Macrophage-like Differentiation Research Articles

Exploring the Therapeutic Potential of Ganoderma lucidum in Cancer.

Triterpenoids, such as ganoderic acid, and polysaccharides, including β-D-glucans, α-D-glucans, and α-D-mannans, are the main secondary metabolites of the medicinal fungus Ganoderma lucidum. There is evidence of the effects of ganoderic acid in hematological malignancies, whose mechanisms involve the stimulation of immune response, the macrophage-like differentiation, the activation of MAP-K pathway, an IL3-dependent cytotoxic action, the induction of cytoprotective autophagy, and the induction of apoptosis. In fact, this compound has been tested in twenty-six different human cancer cell types and has shown an anti-proliferative activity, especially in leukemia, lymphoma, and myeloma lines. Moreover, research clarified the capability of molecules from Ganoderma lucidum to induce mitochondrial damage in acute promyelocytic leukemia cells, without cytotoxic effects in normal mononuclear cells. Active lipids extracted from the spores of this fungus have also been shown to induce apoptosis mediated by downregulation of P-Akt and upregulation of caspases-3, -8, and -9. Among in vivo studies, a study in BALB/c mice injected with WEHI-3 leukemic cells suggested that treatment with Ganoderma lucidum promotes differentiation of T- and B-cell precursors, phagocytosis by PBMCs, and NK cell activity. Our review presents data revealing the possibility of employing Ganoderma lucidum in hematological malignancies and incorporating it into clinical practice.

The mechanism by which hsa_circRNA_103124 highly expressed in peripheral blood of patients with active Crohn's disease regulates macrophage differentiation, pyroptosis and inflammation

Objective: To investigate the role and related mechanism of the highly expressed circular RNA molecule 103124 (hsa_circRNA_103124) in macrophage differentiation, pyroptosis and inflammation in peripheral blood mononuclear cells (PBMC) of patients with active Crohn's disease (CD). Methods: Patients with active CD (CD group) admitted to the Affiliated Suzhou Hospital of Nanjing Medical University from April to September 2018 and healthy people (control group) from the physical examination center of the hospital from July to October 2018 were retrospectively selected. The levels of hsa_circRNA_103124 and Toll-like receptor 4 (TLR4) in PBMC of the two groups were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Tohoku hospital pediatrics-1 (THP1) cell line was used as a model for the study of hsa_circRNA_103124 regulating macrophage differentiation. Lentivirus infection was used to construct hsa_circRNA_103124 overexpressed or down-regulated THP1 cells to induce macrophage-like differentiation. According to the expression level of hsa_circRNA_103124, THP1 cell lines were divided into the following four groups: pLC5-ciR was overexpression control group; hsa_circRNA_103124 OE was the overexpression group; ShRNActrl was down-regulated expression control group; hsa_circRNA_103124 ShRNA was the down-regulated expression group. Flow cytometry was used to detect levels cluster of differentiation (CD) 68, CD80, interleukin (IL)-6, tumor necrosis factor α (TNF-α) and reactive oxygen species (ROS). The expression levels of IL-6, TNF-α, IL-1β, TLR4 and myeloid differentiation factor 88 (MyD88) were detected by RT-qPCR. The levels of gasdermin D (GSDMD), IL-18 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) were determined by immunofluorescence and RT-qPCR. Pearson correlation analysis was used to analyze the correlation between the abundance of hsa_circRNA_103124 and TLR4 expression level or Crohn's disease activity index (CDAI). Results: A total of 50 patients were included in the CD group, including 36 males and 14 females, aged (35±10) (19-64) years. A total of 30 subjects were included in the control group, including 22 males and 8 females, aged (38±9) (24-64) years. hsa_circRNA_103124 [(0.009±0.016) vs (0.003±0.002), P=0.042] and TLR4 [(0.005±0.003) vs (0.001±0.001), P<0.001] were all upregulated in the PBMC of patients in the CD group, compared with the control group. And hsa_circRNA_103124 was positively correlated with TLR4 (r=0.40, P=0.004). hsa_circRNA_103124 level was positively correlated with CDAI (r=0.32, P=0.024). The expression of CD68 (P=0.002) and CD80 (P<0.001) were enhanced. hsa_circRNA_103124 promoted production of ROS and the expression of IL-6, TNF-α, IL-1β, TLR4, MyD88, GSDMD, IL-18 and NLRP3 in macrophage-like M1 differentiated THP1 cells (all P<0.05). Conclusion: High expresion of hsa_circRNA_103124 in PBMC of patients with active CD may promote macrophage M1 differentiation, pyroptosis and inflammation through enhancing the expression of TLR4, MyD88, NLRP3 and GSDMD.

