Abnormal Epigenetic Changes Research Articles

Evaluation of gene expression level of CDC5L and MACC1 in poor prognosis and progression of osteosarcoma.

Current evidences have indicated that osteosarcoma is strongly associated with abnormal genetic and epigenetic changes that lead to the abnormal expression of oncogenes or methylation of tumor suppressor genes. In the present study, MACC1 and CDC5L mRNA levels in the patients with osteosarcoma were evaluated using quantitative real-time PCR. Our results demonstrated that CDC5L mRNA levels were higher in tumor tissues than in adjacent normal tissues (2.713 ± 0.738 vs. 1.071 ± 0.629; P < 0.05). Moreover, MACC1 was upregulated in tumor bone tissues than in adjacent normal tissues (3.221 ± 0. 624 vs. 1.427 ± 0.456; P < 0.05). Our result demonstrated that high expression of CDC5L was significantly related to advanced TNM stage (P = 0.032). No significant difference was determined between CDC5L mRNA expression and other clinicopathological parameters including age, gender, tumor diameter, location, tumor grade, and histological type. In addition, overexpression of MACC1 was strongly correlated with advanced TNM stage (P = 0.027) and high tumor grade (P = 0.035). Our findings indicated that mRNA level of CDC5L is correlated with advanced TNM stage, and MACC1 may be involved in progression of osteosarcoma.

Overexpression of Cancer-Associated Genes via Epigenetic Derepression Mechanisms in Gynecologic Cancer

Like other cancers, most gynecologic cancers are caused by aberrant expression of cancer-related genes. Epigenetics is one of the most important gene expression mechanisms, which contribute to cancer development and progression by regulating cancer-related genes. Since the discovery of differential gene expression patterns in cancer cells when compared with normal cells, extensive efforts have been made to explore the origins of abnormal gene expression in cancer. Epigenetics, the study of inheritable changes in gene expression that do not alter DNA sequence is a key area of this research. DNA methylation and histone modification are well-known epigenetic mechanisms, while microRNAs and alternative splicing have recently been identified as important regulators of epigenetic mechanisms. These mechanisms not only affect specific target gene expression but also regulate the functioning of other epigenetic mechanisms. Moreover, these diverse epigenetic regulations occur simultaneously. Epigenetic regulation of gene expression is extraordinarily complicated and all epigenetic mechanisms to be studied at once to determine the exact gene regulation mechanisms. Traditionally, the contribution of epigenetics to cancer is thought to be mediated through the inactivation of tumor suppressor genes expression. But recently, it is arising that some oncogenes or cancer-promoting genes (CPGs) are overexpressed in diverse type of cancers through epigenetic derepression mechanism, such as DNA and histone demethylation. Epigenetic derepression arises from diverse epigenetic changes, and all of these mechanisms actively interact with each other to increase oncogenes or CPGs expression in cancer cell. Oncogenes or CPGs overexpressed through epigenetic derepression can initiate cancer development, and accumulation of these abnormal epigenetic changes makes cancer more aggressive and treatment resistance. This review discusses epigenetic mechanisms involved in the overexpression of oncogenes or CPGs via epigenetic derepression in gynecologic cancers. Therefore, improved understanding of these epigenetic mechanisms will provide new targets for gynecologic cancer treatment.

Epigenetic and immunohistochemical characterization of the Clusterin gene in ovarian tumors

To characterize abnormal epigenetic changes and protein expression of the clusterin gene in a large series of ovarian malignant and borderline tumors. Protein expression and promoter methylation of clusterin gene in 181 primary ovarian epithelial cancer, 40 borderline ovarian tumors, 54 ovarian cancer mesenteric metastasis, and 10 normal ovarian samples were analyzed by immunohistochemical staining and methylation-specific PCR. Overexpression of clusterin protein was frequently seen in various ovarian epithelial tumors, being detected in 102 of 181 (56%) primary ovarian epithelial cancers, 21 of 37 (57%) borderline ovarian tumors. Surprisingly, clusterin protein expression was significantly reduced in mesenteric metastasis (20 of 54; 37% cases), as compared to primary ovarian carcinoma (p=0.01). Overexpression of clusterin protein was significantly correlated with high-grade histology (p=0.002) and high FIGO stages (p=0.05). Clusterin promoter hypermethylation was detected in 24 of 181 (13%) primary ovarian epithelial cancer, 8 of 54 (14%) mesenteric metastasis, and 10 of 37 (27%) borderline ovarian tumors. Overall, clusterin promoter hypermethylation was significantly correlated with decreased protein expression in these samples (p<0.001). Increased clusterin expression is correlated with more aggressive biologic behavior in ovarian cancer. Promoter methylation of the clusterin gene can be readily detected, though at low frequencies, in ovarian epithelial tumors and is significantly associated with decreased protein expression of the gene.

