Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5045-5045
Author(s):  
Manuel Hein ◽  
Dominik Schnerch ◽  
Andrea Schmidts ◽  
Julia Felthaus ◽  
Dagmar Wider ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Potential Role ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Hl60 Cell ◽  
Cd11b Expression ◽  
Hl60 Cells ◽  
Differentiation Block

Abstract Abstract 5045 Introduction The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and Results We have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. Conclusion This is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. Disclosures No relevant conflicts of interest to declare.

Knockdown of Adhesion-Regulating Molecule 1 Inhibits Proliferation in HL60 Cells

2015 ◽  
Vol 134 (2) ◽  
pp. 88-100 ◽  
Author(s):  
Xiaohui Zheng ◽  
Yafei Guo ◽  
Yingying Chen ◽  
Meilin Chen ◽  
Zhenxin Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Colony Formation ◽  
Nuclear Translocation ◽  
Leukemia Cell ◽  
26S Proteasome ◽  
Biological Behavior ◽  
Hl60 Cell ◽  
Hl60 Cells ◽  
Leukemia Cell Lines

Background/Aims: Adhesion-regulating molecule 1 (ADRM1), a receptor located on the 26S proteasome, is upregulated in many solid cancers. However, little is known about its role in acute leukemia (AL). Methods: We determined ADRM1 expression levels in both untreated AL samples and leukemia cell lines using real-time polymerase chain reaction or Western blot analysis. Growth curves, colony formation assays, cell cycle and apoptosis analyses, cell migration and invasion assays and NF-κB p65 nuclear translocation assays via Western blotting were used to examine the biological behavior of HL60 cells and the underlying mechanism. Results: ADRM1 was upregulated in both untreated AL samples and leukemia cell lines. ADRM1 knockdown significantly suppressed HL60 cell proliferation (48.82 ± 12.58%) and colony formation and caused cell cycle arrest in the G0/G1 phase. Furthermore, we confirmed that ADRM1 knockdown suppressed p65 nuclear translocation. Conclusion: Our study revealed that ADRM1 was overexpressed in AL, especially in CD34+ leukemia stem and progenitor cells. ADRM1 may play a role in AL via the proteasome-ubiquitin pathway by potentially sustaining the activation of NF-κB signaling.

STUDY ON ANTIANGIOGENENIC EFFECT OF ACANTHOPANAX SENTICOSUS IN HL60 CELL LINES

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4857-4857
Author(s):  
Chen Fangyuan ◽  
Zhang Minyue ◽  
Cai Jiayi ◽  
Shen Lijing
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Concentration Dependence ◽  
Cell Lines ◽  
Western Blotting ◽  
Leukemia Cell ◽  
Hl60 Cell ◽  
Vegf Receptor ◽  
Acanthopanax Senticosus ◽  
Hl60 Cells

Abstract Introduction Many studies have been confirmed that neovascularization, the formation of new blood vessels from existing vasculature, plays an essential role in growth, development and metastasis of acute leukemia. At present, antiangiogenic therapy of leukemia become the new hot spot. Acanthopanax senticosus(Chianese name Ci Wu Jia ,CWJ) is a kind of Chinese herb, which contain natural flavonoid compounds and have been proven to inhibit leukemia cell proliferation. But there is no detailed report about the relationship with the inhibition of leukemia cells and the inhibition of angiogenesis effect. In this study, we should demonstrate the inhibition of leukemia cell growth and antiangiogenic mechanism through HL60 cell lines, further confirm the inhibitory effect on leukemia and antiangiogenic effect of Acanthopanax senticosus, Methods HL60 cells were treated with different concentrations of Acanthopanax senticosus (25°¢50°¢75°¢100°¢200µg/ml). Cell proliferation were detected using Cell Counting Kit-8. Kinds of transcription factors in Dll4/Notch (Delta-like 4 is the only ligand of Notch expressing in endothelium) and VEGF(R) signaling pathway were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting. Results Acanthopanax senticosus inhibited the growth of HL60 cells, and the time and concentration dependence(Fig.1). We extracted RNA and protein from these cells at 12hr, 24hr and 48hr respectively, found that Acanthopanax senticosus remarkably results in VEGF, VEGFR2(VEGF Receptor 2), DLL4 down-regulation based on the time and the concentration dependence, and mild inhibit VEGFR1(VEGF Receptor 1) and Notch1 factors gene expression(Fig. 2). Western blotting also showed a significant inhibition protein of VEGFR2, DLL4 and Notch1, mild inhibited the expression of VEGF and VEGFR1 protein, and with time and concentration dependence (Fig. 3). Summary Acanthopanax senticosus can inhibited proliferation of HL60 cells in vitro and anti-angiogenesis effect mainly via inhibition of VEGFR2-mediated signaling. It has an instantaneous effect on Dll4/ Notch signaling pathway. The data have elucidated the potential roles of several key signaling pathways in angiogenesis. Disclosures: No relevant conflicts of interest to declare.

