Browsing by Subject "DNA methylation"

Now showing 1 - 14 of 14

Open Access
Analysis of 45S rDNA promoter methylation and expression of rRNA transcripts in breast cancer
(2015-06) Karahan, Gurbet
Ribosome biogenesis has a central role in cell growth and proliferation that is usually disrupted in tumor cells by the inactivation of tumor suppressor genes and activation of oncogenes. Ribosomal RNA (rRNA) gene expression is one of the most important factors regulating ribosome production, which is controlled by CG rich 45S ribosomal DNA (rDNA) promoter. The effect of DNA methylation at 45S rDNA promoter on rRNA gene expression is a subject of controversy in the literature. In this thesis, a 434 bp region (-380 bp to +54 bp) spanning both upstream control element (UCE) and core promoter located in 45S rDNA promoter containing 54 CpGs was analysed in breast cancer. We also analysed the related rRNA expression levels in the same samples in order to clarify the role of 45S rDNA promoter methylation on rRNA gene expression. 45S rDNA promoter region was highly methylated (74%-96%) in all cell lines including non-tumorigenic breast cell line (MCF10A). Even though 45S rDNA promoter region of breast cancer cell lines are extensively methylated, rRNAs (18S, 28S, 5.8S and 45S ETS) were expressed independent of the heavy methylation. Expression levels of rRNAs are assessed either using housekeeping genes (ACTB, TBP, ACTB&TBP) or geometric mean of rRNAs (GM-rRNAs). We propose GM-rRNA normalization as a new method to identify relative expression differences between rRNA transcripts. Epigenetic drugs 5-Aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) were used to determine the effect of DNA methylation and histone acetylation on rRNA expression. Demethylation with 5-AZA resulted in an unexpected decrease in the expression of all rRNA. TSA treatment did not lead to any significant expression difference in cell lines. To better evaluate the effect of DNA methylation on the expression of rRNA transcripts we analysed the methylation status of 19 breast tumor and matched normal frozen tissue samples. The results showed that majority of the tumors (13/19) have significantly higher methylation levels than their normal pairs. Using the GM-rRNA as reference helped us to determine significant differences in the proportionate expression of rRNAs in these tissue samples. The 5.8S rRNA ratio was significantly lower whereas the 18S rRNA ratio was significantly higher in breast tumor samples. Furthermore, the 45S rDNA promoter methylation levels in normal breast tissue samples were negatively correlated with the18S rRNA ratio but this correlation was disrupted in breast tumors. Similarly, rRNA transcript levels were significantly correlated with each other in normal samples, were lost in tumor samples. It is clear that, there is a dysregulation both in rDNA methylation levels and spliced rRNA transcripts specific to breast tumor samples, which was not observed in normal breast tissues. rRNA gene expression is controlled by mechanisms other than promoter DNA methylation. Tumorigenesis may cause disruption of many control mechanisms that are required for proper rRNA expression, splicing and maturation, resulting in a dysregulation of the correlation between spliced rRNA expression levels, which should be investigated further.
Open Access
Cancer-testis gene expression is associated with the methylenetetrahydrofolate reductase 677 C > T polymorphism in non-small cell lung carcinoma
(BioMed Central, 2013) Senses, K. M.; Gonen, M.; Barutcu, A. R.; Kalaylioglu, Z.; Isbilen, M.; Konu, O.; Chen, Y. T.; Altorki, N. K.; Gure, A. O.
Background: Tumor-specific, coordinate expression of cancer-testis (CT) genes, mapping to the X chromosome, is observed in more than 60% of non-small cell lung cancer (NSCLC) patients. Although CT gene expression has been unequivocally related to DNA demethylation of promoter regions, the underlying mechanism leading to loss of promoter methylation remains elusive. Polymorphisms of enzymes within the 1-carbon pathway have been shown to affect S-adenosyl methionine (SAM) production, which is the sole methyl donor in the cell. Allelic variants of several enzymes within this pathway have been associated with altered SAM levels either directly, or indirectly as reflected by altered levels of SAH and Homocysteine levels, and altered levels of DNA methylation. We, therefore, asked whether the five most commonly occurring polymorphisms in four of the enzymes in the 1-carbon pathway associated with CT gene expression status in patients with NSCLC.Methods: Fifty patients among a cohort of 763 with NSCLC were selected based on CT gene expression status and typed for five polymorphisms in four genes known to affect SAM generation by allele specific q-PCR and RFLP.Results: We identified a significant association between CT gene expression and the MTHFR 677 CC genotype, as well as the C allele of the SNP, in this cohort of patients. Multivariate analysis revealed that the genotype and allele strongly associate with CT gene expression, independent of potential confounders.Conclusions: Although CT gene expression is associated with DNA demethylation, in NSCLC, our data suggests this is unlikely to be the result of decreased MTHFR function. © 2013 Senses et al.; licensee BioMed Central Ltd.
Open Access
DNA methylation in eukaryotes
(TÜBİTAK, 1997) Pınarbaşı, Ergün
In prokaryotes, DNA methylation protects the bacteriaÕs DNA against degradation by restriction enzymes and corrects error in DNA replication by means of the mismatch repair system. In 1964, the first DNA methyltransferase was identified in E. coli. The avaiability of sequence-specific restriction enzymes and methyltransferases led to a major breakthrough in the field of molecular biology. In eukoryotes, DNA methylation appears to have a different role. It has been implicated in the control of several cellular processes, including differentiation, gene regulation and embryonic development. A series of discoveries over the past few years has generated new interest in DNA methylation. It has been found that in mice in particular DNA methyltransferase is essential for normal embryonic development. Cytosine methylation may also contribute to C-T transition mutations, accounting for about one-third of all somatic and germline mutations in humans.
Open Access
Evaluation of X chromosome inactivation with respect to HLA genetic susceptibility in rheumatoid arthritis and systemic sclerosis
(Public Library of Science, 2016) Kanaan, S. B.; Onat, O. E.; Balandraud, N.; Martin, G. V.; Nelson, J. L.; Azzouz, D. F.; Auger, I.; Arnoux, F.; Martin, M.; Roudier, J.; Ozcelik, T.; Lambert, N. C.
Background: Autoimmune diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc) are characterized by a strong genetic susceptibility from the Human Leucocyte Antigen (HLA) locus. Additionally, disorders of epigenetic processes, in particular non-random X chromosome inactivation (XCI), have been reported in many female-predominant autoimmune diseases. Here we test the hypothesis that women with RA or SSc who are strongly genetically predisposed are less susceptible to XCI bias. Methods: Using methylation sensitive genotyping of the androgen receptor (AR) gene, XCI profiles were performed in peripheral blood mononuclear cells from 161 women with RA, 96 women with SSc and 100 healthy women. HLA-DRB1 and DQB1 were genotyped. Presence of specific autoantibodies was documented for patients. XCI skewing was defined as having a ratio ≥ 80:20 of cells inactivating the same X chromosome. Results: 110 women with RA, 68 women with SSc, and 69 controls were informative for the AR polymorphism. Among them 40.9% of RA patients and 36.8% of SSc patients had skewed XCI compared to 17.4% of healthy women (P = 0.002 and 0.018, respectively). Presence of RA-susceptibility alleles coding for the "shared epitope" correlated with higher skewing among RA patients (P = 0.002) and such correlation was not observed in other women, healthy or with SSc. Presence of SSc-susceptibility alleles did not correlate with XCI patterns among SSc patients. Conclusion: Data demonstrate XCI skewing in both RA and SSc compared to healthy women. Unexpectedly, skewed XCI occurs more often in women with RA carrying the shared epitope, which usually reflects severe disease. This reinforces the view that loss of mosaicism in peripheral blood may be a consequence of chronic autoimmunity. © 2016 Kanaan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Open Access
