Differentiation Of HL-60 Cells Research Articles

Brain-Derived Neurotrophic Factor Inhibits Intercellular Adhesion Molecule-1 Expression in Interleukin-1β-Treated Endothelial Cells.

Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation, which directs the quality of tissue repair. The objective of this study is to propose the relevance of BDNF to inflammation. In this study, we investigated the effect of BDNF on intercellular adhesion molecule (ICAM)-1, which is an inflammatory marker, expressed in interleukin (IL)-1β-treated human microvascular endothelial cells (HMVECs). In addition, we studied the effect of BDNF on the adhesion of neutrophil-like differentiated HL-60 cells to HMVECs in a cell adhesion assay. We demonstrated that BDNF attenuates the IL-1β-induced ICAM-1 mRNA and protein expression. Treatment of HMVECs with IL-1β led to an increase in the number of adherent neutrophil-like HL-60 cells. BDNF significantly decreased the number of neutrophil-like HL-60 cells attached to HMVECs. In conclusion, BDNF may reduce excess inflammation through reduced neutrophil recruitment by regulating ICAM-1 expression.

Critical role of neutrophil alkaline phosphatase in the antimicrobial function of neutrophils

AimsTo investigate the roles of neutrophil alkaline phosphatase (NAP) in the migration, reactive oxygen species (ROS) generation and apoptosis of neutrophils. Main methodsNAP was overexpressed in neutrophil-like differentiated HL-60 cells via transfecting coding sequence of NAP by lentivirus. NAP overexpression in HL-60 cells was confirmed by the methods of quantitative RT-PCR and Western blotting. HL-60 cells were induced to differentiate into neutrophil-like cells by exposure to 1.5% dimethylsulfoxide (DMSO). The migration of neutrophil-like cells were detected by Transwell migration assay. ROS generation of neutrophil-like cells were determined by flow cytometry. Neutrophil-like cells continued to be cultured for 24h, and were then harvested for apoptosis and Western blotting. Key findingAfter GFP-NAP infection by lentivirus, the expression of NAP was up-regulated in HL-60 cells. HL-60 cells were allowed to differentiate into neutrophil-like cells after 5-day exposure to 1.5% DMSO. Overexpression of NAP in neutrophil-like cells resulted in an increase in the number of migrated cells, intracellular ROS and cell apoptosis followed by a rise in the expression of Caspase 3, Caspase 9 and Bax, while those results were reversed in the NEG and CON group. SignificanceNAP might play a critical role in the anti-microbial function of neutrophils by promoting its migration and ROS generation, as well as accelerating apoptosis in neutrophils.

PM427. Histamine H4 receptor is involved in clozapine-induced hematopoietic toxicity: vulnerability under granulocytic differentiation of HL-60 cells

PM427. Histamine H4 receptor is involved in clozapine-induced hematopoietic toxicity: vulnerability under granulocytic differentiation of HL-60 cells

Lyn, a tyrosine kinase closely linked to the differentiation status of primary acute myeloid leukemia blasts, associates with negative regulation of all-trans retinoic acid (ATRA) and dihydroxyvitamin D3 (VD3)-induced HL-60 cells differentiation.

