DNA methylation is an epigenetic process affecting gene expression and chromatin organization. It can heritably silence or activate transcription of genes without any change in their nucleotide sequences, and for a long time was not recognized as an important regulatory mechanism. However, during the recent years it has been shown that improper methylation, especially hypermethylation of promoter regions, is observed in nearly all steps of tumorigenesis. Aberrant methylation is also the cause of several major pathologies including developmental disorders involving chromosome instabilities and mental retardation. A great progress has been made in our understanding of the enzymatic machinery involved in establishing and maintaining methylation patterns. This allowed for the development of new diagnostic tools and epigenetic treatment therapies. The new approaches hold a great potential; several inhibitors of DNA methyltransferases have already shown very promising therapeutic effects.
Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m5Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m5C) in the DNA. The technique is based on conversion of m5C into fluorescent 3,N4-etheno-5-methyl-2'deoxycytidine (εm5C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m5C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.
Recent evidences indicate that epigenetic changes play an important role in the transcriptional reprogramming of gene expression that characterizes cardiac hypertrophy and failure and may dictate response to therapy. Several data demonstrate that microRNAs (miRNAs) play critical roles both in normal cardiac function and under pathological conditions. Here we assessed, in in vivo rat models of myocardial infarction (MI) and ischemia-reperfusion (IR), the relationship between two miRNAs (miR-29a and miR-30c) and de novo methyltransferase (DNMT3a) which, altering the chromatin accessibility for transcription factors, deeply impacts gene expression. We showed that the levels of members of miR-29 and miR- 30 families were down regulated in ischemic tissues whilst the protein levels of DNMT3a were increased, such a relation was not present in healthy tissues. Furthermore, by an in vitro assay, we demonstrated that both miRNAs are able to down regulate DNMT3a by directly interacting with DNMT3a 3’UTR and that miR-29a or miR-30c overexpression in the cardiac HL1 cell line causes decrease of DNMT3a enzyme both at the mRNA and protein levels. Our data, besides confirming the down regulation of the miR-29a and miR-30c in infarcted tissues, envisage a cross-talk between microRNAs and chromatin modifying enzymes suggesting a new mechanism that might generate the alterations of DNA methylation often observed in myocardial pathophysiology.
Background: Folate metabolism dysfunctions can result in DNA hypomethylation and abnormal chromosome segregation. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) encoding gene (C677T and A1298C) reduce MTHFR activity, but when associated with aneuploidy, the results are conflicting. Turner Syndrome (TS) is an interesting model for investigating the association between MTHFR gene polymorphisms and nondisjunction because of the high frequency of chromosomal mosaicism in this syndrome. Objective: To investigate the association of MTHFR gene C677T and A1298C polymorphisms in TS patients and their mothers and to correlate these polymorphisms with maternal risk of TS offspring. Subjects and Methods: MTHFR C677T and A1298C polymorphisms were genotyped in 33 TS patients, their mothers and 15 healthy females with their mothers as controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing technique. Results: Genotype and allele frequencies of both C677T and A1298C were not significantly different between TS cases and controls. There were no significant differences in C677T genotype distribution between the TS mothers and controls (p=1). The MTHFR 1298AA and 1298AC genotypes were significantly increased in TS mothers Vs. control mothers (p=0.002). The C allele frequency of the A1298C polymorphism was significantly different between the TS mothers and controls (p=0.02). The association of A1298C gene polymorphism in TS patients was found to increase with increasing age of both mothers (p=0.026) and fathers (p=0.044) of TS cases. Conclusion: Our findings suggest a strong association between maternal MTHFR A1298C and risk of TS in Egypt.
Cytosine methylation patterns in higher eukaryotes are important in gene regulation. Along with 5-methylcytosine (5-mC), a newly discovered constituent of mammalian DNA, 5-hydroxymethylcytosine (5-hmC), is the other modified base in higher organisms. In this study we detected 5-hmC in plant protoplast DNA and demonstrated its increasing content during the first 72 hrs. of protoplast cultivation. In contrast to 5-hmC, the amount of 5-mC decreased during protoplast cultivation. It was also found that 5-hmC did not primarily arise as a product of oxidative DNA damage following protoplast culture.