EGCG Prevents the Transcriptional Reprogramming of an Inflammatory and Immune-Suppressive Molecular Signature in Macrophage-like Differentiated Human HL60 Promyelocytic Leukemia Cells.

Simple SummaryScientists are increasingly harnessing the power of the immune system to prevent cancer. While macrophages are a major component of the tumor microenvironment where they orchestrate various aspects of immunity, dysregulated immune and inflammatory responses will play a key role in cancer initiation and progression. Depending on their activation status, macrophages can have a dual impact on tumorigenesis by either antagonising cytotoxic immune cells or enhancing antitumor responses. Increased infiltration of tumor-associated macrophages has long been associated with poor patient prognosis in most solid cancers, highlighting their value as potential diagnostic and prognostic biomarkers in cancer. A variety of macrophage-centered approaches to cancer therapy have been investigated, including strategies to prevent tumor-promoting activities. In this study, we highlight the chemopreventive properties of EGCG derived from green tea that underpin its ability to re-program macrophage molecular signature and function.Background: The promyelocytic leukemia cell differentiation process enables recapitulation of the polarized M1 or M2 macrophage-like phenotype with inflammatory and immune-suppressive properties. While evidence supports the anti-inflammatory effect of dietary-derived epigallocatechin-3-gallate (EGCG), its impact on the onset of immune phenotype molecular signature remains unclear. Methods: Human HL60 promyelocytic cells grown in suspension were differentiated into CD11bHigh/CD14Low adherent macrophages with phorbol 12-myristate 13-acetate (PMA). Gelatin zymography was used to assess the levels of matrix metalloproteinase (MMP)-9, and total RNA was isolated for RNAseq and RT-qPCR assessment of differentially expressed gene levels involved in inflammation and immunity. Protein lysates were used to assess the phosphorylation status of signaling intermediates involved in macrophage-like cell differentiation. Results: Cell adhesion and induction of MMP-9 were indicative of HL60 cell differentiation into a macrophage-like phenotype. The extracellular signal-regulated kinase (ERK), glycogen synthase kinase (GSK)-3, p90 ribosomal S6 kinases (RSK), and cAMP-response-element-binding protein (CREB) were all phosphorylated, and EGCG reduced such phosphorylation status. Increases in inflammation and immunity genes included, among others, CCL22, CSF1, CSF2, IL1B, and TNF, which inductions were prevented by EGCG. This was corroborated by unbiased transcriptomic analysis which further highlighted the capacity of EGCG to downregulate the hematopoietic stem cell regulator CBFA2T3. Conclusion: EGCG inhibits inflammatory signaling crosstalk and prevents the onset of an immune phenotype in macrophage-like differentiated cells.

