R. G. Hertzog 1, D. I. Hertzog 2, B. Patrinichi 1
1 Army Center for Medical Research, Bucharest – Romania
2 UMF Craiova, Faculty of Dental Medicine, Department of Pharmacology, Romania
Abstract
Objectives. Trichostatin A (TSA) is an antifungal antibiotic and a potent reversible inhibitor of histone deacetylase (HDAC). Pharmacologically, TSA is an antitumoral agent, but it is also a therapeutic agent in some non-tumoral diseases. The molecular mechanisms through which TSA inhibits the proliferation of cancerous cells are not well understood. Because the main target of HDAC inhibitors (HDI) is nuclear chromatin, we proposed to determine the eff ects of TSA morphologically, genetically and immunochemically in HeLa (Henrietta Lacks) cells, derived from cervix cancer cells. Material and methods. As biologic material, we used HeLa cell line that has an unlimited proliferative ability in vitro. After subcultivation (1:5), the cultures were treated with TSA in four fi nal concentrations (10, 50, 200, 400 μg/ml) for 8, 16 and 24 hours. We used morphometric, genetic methods, nucleolus organizers staining (that refl ect the transcription function of ribosomal genes) and the study of chromosomal alterations, analysis of the condensed and decondensed state of the nuclear chromatin, apoptosis and visualization of acetylated nuclear proteins by an indirect immunocytochemistry. Results. By means of morphometric measurements, we emphasized that the cell nuclei treated with TSA were slightly larger than those in control samples. Nucleolus organizer regions (NOR), visualized with AgNO3 and localized in second constrictions level of acrocentric chromosomes, exposed various sizes. Th e analysis of chromosomal damages shows that TSA often induces mono- and bichromatid breaks whose frequency increased in a concentration and time of treatment – dependent manner. Ata small frequency deletions and dicentric chromosomes were identifi ed. TSA induced a decondensed chromatin that was observed both on the chromosomal preparations and interphase nuclei. By immunofl uorescence, we observed that chromatin was uniformly distributed in the nucleus, and no chromatic dense ring was seen at nuclear periphery in every examined cell. By acridin orange-ethidium bromide approach, we emphasized apoptotic cells whose frequency increased in a concentration and time of treatment – dependent manner. The nuclear proteins were strongly acetylated in TSA presence. Conclusions. The increase in HeLa cells’ nuclei size is consistent with chromatin decondensation which is also preserved during the cell division phases. The presence of NOR in most of the cells suggests that ribosomal gene transcription is mostly active and that protein biosynthesis is not aff ected. The chromosome breaks might cause cell death by apoptosis. The overacetylation of nuclear proteins leads to the active state of chromatin which is also preserved during the cell division phases.