Perturbations in early life environments, including intrauterine exposure to maternal gestational diabetes (GDM), are hypothesized to lead to metabolic imprinting resulting in increased risk of cardiometabolic outcomes later in life. We aimed to 1) identify candidate genes and biological pathways associated with differentially methylated regions (DMRs) in relation to exposure to GDM in utero and, 2) using mediation analysis, more definitively investigate the potential for mediation of the effect of exposure to maternal diabetes in utero on cardiometabolic traits in childhood risk through our identified DMRs. Genome-wide methylation analysis of peripheral blood mononuclear cell’s DNA was conducted in 21 healthy children, ages 8-12 years. P-values from multiple linear regression analyses for >27,000 CpG sites were ranked to identify DMRs between the exposure groups. Among the top 10 ranked DMRs, we identified several genes, including NPR1, PANK1, SCAND1, and GJA4, which are known to be associated with cardiometabolic traits. Gene enrichment analysis of the top 84 genes, each with p<=0.005, identified the ubiquitin proteasome system (UPS) as the most enriched biological pathway (p = 0.07). The UPS pathway reflects biological processes known to be associated with endothelial function, inflammation, lipid metabolism, insulin resistance and b-cell apoptosis, whose derangements are central to the pathogenesis of cardiometabolic diseases. Increased methylation of PYGO1 and CLN8 had the greatest relative mediation effect (RME = 87%, p=0.005 and RME=50%, p=0.01) on the impact of exposure to maternal diabetes in utero on VCAM-1 levels in the offspring. Multiple candidate genes and the UPS were identified for future study as possible links between exposure to maternal gestational diabetes in utero and adverse cardiometabolic traits in the offspring. In particular, increased methylation of PYGO1 and CLN8 may be biological links between intrauterine exposure to maternal diabetes and significantly increased VCAM-1 levels in the offspring.
Introduction: αKlotho gene was originally identified as a putative agesuppressing gene in mice. Recently it is known that αKlotho gene functions as a tumor suppressor in many types of cancer, including breast, pancreas, gastric, colon, lung and cervical cancer. The downregulation of αKlotho expression was associated with CpG hipermethylation of promoter region and histone deacetylation. Bladder cancer is the most common cancer of the urinary tract in Polish population. The aim of this study was the analysis of the effect of DNA methyltransferase and histone deacetylase inhibitors on αKlotho gene expression in bladder cancer cells. Materials and methods: In this study T24 bladder cancer cell line was used. The analysis of the effect of DNA methyltransferase and histone deacetylase inhibitors on αKlotho gene expression was performed using Real Time PCR method with TaqMan probes. To determine the methylation profile of αKlotho gene promoter region quantitive Methylation-Specific Polymerase Chain Reaction technique was used. Results: The treatment of T24 cells with DNA methyltransferase inhibitor (AZA) restored the expression of αKlotho gene. After AZA treatment, the methylation level of CpG island in the promoter region of αKlotho gene was nearly half lower (p<0.05) than control cells. In case of the treatment of T24 cells with histone deacetylase inhibitor (TSA) we did not observe any changes in αKL gene expression in respect to the control cells. Treatment cells with both the inhibitors led to the significant increase of mRNA αKlotho gene expression level (p<0.001). Conclusions: The changes in αKlotho gene expression on mRNA level are associated with epigenetic changes.
PL
Wstęp: Gen αKlotho pierwotnie zidentyfikowany został u myszy jako gen, którego ekspresja wpływa na długość ich życia. Obecnie wiadomo, że αKlotho spełnia funkcję genu supresorowego w przypadku wielu typów nowotworów, m.in. w raku piersi, trzustki, żołądka, płuc, okrężnicy i raku szyjki macicy. Wykazano, że spadek ekspresji genu αKlotho związany jest z hipermetylacją wysp CpG w obrębie regionu promotorowego oraz deacetylacją histonów. Rak pęcherza moczowego jest najczęściej występującym nowotworem układu moczowego w Polsce. Celem prowadzonych badań była analiza wpływ inhibitorów metylotransferaz DNA oraz deacetylaz białek histonowych na ekspresję genu αKlotho w komórkach raka pęcherza moczowego. Materiały i metody: Materiał do badań stanowiła linia komórek raka pęcherza moczowego T24. Analizę wpływu inhibitorów metylotransferaz DNA oraz deacetylaz białek histonowych na ekspresję genu αKlotho prowadzono techniką Real Time PCR z użyciem sond fluorescencyjnych TaqMan. Ocenę stopnia metylacji regionu promotorowego badanego genu prowadzono przy użyciu techniki ilościowego MSP-PCR (ang. Methylation-Specific Polymerase Chain Reaction). Wyniki: W wyniku traktowania komórek linii T24 inhibitorem metylotransferaz DNA (AZA) obserwowano przywrócenie ekspresji genu αKlotho. W porównaniu do komórek kontrolnych, komórki traktowane 5-aza-2′-deoksycytydyną wykazywały blisko o połowę niższy stopień metylacji wysp CpG w regionie promotorowym genu αKlotho (p<0,05). W wyniku traktowania komórek inhibitorem deacetylaz białek histonowych (TSA) nie obserwowano zmian w ekspresji genu αKL. Zastosowanie obu inhibitorów prowadziło do istotnego wzrostu ekspresji genu αKlotho na poziomie mRNA (p<0,001). Wnioski: Zmiany ekspresji genu αKlotho na poziomie mRNA związane są ze zmianami we wzorze modyfikacji epigenetycznych.
Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assay we showed that the drugs at 0.01-10 μM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 μM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.
Epigenetics represents the mechanisms that infl uence the regulation and modifi cation of the expression of genetic material not related to the alterations in DNA sequences. These mechanisms include both DNA methylation and histone modifi cations. In the present article, we review current views on the role of aberrations of DNA hyper- and hypomethylation processes and the acetylation of histones, associated with genes that control the cell cycle, cell diff erentiation, DNA repair, apoptosis, cell signaling, angiogenesis, metabolism of xenobiotics and invasion, in the pathogenesis of melanoma. In addition, new strategies for treatment of melanoma associated with epigenetics are presented.
PL
Przez pojęcie epigenetyka należy rozumieć mechanizmy wpływające na regulację i modyfi kację ekspresji materiału genetycznego, jednocześnie niezmieniające sekwencji nukleotydów. Mechanizmy te obejmują zarówno metylację DNA, jak i modyfi kacje histonów. W artykule dokonano przeglądu aktualnych poglądów dotyczących zaburzeń procesów hiperi hipometylacji DNA oraz acetylacji histonów w patogenezie czerniaka, związanych z genami kontrolującymi cykl komórkowy, różnicowanie, naprawę DNA, apoptozę, sygnalizację komórkową, angiogenezę, metabolizm ksenobiotyków i powstawanie przerzutów. Ponadto przedstawiono nowe strategie leczenia czerniaka związane z epigenetyką.
Modyfikacje epigenetyczne są zmianami regulującymi ekspresję genów. Spośród tych modyfikacji metylacja DNA w regionach promotorowych genów jest najlepiej poznaną zmianą. Za metylację DNA odpowiada rodzina metylo-transferaz DNA. Proces ten jest odwracalny w wyniku reakcji demetylacji, w których pośrednią rolę odgrywają białka TET. Hipometylacja DNA oraz hipermetylacja regionów promotorowych genów bogatych w wyspy CpG należy do epigenetycznych mechanizmów powszechnie występujących w wielu typach nowotworów. Epigenetyczny mechanizm transformacji nowotworowej związany jest nie tylko ze zmianami w poziomie metylacji poszczególnych onkogenów czy też genów supresorowych, ale także z potranslacyjnymi modyfikacjami białek histonowych wymuszających zmia-ny w strukturze chromatyny. Określone modyfikacje, takie jak: metylacja, acetylacja, fosforylacja, ubikwitynacja, biotynylacja, ADP-rybozylacja oraz sumoilacja, mogą wpływać na kondensację chromatyny oraz na białka i kom-pleksy enzymatyczne decydujące o dostępności DNA, co z kolei wpływa na upakowanie, replikację, rekombinację, procesy naprawy oraz ekspresję DNA. W mechanizmach modulacji ekspresji genów zaangażowanych w procesy prowadzące do rozwoju nowotworów znaczącą rolę odgrywają dwa główne rodzaje małych interferencyjnych RNA siRNA oraz miRNA. Uzyskiwane dane z prowadzonych badań pokazują, że mechanizmy epigenetyczne uczestniczą w procesach pro- wadzących do rozwoju nowotworów, a poszukiwanie epigenetycznych biomarkerów może być przydatne w terapii nowotworów.
PL
Epigenetic modifications are changes which can regulate gene expression. DNA methylation in gene promoter regions is the most well-known change among epigenetic modifications. The family of DNA methyltransferases is responsible for DNA methylation. Methylation is reversible due to the demethylation reaction, executed by TET proteins. DNA hypomethylation and hypermethylation of gene promoter regions rich in CpG islands belonging to epigenetic mechanisms commonly occur in many tumors. The epigenetic mechanism of malignant transformation is related not only to changes in the level of methylation of oncogenes or tumor suppressor genes, but also to post-translational modifications of histone proteins, forcing changes in the chromatin structure. Certain modifications, such as methy-lation, acetylation, phosphorylation, ubiquitination, biotinylation, ADP–ribosylation, and sumoylation may affect chro-matin condensation, protein and enzyme complexes that determine the availability of DNA, which then affects the condensation, replication, recombination and repair processes, as well as gene expression. Among the modulatory mechanisms of the expression of genes involved in the processes leading to cancer development, two main types of small interfering RNA play an important role: siRNA and miRNA. Research data Show that epigenetic mechanisms are involved in the processes leading to tumor development, and searching for epigenetic biomarkers may be useful in epigenetic cancer therapy.
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