Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus-diseased gestation.

Human mesenchymal stem cells isolated from amniotic fluid (AF-MSCs) demonstrate the potency for self-renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF-MSCs from two cell sources and the expression of the cell surface-specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes' expression specific for adipogenic (peroxisome proliferator-activated receptor-É£, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage-specific differentiation was related to differential expression of miR-17, miR-21, miR-34a, and miR-146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF-MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF-MSCs differentiation in fetus-diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.

Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus-affected gestations: A focus on myogenic and neural differentiations.

Amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are autologous to the fetus and represent a potential alternative source for the regenerative medicine and treatment of perinatal disorders. To date, AF-MSCs differentiation capacity to non-mesodermal lineages and epigenetic regulation are still poorly characterized. The present study investigated the differentiation potential of AF-MSCs toward neural-like cells in comparison to the mesodermal myogenic lineage and assessed epigenetic factors involved in tissue-specific differentiation. Myogenic and neural differentiation assays were performed by the incubation with specific induction media. Typical MSCs markers were determined by flow cytometry, the expression of lineage-specific genes, microRNAs and chromatin modifying proteins were examined by RT-qPCR and Western blot, respectively. AF-MSCs of normal and fetus-affected gestations had similar stem cells characteristics and two-lineage potential, as characterized by cell morphology and the expression of myogenic and neural markers. Two-lineage differentiation process was associated with the down-regulation of miR-17 and miR-21, the up-regulation of miR-34a, miR-146a and DNMT3a/DNMT3b along with the gradual decrease in the levels of DNMT1, HDAC1, active marks of chromatin (H4hyperAc, H3K9ac, H3K4me3) and the repressive H3K9me3 mark. Differentiation was accompanied by the down-regulation of PRC1/2 proteins (BMI1/SUZ12, EZH2) and the retention of the repressive H3K27me3 mark. We report that both AF-MSCs of normal and fetus-affected gestations possess differentiation capacity toward myogenic and neural lineages through rather similar epigenetic mechanisms that may provide potential applications for further investigation of the molecular basis of prenatal diseases and for the future autologous therapy.

Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus-Affected Gestations.

Human amniotic fluid (AF)-derived mesenchymal stem cells (MSCs) sharing embryonic and adult stem cells characteristics are interesting by their multipotency and the usage for regenerative medicine. However, the usefulness of these cells for revealing the fetal diseases still needs to be assessed. Here, we have analyzed the epigenetic environment in terms of histone modifications in cultures of MSCs derived from AF of normal pregnancies and those with fetal abnormalities. The comparison of MSCs samples from AF of normal pregnancies (N) and fetus-affected (P) revealed two distinct cultures by their proliferation potential (P I and P II). Cell populations from N and P I samples had similar growth characteristics and exhibited quite similar cell surface (CD44, CD90, CD105) and stemness markers (Oct4, Nanog, Sox2, Rex1) profile that was distinct in slower growing and faster senescent P II cultures. Those differences were associated with changes in 5-Cyt DNA methylation and alterations in the expression levels of chromatin modifiers (DNMT1, HDAC1/2), activating (H4ac, H3K4me3), and repressive (H3K9me2/me3, H3K27me3) histone marks. MSCs isolated from AF with the genetic or multifactorial fetal diseases (P II samples) were enriched with repressive histone marks and H4K16ac, H3K9ac, H3K14ac modifications. This study indicates that differential epigenetic environment reflects a state of AF-MSCs dependently on their growth, phenotype, and stemness characteristics suggesting a way for better understanding of epigenetic regulatory mechanisms in AF-MSCs cultures in normal and diseased gestation conditions. J. Cell. Biochem. 118: 3744-3755, 2017. © 2017 Wiley Periodicals, Inc.

Fatty acid profiles of monofloral clover beebread and pollen and proteomics of red clover (Trifolium pratense) pollen.

