A Targeted Resequencing Study Allows the Detection of a Common Polymorphism At Mirna-223 Binding Site in 3′Untranslated Region Which Deregulates HSP90B1 Expression in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1776-1776
Author(s):  
Ana E Rodríguez ◽  
Dalia Qwaider ◽  
Rocío Benito ◽  
Irena Misiewicz-Krzeminska ◽  
María Hernández-Sánchez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cell Line ◽  
B Lymphocytes ◽  
Prognostic Significance ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Wild Type ◽  
Single Nucleotide ◽  
Custom Made

Abstract Abstract 1776 Array-based sequence capture (Roche NimbleGen) followed by next-generation sequencing (Roche GS FLX Titanium sequencing platform) was used to analyze genetic variations in 93 genes relevant in CLL and two chromosomal regions: 13q14.3 and 17p13.1. CD19+ cells from 4 patients with CLL and 4 patients with other hematological malignancies (used as controls) were studied. A custom-made data analysis pipeline was used to annotate detected variants, including known single-nucleotide polymorphisms (SNPs), amino acid consequences, genomic location and miRNA binding sites. The enrichment assay followed by NGS allowed the detection of over 1600 variations/sample (median 1721, range 1618–1823). All putative variants were first compared with published single nucleotide polymorphism (SNP) data (dbSNP build 130) and most of the variants detected were identified as known SNPs. Overall, 10% of variants detected in each sample were variations not previously described. Interestingly, a 4bp insertion/deletion polymorphism (rs2307842) in the 3′UTR of HSP90B1, target site for miR-223, was detected. There is an increasing evidence suggesting that SNPs in the 3′UTR targeted by miRNAs (known as miRSNPs) are associated with diseases by affecting gene expression. We hypothesized that this ‘GACT’ deletion disrupts the binding site for miR-223 thereby increasing the translation of HSP90B1 and we confirmed that miR-223 regulates HSP90B1 expression by 3′UTR reporter assays. The relative luciferase activity of the construct with wild-type 3′UTR (WT-3′UTR) was significantly repressed by 31% following miR-223 transfection (p<0.05). However, the presence of rs2307842 polymorphism in 3′UTR of HSP90B1 (VAR-3′UTR) abolished this suppression, suggesting that miR-223 directly binds to this site. We also validated HSP90B1 as a target gene of miR223 by transfecting MM1S and H929 cell lines with miR-223/NC mimics and then measuring HSP90B1 expression by semi-quantitative PCR and Western blot. Exogenous expression of miR-223 downregulated the expression levels of HSP90B1 in H929 cell line (WT-3′UTR) in both mRNA (p<0.05) and protein levels. By contrast, HSP90B1 expression was not modified in MM1S cell line (VAR-3′UTR). To evaluate the clinical impact of HSP90B1 3′UTR polymorphism, we expanded the study to 109 additional patients with CLL and 32 healthy controls. Sequencing of the HSP90B1 3′UTR region was performed by pyrosequencing (PyroMark Q24 system, Qiagen). The rs2307842 was detected in 27/109 (25%) patients and 8/32 (25%) healthy controls, as expected. Overall, we did not find any significant relationship between rs2307842 and clinical characteristics of CLL patients. To gain insight into its influence on gene expression, we measured HSP90B1 mRNA levels in paired samples (tumoral and normal) from CLL patients with rs2307842 (VAR-CLLs, n=6) and wild-type (WT-CLLs, n=12). PCR results showed that B lymphocytes (tumoral fraction) from VAR-CLLs have a higher expression of HSP90B1 than B lymphocytes from WT-CLLs (P=0.002) and also from the normal cells of the same patients (VAR-CLLs) (P=0.011). However, in WT-CLLs, no changes in mRNA expression were observed between tumor and normal fractions, being HSP90B1 mRNA levels similar to the normal fraction of VAR-CLLs. Thus, rs2307842 determined HSP90B1 overexpression only in the tumor fraction of the CLL patients with the polymorphism. Downregulation of miR-223 has prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients, and no target has been proposed or validated for miR-223 in CLL until date. Thus, this work provides novel information about how the downregulation of miR-223 can be determining the poor outcome of CLL patients, maybe through upregulation of HSP90B1 expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Significant Associations of lncRNA H19 Genotypes with Susceptibility to Childhood Leukemia in Taiwan

2021 ◽  
Vol 14 (3) ◽  
pp. 235
Author(s):  
Jen-Sheng Pei ◽  
Chao-Chun Chen ◽  
Wen-Shin Chang ◽  
Yun-Chi Wang ◽  
Jaw-Chyun Chen ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Childhood Leukemia ◽  
Healthy Controls ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Genotypic Distribution ◽  
Heterozygous Variant ◽  
Significant Difference ◽  
The Difference