EphrinB1 Activation As a Potential New Treatment Option in AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5235-5235
Author(s):  
Arja ter Elst ◽  
Kim R Kampen ◽  
Sander H Diks ◽  
Steven M. Kornblau ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Hl60 Cells ◽  
Ephrin Receptors ◽  
Apoptotic Protein ◽  
Ephb Receptors ◽  
New Treatment

Abstract Abstract 5235 Aberrant Ephrin signaling has been shown to be an important pathway that contributes to the pathogenesis of many solid tumors (Surawska et al. Cytokine & Growth factor reviews 2004). Deregulated ephrin receptor (Eph) and ligand (Efn) expression is often associated with poor prognosis in solid tumors. Ephrin receptor and ligand overexpression can result in tumorigenesis through induced tumor growth, tumor cell survival, angiogenesis and metastasis (Surawska et al. Cytokine & Growth Factor Reviews 2004; Campbell et al. Curr. Isues Mol. Biol. 2008; Chen et al. Cancer Research 2008). In normal cells Eph receptors and ligands play key roles in vascular patterning, where they function in endothelial cell migration, and proliferation (Adams et al, Genes Dev. 1999; Zhang et al., Blood 2001). Thus far particularly EphB4 receptor and ephrin-B2 ligand have been implicated in the process of normal angiogenesis. In acute myeloid leukemia (AML) patients it was found that bone marrow biopsies at diagnosis exhibited enhanced microvessel density (MVD) (de Bont ES et al., BJH 2001; Byrd JC et al., Blood 2002; Padro et al., Blood 2000). Normal hematopoietic stem cells (HSCs) express the following mRNA transcripts ephrin receptors EphA1, EphA2, EphB2, and EphB4 and ephrin ligands EfnA3, EfnA4, and EfnB2. Moreover, overexpression of EphB4 receptor in HSCs (from cord blood) resulted in enhanced differentiation towards megakaryocytes (Wang et al. Blood 2002). In AML cell lines there is a common co-expression on protein level observed between EphB4 receptor and ephrin-B2 ligand. Recently, an aberrant DNA methylation of ephrin receptors and ligands was described in acute lymphocytic and myelocytic leukemia cell lines (Kuang et al. Blood 2010). In addition, restoration of EphB4 expression in an acute lymphoid leukemia cell line resulted in reduced proliferation and apoptotic cell death. These data suggests that the ephrin signaling pathway might play an important role in leukemia. In a previous study we have found high kinase activity of EphB receptors and high phosphorylation levels of EphB receptors in AML samples, as measured using kinase arrays and proteome profiler arrays. In this study, we have found extensive membrane expression of EphB1 on AML cell lines and primary AML blasts. To identify the role of Ephrin signaling in AML, two AML cell lines THP-1 and HL60 with an EphB1 membrane expressing cell percentage of 70% and 20% respectively were chosen for stimulation with Ephrin-B1 ligand. Treatment of these cell lines with Ephrin-B1 ligand resulted in a decreased proliferation 30% in THP-1 cells versus 22% in HL60 cells and increased apoptosis 23% in THP-1 cells and 4% in HL60 cells. Of note, the most prominent effect of Ephrin-B1 stimulation was found in THP-1 cells, this cell line contained a higher percentage of EphB1 membrane expressing cells. We further investigated the mechanism through which EphB1 reduces leukemic cell growth and induces leukemic cell death in THP-1 cells. Westernblot analysis of cell cycle regulators showed that expression of the anti-apoptotic protein BCL2 is reduced upon Ephrin-B1 ligand stimulation and the expression of the pro-apoptotic protein BAX is induced. In addition, mRNA expression of the cell cycle inhibitor of cell cycle progression p21 was found to be 2,5 fold upregulated in ephrin-B1 ligand treated cells compared to untreated control cells. MGG stainings of Ephrin-B1 treated cells revealed multiple cells with two nuclei in both THP-1 and HL60 cells. These results indicate that a high percentage of AML cells express EphB1 receptor on the membrane and that stimulation of these cells with Ephrin-B1 ligand results in reduced leukemic growth and increased cell death. EphrinB1 activation in AML deserves further investigation considering EphB1 as a putative new treatment option for AML patients. Disclosures: No relevant conflicts of interest to declare.

