CD14 Mediates Phagocytic Activity during the Granulocytic Differentiation Process in Acute Promyelocytic Leukemia Cells

Abstract All-trans retinoic acid (ATRA) can induce acute promyelocytic leukemia (APL) cells differentiation into mature granulocytes. CD14 and Toll-like receptor 4 (TLR-4) play an important role in the phagocytic activity of macrophage, however, their role during granulopoiesis is still unclear. In this study, we determined the role of CD14/TLR-4 in the development of phagocytic activity in NB4 APL cells after induction into the process of granulocytic differentiation by ATRA. Flow cytometry analysis demonstrate that, during ATRA treatment for 6 days, the phagocytic activity of NB4 cells in engulfing either fluorescein-latex beads or idarubicin-induced apoptotic cells increased in a time-dependent manner, and the level of CD14 expression on NB4 cells was also significantly increased in a time dependent manner, though its level was only minimally expressed in ATRA-untreated NB4 cells. However, TLR-4 was constitutionally expressed in ATRA-untreated cells and its level did not changed significantly during the first 5 days of ATRA treatment. Further study demonstrates that the phagocytic activity of ATRA-NB4 cells was significantly inhibited by pre-treating cells with antibodies specific to either CD14 or TLR-4 before phagocytosis assay. In exploring the role of CD14/TLR4 associated signal transduction mediators, NF-κB and IRF-3, we further demonstrate that the phagocytic activity of ATRA-NB4 cells in engulfing beads was significantly inhibited when cells were pretreated with either a NF-κB inhibitor (BAY 11-7082) or an IRF-3 inhibitor (SP600125). However, this activity in engulfing apoptotic cells was only significantly inhibited by pretreatment with BAY11-7082, but not by pre-treatment with SP600125. Finally, our results indicate that the level of CD14(+) microparticles (MPs) released by ATRA-NB4 cells was significantly enhanced when those cells were induced into the process of apoptosis by pre-treatment with idarubicin. Moreover, by incubation with MPs derived from apoptotic ATRA-NB4 cells, the phagocytic activity of living ATRA-NB4 cells in engulfing apoptotic cells was significantly enhanced, and this phagocytic activity was also significantly inhibited by pre-treating MPs with antibody specific to CD14 before phagocytic assay. We conclude that CD14 contributes to the phagocytic activity of APL cells during the process of granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

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PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Blood ◽

10.1182/blood-2020-136230 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 43-43

Author(s):

Pavithra Shyamsunder ◽

Shree Pooja Sridharan ◽

Pushkar Dakle ◽

Zeya Cao ◽

Vikas Madan ◽

...

Keyword(s):

Retinoic Acid ◽

Regulatory Element ◽

Promyelocytic Leukemia ◽

Luciferase Reporter ◽

Myeloid Differentiation ◽

Atra Treatment ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Acute Myeloid

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

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Molecular Mechanisms Involved in the Development of Extramedullary Disease Following the Administration of All-Trans Retinoic Acid to Patients with Acute Promyelocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.1596.1596 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1596-1596

Author(s):

Adelina Ovcharenko ◽

Galit Granot ◽

Ofer Shpilberg ◽

Pia Raanani

Keyword(s):

Promyelocytic Leukemia ◽

Time Dependent ◽

Mrna Levels ◽

Dependent Manner ◽

Atra Treatment ◽

Nb4 Cells ◽

Adhesion Ability ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Associated Proteins

