Background Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. Methods LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules’ in situ level (n = 40). Results Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). Conclusion Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Napjainkban a genetikai kutatások mellett egyre inkább előtérbe kerülnek az epigenetikai vizsgálatok, ugyanis az epigenetikai jelenségek -köztük a DNS-metiláció is -részt vesznek a fenotípust meghatározó gének expressziójának szabályozásában, így számos betegség mechanizmusával összefüggenek. Jelen összefoglaló közleményünk célja az epigenetikai mechanizmusok közül a DNS-metiláció evolúció során történő megjelenésének, funkciói változatosságának, illetve az öregedésben és a rákos megbetegedésekben betöltött szerepé-nek bemutatása. A DNS-metiláció a prokarióták, az eukarióták, illetve a vírusok körében is megfigyelhető epigenetikai módosulás. A prokarióták és vírusok esetén idegen DNS-sel szembeni védelmi funkciót lát el. A DNS-metiláció prokariótáknál jelentős szereppel bír a transzkripció regulációjában, a replikáció iniciációjában, illetve a Dam-irányított hibajavítás-ban. A vírusoknál a védelmi funkció mellett a terjedésükhöz szükséges kapszid formálásában is részt vesz. Az eukarióták esetén a DNS-metiláció szerepet játszik a kromatinstruktúra és a transzkripció szabályozásában, a rekombináci-óban, a replikációban, az X-kromoszóma inaktivációjában, a transzpozonok szabályozásában és az imprinting jelenség létrehozásában. A fenti tulajdonságok mellett evolúciós szereppel is rendelkezik azáltal, hogy megváltoztatja a DNS mutációs rátáját. Az öregedés során és a rákos megbetegedésekben kialakuló globális hipometilációs eltérések genetikai instabilitáshoz és spontán mutációs eltérésekhez vezethetnek a transzpozonok szabályozásában betöltött funkciójuk révén. A lokális hipermetilációs (például az SFRP1, az SFRP2, a DKK1 és az APC promóterének hipermetilációja) változásoknak a fehérjeexpressziós változások létrehozásában, ezáltal a rák fenotípus kialakulásában van jelentős szerepe. Az elválto-zások általános jellege alapján a fenti eredmények a biológiai kor és a betegségek epigenetikai változások kimutatásán alapuló diagnosztikai és prognosztikai módszerei kutatásának fontosságát támasztják alá. Orv Hetil. 2018; 159(1): 3-15. Kulcsszavak: epigenomika, DNS-metiláció, 5-metilcitozin, öregedés, daganatos megbetegedések Role and alterations of DNA methylation during the aging and cancerBesides the genetic research, increasing number of scientific studies focus on epigenetic phenomena -such as DNA methylation -regulating the expression of genes behind the phenotype, thus can be related to the pathomechanism of several diseases. In this review, we aim to summarize the current knowledge about the evolutionary appearance and functional diversity of DNA methylation as one of the epigenetic mechanisms and to demonstrate its role in aging and cancerous diseases. DNA methylation is also characteristic/also appear to prokaryotes, eukaryotes and viruses. In prokaryotes and viruses, it provides defence mechanisms against extragenous DNA. DNA methylation in prokaryotes plays a significant role in the regulation of transcription, the initiation of replication and in Dam-directed mismatch repair. In viruses, it participates not onl...

No abstract

The incidence and mortality of colorectal cancer (CRC) are considerably high in Central European countries, it is the second most common cancer in both men and women in Hungary with 10,000 newly registered patients per year. These data indicate the necessity of new screening methods that are more comfortable for patients, hence the compliance can be increased. Cell-free DNA (cfDNA) level in blood is elevated in certain physiological conditions, such as pregnancy or high physical activity. Furthermore, cfDNA concentration alterations can also be detected in some pathological processes; increased cfDNA amount was observed in autoimmune and inflammatory diseases, as well as in various cancers including CRC. Numerous studies about origin, function, and mechanism of cfDNA can be found in the scientific literature. In this review, we aimed to describe the quantitative and qualitative changes of cfDNA, to present its functions, and to provide an overview of the available diagnostic applications for CRC. CfDNA can be released to the circulatory system via apoptosis, necrosis or by direct secretions by living cells. In cancer patients, cfDNA can originate from healthy and cancer cells, hence genetic (e.g. mutations in APC, KRAS, BRAF) and epigenetic (e.g. methylation in SEPT9, SFRP1) alterations of tumor cells can be examined in cfDNA fraction. Several high-throughput, sensitive and even automated methods are available providing opportunity to perform standardized sample preparation and to analyse biomarker candidates quantitatively. These enhancements can help to develop alternative screening methods that can be easily integrated into the clinical practice and can contribute to early cancer detection. Orv Hetil. 2019; 160(30): 1167–1177.

Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.