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Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3–72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML). [ABSTRACT FROM AUTHOR]

Membranous CD14 is crucial in the phagocytic activity of neutrophils. However, the role of CD14(+) microparticles (MPs) derived from apoptotic neutrophils (apo-MP) during the phagocytic process is not clear. All trans-retinoic acid (ATRA) induces acute promyelocytic leukemic NB4 cells along granulocytic differentiation. In this study, we investigated the role of CD14(+)apo-MP in the cell–cell interaction during the phagocytic process of apoptotic cells by viable ATRA-NB4 cells. We firstly demonstrate that CD14 expression and phagocytic activity of NB4 cells were upregulated simultaneously after ATRA treatment in a time-dependent manner, and both were significantly enhanced via concurrent lipopolysaccharide treatment. The phagocytic activity of ATRA-NB4 cells and lipopolysaccharide-treated ATRA-NB4 cells were both significantly attenuated by pre-treating cells with an antibody specific to either CD14 or TLR4. Further flow cytometric analysis demonstrates that apoptotic ATRA-NB4 cells release CD14(+)apo-MP in an idarubicin dosage-dependent manner. Both CD14 expression and the phagocytic activity of viable ATRA-NB4 cells were significantly enhanced after incubation with apo-MP harvested from apoptotic ATRA-NB4 cells, and the apo-MP-enhanced phagocytic activity was significantly attenuated by pre-treating apo-MP with an anti-CD14 antibody before incubation with viable cells. We conclude that CD14(+)apo-MP derived from apoptotic ATRA-NB4 cells promotes the phagocytic activity of viable ATRA-NB4 cells in engulfing apoptotic cells. [ABSTRACT FROM AUTHOR]

Membranous CD14 is crucial in the phagocytic activity of neutrophils. However, the role of CD14(+) microparticles (MPs) derived from apoptotic neutrophils (apo-MP) during the phagocytic process is not clear. All trans-retinoic acid (ATRA) induces acute promyelocytic leukemic NB4 cells along granulocytic differentiation. In this study, we investigated the role of CD14(+)apo-MP in the cell–cell interaction during the phagocytic process of apoptotic cells by viable ATRA-NB4 cells. We firstly demonstrate that CD14 expression and phagocytic activity of NB4 cells were upregulated simultaneously after ATRA treatment in a time-dependent manner, and both were significantly enhanced via concurrent lipopolysaccharide treatment. The phagocytic activity of ATRA-NB4 cells and lipopolysaccharide-treated ATRA-NB4 cells were both significantly attenuated by pre-treating cells with an antibody specific to either CD14 or TLR4. Further flow cytometric analysis demonstrates that apoptotic ATRA-NB4 cells release CD14(+)apo-MP in an idarubicin dosage-dependent manner. Both CD14 expression and the phagocytic activity of viable ATRA-NB4 cells were significantly enhanced after incubation with apo-MP harvested from apoptotic ATRA-NB4 cells, and the apo-MP-enhanced phagocytic activity was significantly attenuated by pre-treating apo-MP with an anti-CD14 antibody before incubation with viable cells. We conclude that CD14(+)apo-MP derived from apoptotic ATRA-NB4 cells promotes the phagocytic activity of viable ATRA-NB4 cells in engulfing apoptotic cells.

Background/Aims: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. Methods: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. Results: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. Conclusions: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

The promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion gene is present in 98% of the patients with acute promyelocytic leukemia (APL), the M3 subtype of acute myeloid leukemia (AML). All-trans retinoic acid (ATRA) is widely used to treat patients with APL. Nucleolar Protein 7 (NOL7) is a tumor suppressor gene regulated by retinoid X receptor (RXR). The 6p23 chromosomal region, where NOL7 is located, is associated with many cancers, including AML. Here, we aimed to investigate the effect of NOL7 in two AML cell lines (the PML-RARA-positive NB4 cell line and the PML-RARA-negative HL60 cell line) and in the resistance to ATRA. For this purpose, we knocked down NOL7 expression by using short interfering RNA (siRNA) and stimulated the cells with ATRA. Apoptosis, cell viability, proliferation, and granulocytic differentiation analyses were performed. Our findings show that granulocytic differentiation was blocked in ATRA-treated NB4 cells 24 hours after NOL7 siRNA transfection when the expression of NOL7 had been reduced by 81%. Downregulation of NOL7 had no apparent effect on the cellular proliferation and apoptosis. Our study suggests that ATRA-induced granulocytic differentiation is inhibited in NOL7-downregulated NB4 cells. These results suggest that NOL7 expression contributes to ATRA-induced granulocytic differentiation and loss of NOL7 might be involved in the development of ATRA resistance. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) with various sulfation patterns is one of important modulators of cancer cell fate through interacting with numerous growth factors. Here we found HS 2-O sulfotransferase 1 (HS2ST1) was downregulated at both mRNA and protein levels during granulocytic differentiation of SKM-1 leukemia cells and also HS (glucosamine) 3-O sulfotransferase 3A (HS3ST3A) in epithelial-mesenchymal transition (EMT) of A549 lung cancer cells, meanwhile, cell-surface HS recognized by anti-HS antibody was also changed in both cancer cell lines. Further, HS3ST3A was negatively correlated with the in vitro cell metastasis capability of A549 cells confirmed by RNA interference technology, wound-healing assay and in vitro Matrigel invasion assay. Together, specific HS sulfotransferases may serve as molecular markers and targets for cancer treatment. [ABSTRACT FROM AUTHOR]

Leukocyte differentiation antigens (LDAs) play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 (leucine rich repeat-containing 25) was predicted to have a role in the function of myeloid cells by a large-scale “omics” data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia (AML) cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid (ATRA)-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia (APL; AML-M3, FAB classification) cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced terminal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation. [ABSTRACT FROM AUTHOR]

Emerging evidences have shown that long non-coding RNAs (lncRNAs) play critical roles in cancer development and cancer therapy. LncRNA Nuclear Enriched Abundant Transcript 1 (NEAT1) is indispensable during acute promyelocytic leukemia (APL) cell differentiation induced by all- trans retinoic acid (ATRA). However, the precise mechanism of NEAT1 upregulation has not been fully understood. In this study, we performed chromatin immunoprecipitation and luciferase reporter assays to demonstrate that C/EBP family transcription factor C/EBPβ bind to and transactivate the promoter of lncRNA NEAT1 through the C/EBPβ binding sites both around −54 bp and −1453 bp upstream of the transcription start site. Moreover, the expression of C/EBPβ was increased after ATRA treatment, and the binding of C/EBPβ in the NEAT1 promoter was also dramatically increased. Finally, knockdown of C/EBPβ significantly reduced the ATRA-induced upregulation of NEAT1. In conclusion, C/EBPβ directly activates the expression of NEAT1 through binding to the promoter of NEAT1. Knockdown of C/EBPβ impairs ATRA-induced transcriptional activation of NEAT1. Our data indicate that C/EBPβ contributes to ATRA-induced activation of NEAT1 during APL cell differentiation. Our results enrich our knowledge on the regulation of lncRNAs and the regulatory role of C/EBPβ in APL cell differentiation. [ABSTRACT FROM AUTHOR]

Background/Aims: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. Methods: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. Results: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. Conclusions: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells. [ABSTRACT FROM AUTHOR]

