scholarly journals Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

2002 ◽  
Vol 13 (11) ◽  
pp. 3989-4000 ◽  
Author(s):  
Rodrigo Bermejo ◽  
Nuria Vilaboa ◽  
Carmela Calés
Keyword(s):  
Cell Lines ◽  
Cyclin E ◽  
Terminal Differentiation ◽  
K562 Cells ◽  
Active Role ◽  
Key Factors ◽  
Rna Transcription ◽  
Megakaryocytic Differentiation ◽  
Hel Cells

Endomitosis is the process by which mammalian megakaryocytes become polyploid during terminal differentiation. As in other endoreplicating cells, cyclin-cdk complexes are distinctly regulated, probably to overcome the strict mechanisms that prevent rereplication in most somatic cells. We have asked whether key factors involved in the assembly and licensing of replication origins are equally regulated during endomitosis. Cdc6, cdt1, and geminin expression was analyzed during differentiation of two human megakaryoblastic cell lines, HEL and K562, which respectively do and do not establish endoreplication cycles. Geminin was downregulated, whereas cdt1 levels were maintained upon differentiation of both cell lines, independently of whether cells entered extra S-phases. In contrast, cdc6 was present and remained nuclear only in differentiated endoreplicating cells. Interestingly, cdc6 protein expression was reestablished in K562 cells that underwent endomitosis after transient or stable cyclin E overexpression. The high levels of cyclin E reached in these cells appeared to influence the stabilization of cdc6 protein rather than its RNA transcription rate. Finally, cdc6 overexpression drove HEL cells into endoreplication cycles in the absence of differentiation stimuli. Our results show that both cdt1 and cdc6 are differentially regulated during megakaryocytic differentiation and suggest an active role of cdc6 in endomitosis.

Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation of Bcr-Abl+ K562 leukemic cells by Gab2

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1388-1397 ◽  
Author(s):  
Jay F. Dorsey ◽  
Jess M. Cunnick ◽  
Shrikant M. Mane ◽  
Jie Wu
Keyword(s):  
Map Kinase ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Blast Crisis ◽  
Terminal Differentiation ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Megakaryocytic Differentiation

In the blast crisis phase of chronic myelogenous leukemia (CML), Bcr-Abl+ myeloblasts fail to undergo terminal maturation. The extracellular signal–regulated kinase (Erk) mitogen-activated protein (MAP) kinase has been shown to mediate terminal differentiation of myeloid cells. Interestingly, Bcr-Abl+ CML cell lines established from blast crisis were found to have low Erk MAP kinase activity. In this study, we analyzed the role of the Gab2 docking protein in regulation of the Erk MAP kinase in Bcr-Abl+K562 human CML cells. Overexpression of Gab2 in K562 cells resulted in transcriptional activation of the c-fos serum response element (SRE) promoter, whereas overexpression of SHP2, Grb2, and CrkL had no effect. Activation of the c-fos SRE transcriptional activity by Gab2 required tyrosine 604, which is a SHP2 docking site on Gab2, and the SHP2 tyrosine phosphatase activity. Elk1, c-Jun, and CHOPtrans-reporting assays indicated that overexpression of Gab2 selectively activated the Erk2-Elk1 signaling pathway. To determine cellular consequences of elevating the Gab2 level in K562 cells, stable cell lines for doxycycline-inducible expression of the wild-type Gab2 (Gab2WT) and an SHP2-binding defective Gab2 (Gab2Tyr604Phe) were established. Analysis of these cell lines indicated that induction of Gab2WT expression, but not Gab2Tyr604Phe expression, led to Erk activation, growth arrest, cell spreading, and enlargement; expression of megakaryocyte/platelet lineage–specific integrins αIIb/β3 (CD41/CD61); and upregulation of RNA for megakaryocyte/platelet proteins. All of these changes are characteristics of megakaryocytic differentiation. Together, these results reveal Gab2 as a limiting signaling component for Erk MAP kinase activation and terminal differentiation of K562 CML cells.

PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2393-2393 ◽  
Author(s):  
Rabindranath Bera ◽  
Der-Cherng Liang ◽  
Ming-Chun Chiu ◽  
Ying-Jung Huang ◽  
Sung-Tzu Liang ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Blast Crisis ◽  
K562 Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Megakaryocytic Differentiation ◽  
Deletion Mutations ◽  
Phd Domain

Abstract Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.

hDLG Associated Protein 1 (DLGAP1) Supports Mpl-Driven Cell Proliferation and Differentiation Through Its Centrosomal Function,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3402-3402
Author(s):  
Boguslaw A. Kwiatkowski ◽  
Robert E. Richard
Keyword(s):  
Cell Lines ◽  
Myeloproliferative Neoplasms ◽  
K562 Cells ◽  
Full Length ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Minimal Effect ◽  
Hematopoietic Stem ◽  
Discs Large

Abstract Abstract 3402 The Mpl proto-oncogene functions as a basic factor in megakaryocytic development and platelet production as well as contributing to hematopoietic stem cell (HSC) homeostasis and self-renewal. Several mutations in Mpl have been shown to be associated with the myeloproliferative neoplasms. We used retroviral insertional mutagenesis to screen for factors that might give a proliferative and/or survival advantage for cells dependent on Mpl signaling. We developed a retroviral construct expressing a dimerizable form of Mpl that can interrupt genes through insertion, and can activate adjacent genes with its long terminal repeats. We transduced the human leukemia cell line K562 with this construct and blocked the endogenous transforming BCR-ABL kinase using Imatinib. Cells dependent on Mpl signaling were selected by the addition of the dimerizing drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. Cells that acquired a proliferative advantage and were dependent on Mpl function were expanded. Cloning of retroviral integration sites (RIS) from the selected populations allowed the identification of recurrent RIS that co-localized in the genome. We have identified 668 RIS from 36 independent transductions with a presumed ∼ 1.9 × 106 independent insertions in the initial non-selected, cell population. Among these, 203 RIS represented independent insertion events. Three independent RIS were located in the fourth intron of the Discs large homolog-associated protein 1 (DLGAP1). This protein is a member of the Discs-large/Scribble/Lethal Giant Larvae pathway. DLGAP1, in cooperation with DLG1 and CDC42, has recently been shown to control centrosome positioning and cell polarity in astrocytes (Manneville J-B et al. 2010, JBC, v.191, no 3, pp. 585–598). We investigated the role of DLGAP1 in MPL signaling. Overexpression of full length DLGAP1 had minimal effect on plain K562 cells, but it significantly slowed down the proliferation of K562 cells switched to Mpl signaling. The identified insertion sites would predict the increased expression of an amino-end truncated isoform. When this truncated form was overexpressed in K562 cells and Mpl dependent K562 cells, the cells proliferated at an increased rate. Immunofluorescent studies revealed that the full length DLGAP1 colocalized with major centrosomal markers including gamma-tubulin, PCM1, and APC in K562, HEL, UT7/TPO and Mo7e cell lines. We did not observe colocalization with centrosomes when the truncated isoform of DLGAP1 was overexpressed. Immunoflourescent microscopy revealed that endogenous DLGAP1 in myeloid cell lines was localized in the centriolar satellites, a cellular structure essential for centrosome integrity and function. The immunoflourescent pattern using an anti DLGAP1 antibody showed cell cycle dependent assembly and disassembly. Protein sequence analyses of DLGAP1 indicated consensus sites for presumptive Jak2 phosphorylation as well as consensus sites for SRC tyrosine kinases. To test the possible influence of the two types of kinases on DLGAP1 we treated K562, HEL and UT7/TPO cell lines with an inhibitor of Jak2 (AG490) and independently with an inhibitor of SRC (SU6656). In each case the centrosomal staining of DLGAP1 was diminished with minimal effect on the cytoplasmic fraction of DLGAP1. Interestingly, treatment with SU6656 of the K562 cells switched to Mpl signaling and overexpressing full length GFP-DLGAP1 fusion resulted in a significant increase in the number of polyploid cells. The changes correlated with upregulation of the megakaryocytic marker CD41a and downregulation of the erythroid marker Glycophorin A as assessed by FACS analyses. As centrosomal aberrations are frequent markers of the myeloproliferative neoplasms, we intend to study the role of DLGAP1 in these bone marrow disorders. Disclosures: No relevant conflicts of interest to declare.

Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1470-1470
Author(s):  
Jueqiong Wang ◽  
Chung Hoow Kok ◽  
Richard J. D'Andrea ◽  
Timothy P. Hughes ◽  
Deborah L. White
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
K562 Cells ◽  
Vehicle Control ◽  
Peroxisome Proliferator ◽  
Advisory Committees ◽  
Peroxisome Proliferator Activated Receptor

Abstract Introduction The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. Methods OA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. Results A significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05). Conclusion Ligand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy. 1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures: Hughes: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

scholarly journals Myeloblastic leukemia cells conditionally blocked by myc-estrogen receptor chimeric transgenes for terminal differentiation coupled to growth arrest and apoptosis

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2257-2262 ◽  
Author(s):  
M Selvakumaran ◽  
D Liebermann ◽  
B Hoffman-Liebermann
Keyword(s):  
Cell Lines ◽  
Culture Medium ◽  
Target Genes ◽  
Growth Arrest ◽  
Terminal Differentiation ◽  
Intermediate Stage ◽  
Myeloid Differentiation ◽  
Differentiation Inducers ◽  
Differentiation Inducer

Abstract Conditional mutants of the myeloblastic leukemic M1 cell line, expressing the chimeric mycer transgene, have been established. It is shown that M1 mycer cells, like M1, undergo terminal differentiation coupled to growth arrest and programmed cell death (apoptosis) after treatment with the physiologic differentiation inducer interleukin-6. However, when beta-estradiol is included in the culture medium, M1 mycer cells respond to differentiation inducers like M1 myc cell lines, where the differentiation program is blocked at an intermediate stage. By manipulating the function of the mycer transgene product, it is shown that there is a 10-hour window during myeloid differentiation, from 30 to 40 hours after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, where activation of c-myc has no apparent effect on mature macrophages. M1 mycer cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal myelopoiesis and in leukemogenesis, also providing a strategy to clone c-myc target genes.

scholarly journals Myeloblastic leukemia cells conditionally blocked by myc-estrogen receptor chimeric transgenes for terminal differentiation coupled to growth arrest and apoptosis

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2257-2262 ◽  
Author(s):  
M Selvakumaran ◽  
D Liebermann ◽  
B Hoffman-Liebermann
Keyword(s):  
Cell Lines ◽  
Culture Medium ◽  
Target Genes ◽  
Growth Arrest ◽  
Terminal Differentiation ◽  
Intermediate Stage ◽  
Myeloid Differentiation ◽  
Differentiation Inducers ◽  
Differentiation Inducer

Conditional mutants of the myeloblastic leukemic M1 cell line, expressing the chimeric mycer transgene, have been established. It is shown that M1 mycer cells, like M1, undergo terminal differentiation coupled to growth arrest and programmed cell death (apoptosis) after treatment with the physiologic differentiation inducer interleukin-6. However, when beta-estradiol is included in the culture medium, M1 mycer cells respond to differentiation inducers like M1 myc cell lines, where the differentiation program is blocked at an intermediate stage. By manipulating the function of the mycer transgene product, it is shown that there is a 10-hour window during myeloid differentiation, from 30 to 40 hours after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, where activation of c-myc has no apparent effect on mature macrophages. M1 mycer cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal myelopoiesis and in leukemogenesis, also providing a strategy to clone c-myc target genes.

