EphrinB2/EphB4 Interaction Promotes Myeloid Leukemia Cell Invasion through RhoA-Mediated Mechanism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1018-1018 ◽  
Author(s):  
Xuan Zhou ◽  
Liu Xiaoli ◽  
Na Xu ◽  
Lin Li ◽  
Qisi Lu ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Immunoblot Analysis ◽  
Receptor Activation ◽  
Leukemia Cells ◽  
Migration And Invasion ◽  
Mmp9 Expression ◽  
Myeloid Leukemia Cell

Abstract Background and Objective: Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrinB ligands in a variety of tumors, suggesting a functional relation between EphB/ephrinB signaling and tumor progression. However, how they regulate the invasiveness of myeloid leukemia cells were still unknown. Our previously study suggested that EphB4 were highly expressed in patients with extramedullary leukemia compared with patients without extramedullary leukemia, which indicated that the expression of EphB4 was related with myeloid leukemia cell invasion. To address the molecular mechanism, we aimed to characterize the role of EphB4 and ephrinB2 ligands in the interaction of myeloid leukemia cells. Methods: To clarify the question, myeloid leukemia cell lines (K562 cells and THP-1 cells) treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins were cultured in vitro, then migration and invasion were determined by transwell assay according to different time. Pulldown western immunoblot analysis were used to detect the level of GTP-RhoA and total RhoA; the phosphorylation of EphB4 and MMP9 expression were also determined by immunoblot analysis before and after the treatment of different clustered Fc proteins. Results: The results showed that after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.85-fold, P=0.033), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.46 -fold, P=0.025). However, the numbers of K562 cells migrating through transwell chamber after ephrinA1–Fc stimulation didn’t significantly increase compared to Fc proteins stimulation (P=0.411), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P=0.072) after ephrinA1–Fc stimulation. Moreover, after ephrinB2–Fc stimulation, the numbers of THP-1 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (2.25-fold, P<0.01), meanwhile, the numbers of THP-1 cells invading the matrigel also enhanced (1.66 -fold, P<0.01). However, the numbers of THP-1 cells migrating through transwell chamber and the numbers of THP-1 cells invading the matrigel didn’t significantly enhanced (P>0.05, P>0.05) after ephrinA1–Fc stimulation. Furthermore, EphB4 immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody revealed that EphB4 is phosphorylated on tyrosine in K562 cells after ephrinB2–Fc stimulation. Additionally, the level of active RhoA (GTP-RhoA) and MMP9 in K562 cells were both significantly increased in response to EphB4 receptor activation with its ligand ephrin-B2-Fc ( P<0.05). Conclusions: These findings suggested that EphB4/EprinB2 signaling played an important role in myeloid leukemia cells progression by promoting their migratory ability, activating RhoA activity and increasing MMP9 expression. Our findings reveal a novel regulation of this intriguing receptor/ligand family that contributes to the cell invasiveness of myeloid leukemia cells. Disclosures No relevant conflicts of interest to declare.

Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1360-1360
Author(s):  
Xuan Zhou ◽  
Liu Xiaoli ◽  
Na Xu ◽  
Yajuan Xiao ◽  
Jinfang Zhang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Migration And Invasion ◽  
Cell Migration And Invasion ◽  
Healthy Donors ◽  
Myeloid Leukemia Cell

Abstract Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn’t significantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy. Disclosures: No relevant conflicts of interest to declare.

JQ1, a Potential Therapeutic Molecule for Myeloid Leukemia with PTEN Deficiency

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5899-5899 ◽  
Author(s):  
Nicholas J Baltz ◽  
Natalia C Colorado ◽  
Yan Yan ◽  
Shelly Lensing ◽  
Delli Robinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Jak Inhibitors ◽  
Facs Analysis ◽  
Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys' 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p<0.0001), and 250-500nM for both CB and K562 cells, respectively. Proliferation assay results also supported that TF1a cells are sensitive to JQ1 with an IC50 of 125-250nM, whereas neither CB nor K562 reached the IC50 in the tested concentration range. This suggests that the IC50 of JQ1 for TF1a cells is achievable at concentrations that are mostly nontoxic to normal CB cells, but K562 cells are not sensitive to JQ1. FACS analysis revealed that pSTAT5 is constitutively activated in K562 cells but not in TF-1a cells. Interestingly, the levels of pSTAT5 in both TF-1a and K562 cells were not altered by JQ1 treatment at tested concentrations, which was confirmed by western blot. Conclusions: Our data suggest: 1) JQ1 and other bromodomain inhibitors could be potential therapeutic molecules for selected myeloid leukemias; 2) JQ1 inhibition on colony formation and proliferation in TF-1a cells is not pSTAT5 related. Further studies are underway to test whether JQ1 is effective in primary mouse leukemia cells with Pten deficiency. Disclosures No relevant conflicts of interest to declare.