Abstract Abstract 1596 Poster Board I-622 The administration of all-trans retinoic acid (ATRA) has been associated with the occurrence of extramedullary disease (EMD), despite improvement in the prognosis of patients with acute promyelocytic leukemia (APL) treated with ATRA. EMD has been reported to occur in 3%–8% of patients with APL primarily in the central nervous system and skin. We postulate that, similar to the processes responsible for the development of metastasis, changes in the expression of proteins participating in adhesion, migration and homing may enable malignant hematopoietic cells to inhabit extramedullary sites. Our objective is to identify the molecular and cellular changes associated with exposure of the APL cell lines, NB4 and HL60 to ATRA and to establish the role of these changes in the development of EMD associated with ATRA in APL. We found that 30% of NB4 and HL60 cells, treated with ATRA adhere to fibronectin as opposed to untreated cells which show virtually no adhesion ability. A microarray screen revealed that many of the genes whose expression was altered following ATRA treatment participate in migration and adhesion processes. Among them was PYK2 whose expression was increased by 3-fold. PYK2 is an intracellular non-receptor tyrosine kinase which plays a role in intracellular signaling pathways that regulate processes such as cell adhesion and migration, which have been shown to correlate with tumor development and aggression. It is established that upon stimulation, PYK2 migrates to the membrane, where it is phosphorylated and activated leading to recruitment of additional proteins ultimately initiating cell adhesion. We found that pyk2 mRNA levels were upregulated in a time-dependent and ATRA-dependent manner. pyk2 mRNA levels were reduced to their basal level following ATRA withdrawal. The increase detected in PYK2 mRNA expression was due to enhanced transcription. In addition, PYK2 protein expression and phosphorylation levels were also upregulated in a time-dependent and ATRA-dependent manner. Interestingly, PYK2 protein expression level remained high even 5 days after ATRA withdrawal. Accordingly, NB4 cells maintained their adhesion ability for at least 5 days after ATRA depletion. Several additional PYK2-associated proteins: paxillin, vinculin, talin, GSK3a, integrin β7 and integrin β2 were upregulated at least 2-fold following treatment. Unexpectedly, we found 2 PYK2 isoforms in NB4 cells - the 116KDa known PYK2 isoform and a novel ∼80kDa isoform. Although both isoforms were upregulated and significantly phosphorylated following ATRA treatment, only the 80kDa isoform co-precipitated with paxillin following treatment. In conclusion, we show that following ATRA treatment, the expression of PYK2 and many PYK2-associated proteins is upregulated; PYK2 is extensively phosphorylated, initiating its activation and association with paxillin, possibly leading to the observed adherence of NB4 cells to fibronectin. These findings may contribute to our understanding of the molecular events associated with EMD in APL patients treated with ATRA. This in turn might help in the prevention and treatment of this phenomenon. Disclosures No relevant conflicts of interest to declare.

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ATRA-Induced Activation of the Autophagy Regulator Tfeb Regulates Myeloid Differentiation in Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v124.21.1057.1057 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1057-1057

Author(s):

Nina M. Orfali ◽

Nigel P. Mongan ◽

Mary R Cahill ◽

Sharon L. McKenna ◽

Lorraine J. Gudas

Keyword(s):

Gene Expression ◽

Combined Therapy ◽

Promyelocytic Leukemia ◽

Atra Treatment ◽

Granulocytic Differentiation ◽

Educational Support ◽

Nb4 Cells ◽

Retinoid Signaling ◽

Qrt Pcr ◽

Retinoid Therapy

Abstract Introduction Acute promyelocytic leukemia (APL) is characterized by gene fusions involving RARα, which disrupt retinoid signaling and lead to the clonal expansion of myeloid precursors halted at the promyelocyte stage of maturation. Treatment with all-trans-retinoic acid (ATRA) can overcome this differentiation block, restore granulocytic differentiation in APL cells and improve APL patient outcome. ATRA treatment of APL cells induces autophagy, a catabolic process whereby redundant proteins and organelles are degraded using lysosomal machinery. ATRA-induced autophagy is important for successful granulocytic differentiation of APL cells through the degradation of fusion oncoproteins and executing the structural protein remodeling necessary for cellular differentiation. The molecular mechanisms linking retinoid signaling and autophagy pathways are poorly understood and are the focus of our study. By integrating available chromatin immunoprecipitation, coupled with next-generation sequencing (ChIPseq) datasets and transcriptomic approaches, we examined the transcriptional effects of ATRA treatment on autophagy-related genes during APL cell differentiation. We identified transcription factor EB (TFEB), a master transcriptional regulator of autophagy, as a retinoid target and have evaluated the consequences of TFEB knockdown on both ATRA-induced autophagy and APL cell differentiation. Methods & Results A list of 557 autophagy-related genes was generated from 3 public autophagy databases. Cluster analysis of expression of these 557 genes in the APL TCGA RNAseq dataset, suggest autophagy is associated with patient survival (Cluster 1 = 26.4 months / Cluster 2 = 60 months). We therefore examined whether the 557 autophagy related genes may be direct ATRA targets. Accessing a public ChipSeq database (GSE18886), increased RARα binding was detected within the TFEB gene in NB4 cells (APL cell line, t15:17) treated with ATRA for 24 hours. Increased RNAPolII binding and transcriptionally active histone markers were also detected, consistent with a direct transcriptionally activating retinoid effect. To test the effects of retinoid therapy on APL gene expression, we treated NB4 cells with ATRA or ethanol control for 72 hours. Isolated RNAs were sequenced using the Illumina HiSeq 2000 platform. Reads were aligned and annotated to the hg19 genome using Tophat and subsequent differential expression analysis performed using Cufflinks/Cuffdiff and Cluster/Treeview. We observed changes in autophagy gene expression upon ATRA treatment and recorded a significant >2 fold increase in expression in >50 autophagy-related genes including TFEB, p62 and DRAM1. We then carried out lentivirally-mediated shRNA knockdown of TFEB in NB4 cells and treated both knockdown and scrambled control cells with ATRA for 72 hours. Transcriptomic analysis demonstrated that TFEB knockdown blocked ATRA-induced TFEB induction, affected the regulation of key autophagy-related genes and impaired CD11b expression. These results were validated by qRT-PCR (n=6)(P<0.001). We confirmed TFEB upregulation by qRT-PCR in primary human APL samples treated with ATRA ex vivo (n=1) and in primary peripheral blood mononuclear cells isolated from tumour patients treated with ATRA as part of clinical trial NCT00195156 (n=3). We also found that TFEB was only minimally induced by ATRA in the differentiation-resistant NB4R cell line (qRT-PCR n=6), suggesting an important role for TFEB in ATRA-induced APL differentiation. We evaluated the ability of two clinically used differentiating agents - valproic acid (VPA) and arsenic trioxide (ATO), alone and in combination with ATRA, to induce TFEB expression in NB4 cells. While VPA alone induced TFEB (5 fold), combined therapy with ATRA and VPA resulted in a >20 fold induction of TFEB expression, which correlated with enhanced differentiation (n=6)(P<0.001). ATO alone did not induce TFEB nor did it enhance the induction seen with ATRA. Notably, combined therapy with ATRA and ATO did not significantly enhance differentiation over levels seen with ATRA alone. Conclusion Retinoid signaling promotes APL differentiation in part through transcriptionally activating autophagy. Novel or existing therapies, which enhance autophagy signaling, may promote retinoid-induced differentiation and circumvent resistance to retinoid therapy in acute myeloid leukemia. Disclosures Orfali: MSD: Unrestricted educational support Other; Amgen: Unrestricted educational support, Unrestricted educational support Other; BMS: Unrestricted educational support, Unrestricted educational support Other; Pfizer: Unrestricted educational support, Unrestricted educational support Other; Novartis: Unrestricted educational support Other.

