Identification of a CpG Island in the Human LRP-2 Gene and Analysis of Its Methylation Status in Parathyroid Adenomas

2000 ◽  
Vol 381 (5-6) ◽  
pp. 433-438 ◽  
Author(s):  
A. Knutson ◽  
P. Lillhager ◽  
G. Westin
Keyword(s):  
Cell Line ◽  
Transcriptional Repression ◽  
Cpg Island ◽  
Methylation Status ◽  
Parathyroid Tissue ◽  
Cellular Functions ◽  
Gene Encoding ◽  
Parathyroid Adenomas ◽  
Multiple Ligands

Abstract Methylation of CpG residues in mammalian genomes is a mechanism of vital importance for many cellular functions, which all relate to gene expression. In this study we describe the identification of a CpG island in the 5′-region of the gene encoding human megalin/LRP-2, a receptor capable of binding multiple ligands, which is involved in the regulation of calcium metabolism. Southern blot analysis and genomic bisulfite sequencing revealed that the CpG island is methylated in a non-expressing cell line, largely unmethylated in an expressing cell line and unmethylated in human parathyroid tissue. In addition, we show that artificial methylation of LRP-2 promoter reporter plasmids leads to strong transcriptional repression, in vitro as well as in transfected cells. No evidence for aberrant LRP-2 gene methylation in parathyroid adenomas, in which the LRP-2 protein is generally down-regulated, was found.

hSRBC promoter CpG island hypermethylation as resistant predictive biomarker of oxaliplatin based chemotherapy in metastasic colorectal cancer patients.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e14609-e14609
Author(s):  
Catia Moutinho ◽  
Anna Martinez-Cardus ◽  
Cristina Santos ◽  
Valentín Navarro-Perez ◽  
Eva Martinez-Balibrea ◽  
...  
Keyword(s):  
Cox Regression ◽  
Cpg Island ◽  
Methylation Status ◽  
Stage Iv ◽  
Predictive Biomarker ◽  
Promoter Hypermethylation ◽  
Chemotherapeutic Agents ◽  
P Value ◽  
Potential Biomarker

e14609 Background: Resistance acquisition to chemotherapeutic agents is one of the main problems that come up during cancer treatment. hSRBC is a tumor suppressor gene whose inactivation has been associated with malignant tumor progression. In a previous work, we investigated the influence of hSRBC promoter methylation alterations in oxaliplatin (OXA) resistance acquisition by using a CRC “in vitro” model, detecting an hSRBC promoter hypermethylation in OXA–resistant derived cells when compared with the sensitive counterpart. These results were validated by functional analyses in the same model. Taking this into account, our aim in the present work is to determine the role of hSRBC methylation status as a potential biomarker of OXA resistance, in metastatic CRC patients, treated at first line with fluouropirimidines plus OXA based chemotherapy. Methods: hSRBC promoter hypermethylation was analyzed in DNA extracted from paraffin embebbed tissue of 111 metastatic CRC tumors by using Methylation Specific PCR. Methylation data was correlated to overall response (OR) and progression free survival (PFS) by using F-Fisher test and Kaplan-Meyer Survival curves respectively. A multivariate analysis was carried out by Cox regression. p-values under 0.05 were considered statistic significant. Results: Two independent cohorts of stage IV CRC tumors were included. Twenty-two out of 111 patients received radical surgery for metastasis, that became to be a positive prognostic factor (p-value = 0.04). Gene hypermethylation was detected in a 33% of cases. Although OR was not associated with hSRBC methylation, we observed a significant correlation between hypermethylation of gene and a worse PFS in patients without metastasis surgery (Log Rank; p-value = 0.04). Conclusions: Remarkably, hSRBC promoter hypermethylation is associated with worse PFS in metastatic CRC patients. We suggest hSRBC methylation status as a predictive biomarker of OXA-based treatment outcome in metastatic CRC patients. However, further studies are warranted in order to elucidate the clinical application of these findings.