Fatty acids were identified in monofloral beebread (BB) and bee pollen (BP) loads collected from Trifolium pratense L. A gas chromatography method was used to identify and quantify fatty acids: Thirty-five fatty acids were identified in BB and 42 in BP. A high amount of the healthy n-3 fatty acids was found. The ratio of polyunsaturated fatty acids n-3 to n-6 reached a value of 8.42 and 3.35 in the latter products. The proteomic analysis also was performed on the manually collected T. pratense pollen, and the most abundant protein groups were subjected to mass spectrometry analysis. Proteins identified in T. pratense pollen are involved in the main cellular functions (cell membrane formation, organelles traffic, and mainly metabolic processes). Because of the composition of fatty acids in BB and BP and a variety of proteins present in pollen, these products are considered to be favorable for human nutrition and health.

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.

Epigenetic and molecular mechanisms underlying the antileukemic activity of the histone deacetylase inhibitor belinostat in human acute promyelocytic leukemia cells.

Therapeutic strategies targeting histone deacetylase (HDAC) inhibition have become promising in many human malignancies. Belinostat (PXD101) is a hydroxamate-type HDAC inhibitor tested in phase I and II clinical trials in solid tumors and hematological cancers. However, little is known about the use of belinostat for differentiation therapy against acute myelogenous leukemia. Here, we characterize the antileukemia activity of belinostat as a single drug and in combination with all-trans-retinoic acid (RA) in promyelocytic leukemia HL-60 and NB4 cells. Belinostat exerted dose-dependent growth-inhibitory or proapoptotic effects, promoting cell cycle arrest at the G0/G1 or the S transition. Apoptosis was accompanied by activation of caspase 3, degradation of PARP-1, and cell cycle-dependent changes in the expression of survivin, cyclin E1, and cyclin A2. Belinostat induced a dose-dependent reduction in the expression of EZH2 and SUZ12, HDAC-1, HDAC-2, and histone acetyltransferase PCAF (p300/CBP-associated factor). Belinostat increased acetylation of histone H4, H3 at K9 and H3 at K16 residues in a dose-dependent manner, but did not reduce trimethylation of H3 at K27 at proapoptotic doses. Combined treatment with belinostat and RA dose dependently accelerated and reinforced granulocytic differentiation, accompanied by changes in the expression of CD11b, C/EBPα (CCAAT/enhancer binding protein-α), and C/EBPε. Our results concluded the usefulness of belinostat, as an epigenetic drug, for antileukemia and differentiation therapy.

Epigenetic changes by zebularine leading to enhanced differentiation of human promyelocytic leukemia NB4 and KG1 cells.

Aberrant DNA methylation is a critical epigenetic process involved in gene expression of tumor cells. Diverse DNA methyltransferase inhibitors are being studied as potential anticancer drugs, and there is interest in developing novel and more effective DNMTIs. We evaluated zebularine, a stable and low-toxic cytidine analog, effects on human promyelocytic leukemia cell lines, NB4 and KG1. Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth inhibition, did not induce myeloid differentiation but triggered concentration-dependent apoptosis as manifested by procaspase-3 and PAR-1 cleavage and the occurrence of early apoptosis detected by Annexin-V-propidium iodide. Zebularine co-treatment with all-trans retinoic acid (RA) at pharmacological dose (1 µM for NB4 cells) and higher (3 µM for KG1 cells) increased granulocytic differentiation in both cell lines. Pretreatment for 24 or 48 h with zebularine before the treatment with different doses of RA alone or RA with histone deacetylase inhibitors, phenyl butyrate, and BML-210, resulted in significant acceleration and enhancement of differentiation and cell cycle arrest at G0/1. Zebularine alone or in sequential combination with RA decreased expression of DNMT1, caused fast and time-dependent expression of pan-cadherin and partial demethylation of E-cadherin but not tumor suppressor p15. When used in combination with RA, zebularine increased expression of both genes transcript and protein. Zebularine induced regional chromatin remodeling by local histone H4 acetylation and histone H3-K4 methylation in promoter sites of methylated E-cadherin and also in the promoter of unmethylated p21 as evidenced by chromatin immunoprecipitation assay. Our results extend the spectrum of zebularine effects and the evaluation its utility in acute myeloid leukemia therapy based on epigenetics.

DNA methyltransferase inhibitor RG108 and histone deacetylase inhibitors cooperate to enhance NB4 cell differentiation and E-cadherin re-expression by chromatin remodelling.