BackgroundLyn, an import member of Src family kinases (SFKs), is supposed to be implicated in acute myeloid leukemia (AML) pathogenesis and development by participation in AML differentiation, yet the details still remain incompletely understood. The expression status of Lyn and its correlation with multiple clinical parameters including cell differentiation degree, different cytogenetic risk classification, and the activity of myeloperoxidase (MPO) were thus investigated. To address the mechanisms underlying the involvement of Lyn in differentiation induction, the effects of dasatinib, an inhibitor for SFKs including Lyn, on the alterations of all-trans retinoic acid (ATRA)- or dihydroxyvitamin D3 (VD3)-induced differentiation, and c-Myc protein expression were investigated.MethodsPrimary AML blasts were obtained from 31 newly diagnosed AML patients with different French-American-British (FAB) subtypes. The expression of phosphorylated and total Lyn, c-Myc, and CD11b, CD11c and CD15 was analyzed by flow cytometry. The activation of Akt and Erk known to be involved in the regulation of c-Myc expression was investigated using western blotting.ResultsSignificant higher expression levels of total Lyn were observed in AML patients with favorable cytogenetics, higher MPO activity and FAB M2 subtype. A clear positive correlation between the expression levels of Lyn and differentiation status of primary AML blasts was observed. Dasatinib inhibited the expression of phosphorylated Lyn, and further enhanced the differentiation-inducing activity of ATRA and VD3 in HL-60 cells. Augmented downregulation of c-Myc protein expression was observed in the combination treatment with ATRA, VD3 and dasatinib compared to treatment with each reagent alone in HL-60 cells. The suppression of the activation of Akt and Erk was also observed concomitantly.ConclusionsThe expression level of total Lyn is closely linked to the differentiation status of AML blasts. The enhancement of differentiation-inducing activity of ATRA/VD3 by dasatinib suggested that Lyn was associated in the negative regulation of ATRA/VD3-induced HL-60 cells differentiation. The enhancement probably was attributed to the downregulation of c-Myc implicated with the suppression of the activation of Akt and Erk. These results provide novel insights into a possible combinational therapeutic approach by targeting Lyn for AML patients, and offer new possibilities for the combination therapy with VD3 and dasatinib.

Synthesis of Resveratrol Derivatives and In Vitro Screening for Potential Cancer Chemopreventive Activities.

New resveratrol (trans-3,4',5-trihydroxystilbene) analogs were synthesized and screened for their in vitro cancer chemopreventive potential using various bioassays relevant for the prevention of carcinogenesis in humans: two assays to detect modulators of carcinogen metabolism (Cyp1A inhibition; determination of NAD(P)H/quinone reductase (QR) activity), three assays to identify radical scavenging and antioxidant properties (DPPH, ORAC, superoxide anion radicals in differentiated HL-60 cells), four assays to determine anti-inflammatory and anti-hormonal effects (iNOS, Cox-1 and aromatase inhibition, anti-estrogenic potential). 3,4',5-Tri-O-methyl resveratrol 1a was about sevenfold more active than resveratrol in inhibiting Cyp1A activity, it was a potent inducer of QR activity, and it showed pure anti-estrogenic activity (whereas resveratrol is a known mixed estrogen (ant)agonist with both estrogenic and anti-estrogenic properties). Dual estrogen ant-/agonist activity was restored in the mono-O-benzyl-substituted derivatives 4b (4'-O-benzyl resveratrol) and 5b (3-O-benzyl resveratrol). With respect to aromatase inhibition (Cyp19), which provided the highest number of actives, the benzyl-substituted series was more potent than the methyl-substituted derivatives of resveratrol, and 3-O-benzyl resveratrol 5b was about eightfold more active than resveratrol. Overall, 3,4',5-tri-O-pivaloyl resveratrol oxide 7c was identified as a potent inducer of phase 2 enzymes concomitant with inhibition of LPS-mediated iNOS induction.

Hybrid mechanosensing system to generate the polarity needed for migration in fish keratocytes

ABSTRACTCrawling cells can generate polarity for migration in response to forces applied from the substratum. Such reaction varies according to cell type: there are both fast- and slow-crawling cells. In response to periodic stretching of the elastic substratum, the intracellular stress fibers in slow-crawling cells, such as fibroblasts, rearrange themselves perpendicular to the direction of stretching, with the result that the shape of the cells extends in that direction; whereas fast-crawling cells, such as neutrophil-like differentiated HL-60 cells and Dictyostelium cells, which have no stress fibers, migrate perpendicular to the stretching direction. Fish epidermal keratocytes are another type of fast-crawling cell. However, they have stress fibers in the cell body, which gives them a typical slow-crawling cell structure. In response to periodic stretching of the elastic substratum, intact keratocytes rearrange their stress fibers perpendicular to the direction of stretching in the same way as fibroblasts and migrate parallel to the stretching direction, while blebbistatin-treated stress fiber-less keratocytes migrate perpendicular to the stretching direction, in the same way as seen in HL-60 cells and Dictyostelium cells. Our results indicate that keratocytes have a hybrid mechanosensing system that comprises elements of both fast- and slow-crawling cells, to generate the polarity needed for migration.