scholarly journals p38β - MAPK11 and its role in female cancers

2020 ◽  
Author(s):  
Periklis Katopodis ◽  
Rachel Kerslake ◽  
Athanasios Zikopoulos ◽  
Nefeli Eirini Beri ◽  
Vladimir Anikin
Keyword(s):  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Her2 Status ◽  
Tissue Specific ◽  
Cancer Data ◽  
Signalling Molecules ◽  
Significant Difference

Abstract Background The p38MAPK family of Mitogen Activated Protein Kinases are a group of signalling molecules involved in cell growth, survival, proliferation and differentiation. The widely studied p38α isoform is ubiquitously expressed and is implicated in a number of cancer pathologies, as are p38γ and p38δ. However, the mechanistic role of the isoform, p38β, remains fairly elusive. Recent studies suggest a possible role of p38β in both breast and endometrial cancer with research suggesting involvement in bone metastasis and cancer cell survival. Female tissue specific cancers such as breast, endometrial, uterine and ovary account for over 3,000,000 cancer related incidents annually; advancements in therapeutics and treatment however require a deeper understanding of the molecular aetiology associated with these diseases. This study provides an overview of the MAPK signalling molecule p38β (MAPK11) in female cancers using an in-silico approach. Methods A detailed gene expression and methylation analysis was performed using datasets from cBioportal, CanSar and MEXPRESS. Breast, Uterine Endometrial, Cervical, Ovarian and Uterine Carcinosarcoma TCGA cancer datasets were used and analysed.Results Data using cBioportal and CanSAR suggest that expression of p38β is lower in cancers: BRCA, UCEC, UCS, CESC and OV compared to normal tissue. Methylation data from SMART and MEXPRESS indicate significant probe level variation of CpG island methylation status of the gene MAPK11. Analysis of the genes’ two CpG islands shows that the gene was hypermethylated in the CpG1 with increased methylation seen in BRCA, CESC and UCEC cancer data sets with a slight increase of expression recorded in cancer samples. CpG2 exhibited hypomethylation with no significant difference between samples and high levels of expression. Further analysis from MEXPRESS revealed no significance between probe methylation and altered levels of expression. In addition, no difference in the expression of BRCA oestrogen/progesterone/HER2 status was seen. Conclusion This data provides an overview of the expression of p38β in female tissue specific cancers, showing a decrease in expression of the gene in BRCA, UCEC, CESC, UCS and OV, increasing the understanding of p38β MAPK expression and offering insight for future in-vitro investigation and therapeutic application.

Dabrafenib, a selective BRAFV600E inhibitor, is not able to reverse the CpG island methylator phenotype in RKO colorectal cell line

2021 ◽  
Author(s):  
Ioannis Tsomidis
Keyword(s):  
Colorectal Cancer ◽  
Cell Line ◽  
Cancer Cell ◽  
Cpg Island ◽  
Methylation Status ◽  
Cancer Cell Line ◽  
Developed Countries ◽  
Braf V600e ◽  
Pcr Analysis ◽  
Cpg Island Methylator Phenotype

Background: Colorectal cancer is considered as one of the most common death causes among cancer types in the developed countries. Methylation in the promoter of genes shows specific patterns, which define the molecular pathogenesis and prognosis of the cancer. Therefore, reversal of DNA methylation constitutes a potential therapeutic target. Coexistence of B-RAF V600E mutation with hypermethylation in the promoter of specific genes and chromosomal instability characterize the serrated pathway of carcinogenesis in colorectal cancer and has been associated with poor prognosis. The purpose of this study was to investigate if inhibition of BRAF V600E mutation by the selective inhibitor Dabrafenib in the RKO cell line has any effect on the methylation phenotype of the Weisenberger’s CIMP panel genes. Materials and methods: RKO cancer cell line was cultured under various conditions of Dabrafenib concentrations, time of treatment, cell passage and culture medium provision. Cells from every condition were counted and the subsequently extracted DNA was modified using sodium bisulfate. The characterization of the methylation phenotype was performed by MS-PCR analysis. Modified genomic DNA from Caco2 cancer cell line was used as a control. Results: Dabrafenib treatment resulted in a 50% inhibition of cell growth rate, independent of the concentration used and has no effect on the methylation status of the genes tested under all conditions. Conclusions: Inhibition of the B-RAFV600E by Dabrafenib was not able to reverse the CIMP phenotype in the RKO cell line.

