Differentiation Markers CD14 Research Articles

BCR-ABL1 Dislocates NPM1, PU.1 and KLF1 into Cytoplasm to Thereby Skew Granulo-Monocytic and Impede Erythroid Differentiation

BCR-ABL1 causes chronic myeloid leukemia (CML) but the molecular mechanisms by which it skews granulo-monocytic commitment towards granulocytes, increases erythroid precursors yet causes anemia (Bruns et al, Leukemia 2009), and positions for specific lineage-phenotypes of blast crises (granulocytic, erythroid, B-lymphocytic), are unknown. To investigate these mechanisms, we reasoned that distinctly altered myeloid differentiation suggests specific impacts of BCR-ABL1 on master transcription factors (MTFs) that specify and drive myeloid lineage fates. Although wildtype ABL1 is nuclear, BCR-ABL1 is cytoplasmic. We therefore examined cellular location of lineage MTF in CML blast crisis cells K562 and LAMA84. These cells, like 7/14 (50%) of CML cell lines in the CCLE repository, highly express the lineage MTFs GATA1 and KLF1, that specify and drive to terminal erythroid fates. Incongruously, K562 and LAMA84 also highly express PU.1 essential for monocyte or B-cell fates. Using immunofluorescence microscopy (IF) of methanol-fixed K562 ( Fig.1A) and LAMA84, we found GATA1 in nuclei, but KLF1 and PU.1 were in cytoplasm. We corroborated these results by Western blot for these MTF in nuclear vs cytoplasmic fractions. Kinase functions of ABL1 in BCR-ABL1 are essential for BCR-ABL1 mediated oncogenesis. Therefore, we investigated if these kinase functions mediate KLF1 and PU.1 dislocation: one hour (1h) exposures to ABL1-kinase inhibitors imatinib 250nM or dasatinib 1nM relocated KLF1 and PU.1 into nuclei, seen both by IF ( Fig.1A) and by Western blot of nuclear/cytoplasmic fractions. We then examined cell fate impacts of the KLF1 and PU.1 nuclear relocations. Imatinib and dasatinib upregulated terminal erythroid lineage-differentiation markers transferrin (CD71) or glycophorin A (CD235a) (~10-fold increase in median fluorescence intensity, flow cytometry at 72h) ( Fig.1B), and increased hemoglobinization, evident by cell pellet reddening to the naked eye. The monocyte differentiation marker CD14 was not increased. As expected from resumed lineage-maturation, proliferation measured at 72h was terminated by imatinib 250nM or decreased ~50% by dasatinib 1nM vs vehicle, even though these drug concentrations did not increase apoptosis vs vehicle, measured by annexin staining at 24h. To further investigate the pathway by which BCR-ABL1 kinase dislocates KLF1 and PU.1 into cytoplasm, we then looked at NPM1, because: (a) NPM1 chaperones PU.1, and mutant-NPM1 that causes acute myeloid leukemia (AML) dislocates into cytoplasm, dislocating PU.1 with it (Gu et al, J Clin Invest 2018); and (b) NPM1 is neither mutated nor deleted (by 5q-) in CML blast crises, even though such alterations are highly recurrent in AMLs, consistent with NPM1 participating in the BCR-ABL1 pathway to begin with. NPM1 was accordingly mostly in cytoplasm in K562 and LAMA84 cells, and 1h of imatinib or dasatinib relocated NPM1 into nuclei, with NPM1/KLF1 colocalizing ( Fig.1A). These results were corroborated by Western blot of nuclear/cytoplasmic fractions. We are presently immunoprecipitating endogenous NPM1 from nuclear/cytoplasmic fractions of K562 cells and analyzing by mass spectrometry (LCMS/MS) to examine: (a) NPM1 chaperone function for KLF1 as well as PU.1; and (b) NPM1 phosphorylation in the different fractions and kinase-inhibitor impacts on this. Taken altogether, these results outline a pathway in which BCR-ABL1 phosphorylates NPM1 to dislocate it into cytoplasm, to hence also dislocate chaperoned KLF1 and PU.1. Interference with specific lineage MTFs in this way can: (a) expand erythroid precursors but block their maturation, because nuclear GATA1 allows erythroid commitment, but KLF1 (in nuclei) is needed for maturation; (b) skew commitment decisions to granulocytes over monocytes, via decreased nuclear PU.1/CEBPA ratio; (c) expand mature granulocytes, because PU.1 contributes to, but is not essential for, granulocyte lineage-maturation, and NPM1 is downregulated in the granulocyte lineage, releasing terminal maturation; (d) transform B-cell lineage precursors, since PU.1 is an essential B-cell MTF; and (e) position for specific lineage-phenotypes and genetics of CML blast crises, evolving from these foundations. Since the ABL family kinase ABL2 is amplified in a third of all cancers, these motifs for altering lineage MTF function for oncogenesis may operate beyond CMLs.

