Undifferentiated HL-60 Cells Research Articles

Evaluation of cyclin D3 as a transcriptional regulator in a neutrophil differentiation model.

Abstract Cyclin D3 is known to regulate cell cycle entry during the G1 to S transition by associating with its binding partners CDK4/6 upon mitogenic stimulation. Cyclin D3 has also been implicated as a transcriptional regulator, but the indirect mechanism employed is not known. The HL60 cell line was used as a model to investigate cyclin D3’s role in altered gene transcription as DMSO-driven differentiation into neutrophils is induced. We have validated this system by evaluating changes in gene expression indicative of progression to the terminal neutrophil state. Interestingly, ectopic expression of cyclin D3 in undifferentiated HL60 (uHL60) cells causes changes in gene expression to mirror that seen in differentiated (dHL60) cells without the need to treat with DMSO. We have also assessed a cohort of target genes hypothesized to be sensitive to cyclin D3-impacted gene transcription as cells transition from uHL60 to dHL60. For those genes that are differentially expressed between uHL60 and dHL60, many are also sensitive to ectopic expression of cyclin D3. Forced expression of cyclin D3 mutants in uHL60 cells suggests that a C-terminal domain within cyclin D3 is responsible for this observed regulation of gene expression, as three mutations (L215M, Q244R, P278R) abrogate the wild-type cyclin D3-induced differentiation gene signature. Ongoing studies aim to elucidate transcription factor binding partners that interact in complex with cyclin D3 to cause differential gene expression.

Transcriptomic analysis of lipoteichoic acid‑treated undifferentiated and neutrophil‑like differentiated HL‑60 cells.

Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.

Monocyte/Macrophage - like cell differentiation induced by TPA in HL60 cells leads to loss of Histone H4 Lysine 16 Acetylation

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester and induces monocyte/macrophage like cell differentiation in HL60 cells. The levels and patterns of gene expression differ greatly between differentiated and undifferentiated HL60 cells. Epigenetic histone modifications play a crucial role in transcriptional activation and alterations in the modification levels greatly affect the gene expression pattern. Acetylation of Histone H4 Lysine 16 (H4K16ac) is one such modification and has a significant role in transcriptional activation. Changes in its acetylation level either due to a physiological or pathological effect will have a dramatic effect on cellular gene expression. Here, a study was done to see the effect of TPA induced differentiation on H4K16ac levels in HL60 cells. Results obtained from flow cytometric analysis showed expression of macrophage cell surface marker CD11b on TPA differentiated HL60 cells and the western blots revealed a drastic downregulation of H4K16ac in differentiated cells. Immunoblotting and co-immunoprecipitation assay revealed DNA damage dependent enhancement of H4K16 acetylation and its co-localization with γH2AX in undifferentiated cells. Whereas, TPA differentiated cells (CD11B+ve) didn’t show any such enhancement in H4K16acetylation levels in the presence of DNA damage. The present study shows that TPA induced differentiation of HL60 cells into macrophages leads to the downregulation of H4K16 acetylation.

Toxicity of metamizole on differentiating HL60 cells and human neutrophil granulocytes

Metamizole is an analgesic and antipyretic with a superior analgesic efficacy than paracetamol. Since metamizole can cause neutropenia and agranulocytosis, it is currently used in only few countries. In a previous study, we have shown that N-methyl-4-aminoantipyrine (MAA), the active metamizole metabolite, reacts with hemin and forms an electrophilic metabolite that is toxic for HL60 cells, but not for mature neutrophil granulocytes. In the current study, we investigated the toxicity of hemin (12.5 μM) and MAA (100 μM) on differentiating HL60 cells. In undifferentiated HL60 cells, hemin decreased the viability and this effect was significantly increased by MAA. Similarly, hemin/MAA was more toxic than hemin alone on human cord blood cells. At 3 days (metamyelocyte stage) and 5 days of differentiation (mature neutrophils), hemin/MAA was not toxic on HL60 cells, whereas hemin alone was still toxic. No toxicity was observed on freshly isolated human neutrophils. The protein expression of enzymes responsible for hemin metabolism increased with HL60 cell differentiation. Inhibition of heme oxygenase-1 or cytochrome P450 reductase increased the toxicity of hemin and hemin/MAA in undifferentiated, but only for hemin in differentiated HL60 cells. Similar to the enzymes involved in hemin metabolism, the protein expression of enzymes involved in antioxidative defense and the cellular glutathione pool increased with HL60 cell differentiation. In conclusion, HL60 cells become resistant to the toxicity of hemin/MAA and partly also of hemin during their differentiation. This resistance is associated with the development of heme metabolism and of the antioxidative defense system including the cellular glutathione pool.