Expression of ICAT and Wnt signaling-related proteins in the monocytic differentiation of HL-60 cells induced by a new steroidal drug NSC67657

To investigate the expression of mRNA and proteins of β-catenin, TCF-4 (ICAT) and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657. Wright's staining and α-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of 10 μmol/L NSC67657 treatment. Flow cytometry (FCM) was used to detect the differentiation and cell cycles. The expressions of mRNA and proteins of ICAT and Wnt signaling pathway-related factors, including β-catenin, TCF-4, c-myc, cyclin D1 and TCF-1 before and after differentiation, were detected by RT-PCR and Western blot. Morphological observation showed that NSC67657 induced monocytic differentiation of HL-60 cells. At 5 days after 10 μmol/L NSC67657 treatment, the number of CD14(+) HL-60 cells was (94.37±2.84)%, significantly higher than the (1.31±0.09)% in control group (P<0.01). The flow cytometry assay revealed that NSC67657 induced (76.46±2.83)% of G1/G0 phase arrest, significantly higher than that of (59.40±5.42)% in the control group (P<0.05), while the S phase cells were of (18.76±0.98)%, significantly lower than that of (34.38±2.61) % in the control group (P<0.05). The NSC67657 treatment also up-regulated the expression of ICAT mRNA and protein, and down-regulated the expression of β-catenin mRNA and protin (P<0.01 for all). However, the nuclear expression of β-catenin was down-regulated (P<0.01). The NSC67657 treatment induced nonsignificant alterations of TCF-4 mRNA, total protein and nuclear protein in the HL-60 cells (P>0.05 for all). The target genes of Wnt signaling pathway, including c-myc, cyclinD1 and TCF-1 mRNA and proteins in the HL-60 cells were significantly down-regulated after NSC67657 treatment (P<0.05). The new steroidal drug NSC67657 induces monocytic differentiation of HL-60 cells, and down-regulates the expression of β-catenin and target genes of Wnt signaling pathway. These results indicate that Wnt signaling pathway may be directly or indirectly involved in the monocytic differentiation process of HL-60 cells.

Effect of Inhibiting and Activating Wnt Signalling Pathway on NSC67657-inducing Monocytic Differentiation of HL-60 Cells

To investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism. The HL-60 cells were treated with 5, 10 and 20 µmol/L XAV-939 (inhibitor of Wnt signalling pathway) for 3 days, and with 10, 20 and 30 mmol/L LiCl (activator of Wnt signalling pathway) for 1 day; the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot, respectively; the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry, moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined. Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 µmol/L for 3 days; the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control (culture mediam) group, simple NSC67657-treated group, NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed. 20 µmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively, could significantly down- and up-regulate the expression of cyclinD1, TCF1 and c-Jun genes (P < 0.05) and proteins (P < 0.05); moreover, the number of CD10(+) HL-60 cells in these conditions was below 1%, no early apoptosis of HL-60 cells was found. In the simple NSC67657-treated groups, the expression of cyclinD1, TCF1 and c-Jun proteins was down-regulated (P < 0.05), and the percentage of CD14(+) HL-60 cells accounted for 62.13 ± 9.44; after the HL-60 cells were treated with XAV-939, the NSC67657 could more significantly down-regulate the expression of cyclinD1, TCF1 and c-Jun proteins and the percentage of CD14(+) HL-60 cell accounted for 84.17 ± 5.39%, as compared with simple NSC67657-treated group; as compared with blank controls group, the expression of cyclinD1, TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14(+) HL-60 cells decreased to 33.99 ± 8.37% in NSC67657 combined LiC1 streated group, but which were higher than those in simple NSC67657-treated group (P < 0.05). 20 µmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins. The influence of XAV-939 and LiC1 on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.

Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching

ABSTRACTTo investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.

Population variation in anti-S. aureus IgG isotypes influences surface protein A mediated immune subversion

BackgroundStaphylococcus aureus is a pathogen which causes life-threatening infection, the incidence of which rises during adult life. This, together with the emergence of drug-resistant strains and the expansion of more susceptible elderly populations, represents the rationale for the ongoing development of S. aureus vaccines targeting adult populations. Humoral responses to S. aureus naturally develop early in life, influence susceptibility to infection, and potentially influence the effect of vaccination. Despite this, the nature of pre-existing anti-S. aureus antibodies in healthy adult populations is not fully characterised. MethodsImmunoglobulin levels against S. aureus surface antigens were measured by a filter membrane enzyme-linked immunosorbent assay using fixed ΔSpA S. aureus as an antigen in serum samples obtained from three clinical cohorts comprising 133 healthy adult volunteers from 19 to 65 years of age. Functional capacity of antibody was also assessed, using antibody-mediated attachment of FITC-stained S. aureus to differentiated HL-60 cells. ResultsWide variation in the concentrations of immunoglobulins recognising S. aureus surface antigens was observed among individuals in all three cohorts. There was a decline of anti-S. aureus IgG1 with age, and a similar trend was observed in IgM, but not in IgA or other IgG sub-classes. Antibody mediated bacterial attachment to cells was associated with IgG1 and IgG3 concentrations in serum. The presence of SpA on the bacterial cell surface reduced antibody-mediated binding of bacteria to phagocytes in serum with low, but not high, levels of naturally occurring anti-S. aureus IgG3 antibodies. ConclusionsNaturally acquired immunoglobulin responses to S. aureus are heterogeneous in populations and their concentrations alter during adulthood. Elevated IgG1 or IgG3 titres against S. aureus enhance S. aureus recognition by phagocytosis and may be correlates of natural protection and/or vaccine efficacy in adult populations.

Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells

Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2”,3”,4”,6”-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains.

Pulsatilla saponin A induces differentiation in acute myeloid leukemia in vitro

Objectives: To identify whether Pulsatilla saponin A (PsA), an active molecule extracted from Pulsatilla chinensis regel, can induce acute myeloid leukemia (AML) cells differentiate.Methods: PsA was isolated from P. chinensis, and its effects of differentiation induction on both AML cell lines and the primary leukemia cells were investigated.Results: Compared with the untreated control, PsA induced the differentiation of U937 cells, K562 cells and HL-60 cells, represented as the increased CD15+ cells in a dose- and time-dependent manner in all the three AML cell lines, after PsA treatment. As the same time, the cell morphology of these AML cells was changed correspondingly; the cytoplasm/nuclei ratio was increased, basophilic cytoplasm was decreased, and eccentric nucleus and granules were also observed. Also, the same effects of differentiation induction by PSA were confirmed in the primary leukemia cells. However, the specific MEK/ERK inhibitor U0126 effectively abrogated the differentiation induced by PsA in vitro.Conclusions: PsA can modify the differentiation activity of AML cells, probably though the MEK/ERK signaling pathway.

A highly sensitive and selective impedimetric aptasensor for interleukin-17 receptor A

Interleukin-17 receptor A (IL-17RA) has been recognized as a valuable biomarker for diverse diseases, including autoimmune diseases. In this work, an electrochemical biosensor with great sensitivity and selectivity toward IL-17RA was fabricated using an IL-17RA aptamer (Kd=14.00nM) for the first time. The aptasensor was manufactured using electrodeposition of gold nanoparticles, and then quantitative detection of IL-17RA was performed based on impedimetry. The developed sensor exhibited a superior analytical performance for IL-17RA with a wide dynamic range of 10–10,000pg/mL in buffer and a detection limit of 2.13pg/mL, which is lower than that of commercially available ELISA kits. In addition, we validated the high specificity of the designed aptasensor to only IL-17RA, which showed good sensitivity even in human serum solution. Furthermore, the detection of the differentiated HL-60 cells expressing IL-17RA was successfully performed. Clinical applicability of the sensor was also demonstrated utilizing neutrophils separated from asthma patients. It is expected that the fabricated aptasensor will become an excellent diagnostic platform for IL-17RA-mediated diseases.