Abstract 730: The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages

Abstract Tumor-associated macrophages (TAMs) have important roles in the angiogenesis, tumor immunosuppression and tumor infiltration of various cancers. Previously, we reported that a large number of infiltrated tumor promoting CD204+ TAMs within esophageal squamous cell carcinomas (ESCCs) contributed to the significant association of clinicopathological malignancy and poor disease free survival. However, the relevant malignant progression mechanisms have not been studied explicitly yet. To elucidate roles of TAMs in ESCCs, we established an in vitro macrophage model using human peripheral blood acquired by healthy volunteer donors. Peripheral blood monocytes were treated with 25 ng/mL recombinant human M-CSF for 6 days to induce macrophage-like differentiation (macrophage-like cells), then exposed to the conditioned media of TE series ESCC cell lines (TECM) for 2 days to induce TAM-like cells. From the cDNA microarray data between macrophages-like cells treated with and without TECM, we interested in upregulated genes. Among highly expressed genes in TAM-like cells, we focused on neural cell adhesion molecule (NCAM) which was a possibility to act on the regulation between tumor and its surrounding stroma. Gene knockdown of NCAM by siRNA in TAM-like cells revealed significant suppression of cell survival and migration via decrease of PI3K/Akt signaling and downregulation of fibroblast growth factor receptor 1 (FGFR1) expression. Interestingly, fibroblast growth factor-2 (FGF-2) derived from ESCCs promotes FGF2/FGFR1 autocrine loop, and activates FGFR1 signaling in TAM-like cells. Furthermore, we analyzed 70 ESCC tissue samples by immunohistochemistry whether the expression levels of FGF-2 were associated with clinicopathological background factors of ESCC patients. Relationships of the expression levels of FGF-2 in human ESCC stromal cells including macrophages had a correlation with the depth of invasion, blood vessel invasion, TNM classification UICC cancer staging and high number of infiltrating CD163 positive and CD204 positive macrophages as a marker for the M2 macrophage phenotype. These findings may indicate novel roles of FGF-2-NCAM/FGFR1 interplay in ESCCs and its microenvironment including TAMs. Citation Format: NOBUHISA TAKASE, Yuichiro Koma, Noriaki Arai, Himiko Kodaira, Masayoshi Hosono, Yumi Ichihara, Mari Nishio, Manabu Shigeoka, Hiroshi Yokozaki. The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 730.

Attenuation of IL-32-induced caspase-1 and nuclear factor-κB activations by acteoside

Acteoside has anti-inflammatory and antioxidant potentials. Nevertheless, little information is available about the pharmacological mechanism of acteoside. Here, we report the regulatory effects and underlying mechanisms of acteoside on interleukin (IL)-32-induced inflammatory reactions using human monocytes cells line, THP-1 cells. Acteoside suppressed IL-32-induced macrophage-like cells differentiation. Levels of thymic stromal lymphopoietin, tumor necrosis factor (TNF)-α, IL-1β, and IL-8 increased by IL-32 or LPS were significantly reduced by treatment with acteoside in THP-1 cells. Acteoside attenuated IL-32-induced caspase-1 and nuclear factor-κB activations in THP-1 cells. In IL-32-induced macrophages, acteoside significantly reduced LPS-induced TNF-α, IL-1β, IL-6, and IL-8 production. In addition, production of nitric oxide (NO) and expression of inducible NO synthase increased by LPS were significantly decreased by treatment with acteoside in IL-32-induced macrophages. Our data suggest that acteoside exhibits an anti-inflammatory activity by suppressing IL-32 signaling pathway. Collectively, the results indicate that acteoside may act as a regulator of the IL-32 induced immune responses.

MiR-181 family: regulators of myeloid differentiation and acute myeloid leukemia as well as potential therapeutic targets.

MicroRNAs have been shown to play an important role in normal hematopoisis and leukemogenesis. Here, we report function and mechanisms of miR-181 family in myeloid differentiation and acute myeloid leukemia (AML). The aberrant overexpression of all the miR-181 family members (miR-181a/b/c/d) was detected in French-American-British M1, M2 and M3 subtypes of adult AML patients. By conducting gain- and loss-of-function experiments, we demonstrated that miR-181a inhibits granulocytic and macrophage-like differentiation of HL-60 cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) by directly targeting and downregulating the expression of PRKCD (which then affected the PRKCD-P38-C/EBPα pathway), CTDSPL (which then affected the phosphorylation of retinoblastoma protein) and CAMKK1. The three genes were also demonstrated to be the targets of miR-181b, miR-181c and miR-181d, respectively. Significantly decreases in the expression levels of the target proteins were detected in AML patients. Inhibition of the expression of miR-181 family members owing to Lenti-miRZip-181a infection in bone marrow blasts of AML patients increased target protein expression levels and partially reversed myeloid differentiation blockage. In the mice implanted with AML CD34+ HSPCs, expression inhibition of the miR-181 family by Lenti-miRZip-181a injection improved myeloid differentiation, inhibited engraftment and infiltration of the leukemic CD34+ cells into the bone marrow and spleen, and released leukemic symptoms. In conclusion, our findings revealed new mechanism of miR-181 family in normal hematopoiesis and AML development, and suggested that expression inhibition of the miR-181 family could provide a new strategy for AML therapy.