Over-Expression of RPS3 Promotes Acute Lymphoblastic Leukemia Growth and Progress By Down-Regulating COX-2 through NF-κb Pathway

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3927-3927
Author(s):  
Wang Hua ◽  
Lu Yue ◽  
Shi Dingbo
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Mrna And Protein Expression ◽  
Cox 2

Abstract The Ribosome protein S3 (RPS3) is a component of 40S ribosomal subunit, which is important in ribosomal maturation.In addition, RPS3 plays a central role in the regulation of cell cycle,proliferation,migration,DNA repair,and apoptosis.Recent study has also been reported that RPS3 is secreted as a homodimer in cancer cells. The increased level of secreted RPS3 was detected in more malignant cells.These findings suggest that the RPS3 protein is an indicator of malignant tumors.Therefore, we studied the roles and the functions mechanisms of RPS3 in leukemia in order to understand whether RPS3could be a key target for leukemia therapy. qRT-PCR and western blot analysis were carried out in a small cohort of acute lymphoblastic leukemia patients(ALL) and multiple leukemia cell lines to evaluate RPS3 mRNA and protein expression levels.To assess its biological functions relevance, its expression was down modulated by transient RNA interference in ALL cell lines.Our results show that RPS3 mRNA and protein expression is higher in both ALL patients and the ALL cell lines when compared to the healthy donors peripheral blood mononuclear cell or myeloid leukemia cell lines. Correspondence with this, the ALL patients with higher expression of RPS3 had shorter overall survival than those with lower expression of RPS3 (25.1% vs. 63.4%, P<0.001, for 5 year-OS).Furthermore,blocking RPS3 activity in four ALL cell lines, by either knockdown or treatment with the RPS3 inhibitor, causes significant decrease in their cell proliferation.This decrease in cell proliferation was coupled with both an induction of the G1/S cell cycle arrest and with an increase of apoptosis induced in the leukemia population. In vivo,we also found that knockdown of RPS3 significantly inhibited tumor growth in a ALL xenograft mouse model. Finally, mechanism studies showed that RPS3 knockdown in ALL cells triggered suppression of COX-2 expression and its down-stream targets PGE2 release,inhibited COX-2 promoter activity by decreased P50 /P65 Binding to cox2 promotor. In conclusion,our results suggest that overexpression of RPS3 promotes acute lymphoblastic leukemia growth and progress by up-regulating COX-2 through NF-κB pathway. and that targeting RPS3could be an attractive strategy for ALL therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance Via Blocking Receptor Auto-Induction and BIM/Bmf Expression in ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3758-3758
Author(s):  
Jianping Li ◽  
Catalina Troche ◽  
Julia Hlavka Zhang ◽  
Jonathan Shrimp ◽  
Jacob S. Roth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Mutant Allele ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Pediatric All

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

Cleavage and polyadenylation-specific factor 3 induces cell cycle arrest via PI3K/Akt/GSK-3β signaling pathways and predicts a negative prognosis in hepatocellular carcinoma

2021 ◽  
Vol 15 (5) ◽  
pp. 347-358
Author(s):  
Ning Li ◽  
Shaotao Jiang ◽  
Rongdang Fu ◽  
Jin Lv ◽  
Jiyou Yao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Lines ◽  
Potential Role ◽  
Control Group ◽  
Specific Factor ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Cleavage And Polyadenylation ◽  
Gsk 3Β