Development of acute myeloid leukemia is usually sustained by deregulated epigenome. Alterations in DNA methylation and histone modifications are common manifestations of the disease. Acute promyelocytic leukemia (APL) is not an exception. Therefore, drugs that target epigenetic processes suggest an appealing strategy for APL treatment. In this study we tested the anti-leukemic activity of histone deacetylase inhibitor (HDACi) Belinostat (PXD101, (2 E )- N -Hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide), and histone methyltransferase inhibitor (HMTi) 3-Deazaneplanocin A (DZNep, 5R-(4-amino-1H-imidazo[4,5- c ]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1S,2R-diol) combined with retinoic acid (RA) in APL cells NB4 and HL-60. We demonstrated that APL cell treatment with combinations of differentiation inductor RA, HDACi Belinostat and HMTi DZNep caused a depletion of leukemia cell growth and viability, initiated apoptosis and exaggerated RA induced granulocytic differentiation. Also an increased expression of transcription factors C/EBPε and PPARγ was demonstrated, while no significant reduction in C/EBPα gene level was detected. Furthermore, combined treatment depleted gene expression levels of EZH2 and SUZ12, especially in HL-60 cells, and diminished protein levels of Polycomb Repressive Complex 2 (PRC2) components EZH2, SUZ12 and EED. In addition, our study has shown that Belinostat and DZNep together with RA caused a depletion in HDAC1 and HDAC2 protein levels, HDAC2 gene expression and increased hyperacetylation of histone H4 in both leukemia cell lines. Using ChIP method we also demonstrated the increased association of hyperacetylated histone H4 with the C/EBPα and C/EBPε promoter regions in HL-60 cells. Summarizing, these findings indicate that combined treatment with RA, Belinostat and 3-Deazaneplanocin A is an effective epigenetic inducer for leukemia cell differentiation. [ABSTRACT FROM AUTHOR]

Today, cancer is understood as an epigenetic as well as genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. Proteins such as histone deacetylases (HDACs) that cause modifications of histones and other proteins can be targets for novel anticancer agents. Recently, interest in compounds that can inhibit HDACs increased, and now there are many HDACs inhibitors (HDACIs) available with different chemical structures, biological and biochemical properties; hopefully some of them will succeed, probably in combination with other agents, in cancer therapies. In our study we focused on the novel HDACI-BML-210. We found that BML-210 (N-phenyl-N'-(2-Aminophenyl)hexamethylenediamide) inhibits the growth of NB4 cells in dose-and time-dependent manner. In this study we also examined how expression and activity of HDACs are affected after leukemia cell treatment with BML-210. Using a mass spectrometry method we identified proteins that changed expression after treatment with BML-210. We prepared RT-PCR analysis of these genes and the results correlated with proteomic data. Based on these and other findings from our group, we suggest that HDACIs, like BML-210, can be promising anticancer agents in promyelocytic leukemia treatment. [ABSTRACT FROM AUTHOR]

Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases ( HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia ( APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid ( RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers ( HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells. [ABSTRACT FROM AUTHOR]

A series of α-branched α,β-unsaturated ketones were prepared via boron trifluoride etherate mediated reaction between arylalkynes and carboxaldehydes. The evaluation of the antiproliferative activity over hematological (NB4) and solid cancer (A549, MCF-7) cell lines provided a structure-activity relationship. 5-Parameter QSAR equations were built which were able to explain 80%–92% of the variance in activity. The resulting selective lead compound showed IC 50 value 0.6 μM against the hematological cell line and did not cause apoptosis, but blocked cell cycle in G0/G1. Moreover, it was demonstrated that this compound enhances and accelerates retinoic acid induced granulocytic differentiation. [ABSTRACT FROM AUTHOR]

Abstract: We explored the potential involvement of FoxO3a activation in erythroid and granulocytic differentiation for Ph+ cells of chronic myeloid leukemia blast crisis (CML BC). We demonstrate that FoxO3a activation in CML blast crisis (BC) cells by overexpressing FoxO3a leads to the maturation of CML BC cells. Hemoglobin production significantly increased upon FoxO3a activation in CML BC cells. FoxO3a activation upregulated erythroid surface protein (glycophorin A, GPA), but did not significantly modulate granulocytic markers (CD11b). Additionally, FoxO3a activation reduced the mRNA and protein expression of Tal1. Similar results were observed in cells that were given nilotinib. Our results indicate that FoxO3a activation may promote erythroid differentiation of BC cells via down-regulating Tal1 expression. [Copyright &y& Elsevier]