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Differentiated Acute Promyelocytic Leukemic Cells-Derived Microparticles Inhibit Cell Migration through Annexin A1

Blood ◽

10.1182/blood.v116.21.2750.2750 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2750-2750

Author(s):

Hui-Chi Hsu ◽

Wen-Hui TSai ◽

Hon-Yu Chien ◽

Shau-Chieh Hsu

Keyword(s):

Specific Antibody ◽

Conflicts Of Interest ◽

Control Cell ◽

Leukemic Cells ◽

Dependent Manner ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Mediators Of Inflammation ◽

Anti Inflammatory

Abstract Abstract 2750 Neutrophil-derived microparticles (MP) have become important mediators of inflammation, coagulation and vascular homeostasis. MP carry surface antigens, proteins and adhesion molecules from their originating cell and can mediate intercellular cross-talk. Human neutrophil contain high levels of the anti-inflammatory protein annexin 1 (AnxA1), which contributes to mechanisms activated in the host to keep under control cell activation and trafficking in the resolution of inflammation. We investigated the role of MP released by all-trans retinoic acid (ATRA)-treated acute promyelocytic leukemic (APL; NB4) cells during the process of granulocytic differentiation. Materials & Methods: We determined the expression of AnxA1 and its receptor (FPRL1) on the NB4 cells and their MP by flowcytometry, real-time PCR and western blotting. The anti-inflammatory effect of AnxA1was determined by the transmigration assay. Results: AnxA1 was constitutively expressed on the surface of ATRA-untreated NB4 cells. ATRA treatment of NB4 cells can significantly enhance their surface expression of Anx-A1 in a time dependent manner, but did not change their mRNA expression or release of free AnxA1 into the conditioning medium. We further determined the amount of MP in the CM by flowcytometry. Significantly higher number of MP was released by the ATRA-NB4 cells, as compared with those by ATRA-untreated NB4 cells (p<0.05). Further study demonstrated that AnxA1was expressed on the surface of MP, and ATRA can also enhance the release of Anx-A1 (+) MP from ATRA-NB4 cells. Transmigration studies indicated that exogenous Anx-A1 protein can inhibit the transmigration of ATRA-NB4 cells in a time- and dose-dependent manner (p<0.05 & p<0.05; respectively). AnxA1 receptor (FPRL-1) was expressed on the surface of both ATRA-treated and ATRA-untreated NB4 cells. Blocking the FPRL1 on the surface of ATRA-NB4 cells with its specific antibody can further enhance their transmigration activity, indicating that the important anti-migratory role of AnxA1-FPRL1 axis on the ATRA-treated NB4 cells. We further demonstrated that ATRA-treated NB4 cells-derived MP can also significantly inhibit the transmigration of ATRA-NB4 cells (p<0.05). Similarly, anti-migratory effect of MP can be attenuated when their surface Anx-A1 was blocked with its specific antibody, implying that AnxA1 mediates the rapid anti-migratory effects of MP. We further determined the mode of action that Anx-A1(+) MP acted on the recipient cells. MPs were pre-labeled with FITC-conjugated Anx-A1 monoclonal antibody before incubating with recipient ATRA-treated NB4 cells. Flowcytometry demonstrated that FITC-labeled Anx-A1 was detected on 87% of recipient ATRA-treated NB4 cells, indicating that Anx-A1 (+) MP can fuse into the surface of the recipient ATRA-treated NB4 cells. Conclusion: differentiated APL cells-derived microparticles contain functionally active AnxA1 that confers them anti-migratory properties which contributes to the control mechanism of cell recruitment. Disclosures: No relevant conflicts of interest to declare.