Arsenic trioxide augments all-trans retinoic acid-induced differentiation of HL-60 cells

AimsAll-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukemia (APL) due to its ability to resume the differentiation of APL cells. Recently, clinical trials have been started to evaluate ATRA plus arsenic trioxide (ATO) as a combination treatment for APL patients. However, little is known about the detailed mechanisms underlying its efficacy. We therefore investigated the effects of this combination on the differentiation and differentiation-related gene expression. Main methodsHuman leukemia HL-60 cells differentiation was examined using nitro blue tetrazolium and CD11b. The levels of mRNA and protein were determined by RT-qPCR, microarray, western blot and ELISA, respectively. The promoter activity was assessed by luciferase activity. The arsenic concentration was determined by ICP-MS. Key findingsATRA-induced HL-60 differentiation was augmented by co-treatment with ATO. A microarray analysis showed that ATRA plus ATO treatment markedly down-regulated the expression of proteinase 3 (PRTN3), which is involved in the differentiation arrest of leukemia cells, compared with treatment with ATRA alone. The PRTN3 mRNA level was suppressed by treatment with ATRA alone, and then further suppressed by co-treatment with ATO, accompanied by a concomitant increase in Sp1 protein, which is known to facilitate differentiation. The expression levels of azurocidin, telomerase reverse transcriptase, ferritin, and interleukin-1β were also altered by co-treatment with ATO. SignificanceCo-treatment with ATO enhances ATRA-induced HL-60 differentiation by altering the expression of genes involved in cell differentiation, providing the molecular basis for a combination therapy using ATO plus ATRA to treat leukemia patients.

Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D₂ as Potential Anti-Leukemic Agents.

Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D2 (1,25D2) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D2. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D2 and 1,25D3, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D2. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist.

Total synthesis of natural derivatives and artificial analogs of 13-oxyingenol and their biological evaluation.

We have established an efficient synthetic methodology for the 13-oxyingenol natural derivative (13-oxyingenol-13-dodecanoate-20-hexanoate), featuring a ring-closing olefin metathesis reaction for the "direct" construction of a highly strained inside-outside framework and a Mislow-Evans-type [2,3]-sigmatropic rearrangement for the stereoselective introduction of the hydroxy group at C5. We also synthesized artificial analogs of 13-oxyingenol and ingenol by using our synthetic strategy. In vitro activation assays of protein kinase C (PKC) α and δ revealed that the dodecanoyl group at O13 on 13-oxyingenol analogs had a significant role in PKCδ activation. The PKCα- or PKCδ-activating 13-oxyingenol and ingenol analogs induced both distinct morphological changes and increases of CD11b expression in HL-60 cells, which would be typical signs of HL-60 cell differentiation to macrophage-like cells, as expected by previous reports. Intriguingly, however, similar differentiation phenotypes were observed with the use of 13-oxyingenol natural derivatives and 13-oxyingenol-13-dodecanoate showing a remarkably less potent PKCα or PKCδ activation ability, which the PKC inhibitor Gö6983 diminished. This indicated the involvement of other PKC isozymes or related kinase activities. 13-Oxyingenol analogs, which induced HL-60 cell differentiation, also induced HL-60 cell death, similar to the action of a phorbol ester, a strong PKC activator.

The Effects of Purified Artemia Extract Proteins on Proliferation, Differentiation and Apoptosis of Human Leukemic HL-60 Cells

There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to 100 µg/mL), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of 10 µg/ml and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with 100 µg/mL of fraction E or F for 96 hr increased the fraction of differentiated cells up to 14.8 ± 3.56% and 16.5 ± 2.08% respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells (56.8 ± 3.7% and 67.4 ± 4.2%, p≤0.01). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells.

