scholarly journals Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2678
Author(s):  
Judit Vágó ◽  
Katalin Kiss ◽  
Edina Karanyicz ◽  
Roland Takács ◽  
Csaba Matta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
In Situ Hybridization ◽  
Marker Genes ◽  
Specific Gene ◽  
Cartilage Formation ◽  
Methylation Inhibitor ◽  
Dna Methylation Inhibitor

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

scholarly journals Modulation of DNA Methylation Influences Cartilage Formation in Murine Chondrogenic Models

2021 ◽  
Author(s):  
Judit Vágó ◽  
Katalin Kiss ◽  
Edina Karanyicz ◽  
Roland Takács ◽  
Csaba Matta ◽  
...  
Keyword(s):  
Dna Methylation ◽  
In Situ Hybridization ◽  
Dna Methyltransferase ◽  
Mouse Cell ◽  
Dna Demethylation ◽  
Specific Gene ◽  
Cartilage Formation

The aim of this study was to investigate the role of DNA methylation in the regulation of in vitro and in vivo cartilage formation. Based on the data of an RNA chip-assay performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expression of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3) and Ogt (O-linked N-acetylglucosamine transferase) genes was examined with RT-qPCR in mouse cell-line based and primary micromass cultures. RNA probes for in situ hybridization were used on frozen sections of 15-day-old mouse embryos. DNA methylation was inhibited with 5-azacytidine during culturing. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. Inhibition of DNA methylation applied during early stages of differentiation reduced cartilage-specific gene expression and cartilage formation. In contrast, it had stimulatory effect when added to differentiated chondrocytes. Our results indicate that the DNA demethylation-inducing Tet1 is a significant epigenetic factor of chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

scholarly journals Gene expression analysis by non-radioactive RNA-RNA in situ hybridization techniques

2004 ◽  
Vol 23 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Danijela Drakulic ◽  
Milena Stevanovic ◽  
Gordana Nikcevic
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cultured Cells ◽  
Specific Gene ◽  
Rna In Situ Hybridization ◽  
Sox Gene ◽  
Labeled Rna ◽  
Set Up

RNA-RNA in situ hybridization is a reliable method for studying tissue and cell specific gene expression, which enables visualization of labeled antisense RNA probe hybridized to specific mRNA. In this study we employed non-radioactive RNA-RNA in situ hybridization using biotin- or digoxigenin-labeled RNA probes in order to detect SOX gene expression in carcinoma cell lines. By this approach we confirmed results obtained by Northern blot analysis, where the presence of SOX2 mRNA in NT2/D1 and SOX14 mRNA in HepG2 cells has been established. Our aim was to set up RNA-RNA in situ hybridization method in in vitro cultured cells in order to perform further analyses of SOX gene expression on various normal and cancer tissues.

167 EFFECT OF DNA METHYLATION INHIBITOR ON HETEROCHROMATIN IN BOVINE EMBRYOS DERIVED FROM HEAT-SHOCKED OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 213
Author(s):  
T. D. Araujo ◽  
J. Jasmin ◽  
C. C. R. Quintao ◽  
E. D. Souza ◽  
J. H. M. Viana ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Shock ◽  
In Vitro Maturation ◽  
Methylation Status ◽  
Control Groups ◽  
Methylation Inhibitor ◽  
Response To Stress ◽  
Dna Methylation Inhibitor ◽  
And Control

The cell response to stress involves epigenetic modifications in order to regulate the gene expression, which is dependent of chromatin structure and DNA methylation status. On the other hand, changes on DNA methylation can have an effect on chromatin organisation (Espada and Esteller 2010 Semin. Cell. Dev. Biol. 21, 238–246). In this study we evaluated the effect of 5-aza-2′-deoxycytidine (5-aza; Sigma, St. Louis, MO, USA), a DNA methylation inhibitor, on heterochromatin 1 β formation of bovine pre-implantation embryos derived from oocytes that did or did not undergo heat shock during in vitro maturation (IVM). Oocytes were IVM under 38.5°C for 24 h (non-heat-shock: NHS group) or under 41.5°C for 12 h followed by 38.5°C for 12 h (heat-shock: HS group). Oocytes were IVF for 20 h and the denuded presumptive zygotes from NHS or HS groups were cultured with 0 (nontreated controls) or 10 nM of 5-aza for 24 h or 48 h in CR2aa plus 2.5% FCS at 38.5°C with 5% CO2, 5% O2 and 90% N2. Embryos with 4–7 cells at 44 h post-insemination (hpi) and embryos with 8–16 cells at 68 hpi were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β first antibody, then immunofluorescence was evaluated by confocal microscopy (Leica TCS SP5II) and images were processed by ImageJ 1.49 (NIH, Bethesda, MD, USA). Fluorescence of nuclei and of background area (fluorescence/unit area) were measured and then the corrected relative fluorescence per nucleus was calculated. We analysed 129 and 149 nuclei at 44 hpi from 29 and 34 embryos as well as 268 and 182 nuclei at 68 hpi from 37 and 22 embryos of the NHS and HS groups, respectively, obtained from 3 replicates. Data underwent log-transformation and was analysed by ANOVA, and means compared by Student-Newman-Keuls. Embryos with 8–16 cells derived from NHS oocytes and treated with 5-aza for 24 h or for 48 h had nuclei with lower HP1 fluorescence than their respective NHS (nontreated) control (P < 0.01). In contrast, 8–16-cells embryos derived from HS and treated with 5-aza displayed nuclei with the same HP1 fluorescence of their respective HS control (P > 0.05). When embryos derived from HS and NHS (nontreated) control groups were compared, higher HP1 fluorescence was found in those with 4–7 cells of HS group (P < 0.05); however, embryos with 8–16 cells displayed similar HP1 fluorescence between both NHS and HS control groups (P > 0.05). There was no difference on HP1 fluorescence between nuclei of embryos with 4–7 cells treated with 5-aza for 24 h and control (nontreated) in both HS and NHS groups. These data suggest that embryos derived from heat-shocked oocytes can accumulate more heterochromatin at earlier stages than those from non-heat-shocked oocytes and that the effect of DNA methylation inhibition by 5-aza on embryo heterochromatin can vary accordingly to the exposure of the oocyte to heat shock during in vitro maturation. Financial support from CNPq, Fapemig, and CAPES is acknowledged.