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Role of ROS in Aβ42 Mediated Activation of Cerebral Endothelial Cells

Central Asian Journal of Global Health ◽

10.5195/cajgh.2014.179 ◽

2014 ◽

Vol 3 ◽

Author(s):

Andrey Tsoy ◽

Bauyrzhan Umbayev ◽

Tamara Shalakhmetova ◽

Sholpan Askarova

Keyword(s):

Endothelial Cells ◽

Actin Polymerization ◽

Inflammatory Responses ◽

Time Dependent ◽

Ros Generation ◽

Dependent Manner ◽

Ros Production ◽

Cerebral Endothelial Cells ◽

Pre Treatment

Introduction. There is substantial evidence that the deposition of aggregated amyloid-beta peptide (Aβ) in brain parenchyma and brain vessels is the main cause of neuronal dysfunction and death in Alzheimer’s disease (AD). Aβ exhibits multiple cytotoxic effects on neurons and glial cells and causes dysfunction of the blood brain barrier (BBB). In AD brains, an increased deposition of Aβ in the cerebral vasculature has been found to be correlated with increased transmigration of blood-borne inflammatory cells and neurovascular inflammation. However, regulatory mediators of these processes remain to be elucidated. In this study, we examined the role of ROS in actin polymerization and expression of adhesion molecules (P-selectin) on the surface of the cerebral endothelial cells (CECs) that are activated by Aβ42.Materials and methods. Mouse BEnd3 line (ATCC) was used in this research. BEnd3 cells respond to Aβ treatment similarly to human primary CECs and are a common model to investigate CECs’ function. We used immortalized bEnd3 cells as the following: controls; cells incubated with Aβ42 for 10, 30, and 60 minutes; cells incubated with 30 mM of antioxidant N-acetylcysteine (NAC) for 1 hr; and, cells pre-treated with NAC followed by Aβ42 exposure. We measured DHE fluorescence to investigate intracellular ROS production. Immunofluorescent microscopy of anti-P-selectin and oregon green phalloidin was used to quantify the surface P-selectin expression and actin polymerization, and Western blot analysis was used to analyze total P-selectin expression.Results. The results of this study have demonstrated a significant time-dependent ROS accumulation after 10 minutes, 30 minutes, and 60 minutes of Aβ42 treatment, while Aβ42 stimulated ROS production in CECs was attenuated by pre-treatment with the NAC antioxidant. We also found that Aβ42 increased P-selectin fluorescence at the surface of bEnd3 cells in a time dependent manner in parallel to ROS elevation. However, total expression levels of P-selectin were not changed following exposure to Aβ42. Pre-treatment with NAC attenuated Aβ42 induced P-selectin localization, while NAC alone did not significantly affect P selectin localization. As a positive control, H2O2 also increased P-selectin expression on the cell surface, which peaked after 30 minutes of H2O2 treatment. Exposure of CECs with Aβ42 promoted actin polymerization, which peaked after 10 minutes of Aβ42 treatment, while no significant increase of F-actin intensity was observed when cells were pre-treated with NAC. H2O2 was able to mimic Aβ42 induced oxidative stress, causing increased actin polymerization with similar timing.Conclusions. The results of our study have indicated that Aβ42 induced accumulation of P-selectin on the surface of bEnd3 cells and promoted actin polymerization, and all these events were correlated with ROS generation. The rapid post-translational cell signaling response mediated by ROS may well represent an important physiological trigger of the microvascular inflammatory responses in AD and requires further investigations.

