engleski

Suppression of experimental germ cell tumor development by epigenetic modulators in vitro

Testicular germ cell tumors are the most common malignancies among young male population. They arise from primordial germ cells or gonocytes that had impaired maturation to prespermatogonia and spermatogonia. Somatic mutations are unusual and it is therefore likely that epimutations, that are reversible, are involved in development. Mouse teratoma is a well-established in vitro model in which two weeks long cultivation of embryonic disc results in developing teratoma-like structures. We isolated embryonic discs from 7, 5 days old mouse embryos, treated them for two hours with epigenetic modulators that influence methylation (5-azacytidine), histone acetylation (Trichostatin A, Valproate) and translation of mRNA (esiNanog, esiOct3/4 and esiTrrap) and then cultivated them in MEM enriched with rat serum. Growth of embryonic discs/teratomas was followed by measuring their size at day 0 and for the next 7 days of cultivation whereupon were used for analysing DNA methylation and gene expression. To investigate effect of used epigenetic modulators on stamness DNA methylation status of the promoter region of Nanog, Oct3/4 and Sox2 genes as well as quantification of mRNA of these genes was performed. Furthermore, the expressions of endoderm (Sox17, Gata4), mesoderm (Brachyury , Eomes), neuroectoderm markers (Nestin, Sox2, Six3), marker of primitive ectoderm (Fgf5) and marker of myogenic commitment (MyoD) were determined. Epigenetic agents significantly reduced embryonic discs/teratomas growth. The strongest decrease was detected in esiOct3/4 treated group followed by 5-azacytidine, Valproate and TrichostatinA treated groups. 5-azacytidine decreased methylation in promotor regions of stemness genes.The highest increment of DNA methylation in promotor regions was achieved by esiNanog for Nanog gene and by esiOct3/4 for Oct3/4 gene. Expression of analysed stemness markers was significantly reduced by Valproate which also reduced expression of mesodermal, neuroectodermal markers and marker of myogenic commitment. EsiOct3/4 treated group showed the most prominent effect on mesoderm and primitive ectoderm marker expression. Taking everything into account, various epigenetic modulators impact experimental germ cell tumor development via methylation alternations in promotors of stemness and differentiation genes and their expression. Epigenetic modulators have potential for reverting epimutations and therefore restoring propriate gene expression.

experimental germ cell tumor ; epigenetic modulators

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