The purpose of our study was to investigate whether genetic variations in lncRNA H19 were associated with susceptibility to childhood leukemia. Two hundred and sixty-six childhood leukemia patients and 266 healthy controls were enrolled in Taiwan, and two single nucleotide polymorphisms (SNPs), rs2839698 and rs217727, in H19 were genotyped and analyzed. There was a significant difference in the genotypic distribution of rs2839698 between patients and healthy controls (p = 0.0277). Compared to the wild-type CC genotype, the heterozygous variant CT and homozygous variant TT genotypes were associated with significantly increased risks of childhood leukemia with an adjusted odd ratio (OR) of 1.46 (95% confidence interval (CI), 1.08–2.14, p = 0.0429) and 1.94 (95%CI, 1.15–3.31, p = 0.0169), respectively (pfor tread = 0.0277). The difference in allelic frequencies between childhood leukemia patients and controls was also significant (T versus C, adjusted OR = 1.53, 95%CI, 1.13–1.79, p = 0.0077). There were no significant differences in the genotypic and allelic distributions of rs217727 between cases and controls. Interestingly, the average level of H19 rs2839698 was statistically significantly higher for patients with CT and TT genotypes than from those with the CC genotype (p < 0.0001). Our results indicate that H19 SNP rs2839698, but not rs217727, may serve as a novel susceptibility marker for childhood leukemia.

DDR1 methylation is associated with bipolar disorder and the isoform expression and methylation of myelin genes

Epigenomics ◽  
2021 ◽  
Author(s):  
Beatriz Garcia-Ruiz ◽  
Manuel Castro de Moura ◽  
Gerard Muntané ◽  
Lourdes Martorell ◽  
Elena Bosch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bipolar Disorder ◽  
Mrna Expression ◽  
Gene Expression Analysis ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Genome Wide ◽  
Isoform Expression

Aim: To investigate DDR1 methylation in the brains of bipolar disorder (BD) patients and its association with DDR1 mRNA levels and comethylation with myelin genes. Materials & methods: Genome-wide profiling of DNA methylation (Infinium MethylationEPIC BeadChip) corrected for glial composition and DDR1 gene expression analysis in the occipital cortices of individuals with BD (n = 15) and healthy controls (n = 15) were conducted. Results: DDR1 5-methylcytosine levels were increased and directly associated with DDR1b mRNA expression in the brains of BD patients. We also observed that DDR1 was comethylated with a group of myelin genes. Conclusion: DDR1 is hypermethylated in BD brain tissue and is associated with isoform expression. Additionally, DDR1 comethylation with myelin genes supports the role of this receptor in myelination.

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

scholarly journals Association between matrix Gla protein and ulcerative colitis according to DNA microarray data

2019 ◽  
Vol 8 (1) ◽  
pp. 66-75 ◽  
Author(s):  
Xu-Yang Dong ◽  
Mei-Xu Wu ◽  
Hui-Min Zhang ◽  
Hong Lyu ◽  
Jia-Ming Qian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ulcerative Colitis ◽  
Binding Site ◽  
Dna Microarray ◽  
Bioinformatics Analysis ◽  
Matrix Gla Protein ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Healthy Controls ◽  
Microarray Profiling

Abstract Background Matrix Gla protein (MGP) is a secreted protein contributed to the immunomodulatory functions of mesenchymal stromal cells. Microarray profiling found a significantly higher expression level of the extracellular matrix gene MGP in patients with ulcerative colitis (UC). However, little is known about the role of MGP in UC and its upstream signaling regulation. This study aimed to identify the expression of MGP in UC and its upstream regulator mechanism. Methods Colonic mucosa biopsies were obtained from patients with UC and healthy controls. DNA microarray profiling was used to explore underlying genes correlating with UC development. Mice were fed with water containing different concentrations of dextran sodium sulfate (DSS) to induce an experimental colitis model. Colonic tissues were collected and evaluated using immunohistochemistry, immunoblot, real-time polymerase chain reaction, and chromatin immunoprecipitation assay. Bioinformatics analysis was performed to identify candidate MGP gene-promoter sequence and transcription-initiation sites. Luciferase-reporter gene assay was conducted to examine the potential transcription factor of MGP gene expression. Results The expression of MGP was significantly increased in colonic tissues from UC patients and DSS-induced colitis models, and was positively correlated with disease severity. Bioinformatics analysis showed a conserved binding site for Egr-1 in the upstream region of human MGP gene. The significantly higher level of Egr-1 gene expression was found in UC patients than in healthy controls. The activity of luciferase was significantly enhanced in the Egr-1 expression plasmid co-transfected group than in the control group and was further inhibited when co-transfected with the Egr-1 binding-site mutated MGP promoter. Conclusions Up-regulated expression of MGP was found in UC patients and DSS-induced colitis. The expression of MGP can be regulated by Egr-1.