Inhibition of IL-32 signaling by bamboo salt decreases pro-inflammatory responses in cellular models of allergic rhinitis.

Previously, we showed the antiallergic effect of bamboo salt (BS) in allergic rhinitis (AR). We also demonstrated that interleukin (IL)-32 is an important mediator of AR. The aim of this study was to evaluate the effect and specific underlying mechanism of BS, NaCl, and the mineral mixture (components of BS other than NaCl, including zinc, magnesium, and potassium, Mix) on IL-32 signaling using the human monocyte cell line, THP-1. Here, we documented for the first time that BS significantly decreased IL-32-induced thymic stromal lymphopoietin protein and mRNA expression in THP-1 cells. BS treatment significantly inhibited IL-32-induced proinflammatory cytokine production including IL-1β, IL-8, and tumor necrosis factor (TNF)-α by suppressing nuclear factor-κB, p38 mitogen-activated kinase, and caspase-1 pathways. The presence of BS or Mix effectively suppressed IL-32-induced macrophage-like cell differentiation but NaCl exhibited no effect on monocyte-to-macrophage-like cell differentiation. In IL-32-induced macrophages, the production of IL-1β, IL-6, IL-8, and TNF-α, and expression of inducible nitric oxide synthase and cyclooxygenase-2, induced by lipopolysaccharide was dramatically decreased in a dose-dependent manner after BS treatment. BS also significantly decreased IL-32-induced nitric oxide, IL-8, and TNF-α production. Furthermore, BS inhibited granulocyte-macrophage colony-stimulating factor-induced IL-32 and IL-8 protein and mRNA expression in EOL-1 cells. Taken together, BS suppressed inflammatory activity by inhibiting the IL-32 signaling pathway in AR.

Erratum to: Development of a lentiviral vector and an efficient infection method for gene therapy for p22phoxdefective chronic granulomatous disease

Chronic granulomatous disease (CGD) is caused by impaired antimicrobial activity in phagocytes due to the absence or malfunction of the respiratory burst NADPH oxidase. In a previous study, we found that 12 patients from 10 unrelated families on Jeju Island had an identical homozygous single-base C-to-T substitution in exon 1 (c.7C > T) of CYBA, which encodes p22phox. Autosomal recessive p22phox-defective CGD carrierderived white blood cells were efficiently transduced by the elongation factor 1-alpha lentivirus constructs, as up to 90% of cells were green fluorescent protein (eGFP)-positive at 3 days post-transduction. pLL3.7-driven eGFP expression was stable for at least 4 weeks after transduction and persisted after CGD carrierderived cells were immortalized by human telomerase reverse transcriptase (hTERT) and B lymphoma Mo-MLV insertion region 1 (Bmi-1). Upon macrophage-like differentiation of the transduced HL-60 cells by dimethyl sulfoxide, up to 28% of the cells had higher mean levels of superoxide production than undifferentiated cells, and lentivirus efficiently transduced cells and induced the expression of genes for extended periods.