Dystrobrevins (DBs) bind directly to dystrophin and are prominent components of the dystrophin-associated protein complex (DAPC) that links the cytoskeleton to the extracellular matrix. They are involved in brain development, synapse formation and plasticity, as well as water and ion homeostasis. However, the role of DB in non-muscular cells is not clear. In this study, we show that different α-dystrobrevin isoforms are present in promyelocytic leukemia (NB4) cells. Only the biggest α-dystrobrevin isoform (DB-α), which can be important for its function, was expressed in the membrane fraction of NB4 cells; the other α-DB isoforms were found in the hydrophilic cell fractions. Employing the immunoprecipitation and mass spectrometry, we identified novel α-DB-interacting proteins involved in cytoskeleton reorganization (actin, tropomyosin, gelsolin, tubulin) and signal transduction process (stathmin, prohibitin, RIBA) during proliferation and differentiation of NB4 cells. Our results suggest that α-DB isoforms play a central role in cytoskeleton reorganization via their multiple interactions with actin and actin-associating proteins and may participate in signal transduction process during NB4 cell granulocytic differentiation via directly and non directly associated proteins. [ABSTRACT FROM AUTHOR]

Abstract: The present study was undertaken to provide more information on the differentiation and maturation of human granulocytes using computer-assisted image RNA densitometry at single-cell level. The bone marrow of patients suffering from chronic phase of chronic myeloid leukemia represents a very convenient model for such measurements because of the satisfactory number of early stages, as well as advanced stages, of the granulocytic cell lineage represented by neutrophils. In contrast to the erythroid cell lineage, similar nucleolar and cytoplasmic RNA density–concentration values were found only in early granulocytic progenitors such as myeloblasts and promyelocytes. In advanced stages of the granulocytic development starting with myelocytes, these cells were characterized by a larger decrease in the cytoplasmic RNA concentration in comparison with that of the nucleoli. Thus, the nucleolar to cytoplasmic RNA concentration ratio in these cells was above 1. On the other hand, it should be pointed out that late differentiation stages of granulocytes, starting with myelocytes, possessed nucleolar bodies (nucleoli without surrounding perinucleolar chromatin) of a markedly reduced size. [ABSTRACT FROM AUTHOR]

The biological effects of low-dose radiation have attracted attention, but data are currently insufficient to fully understand the beneficial role of the phenomenon. In the present study, we have investigated the effects of low doses of gamma-irradiation alone and in combination with all- trans-retinoic acid (RA) on proliferation, apoptosis and differentiation of the human promyelocytic leukemia HL-60 cells. Changes in cell behavior and protein expression were determined with the use of light and fluorescent microscopy, immunocytochemical and Western blot analysis. Low-dose irradiation with 1–100 cGy caused a dose-dependent inhibition of HL-60 cell proliferation, and induced apoptosis and differentiation to granulocytes with an increase in the number of CD15-positive cells. Pre-irradiation with 1–100 cGy for 24 h before treatment with RA promoted apoptosis but did not impair RA-induced differentiation. Both processes were associated with a decrease in the expression of the proliferating cell nuclear antigen (PCNA), BCL-2, c-MYC, and changes in both cytosolic and nuclear levels of protein tyrosine-phosphorylation as well as protein kinase C alpha or beta isoforms. These results demonstrate the beneficial role of low-dose irradiation in modulating leukemia cell proliferation, differentiation and apoptosis. [ABSTRACT FROM AUTHOR]

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all- trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl- l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-κB. [ABSTRACT FROM AUTHOR]

Abstract: Expression of N-myc downregulated gene 1 (NDRG1) is associated with growth arrest and differentiation of tumor cells. In hematopoietic cells, NDRG1 was identified in a screen for differentiation-related genes in human myelomonocytic leukemic U937 cells. In the present study, we found significantly higher NDRG1 mRNA levels in granulocytes of healthy donors than in primary acute myeloid leukemia (AML) cells. Another NDRG family member, NDRG2, was significantly higher expressed in normal macrophages compared to primary AML cells. Moreover, NDRG1 mRNA levels increased in two acute promyelocytic leukemia (APL) patients as well as in NB4 and HT93 APL cells upon all-trans retinoic acid (ATRA) therapy. In line with these observations, silencing of NDRG1 diminished neutrophil differentiation of leukemic cell lines. In conclusion, we found an association of low NDRG1 levels with an immature cell phenotype and provide evidence that NDRG1 is functionally involved in neutrophil maturation. [Copyright &y& Elsevier]