Effect of LCR 5′HS4 and 5′HS1 Deletions on β-Like Globin Gene Expression in β-Yac Transgenic Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1209-1209
Author(s):  
Susanna Harju ◽  
Halyna Fedosyuk ◽  
Kenneth R. Peterson
Keyword(s):  
Gene Expression ◽  
Homologous Recombination ◽  
Globin Gene ◽  
Developmental Expression ◽  
Mrna Levels ◽  
Wild Type ◽  
Rnase Protection ◽  
Transgenic Lines ◽  
Definitive Erythropoiesis ◽  
Globin Gene Expression

Abstract A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently. To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.

Gremlin-1 Is Overexpressed in Endothelial Cells of Patients with Loeys-Dietz Syndrome Due to Dysregulation of TGF-β Signalling,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3269-3269
Author(s):  
Jasmin Wellbrock ◽  
Sara Sheikhzadeh ◽  
Veronika Bonk ◽  
Leticia Oliveira-Ferrer ◽  
Kristin Klaetschke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
T Test ◽  
Aortic Aneurysms ◽  
Angiogenic Factor ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Ehlers Danlos Syndrome ◽  
Bone Morphogenic Proteins ◽  
Gremlin 1

Abstract Abstract 3269 The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder with symptoms similar to those of Marfan syndrome and the vascular type of Ehlers-Danlos syndrome. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. Patients with LDS harbour a mutation in the transforming growth factor β (TGF-β) receptors TGFBR1 (also named ALK-5) or TGFBR2. Since the TGF-β pathway plays a crucial role in many cellular processes including angiogenesis, we focussed our analyses on endothelial cell dysfunction in patients with Loeys-Dietz syndrome. We isolated circulating outgrowth endothelial cells (OEC) from the peripheral blood of two LDS patients (one female, 54 years; one male, 26 years old) both harbouring a mutation in the TGFBR2 gene. Gene expression profiles of OEC clones were performed using Affymetrix Human Genome U133 Plus 2.0 Arrays and confirmed by quantitative PCR analysis for genes of interest. OEC clones isolated from age- and sex-matched healthy controls served as reference subjects. We demonstrate that several genes belonging to the TGF-β pathway had altered expression in OECs isolated from LDS patients compared to those from healthy controls. For example, mRNA levels of bone morphogenic proteins (BMP) 2 and 4 were decreased in both LDS OEC clones (mean decrease 4 and 6 fold, respectively) whereas gene expression of inhibitory downstream molecule SMAD-6 was increased 2-fold. In both analysed OEC clones from LDS patients, gene expression of BMP antagonist Gremlin-1 (also known as Drm) showed the most prominent dysregulation with a 1136-fold and 164-fold higher expression in LDS OECs compared to healthy controls, respectively. Interestingly, in OECs isolated from healthy donors, Gremlin-1 expression was significantly down-regulated after incubation with SB431542 (5 μM), a small molecule inhibitor of the TGF-β receptor complex (mean decrease 4 fold; t-test: p = 0.002; n = 6). In contrast, the stimulation of OEC clones with TGF-β1 (1 ng/ml) resulted in significant up-regulation of Gremlin-1 mRNA levels (mean increase 7 fold; t-test: p = 0.014; n = 6). Apparently, the up-regulation of Gremlin-1 in LDS OECs seems to mirror an activated TGF-β signalling cascade in outgrowth endothelial cells. These findings are in line with other studies published on LDS where hyperactivity of the TGF-β downstream signalling was demonstrated by higher phosphorylation levels of SMAD-2 in the aortic media of LDS patients (Loeys et al., Nat Genet. 2005 Mar;37(3):275–81). Gremlin-1 might represent a second gene supporting the concept of increased TGF-β signalling in Loeys-Dietz syndrome. Gremlin-1 itself displays opposing effects on angiogenesis. First, it is known as a pro-angiogenic factor and was recently shown to stimulate angiogenesis via direct binding to the VEGF receptor 2 (Mitola et al., Blood. 2010 Nov 4;116(18):3677–80). On the other hand, as antagonist of bone morphogenic proteins, Gremlin-1 possesses anti-angiogenic properties by suppressing pro-angiogenic effects of BMP-2 and BMP-4. In summary, we believe that due to its drastic up-regulation in OECs of LDS patients, Gremlin-1 represents a crucial effector of dysregulated TGF-β signalling in endothelial cells inducing vascular pathology in Loeys-Dietz syndrome. Disclosures: Fiedler: Pfizer: Research Funding.

