Epigenetic changes during hematopoietic cell granulocytic differentiation--comparative analysis of primary CD34+ cells, KG1 myeloid cells and mature neutrophils.

BACKGROUND: Epigenetic regulation is known to affect gene expression, and recent research shows that aberrant DNA methylation patterning and histone modifications may play a role in leukemogenesis. In order to highlight the co-operation of epigenetic mechanisms acting during the latter process it is important to clarify their potential as biomarkers of granulocytic differentiation. RESULTS: In this study we investigated epigenetic alterations in human hematopoietic cells at a distinct differentiation stages: primary hematopoietic CD34+ cells, KG1 myeloid leukemic cells, whose development is stopped at early stage of differentiation, and mature neutrophils. We focused on the epigenetic status of cell cycle regulating (p15, p16) and differentiation related (E-cadherin and RARß) genes. We found that the methylation level in promoter regions of some of these genes was considerably higher in KG1 cells and lower in CD34+ cells and human neutrophils. As examined and evaluated by computer-assisted methods, histone H3 and H4 modifications, i.e. H3K4Me3, H3K9Ac, H3K9Ac/S10Ph and H4 hyperAc, were similar in CD34+ cells and human mature neutrophils. By contrast, in the KG1 cells, histone H3 and H4 modifications were quite high and increased after induction of granulocytic differentiation with the HDAC inhibitor phenyl butyrate. CONCLUSIONS: We found the methylation status of the examined gene promoters and histone modifications to be characteristically associated with the hematopoietic cell progenitor state, induced to differentiate myeloid KG1 cells and normal blood neutrophils. This could be achieved through epigenetic regulation of E-cadherin, p15, p16 and RARß genes expression caused by DNA methylation/demethylation, core and linker histones distribution in stem hematopoietic cells, induced to differentiation KG1 cells and mature human neutrophils, as well as the histone modifications H3K4Me3, H3K9Ac, H3K9Ac/S10Ph and H4 hyperAc in relation to hematopoietic cell differentiation to granulocyte. These findings also suggest them as potentially important biomarkers of hematopoietic cell granulocytic differentiation and could be valuable for leukemia induced differentiation therapy.

Histone deacetylase inhibitor BML-210 induces growth inhibition and apoptosis and regulates HDAC and DAPC complex expression levels in cervical cancer cells.

Histone deacetylase inhibitors (HDACIs) represent a new class of targeted anti-cancer agents and different other diseases, like muscular disorders. A number of studies have shown that extracellular signal-activated kinases can target chromatin-modifying complexes directly and regulate their function. The molecular connection between the dystrophin-associated protein complex (DAPC) and chromatin has been described, by showing that NO signaling regulates histone deacetylase (HDAC) activity and influences gene expression in different cell types. In present study, we investigated HDACs changes in HeLa cells undergoing growth inhibition and apoptosis, caused by HDACI BML-210 and retinoic acid (ATRA). Cell cycle analysis indicated that HeLa cell treatment with 20 and 30 µM concentration of BML-210 increased the proportion of cells in G0/G1 phase, and caused accumulation in subG1, indicating that the cells are undergoing apoptosis. We determined down-regulation of HDAC 1-5 and 7 after treatment with BML-210. Also, we demonstrated expression of different isoforms of alpha-dystrobrevin (α-DB) and other components of DAPC such as syntrophin, dystrophin, beta-dystrobrevin (ß-DB) and NOS in HeLa cells after treatments. We determined changes in protein expression level of dystrophin, NOS1, α- and ß-DB and in subcellular localization of α-DB after treatments with BML-210 and ATRA. In conclusion, these results suggest that HDACI BML-210 can inhibit cell growth and induce apoptosis in cervical cancer cells, what correlates with down-regulation of HDAC class I and II and changes in the DAPC expression levels. This can be important for identifying target proteins in DAPC signaling to HDACs, as a target of pharmacological intervention for treatment of muscular dystrophies and other diseases.

Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.

Retinoic acid and histone deacetylase inhibitor BML-210 inhibit proliferation of human cervical cancer HeLa cells.

Human papillomavirus (HPV) infection is believed to be the central cause of cervical cancer. The viral proteins E6 and E7 from high-risk HPV types prevent cells from differentiating apoptosis and inducing hyperproliferative lesions. Human cervical carcinoma HeLa cells contain integrated human papillomavirus type 18 (HPV-18). Retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HeLa cervical carcinoma cells. Cellular responses to RA are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors. On the other hand, histone deacetylase inhibitors have been shown to be chemopreventive agents for the treatment of cancer cells. In this article, we have examined the antiproliferative effect of RA and histone deacetylase inhibitor BML-210 on HeLa cells, and particularly the effects on protein expression that may be involved in the cell cycle control and apoptosis. Our data suggest that a combination of RA and BML-210 leads to cell growth inhibition with subsequent apoptosis in a treatment time-dependent manner. We confirm that BML-210 alone or in combination with RA causes a marked increase in the level of p21. The changes in the p53 level are under the influence of p38 phosphorylation. We also discovered that the histone deacetylase inhibitor BML-210 causes increased levels of anti-apoptotic protein Bcl-2 and phosphorylated p38 MAP Kinase; the latter link in cell cycle arrest with response to extracellular stimuli. Our results suggest that RA and BML-210 are involved in different signaling pathways that regulate cell cycle arrest and lead to apoptosis of HeLa cells.