scholarly journals RNA Binding Protein Rbmx Is Required in Acute Myeloid Leukemia By Regulating the Transcriptional Activity of the Heterochromatin Protein HP1α

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 883-883
Author(s):  
Camila Prieto ◽  
Diu Nguyen ◽  
Ly P Vu ◽  
Alexendar Perez ◽  
Saroj Gourkanti ◽  
...  
Keyword(s):  
Colony Formation ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Flow Analysis ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Heterochromatin Protein ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell

Abstract Acute myeloid leukemia (AML) is characterized by a block in the development of myeloid cells, often due to dysregulation of genes involved in key processes including self-renewal, proliferation, and differentiation. Somatic mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to be important in hematological malignancies. For example, our group and others have recently determined that increased expression of MUSASHI-2 and SYNCRIP drives aggressive leukemia. To discover novel RBP regulators of leukemia, we performed an in vivo pooled shRNA screen of 127 MSI2 direct protein interactors and associated genes (Vu et al. Nat Gen. 2017). In this screen, shRNAs specific to the RBP RBMX (RNA binding motif protein, X-linked) were selectively depleted in murine MLL-AF9 driven leukemia. RBMX has been implicated in regulating alternative splicing, chromatin cohesion, and DNA-damage response, but its function in hematopoiesis and leukemia is not known. We confirmed that depletion of RBMX with shRNAs in murine MLL-AF9 leukemia cells resulted in reduced myeloid colony formation, increased apoptosis, and increased differentiation as determined by flow analysis of myeloid cell surface markers Gr-1 and Mac-1 (mean of 61-65% shRNA versus mean of 12.95% control). Furthermore, RBMX is highly expressed among human myeloid leukemia cell lines (n=10/11) and primary AML patient samples (n=2/4). Depletion of RBMX with shRNAs led to a dramatic decrease in cell proliferation and 3-fold induction of apoptosis in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX depletion in AML cells induced myeloid differentiation and significantly delayed leukemogenesis cells in vivo (median survival of 51.5 days in control versus median 'not reached' in shRNA1 and shRNA2). To determine if there is a differential requirement of RBMX in survival of leukemia cells compared to normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBMX with shRNAs in normal murine bone marrow c-Kit+ cells and found no significant changes in colony formation. Depleting RBMX with shRNAs in human cord blood derived CD34+ HSPCs resulted in reduced colony formation but no increase in apoptosis. Thus, these data suggest that there is a differential requirement for RBMX in myeloid leukemia cells compared to normal cells. To uncover the mechanism of RBMX function, we performed RNA-sequencing of human AML cells (MOLM-13) depleted for RBMX. Gene set enrichment analysis demonstrated a loss of cell cycle and DNA repair associated programs in RBMX depleted cells. Complex chromosomal karyotyping analysis of these cells revealed increased metaphases with breaks and gaps (mean of 30.67% shRNA versus mean of 13.33% control) and irregular chromatin compaction (mean of 47.67%shRNA versus mean of 20% control), while cell cycle analysis showed significantly increased S-phase arrest as determined by flow analysis of Hoechst stained cells (mean of 37-40% shRNA versus of 24.18% control). Reanalysis of RBMX transcriptome-wide binding sites in 293T cells revealed that RBMX directly binds to heterochromatin protein HP1α transcripts (Liu et al. Nucleic Acids Res. 2017). HP1α, also called CBX5, is a key heterochromatin protein that binds to histone H3-K9 tri-methylation marks to promote heterochromatin formation, which is critical in chromatin condensation and chromosome segregation. HP1α has also been determined to be required for MLL leukemia stem cell maintenance. We demonstrated that RBMX depletion resulted in a significant decrease of HP1α mRNA expression without affecting its mRNA stability in AML cells. We confirmed that RBMX depletion reduced the protein abundance of HP1α. Moreover, overexpression of HP1α rescued the effect of RBMX depletion on cell growth and apoptosis. Our study finds that RBMX binds to HP1α mRNA and regulates the transcriptional activity of the HP1α locus, which then maintains proper chromatin compaction in leukemia cells. Overall, we determine that RBMX function is critical for myeloid leukemia survival and has potential as a novel therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals JQ1 Inhibits Proliferation and Induces Apoptosis of Leukemia Cells Through BCL-2 Regulated Pathway

2021 ◽  
Author(s):  
Yifan Zeng ◽  
Xing-Hua Liang ◽  
Yong Xia ◽  
Wen-Yin He
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
High Concentration ◽  
Apoptosis Pathway ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell

Abstract Objective To explore the mechanism of JQ1 on leukemia cells. Methods This study takes two myeloid leukemia cell lines as a research model. Cells treated with high concentration of JQ1 were collected for quantitative real-time PCR, immunoblot and flow cytometry to verify the effects of JQ1 on myeloid leukemia tumor cells. Combined with mRNA sequencing of cell lines to identify the differences in mRNA expression of different cell lines. Results Two cell lines changed cell morphology under JQ1 treatment. The cell membrane appeared in varying degrees of wrinkled internal subsidence. K562 cell lines can maintain stable proliferation after being induced by a specific concentration of JQ1. However, JQ1 cannot induce the death of the K562 cells. Although the MYC and BCL2 gene expression decreased, JQ1 did not affect the c-Myc targeted genes to affect the cell cycle, nor did it trigger the BCL2-mediated apoptosis pathway. On the contrary, after JQ1 induced the MV-4-11 cells, the MYC-mediated cell cycle significantly slowed down and arrested at the G0/G1 phase. The death of MV-4-11 tumor cells through the apoptosis pathway regulated by BCL-2 family. Conclusion JQ1 has different pharmacological effects on two myeloid leukemia cell lines. For MV-4-11, JQ1 mainly inhibited cell cycle by regulating MYC pathway and induced BCL-2-mediated apoptosis to kill myeloid leukemia tumor cells and thus perform anti-tumor effects. K-562 cells showed drug resistance to JQ1 which confirmed that the K-562 cell line has a feedback mechanism that prevents JQ1-induced apoptosis.

Wilm's Tumor 1 Protein Confers TRAIL Resistance in Myeloid Leukemia Cells Through Bcl-Xl

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2557-2557
Author(s):  
Hima Bansal ◽  
Theresea Siefert ◽  
Divya Chakravarthy ◽  
Manjeet Rao ◽  
Gail E Tomlinson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Hsp90 Inhibitors ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemias ◽  
Trail Resistance ◽  
Induced Apoptosis ◽  
Myeloid Leukemia Cell

Abstract Abstract 2557 INTRODUCTION: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive therapeutic agent because of its selective potential to induce apoptosis in variety of tumors but not in normal cells. However, many cancers including myeloid leukemias exhibit resistance to TRAIL and the underlying mechanisms are not fully understood. Wilms' tumor 1 (WT1) protein is an aberrantly overexpressed protein that is associated with leukemia blast survival, chemoresistance and shortened disease-free survival in acute myeloid leukemia (AML). Here, we report a previously unknown protective role of WT1 against TRAIL-induced apoptosis in leukemia cells. METHODS: Various myeloid leukemia cell lines with variable WT1 expression levels were tested for TRAIL sensitivity. The effect of pharmacological or short hairpin RNA (shRNA)-mediated inhibition of WT1 on TRAIL-induced apoptosis was evaluated by AnnexinV/PI staining method. The mRNA levels of WT1 and Bcl-xL were measured by reverse transcription-PCR (RT-PCR) and the expression of apoptosis regulators were analyzed by western blotting. RESULTS: We observed a strong correlation between higher levels of WT1 and increased TRAIL resistance in myeloid leukemia cell lines. WT1 downregulation by shRNA significantly sensitized leukemia cells to TRAIL-induced apoptosis and importantly, ectopic expression of shRNA resistant WT1 (WT1SR) in WT1-knockdown cells restored the TRAIL resistance. WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members such as Bcl-2 and Bak. We found for the first time, a positive correlation between WT1 and Bcl-xL expression in leukemia cell lines and primary AML samples. Furthermore, using chromatin immunoprecipitation (ChIP) analysis, we show that Bcl-xL is a bonafide WT1-target gene as WT1 transctivates Bcl-xL by binding to its promoter. We have (Bansal et al., Blood 2010) recently shown that the expression and oncogenic functions of WT1 could be abrogated by heat shock protein 90 (Hsp90) inhibitors in myeloid leukemia cells. In order to establish whether Hsp90- mediated regulation of WT1 has any functional significance in TRAIL-resistance we treated leukemia cell with the Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG). The results confirm that Hsp90 inhibition was able to overcome TRAIL resistance, by reducing levels of WT1 and Bcl-xL and by increasing the levels of the proapoptotic protein Bak. Taken together, our results reveal that increased expression of Bcl-xL, along with low Bak, confer WT1-mediated TRAIL resistance. CONCLUSION: Our data indicate that TRAIL resistance in myeloid leukemia cells is mediated by overexpression of the panleukemic marker WT1 and this can be overcome by either silencing WT1 or by pharmacologic inhibitors such as 17-AAG. of Hsp90. Our study highlights the potential therapeutic benefit of the combining TRAIL and Hsp90 inhibitors for the treatment of myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.

Study of Abnormal NHEJ Function in Human Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4349-4349
Author(s):  
Zheng-zheng Fu ◽  
Zi-xing Chen ◽  
Zhi-min Wang2 ◽  
Jian-nong Cen ◽  
Jun He ◽  
...  
Keyword(s):  
Cell Lines ◽  
Plasmid Dna ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
The Mean ◽  
Plasmid Puc18 ◽  
Myeloid Leukemia Cell

Abstract Non-homologous end joining (NHEJ) is a major mechanism by which eukaryote cells can repair the DNA double-strand break (DSB) and protect the cell from further damages. Evidences have suggested that the genomic instability caused by deficient DNA repair function may contribute to the development of solid tumor, while its role in leukemogenesis has not been adequately studied. To study the NHEJ function in myeloid leukemia, an in vitro system was developed for clinical samples by using the linear plasmid pUC18 DNA digested with EcoR I as assay system.. Nuclear protein extracted from leukemic cells and mononuclear cells (MNCs) from normal BM or PB, was incubated with linear plasmid pUC18 DNA under certain conditions. Plasmid DNA was separated by agarose gel electrophoresis and imaged by SYBR greenIstaining. End-ligation efficiency was assessed by dividing the densitometry readings for the sum of all converted plasmid products by the sum of all products. This assay was performed on 7 myeloid leukemia cell lines, 16 samples of normal BM or PB cells, 20 cases of CML cells and 19 cases of de novo AML cells. E. coli strain DH5α was electrotransformed with pUC18 DNA end-joined by nuclear proteins from normal BM or leukemia cells, and was plated on LB agar, containing X-gal and IPTG. Correct ligation of cut plasmid DNA resulted in blue colonies while faulty repair would result in white colonies. The percentage of white colonies over total colonies gave the frequency of misrepair. Primers around the EcoR I site were designed and colony PCR was performed on blue and white colonies. Sequencing of PCR products was performed. Antibodies against the nuclear repair proteins Ku70, Ku86 and DNA-PKcs were used for antibody abrogation studies. We have found that the mean repair efficiency of normal MNCs was 18.6±13.1% (0~46.6%),. The ligation efficiencies in myeloid leukemic cell lines ranged 10.6%~31.0% (mean 22.4%, P&gt;0.05). The mean ligation efficiency was significantly higher in CML cells as compared with normal MNCs (24.8±14.9% v.s 18.6%, p=0.024). The DNA repair capacity of de novo acute myeloid leukemia cells(7.2%~76.9%)was markedly increased as compared with normal MNCs (mean 41.1±15.4% v.s 18.6%, p&lt;0.0001 ). The mean frequency of misrepair from AML and CML cells were considerable higher (AML, 8.17% and CML, 2.10%) than that of normal BM cells (0.91%). Most misrepair were small deletions near EcoR I site. Large deletions (&gt;100bp) were found from assays with AML cells. Three abnormalities of sequence deletion, inversion and insertion were found in a rare misrepair white colony with AML cells. The DNA end-ligation efficiencies of AML cells could be dramatically inhibited by antibodies against proteins Ku70, Ku86 and DNA-PK. We concluded that the Cell-free NHEJ assay system can be used to examine the clinical leukemic samples. NHEJ efficiencies were slightly enhanced in most myeloid leukemia cell lines compared with normal MNCs. The end-ligation efficiency was significantly higher in primary myeloid leukemia cells. The overactive but more error-prone NHEJ function relied on the activity of Ku and DNA-PK proteins for DSB repair may contribute to genomic instability in AML cells.

Cytotoxic T-Lymphocyte Harnessing CML66 Can Kill Human Myeloid Leukemia Cells, In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2746-2746
Author(s):  
Koichiro Suemori ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Masaki Yasukawa
Keyword(s):  
Cell Line ◽  
Myeloid Leukemia ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Expression Level ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Myeloid Leukemia Cell

Abstract [Purpose & background] CML66 is a newly identified cancer-testis antigen by SEREX method in post-transplant CML patient who had a second remission by DLI for relapse. Thus CML66 is initially considered to be implicated in graft-versus-leukemia (GvL) effect against CML, while its’ physiological function remains unknown. The identification by SEREX means its’ immunogenicity to produce antibody, however the T-cell response specific for CML66, particularly its’ ability to generate cytotoxic T-lymphocyte (CTL) against leukemia still remains to be verified. Thus we explored a CTL-epitope of CML66 to induce epitope-specific CTL which can kill human leukemia cells, because of the exploration of its’ clinical applicability as an anticancer vaccine for the immunotherapy. [Methods] At first, we synthesized a variety of CML66-derived 9 aminoacid peptides (9 mer) that had computedly-predicted high binding affinity to HLA-A*2402 molecule. CD8+ T lymphocytes from an HLA-A*2402+healthy donor were co-cultured with autologous monocyte-derived mature dendritic cells (mDCs). CD8+T lymphocytes were repeatedly stimulated with peptide-loaded mDCs. Thereafter, the target epitope-specificity of growing cells was examined by a standard 51Cr-release assay. Additionally, the blocking tests by using anti-HLA class I and anti-class II monoclonal antibody (mo.ab.) were conducted to confirm its’ HLA-A*2402-restricted fashion. Next, CML66 mRNA expression level of target cells including myeloid leukemia cell line cells and primary leukemia cells was examined by real-time semi-quantitative PCR (RQ-PCR). The relative expression level of CML66 mRNA was determined by comparative Ct method. [Result] We identified two CML66-derived 9 mer epitopes with high binding affinity to HLA-A*2402 measured by using HLA-A*2402 gene transfected T2 (T2-A24) cell. One of 2 epitopes, the epitope of CML66; aa70–78: WIQDSVYYI generated the epitope-specific CTL, in vitro, and those CTL exerted anti-leukemia activity against human myeloid leukemia cell line cells in an HLA-A*2402-restricted fashion, but not any cytotoxicity against normal cells. Furthermore, the HLA-A*2402 restriction was confirmed by blocking test by HLA-class I and II mo.ab. Next CML66 mRNA expression level was revealed high in myeloid leukemia cell line cells but low in normal cells, which were compared to that of K562 cell line cell. In primay leukemia cells, acute myelogenous leukemia(AML) cells and acute lymphoblastic leukemia(ALL) cells showed the high expression level of CML66 mRNA. Regarding to the FAB classification of AML, the expression level of CML66 mRNA tended to be higher in subsets ranging from M1 to M4, particularly M2 cells. Even by small number, it was of interest that the expression level of CML66 mRNA in primary chronic myelogenous leukemia (CML) cells was high in cells from blastic phase, but low in cells from chronic phase. This finding may suggest the correlation between CML66 and growth activity of tumor cells. [Conclusion] We identified the novel HLA-A*2402 restricted CTL-epitope derived from CML66; aa70–78: WIQDSVYYI, which may be a promising and secure target for immunotherapy against acute leukemias and aggressive CML.

Resveratrol Inhibits Myeloid Leukemia Cell Lines With JAK2V617F Via Both Inactivation Of The JAK/STAT Pathway and Upregulation Of The ERK Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3854-3854
Author(s):  
Ly Quoc Trung ◽  
Luis J. Espinoza ◽  
Dao T. T. An ◽  
Shinji Nakao
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Jak2v617f Mutation ◽  
Erk Phosphorylation ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell ◽  
Stat Pathway

