNMT3b gene. To exclude a non-specific effect of ATRA, independent of PML-RARa, we used resistant clones LR2 and MR2 bearing mutations in retinoic acid-binding domain. HOX gene expression together with JMJD3, UTX and DNMT3b expression did not change upon ATRA treatment. These results confirm the PML-RARa-dependent regulation of HOX genes. To test the role of JMJD3 in the HOX gene expression regulation, we cultured NB4 cells with a specific inhibitor of histone demethylases, GSK-J4 (1 uM, 10 uM), in the presence of ATRA. The co-treatment caused significant decrease in the expression of studied HOX genes (HOXA1, HOXA3, HOXA5, HOXA7, HOXA10, HOXB4, HOXB6) in comparison to ATRA alone which supports the role of JMJD3 in the transcription regulation. Further, we performed chromatin immunoprecipitation (ChIP) to investigate if the changes of HOX gene expression upon ATRA and GSK-J4 treatment would correspond with changes of histone code on HOX gene promoter regions. ATRA treatment caused reduction of repressive histone mark (H3K27me3) on particular HOX gene promoters (HOXA1, HOXA3, HOXA5, HOXA7), by contrast, combinational treatment of ATRA and GSK-J4 reversed this effect. Accordingly, we detected that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2. Next we were interested if JMJD3 inhibition would interfere with the differentiation effect of ATRA. As shown previously, ATRA treatment alone caused differentiation of NB4 cell line whereas the combination with GSK-J4 did not reduce the effect. Interestingly, in addition to differentiation it led cells to apoptosis. Combination of drugs (ATRA - 1uM, GSK-J4 - 1, 2, 5uM) increased significantly the percentage of dead cells in comparison to ATRA or GSK treatment alone (GSK-J4 alone vs in combination with ATRA, 1uM - 1.8 fold, 2uM - 2.2 fold, 5 uM - 2.3 fold increase). Next we measured apoptosis in resistant clones LR2 and MR2. In both cases the highest concentration used of GSK-J4 (5uM) in combination with ATRA caused significant increase of dead cells as well (LR2 - 2.1 fold, MR2 - 2.0 fold increase). Our results indicate that JMJD3 is responsible for the regulation of HOX gene expression in PML-RARa positive leukemia since changes of HOX gene expression correspond with histone modifications on the regions of HOX gene promoters. We assume that DNA methylation driven by DNMT3b can also participate in this process. Moreover, our findings demonstrate potential therapeutic implications of GSK-J4 inhibitor in combination with ATRA in patients with acute promyelocytic leukemia who are not responsive to ATRA monotherapy. Supported by P304/12/2214 and GAUK 196616 Disclosures No relevant conflicts of interest to declare.

Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Anita SAHU ◽  
Sromona Mukherjee ◽  
Kate Stenson ◽  
Maradumane L Mohan ◽  
Sathyamangla V Prasad
Keyword(s):  
Hek293 Cells ◽  
Protein Kinase Activity ◽  
Pi3k Inhibitors ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Β Blocker ◽  
Sirna Knockdown ◽  
G Protein Coupled ◽  
Diabetes And Obesity

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β 2 AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.

scholarly journals The first enhancer in an enhancer chain safeguards subsequent enhancer-promoter contacts from a distance

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Wei Song ◽  
Roded Sharan ◽  
Ivan Ovcharenko
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Target Gene ◽  
Regulatory Elements ◽  
Distal Enhancer ◽  
Human Genes ◽  
Multiple Cell ◽  
A Chain ◽  
Target Promoter

Abstract Background Robustness and evolutionary stability of gene expression in the human genome are established by an array of redundant enhancers. Results Using Hi-C data in multiple cell lines, we report a comprehensive map of promoters and active enhancers connected by chromatin contacts, spanning 9000 enhancer chains in 4 human cell lines associated with 2600 human genes. We find that the first enhancer in a chain that directly contacts the target promoter is commonly located at a greater genomic distance from the promoter than the second enhancer in a chain, 96 kb vs. 45 kb, respectively. The first enhancer also features higher similarity to the promoter in terms of tissue specificity and higher enrichment of loop factors, suggestive of a stable primary contact with the promoter. In contrast, a chain of enhancers which connects to the target promoter through a neutral DNA segment instead of an enhancer is associated with a significant decrease in target gene expression, suggesting an important role of the first enhancer in initiating transcription using the target promoter and bridging the promoter with other regulatory elements in the locus. Conclusions The widespread chained structure of gene enhancers in humans reveals that the primary, critical enhancer is distal, commonly located further away than other enhancers. This first, distal enhancer establishes contacts with multiple regulatory elements and safeguards a complex regulatory program of its target gene.

scholarly journals Foxa3 (HNF-3γ) binds to and activates the rat proglucagon gene promoter but is not essential for proglucagon gene expression

2002 ◽  
Vol 366 (2) ◽  
pp. 633-641 ◽  
Author(s):  
Yuanfang LIU ◽  
Wei SHEN ◽  
Patricia L. BRUBAKER ◽  
Klaus H. KAESTNER ◽  
Daniel J. DRUCKER
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Forkhead Box ◽  
Mild Hypoglycaemia ◽  
Element Binding Protein ◽  
Prolonged Fast

