scholarly journals Antibodies to myeloid precursor cells in autoimmune neutropenia

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 625-631 ◽  
Author(s):  
KR Hartman ◽  
VF LaRussa ◽  
SW Rothwell ◽  
TO Atolagbe ◽  
FT Ward ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Cell ◽  
K562 Cells ◽  
Precursor Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Autoimmune Neutropenia ◽  
Igg Antibodies

Abstract Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further studies, antibodies from 12 sera bound to mononuclear CD34+ cells that had been purified from normal human bone marrow by an immunomagnetic separation procedure. Moreover, two of these sera suppressed the growth of granulocyte-macrophage colony- forming units (CFU-GM) in methylcellulose cultures. The presence of antibodies to primitive hematopoietic cells in the sera of some patients with suspected immune neutropenia suggests that these antibodies may have a role in the pathogenesis of the neutropenia observed.

scholarly journals Antibodies to myeloid precursor cells in autoimmune neutropenia

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 625-631 ◽  
Author(s):  
KR Hartman ◽  
VF LaRussa ◽  
SW Rothwell ◽  
TO Atolagbe ◽  
FT Ward ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Cell ◽  
K562 Cells ◽  
Precursor Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Autoimmune Neutropenia ◽  
Igg Antibodies

Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further studies, antibodies from 12 sera bound to mononuclear CD34+ cells that had been purified from normal human bone marrow by an immunomagnetic separation procedure. Moreover, two of these sera suppressed the growth of granulocyte-macrophage colony- forming units (CFU-GM) in methylcellulose cultures. The presence of antibodies to primitive hematopoietic cells in the sera of some patients with suspected immune neutropenia suggests that these antibodies may have a role in the pathogenesis of the neutropenia observed.

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

Bionatura ◽  
2021 ◽  
Vol 6 (2) ◽  
pp. 1725-1732
Author(s):  
Hamdah Alsaeedi ◽  
Rowaid Qahwaji ◽  
Talal Qadah
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Time Dependent ◽  
Leukemia Cell Line ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Anti Cancer

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

scholarly journals Dye-mediated photolysis is capable of eliminating drug-resistant (MDR+) tumor cells

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 793-800 ◽  
Author(s):  
RM Lemoli ◽  
T Igarashi ◽  
M Knizewski ◽  
L Acaba ◽  
A Richter ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Light Exposure ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Drug Resistant ◽  
Colony Forming Unit

Abstract We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.

Microrna Expression Profiling in Acute Myelogenous Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1131-1131
Author(s):  
Fernando J. Suarez Saiz ◽  
Serban San-Marina ◽  
Mark D. Minden
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Erythroid Cell ◽  
Normal Bone Marrow ◽  
Hematopoietic Differentiation ◽  
Normal Bone ◽  
Acute Myelogenous

Abstract Acute myelogenous leukemia (AML) arises due to changes in gene expression that block or alter the normal differentiation program of hematopoietic stem cells. A variety of mutations in protein-encoding genes have been shown to contribute to the development of leukemia. Recently a new class of genes called microRNAs (miRNAs) have been identified. miRNAs are a subgroup of highly conserved, non-coding RNAs found only in eukaryotes. They do not encode proteins, and appear to have a significant effect on the proteome of a cell. Their conservation between species suggests their involvement in important biological functions, and in fact been shown to be involved in hematopoietic differentiation. While the function of most miRNAs is still unknown, it is believed that they regulate expression of target mRNAs by using the siRNA machinery either to promote degradation of the mRNA or to block its translation. To begin to understand the role of miRNAs in AML, we used Quantitative Polymerase Chain Reaction (QPCR) to measure the expression level of 20 miRNA precursors in the pro erythroid cell line K562, the pro-myelocytic cell line NB4, the myelomococytic cell line OCI/AML2, AML patients’ blasts and in normal bone marrow (NBM). The investigated miRNAs included some that are known to be specific for hematopoietic tissues or involved in hematopoietic differentiation, as well as all the miRNAs in chromosome 7, a hot spot for gene deletion in AML. Our findings indicate that miRNAs are differentially expressed in patients and cell lines when compared among themselves and against normal bone marrow. For example pre-miR-142 was expressed in NBM and K562 but was found to be elevated in OCI/AML2, NB4 and in all patient samples. Pre-miR-20 was found to be overexpressed in only a subset of patients. Other miRNAs like pre-miR-335 and pre-miR-148a were expressed in NBM and in some patients and not in the cell lines. In an effort to identify possible regulators of miRNA expression, we analyzed the upstream region of pre-miR-142 and found an LMO2 binding site. In AML, the LMO2 gene can be overexpressed relative to normal bone marrow and healthy lymphocytes. This transcription factor is involved in the regulation of genes important in the development of blood cells. To investigate if LMO2 could be involved in the regulation of miR-142 expression, we performed chromatin immunoprecipitation (ChIP) from K562 using an anti-LMO2 antibody. Only the LMO2 immunoprecipitation, and not those from the pre-immune control, were enriched in promoter DNA for pre-miR-142. This is consistent with the observation that miRNAs and coding RNAs can be regulated by the same environmental signals. Based on this observation we propose that oncogenes regulate in part the phenotype and biological behaviour of leukemia by affecting the expression of miRNAs. This further suggests that different forms of leukemia may be recognized based upon the spectrum of miRNAs they express.