Genetics and epigenetics of liver cancer
(Elsevier, 2013) Özen, Çiğdem; Yıldız, Gökhan; Dağcan, Alper Tunga; Çevik, Dilek; Örs, Ayşegül; Keleş, Umut; Topel, Hande; Öztürk, Mehmet
Hepatocellular carcinoma (HCC) represents a major form of primary liver cancer in adults. Chronic infections with hepatitis B (HBV) and C (HCV) viruses and alcohol abuse are the major factors leading to HCC. This deadly cancer affects more than 500,000 people worldwide and it is quite resistant to conventional chemo- and radiotherapy. Genetic and epigenetic studies on HCC may help to understand better its mechanisms and provide new tools for early diagnosis and therapy. Recent literature on whole genome analysis of HCC indicated a high number of mutated genes in addition to well-known genes such as TP53, CTNNB1, AXIN1 and CDKN2A, but their frequencies are much lower. Apart from CTNNB1 mutations, most of the other mutations appear to result in loss-of-function. Thus, HCC-associated mutations cannot be easily targeted for therapy. Epigenetic aberrations that appear to occur quite frequently may serve as new targets. Global DNA hypomethylation, promoter methylation, aberrant expression of non-coding RNAs and dysregulated expression of other epigenetic regulatory genes such as EZH2 are the best-known epigenetic abnormalities. Future research in this direction may help to identify novel biomarkers and therapeutic targets for HCC.
Open Access
Identification of a novel experimental model to reveal mechanisms leading to epigenetic changes and subsequent activation of cancer testis genes in cancer
(2016-08) Küçükkaraduman, Barış
Epigenetic aberrations are frequently observed in cancer. Tumor-suppressor genes are often repressed with anomalous hypermethylation in cancer, while DNA hypomethylation has been identified in repetitive sequences and promoter regions of cancer testis (CT) genes resulting in genomic instability. Although it has been shown that CT genes are often regulated by dissociation of repressive proteins from promoter-proximal regions and epigenetic mechanisms, including DNA methylation, histone methylation and acetylation, the process leading to epigenetic changes and de-repression of CT genes remains largely unknown. This study aimed to reveal molecular mechanisms which may have role in coordinating CT gene expression. For this purpose, we designed two groups of experiments. The first was based on extending our previous observations related to two genes (ALAS2, CDR1) which showed inverse expression patterns, compared to CT genes in cancer cell lines. The ex vivo analysis of expression patterns of these genes, however, did not support an inverse relation between their expression and that of CT genes. The second approach was based on categorizing cancer cells into CT-high, CT-intermediate and CT-low groups to define differentially expressed non-CT genes that could help explain mechanisms underlying epigenetic changes and subsequent activation of CT genes. Surprisingly, we could not identify any transcripts that differentially expressed between these subgroups. We therefore, hypothesized that non-overlapping and distinct mechanisms could be involved in the upregulation of CT genes in different tumors. As our earlier work suggested a relationship between epithelial to mesenchymal transition (EMT) and CT expression we asked if an EMT based classification could help elucidate these mechanisms. Indeed, differential genes and differentially activated signaling pathways were discovered when cancer cells were first grouped by their EMT status. This helped us identify candidate proteins (BMI1, PCGF2, RB1 and RBL1) and pathways including MAPK/ERK and PTEN/PI3K pathways which can coordinate CT gene expression in cancer. Thirdly, we investigated clinical relevance of high CT gene expression in triple negative breast cancer by attempting to correlate this with drug sensitivity. Drug sensitivity against panobinostat showed correlation with CT gene expression. In summary, this study suggests new approaches to elucidate mechanisms which coordinate epigenetic aberrations in cancer and how these can be utilized for cancer therapy.
Open Access
Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal
(American Association for the Advancement of Science (A A A S), 2013) Gao J.; Aksoy, B. A.; Dogrusoz, U.; Dresdner, G.; Gross, B.; Sumer, S. O.; Sun, Y.; Jacobsen, A.; Sinha, R.; Larsson, E.; Cerami, E.; Sander, C.; Schultz, N.
