scholarly journals Photocytotoxic effect of c60 fullerene against l1210 leukemic cells is accomPanied by enhanced nitric oxide Production and p38 maPk activation

2016 ◽  
Vol 38 (2) ◽  
pp. 89-93 ◽  
Author(s):  
D V Franskevych ◽  
I I Grynyuk ◽  
S V Prylutska ◽  
G V Pasichnyk ◽  
D M Petukhov ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Cycle ◽  
P38 Mapk ◽  
No Synthase ◽  
Leukemic Cells ◽  
C60 Fullerene ◽  
Cell Cycle Distribution ◽  
Nitrite Level ◽  
Mapk Activity ◽  
L1210 Cells

Aim: To estimate the combined action of C60 fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO) generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. Methods: Cell viability was assessed by MTT test. Light-emitting diode lamp (λ = 410–700 nm, 2.45 J/cm2) was used for C60 fullerene photoexcitation. Nitrite level and NO-synthase activity were measured by Griess reaction and by conversion of L-arginine to L-citrulline, respectively. p38 MAPK activity was assessed by Western blot analysis. Cell cycle distribution was determined by flow cytometry. Results: It was shown that light irradiation of C60 fullerene-treated L1210 cells was accompanied by 55% decrease of their viability at 48 h of culture. Nitrite level measured as an index of reactive NO generation was increased at the early period after C60 fullerene photoexcitation due to activation of both constitutive and inducible NO-synthase isoforms. The simultaneous activation of p38 MAPK was detected. Accumulation of L1210 cells in sub-G1 phase of cell cycle was observed after C60 fullerene photoexcitation. Conclusion: Photoexcited C60 fullerene exerts cytotoxic effect, at least in part, through triggering production of reactive NO species and activation of p38 kinase apoptotic pathways in L1210 leukemic cells.

scholarly journals Mode of photoexcited C60 fullerene involvement in potentiating cisplatin toxicity against drug-resistant L1210 cells

Bioimpacts ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 211-217
Author(s):  
Daria Franskevych ◽  
Svitlana Prylutska ◽  
Iryna Grynyuk ◽  
Ganna Pasichnyk ◽  
Liudmyla Drobot ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Molecular Mechanisms ◽  
Combined Treatment ◽  
Leukemic Cells ◽  
C60 Fullerene ◽  
Colloid Solution ◽  
Cell Cycle Distribution

Introduction: C60 fullerene has received great attention as a candidate for biomedical applications. Due to unique structure and properties, C60 fullerene nanoparticles are supposed to be useful in drug delivery, photodynamic therapy (PDT) of cancer, and reversion of tumor cells’ multidrug resistance. The aim of this study was to elucidate the possible molecular mechanisms involved in photoexcited C60 fullerene-dependent enhancement of cisplatin toxicity against leukemic cells resistant to cisplatin.Methods: Stable homogeneous pristine C60 fullerene aqueous colloid solution (10-4 М, purity 99.5%) was used in the study. The photoactivation of C60 fullerene accumulated by L1210R cells was done by irradiation in microplates with light-emitting diode lamp (420-700 nm light, 100 mW·cm-2). Cells were further incubated with the addition of Cis-Pt to a final concentration of 1 μg/mL. Activation of p38 MAPK was visualized by Western blot analysis. Flow cytometry was used for the estimation of cells distribution on cell cycle. Mitochondrial membrane potential (Δψm) was estimated with the use of fluorescent potential-sensitive probe TMRE (Tetramethylrhodamine Ethyl Ester). Results: Cis-Pt applied alone at 1 μg/mL concentration failed to affect mitochondrial membrane potential in L1210R cells or cell cycle distribution as compared with untreated cells. Activation of ROS-sensitive proapoptotic p38 kinase and enhanced content of cells in subG1 phase were detected after irradiation of L1210R cells treated with 10-5M C60 fullerene. Combined treatment with photoexcited C60 fullerene and Cis-Pt was followed by the dissipation of Δψm at early-term period, blockage of cell transition into S phase, and considerable accumulation of cells in proapoptotic subG1 phase at prolonged incubation.Conclusion: The effect of the synergic cytotoxic activity of both agents allowed to suppose that photoexcited C60 fullerene promoted Cis-Pt accumulation in leukemic cells resistant to Cis-Pt. The data obtained could be useful for the development of new approaches to overcome drug-resistance of leukemic cells.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 210-215 ◽  
Author(s):  
Mona Diab-Assaf ◽  
Josiane Semaan ◽  
Marwan El-Sabban ◽  
Soad K. Al Jaouni ◽  
Rania Azar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Dependent Manner ◽  
Apoptosis Induction ◽  
Inhibition Of Proliferation ◽  
Human T Cell

Introduction: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. Objective: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Materials and Methods: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. Results: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Conclusion: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

p38 MAPK-dependent eNOS upregulation is critical for 17β-estradiol-mediated cardioprotection following trauma-hemorrhage