Members of the Forkhead box a (Foxa) transcription factor family are expressed in the liver, pancreatic islets and intestine and both Foxa1 and Foxa2 regulate proglucagon gene transcription. As Foxa proteins exhibit overlapping DNA-binding specificities, we examined the role of Foxa3 [hepatocyte nuclear factor (HNF)-3γ] in control of proglucagon gene expression. Foxa3 was detected by reverse transcriptase PCR in glucagon-producing cell lines and binds to the rat proglucagon gene G2 promoter element in GLUTag enteroendocrine cells. Although Foxa3 increased rat proglucagon promoter activity in BHK fibroblasts, augmentation of Foxa3 expression did not increase proglucagon promoter activity in GLUTag cells. Furthermore, adenoviral Foxa3 expression did not affect endogenous proglucagon gene expression in islet or intestinal endocrine cell lines. Although Foxa3-/- mice exhibit mild hypoglycaemia during a prolonged fast, the levels of proglucagon-derived peptides and proglucagon mRNA transcripts were comparable in tissues from wild-type and Foxa3-/- mice. These findings identify Foxa3 as a member of the proglucagon gene G2 element binding-protein family that, unlike Foxa1, is not essential for control of islet or intestinal proglucagon gene expression in vivo.

Role of FOXA and Sp1 in mitochondrial acylcarnitine carrier gene expression in different cell lines

2011 ◽  
Vol 404 (1) ◽  
pp. 376-381 ◽  
Author(s):  
Paolo Convertini ◽  
Vittoria Infantino ◽  
Faustino Bisaccia ◽  
Ferdinando Palmieri ◽  
Vito Iacobazzi
Keyword(s):  
Gene Expression ◽  
Cell Lines

scholarly journals Feedback inhibition of immunoglobulin gene rearrangement by membrane mu, but not by secreted mu heavy chains.

1988 ◽  
Vol 168 (4) ◽  
pp. 1363-1381 ◽  
Author(s):  
J Manz ◽  
K Denis ◽  
O Witte ◽  
R Brinster ◽  
U Storb
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Lines ◽  
Gene Rearrangement ◽  
Feedback Inhibition ◽  
Allelic Exclusion ◽  
Immunoglobulin Gene ◽  
Heavy Chains

Previous work (6-10) has shown that allelic exclusion of Ig gene expression is controlled by functionally rearranged mu and kappa genes. This report deals with the comparison of membrane mu (micron) and secreted mu (microsecond) in promoting such feedback inhibition. Splenic B cell hybridomas were analyzed from transgenic mice harboring a rearranged kappa gene alone or in combination with either an intact rearranged mu gene or a truncated version of the mu gene. The intact mu gene is capable of producing both membrane and secreted forms of the protein, while the truncated version can only encode the secreted form. The role of the microsecond was also tested in pre-B cell lines. Analysis of the extent of endogenous Ig gene rearrangement revealed that (a) the production of micron together with kappa can terminate Ig gene rearrangement; (b) microsecond with kappa does not have this feedback effect; (c) microsecond may interfere with the effect of micron and kappa; and (d) the feedback shown here probably represents a complete shutoff of the specific recombinase by micron + kappa; the data do not address the question of mu alone affecting the accessibility of H genes for rearrangement.

scholarly journals Dual Role for Transforming Growth Factor β-Dependent Signaling in Trypanosoma cruzi Infection of Mammalian Cells

2000 ◽  
Vol 68 (4) ◽  
pp. 2077-2081 ◽  
Author(s):  
Belinda S. Hall ◽  
Miercio A. Pereira
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Growth Arrest ◽  
Transforming Growth Factor Β

ABSTRACT Expression of functional transforming growth factor β (TGF-β) receptors (TβR) is required for the invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi. However, the precise role of this host cell signaling complex in T. cruzi infection is unknown. To investigate the role of the TGF-β signaling pathway, infection levels were studied in the mink lung epithelial cell lines JD1, JM2, and JM3. These cells express inducible mutant TβR1 proteins that cannot induce growth arrest in response to TGF-β but still transmit the signal for TGF-β-dependent gene expression. In the absence of mutant receptor expression, trypomastigotes invaded the cells at a low level. Induction of the mutant receptors caused an increase in infection in all three cell lines, showing that the requirement for TGF-β signaling at invasion can be divorced from TGF-β-induced growth arrest. TGF-β pretreatment of mink lung cells expressing wild-type TβR1 caused a marked enhancement of infection, but no enhancement was seen in JD1, JM2, and JM3 cells, showing that the ability of TGF-β to stimulate infection is associated with growth arrest. Likewise, expression of SMAD7 or SMAD2SA, inhibitors of TGF-β signaling, did not block infection by T. cruzi but did block the enhancement of infection by TGF-β. Taken together, these results show that there is a dual role for TGF-β signaling in T. cruzi infection. The initial invasion of the host cell is independent of both TGF-β-dependent gene expression and growth arrest, but TGF-β stimulation of infection requires a fully functional TGF-β signaling pathway.

Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2390-2390
Author(s):  
Yanjuan He ◽  
Joan Cain ◽  
Lee Ratner ◽  
Leon Bernal-Mizrachi
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Fold Increase ◽  
Daudi Cell ◽  
Xiap Expression ◽  
Apoptotic Effect ◽  
Effective Doses ◽  
Induction Of Apoptosis ◽  
Apoptotic Pathways

Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99

Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3526-3526
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Sophia Adamia ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Genomic Alteration ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Cell Growth And Survival

Abstract Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

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