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and β-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)(n) repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Recognition of secretory IgA by DC-SIGN: Implications for immune surveillance in the intestine

Secretory IgA (SIgA), the predominant class of antibody in intestinal secretions, serves as the first line of defense against enteric infections. SIgA has also been proposed to function in immune surveillance, given that both SIgA and SIgA-antigen complexes are actively transported by Peyer's patch M cells from the intestinal lumen to sub-epithelial dendritic cells (DCs). The goal of the present study was to identify the receptor(s) potentially utilized by mucosal DCs to recognize and internalize SIgA. We demonstrate that human colostral SIgA is recognized by purified recombinant human DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) in a solid phase binding assay, as well as by DC-SIGN ectopically expressed on the surface of Chinese hamster ovary (CHO-S) cells. The interaction between SIgA and DC-SIGN was specific, given that it was Ca 2+-dependent and inhibited by mannan. Moreover, SIgA bound to, and was internalized by, endogenous DC-SIGN expressed on THP-1 cells following monocyte to macrophage-like cell differentiation by stimulation with phorbol ester and interleukin-4. These data identify DC-SIGN as a putative receptor for SIgA, and reveal a mechanism by which DCs could collaborate with M cells in immune surveillance at mucosal surfaces.

Characterization of various promoter regions of the human DNA helicase-encoding genes and identification of duplicated ets (GGAA) motifs as an essential transcription regulatory element

DNA helicases are important in the regulation of DNA transaction and thereby various cellular functions. In this study, we developed a cost-effective multiple DNA transfection assay with DEAE-dextran reagent and analyzed the promoter activities of the human DNA helicases. The 5'-flanking regions of the human DNA helicase-encoding genes were isolated and subcloned into luciferase (Luc) expression plasmids. They were coated onto 96-well plate and used for co-transfection with a renilla-Luc expression vector into various cells, and dual-Luc assays were performed. The profiles of promoter activities were dependent on cell lines used. Among these human DNA helicase genes, XPB, RecQL5, and RTEL promoters were activated during TPA-induced HL-60 cell differentiation. Interestingly, duplicated ets (GGAA) elements are commonly located around the transcription start sites of these genes. The duplicated GGAA motifs are also found in the promoters of DNA replication/repair synthesis factor genes including PARG, ATR, TERC, and Rb1. Mutation analyses suggested that the duplicated GGAA-motifs are necessary for the basal promoter activity in various cells and some of them positively respond to TPA in HL-60 cells. TPA-induced response of 44-bp in the RTEL promoter was attenuated by co-transfection of the PU.1 expression vector. These findings suggest that the duplicated ets motifs regulate DNA-repair associated gene expressions during macrophage-like differentiation of HL-60 cells.

Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage

Tescalcin is a 25-kDa EF-hand Ca2+-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16–48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15–30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

Nuclear Translocation of β-Actin Is Involved in Transcriptional Regulation during Macrophage Differentiation of HL-60 Cells

Studies have shown that nuclear translocation of actin occurs under certain conditions of cellular stress; however, the functional significance of actin import remains unclear. Here, we demonstrate that during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells toward macrophages, beta-actin translocates from the cytoplasm to the nucleus and that this process is dramatically inhibited by pretreatment with p38 mitogen-activated protein kinase inhibitors. Using chromatin immunoprecipitation-on-chip assays, the genome-wide maps of beta-actin binding to gene promoters in response to PMA treatment is analyzed in HL-60 cells. A gene ontology-based analysis shows that the identified genes belong to a broad spectrum of functional categories such as cell growth and differentiation, signal transduction, response to external stimulus, ion channel activity, and immune response. We also demonstrate a correlation between beta-actin occupancy and the recruitment of RNA polymerase II at six selected target genes, and beta-actin knockdown decreases the mRNA expression levels of these target genes induced by PMA. We further show that nuclear beta-actin is required for PMA-induced transactivation of one target gene, solute carrier family 11 member 1, which is important for macrophage activation. Our data provide novel evidence that nuclear accumulation of beta-actin is involved in transcriptional regulation during macrophage-like differentiation of HL-60 cells.