Epigenetic silencing of cancer-related genes by abnormal methylation and the reversal of this process by DNA methylation inhibitors represents a promising strategy in cancer therapy. As DNA methylation affects gene expression and chromatin structure, we investigated the effects of novel DNMT (DNA methyltransferase) inhibitor, RG108, alone and in its combinations with structurally several HDAC (histone deacetylase) inhibitors [sodium PB (phenyl butyrate) or BML-210 (N-(2-aminophenyl)-N'phenyloctanol diamine), and all-trans RA (retinoic acid)] in the human PML (promyelocytic leukaemia) NB4 cells. RG108 at different doses from 20 to 100 µM caused time-, but not a dose-dependent inhibition of NB4 cell proliferation without cytotoxicity. Temporal pretreatment with RG108 before RA resulted in a dose-dependent cell growth inhibition and remarkable acceleration of granulocytic differentiation. Prolonged treatments with RG108 and RA in the presence of HDAC inhibitors significantly increased differentiation. RG108 caused time-dependent re-expression of methylation-silenced E-cadherin, with increase after temporal or continuous treatments with RG108 and RA, or RA together with PB in parallel, in cell maturation, suggesting the role of E-cadherin as a possible therapeutic marker. These processes required both PB-induced hyperacetylation of histone H4 and trimethylation of histone H3 at lysine 4, indicating the cooperative action of histone modifications and DNA methylation/demethylation in derepression of E-cadherin. This work provides novel experimental evidence of the beneficial role of the DNMT inhibitor RG108 in combinations with RA and HDACIs in the effective differentiation of human PML based on epigenetics.

Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells.

DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.

C/EBPα and PU.1 are involved in distinct differentiation responses of acute promyelocytic leukemia HL-60 and NB4 cells via chromatin remodeling.

C/EBPα and PU.1 are the basic transcription factors that control differentiation-related genes, including granulocyte- colony-stimulating factor (G-CSFR) and human neutrophil elastase (HNE). Here, we analyzed a role of C/EBPα and PU.1 in human acute leukemia cell lines, HL-60 and NB4, in association with a modified chromatin structure by histone deacetylase inhibitors, FK228, sodium phenyl butyrate and vitamin B3. We found that sodium phenyl butyrate alone and 6h-pretreatment with phenyl butyrate or FK228 before the induction of differentiation with all-trans-retinoic acid in the presence of vitamin B3 effectively accelerated and enhanced differentiation to granulocytes in HL-60 but not in NB4 cells as detected by NBT test and the expression of CD11b and CD114 (G-CSFR) using flow cytometric analysis. HDACIs induced a time- and dose-dependent accumulation of hyper-acetylated histone H4 in both cell lines with the delay in NB4 cells. Time-dependent different induction of HL-60 and NB4 cell differentiation was paralleled by the activation of C/EBPα and PU.1 binding to the G-CSFR and the HNE promoters in electrophoretic mobility shift assay. Chromatin immunoprecipitation analysis revealed histone H4 acetylation in the G-CSF receptor promoter at the C/EBPα binding site in HL-60 but not in NB4 cells under the combined treatment. The results indicate that epigenetic events, such as histone acetylation, are involved in the activity modulation of the key transcription factors responsible for the induction of granulocytic differentiation in promyelocytic leukemia cells.

Comparative Proteomic Assessment of Normal vs. Polyhydramnios Amniotic Fluid Based on Computational Analysis.