Effect of iron overload from multi walled carbon nanotubes on neutrophil-like differentiated HL-60 cells

Multi walled carbon nanotubes (MWCNTs) are one of the most intensively explored nanomaterials because of their unique physical and chemical properties. Due to the widespread use of MWCNTs, it is important to investigate their effects on human health. The precise mechanism of MWCNT toxicity has not been fully elucidated. The present study was designed to examine the mechanisms of MWCNT toxicity toward human promyelocytic leukemia HL-60 cells. First, we found that MWCNTs decreased the viability of neutrophil-like differentiated HL-60 cells but not undifferentiated HL-60 cells. Because neutrophil-like differentiated HL-60 cells exhibit enhanced phagocytic activity, the cytotoxicity of MWCNTs is dependent on the intracellularly localized MWCNTs. Next, we revealed that the cytotoxicity of MWCNTs is correlated with the intracellular accumulation of iron that is released from the engulfed MWCNTs in an acidic lysosomal environment. The intracellular accumulation of iron was repressed by treatment with cytochalasin D, a phagocytosis inhibitor. In addition, our results indicated that iron overload enhanced the release of interleukin-8 (IL-8), a chemokine that activates neutrophils, and subsequently elevated intracellular calcium concentration ([Ca2+]i). Finally, we found that the sustained [Ca2+]i elevation resulted in the loss of mitochondrial membrane potential and the increase of caspase-3 activity, thereby inducing apoptotic cell death. These findings suggest that the iron overload caused by engulfed MWCNTs results in the increase of IL-8 production and the elevation of [Ca2+]i, thereby activating the mitochondria-mediated apoptotic pathway.

Differentiated and exponentially growing HL60 cells exhibit different sensitivity to some genotoxic agents in the comet assay

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation.A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status.Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing.

Involvement of the histamine H4 receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100μM) and doxorubicin (0.2µM) decreased the cell survival rate, but olanzapine (1–100µM) did not. Under granulocytic differentiation for 5days, clozapine, even at a concentration of 25μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H4 receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H4 receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H4 receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H4 receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation.

Effects of PCB126 and PCB153 on telomerase activity and telomere length in undifferentiated and differentiated HL-60 cells.

PCBs are persistent organic pollutants that are carcinogenic and immunotoxic and have developmental toxicity. This suggests that they may interfere with normal cell maturation. Cancer and stem/progenitor cells have telomerase activity to maintain and protect the chromosome ends, but lose this activity during differentiation. We hypothesized that PCBs interfere with telomerase activity and the telomere complex, thereby disturbing cell differentiation and stem/progenitor cell function. HL-60 cells are cancer cells that can differentiated into granulocytes and monocytes. We exposed HL-60 cells to PCB126 (dioxin-like) and PCB153 (nondioxin-like) 6 days before and during 3 days of differentiation. The differentiated cells showed G0/G1 phase arrest and very low telomerase activity. hTERT and hTR, two telomerase-related genes, were downregulated. The telomere shelterins TRF1, TRF2, and POT1 were upregulated in granulocytes, and TRF2 was upregulated and POT1 downregulated in monocytes. Both PCBs further reduced telomerase activity in differentiated cells, but had only small effects on the differentiation and telomere-related genes. Treatment of undifferentiated HL-60 cells for 30 days with PCB126 produced a downregulation of telomerase activity and a decrease of hTERT, hTR, TRF1, and POT1 gene expression. With PCB153, the effects were less pronounced and some shelterin genes were increased after 30 days of exposure. With each PCB, no differentiation of cells was observed and cells continued to proliferate despite reduced telomerase activity, resulting in shortened telomeres after 30 days of exposure. These results indicate cell-type and PCB congener-specific effects on telomere/telomerase-related genes. Although PCBs do not seem to strongly affect differentiation, they may influence stem or progenitor cells through telomere attrition with potential long-term consequences for health.