scholarly journals Downregulation of GATA-1 expression during phorbol myristate acetate- induced megakaryocytic differentiation of human erythroleukemia cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1214-1221 ◽  
Author(s):  
W Dai ◽  
MJ Jr Murphy
Keyword(s):  
Transcription Factor ◽  
Steady State ◽  
Phorbol Myristate Acetate ◽  
Mrna Level ◽  
Terminal Differentiation ◽  
Specific Gene ◽  
Glycophorin A ◽  
Myristate Acetate ◽  
Megakaryocytic Differentiation ◽  
Hel Cells

Phorbol myristate acetate (PMA) induces the expression of megakaryocyte and/or platelet proteins during terminal differentiation of human erythroleukemia (HEL) cells. However, it is not established whether megakaryocytic differentiation is accompanied by the downregulation of the major erythroid transcription factor GATA-1 and the concomitant loss of the erythrocytic phenotype. Studies of the molecular mechanism of PMA-induced differentiation in HEL cells showed that when HEL cells are treated with PMA, they dramatically decrease the expression of the erythroid-specific gene glycophorin A at the mRNA level but apparently not at the steady-state protein level. In addition, a gel mobility shift assay was used to demonstrate that GATA-1, a major erythroid transcription factor normally present at high levels in HEL cells is downregulated after treatment with PMA. In contrast, the DNA-binding activities of transcription factors AP-1 and SP-1 are upregulated by PMA treatment of HEL cells. Furthermore, Northern blot analysis shows that PMA also downregulates the steady-state level of GATA-1 mRNA in HEL cells. The coordinated negative regulation of glycophorin A mRNA and GATA-1 expression after PMA treatment suggests that downregulation of GATA-1 expression may be partially responsible for the loss of the erythroid phenotype during megakaryocytic differentiation. The reported data also suggest that GATA-1 activity may not be essential for obtaining megakaryocytic phenotype during terminal differentiation in HEL cells.

Homozygous JAK2 V617P Gain-of-Function Mutation Is Responsible for Constitutive JAK2/STAT5 Activation and Proliferation of HEL.92.1.7. Cell Line.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4377-4377
Author(s):  
Myka L. Estes ◽  
Yuki Ozawa ◽  
Ann H. Williams ◽  
Alan F. List ◽  
Lubomir Sokol
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cell ◽  
K562 Cells ◽  
Constitutive Activation ◽  
Jak2 Inhibitor ◽  
Hel Cells

Abstract HEL (Human erythroleukemia) is a growth factor independent erythroleukemic cell line established from the bone marrow of a patient with relapsed Hodgkin disease after autologous bone marrow transplantation (Martin P & Papayannopoulou T: Science1982;216:1233–1235). HEL cells display a block in differentiation at the level of common erythroid-megakaryocytic progenitor and have been commonly used as a model to study erythroid and megakaryocytic differentiation. Liu RY et al. (Blood1999;93:2369–2379) reported that constitutive activation of JAK2 tyrosine kinase in Dami/HEL cell line correlated with factor independent growth. Recently, several groups of investigators described a point mutation V617P in the autoinhibitory regulatory (JH2) domain of JAK2 tyrosine kinase in patients with myeloproliferative disorders (MPD). This acquired mutation results in constitutive activation of the JAK2/STAT5 pathway in hematopoietic cells. To date, the V617P mutation represents the most common causative genetic defect detected in patients with Philadelphia chromosome negative MPD. Interestingly, rare cases of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) were found to have the same mutation. We tested several hematopoietic cell lines including HL-60, HEL, KG-1, K-562, TF-1, U937 and UT-7 for the presence of V617P mutation using RFLP and automatic DNA sequencing. We found the homozygous mutation V617P only in HEL cells. This cell line has a very complex karyotype suggesting that a defect of multiple oncogenes or antioncogenes could be responsible for sustained cell proliferation. We employed the JAK2 inhibitor, AG490 at concentrations ranging from 0 to 100 mM and measured proliferation of HEL and control K562 cell lines using MTT assay. We observed dose-dependent inhibition of HEL cell proliferation with low micromolar concentrations of AG490 in contrast to K562 control cell line, harboring bcr-abl fusion tyrosine kinase. IC50 was 13 mM for HEL cells and 65 mM for control K562 cells, respectively. Low concentrations of AG490 significantly decreased phosphorylation of JAK2 and STAT5 in HEL cells but not in control K562 cell line, where bcr-abl activates separately JAK2 and STAT5 pathway. Exposure of HEL cells to AG490 [0–50mM] induced apoptosis as measured by annexin V labeling in a concentration dependent fashion. No significant increase in apoptosis was detected in K562 cells using similar concentrations of the JAK2 inhibitor. Overall, these results suggest that the mechanism of proliferation of the HEL cell line is driven by constitutive activation of JAK2 tyrosine kinase secondary to V617P gain-of-function mutation. Our data provides further evidence that the JAK2/STAT5 intracellular signaling pathway is preserved in this cell line. Thus, HEL cells can serve as a model to test novel JAK2 specific inhibitors in preclinical studies.