Abstract: Exocytosis of neutrophil granules is a major event that converts circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis and destruction of pathogens. The PLB-985 cell line is a suitable neutrophilic cellular model which is utilised to study the different functional responses of neutrophils. In this study, we characterised the differentiation of PLB-985 cells toward the granulocytic pathway, using three different inducing agents: dbcAMP, DMSO and DMF. The differentiation efficiency was monitored by observation of cell morphology with electron microscopy, and by analysis of the expression of receptors such as FPRL1 and FcγRIIA, the distribution or release of granule markers, phagocytic capacity, as well as measurement of fMLF-induced calcium fluxes. Exocytosis and phagocytosis in differentiated cells were weaker as compared to neutrophils. fMLF stimulated primary granule exocytosis in cells differentiated with dbcAMP, DMSO and DMF, whereas the release of the contents of tertiary granules, as well as that of secretory vesicles, was only observed in dbcAMP-differentiated cells. DMSO-differentiated cells exhibited the highest phagocytic capacity. Altogether our results reinforce the fact that depending on the differentiating agent used, PLB-985 cells represent a useful model to study neutrophil functions and to bypass difficulties inherent to these primary cells. [Copyright &y& Elsevier]

The human HL-60 promyelocytic leukemia cell line has been widely used as a model for studying granulocytic differentiation. All- trans retinoic acid (ATRA) treatment of HL-60 cells promotes granulocytic differentiation and is effective as differentiation therapy for patients with acute promyelocytic leukemia. The identification of genes that are transcriptionally regulated by ATRA has provided insight into granulocytic differentiation and differentiation therapy. The Asb-2 (ankyrin repeat SOCS box 2) gene has previously been identified as a transcriptional target in ATRA-treated HL-60 cells. The ASB-2 protein forms an E3 ubiquitin ligase complex with the proteins, Cul5, regulator of cullin 2 (ROC2), and elongin B and C. The purpose of this study was to determine if there is increased expression of Cul5 during granulocytic differentiation of HL-60 cells. To induce granulocytic differentiation, HL-60 cells were treated for 5 d with ATRA and differentiation was confirmed by examining superoxide anion production, nuclear morphology, and changes in the expression of CD11b, CD13, and CD15. Quantitative real-time RT-PCR was used to measure Cul5 mRNA expression and also the expression of other components of the E3 ubiquitin ligase (ASB-2, ROC2, elongin B and C). Granulocytic differentiation of HL-60 cells was associated with a 1.6-, 1.7-, and 23-fold statistically significant ( P ≤ 0.05) increase in mRNA expression for Cul5, ROC2, and ASB-2, respectively. No significant change was found in elongin B and C mRNA expression. Using Western blot analysis, the expression of Cul5 protein was increased 6.5-fold with granulocytic differentiation of the HL-60 cells. Increased expression of multiple components of the Cul5-containing E3 ubiquitin ligase complex with ATRA treatment of HL-60 cells indicates that this complex may play an important role in granulocytic differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Wogonin, a natural monoflavonoid, has been shown to have tumor therapeutic potential in vitro and in vivo. Recently many studies have focused on the induction of apoptosis of tumor cells by wogonin. In this study, we found that wogonin could induce differentiation and G1 phase arrest of human U-937 leukemia cells. The growth of U-937 cells incubated with wogonin was inhibited in a time- and concentration-dependent manner. After treatment with wogonin, U-937 cells exhibited the characteristics of mature granulocytes, such as increased cytoplasmic-to-nuclear ratio, enhanced prominence of cytoplasmic granules, membrane ruffling, a higher NBT-reducing ability, and an increased expression of CD11b. Moreover, wogonin could induce G1 phase arrest and influenced the expression of associated proteins. For example, the expression of phorsphorylated protein kinase C (PKC) δ, p21 increased, while that of cyclin D1/cyclin-dependent kinase (CDK) 4, p-Rb decreased. The upregulation of p21 could be reversed by rottlerin, an inhibitor of PKCδ. Taken together, wogonin induced U-937 cells to undergo granulocytic differentiation and G1 phase arrest via PKCδ phosphorylation-induced upregulation of p21 proteins. [Copyright &y& Elsevier]

