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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 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.

Acute promyelocytic leukemia KG1 cells with t(11;17) PLZF-RARalpha respond poorly to the differentiation inducer all-trans retinoic acid (RA), and the reason for the RA resistance is the recruitment of histone deacetylase by PLZF-RARalpha. Here, we demonstrate that histone deacetylase inhibitors (HDACIs), FK228, BML-210, phenyl butyrate, and vitamin B3, in different combinations with RA, act as KG1 cell growth inhibitors. Partial differentiation to granulocytes was induced by 3 micromol/L RA, and its combination with HDAC inhibitors did not enhance RA-induced but potentiated apoptosis. HDACIs induced accumulation of hyperacetylated histone H4. Chromatin immunoprecipitation analysis has revealed phenyl butyrate and its combinations with RA and vitamin B3 cause histone H4 acetylation in the p21 promoter regions corresponding to p53 and/or Sp1 sites. This was coincident with the activation of the transcription factor p53-binding activity to the p21 promoter in electrophoretic mobility shift assay. The results indicate the possibility of using the combination of agents for therapeutic strategy in RA-resistant acute myeloid leukemia to produce both differentiation and apoptosis.

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.

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.

A main shortcoming of using HL-60 cells as a model of granulocyte-macrophage differentiation is that some cells in the differentiating population undergo apoptosis. To address this issue, we have identified which tyrosine-phosphorylated proteins are involved in apoptosis and differentiation, respectively. HL-60 cells were induced specifically to undergo apoptosis with 68 microM etoposide, and to undergo granulocytic differentiation with 1 microM retinoic acid (RA). The corresponding two-dimensional electrophoretic maps of tyrosine-phosphorylated proteins from treated cells were compared. In the 8 h etoposide-treated HL-60 cell population, 83% of the cells were apoptotic. In the 120 h RA-treated cells, 50% of the cells were apoptotic. Eighteen cytosolic and nuclear tyrosine-phosphorylated proteins were found in both the 8 h etoposide- and the 120 h RA-treated cells, but not in the proliferating HL-60 cell population. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses suggested that some of the proteins may be involved in signal transduction pathways (NFkappaB, GTP-binding protein, protein disulfide isomerase, Cyclophilin A), others in cell transcriptional and translational control (hnRNP H, hnRNP L, Hsp60, Hp1, Hcc-1, 26S proteasome beta-subunit, ATP synthase beta-chain), and a third group in cell cytoskeleton organization and receptor cycling (profilin, caveolin-1). An understanding of signal transduction in apoptosis initiation by screening for tyrosine-phosphorylated proteins associated with apoptosis may provide new targets for the treatment of leukemia.

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.

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.

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 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.

Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPβ and c-Myb) and for survival of differentiated cells (STATs and NFκB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid. c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPβ, which suggests a combinatorial interaction of these transcription factors in the granulocytic signalling pathway. Expression of STAT5a and STAT5b varied during differentiation, whereas no significant changes were seen in STAT3 levels. Increased cytosolic level of NFκB p65 during precommitment and commitment stages of granulocytic differentiation coincided with augmentation of the STAT5a protein level, which could be evidence of their possible cooperation during granulocytic-lineage commitment of HL-60 cells. Our results suggest that the studied transcription factors cooperatively promote signalling in the differentiating promyelocytic HL-60 cell line in response to retinoic acid.Key words: C/EBP, Myb, STAT, NFκB, phosphorylation.

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6–24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48–120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48–120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N6,O2-dibutyryl adenosine 3':5'-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12-72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35-110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus.