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

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

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

Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 873-873
Author(s):  
Maria Eugenia Riveiro ◽  
Lucile Astorgues-Xerri ◽  
Charlotte Canet-jourdan ◽  
Mohamed Bekradda ◽  
Esteban Cvitkovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mrna Levels ◽  
Protein Levels ◽  
V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Akt Activation Is Important In KRAS-Mediated Multistep Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4200-4200
Author(s):  
Ashley F. Ward ◽  
Angell Shieh ◽  
Emily Rose Harding-Theobald ◽  
Gideon Bollag ◽  
Kevin Shannon
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Partial Loss ◽  
Ras Mutations ◽  
Downstream Effector ◽  
Effector Pathways

Abstract Abstract 4200 Activating mutations of Ras are found in approximately 30% of all human malignancies, and 85% of these mutations are in the K-ras isoform. These mutations dysregulate cell cycle progression, proliferation and apoptosis, and have been implicated in both initiation and maintenance of tumors. While mutant K-ras thus represents an attractive therapeutic target, attempts to develop a Ras inhibitor have been unsuccessful to date. K-Ras has three canonical downstream effector pathways: MEK/Erk, PI3K/Akt, and Ral. One or more of these pathways may represent an alternative drug target for Ras-driven malignancies, but it is not yet understood how each of these pathways contributes to leukemogenesis. Partial loss-of-function Ras mutations have been identified that render oncogenic K-Ras (K-RasG12D) unable to interact with one or more downstream effectors. Our lab has previously demonstrated that one such partial loss-of-function mutation, K-RasG12D,Y64G, does not activate the PI3K/Akt pathway. Mice transplanted with hematopoietic progenitor cells transduced with MSCV vectors encoding K-RasG12D,Y64G develop an aggressive T-lineage acute lymphoblastic leukemia (T-ALL) with a median survival of 112 days (Shieh and Shannon, Blood (ASH Annual Meeting Abstracts) 2007 110: Abstract 1617). To determine if the “missing” Ras effector pathway is deregulated during multistep tumorigenesis, we generated cell lines from K-RasG12D,Y64G leukemias (n=6). Western blot analysis revealed low Ras levels and absent PTEN protein expression in 5 of 6 K-RasG12D,Y64G leukemia cell lines. Quantitative PCR analysis revealed reduced PTEN mRNA levels in these cell lines, which was not due to somatic Pten mutations. As expected, cell lines without detectable PTEN showed high pAkt levels that persisted during serum and cytokine deprivation. One K-RasG12D,Y64G leukemia cell line was remarkable because it contained high levels of Ras and retained PTEN expression. DNA sequence analysis of this cell line unexpectedly revealed both the Y64G substitution and a de novo in frame insertion of two amino acids (arginine and aspartic acid) within the switch II domain of K-Ras, between codons 69 and 70. This insertion was also identified in the primary T-ALL. Murine fetal liver cells engineered to express K-RasG12D,Y64G, 69RN70 induced a dramatic pattern of hypersensitive progenitor growth characterized by cytokine-independent colony formation and large and aberrant CFU-GM morphology in the presence of GM-CSF that was indistinguishable from cells expressing K-RasG12D. Phospho-FACS analysis of these cells revealed markedly increased expression of pAkt when compared to cells expressing K-RasWT or K-RasG12D,Y64G, and similar to pAkt levels in cells expressing K-RasG12D. Preliminary in silico structural analysis of K-RasG12D,Y64G,69RN70 suggests that this novel insertion may restore a critical salt bridge between K-Ras and PI3Kγ. These data suggest that K-RasG12D oncogenes defective in PI3K signaling are still able to cause dysregulated growth of hematopoietic cells in vitro and in vivo via the acquisition of additional mutations that restore signaling through the PI3K pathway, and strongly support simultaneously targeting multiple downstream effector pathways as a general therapeutic strategy for the substantial fraction of human cancers that contain RAS mutations. Disclosures: Bollag: Plexxicon, Inc: Employment, Equity Ownership, Patents & Royalties.

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

1988 ◽  
Vol 6 (3) ◽  
pp. 209-220 ◽  
Author(s):  
Hiroyuki Tsuda ◽  
Mamoru Sakaguchi ◽  
Makoto Kawakita ◽  
Shimpei Nakazawa ◽  
Taijiro Mori ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Cycle Progression ◽  
Human Leukemia Cell ◽  
Human Leukemia Cell Line

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

1994 ◽  
Vol 57 (4) ◽  
pp. 522-528 ◽  
Author(s):  
John R. Zalcberg ◽  
Xiu F. Hu ◽  
Dominic M. Wall ◽  
Shelagh Mirski ◽  
Susan Cole ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Resistant Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Human Leukemia Cell Line

A Novel Function of Stat1α and Stat3 Proteins in Erythropoietin-Induced Erythroid Differentiation of a Human Leukemia Cell Line