The cBioPortal for Cancer Genomics (http://cbioportal.org) provides a Web resource for exploring, visualizing, and analyzing multidimensional cancer genomics data. The portal reduces molecular profiling data from cancer tissues and cell lines into readily understandable genetic, epigenetic, gene expression, and proteomic events. The query interface combined with customized data storage enables researchers to interactively explore genetic alterations across samples, genes, and pathways and, when available in the underlying data, to link these to clinical outcomes. The portal provides graphical summaries of gene-level data from multiple platforms, network visualization and analysis, survival analysis, patient-centric queries, and software programmatic access. The intuitive Web interface of the portal makes complex cancer genomics profiles accessible to researchers and clinicians without requiring bioinformatics expertise, thus facilitating biological discoveries. Here, we provide a practical guide to the analysis and visualization features of the cBioPortal for Cancer Genomics. © 2013 American Association for the Advancement of Science.
Open Access
Oligonucleotide-based label-free detection with optical microresonators: strategies and challenges
(Royal Society of Chemistry, 2016) Toren, P.; Ozgur E.; Bayındır, Mehmet
This review targets diversified oligonucleotide-based biodetection techniques, focusing on the use of microresonators of whispering gallery mode (WGM) type as optical biosensors mostly integrated with lab-on-a-chip systems. On-chip and microfluidics combined devices along with optical microresonators provide rapid, robust, reproducible and multiplexed biodetection abilities in considerably small volumes. We present a detailed overview of the studies conducted so far, including biodetection of various oligonucleotide biomarkers as well as deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs) and proteins. We particularly advert to chemical surface modifications for specific and selective biosensing.
Open Access
SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation
(2011) Acun, T.; Oztas, E.; Yagci, T.; Yakicier, M.C.
Background: Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.Methods: SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.Results: We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.Conclusions: Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis. © 2011 Acun et al; licensee BioMed Central Ltd.
Open Access
Skewed X chromosome inactivation in blood cells of women with scleroderma
(John Wiley & Sons, Inc., 2005) Özbalkan, Z.; Baǧişlar, S.; Kiraz, S.; Akyerli, C. B.; Özer H. T. E.; Yavuz, Ş.; Birlik, A. M.; Çalgüneri, M.; Özçelik, T.
Objective. Scleroderma (SSc) is an autoimmune disease of unknown etiology. The disease is 3-8 times more frequent in women than in men. The role of X chromosome inactivation (XCI) in the predisposition of women to autoimmunity has been questioned. Until now this has not been illustrated experimentally. This study was undertaken to test the hypothesis that disturbances in XCI mosaicism may be involved in the pathogenesis of the disease in female patients with SSc. Methods. Seventy female SSc patients and 160 female controls were analyzed for the androgen receptor locus by the Hpa II/polymerase chain reaction assay to assess XCI patterns in DNA extracted from peripheral blood cells. Furthermore, skin biopsy samples were obtained from 5 patients whose blood revealed an extremely skewed pattern of XCI, and the analysis repeated. Since microchimerism in SSc was reported, Y chromosome sequences were investigated in all samples. Results. Skewed XCI was observed in DNA from peripheral blood cells in 35 of 55 informative patients (64%), as compared with 10 of 124 informative controls (8%) (P < 0.0001). Extreme skewing was present in 27 of the patient group (49%), as compared with only 3 of the controls (2.4%) (P < 0.0001). However, XCI was random in all skin biopsy samples. The potential contribution of microchimerism to the random XCI pattern is highly unlikely based on the medical histories of the patients. Conclusion. Skewed XCI mosaicism may play a significant role in the pathogenesis of SSc.
Open Access
Skewed X inactivation in an X linked nystagmus family resulted from a novel, p.R229G, missense mutation in the FRMD7 gene
(BMJ Group, 2008) Kaplan, Y.; Vargel, I.; Kansu, T.; Akin, B.; Rohmann, E.; Kamaci, S.; Uz, E.; Ozcelik, T.; Wollnik, B.; Akarsu, N. A.