Abnormal Genetic and Epigenetic Changes in Signal Transducer and Activator of Transcription 4 in the Pathogenesis of Inflammatory Bowel Diseases

Changes in the expression of signal transducer and activator of transcription 4 (STAT4) contribute to the development of a variety of autoimmune diseases including inflammatory bowel diseases (IBDs). Moreover, epigenetic modifications, including DNA methylation, are considered a basis for differentiation of T helper cells and regulation of cytokines. In this study, we investigated the methylation status of STAT4 gene in IBD patients and the associations between its genetic and epigenetic alterations in IBD patients. Blood and colonic mucosa samples were obtained from Korean patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated, and total RNA and genomic DNA were isolated from the PBMCs and colon mucosa tissues. The mRNA level and DNA methylation status of the promoter were determined by real-time RT-PCR and pyrosequencing, respectively. The chosen SNPs (rs11889341, rs7574865, rs8179673, rs6752770, rs925847, rs10168266, rs10181656, and rs11685878) were genotyped using the TaqMan nuclease assay. Elevated expression of STAT4 was observed in the colonic mucosa and PBMCs of IBD patients. IBD patients showed a lower degree of methylation of the STAT4 promoter than did the healthy controls. Moreover, a significant correlation between risk alleles and methylation status at -172 of the STAT4 promoter was observed, and mRNA levels of STAT4 in IBD patients were correlated inversely with the T-risk allele (rs7574865). Our data demonstrated that the DNA methylation status of STAT4 is associated with genetic polymorphisms, providing insights into the interactions between genetic and epigenetic aberrances in STAT4 that contribute to the development of IBD.

Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

Background-Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR), apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR) and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology.Results-The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P = 0.001), DCR1 (P = 0.00001), DCR2 (P = 0.0000000005) and BRCA2 (P = 0.007) and hypomethylation of DR4 (P = 0.011) in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P = 0.047) and DNA damage repair potential (P = 0.004) in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors.Conclusion-Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing tumors result in aberrant DDR-apoptotic pathway thereby promoting tumor development. We propose, since pathological epigenetic changes of the DDR-apoptotic genes are reversible modifications, these could further be targeted for therapeutic interventions.

Aberrant Epigenetic Silencing of Tumor Suppressor Genes Is Reversed by Direct Reprogramming

Direct reprogramming procedures reset the epigenetic memory of cells and convert differentiated somatic cells into pluripotent stem cells. In addition to epigenetic memory of cell identity, which is established during development, somatic cells can accumulate abnormal epigenetic changes that can contribute to pathological conditions. Aberrant promoter hypermethylation and epigenetic silencing of tumor suppressor genes (TSGs) are now recognized as an important mechanism in tumor initiation and progression. Here, we have studied the fate of the silenced TSGs p16(CDKN2A) during direct reprogramming. We find that following reprogramming, p16 expression is restored and is stably maintained even when cells are induced to differentiate. Large-scale methylation profiling of donor cells identified aberrant methylation at hundreds of additional sites. Methylation at many, but not all these sites was reversed following reprogramming. Our results suggest that reprogramming approaches may be applied to repair the epigenetic lesions associated with cancer.

Enzymatic incorporation of multiple dyes for increased sensitivity in QD-FRET sensing for DNA methylation detection.