Loss of CCAAT‐Enhancer Binding Protein Delta Promotes Acute Myeloid Leukemia Cell Proliferation and Survival By Upregulating Cyclin D1 Expression

Despite improvements in the treatment options, acute myeloid leukemia (AML) remains a fatal malignancy with an estimated 5-year relative survival rate of 31.7%. There is continued need for identification of new disease drivers to develop novel therapeutic options and improve clinical outcomes. Dysregulated transcriptional landscapes are associated with disease pathogenesis and treatment responses. To identify new disease drivers of AML, we analyzed RNA-Seq data from 59 patient-matched diagnosis and relapse (PAML) specimens (study cohort). A set of five differentially expressed genes (DEGs; STAT4, XRCC2, USP3, CEBPD, RBM47) with concordant changes in expression between diagnosis and relapse (Figure 1A) were selected for study using the following criteria: (a) concordant differential expression (DESeq2: log2FC > |1.2| and q < 0.05) in at least 25% of the relapsed patients in the study cohort (b) concordant differential expression in independent datasets of RNA-Seq without any experimental validation, and (c) high cancer driver potential based on their molecular function. Binding analysis for regulation of transcription (BART) on DEGs between normal CD34 + cells, diagnosis and relapsed AML patient specimens suggested that CEBPD could function as a transcriptional regulator of a subset of the DEGs. We hypothesized that dysregulated expression of these genes promotes AML disease progression. To examine their role in AML, we altered their expression in AML cell line models and measured changes in AML cell phenotypes. Of these genes, CRISPR-mediated knockdown of the transcription factor CCAAT/enhancer-binding protein delta (CEBPD) enhanced AML cell proliferation, self-renewal capacity, and survival of the relapsed AML cell line OCI-AML5. CEBPD knockdown also conferred OCI-AML5 resistance to cytarabine - a common chemotherapy drug used for AML treatment. Short hairpin RNA (shRNA)-mediated knockdown of CEBPD in OCI-AML5 validated these results. CRISPR-mediated upregulation of CEBPD expression induced monocyte/macrophage differentiation marker CD14 expression and enhanced apoptosis rates in OCI-AML5 cells. CEBPD upregulation also induced differentiation in the diagnosis AML cell line OCI-AML2. Survival analyses showed that lower expression of CEBPD was significantly associated with poor survival in pediatric AML patients, and there was a trend of lower CEBPD expression associating with worse survival in adult AML patients (UCSC Xena browser). These results altogether suggest that CEBPD plays a tumor suppressor role in AML. Gene set enrichment analyses (GSEA) on RNA-Seq data revealed significant positive enrichment of cell proliferation pathways upon CEBPD knockdown in OCI-AML5 cells as well as in a subset of the PAML patients with downregulated CEBPD expression at relapse. Differential gene expression and GSEA analyses altogether suggested upregulated cyclin D1 as one of the downstream drivers of enhanced growth of AML cells with repressed CEBPD expression (Figure 1B). RT-qPCR and Western Blot experiments confirmed upregulation of CCND1 expression in CEBPD knockdown OCI-AML5 and OCI-AML2 cells. Next, we sought to understand the molecular mechanisms repressing CEBPD expression during AML disease progression. We found an inverse association between CEBPD and DNMT3 methyltransferases mRNA expression in PAML and independent datasets. Treatment with the hypomethylating agent azacitidine upregulated CEBPD mRNA expression in multiple AML cell lines including OCI-AML5 and OCI-AML2, suggesting that hypermethylation represses CEBPD gene expression in these AML cells. Integrative analysis of gene expression with DNA methylation at the CEBPD promoter identified hypermethylation of multiple CpGs within the binding region of the transcription factor Sp1 in a subset of patients with CEBPD downregulation. CpG methylation has been shown to interfere with Sp1 binding at the CEBPD promoter. Since Sp1 binding promotes CEBPD transcription, hypermethylation at these sites may repress CEBPD expression during AML disease progression. Our study suggests that CEBPD can function as tumor suppressor in AML. Results suggest a role for DNA methylation in repressing CEBPD expression during AML disease progression. Furthermore, we show that CEBPD repression promotes AML cells' growth potentially via inducing the expression of the cell cycle driver CCND1.