Aims: This study aimed to identify the underlying genetic defect of a large Turkish X linked nystagmus (NYS) family. Methods: Both Xp11 and Xq26 loci were tested by linkage analysis. The 12 exons and intron-exon junctions of the FRMD7 gene were screened by direct sequencing. X chromosome inactivation analysis was performed by enzymatic predigestion of DNA with a methylation-sensitive enzyme, followed by PCR of the polymorphic CAG repeat of the androgen receptor gene. Results: The family contained 162 individuals, among whom 28 had NYS. Linkage analysis confirmed the Xq26 locus. A novel missense c.686C>G mutation, which causes the substitution of a conserved arginine at amino acid position 229 by glycine (p.R229G) in exon 8 of the FRMD7 gene, was observed. This change was not documented in 120 control individuals. The clinical findings in a female who was homozygous for the mutation were not different from those of affected heterozygous females. Skewed X inactivation was remarkable in the affected females of the family. Conclusions: A novel p.R229G mutation in the FRMD7 gene causes the NYS phenotype, and skewed X inactivation influences the manifestation of the disease in X linked NYS females.
Open Access
The SOCS-1 gene methylation in chronic myeloid leukemia patients
(John Wiley & Sons, Inc., 2007) Hatirnaz, O.; Ure, U.; Ar, C.; Akyerli, C.; Soysal, T.; Ferhanoǧlu, B.; Özçelik, T.; Ozbek, U.
SOCS-1, an important protein in the JAK/STAT pathway, has a role in the down stream of BCR-ABL protein kinase. We investigated 56 CML patients and 16 controls for the methylation status of SOCS-1 gene promoter and Exon 2 regions. Exon 2 was found to be methylated in 58.9% of the patients and 93.8% of the controls [P = 0.020, OR = 0.121(0.015-0.957)%95CI]. The promoter region was found unmethylated in all patient samples and controls. Although previous studies revealed a relation between SOCS1 gene Exon-2 hypermethylation and CML development or progression, the results of this study showed no such correlation. On the contrary, our results might be indicating hypomethylation in CML patients, this hypothesis need to be studied in larger study population.
Open Access
TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas
(Taylor & Francis Inc., 2022-08-23) Şerifoğlu, Naz; Adams, Michelle; Erbaba, Begün; Arslan-Ergül, Ayça
Telomerase is reactivated in the majority of cancers. For instance, in gliomas, it is common that the TERT promoter is mutated. Research on telomere promoter GC islands have been focused primarily on proximal TERT promoter but little is known about the distal promoter. Therefore, in this study, we investigated the proximal and distal TERT promoter, in terms of DNA methylation. We did bisulfite sequencing in zebrafish tissue samples for the distal tert promoter. In the zebrafish brain tissues, we identified a hypomethylation site in the tert promoter, and found that this hypomethylation was associated with aging and shortened telomeres. Through site directed mutagenesis in glioma cell lines, we changed 10 GC spots individually, cloned into a reporter vector, and measured promoter activity. Finally, we silenced DNMT3B and measured telomerase activity along with vidaza and adriamycin treatments. Site directed mutagenesis of glioma cell lines revealed that each of the 10 GC spots are critical for telomerase activity. Changing GC to AT abolished promoter activity in all spots when transfected into glioma cell lines. Then, through silencing of DNMT3B, we observed a reduction in hTERT expression levels, while hTR remained the same, and a major increase in senescence-associated beta-galactosidase activity. Finally, we propose a model regarding the efficacy of two chemotherapeutic drugs, adriamycin and azacytidine, on gliomas. Here, we show that distal TERT promoter is critical; changing even one GC to AT abolishes TERT promoter activity. DNMT3B, a de novo methyltransferase, together with GC islands in distal TERT promoter plays an important role in regulation of telomerase expression and senescence.
Open Access
Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer
(Springer, 2015-09-28) Sayar, N.; Karahan, G.; Konu, O.; Bozkurt, B.; Bozdogan, O.; Yulug, I. G.
Background: CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2′-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Results: Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. Conclusions: TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.