Overexpression of the EphB4 Receptor Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Through Regulating Mlcp and VAV1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4421-4421
Author(s):  
Liu Xiaoli ◽  
Jinfang Zhang ◽  
Qingfeng Du ◽  
Na Xu ◽  
Lulu Xu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Differential Expression Genes

Abstract Abstract 4421 Objective: To study the role of EphB4 in imatinib (IM) resistant chronic myeloid leukemia (CML) and investigate the mechanism. Methods: We derived IM-resistant cells, K562-R cells, from wild K562 cells under gradually increasing IM concentrations. We analysed expression level of EphB4 in CML patients, wild K562 and K562-R cell lines by real-time reverse transcription PCR and Western blot analysis. Then we established stable under-expressing EphB4 cell (K562-R-EphB4-sh) lines. We analysed the sensitive for IM of K562, K562-R, K562-R-EphB4-sh cell lines by CCK8 assay. Microarray analysis was used to screen differential expression genes between K562-R and K562-R-EphB4-sh cell lines. Results: The mRNA and protein of EphB4 were significantly increased in IM resistant CML patients compared to IM sensitive CML patients (p<0.05). The Similar results were observed in K562-R and K562 cells (p<0.01). To analyze the role of EphB4 in IM resistance, EphB4 was knocked down with shRNA expressed by pLL3.7 lentivirus vector. We established stable under-expressing EphB4 cell line K562-R-EphB4-sh. RT-PCR and western blot analysis showed that mRNA and protein expression of EphB4 in K562-R-EphB4-sh cells were reduced (p<0.05). CCK8 assay found K562 cells (IC50 0.1207±0.0234μM), K562-R-EphB4-sh cells (IC50 0.7228±0.04752μM) were sensitive to IM but K562-R (IC50 2.8101±0.04674μM) still showed IM resistance (p<0.05). Those suggested K562-R-EphB4-sh cells resensitize to IM when the expression of EphB4 was down regulated. However, these cells were still less sensitive than K562 cells. Microarray analysis between K562-R and K562-R-EphB4-sh cell lines found 641 differential expression genes, most of them were related to cell adhesion and cell cytoskeleton. We confirmed MLCP and VAV1 were down regulated in K562-R-EphB4-sh cells compared to K562-R cell lines by western blot analysis. Conclusion: Our study suggest EphB4 receptor contributes to IM-resistant in CML through regulating cell adhesion molecular MLCP and VAV1, which may provide new biomarkers and contribute to] developping new drugs for the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting Aberrant Ncam (neural cell adhesion molecule; CD56) Expression in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 311-311 ◽  
Author(s):  
Daniel Sasca ◽  
Andrea Schueler ◽  
Jakub Szybinski ◽  
Oliver Kriege ◽  
Kerstin Kunz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Neural Cell Adhesion ◽  
Cd56 Expression