BCL6 Overexpression – Regulated By AhR/ARNT and Wild-Type MEF2B – Drives Expression of Germinal Center Markers MYBL1 and LMO2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 865-865
Author(s):  
Hilmar Quentmeier ◽  
Jie Ding ◽  
Willy Dirks ◽  
Stefan Ehrentraut ◽  
Robert Geffers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Differentially Expressed Genes ◽  
Germinal Center ◽  
Copy Number ◽  
Specific Gene ◽  
Wild Type ◽  
Expression Levels ◽  
Numerical Aberrations ◽  
Bcl6 Expression

Abstract The evolution of tumor clones and the clonal architecture of tumors can be followed by the analysis of clone-specific mutations. The diffuse large B-cell lymphoma (DLBCL)-derived cell line U-2932 comprises two subclones (R1 and R2). Immunoglobulin gene hypermutation analysis showed that R1 and R2 represent subclones of the original tumor. Thus, the two U-2932 subclones seemed to be ideal to study the cellular consequences of clonal evolution. Both clones were derived from a presumptive mother clone with genomic BCL2 amplification, which acquired distinct sets of secondary rearrangements leading alternatively to the overexpression of BCL6 (R1) and MYC (R2) in the respective daughter clones. R2 carries t(8;14) a classical activating rearrangement of MYC in B-cells. R1 did not show any of the typical BCL6 translocations responsible for aberrant BCL6 expression. We applied a whole genome array to find out whether numerical aberrations might explain BCL6 expression in R1 and to investigate if subclone-specific gene expression might be attributable to numerical aberrations in general. More than 200 genes showed >10 fold expression differences between R1 and R2. Statistical analysis of results from copy number aberration and expression data analysis revealed that for 58/221 of the differentially expressed genes, numerical differences between the two subclones effectively predict differences in gene expression (sensitivity 0.64; specificity 0.94; accuracy 0.78). Thus, for a sizeable minority of genes numerical aberrations provided an explanation for the differences in gene expression between the U-2932 subclones. However, BCL6 was none of these genes. Thus, we searched for an alternative explanation for the R1-restricted overexpression of this germinal center oncogene. MEF2B point mutations occur in 11% of DLBCL contributing to the genesis of BCL6 positive lymphomas. The U-2932 subclones did not carry MEF2B mutations. Interestingly however, expression levels of MEF2B paralleled those of BCL6 in the U-2932 subclones. Knockdown experiments showed that wild-type MEF2B controlled BCL6 transcription. To test whether MEF2B and BCL6 showed coordinated expression in general, we analyzed the expression and the mutational status of these genes in 23 DLBCL cell lines. Confirming a positive correlation, independence of MEF2B and BCL6 expression levels could be rejected with a p-value according to Fisher´s exact test of 0.0001 against a level of significance of 0.05 (sensitivity 0.92, specificity 0.9, accuracy 0.91). The MEF2B promoter carries binding sites of the AhR/ARNT transcriptional complex. AhR inhibition and ARNT knockdown experiments with the U-2932 subclones revealed that MEF2B is a downstream target of AhR/ARNT signalling. A positive correlation between AhR and MEF2B expression levels could be shown for the majority of 23 DLBCL cell lines tested (sensitivity 0.61, specificity 1.0, accuracy 0.78). These results indicate that the AhR/ARNT-induced expression of wild-type MEF2B might be an independent regulator for BCL6 expression in DLBCL, besides canonical BCL6 translocations, BCL6 promoter hypermutation and MEF2B mutations. To find out the extent to which BCL6 contributed to the subclone-specific gene expression in the U-2932 subclones, we ectopically expressed BCL6 in subclone R2: 48/221 differentially expressed genes were affected. Interestingly, 28/48 genes were upregulated by BCL6 inducing the germinal center markers MYBL1 and LMO2, although BCL6 is believed to act as a transcriptional repressor. In summary, the two subclones of the DLBCL cell line U-2932 faithfully model tumor heterogeneity. Significant expression differences were shown for 221 genes, more than half of which were attributable to genomic copy number differences or to clone-specific expression of the signal transducer AhR and the oncogene BCL6. Moreover, we could show that BCL6 overexpression – regulated by AhR/ARNT and wild-type MEF2B – drives expression of subclone-specific germinal center markers MYBL1 and LMO2 in the DLBCL cell line U-2932. Disclosures No relevant conflicts of interest to declare.