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 ( HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis. [ABSTRACT FROM AUTHOR]

Promyelocytic leukemia (PML) is a nuclear protein that functions as a regulator of transcription, cell proliferation, apoptosis and myeloid cell differentiation. PML is subjected to post-translational modifications such as sumoylation and phosphorylation. However, the physiological significance of these modifications, especially for myeloid cell differentiation, remains unclear. In this report, we found that four serine residues in the PML C-terminal region are highly phosphorylated in a myeloid cell line. Wild-type PML accelerated G-CSF-induced granulocytic differentiation, but a phosphorylation-deficient PML mutant failed. PML interacted with C/EBP epsilon, a transcription factor essential for granulopoiesis, activated C/EBP epsilon-mediated transcription in concert with p300 and accelerated C/EBP epsilon-induced granulocytic differentiation. Phosphorylation of PML was required for stimulating C/EBP epsilon-dependent transcription and accelerating C/EBP epsilon-induced granulocytic differentiation. We also found that PML phosphorylation was required for stimulation of PU.1-dependent transcription and acceleration of PU.1-induced granulocytic differentiation. These results suggest that phosphorylation plays essential roles in the regulation of PML to accelerate granulocytic differentiation through multiple pathways. [ABSTRACT FROM AUTHOR]

Abstract: A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a–i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a–l). Compounds 3a–i and 4a–l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2. Tested at 5μM against human HDAC1 and HDAC4, 3b, 4a, and 4j showed significant HDAC1 inhibition, whereas on HDAC4 only 4a was highly effective. On the human leukemia U937 cell line, the same compounds did not alter the cell cycle phases and failed in inducing apoptosis. However, they displayed granulocytic differentiation at 5μM, with 3b being the most potent (76% CD11c positive cells). Tested to evaluate their effects on histone H3 and α-tubulin acetylation, 3b and 4a showed high H3 acetylation, whereas 4a and 4b were the most potent with α-tubulin as a substrate. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: All-trans-retinoic acid (RA) and 1α,25-dihydroxyvitamin D3 (1,25D3) are involved in the control of hematopoiesis and have been suggested to play a role in cellular differentiation and are as such potent inducers of differentiation of myeloid leukemia cells. In this study, we show that, in promyelocytic NB4 cells, addition of 1,25D3 enhances terminal granulocytic RA-dependent differentiation concomitant with the enhanced activation of the RA transcriptional activity through an RARβ promoter. By EMSA and ChIP assays, we further demonstrate that, while both VDR and RAR are bound to the RARβ promoter in NB4 cells, addition of 1,25D3 increases VDR binding to this promoter, while that of RA induces the release of VDR and increases the binding of RAR. Thus, contrary to normal myeloid cells, 1,25D3 does not act as a transrepressor of RA transcriptional activity in leukemic cells, suggesting that transcriptional regulation of RA-target genes may be modified in malignant cells. In promyelocytic leukemic cells, the combination of 1,25D3 and RA results in both enhanced transactivation and differentiation. [Copyright &y& Elsevier]

Abstract: Vav is one of the genetic markers that correlate with the differentiation of hematopoietic cells. In T and B cells, it appears crucial for both development and functions, while, in non-lymphoid hematopoietic cells, Vav seems not involved in cell maturation, but rather in the response of mature cells to agonist-dependent proliferation and phagocytosis. We have previously demonstrated that the amount and the tyrosine phosphorylation of Vav are up-regulated in both whole cells and nuclei of tumoral promyelocytes induced to granulocytic maturation by ATRA and that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity. In this study, we report that Vav accumulates in nuclei of ATRA-treated APL-derived cells and that the down-modulation of Vav prevents differentiation of tumoral promyelocytes, indicating that it is a key molecule in ATRA-dependent myeloid maturation. On the other hand, the overexpression of Vav induces an increased expression of surface markers of granulocytic differentiation without affecting the maturation-related changes of the nuclear morphology. Consistent with an effect of Vav on the transcriptional machinery, array profiling shows that the inhibition of the Syk-dependent tyrosine phosphorylation of Vav reduces the number of ATRA-induced genes. Our data support the unprecedented notion that Vav plays crucial functions in the maturation process of myeloid cells, and suggest that Vav can be regarded as a potential target for the therapeutic treatment of myeloproliferative disorders. [Copyright &y& Elsevier]