scholarly journals Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine

2005 ◽  
Vol 11 (12) ◽  
pp. 4571-4579 ◽  
Author(s):  
Hideaki Dote ◽  
David Cerna ◽  
William E. Burgan ◽  
Donna J. Carter ◽  
Michael A. Cerra ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Cell ◽  
Methylation Inhibitor ◽  
Dna Methylation Inhibitor

scholarly journals Genome-wide epigenetic analysis delineates a biologically distinct immature acute leukemia with myeloid/T-lymphoid features

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2795-2804 ◽  
Author(s):  
Maria E. Figueroa ◽  
Bas J. Wouters ◽  
Lucy Skrabanek ◽  
Jacob Glass ◽  
Yushan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Cpg Islands ◽  
Specific Gene ◽  
Molecular Heterogeneity ◽  
Dna Hypermethylation ◽  
Genome Wide

Abstract Acute myeloid leukemia is a heterogeneous disease from the molecular and biologic standpoints, and even patients with a specific gene expression profile may present clinical and molecular heterogeneity. We studied the epigenetic profiles of a cohort of patients who shared a common gene expression profile but differed in that only half of them harbored mutations of the CEBPA locus, whereas the rest presented with silencing of this gene and coexpression of certain T-cell markers. DNA methylation studies revealed that these 2 groups of patients could be readily segregated in an unsupervised fashion based on their DNA methylation profiles alone. Furthermore, CEBPA silencing was associated with the presence of an aberrant DNA hypermethylation signature, which was not present in the CEBPA mutant group. This aberrant hypermethylation occurred more frequently at sites within CpG islands. CEBPA-silenced leukemias also displayed marked hypermethylation compared with normal CD34+ hematopoietic cells, whereas CEBPA mutant cases showed only mild changes in DNA methylation compared with these normal progenitors. Biologically, CEBPA-silenced leukemias presented with a decreased response to myeloid growth factors in vitro.

scholarly journals Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

1989 ◽  
Vol 37 (5) ◽  
pp. 697-701 ◽  
Author(s):  
F J Tang ◽  
P O Ts'o ◽  
S A Lesko
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Specific Gene ◽  
Whole Cells ◽  
Beta Interferon ◽  
Ht1080 Cells ◽  
In Situ Detection

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

Gene expression profiling of microdissected Hodgkin Reed-Sternberg cells correlates with treatment outcome in classical Hodgkin lymphoma

Blood ◽  
2012 ◽  
Vol 120 (17) ◽  
pp. 3530-3540 ◽  
Author(s):  
Christian Steidl ◽  
Arjan Diepstra ◽  
Tang Lee ◽  
Fong Chun Chan ◽  
Pedro Farinha ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Treatment Failure ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Specific Gene ◽  
Classical Hodgkin Lymphoma

Abstract In classical Hodgkin lymphoma (CHL), 20%-30% of patients experience relapse or progressive disease after initial treatment. The pathogenesis and biology of treatment failure are still poorly understood, in part because the molecular phenotype of the rare malignant Hodgkin Reed-Sternberg (HRS) cells is difficult to study. Here we examined microdissected HRS cells from 29 CHL patients and 5 CHL-derived cell lines by gene expression profiling. We found significant overlap of HL-specific gene expression in primary HRS cells and HL cell lines, but also differences, including surface receptor signaling pathways. Using integrative analysis tools, we identified target genes with expression levels that significantly correlated with genomic copy-number changes in primary HRS cells. Furthermore, we found a macrophage-like signature in HRS cells that significantly correlated with treatment failure. CSF1R is a representative of this signature, and its expression was significantly associated with progression-free and overall survival in an independent set of 132 patients assessed by mRNA in situ hybridization. A combined score of CSF1R in situ hybridization and CD68 immunohistochemistry was an independent predictor for progression-free survival in multivariate analysis. In summary, our data reveal novel insights into the pathobiology of treatment failure and suggest CSF1R as a drug target of at-risk CHL.

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