1,25-Dihydroxyvitamin D3 induces human myeloid cell differentiation via the mTOR signaling pathway

1,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 is known to play an important role in the differentiation of human myeloid cells. However, the molecular mechanism underlying the 1,25(OH)2D3-mediated differentiation of human myeloid cells is incompletely understood. Here, we report that 1,25(OH)2D3 induces differentiation of human myeloid cell lines such as U937 and THP-1 cells via the mammalian target of rapamycin (mTOR) signaling pathway. Both the expression of the differentiation marker CD14 and activation of the mTOR signaling pathway were induced by 1,25(OH)2D3 in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 and THP-1 cells. The 1,25(OH)2D3-induced expression of CD14 in PMA-differentiated U937 and THP-1 cells was prevented by mTOR inhibitors, PP242 and Torin1. The 1,25(OH)2D3-induced morphological changes as characteristics of differentiated myeloid cells were also reversed after PP242 and Torin1 treatment. Silencing of either regulatory-associated protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) in PMA-differentiated THP-1 cells with small-interfering RNA resulted in the inhibition of CD14 expression and morphological changes induced by 1,25(OH)2D3, indicating that both mTORC1 and mTORC2 were important for the differentiation of myeloid THP-1 cells. Previous studies have shown that phosphatidic acid (PA) maintains the stability of the mTOR complex. Here we found that the attenuation of PA production with 1-butanol or a PLD inhibitor prevented the 1,25(OH)2D3-induced upregulation of CD14. Taken together, our results show that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway.

Inhibition of DOT1L and PRMT5 Promotes Anti-Tumor Activity in a Human MLL Leukemia Model Induced By CRISPR/Cas9

MLL rearrangements play a crucial role in leukemogenesis. They are associated with a poor prognosis and new treatment strategies are urgently needed. Here, we used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4/-AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient leukemic cells faithfully mimicking the nature of the disease. Thus, it serves as ideal fundamental basis for pharmacological studies.The catalytic activity of disruptor of telomeric silencing 1-like histone methyltransferase (DOT1L) is critical for the transforming potential of MLL oncogenes. Therefore, inhibition of DOT1L deals as one specific therapeutic approach for MLL-rearranged leukemias currently tested in clinical trials. However, the previous success was limited by restricted response warranting further investigation of DOT1L combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines in vitro and in MLL mouse models. Here, we used DOT1L (EPZ004777) and PRMT5 (EPZ015666) inhibitors alone or in combination in our human MLL-rearranged model demonstrating a dose-dependent reduced proliferation, impairment of cell cycle, upregulation of differentiation marker CD14 and apoptosis measured by flow cytometry and downregulation of known MLL target genes determined by qPCR. Strikingly, the combination of both compounds led to complementary anti-tumor effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias. DisclosuresNo relevant conflicts of interest to declare.