Mass spectrometry-based proteomics have become a valued tool for conducting comprehensive analyses in amniotic fluid samples with pathologies. Our research interest is the finding and characterization of proteins related to normal vs. polyhydramnios (non-immune hydrops) pregnancy. Proteomic analysis was performed on proteins isolated from fresh amniotic fluid samples. Proteins were fractionated by 2DE using a different pI range (pI 3-11, pI 4-7) and analyzed with MALDI-TOF-MS. Furthermore, by using computational analysis, identified proteins in protein maps specific to normal vs. polyhydramnios pregnancy were compared and the quantities of expressed proteins were evaluated mathematically. Comparative analysis of proteome characteristic for the same polyhydramnios pregnancy fractionated by 2DE in different pI range (3-11 and 4-7) was performed and particular protein groups were evaluated for the quantification of changes within the same protein level. Proteins of normal and polyhydramnios pregnancies were fractionated by 2DE in pI range 3-11 and in pI range 4-7. Mass spectrometry analysis of proteins has revealed that the quantity changes of the main identified proteins in normal vs. polyhydramnios pregnancy could be assigned to immune response and inflammation proteins, cellular signaling and regulation proteins, metabolic proteins, etc. Specifically, we have identified and characterized proteins associated with heart function and circulatory system and proteins associated with abnormalities in prenatal medicine. The following are: serotransferrin, prothrombin, haptoglobin, transthyretin, alpha-1-antitrypsin, zinc-alpha-2-glycprotein, haptoglobin kininogen-1, hemopexin, clusterin, lumican, afamin, gelsolin. By using computational analysis, we demonstrated that some of these proteins increased a few times in pathological pregnancy. Computer assistance analysis of 2DE images suggested that, for the better isolation of the proteins' isoforms, those levels increased/decreased in normal vs. polyhydramnios pregnancy, and the fractionation of proteins in pI rage 3-11 and 4-7 could be substantial. We analyzed and identified by MS proteins specific for normal and polyhydramnios pregnancies. Identified protein levels increased and/or modification changed in case of non-immune hydrops fetus and in cases of cardiovascular, anemia, growth restriction, and metabolic disorders. Computational analysis for proteomic characterization empower to estimate the quantitative changes of proteins specific for normal vs. polyhydramnios pregnancies.

Characterization of human alpha-dystrobrevin isoforms in HL-60 human promyelocytic leukemia cells undergoing granulocytic differentiation.

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.

Effects of histone deacetylase inhibitors, sodium phenyl butyrate and vitamin B3, in combination with retinoic acid on granulocytic differentiation of human promyelocytic leukemia HL-60 cells.

Water-soluble vitamin B3, niacin, and its related compounds were suggested to be applicable for medical use. In this article, we examined the anti-leukemic effects of two distinct histone deacetylase (HDAC1 and Sir2) inhibitors, sodium phenyl butyrate (PB) and vitamin B3, respectively, on human promyelocytic leukemia cells HL-60, using HDACIs alone and in combination with all trans retinoic acid (RA). We demonstrated that the HDACI combinations exert different effects on cell cycle arrest and differentiation as determined by nitro blue reduction and the expression of the early myeloid differentiation marker CD11b. The most beneficial effects were found by use of 6-h pretreatment with PB and vitamin B3 before the exposition to RA alone or in combination with vitamin B3, showing significant acceleration and a high level of granulocytic differentiation. The effects were associated with a rapid histone H4 acetylation and later histone H3 modifications. Our results suggest that the use of two HDACI altogether before the induction of differentiation and acting via chromatin remodeling may be promising for the treatment of acute promyelocytic leukemia.

The histone deacetylase inhibitor FK228 distinctly sensitizes the human leukemia cells to retinoic acid-induced differentiation.

FK228 (depsipeptide) is a novel histone deacetylase inhibitor (HDACI) that has shown therapeutical efficacy in clinical trials for malignant lymphoma. In this article, we examined in vitro effects of FK228 on human leukemia cell lines, NB4 and HL-60. FK228 alone (0.2-1 ng/mL) inhibited leukemia cell growth in a dose-dependent manner and induced death by apoptosis. FK228 had selective differentiating effects on two cell lines when used for 6 h before induction of granulocytic differentiation by retinoic acid (RA) or in combination with RA. These effects were accompanied by a time- and dose-dependent histone H4 hyper-acetylation or histone H3 dephosphorylation and alterations in DNA binding of NF-kappaB in association with cell death and differentiation. Pifithrin-alpha (PFT), an inhibitor of p53 transcriptional activity, protected only NB4 cells with functional p53 from FK228-induced apoptosis and did not interfere with antiproliferative activity in p53-negative HL-60 cells. In NB4 cells, PFT inhibited p53 binding to the p21 (Waf1/Cip1) promotor and induced DNA binding of NF-kappaB leading to enhanced cell survival. Thus, beneficial effects of FK228 on human promyelocytic leukemia may be exerted through the induction of differentiation or apoptosis via histone modification and selective involvement of transcription factors, such as NF-kappaB and p53.

The novel histone deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines.

Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 (N-(2-Aminophenyl)-N' phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents - all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-kappaB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-kappaB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.

Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy.

