br Materials and methods br Results

Materials and methods
Results
Discussion Characterization of the karyotypically abnormal cells and culture adaptation process is important for stem cell biology and can provide valuable insights into the mechanisms regulating transformation of cells towards malignant fate. In this study we show that the levels of the epigenetic regulators, HDAC1 and HDAC2 proteins, are increased in the hESCs with genomic abnormalities. Importantly, we further demonstrate that, similar to many cancer cell lines, HDAC inhibitors repress proliferation of the karyotypically abnormal hESCs, whereas normal cells are more resistant to the treatment. The reduced growth of abnormal hESCs in response to HDAC inhibition correlated with increased levels of AcH3 and CDKN1A and with altered regulation of RB1. Interestingly, levels of both HDAC1 and HDAC2 proteins were decreased in response to both VPA and CBHA treatments. Valproic Romidepsin prevents catalytic activity of type I HDACs; however, it does not necessarily induce degradation of the target protein. Previously, in HEK293T human embryonic kidney carcinoma cells and mouse F9 teratocarcinoma cells VPA has been shown to specifically induce proteosomal degradation of HDAC2, whereas HDAC1 levels were not reduced (Gottlicher et al., 2001; Kramer et al., 2003). Taken together, our findings suggest that HDACs are implicated in the stem cell transformation and support self-renewal of abnormal cells. Although HDAC1 has been reported to be crucial for the growth and normal development of the mouse embryos (Brunmeir et al., 2009), our observation is that HDAC depletion with valproic acid, at least up to the concentration of 1.5mM, does not have any major impact on the self-renewal and proliferation of the karyotypically normal hESCs. This is consistent with the findings in a recent study where HDAC1 deficiency did not affect proliferation of the mouse embryonic stem cells, but rather was required for the efficient differentiation of the cells (Dovey et al., 2010). HDACs are indeed known to be key factors in directing differentiation of the stem cells to certain lineages, such as neuronal and hematopoietic lineages (Brunmeir et al., 2009; Cunliffe and Casaccia-Bonnefil, 2006; Dovey et al., 2010; Humphrey et al., 2008; Wada et al., 2009), probably indirectly by targeting non-histone proteins and directly through epigenetic modifications of the regulatory areas of genes important for differentiation and development. While our study was in progress another study by Ware et al. (2009) reported that HDAC inhibitors support self-renewal of both human and mouse ESCs in the absence of FGF2 or LIF, respectively. In contrast, they also reported that karyotypic abnormalities were observed at equal frequencies under HDAC inhibitor when using butyrate, vorinostat, trichostatin or butyryl than in the absence of these compounds. However, the karyotypic results for cells cultured under valproic acid were not reported and importantly it did not become clear whether changing the concentrations of HDAC inhibitors under well controlled setup would reveal a concentration for selective suppression of abnormal cell growth. Ware et al. used only 0.5mM valproic acid, which in our hands is too low concentration to prevent growth of abnormal cells. Our data shows that HDACs communicate with well known proteins implicated in oncogenic processes, such as RB1 and POU5F1 in hESCs. Possibly through co-operation with RB1 and other factors, HDACs modulate activity of genes implicated in development, cell growth and transformation enabling survival of cells with genomic abnormalities. According to our results HDAC inhibition is cytotoxic to abnormal hESCs, whereas normal cells are more resistant to the treatment. This selective sensitivity of the transformed cells is similar to the cells in somatic tissues as reported by several preclinical studies for a wide range of different cancer types. HDAC inhibitor vorinostat has been approved for clinical use in the treatment of cutaneous T cell lymphoma and several others are currently under evaluation (Chateauvieux et al., 2010). We have also here confirmed the growth inhibitory effect of HDAC inhibition on human acute lymphocytic leukemia cell line (CCRF-CEM) and embryonal carcinoma cells (NT2D1) derived from germ cell tumor. The mechanism behind the antitumor effect of HDAC inhibition has remained unclear.