DNA methylation is an epigenetic modification that occurs at 5′ cytosine in CpG dinucleotides and is observed in high frequency in the transcription promoter regions of tumor-suppressor genes.[1,2] Such changes account for the loss of function of tumor-suppressor genes for almost every type of cancer;[3] and more genes are inactivated by promoter hyper-methylation than by genetic mutations.[4] These abnormal epigenetic changes appear to be an early event that precedes the occurrence of genetic mutations.[5-8] Hence, detecting DNA promoter methylation has significant clinical and prognostic implications for early cancer detection. Methylation-specific PCR (MSP) remains one of the most widely used methods for the sensitive detection of DNA methylation,[9] but requires gel electrophoresis and is limited to qualitative analysis. Fluorescence-based MSP variants[10,11] utilize fluorescent probes for real-time detection, thereby facilitating high-throughput and quantitative detection of DNA methylation. In recent years, quantum dots (QDs) have emerged as promising fluorophores for biological sensing due to their unique spectroscopic properties, such as broad excitation spectra, narrow size-tunable emission wavelengths, and high photostability. QDs have also been used as FRET donors to detect biomolecular targets.[12-15] The large Stokes shift and narrow emission spectra of QDs allow for FRET with a significantly reduced acceptor excitation and spectral crosstalk, and thus permits the design of QD-FRET biosensors with the minimal background necessary for detecting targets at low-concentration.[13,16] Recently, this feature of low intrinsic background of QD-FRET facilitated the development of a methylation-detection method that achieves greater sensitivity over conventional MSP methods, as exemplified by analyzing sputum samples from lung cancer patients.[16]

Downregulation of Dicer and micro RNA implicates aberrant epigenetic marks and transcriptional factor expression in early embryogenesis

OBJECTIVE: To investigate the impact of Dicer and miRNA on epigenetic modification and expression profile of transcriptional factors during embryogenesis. DESIGN: Genome-wide DNA methylation patterns and expression profiles of pluripotency factors were assessed in both normal and Dicer-silenced mouse embryos. MATERIALS AND METHODS: Mouse sperm-heads mixed with Dicer1 siRNA (Group A) or negative control siRNA (Group B) were co-injected into MII oocytes and cultured to the blastocyst stage. Reversibility of Dicer silencing effect was tested by injection of miR-290 cluster mimics into zygotes of Dicer1 silenced (Group C) and negative control (Group D). DNA methylation patterns as well as expressions of miR-290, de novo DNA methyltransferases (Dnmt3a, Dnmt3b), and pluripotency factors (Oct4, Sox2 and Nanog) were assessed. RESULTS: Transcripts of Dicer decreased sharply to 17% (Group A) after injection, and the levels remained low throughout preimplantation development. Depletion of Dicer1 (Group A) significantly reduced miR-290 transcripts (to 18%), and downregulated Dnmt3a and Dnmt3b (to 27% and 39%, respectively) which led to a significant hypomethylation at the blastocyst stage. Reintroduction of miR-290 cluster mimics into zygotes (Group C) rescued the expression of Dnmt3a and Dnmt3b (74% and 67%), and regained normal methylation patterns in blastocytes. Oct4, Sox2 and Nanog were significantly downregulated in Dicer1-silenced blastocysts (36%, 52% and 47%), yet were immune to reintroduction of miR-290 mimics. CONCLUSIONS: We have demonstrated that miRNA and epigenetics cooperate in controlling gene expression during embryogenesis. The aberrant genome-wide DNA methylation could be attributed by decreasing the expression of de novo DNA methyltransferases which occured upon the loss of miR-290. Abnormal epigenetic changes can be reversed by introduction of miR-290 mimics. Therefore, regulation of miRNA and epigentics may be new strategies in controlling embryonic cell differentiation and improving embryo quality.

DNA Methylation Profiles of Donor Nuclei Cells and Tissues of Cloned Bovine Fetuses

Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissue-specific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.

Hypermethylation of Retinoic Acid Receptor Alpha 2 (RARα2) Promoter in Human AML Cells Correlates with Poor RARα2 Expression and Response to Differentiation Inducers.