Transcriptome Profiling of Lenalidomide Treated Myeloma Patients Identifies an Interferon Signature Gene Response and a Novel IRF4/MYC Independent Mechanism of Resistance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 170-170
Author(s):  
Paola E. Neri ◽  
Ines Tagoug ◽  
Li Ren ◽  
Ranjan Maity ◽  
Justin Simms ◽  
...  
Keyword(s):  
Cell Line ◽  
Transcriptional Repression ◽  
Sequence Data ◽  
Transcriptome Profiling ◽  
Proteasomal Degradation ◽  
Response To Therapy ◽  
Differentially Expressed ◽  
Interferon Response ◽  
Type I

Abstract Background: Immunomodulatory (IMiDs) drugs are now recognized as modifiers of the degrons targeted by the CLR4-CRBN E3 ligase. Lenalidomide binding to CRBN promotes the proteasomal degradation to the B cell specific zinc finger transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) and the transcriptional repression of IRF4 and MYC. Loss of CRBN thalidomide binding domain as well as the expression of mutants IKZF1Q146H or IKZF3Q147H confer resistance to IMiDs in vitro; however these events are rare in primary MM cells. In addition over-expression of IRF4 only partially protects MM cells from the anti-proliferative effects of IMiDs suggesting that a yet unidentified Aiolos dependent mechanism(s) regulate IMiDs sensitivity. Methods and Results: In order to identify novel mechanisms of resistance to IMiDs we profiled the transcriptome of IMiDs treated patients (sensitive and resistant), MM cell line (MM1S) exposed to lenalidomide and Aiolos silenced MM cells (OPM2). In primary samples, RNA-seq analysis was performed on paired CD138 selected cells sequentially collected from patients’ BM prior to lenalidomide treatment initiation (n=15) and at the time of acquired resistance (n=12) or ongoing response to therapy (n=3). Transcriptome sequence data was generated on the Ion Torrent Proton platform with a minimum of 70x106 reads per sample. Filtered Fastq files were mapped with the TopHat2 splice aligner against hg19. DESeq2 was used to detect differentially expressed (DE) genes. Amongst lenalidomide sensitive patients a total of 870 genes were identified as differentially expressed (FDR <0.1) between the pre- and post-lenalidomide paired samples. Functional annotation of these DE genes using DAVID revealed enrichment of genes involved in immune mediated responses (Gene-Ontology). Of interest interferon γ (IFNγ) was upregulated 3.3 fold in the post-lenalidomide sensitive cohort and 20.4% of the DE genes are type I and II interferon regulated genes (Interferome v2.01). A similar Interferon type of response was also observed in MM1S (Len-sensitive MM cell line) and post Aiolos knockdown of OPM2 cells but not in the lenalidomide resistant cohort. Notably, while several genes that are required for the induction of an interferon response (IL1α, IL1β, TBK1, NLRP3) were upregulated post-lenalidomide in the sensitive cohort, they were downregulated in resistant patients. Of particular interest, two genes that play a key role in modulating IFN response were differentially expressed in the Len resistant cohort: 1) NLRP4 a member of the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) was significantly upregulated in lenalidomide resistant patients. NLRP4 negatively regulates type I IFN signaling by targeting the kinase TBK1 for proteasomal degradation and is also recognized to suppress autophagy through Beclin1; 2) NFKBIZ (IkBζ), an atypical IkB kinase required for the induction of IFN response was significantly reduced in lenalidomide-resistant patients. Validation of this lenalidomide induced IFN response was carried out in vitro in MM cells exposed to lenalidomide 10 μM for 24 and 72 hours. A significant increase in IFN stimulated genes (ISGs) such as XAF1, DDX58, IFIT3 was observed following lenalidomide treatment. Similar changes were observed in Aiolos knockdown MM cells. Functionally, silencing of NFKBIZ (through lentiviral shRNA or transient siRNA expression) in MM1S cells resulted in 30% reduction in lenalidomide induced cell death and supressed p21 upregulation but had no effect on the downregulation Aiolos, IRF4 and MYC. Similarly stable overexpression of NLRP4 in MM cells, conferred resistance to lenalidomide. Conclusions: Through comparative transcriptome profiling of lenalidomide resistance and sensitive patients we have identified an Aiolos-dependent induction of interferon stimulated genes as a novel mechanisms of IMiDs mediated cytotoxicity and identified NLRP4 and NFKBIZ as potential mediators of IMiDs resistance. Disclosures Bahlis: Celgene: Honoraria, Research Funding.