scholarly journals Orlistatand and Rosuvastatin Suppressed Proliferation of K562 Human Myelogenous Leukemia Cell Line through AMPK/Akt/c-Myc Signaling

2020 ◽  
Author(s):  
Behnam Mojjarad ◽  
Yaghub Pazhang
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Mrna Levels ◽  
Hsp 70

Abstract Background: Chronic myeloid leukemia is a myeloproliferative cancer with worldwide incidence, has become as a clinical concern due to chemoresistance in the patients received chemotherapy. Here, we investigated the effect of Orlistat and Rosuvastatin on K562 human myelogenous leukemia cell line in vitro and attempted to illuminate their possible underlying mechanisms. Methods: Cells were exposed to Orlistat and Rosuvastatin, the inhibitors of lipogenesis, then survival and apoptosis rate of K562 cells were examined by MTT assay and flow cytometric analysis respectively. The real time-PCR analysis was used to quantify mRNA levels of Bax, Bcl-2, and Hsp-70 genes. Cell cycle analysis was performed using flow cytometry, whereas the subcellular distribution of c-Myc was measured via immunofluorescence imaging technique. Additionally, the protein level of AMPK, p-AMPK Akt-1, and p-Akt-1 were studied by western blotting. Results: The results showed Orlistat and Rosuvastatin had synergistic anticancer effects on cells and in comparison with the control group, viability and apoptosis rate decreased and increased in treated cells respectively in a dose/time-dependent manner (P<0.05). The mRNA levels of Bax increased while expression of Hsp-70 decreased (P< 0.05). K562 cells treated with Orlistat and Rosuvastatin showed a cell cycle arrest in sub-G1 phase and a decreased level of c-Myc positive cells. Upon outlining the mechanism, it was revealed that AMPK/p-AMPK and p-Akt-1/Akt-1 ratio decreased in treated cells (P< 0.05). Conclusions: Data suggest Orlistat and Rosuvastatin could synergically suppress proliferation of K562 cells through AMPK/Akt/c-Myc axis, proposing a theoretical basis for upcoming application in the treatment of chronic myeloid leukemia

scholarly journals Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1124-1130
Author(s):  
AL Epstein ◽  
M Samoszuk ◽  
E Stathopoulos ◽  
GS Naeve ◽  
CV Clevenger ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Dna Binding ◽  
Cell Lines ◽  
Solid Phase ◽  
Specific Antigen ◽  
Precursor Cells ◽  
Myelogenous Leukemia ◽  
Nuclear Antigen ◽  
Cortex Of The Thymus

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Nucleophosmin Protects Hematopoietic Cells from Stress-Induced Apoptosis through Inhibition of P53.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2558-2558
Author(s):  
Qishen Pang ◽  
June Li ◽  
Xiaoling Zhang ◽  
Daniel P. Sejas ◽  
Grover C. Bagby
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Induced Apoptosis