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Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00527.2016 ◽

2017 ◽

Vol 313 (5) ◽

pp. L899-L915 ◽

Cited By ~ 8

Author(s):

Fumiaki Kato ◽

Seiichiro Sakao ◽

Takao Takeuchi ◽

Toshio Suzuki ◽

Rintaro Nishimura ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cell ◽

Vascular Remodeling ◽

Time Dependent ◽

Causative Role ◽

Dependent Manner ◽

Vascular Endothelial ◽

Pulmonary Vascular Remodeling ◽

Pulmonary Arterial

Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of cross talk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.

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CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Blood ◽

10.1182/blood-2019-129873 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1734-1734

Author(s):

Orit Uziel ◽

Zinab Sarsur- Amer ◽

Einat Beery ◽

Pia Raanani ◽

Uri Rozovski

Keyword(s):

B Cells ◽

Time Dependent ◽

Dependent Manner ◽

Environmental Resources ◽

Type B ◽

Wild Type ◽

Cell Competition ◽

Western Immunoblotting ◽

Competition Theory

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

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Induction of apoptosis by Kola nut extract as a recent and promising treatment strategy for Leukemia

Bionatura ◽

10.21931/rb/2021.06.02.10 ◽

2021 ◽

Vol 6 (2) ◽

pp. 1725-1732

Author(s):

Hamdah Alsaeedi ◽

Rowaid Qahwaji ◽

Talal Qadah

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Leukemia Cell ◽

K562 Cells ◽

Myelogenous Leukemia ◽

Time Dependent ◽

Leukemia Cell Line ◽

Apoptotic Cells ◽

Dependent Manner ◽

Anti Cancer

Kola nut extracts have recently been reported to contain chemopreventive compounds providing several pharmacological benefits. This study investigated Kola nut extracts' anti-cancer activity on human immortalized myelogenous leukemia cell line K562 through apoptosis and cell cycle arrest. Fresh Kola nuts were prepared as powder and dissolved in DMSO. Different concentrations (50, 100, 150, 200, and 250 μg/ml) of working solutions were prepared. The K562 cells were treated with the different concentrations of Kola nut extract or vehicle control (10% DMSO) followed by incubation at 37°C for 24, 48, and 72 hours, respectively. Treatment activity was investigated in K562 cells; by Resazurin, and FITC/Propidium Iodide and 7-AAD stained cells to evaluate apoptotic cells and the cell cycle's progression. Inhibition of leukemia cell proliferation was observed. The extract effectively induced cell death, early and late apoptosis by approximately 30% after 24 and 48 hours incubation, and an increase in the rate of dead cells by 50% was observed after 72 hours of incubation. Also, cell growth reduction was seen at high dose concentrations (150 and 200 µg/ml), as evident by cell count once treated with Kola nut extract. The total number of apoptotic cells increased from 5.8% of the control group to 27.4% at 250 µg/ml concentration. Moreover, Kola nut extracts' effects on K562 cells increased gradually in a dose and time-dependent manner. It was observed that Kola nut extracts could arrest the cell cycle in the G2/M phase as an increase in the number of cells by 29.8% and 14.6 % were observed from 9.8% and 5.2% after 24 and 48 hours of incubation, respectively. This increase was detected in a dose and time-dependent manner. Kola nut extracts can be used as a novel anti-cancer agent in Leukemia treatment as it has shown significant therapeutic potential and therefore provides new insights in understanding the mechanisms of its action. Keywords: Kola nut extracts, Leukemia, K562 cell line, Apoptosis, Cancer.

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Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors In Vitro: Antitumoral Effects

Cancers ◽

10.3390/cancers12020345 ◽

2020 ◽

Vol 12 (2) ◽

pp. 345

Author(s):

Xi-Feng Jin ◽

Gerald Spöttl ◽

Julian Maurer ◽

Svenja Nölting ◽

Christoph Josef Auernhammer

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Lines ◽

Neuroendocrine Tumors ◽

Density Lipoprotein ◽

Time Dependent ◽

Dependent Manner ◽

Antitumor Properties

Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target.

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PML/RARa Represses Transactivation of PSMB10 Via a PU.1-Dependent Manner In Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v116.21.3866.3866 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3866-3866

Author(s):

Xianwen Yang ◽

Ping Wang ◽

Xujie Zhao ◽

Huahua Zhu ◽

Sai-Juan Chen ◽

...

Keyword(s):

Transcriptional Regulation ◽

Retinoic Acid ◽

Immune System ◽

Acute Promyelocytic Leukemia ◽

Target Genes ◽

Promyelocytic Leukemia ◽

Class I ◽

Luciferase Reporter ◽

Atra Treatment ◽

Nb4 Cells

Abstract Abstract 3866 Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner. Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures: No relevant conflicts of interest to declare.

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