Pan-Mutant-IDH1 Inhibitor Bay-1436032 Is Highly Effective Against Human IDH1 Mutant Acute Myeloid Leukemia In Vivo

Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are frequently found in patients with acute myeloid leukemia (AML) and several other tumors. Mutant IDH1 produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, and leads to a block in differentiation to promote tumorigenesis.Methods: We developed a novel, highly active oral pan-IDH1 inhibitor, BAY-1436032, for clinical evaluation. Its inhibitory potency was evaluated in primary human AML cells in vitro for the five major IDH1R132 mutation types and in two patient derived AML xenograft (PDX) models in vivo, in which BAY-1436032 cleared leukemic blasts in peripheral blood and prolonged survival by induction of differentiation and inhibition of leukemia stem cell self-renewal.Results: R-2HG production by mutant IDH1 was effectively inhibited in patient derived AML cells with all reported IDH1R132 mutations ex vivo by BAY-1436032 with an IC50 between 3 to 16 nM. AML cells cultured ex vivo showed morphologic differentiation and marked upregulation of the myeloid differentiation markers CD14 and CD15.For in vivo experiments, human AML cells from two patients were transplanted into sublethally irradiated NSG mice. After stable engraftment at 17 (PDX1) or 90 (PDX2) days post transplantation, mice were treated with BAY-1436032 orally every day at a dose of 150 mg/kg or vehicle for 100-150 days (n=10 per group). The R/S-2HG ratio in serum was reduced to near normal levels by BAY-1436032. Leukemic cell counts in peripheral blood constantly increased in control mice, while leukemic cells declined from day 30 of BAY-1436032 treatment onwards with morphologic and immunophenotypic evidence of differentiation (Figure). Importantly, all BAY-1436032 treated PDX1 mice survived until the end of treatment at 150 days. In contrast, vehicle-treated mice died with a median latency of 91 days (range 70-95, P<.001). In an independent second model (PDX2) 6 of 10 BAY-1436032 treated mice survived until the end of treatment at day 100 with a median of 15% leukemic cells in peripheral blood, while all vehicle-treated mice suffered from high leukemic burden and died from leukemia with a median survival of 62 days (P=.014). Early mortality was increased with 4 mice dying in the BAY-1436032 group reminiscent of clinical differentiation syndrome in AML patients treated with the IDH1 inhibitor AG-120.To assess the effect of BAY-1436032 on leukemic stem cell self-renewal we treated PDX1 mice with 150 mg/kg BAY-1436032 or vehicle for 4 weeks and performed a limiting dilution transplantation experiment in secondary recipient mice. LSC frequency was 100-fold lower in BAY-1436032 treated compared to control mice. Gene expression profiling showed that stemness associated genes were downregulated, while genes associated with myeloid differentiation like PU.1 and CEBPA were upregulated upon treatment with BAY-1436032. In addition, cell cycle progression was slowed and E2F transcription factors concomitantly inhibited. In accordance with gene expression profiling results, methylation of the PU.1 promoter decreased, while E2F1 promoter methylation increased upon treatment with BAY-1436032. Finally, histone trimethylation levels at residues H3K4, H3K9, H3K27, and H3K36 decreased in both IDH1R132C and IDH1R132H mutant AML cells but not in IDH1 wildtype cells upon BAY-1436032 treatment.Conclusion: In summary, the novel oral pan-mutant IDH1 inhibitor BAY-1436032 is active against all IDH1R132 mutation types and shows strong anti-leukemic activity in two independent AML PDX mouse models. Clinical development is ongoing with a first in man study with BAY-1436032 in IDH1 mutant solid tumors.* M. Heuser and L. Herbst contributed equally to this article#A. Krämer and A. Chaturvedi share senior authorship [Display omitted] DisclosuresHeuser:Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Pusch:German Cancer Research Center: Patents & Royalties: WO2013/127997A1. Kaulfuss:Bayer Pharma AG: Employment. Panknin:Bayer Pharma AG: Employment. Zimmermann:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210 . Toschi:Bayer Pharma AG: Employment. Neuhaus:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210. Haegebarth:Bayer Pharma AG: Employment, Equity Ownership. Rehwinkel:Bayer Pharma AG: Employment, Equity Ownership, Patents & Royalties: WO2015/121210. Hess-Stumpp:Bayer Pharma AG: Employment. Bauser:Bayer Pharma AG: Employment. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. von Deimling:German Cancer Research Center: Patents & Royalties: IDH1R132H mutant specific antibody H09; BRAF V600E mutant specific antibody VE1; BAY-1436032 patent.