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 462-471 ◽  
Author(s):  
Keita Kirito ◽  
Mie Uchida ◽  
Masaaki Takatoku ◽  
Koichi Nakajima ◽  
Toshio Hirano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Leukemia Cell ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
A Cell ◽  
Macrophage Colony

We recently determined that erythropoietin (EPO) activates 3 members of the signal transducer and activator of transcription (STAT) family, Stat1α, Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7 and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In addition, we have shown that Stat1α, but not Stat3, is involved in EPO-induced cellular proliferation. In this study, we examined the roles of Stat1α and Stat3 in EPO-induced erythroid differentiation. UT-7/GM was used as a model system, because this cell line can differentiate into erythroid-lineage cells with EPO treatment (Komatsu et al, Blood 89:4021, 1997). We found that EPO did not activate Stat1α or Stat3 in UT-7/GM cells. Transfection experiments showed that both Stat1α and Stat3 inhibited the induction by EPO of γ-globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of hemoglobin-positive cells. Dominant negative forms of Stat1α or Stat3 promoted the EPO-induced erythroid differentiation of UT-7/GM cells, even in the presence of granulocyte-macrophage colony-stimulating factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis showed that the constitutive activation of Stat1α, but not Stat3, shortened the period of G0/G1 prolongation caused by EPO stimulation. Taken together, our data suggest that Stat1α and Stat3 act as negative regulators in EPO-induced erythroid differentiation. Specifically, Stat1α may activate a cell cycle-associated gene(s), leading to the entry of cells into the cell cycle.

A Novel Function of Stat1α and Stat3 Proteins in Erythropoietin-Induced Erythroid Differentiation of a Human Leukemia Cell Line

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 462-471 ◽  
Author(s):  
Keita Kirito ◽  
Mie Uchida ◽  
Masaaki Takatoku ◽  
Koichi Nakajima ◽  
Toshio Hirano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Leukemia Cell ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
A Cell ◽  
Macrophage Colony

Abstract We recently determined that erythropoietin (EPO) activates 3 members of the signal transducer and activator of transcription (STAT) family, Stat1α, Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7 and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In addition, we have shown that Stat1α, but not Stat3, is involved in EPO-induced cellular proliferation. In this study, we examined the roles of Stat1α and Stat3 in EPO-induced erythroid differentiation. UT-7/GM was used as a model system, because this cell line can differentiate into erythroid-lineage cells with EPO treatment (Komatsu et al, Blood 89:4021, 1997). We found that EPO did not activate Stat1α or Stat3 in UT-7/GM cells. Transfection experiments showed that both Stat1α and Stat3 inhibited the induction by EPO of γ-globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of hemoglobin-positive cells. Dominant negative forms of Stat1α or Stat3 promoted the EPO-induced erythroid differentiation of UT-7/GM cells, even in the presence of granulocyte-macrophage colony-stimulating factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis showed that the constitutive activation of Stat1α, but not Stat3, shortened the period of G0/G1 prolongation caused by EPO stimulation. Taken together, our data suggest that Stat1α and Stat3 act as negative regulators in EPO-induced erythroid differentiation. Specifically, Stat1α may activate a cell cycle-associated gene(s), leading to the entry of cells into the cell cycle.

scholarly journals Design, Synthesis of Novel Tetrandrine-14-l-Amino Acid and Tetrandrine-14-l-Amino Acid-Urea Derivatives as Potential Anti-Cancer Agents

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1738
Author(s):  
Sheng-Cao Hu ◽  
Jin Yang ◽  
Chao Chen ◽  
Jun-Rong Song ◽  
Wei-Dong Pan
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Urea Derivatives ◽  
Anti Cancer

Tetrandrine, a dibenzyltetrahydroisoquinoline alkaloid isolated from the root of the traditional Chinese medicinal plant Stephania tetrandra S. Moore, a member of the Menispermaceae, showed anti-cancer activity by inhibiting cell proliferation, preventing cell cycle progress and induction of cell death and autophagy. In this study, twelve tetrandrine-l-amino acid derivatives and twelve tetrandrine-14-l-amino acid-urea derivatives were designed and synthesized, using C14-aminotetrandrine as raw material. Then the preliminary in vitro anti-cancer activities of these derivatives against human breast cancer cell line MDA-MB-231, human leukemia cell lines HEL and K562 were evaluated. The in vitro cytotoxicity results showed that these derivatives exhibited potent inhibitory effects on cancer cell growth, and the primary structure-activity relationships were evaluated. Notably, compound 3f exhibited satisfactory anticancer activity against all three cancer cell lines, especially the HEL cell line, with the IC50 value of 0.23 µM. Further research showed that 3f could induce G1/S cycle arrest and apoptosis in a dose- and time- dependent manner on the leukemia cell line HEL. The results suggested that 3f may be used as a potential anti-cancer agent for human leukemia.

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