scholarly journals Heat shock of human synovial and dermal fibroblasts induces delayed up-regulation of collagenase-gene expression

1995 ◽  
Vol 308 (3) ◽  
pp. 743-747 ◽  
Author(s):  
E G Hitraya ◽  
J Varga ◽  
S A Jimenez
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Human Fibroblasts ◽  
Dermal Fibroblasts ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Mobility Shift ◽  
Collagenase Gene

We investigated the effect of heat shock on the expression of the collagenase gene in normal human synovial and dermal fibroblasts. Heat shock (42-44 degrees C for 1 h) caused a marked increase in heat-shock protein 70 (HSP-70) mRNA levels, followed by a delayed increase in collagenase mRNA levels, in both cell types. Pretreatment with cycloheximide had no effect on the heat-shock-induced increase in HSP-70 mRNA expression, but abrogated the induction of collagenase mRNA during the recovery. To study the mechanisms of collagenase-gene induction by heat shock, the transcriptional activity of a collagenase-promoter-driven chloramphenicol acetyltransferase (CAT) reporter gene was examined in transient transfection experiments. Heat shock was followed by a > 2-fold increase in CAT activity driven by a 3.8 kb fragment of the collagenase promoter, or by a construct containing an AP-1 binding site. A mutation in the AP-1 binding site abolished the effect of heat shock. Electrophoretic-mobility-shift assays revealed a marked increase in DNA-binding activity specific for the AP-1 binding site in nuclear extracts prepared from synovial fibroblasts recovering from heat shock. These results indicate that heat shock causes a delayed increase in collagenase-gene expression in human fibroblasts, and suggests that this stimulation involves, at least in part, transcriptional activation through an AP-1 binding site. Heat shock appears to initiate a programme of cellular events resulting in collagenase-gene expression, and therefore may contribute to connective-tissue degradation in disease states.

The role of a single nucleotide polymorphism of MDM2 in glioblastoma multiforme

2008 ◽  
Vol 109 (5) ◽  
pp. 842-848 ◽  
Author(s):  
Rina G. Khatri ◽  
Kapila Navaratne ◽  
Robert J. Weil
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Glioblastoma Multiforme ◽  
Age Of Onset ◽  
Polymerase Chain Reaction Analysis ◽  
Genetic Alterations ◽  
Primary Brain Tumor ◽  
Healthy Controls ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

Object Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a 5-year survival rate of < 5%. Aberrant function of TP53 is common in GBM. Although mutational inactivation of p53 is found in many cases, there remain tumors in which genetic alterations of p53 are absent. Negative regulators of the TP53 pathway such as MDM2, which directly inhibits TP53 expression and activity, may influence the pathogenesis of GBM. To understand its potential function in gliomagenesis, the authors analyzed a novel single nucleotide polymorphism (SNP) in the MDM2 promoter that enhances MDM2 expression. Methods The investigators isolated DNA from 98 patients with GBM and 102 healthy, cancer-free controls. A polymerase chain reaction analysis was performed to determine the MDM2 SNP309 genotype by using distinct primer pairs for the wild-type (T) and mutant (G) alleles. Results The frequency of the mutant MDM2 polymorphism was found to be higher (p = 0.0092) in patients with GBM (54.6%) compared with healthy controls (41.2%); the TT and GG genotypes were more common in healthy controls and patients with GBM (p = 0.0004 and p = 0.02, respectively). Although there was no association between the MDM2 SNP309 and overall survival, the GG genotype was associated with development of GBM at a younger age in patients with tumors harboring wild-type p53, which may mitigate the effect of the MDM2 SNP. Conclusions Although the MDM2 SNP309 does not portend decreased survival, the increased incidence of the mutant G allele in patients with GBM and its influence on age of onset suggest a potential role in the molecular pathogenesis of GBM, and may be a therapeutic target.

scholarly journals Accelerated Prion Replication in, but Prolonged Survival Times of, Prion-Infected CXCR3−/− Mice

2008 ◽  
Vol 82 (24) ◽  
pp. 12464-12471 ◽  
Author(s):  
Constanze Riemer ◽  
Julia Schultz ◽  
Michael Burwinkel ◽  
Anja Schwarz ◽  
Simon W. F. Mok ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prion Diseases ◽  
Proteinase K ◽  
Prolonged Survival ◽  
Disease Development ◽  
Mrna Levels ◽  
Wild Type ◽  
Survival Times ◽  
Cytokines And Chemokines ◽  
Asymptomatic Stage

ABSTRACT Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3−/−) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3−/− mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrPSc and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3−/− compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrPSc in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrPSc.

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