Ornithine decarboxylase interferes with macrophage-like differentiation and matrix metalloproteinase-9 expression by tumor necrosis factor alpha via NF-κB

Ornithine decarboxylase (ODC), a tumor promoter, provokes cell proliferation, and inhibits cell death; but the mechanism involved in cell differentiation remains unknown. Herein, we examine whether it functions during macrophage-like differentiation. Previous studies reveal that ODC, a rate-limiting enzyme of polyamine biosynthesis, and polyamines are involved in restraining immune response in activated macrophage. By using 12- O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human promyelocytic HL-60 and promonocytic U-937 cells, we discover that polyamines block the expression, secretion and activation of MMP-9. Meanwhile conventional expression of ODC represses tumor necrosis factor-alpha (TNF-α) expression and nuclear factor-kappaB (NF-κB) activation as well as MMP-9 enzyme activity. Following stimulation by TNF-α, the secretion of MMP-9 is restored in ODC-overexpressed cells. In addition, the NF-κB inhibitors (pyrrolidinedithiocarbamate, BAY-11-7082 and lactacystin) suppress the TPA-induced MMP-9 enzyme activity. Concurrently, both the irreversible inhibitor of ODC, α-difluoromethylornithine, and TNF-α could not recover MMP-9 activation following NF-κB inhibitor treatment in parental cells. Furthermore, ODC could directly inhibit and attenuate NF-κB DNA binding and transcriptional activation. Therefore, we suggest that ODC inhibits the TNF-α-elevated MMP-9 activation via NF-κB as TPA-induced macrophage-like differentiation and this interrupting mechanism may provide a new conceivable resolution why leukemia is poorly differentiated besides atypical growth.

Bystander Effect Studies in HL60 Human Promyelocytes

In the framework of the NOTE Integrated Project (FP6-36465, Euratom), we used for the first time HL60 human promyelocytes for bystander effects studies. This cell line is capable to differentiate in vitro towards either granulocytes or monocytes/macrophages and, therefore, one of the major interest is that it is possible to investigate the nature of signals released from the same cell type in various differentiating conditions and also to evaluate if cell differentiation can be induced through bystander mechanisms. The experiments have been focused (i) to the characterization of the biological system using the appropriate cluster of differentiation (CD) of cell surface molecules for the different cells of interest (i.e., promyelocytes, monocyte-and macrophage-like cells) and (ii) to obtain information about the response to signalling factors from sham/irradiated HL60 promyelocytes in terms of micronuclei (MN) induction and cell killing. Experiments were also performed to characterize the basal levels of ROS and RNS in cells at different stages of differentiation. Monocyte- and macrophage-like differentiation was obtained using vitamin D3 and phorbol diester (12-O-tetradecanoylphorbol-13-acetate, TPA) as inducers, respectively. Irradiation was performed with gamma rays and bystander experiments were carried out using the medium transfer approach. The experiments performed to characterize the differentiation status in the HL60 clone we used show that CD11b and CD11c appear both suitable for identifying macrophage-like and monocyte-like cells. The former are negative while the latter are positive to CD14. Actively proliferating promyelocytes present some positivity to CD95 but not to CD11b and CD11c or CD14. At the present experiments are planned aimed at distinguishing promyelocytes from macrophage-like cells in a mixed population. The results on MN induction in unirradiated promyelocytes by the medium collected from promyelocytes irradiated with 0.5 Gy and incubated for different times at 37°C show an increase after 2 h incubation that disappears at longer incubation times. Measurements of cell killing show that incubation with “conditioned” medium from 1 Gy irradiated promyelocytes leads to an increase in the cloning efficiency of the bystander promyelocytes, giving surviving fractions higher than 1. However, this effect seems more related to factors physiologically released by the cells than to factors induced by irradiation, as indicated by the comparison with medium from sham-irradiated cells. These results suggest that growth stimulating factors, instead of growth inhibiting factors, are released by HL60 promyelocytes.

Ceramide kinase expression is altered during macrophage-like cell differentiation of the leukemia cell line HL-60

Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes, so its expression levels should be strictly regulated. Here, we report that the mRNA expression and enzyme activity of CerK were decreased during macrophage-like cell differentiation of the leukemia cell line HL-60, yet neither was altered during granulocytic differentiation of the same cells. Our findings demonstrate that HL-60 cells are useful for studying CerK functions in leukocyte differentiation, and they also suggest that CerK might have an important role in such differentiation.