Background: Recent studies have shown that cleavage and polyadenylation-specific factor 3 (CPSF3) is a promising antitumor therapeutic target, but its potential role in hepatocellular carcinoma (HCC) has not been reported. Materials & methods: We explored the expression pattern of CPSF3 in HCC through bioinformatics analysis, quantitative polymerase chain reaction (qPCR) and western blot. The potential role of CPSF3 as a biomarker for HCC was evaluated by Kaplan–Meier analysis. Next, changes in HCC cell lines in the CPSF3 knockdown model group and the control group were assessed by Cell Counting Kit-8, clonal formation, flow cytometry and EdU staining. Western blot detected changes in protein levels of the PI3K/Akt/GSK-3β axis of two HCC cell lines in the knockdown group and the control group. Results: The results showed that the transcription and protein levels of CPSF3 were significantly higher in HCC tissues than in adjacent normal tissues (p < 0.05). The HCC cohort with increased expression of CPSF3 is associated with advanced stage and differentiation and predicts poorer prognosis (p < 0.05). CPSF3 knockdown significantly inhibited proliferation and clone formation of HepG2 and SMMC-7721 cell lines. Flow cytometry analysis showed G1–S cell cycle arrest in the CPSF3 knockdown group, and the results of EdU staining were consistent with this. Compared with the control group, p-Akt and cyclin D1 were decreased, and GSK-3β was increased in the knockdown group. Conclusion: CPSF3 may be a potential diagnostic biomarker and candidate therapeutic target for HCC.

Tumor Necrosis Factor-a Enhances Human Leukemia Cell Differentiation Induced by DMSO, but Not Retinoic Acid.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3893-3893
Author(s):  
Hong-Nu Yu ◽  
Young-Rae Lee ◽  
Hyun-Jaung Shim ◽  
Myung-Kwan Han ◽  
Eun-Kyung Song ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Tumor Necrosis ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Induced Differentiation ◽  
Cd11b Expression ◽  
Hl60 Cells ◽  
Reducing Activity ◽  
Necrosis Factor

Abstract One of the human leukemia treatment methods is to differentiate leukemia cells into mature cells. Because differentiated cells lose their proliferative and tumor-forming abilities, differentiation inducers may be useful for the treatment of leukemia. Differentiation of leukemia cells has been studied using HL60 cells, a human promyelocytic leukemia cell line, which can be differentiated into granulocyte-like or monocyte/macrophage-like cells by various pharmacological agents such as dimethyl sulfoxide (DMSO), retinoic acid and phorbol myristic acetate (PMA). We previously reported that nuclear factor - kB (NF-kB) activation plays the important role in DMSO-induced differentiation of HL60 cells. Thus, we hypothesized that NF-kB activators could enhance DMSO-induced differentiation of HL60 cells. Here we examine whether tumor necrosis factor-a (TNF-a), a potent NF-kB inducer, enhance DMSO-induced differentiation of HL60 cells. TNF-a was found to enhance HL60 cell differentiation induced by DMSO. CD11b, a differentiation marker, was increased in 0.5 % DMSO-treated cells compared to control cells. When TNF-a was added to the same condition, CD11b expression was further enhanced in a dose and a time dependent manners. We also found that nitro blue tetrazolium (NBT) reducing activity, a marker for granulocytic differentiation, was further increased in DMSO plus TNF-a treated cells compared to only DMSO- treated cells. However, TNF-a alone had no effect on CD11b expression and NBT reducing activity. The enhancement of DMSO-induced HL60 differentiation by TNF-a was offset by NF-kB inhibition. Interestingly, retinoic acid- induced differentiation of HL60 cells showed no enhancing effect of TNF--a on the differentiation. These findings indicate that TNF--a might affect only NF-kB dependent differentation of HL60 cells. Taken together, we demonstrated that TNF-a enhances DMSO-induced differentiation of HL60 cells by stimulating NF-kB activation. Our results suggest that NF-kB inducers such as TNF-a are useful for the treatment of leukemia in combination with DMSO.

Analysis of Aurora Kinase Expressions and Cell Cycle Regulation by Aurora-C in Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1366-1366 ◽  
Author(s):  
Miki Kobayashi ◽  
Satoki Nakamura ◽  
Takaaki Ono ◽  
Yuya Sugimoto ◽  
Naohi Sahara ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Aurora A ◽  
Aurora Kinases