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease of the hematopoietic progenitor cell driven by the subsequent acquisition of genetic alterations. Approximately 20% of AML patients show strong expression of CD56 (neural cell adhesion molecule; NCAM). Expression of NCAM is associated with poor overall survival; however, the functional role of aberrant NCAM expression has not been investigated to date. The goal of this study is to examine the biological role of NCAM in AML and to explore whether NCAM represents a potential therapeutic target. Results In order to evaluate the clinical significance of elevated NCAM expression in AML, we screened a panel of human cell lines for CD56 expression. Most cell lines were positive and cell surface expression correlated with mRNA levels. Knockdown of NCAM with three different doxycycline-inducible shRNAs suppressed cell growth and MTT activity in all positive cell lines. Propidium iodide staining demonstrated an increase in G1 arrest. Importantly, there was a marked apoptosis after NCAM suppression and this effect was proportional to the knockdown efficiency. Survival of NOD-SCIDgamma chain mice in a leukemia engraftment model was significantly prolonged upon NCAM knockdown. Suppression of NCAM sensitized leukemic blasts to treatment with Ara-C or Daunorubicin in vitro and in xenotransplantation experiments. To test the consequences of NCAM overexpression in negative leukemic cell lines we transduced the NCAM transcript from Nomo-1 into HL60 and K562 cells. HL60 cells had a significantly lower sensitivity towards Ara-C or Daunorubicin treatment. IC50 for the BCR-ABL inhibitor Dasatinib in K562 cells increased from 0.95 nM (EV, empty vector) to 2.2 nM in NCAM overexpressing cells. To dissect possible upstream regulation mechanisms of NCAM expression we performed DNAseI hypersensitivity assays coupled to qRT-PCR mapping of known putative sites in the NCAM promoter and observed open chromatin for the binding sites of Meis1, Mef2 and Stat1. shRNA-mediated knockdown of MEIS1, MEF2c and MLL-AF9 resulted in significant suppression of NCAM cell surface expression, suggesting an upstream regulatory role for MLL-AF9. To gain insights into the mechanisms underlying the NCAM function in AML we performed gene expression comparisons of the 30 highest versus 30 lowest expressing samples in the GSE8043 dataset. Fifty-seven Biocharta pathways were differentially expressed between NCAMhigh and NCAMlow samples, while expression changes predicted abnormal cell-cycle regulation, stress and DNA damage response, cell survival, renewal and adhesion. Western blot, protein array and qRT-PCR analyses of candidate downstream signaling pathways upon knockdown of NCAM demonstrated enhanced degradation of BetaCatenin, decreased expression of BCL-2 and increased levels of p21 and p27. The upstream regulation mechanism described above revealed MLL-AF9 (M/A9) as a top candidate for NCAM regulation. Subsequent analysis of M/A9 L-GMPs (Lin- cKit+ CD34+ FcgR+) demonstrated strong surface expression of NCAM, whereas normal HSCs (Lin-cKit+ Sca1+) were NCAM-negative. This could be validated by gene expression analyses of M/A9 L-GMPs compared with normal HSCs. In order to elucidate the role of NCAM on leukemic cell function in a mouse model, NCAM-/- and control wildtype (WT) bone marrow cells were transformed with a retroviral construct of M/A9 and transplanted into lethally irradiated littermates. Recipients of NCAM-/- M/A9 cells developed acute leukemia with prolonged disease latency. NCAM-/- M/A9 cells had lower CD117 and Gr-1 expression, but higher expression of Mac-1 and, in some samples, aberrant B220 co-expression. Importantly, there was a reduced representation of L-GMPs in the NCAM-/- M/A9 group and limited dilution retransplantation assays revealed a significantly prolonged survival of NCAM-/- M/A9 mice. Replating activity in methylcellulose was diminished and could be eradicated with sublethal doses of Cytarabine. Summary Targeting aberrant expression of NCAM demonstrated strong antileukemic activity in vitro and sensitized leukemic blasts to genotoxic stress. In vivo, depletion of NCAM resulted in prolonged disease survival in syngeneic and xenotransplantation experiments and diminished self-renewal capacities. Our data suggest that NCAM represents a promising therapeutic strategy and likely targets AML cells at the LSC level. Disclosures No relevant conflicts of interest to declare.

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