engleski

Screening for the activity of histone deacetylation inhibitors in the gastrulating embryo-derived teratoma biological system

In our in vitro natural 3D biological system, rodent embryos explanted at the time of formation of the three germ-layers (gastrulation), develop a teratoma-like structure. We present the results of screening for the activity of Histone Deacetylation Inhibitors (HDIs) in this biological system. 9.5-days-old Fisher rat embryos-proper were cultivated for 14 days in Eagle’s MEM and 50% rat serum with 2mM or 1mM valproate or trichostatin A (66 nM) at the air-liquid interface. Histone acetylation was assessed by western blotting and apoptosis and cell proliferation by immunohistochemistry and stereology. Spent media metabolomes were analyzed by IR-spectroscopy (FTIR). Valproate and TSA both negatively influenced growth of explants and differentiation of the neural tissue. Valproate 2mM significanty increased histone acetylation and apoptosis in explants. In spent media metabolomes the amide I α-helices and the elevated ratio of A(CH3)/A(CH2, as biomarkers for histone acetylation, and a higher intensity of the 1625 cm-1 absorption band for the parallel β-strand structure and CH2 vibrations of lipids at 2853 cm- 1, as biomarkers of apoptosis, were assessed. Our in vitro biological screening system detected embryotoxic/anti-tumor impact of both HDIs. FTIR spectroscopy was able to discern biomarkers of histone acetylation and apoptosis in spent media metabolomes, thus upgrading our biological in vitro system for faster screening of embryotoxic/antitumor agents.

histone deacethylation ; valproate ; gastrulation ; in vitro

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