Structure-Activity Relationship Study of N(6)-Benzoyladenine-Type BRD4 Inhibitors and Their Effects on Cell Differentiation and TNF-α Production.

Bromodomains are epigenetic 'readers' of histone acetylation. The first potent bromodomain and extra-terminal domain (BET) inhibitors, (+)-JQ1 and I-BET762 (also known as GSK525762), were reported in 2010. Some BET inhibitors are already under clinical trial for the treatment of cancers, but so far, only a few chemical scaffolds are available. We have reported potent N(6)-benzoyladenine-based inhibitors of BRD4, a BET family member that serves as a key mediator of transcriptional elongation. Here we present an analysis of the structure-activity relationships of these inhibitors. Among the compounds examined, 20, 28 and 29 enhanced all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation and inhibited tumor necrosis factor (TNF)-α production by THP-1 cells.

A Methylene Group on C-2 of 24,24-Difluoro-19-nor-1α,25-dihydroxyvitamin D3 Markedly Increases Bone Calcium Mobilization in Vivo.

Four side chain fluorinated analogues of 1α,25-dihydroxy-19-norvitamin D have been prepared in convergent syntheses using the Wittig-Horner reaction as a key step. Structures and absolute configurations of analogues 3 and 5 were confirmed by X-ray crystallography. All analogues showed high potency in HL-60 cell differentiation and vitamin D-24-hydroxylase (24-OHase) transcription as compared to 1α,25-dihydroxyvitamin D3 (1). Most important is that all of the 20S-configured derivatives (4 and 6) had high bone mobilizing activity in vivo. However, in the 20R series, a 2-methylene group was required for high bone mobilizing activity. A change in positioning of the 20R molecule in the vitamin D receptor when the 2-methylene group is present may provide new insight into the molecular basis of bone calcium mobilization induced by vitamin D.

Inflammatory but not apoptotic death of granulocytes citrullinates fibrinogen.

BackgroundNeutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA). Citrullinated fibrinogen is bound by ACPA but it is less well understood how extracellular proteins are citrullinated. The cells that produce fibrinogen, hepatocytes, do not express peptidyl arginine deiminase (PAD) enzymes nor do PAD enzymes include N-terminal signal peptides to direct them into the secretory pathway. We hypothesized that dying neutrophils release PAD in the extracellular space, and that this could cause citrullination of target extracellular antigens relevant to RA such as fibrinogen.MethodsHL60 cells were differentiated into neutrophil-like cells by treatment with all-trans retinoic acid (ATRA). Differentiation was confirmed by CD11b staining, PAD4, PAD2 and myeloperoxidase expression, cell division, and nuclear morphology. Death was induced with various stimuli, including freeze-thaw to induce necrosis, Ionomycin and PMA to induce NETosis, and UV-B to induce apoptosis. Death markers were assessed by immunohistochemistry and flow cytometry. To quantify extracellular citrullination, dying ATRA-differentiated HL60 cells were cultured with fibrinogen for 24 hours and supernatants were probed for fibrinogen citrullination, PAD2 and PAD4 by western blot.ResultsWhile both NETotic and necrotic ATRA differentiated HL60 cells citrullinated fibrinogen, apoptotic cells did not citrullinate fibrinogen, even when allowed to undergo secondary necrosis. Incubation of necrotic neutrophil lysates with fibrinogen also causes fibrinogen citrullination. PAD2 and PAD4 were detected by western blot of supernatants of ATRA-differentiated HL60 cells undergoing necrotic and NETotic death, but not apoptotic or secondarily necrotic cell death.ConclusionWe implicate granulocytes undergoing inflammatory cell death as a mechanism for altering extracellular self-proteins that may be targets of autoimmunity linked to inflammatory diseases such as rheumatoid arthritis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0890-0) contains supplementary material, which is available to authorized users.