Human amniotic-fluid-derived mesenchymal stem cells (AF-MSCs) are interesting for their multilineage differentiation potential and wide range of therapeutic applications due to the ease of culture expansion. However, MSCs undergo replicative senescence. So far, the molecular mechanisms that underlie fetal diseases and cell senescence are still poorly understood. Here, we analyzed senescence-associated morphologic, molecular, and epigenetic characteristics during propagation of MSCs derived from AF of normal and fetus-affected pregnancy. AF-MSCs cultures from both cell sources displayed quite similar morphology and expression of specific cell surface (CD44, CD90, and CD105) and stemness (Oct4, Nanog, Sox2, and Rex1) markers but had interindividual variability in proliferation capability and time to reach senescence. Within passages 4 and 8, senescent cultures exhibited typical morphological features, senescence-associated ß-galactosidase activity, increased levels of p16, and decreased levels of miR-17 and miR-21 but showed differential expression of p21, p53, and ATM dependently on the onset of cell senescence. These differences correlated with changes in the level of chromatin modifiers (DNMT1 and HDAC1) and polycomb group proteins (EZH2, SUZ12, and BMI1) paralleling with changes in the expression of repressive histone marks (H3K9me3 and H3K27me3) and stemness markers (Oct4, Nanog, Sox2, and Rex1). Therefore epigenetic factors are important for AF-MSCs senescence process that may be related with individuality of donor or a fetus malignancy status.

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy.

Xenograft models are suitable for in vivo study of leukemia's pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.

Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis.

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

Alterations in protein expression in HL-60 cells during etoposide-induced apoptosis modulated by the caspase inhibitor ZVAD.fmk.

DNA topoisomerase inhibitors induce a specific signaling cascade that promotes an active apoptotic caspase-dependent cell death process. However, little is known about the initial signals elicited by these agents. In the present study, we compared apoptosis in HL-60 cells treated either with the chemotherapeutic drug etoposide (VP16) alone or combined with the broad caspase inhibitor ZVAD.fmk. Apoptosis was assessed by changes in cell morphology and agarose gel electrophoresis of extracted cell DNA. We found that ZVAD.fmk prevents VP16-induced DNA fragmentation and the appearance of an increased number of apoptotic cells in the culture. We also compared the effects of etoposide alone or together with the pan-caspase inhibitor ZVAD.fmk on proliferating cell nuclear antigen, Bcl-2, and actin expression in human promyelocytic leukemia HL-60 cells. In addition, we screened for proteins that were initially upregulated in a caspase-dependent manner. Indeed, some proteins were induced in the cytoplasm and subsequently accumulated in the nuclei after etoposide treatment. This process was slightly inhibited by the caspase inhibitor ZVAD.fmk. We suggest that these proteins are associated with the induction of specific signaling cascades that characterize the apoptotic cell death process.

Sp1 and NF-kappaB transcription factor activity in the regulation of the p21 and FasL promoters during promyelocytic leukemia cell monocytic differentiation and its associated apoptosis.

Treatment of human acute promyelocytic leukemia cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest and differentiation toward monocytes, which subsequently die by apoptosis. However, the relationship between terminal differentiation and apoptosis remains unclear. Here we have studied Sp1 and nuclear factor kappaB (NF-kappaB) transcription factor activity in controlling promoters of cell cycle-regulating (p21/WAF1/CIP1) and cell death (FasL) genes during monocytic differentiation and apoptosis of the human acute promyelocytic leukemia cell lines NB4 and HL-60. Using the electrophoretic mobility shift assay, we observed that PMA treatment of NB4 cells caused an early response in Sp1 binding to the p21 and FasL promoters at 8 h. The firmly adherent cell phenotype, characteristic of differentiated cells, retained Sp1-binding activity to either promoter, but it was often lost completely in detached, apoptotic cells. The association of Sp1 with the p21 promoter during monocytic differentiation correlated with the levels of expressed p21 in the cytoplasmic fraction, as detected by immunoblotting. In HL-60 cells, very weak or no Sp1 binding to either promoter was observed. Low NF-kappaB affinity for its consensus sites and to the FasL promoter was characteristic of apoptotic cells. The results of this study suggest a positive role of Sp1 and NF-kappaB, as regulators of p21 and FasL genes, in leukemic cell survival and monocytic differentiation and a negative role in apoptotic cell death.