Abstract Background: The conserved Aurora family kinases, a family of mitotic serine/threonine kinases, have three members (Aurora-A, -B and -C) in mammalian cells. The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. Aurora A localizes to the centrosomes during anaphase, and it is required for mitotic entry. Aurora B regulates the formation of a stable bipolar spindle-kinetochore attachment in mitosis. The function of Aurora-C in mammalian cells has not been studied extensively. In this study, we investigated that human leukemia cells expressed all 3 Aurora kinases at both protein and mRNA level, and the mechanisms of cell cycle regulation by knock down of Aurora C in leukemia cells. Methods: In this study, we used the 7 human leukemia cell lines, K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells. The expression levels of mRNA and proteins of Aurora kinases were evaluated by RT-PCR and western blot. The analysis of proliferation and cell cycle were performed by MTT assay and FCM, respectively. Results: The mRNA of Aurora-A and Aurora-B are highly expressed in human leukemia cell lines (K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells), while the mRNA of Aurora C is not only expressed highly in all cells. In contrast, an increase in the protein level of the 3 kinases was found in all cell lines. These observations suggested posttranscriptional mechanisms, which modulate the expression of Aurora C. In cell cycle analysis by flow cytometory, the knock down of Aurora C by siRNA induced G0/G1 arrest and apoptosis in leukemia cells, and increased the protein levels of p27Kip1 and decreased Skp2 by western blot. In MTT assay, it was revealed that the growth inhibition of leukemia cells transfected with siRNA Aurora C compared with leukemia cells untransfected with siRNA Aurora C. Moreover, We showed that Aurora C was associated with Survivin and directly bound to Survivin by immunoprecipitation and western blot. Conclusion: We found that human leukemia cells expressed all 3 members of the Aurora kinase family. These results suggest that the Aurora kinases may play a relevant role in leukemia cells. Among these Aurora kinases, Aurora C interacted with Survivin and prevented apoptosis of leukemia cells, and induced cell cycle progression. Our results showed that Aurora-C may serve as a key regulator in cell division and survival. These results suggest that the Aurora C kinase may play an important role in leukemia cells, and may represent a target for leukemia therapy.

Alternation of Cellular Morphology and Proliferation Induced by microRNA in Human Leukemia Cell Line, UT-7/EPO.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4203-4203
Author(s):  
Nobuyoshi Kosaka ◽  
Yusuke Yamamoto ◽  
Nami Nogawa ◽  
Keiichi Sugiura ◽  
Hiroshi Miyazaki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Macrocytic Anemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Morphological Studies

Abstract Mature microRNA (miRNA) originated from primary miRNA (pri-miRNA) is a new group of potential regulator for cell differentiation, apoptosis, proliferation and oncogenesis. Some miRNAs were recently identified in hematopoietic cells, while the roles of miRNAs in erythrocytic and megakaryocytic cells had not been well examined. As a first step to explore for miRNAs specific for hematopoietic lineage, the expressions of several known primary microRNAs in erythrocytic and megakaryocytic cell lines, such as TF-1, HL-60, HEK293 and UT-7 leukemia cells, were examined by RT-PCR. We consequently focused on the pri-miR-10a, a primary transcript of miR-10a located within Hox gene clusters, and found the significant expression in TF-1 cells and UT-7/EPO cells. The UT-7/EPO cells were a subline established from the original UT-7 cells, as well as UT-7/GM and UT-7/TPO cells; therefore it was suitable for the further comparative analysis. Interestingly, in UT-7/EPO cells, the expression of pri-miR-10a increased under stimulation of erythropoietin (EPO; 1U/mL and 10U/mL). Based on these observations, it was postulated that pri-miR-10a might involve in modulating erythrocyte differentiation or proliferation. To clarify the role of pri-miR-10a in UT-7/EPO, we have established clonal cell lines by transfecting UT-7/EPO cells with either the control vector or the pri-miR-10a expression vector pCMV-pri-miR10a. Overexpression of pri-miR-10a in the UT-7/EPO cell line (miR10a-UT-7/EPO) was confirmed by RT-PCR. MiR10a-UT-7/EPO showed higher proliferation rate even at low concentration of EPO (0.1 mU/mL). Overexpression of pri-miR-10a did not appear to affect HOXB4 and HOXA1 expression, as similar mRNA levels were seen in both cell lines. It was notable that the cellular size of miR10a-UT-7/EPO became larger than its parental cells. Morphological studies of miR10a-UT-7/EPO were performed in detail. It is possible that miR-10a was capable to modulate morphological features particularly in cellular size relating to cell cycle regulation. For instance, loss of the E2F family members result in marked macrocytic anemia with megaloblastic features in adult mice (Mol Cell. 2000 Aug;6(2):281–91., Mol Cell Biol. 2003 May;23(10):3607–22., Blood. 2006 Aug 1;108(3):886–95.). Data presented here hypothesized that the roles of miR-10a in erythroid cells are tightly associated with cell cycle.

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