Silencing of CD47 and SIRPα by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells.

BackgroundIn the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα.In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.MethodsTHP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays.ResultsTHP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.ConclusionsOur data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation

NPM1 and DNMT3A mutations are highly recurrent (∼30% of cases) in de novo acute myeloid leukemia (AML). Besides aberrant cytoplasmic localization of mutated NPM1, and decreased DNA methyltransferase activity of mutated DNMT3A, the molecular mechanisms of pathogenesis are mysterious. Here, novel specific molecular mechanisms are demonstrated. These results build on and are separate from data presented (poster) at ASH 2012.LC-MSMS analysis of murine hematopoietic cells was used to analyze the protein interaction network of the key monocyte/macrophage differentiation-driving transcription factor Pu.1. Npm1 was noted to interact with Pu.1 in these analyses, confirmed by bidirectional co-immunoprecipitation-Western blot assays. This interaction suggested that PU.1 might be dragged into the cytoplasm by mutated NPM1 in AML. Accordingly, in the OCI-AML3 cell line that contains an NPM1 mutation, but not in OCI-AML2 cells with wild-type NPM1, PU.1 was co-dislocated into the cytoplasm together with NPM1, obvious by Western analysis of cellular fractions, and by immunofluorescence (IF) assays. IF analysis of primary AML cells from patients with NPM1 mutated AML confirmed this cytoplasmic dislocation of both NPM1 and PU.1 (n=3), with strong cytoplasmic instead of nuclear staining of both proteins. In contrast, primary AML cells with wild-type NPM1demonstrated the expected strong staining of these proteins in the nucleus but not in the cytoplasm (n=3).NPM1 movement into the cytoplasm is mediated by CRM1 (exportin). Thus, we hypothesized that antagonizing mutated NPM1 interaction with CRM1 would retain mutated NPM1 and PU.1 in the nucleus. Decoy peptides based on NPM1 C-terminal sequences, to hopefully minimize disruption to CRM1 interactions with other proteins, were designed to antagonize NPM1 binding to CRM1. These decoy peptides were combined with different nuclear delivery sequences including the TAT peptide, and with fluorescent tags for tracking. All six peptides entered AML-OCI3 cells (shown by IF), with the strongest signals observed for peptides fused to TAT and TAT-NES (nuclear export signal). The decoy peptides significantly and substantially inhibited cell growth of AML-OCI3 cells (>3-fold reduction), accompanied by an increase in expression of the monocyte differentiation marker CD14 (quantified by flow-cytometry). In contrast, TAT peptide alone, as a control, did not inhibit cell growth or induce monocytic differentiation. Western and IF analyses was used to study PU.1 and NPM1 localization: decoy peptide treatment clearly increased nuclear presence of both PU.1 and NPM1, although these proteins remained detectable in cytoplasmic fractions also.At ASH 2012, we showed that OCI-AML3 cells have high nuclear CEBPA and retain granulocytic differentiation potential, readily induced by ATRA. Thus, we hypothesized that the NPM1-induced differentiation block, that is specific for the monocytic lineage, creates selective pressure for cooperative mutations that derepress monocyte-commitment. DNMT3A interacts with polycomb proteins that repress lineage-programs, and in NPM1/DNMT3A double mutant versus NPM1 mutant AML, double mutation was significantly associated with M4/M5 (14/23 [61%]) versus M1 morphology (4/13 [30%], p<0.001), higher peripheral blood absolute monocyte counts (median 3.96 v 0.37, p=0.04) and bone marrow non-specific esterase positivity (14/21 [67%] v 7/19 [37%], p=0.06)(TCGA). Chromatin-immunoprecipitation (ChIP) analyses in OCI-AML3 (NPM1/DNMT3A double mutant) and THP1 cells (NPM1/DNMT3A wild-type) that have similar DNMT3A protein expression, demonstrated DNMT3A localization at all (n=7) PU.1 proximal promoter and distal enhancer elements quantified by QRT-PCR (2-40 fold enrichment over ChIP with IgG). Consistent with decreased methyl-transferase activity of the recruited mutated DNMT3A in OCI-AML3, methylation levels at 9 CpG in the PU.1proximal promoter (chr11: 47399783–47400031, pyrosequencing) were significantly lower in OCI-AML3 than in THP1 cells (30-60% of methylation level in THP1). PU.1 mRNA expression was strikingly higher (>40-fold, p<0.001) in OCI-AML3 cells compared to THP1, although THP1 cells also have M5 morphology.Thus, specific molecular mechanisms explain the lineage-associations and coincidence of NPM1 and DNMT3A mutations in AML. These mechanisms provide guidance for novel treatment methods. Disclosures:Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Maturation and demise of human primary monocytes by carbon nanotubes

