Sára Zsigrai dr.1, Alexandra Kalmár dr.2, Krisztina Andrea Szigeti1, Barnabás Wichmann dr.2, Gábor Valcz dr.2, Barbara Kinga Barták dr.1, Zsófia Brigitta Nagy1, Orsolya Galamb dr.2, Titanilla Dankó3, Anna Sebestyén dr.3, Gábor Barna dr.3, Zsolt Tulassay dr.2, Péter Igaz dr.2, Béla Molnár dr.2
1 2nd Department of Internal Medicine, Semmelweis University, Budapest
2 Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest & 2nd Department of Internal Medicine, Semmelweis University, Budapest
3 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest
Introduction: Global DNA hypomethylation can be observed along aging of normal cells, and it is also connected to genomic instability. Methylation is a reversible process using of methyl donors, such as folic acid (FA) and S-adenosylmethionine (SAM).
Aims: We compared the FA, and SAM levels, the DNA double-strand break and global methylation status of normal, adenoma and colorectal carcinoma (CRC) biopsy samples, then examined the expression levels of nucleotide synthesis and DNA methylation enzymes. Additionally, we investigated the effect of methyl donor supplementation on tumor cell DNA methylation, genomic integrity, cell cycle and apoptosis.
Method: Immunohistochemical staining was performed on 15 human adenoma, 15 CRC and 15 normal biopsy samples for intracellular FA, SAM, DNA double strand break and DNA methylation level detection. Global DNA methylation level was quantified with pyrosequencing . Gene expression changes of 60 biopsy samples were analysed, using HTA 2.0 microarray chip. HT- 29 colorectal carcinoma cells were treated with methyl donors, then immunofluorescent staining and flow cytometry analysis were assessed.
Results: Global DNA hypomethylation (p<0,001), increased double-strand DNA break levels, however decreased methyl donor levels were noticed in adenoma and CRC biopsies compared to normal samples. Significant elevation (p<0,001) of enzymes, that are needed for nucleotide synthesis were detected, while no remarkable changes in the expression levels of methylation-related enzymes could be seen. After FA and SAM treatment, elevation of DNA methylation and intracellular methyl donor levels, in addition reduced DNA double-strand break level and cell cycle alterations were observed.
Conclusion: FA and SAM levels are in correlation with global DNA methylation pattern and DNA integrity, thereby can contribute to the prevention of tumor development. Methyl donor supplementation has an effect on DNA methylation, therefore its application in proper time and dosage might be a promising approach for preventive DNA remethylation.
Doctoral School: Clinical Medicine
Supervisor: Béla Molnár
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