Regulation of the Interleukin-6 gene expression during monocytic differentiation of HL-60 cells by chromatin remodeling and methylation

The pro-inflammatory cytokine Interleukin (IL)-6 is involved in the proliferation and differentiation of leukocytes and non-immune cells, but its overproduction is associated with inflammatory and autoimmune disorders. The main producers of IL-6 are mature monocytes, whereas progenitor cells and the promyeloid cell line HL-60 do not synthesize IL-6. In contrast, HL-60 cells differentiated into monocytic cells were able to express IL-6 after lipopolysaccharide (LPS) stimulation. This study investigated the chromatin structure of the IL-6 promoter and the effect of methylation on IL-6 gene regulation during monopoiesis. The results show that the proximal IL-6 promoter regions I to III (+13/−329) were inaccessible in undifferentiated HL-60 cells but became significantly accessible in differentiated HL-60 cells stimulated with LPS. Region IL-6 VI (−1099/−1142) remained closed, but the upstream region IL-6 VII (−2564/−2877) relaxed after differentiation and LPS treatment. The opening of IL-6 IV (−309/−521) and IL-6V (−500/−722), containing DNA and histone methylation sites, was differentiation-dependent only. Demethylation experiments using 5-aza-2′-deoxycytidine (AZA) followed by LPS stimulation revealed a significant enhanced IL-6 mRNA expression and protein release by HL-60 cells. AZA treatment resulted in significant increased IL-6 promoter accessibilities, identifying methylation as an important repressor of IL-6 gene regulation in promyeloid cells. The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) had no effect on IL-6 promoter accessibility.Our data indicate that during monopoiesis the proximal IL-6 promoter is reorganized into an accessible conformation allowing transcription of IL-6 after LPS stimulation. DNA methylation appears to be the essential epigenetic mechanism in IL-6 gene expression of mature monocytes and their progenitors by controlling the chromatin structure.

The UDP-sugar-sensing P2Y14 receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils

The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y(14)-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y(14)-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y(14)-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y(14)-R-mediated neutrophil motility via Rho/Rho kinase activation.

Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions

Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin–Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96 h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72 h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96 h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo.

Clarithromycin Accumulation by Phagocytes and Its Effect on Killing of Aggregatibacter actinomycetemcomitans

Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [(3)H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs. Mature human PMNs, HL-60 cells differentiated into granulocytes, and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a K(m) of ≈250 μg/ml and a V(max) of 473 ng/min/10(6) cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half-times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.

Changes in chromatin structure and methylation of the human interleukin‐1β gene during monopoiesis