scholarly journals Food Safety and Nutraceutical Potential of Caramel Colour Class IV Using In Vivo and In Vitro Assays

Foods ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 392 ◽  
Author(s):  
Marcos Mateo-Fernández ◽  
Pilar Alves-Martínez ◽  
Mercedes Del Río-Celestino ◽  
Rafael Font ◽  
Tania Merinas-Amo ◽  
...  
Keyword(s):  
Food Safety ◽  
Cell Line ◽  
Methylation Status ◽  
Lethal Dose ◽  
In Vitro Assays ◽  
Leukaemia Cell Line ◽  
Colour Class ◽  
Class Iv

Nutraceutical activity of food is analysed to promote the healthy characteristics of diet where additives are highly used. Caramel is one of the most worldwide consumed additives and it is produced by heating natural carbohydrates. The aim of this study was to evaluate the food safety and the possible nutraceutical potential of caramel colour class IV (CAR). For this purpose, in vivo toxicity/antitoxicity, genotoxicity/antigenotoxicity and longevity assays were performed using the Drosophila melanogaster model. In addition, cytotoxicity, internucleosomal DNA fragmentation, single cell gel electrophoresis and methylation status assays were conducted in the in vitro HL-60 human leukaemia cell line. Our results reported that CAR was neither toxic nor genotoxic and showed antigenotoxic effects in Drosophila. Furthermore, CAR induced cytotoxicity and hipomethylated sat-α repetitive element using HL-60 cell line. In conclusion, the food safety of CAR was demonstrated, since Lethal Dose 50 (LD50) was not reached in toxicity assay and any of the tested concentrations induced mutation rates higher than that of the concurrent control in D. melanogaster. On the other hand, CAR protected DNA from oxidative stress provided by hydrogen peroxide in Drosophila. Moreover, CAR showed chemopreventive activity and modified the methylation status of HL-60 cell line. Nevertheless, much more information about the mechanisms of gene therapies related to epigenetic modulation by food is necessary.

scholarly journals Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

2004 ◽  
Vol 18 (7) ◽  
pp. 1740-1755 ◽  
Author(s):  
Leo Tsz-On Lee ◽  
Kian-Cheng Tan-Un ◽  
Ronald Ting-Kai Pang ◽  
David Tai-Wai Lam ◽  
Billy Kwok-Chong Chow
Keyword(s):  
Gene Expression ◽  
Hepg2 Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Specific Pcr ◽  
E Box

Abstract To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

scholarly journals Transcriptional Repression by DrosophilaMethyl-CpG-Binding Proteins

2000 ◽  
Vol 20 (19) ◽  
pp. 7401-7409 ◽  
Author(s):  
Karim Roder ◽  
Ming-Shiu Hung ◽  
Tai-Lin Lee ◽  
Tzu-Yang Lin ◽  
Hengyi Xiao ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Binding Proteins ◽  
Genomic Structure ◽  
Chromatin Condensation ◽  
Cellular Functions ◽  
Developmentally Regulated ◽  
Cpg Dinucleotides ◽  
Alternatively Spliced ◽  
Mbd Proteins

ABSTRACT C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m5CpG. We now report the genomic structure of aDrosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Δ. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Δ could preferentially recognize m5CpG-containing DNA through its MBD. Furthermore, dMBD2/3Δ as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Δ. Finally, dMBD2/3Δ also could repress transcription effectively in transfectedDrosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.