Inhibition of HuR and MMP-9 expression in macrophage-differentiated HL-60 myeloid leukemia cells by green tea polyphenol EGCg

Matrix metalloproteinase (MMP)-9 expression is linked with myeloid cell differentiation, as well as inflammation and angiogenesis processes related to cancer progression. MMP-9 secretion and macrophage-like HL-60 myeloid leukemia cells differentiation were triggered by the tumor-promoting agent PMA. The chemopreventive effects of green tea catechins epigallocatechin-gallate, catechin-gallate, and epicatechin-gallate, but not those catechins that lack a 3′-galloyl group, inhibited in a time- and dose-dependent manner MMP-9 secretion. The gene and protein expression of MMP-9 and of the mRNA stabilizing factor HuR were also inhibited, while that of the 67 kDa laminin receptor remained unaffected. Specific catechins may help optimize current chemotherapeutic treatment protocols for leukemia.

Purification and characterization of C-terminal truncated forms of histone H2A in monocytic THP-1 cells

Histones are key components of chromatin. We investigated histone H2A-immunoreactive proteins in acute monocytic leukemia THP-1 cells using three polyclonal antibodies raised against peptides corresponding to distinct regions of H2A. Two unknown immunoreactive proteins (9- and 12-kDa proteins), H2A (14 kDa) and ubiquitinated H2A (23 kDa) were found in the cell lysates prepared by immediate direct addition of SDS-PAGE sample buffer to the cells as well as in the nuclear and chromatin fractions. However, they were not found in the cytoplasmic fraction. The unknown proteins were successfully purified by immunoaffinity chromatography from the cell nucleus extract and identified as 9-kDa H2A 1–87 and 12-kDa H2A 1–114, suggesting that both were produced by limited proteolysis of intact H2A 1–129. The truncated forms of H2A probably persisted as chromatin constituents, since the stability of H2A 1–87 in the chromatin fraction was sensitive to treatment with micrococcal nuclease, and H2A 1–114 was solubilized with lower ionic strength from the chromatin fraction obtained by micrococcal nuclease treatment. Truncated H2A proteins in THP-1 cells were transiently increased in amount by short-term treatment with phorbol 12-myristate 13-acetate or all- trans-retinoic acid, both of which induce macrophage-like differentiation. Furthermore, these increases were suppressed by preceding treatment with carbobenzoxy- l-leucyl- l-leucyl- l-leucinal (MG132) but not with carbobenzoxy- l-isoleucyl-γ- t-butyl- l-glutamyl- l-alanyl- l-leucinal (PSI), both of which are generally known as proteasome inhibitors. Our results suggest that histone H2A is cleaved at least at two sites by protease(s) that remain obscure, and might affect chromatins in the early stage of THP-1 cell differentiation.

Expression of cell kinetics and death during monocyte–macrophage differentiation: effects of Actinomycin D and Vinblastine treatments

The different effects of two cytostatic drugs, Actinomycin D and Vinblastine, during macrophage-like differentiation induced in THP-1 monocytic cell line by phorbol ester phorbol 12-myristate 13-acetate (PMA) (6, 30, and 60 nM), were studied by morpho-cytochemical approaches. In PMA-unstimulated monocytic cells, the cytostatic effects of Actinomycin D (an antimetabolic drug) were characterized by a drastic reduction of the G2/M cells accompanied by dramatic death of the G1 cells; on the contrary, Vinblastine (a microtubule-depolymerizating drug) induced an accumulation of the G2/M cells with the appearance of aneugenic micronuclei and scarce cell death mainly from the G1 cells. After 60 nM PMA stimulation, the culture was mostly composed by macrophagic cells characterized by low proliferation and the appearance of mono-/binucleated polyploid cells; in this condition, the cytotoxicity of the two drugs, more effective for Vinblastine, induced cell death in the different ploidy classes (2c, 4c, 8c). Cell death appeared to be of apoptotic nature, but with some morpho-phenotypic differences due to the action mechanism of the drugs and dependent on cell culture growth and differentiation. As a consequence of the different block-action of the two drugs on the cell cycle phases and in relation to the different subcellular targets, the effects changed during the transition from not-adhering/proliferating monocytes to adhering/low-proliferating differentiated macrophages.