Interleukin-1beta (IL-1beta) induces the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. However, the molecular regulation of IL-1beta expression in myeloid differentiation has not been elucidated. In this study the chromatin structure of the IL-1beta promoter and the impact of methylation on IL-1beta expression in monocytic development were examined. The results revealed that the IL-1beta promoter was inaccessible in undifferentiated promyeloid HL-60 cells but highly accessible in differentiated monocytic cells which additionally acquired the ability to produce IL-1beta. Accessibilities of differentiated cells were comparable to those of primary monocytes. Lipopolysaccharide (LPS) stimulation did not affect promoter accessibility in promyeloid and monocytic HL-60 cells, demonstrating that the chromatin remodelling of the IL-1beta promoter depends on differentiation and not on the transcriptional status of the cell. Demethylation via 5-aza-2'-deoxycytodine led to the induction of IL-1beta expression in undifferentiated and differentiated cells, which could be increased after LPS stimulation. Our data indicate that the IL-1beta promoter is reorganized into an open poised conformation during monopoiesis being a privilege of mature monocytes but not of the entire myeloid lineage. As a second mechanism, IL-1beta expression is regulated by methylation acting independently of the developmental stage of myeloid cells.

MiR-181a Regulates Cap-Dependent Translation of p27kip1 mRNA in Myeloid Cells

p27(kip1) (p27) is a cell cycle inhibitor and tumor suppressor whose expression is tightly regulated in the cell. Translational control of p27 mRNA has emerged as a prominent mechanism to regulate p27 expression during differentiation, quiescence, and cancer progression. The microRNAs miR-221 and miR-222 repress p27 expression in various cancer cells, and this repression promotes tumor cell proliferation. In addition, the presence of an internal ribosome entry site in the 5' untranslated region (UTR) of p27 mRNA has been reported. Here, we show that p27 mRNA is translated via a cap-dependent mechanism in HeLa and HL60 cells and that the previously reported IRES activity can be attributed to cryptic promoters in the sequence corresponding to the p27 5' UTR. Furthermore, cap-dependent translation of p27 mRNA is repressed by miR-181a in undifferentiated HL60 cells. Repression by miR-181a is relieved during differentiation of HL60 into monocyte-like cells, allowing the accumulation of p27, which is necessary to fully block cell cycle progression and reach terminal differentiation. These results identify miR-181a as a regulator of p27 mRNA translation during myeloid cell differentiation.

Effect of 100 mT homogeneous static magnetic field on [Ca2+]c response to ATP in HL‐60 cells following GSH depletion

Calcium is an important molecule in a number of biological systems. Often these systems are signal transduction cascades involving molecules such as ATP. ATP activates second messengers which can interact with ion channels on the endoplasmic/sarcoplasmic reticulum resulting in the emptying of the intracellular calcium stores and an increase in cytosolic free calcium concentration ([Ca2+]c). Changes in [Ca2+]c can be influenced by external factors such as a static magnetic field (SMF). One hypothesis suggests that a SMF affects the cells through the radical pair mechanism. By reducing the number of antioxidant molecules like glutathione (GSH), the proportion of free radicals in the cells is increased and may lead to a greater probability of a biological response to a SMF. The purpose of this study was to determine if the [Ca2+]c response to ATP was affected by depletion of GSH by diethylmaleate (DEM) and the absence or presence of a 100 mT homogeneous SMF. Undifferentiated HL-60 cells were loaded with fura-2 AM. [Ca2+]c was measured in real time using a ratiometric fluorescence spectroscopy system. Various (DEM) ranging from 1 to 15 mM were added to deplete GSH. Cells were either exposed to sham or magnetic field (100 mT) for 13 min (780 s) and challenged with 1 microM ATP. The data show that [Ca2+]c was elevated following treatment with DEM with greater [Ca2+]c at higher [DEM]. The [Ca2+]c response to ATP was decreased as the DEM concentration increased. However, there was no effect of a 100 mT SMF on the average [Ca2+]c peak following ATP activation or the full width at half maximum (FWHM) of the [Ca2+]c response and recovery after ATP activation.

Discovery of Mitocryptide-1, a Neutrophil-activating Cryptide from Healthy Porcine Heart

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing beta-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and G(i2)-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce beta-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.