Abstract Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Here we report that NPM protects hematopoietic cells from stress-induced apoptosis through inhibition of the tumor suppressor p53. Specifically, we forced expression of wild-type (WT) NPM or a mutant variant with a deletion of the C-terminal 120 aa of NPM (NPMΔC) by retroviral gene transfer in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line MO7e (expresses low level of NPM) and the lymphoblast HSC536 cells derived from a Fanconi anemia (FA) patient in the C complementation group (expresses essentially undetectable NPM). Using a flow cytometric method for caspase 3 activation (early apoptosis), we demonstrated that overexpression of NPM but not the mutant NPMΔC confers MO7e and HSC536 cells resistance to apoptosis induced by growth factor deprivation and treatment with the chemotherapeutic drug etoposide. In addition, suppression of NPM expression by small interfering RNA targeting NPM in chronic myelogenous leukemia line K562 and FA-associated acute myelogenous leukemia cell line UoC-M1 increases etoposide-induced apoptosis, thus providing proof of concept evidence that the pathological elevations of NPM found in cancers and leukemias are important for maintaining cell survival and resistance to apoptosis. Because overexpression of the mutant NPMΔC, which lacks the p53-interacting domain, fails to confer cellular resistance to stress-induced apoptosis, we determined whether NPM protects cells from apoptotic cell death through a mechanism involving p53. We used the genetically matched p53 WT and null mouse bone marrow (BM) cells to show that overexpression of WT NPM protects against ionizing irradiation (IR)-induced apoptosis of wild-type but not p53-null BM cells. Moreover, NPM inhibits IR-induced p53 phosphorylation at Ser18 and transactivation, and interacts with p53 in bone marrow hematopoietic cells. Thus, this study not only demonstrates anti-apoptotic function of a proliferation-promoting protein but also suggests that cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.

To Study the Proliferative Inhibition of Anticancer Drug in Two Kinds of Ph (+) Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4110-4110
Author(s):  
Yuping Gong ◽  
Xi Yang ◽  
Ting Niu
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cell Lines

Abstract Abstract 4110 Objective To study the proliferative inhibition of imatinib, daunorubicin and bortezomib in two kinds of Ph(+) leukemia cell lines: chronic myelogenous leukemia cell line K562 expressing P210 protein and acute lymphoblastic leukemia cell line SUP-B15 expressing P190 protein. Methods (1) Cell proliferation with imatinib, daunorubicin and bortezomib for 72 hours was analyzed by the MTT assay and displayed by growth curve and IC50 value. (2) The change of bcr-abl gene mRNA levels after the 48 hours' intervention of imatinib (final concentration at 0μM, 0.35μM, 1 μM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Results (1) The IC50 values of K562 and SUP-B15 cells inhibited by imatinib, daunorubicin and bortezomib for 72 hours was respectively 0.286±0.06 (μmol/L), 0.303±0.009 (μmol/L), 22.127±3.592 (nmol/L) and 1.387±0.180(μmol/L), 0.117±0.017 (μmol/L), 12.350±0.740 (nmol/L), which indicated that the K562 cell line was the more sensitive to imatinib than SUP-B15 cell line, whereas the SUP-B15 cell line had the more sensitivity to daunorubicin and bortezomib. (2) There was no change of bcr-abl gene expression after the 48 hours' intervention of imatinib in both cell lines. Conclusion (1) Imatinib, daunorubicin and bortezomib had good anti-cancer effect to Ph+ leukemia cells in vitro. What's more, the K562 cell was the more sensitive to imatinib and only imatinib will have good effect on chronic myelogenous leukemia. Whereas the SUP-B15 cell had the more sensitivity to daunorubicin and bortezomib and combining imatinib with daunorubicin or bortezomib, the effect will be better on Ph(+) acute lymphoblastic leukemia. (2) The short time intervention of imatinib had no effect on the bcr-abl gene expression and imatinib could need long time to show curative effect for the Ph+ leukemia. Disclosures: No relevant conflicts of interest to declare.

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.

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