scholarly journals Transcriptomic Changes Associated with Loss of Cell Viability Induced by Oxysterol Treatment of a Retinal Photoreceptor-Derived Cell Line: An In Vitro Model of Smith–Lemli–Opitz Syndrome

2021 ◽  
Vol 22 (5) ◽  
pp. 2339
Author(s):  
Bruce A. Pfeffer ◽  
Libin Xu ◽  
Steven J. Fliesler
Keyword(s):  
Cell Line ◽  
Molecular Mechanisms ◽  
Enrichment Analysis ◽  
Negative Control ◽  
Specific Cell ◽  
Cell Type Specificity ◽  
Gene Encoding ◽  
Dna Damage And Repair ◽  
Opitz Syndrome

Smith–Lemli–Opitz Syndrome (SLOS) results from mutations in the gene encoding the enzyme DHCR7, which catalyzes conversion of 7-dehydrocholesterol (7DHC) to cholesterol (CHOL). Rats treated with a DHCR7 inhibitor serve as a SLOS animal model, and exhibit progressive photoreceptor-specific cell death, with accumulation of 7DHC and oxidized sterols. To understand the basis of this cell type specificity, we performed transcriptomic analyses on a photoreceptor-derived cell line (661W), treating cells with two 7DHC-derived oxysterols, which accumulate in tissues and bodily fluids of SLOS patients and in the rat SLOS model, as well as with CHOL (negative control), and evaluated differentially expressed genes (DEGs) for each treatment. Gene enrichment analysis and compilation of DEG sets indicated that endoplasmic reticulum stress, oxidative stress, DNA damage and repair, and autophagy were all highly up-regulated pathways in oxysterol-treated cells. Detailed analysis indicated that the two oxysterols exert their effects via different molecular mechanisms. Changes in expression of key genes in highlighted pathways (Hmox1, Ddit3, Trib3, and Herpud1) were validated by immunofluorescence confocal microscopy. The results extend our understanding of the pathobiology of retinal degeneration and SLOS, identifying potential new druggable targets for therapeutic intervention into these and other related orphan diseases.

scholarly journals Association of allele-specific methylation of the ASNS gene with asparaginase sensitivity and prognosis in T-ALL

2021 ◽  
Author(s):  
Koshi Akahane ◽  
Shunsuke Kimura ◽  
Kunio Miyake ◽  
Atsushi Watanabe ◽  
Keiko Kagami ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Cell Lines ◽  
Cpg Island ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Aberrant Methylation ◽  
Imprinted Gene ◽  
Allele Specific ◽  
Allele Specific Methylation

Asparaginase therapy is a key component of chemotherapy for T-cell acute lymphoblastic leukemia (T-ALL) patients. Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, while leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Since the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite PCR products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from childhood T-ALL patients in Japanese cohorts (n = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In childhood T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively showed ASNS hypomethylation status. These observations demonstrate that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

scholarly journals Molecular Cloning and Biological Characterization of a Novel Murine Lymphoid Growth Factor

2000 ◽  
Vol 192 (5) ◽  
pp. 671-680 ◽  
Author(s):  
John E. Sims ◽  
Douglas E. Williams ◽  
Philip J. Morrissey ◽  
Kirsten Garka ◽  
Diane Foxworthe ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cell ◽  
Biological Characterization ◽  
Gene Encoding ◽  
Interleukin 7 ◽  
Stromal Cell Line ◽  
Thymic Stromal Cell

Using a bioassay consisting of the proliferation of a murine B cell line, a cDNA of a gene whose product supports the growth of that cell line was isolated from a thymic stromal cell line. This factor, termed thymic stromal lymphopoietin (TSLP), is a protein of 140 amino acids. The gene encoding TSLP was mapped to murine chromosome 18. Purified recombinant TSLP supported the growth of pre-B cell colonies in vitro, but had no myelopoietic activity. TSLP had comitogenic activity for fetal thymocytes, but was not as potent as interleukin 7 in lobe submersion cultures. Injection of TSLP into neonatal mice induced the expansion of B220+BP-1+ pre-B cells.

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