All-trans retinoic acid (ATRA) signaling pathway is of critical importance for optimal myelomonocytic differentiation and its disruption by translocations of the RARα gene leads to acute promyelocytic leukemia (APL). APL associated fusion oncoproteins, such as PML-RARα and PLZF-RARα, function through recruitment of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), thus promoting an inactive chromatin state and leading to repression of RARα target genes. Recently, we demonstrated that up-regulation of RARα2 expression by ATRA directly correlates with differentiation in non-APL AML cells. Treatment of ATRA non-responsive cells with the demethylating agent 5-Aza-2′-deoxycytidine (decitabine) restores RARα2 expression and differentiation response to ATRA in different AML subtypes, suggesting abnormal epigenetic changes as a possible mechanism underlying silencing of RARα2 and contributing to a differentiation deficit in these cells. Here we compared the methylation status of ATRA-inducible RARα2 and constitutive RARα1 promoters in 5 human AML cell lines and in normal human CD11b+/CD33+ leukocytes (NLs) by sequencing their respective bisulfite modified CpG islands. Whereas the CpG-island of the RARα2 promoter was completely methylated in all AML samples, it was not methylated in NLs nor in a control preB cell line, NALM-6. The methylation status of the RARα2 promoter directly correlates with the RARα2 expression level, which was shown by real-time RT-PCR to be 100–1000 fold higher in NLs or NALM-6 cells than in AML cell lines. Expression of RARα2 in human primary AML cells was found to be as low as in AML cell lines. In contrast, RARα1 promoter was not methylated in any of the samples and RARα1 expression levels were similar in AML cells and NLs. We have previously shown that stimulation of AML cell differentiation by ATRA and G/GM-CSF (GFs) is associated with a 10 fold induction of RARα2 expression through direct effects on receptor activity. We now show that pre- or co-treatment with decitabine results in up to 100 fold further increase in RARα2 expression levels, rendering the RARα2 mRNA levels similar to these observed in NLs. This synergism in RARα2 induction is paralleled by marked enhancement of leukemic cell differentiation. These results highlight the potential therapeutic use of epigenetic modifiers like decitabine, which may relieve the repression of aberrantly silenced genes required for cellular maturation and amplify the effects of differentiation inducers like ATRA and GFs in AML.

Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to muta-genesis detections

In vitro enumeration of diploid human cell variants that are resistant to purine analogues is a possible method of detecting mutagenesis. Their incidences can be increased by the known mutagens, X-rays and N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG). Usefulness of this method depends on the kinds of hereditary changes that confer analogue-resistance on somatic cells. If resistance usually results from changes in genetic material, in vitro studies could be useful indicators of mutagenic effects on somatic cells and germ cells in vivo . If epigenetic changes are primarily responsible for analogue-resistant variants, their enumeration might not provide information relevant to germinal mutations but would still be a useful way to detect induction of general kinds of stable phenotypic changes that could cause cancer. This article outlines hypothetical epigenetic and genetic causes of somatic cell variation and a prospective genetic analysis of human cell variants that are resistant to 8-azaguanine (AG) or 2,6-diaminopurine ( (DAP). Recent evidences and arguments favoring epigenetic origins of resistance to base-analogues are inconclusive. The often cited high rate of changes causing impermeability to BUdR in hamster cells is based on one improperly executed determination. Comparisons of rates of variation conferring BUdR-resistance on cultured haploid and diploid frog cells included diploid variants that did not behave as mutants and ignored major sources of error in estimating mutation rates. AG-resistance could result from recessive mutations in X-chromosomal genes but comparisons of rates of mutation in hamster cells of different ploidies did not provide information about the numbers of X-chromosomes in the variants. Reports that normal rodent HGPRT reappeared in hybrids of enzyme-deficient rodent cells and HGPRT-containing cells of other species or in the rodent cells alone in response to the conditions of cell hybridization did not include adequate controls for reversions in mutant genes of the rodent cells. Questions about the epigenetic and genetic origins of analogue-resistance are mostly unanswered. It remains possible that some kinds of abnormal epigenetic changes cause somatic disease. Specific methods for detecting their occurrence and responsiveness to environmental factors should be devised by focusing efforts on traits that are normally subject to epigenetic regulation. Derepression of genes on the inactive X-chromosome and of liver phenylalanine hydroxylase production are presented as possible examples of abnormal epigenetic changes that could be quantitatively studied by direct selection in vitro .