The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10–50 nm, length up to 10 μm) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses.

Circulating tumor cells in melanoma patients.

Circulating tumor cells (CTCs) are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X) from blood of melanoma patients using a simple centrifugation device (OncoQuick), and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF) were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively) compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001). There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001), and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50%) stained for both pan-cytokeratin (KRT) markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14). Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA) may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs). The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids, and their role in metastatic progression.

Methanol extract of Antrodia cinnamomea mycelia induces phenotypic and functional differentiation of HL60 into monocyte-like cells via an ERK/CEBP-β signaling pathway

Antrodia cinnamomea (named as Niu-chang-chih), a well-known Taiwanese folk medicinal mushroom, has a spectrum of biological activities, especially with anti-tumor property. This study was carried out for the first time to examine the potential role and the underlying mechanisms of A. cinnamomea in the differentiation of human leukemia HL60 cells. We found that the methanol extract of liquid cultured mycelia of A. cinnamomea (MEMAC) inhibited proliferation and induced G1-phase cell cycle arrest in HL60 cells. MEMAC could induce differentiation of HL60 cells into the monocytic lineage, as evaluated by the morphological change, nitroblue tetrazolium reduction assay, non-specific esterase assay, and expression of CD14 and CD11b surface antigens. In addition, MEMAC activated the extracellular signal-regulated kinase (ERK) pathway and increased CCAAT/enhancer-binding protein β (C/EBPβ) expression. Reverse transcriptase polymerase chain reaction analysis showed that MEMAC upregulated the expression of C/EBPβ and CD14 mRNA in HL60 cells. DNA affinity precipitation assay and chromatin immunoprecipitation analyses indicated that MEMAC enhanced the direct binding of C/EBPβ to its response element located at upstream of the CD14 promoter. Furthermore, inhibiting ERK pathway activation with PD98059 markedly blocked MEMAC-induced HL60 monocytic differentiation. Consistently, the MEMAC-mediated upregulation of C/EBPβ and CD14 was also suppressed by PD98059. These findings demonstrate that MEMAC-induced HL60 cell monocytic differentiation is via the activating ERK signaling pathway, and downstream upregulating the transcription factor C/EBPβ and differentiation marker CD14 gene, suggesting that MEMAC might be a potential differentiation-inducing agent for treatment of leukemia.

Vitamin D3 Induces Autophagy of Human Myeloid Leukemia Cells

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.