Abstract Resveratrol, a naturally-occurring polyphenol that has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities, has shown potential anti-tumor activities against malignant NK cells via JAK2/STAT3 pathway inhibition (Trung, et al. PLoS One, 2012). The inhibitory effect of resveratrol on the JAK2/STAT3 pathway suggests that it may be effective for the treatment of myeloid malignancies that are characterized by excessive activation of this signaling pathway, including myeloproliferative neoplasms with the JAK2V617F mutation. To test this hypothesis, we examined the anti-proliferative effects of resveratrol on JAK2V617F mutant myeloid leukemia cell lines, HEL and SET-2. Resveratrol inhibited the proliferation of these JAK2V617F mutant cell lines in a dose-dependent manner in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny​l tetrazoliumbromide) assay, and its inhibitory effects were 3.3-8.4 times greater than those on other myeloid leukemia cell lines without the JAK2V617F mutation, including K562, THP-1 and TF-1. The inhibitory effects on JAK2V617F(+) cell lines by 50 µM of resveratrol were comparable to those induced by 1000 ng/ml of ruxolitinib, a selective JAK1/2 inhibitor. Incubation of different myeloid leukemia cell lines at a resveratrol concentration of 50 µM or at a concentration of 500 ng/ml of ruxolitinib for 24 hours induced apoptosis in 25.1 ± 2.2%/14.4 ± 1.1% of HEL, 23.7 ± 1.5%/19.1 ± 2.6% of SET-2, 6.9 ± 0.7% /0.34 ± 0.41% of K562, 4.9 ± 1.6%/1.9 ± 0.7% of THP-1 and 7.1 ± 1.0%/0.99 ± 0.58% of TF-1 cells (Fig. 1A). Resveratrol inhibited the proliferation of K562 cells transfected with JAK2V617F (K562JAK2V617F) 1.68-times more than it did wild-type K562 cells (K562JAK2WT cells), and induced apoptosis in 12.9 ± 1.6% of K562JAK2V617F and 6.5 ± 0.5% of K562JAK2WT cells. The level of apoptosis induced in K562JAK2V617F cells by resveratrol (6.3±1.5%) was comparable to that (8.2 ± 2.1%) induced by 500 ng/ml of ruxolitinib. Resveratrol inhibited the phosphorylation of JAK1, JAK2 and Tyk2, but did not affect the phosphorylation of JAK3, PTEN or Akt, or their downstream target proteins, including STAT3 and STAT5 (Fig. 1B). In contrast to the inhibition of ERK phosphorylation by ruxolitinib in JAK2V617F cells, resveratrol dramatically enhanced the phosphorylation of ERK (Fig. 1B), which is known to activate caspase-3. The inhibition of the phosphorylation of JAK2/STAT3 and the upregulation of ERK phosphorylation by resveratrol were also observed in K562JAK2V617F cells (Fig. 1B). Resveratrol also induced robust G1 cell cycle arrest of HEL and SET-2 cells, and led to the downregulation of the anti-apoptotic protein, Mcl-1, as well as the upregulation of the pro-apoptotic protein, Bim, both of which play important roles in prolonging the survival of JAK2V617F cells. Moreover, 25 µM of resveratrol augmented the apoptosis in HEL cells induced by 400 ng/ml of ruxolitinib, leading to 2.7 times more apoptosis than ruxolitinib alone in the annexin V assay. These data suggest that resveratrol exerts a potent anti-tumor effect on malignant myeloid cells with the JAK2V617F mutation via both the inactivation of the JAK/STAT pathway and the upregulation of the ERK pathway. Increased ERK phosphorylation has recently been shown to induce apoptosis in several types of cancer cells (Nguyen, et al. Int J Oncol, 2008; Mustafi, et al. PLoS One, 2010). Thus, resveratrol may have therapeutic potential against myeloproliferative neoplasms with the JAK2V617F mutation, and also against other myeloid malignancies where the JAK/STAT pathway plays an essential role in their development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Changes in contractile proteins during differentiation of myeloid leukemia cells. I. Polymerization of actin.

1980 ◽  
Vol 85 (2) ◽  
pp. 273-282 ◽  
Author(s):  
K Nagata ◽  
J Sagara ◽  
Y Ichikawa
Keyword(s):  
Cell Line ◽  
Conditioned Medium ◽  
Mitotic Activity ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Before And After ◽  
Myeloid Leukemia Cell ◽  
Qualitative Changes

Quantitative and qualitative changes in cellular actin were followed during differentiation of a myeloid leukemia cell line, namely Ml, which was inducible with conditioned medium (CM). During 3 d of incubation with CM, when the Ml cells differentiated to macrophages and lost their mitotic activity, the actin content, F-actin ratio in total actin, and the actin synthesis showed an increase. A greater difference before and after differentiation was found in the ability of G-actin to polymerize. Actin harvested from CM-treated cells showed a greater ability to polymerize, depending on the increased concentration of MgCl2 and/or KCl and proteins, as compared with the actin from untreated Ml cells. Actin harvested from the Mml cell line, a macrophage line, had a particularly high polymerizability with or without CM treatment. In contrast, the actin from the D- subline, which is insensitive to CM, showed almost no polymerization.

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