Real‐time measurement of cytosolic free calcium concentration in HL‐60 cells during static magnetic field exposure and activation by ATP

Calcium ions are involved in a number of important signal transduction pathways in cells. Cytosolic calcium concentration ([Ca(2+)](c)) can be affected by the activation of Ca(2+) channels through the action of ligands such as ATP. The response of [Ca(2+)](c) to ligands may be affected by external factors like magnetic fields. The purpose of this study was to determine if exposure to a static magnetic field (SMF) for 800 s altered the [Ca(2+)](c) response to ATP in undifferentiated HL-60 cells. We sham exposed or field exposed fura-2 loaded HL-60 cells to a SMF of 1, 10, and 100 mT. Cells were activated with ATP 300 s into the exposure. The level of [Ca(2+)](c) was followed before, during, and after field or sham exposure with a ratiometric fluorescence spectroscopy system. It was found that high concentrations of ATP resulted in greater [Ca(2+)](c) responses, but faster recovery to near basal levels. The application of 1, 10, or 100 mT SMF did not affect the [Ca(2+)](c) response to ATP. Future work could examine the effect of a longer SMF exposure on the [Ca(2+)](c) response to ATP. Longer exposures might provide sufficient time for morphological changes in the plasma membrane to occur.

PKC and MEK pathways inhibit caspase-9/-3-mediated cytotoxicity in differentiated cells

Many studies have indicated that differentiated cells inhibit drug-induced cytotoxicity but undifferentiated cells do not, though the mechanisms are unclear. Currently, HL-60 cells are induced to differentiate into macrophage-like cells with Phorbol-12-myristate-13-acetate (TPA) treatment (TPA-differentiated cells). Our study shows that caspase-9/-3-mediated cytotoxicity can be induced in undifferentiated HL-60 cells but not in TPA-differentiated HL-60 cells. However, caspase-9/-3-mediated cytotoxicity can be induced in TPA-differentiated cells if they are pretreated with a protein kinase C (PKC) or a mitogen activated protein kinase (MEK) inhibitor. Taken together, this study demonstrates that TPA-differentiated HL-60 cells inhibit caspases-9/-3-mediated cytotoxicity through the PKC and MEK signaling pathways.

Trimetazidine decreases Ca2+ response to thapsigargin in differentiated and undifferentiated human HL-60 cells

The search for compounds that selectively suppress the activity of plasma membrane Ca 2+ channels in electrically nonexcitable cells is a topical medicobiological problem. This was the first study to demonstrate that the antianginal drug trimetazidine selectively suppressed the activity of Ca 2+ channels activated by a drop in the Ca 2+ level in intracellular calcium stores in HL-60 human promyelocytes, the precursors of blood neutrophils and monocytes. It is principally important that trimetazidine as such did not induce Ca 2+ release from inositol triphosphate-sensitive Ca 2+ stores and did not activate Ca 2+ channels regulated by these stores. The results of this study allowed us to explain in a new way some characteristics of the effect of trimetazidine on human neutrophils and its effect in myocardial

Human GM 3 synthase: a new mRNA variant encodes an NH 2-terminal extended form of the protein

All human GM 3 synthase mRNA variants until now identified predict a protein of 362 amino acids having substrate activity highly restricted to lactosylceramide. In this report we describe the identification of a new GM 3 synthase transcript containing an additional translation start codon, located upstream and in-frame with that up to now considered unique translation initiation site in the human GM 3 synthase gene. In vitro expression studies showed that the new transcript produces a longer form of human GM 3 synthase, that is efficiently translocated into the microsomal lumen and glycosylated. Moreover, stable cDNA transfection into mammalian cells gives rise to a threefold increase of GM 3 synthase activity, associated to a broader substrate specificity. Although this transcript has been initially identified in the human placenta, RT-PCR analyses verified the expression of an identical mRNA also in undifferentiated HL60 cells, but not in the monocytic lineage. Altogether, these results are the first demonstration of the existence of a new isoform of human GM 3 synthase, which could play an important role during HL60 cell differentiation. The functional relevance of the existence of two isoforms of GM 3 synthase is also discussed.