A model system in haematology and immunology: The human monocytic cell line MONO-MAC-1

MONO-MAC-1 is a human cell line with properties of blood monocytes, which can be used as a model system to study monocytic functions in vitro. In the present study, we prepared a karyotype of MONO-MAC-1, analysed the growth behaviour, determined the presence of differentiation-associated antigens and studied the expression and secretion of several cytokines upon stimulation with 12-O-tetradecanoyl phorbol 13-acetate (TPA) and lipopolysaccharide (IPS). The MONO-MAC-1 cells have a near diploid karyotype and contain several recurrent chromosomal rearrangements, in particular the translocation (9;11) commonly found in AML-M5. Stimulation with TPA or LPS induced changes in morphology and gene expression, especially an increase in the level of the differentiation marker CD14 and the production of monocyte-related cytokines. Both biomodulators alone were sufficient to promote TNFα release; however, the combination of TPA and LPS resulted in a synergistic increase of TNFα secretion. Northern blot analysis indicated that upregulated production of TNFα was due to induced synthesis of mRNA. The mRNA accumulation peaked approximately 2 h after stimulation and maximum levels of TNFα were found in the supernatants after 4–8 h of culture. The MONO-MAC-1 cells could not be restimulated with the same inducer to release TNFα when a 48 h pre-treatment was carried out with LPS or TPA. LPS induced the release of granulocyte colony-stimulating factor (G-CSF), while TPA failed to do so. Vice versa, secretion of macrophage CSF (M-CSF) could be induced by TPA, but not by LPS. However, LPS enhanced the TPA-induced M-CSF production. Similarly, incubation of MONO-MAC-1, simultaneously with TPA and LPS, led to granulocyte macrophage CSF (GM-CSF) and interleukin-1β (IL-1β)secretion, while both stimulators alone had almost no (TPA) or only a weak (LPS) effect on the secretion of GM-CSF and IL-1β. Our results demonstrate that MONO-MAC-1 is a unique cell line with distinct monocytic features; certain monocytic properties can be upregulated by activation of intracellular signalling pathway(s). We suggest that, besides the LPS receptor CD14, activation of PKC participates in these process, especially in the production and secretion of cytokines by MONO-MAC-1 cells.

IL-4 Decreases the Expression of the Monocyte Differentiation Marker CD14, Paralleled by an Increasing Accessory Potency

IL-4 has been found to affect the phenotype and a variety of functions of human monocytes and macrophages and has been discussed as a monocyte activating protein along with other cytokines, such as IL-1 and IL-6. In this study we compared the effects of the cytokines IL-1, IL-6, IL-4, and a combination of IL-1 and IL-6 on the expression of the CD14 antigen, the FcIIIg receptor molecule CD16 and the MHC-class II molecules HLA-DR and HLA-DP. These molecules represent characteristic monocyte surface markers. Furthermore, the CD14 molecule has been described as a surface antigen of high in vivo relevance representing an indirect receptor for LPS. We further analyzed the effect of IL-4 on monocytes and macrophages with respect to their accessory function to initiate T-lymphocyte proliferation. Human peripheral blood monocytes strongly express the antigen CD14 and maintain it as a stable surface molecule during their differentiation to macrophages. Flow cytometry analysis of cultured monocytes demonstrated that cells incubated in the presence of IL-4, but not IL-1 and/or IL-6 revealed a reduced expression of the CD14 antigen in a dose- and time-dependent manner. After 3 days IL-4 treated cells were virtually CD14-negative. At the same time the expression of the CD 16 antigen (FcRIIIg) was also strongly reduced, whereas the treatment with IL-4 led to an increased expression of MHC class II antigens such as HLA-DR and HLA-DP. The spontaneous low expression of HLA-DQ antigen on monocytes was not affected by any of the cytokines. Functionally, IL-4 treated CD14-negative monocytes exhibited a more than 2-fold higher activity to stimulate an accessory cell-dependent T cell proliferation. This was found in a mitogenic assay and in MLC when compared to monocytes cultured in the absence of IL-4. These observations provide further evidence that IL-4 is a major modulator of monocyte surface antigen expression. Moreover, IL-4 has an enhancereffect on monocytes as accessory cells and therefore may be of considerable importance as a regulatory factor during monocyte development to accessory cells. Inasmuch as the CD14 molecule functions as a receptor for LPS-binding protein, our results suggest that IL-4 might also play an important regulatory role in processes initiated by bacterial lipopolysaccharides during inflammation and sepsis.