CCAAT/enhancer-binding proteins (C/EBPs) are a family of highly conserved transcription factors that have important roles in normal myelopoiesis as well as associated with myeloid disorders. The chronic myelogenous leukemia (CML) cell lines, KCL22 and K562, express exceptionally low levels of endogenous C/EBPs and provide a good model to test the effects of C/EBPs on myeloid differentiation. To explore the possibility that C/EBPdcan promote differentiation in BCR-ABL-positive cells, we generated stable KCL22 and K562 clones that expressed an inducible C/EBPdgene. C/EBPdexpression resulted in G0/G1 proliferative arrest and a moderate increase in apoptosis of the KCL22 and the K562 cells. Within 4 days of inducing expression of C/EBPd, myeloid differentiation of the CML blast cells occurred as shown by morphologic changes and induction of secondary granule-specific genes. We also showed that during granulocytic differentiation of KCL22 cells, the C/EBPdprotein was detected in immunocomplexes with both Rb and E2F1. Furthermore, expression of C/EBPdwas associated with downregulation of c-Myc and cyclin E and upregulation of the cyclin-dependent kinase inhibitor p27Kip1 in both the KCL22 and K562 cell lines. These results show that expression of C/EBPdin BCR-ABL-positive leukemic cells in blast crisis is sufficient for neutrophil differentiation and point to the therapeutic potential of ectopic induction of C/EBPdin the acute phase of CML.Oncogene (2005) 24, 1589-1597. doi:10.1038/sj.onc.1208393 Published online 10 January 2005 [ABSTRACT FROM AUTHOR]

We report a novel effect of dehydroepiandrosterone (DHEA) on human granulocyte differentiation: DHEA enhances the all-trans-retinoic acid (ATRA)-induced differentiation of promyelocytic NB4 cells. DHEA (100 microM) significantly augmented the respiratory burst activity of NB4 cells treated with 1 nM ATRA, whereas DHEA alone did not induce respiratory burst activity. The protein and message expressions of p67phox, the gene for the dose-limiting component of phagocyte NADPH oxidase, were significantly enhanced by the coexistence of DHEA and ATRA. The protein expression of p47phox, another component of phagocyte NADPH oxidase, was also up-regulated by DHEA and ATRA. Moreover, the ATRA-induced increment of CCAAT/enhancer-binding protein beta (C/EBPbeta) and the reciprocal reduction in C/EBPUalpha expression were also potentiated by DHEA. In contrast, the expression of PU.1, a transcription factor reportedly involved in the basal expression of p67phox in monocytic cells, was only slightly up-regulated by DHEA and ATRA. Interestingly, DHEA sulfate (DHEAS), the sulfate ester of DHEA that exists in peripheral blood at a concentration approximately 3 orders of magnitude larger than that of DHEA, did not stimulate the ATRA-induced differentiation of NB4 cells. Thus, DHEA, but not DHEAS, plays important roles in synergy with ATRA during granulocyte differentiation of human promyelocytic NB4 cells. [ABSTRACT FROM AUTHOR]

Cytokinins are important purine derivatives that act as hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA), kinetin and benzyladenine were very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. The metabolism of cytokinins to their nucleotides was closely associated with cytokinin-induced differentiation and growth inhibition. When the cells were incubated with [14C]-benzyladenine, radioactivity was significantly incorporated into RNA and DNA. However, the radioactive nucleotides in RNA or DNA were adenine nucleotides, not benzyladenine nucleotides, suggesting that cytokinins were not incorporated into RNA and DNA. The benzyladenine nucleotides were not stably released into the medium in intact form. Cytokinins effectively induced a phosphorylated (active) form of mitogen-activated protein kinase (MAPK). MAPK activation was necessary for cytokinin-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by cytokinins. These results suggest that cytokinin nucleotides themselves play an important role in inducing the differentiation of HL-60 cells. [Copyright &y& Elsevier]