2008 ◽  
Vol 294 (6) ◽  
pp. H2627-H2636 ◽  
Author(s):  
Wen-Hong Kan ◽  
Jun-Te Hsu ◽  
Zheng-Feng Ba ◽  
Martin G. Schwacha ◽  
Jianguo Chen ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Cardiac Tissue ◽  
Tissue Injury ◽  
No Synthase ◽  
Myeloperoxidase Activity ◽  
Sprague Dawley ◽  
Enos Expression ◽  
Mapk Activity ◽  
17Β Estradiol ◽  
Synthase Inhibitor

Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17β-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35–40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 μg/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at Ser1177, and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-α), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-1, which was reversed by l-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and l-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation.

scholarly journals The Nitric Oxide-Generating Prodrug Js-K Reverses the Multi-Drug Resistance Phenotype in AML Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5127-5127
Author(s):  
Stephan Morys ◽  
Catherine Gannage ◽  
Moises Terrazas ◽  
Imit Kaur ◽  
Ken M Kosak ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Cycle ◽  
Drug Resistance ◽  
Efflux Pump ◽  
Leukemic Cells ◽  
Multi Drug Resistance ◽  
Variable Expression ◽  
The Difference

Abstract Nitric oxide (NO) is toxic to acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolates class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic®P123 polymers. A major cause of treatment failure in AML is the multi-drug resistance (MDR) phenotype associated with overexpression of the P-glycoprotein (Pgp) by leukemic cells. Here, we investigated the effect of JS-K and its formulation on the MDR phenotype in AML cells using HL-60 cells and HL-60/RV+ cells as a model. HL-60/RV+ are selected from the parent HL-60 line and express the Pgp. They are maintained in culture under vincristine (VCR) selection. We studied the effect of the formulation alone (P123), JS-K without the formulation (free JS-K), and JS-K in the formulation (P123/JS-K). Both VCR and free JS-K were cytotoxic towards HL-60 cells with 50% inhibitory concentrations (IC50) of around 0.4 and 0.1 μM, respectively. By contrast, the IC50 for VCR-treated HL-60/RV+ cells, was greater than 1 μM. Free JS-K was cytotoxic to HL-60/RV+ with an IC50 of around 0.2 μM. Pretreatment of HL-60/RV+ cells for 2 hours with free JS-K (0.1 μM), P123/JS-K (0.1 μM), or an equivalent volume of P123 sensitized HL-60/RV+ cells to the effect of VCR: after 3 days of culture, the VCR IC50went from 1.5 μM in control cells to an estimated 1, 0.9, and 0.75 μM for P123, free JS-K, and P123/JS-K pre-treated cells, respectively. We then conducted cell cycle analyses of HL-60/RV+ cells treated with different combinations of JS-K and VCR using propidium idodide staining. Cells were treated with combinations of VCR (1.75 μM), free JS-K (0.1 μM), P123/JS-K (0.1 μM), or an equivalent volume of P123. After 24 hours, the observed percentage (average of 3 different repeats per variable) of the sub-G0/G1 fraction was 7, 6,14, 14, 25, 26, and 43 for untreated controls, free JS-K, P123/JS-K, VCR, free JS-K + VCR, P123 + VCR, and P123/JS-K + VCR, respectively. At 48 hours, the sub-G0/G1 fraction was 4, 5, 5, 19, 20, 23, and 28% for the same variables, respectively. At 72 hours, the sub-G0/G1fraction was 3, 3, 3, 11, 12, 19, and 19% for the same variables, respectively. Thus, the peak effect on the cell cycle was observed at 24 hours. We also observed an effect of P123 alone. In order to determine whether JS-K affects the efflux pump itself, we investigated the effect of 0.1 μM free JS-K, 0.1 μM P123/JS-K, or an equivalent volume of P123 on Rho-123 accumulation in HL-60/RV+ cells after 6 hours of treatment. As a percent of untreated controls, Rho-123 accumulation (average of 9 repeats) was 143, 159, and 144 for free JS-K, P123, and P123/JS-K, respectively (P < 0.05 for all differences with untreated controls). Using flow cytometry, we also determined whether JS-K affects the expression of the Pgp by HL-60/RV+ cells treated with 0.1 μM free JS-K, 0.1 μM P123/JS-K, or an equivalent volume of P123. After 24 hours, the observed (average of 6 different repeats per variable) expression of Pgp as a percent of untreated controls was 90, 79, and 63, for P123, free JS-K, and P123/JS-K, respectively (P < 0.05 for the difference between P123/JS-K and untreated controls). After 48 hours, the observed (average of 6 different repeats per variable) expression of Pgp as a percent of untreated controls was 83, 79, and 57, for the same variables, respectively (P < 0.05 for the difference between P123/JS-K and untreated controls). After 72 hours, there were no significant differences in the expression of Pgp between the different treatments and untreated controls. We conclude that JS-K in a P123 Pluronic® formulation can reverse the MDR phenotype. The Pluronic® polymers themselves could also affect the MDR phenotype. As such, P123/JS-K could constitute a major addition to our armamentarium for the treatment of AML. Disclosures Shami: JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Antibacterial and Cytotoxic Activity of Compounds Isolated fromFlourensia oolepis

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Mariana Belén Joray ◽  
Lucas Daniel Trucco ◽  
María Laura González ◽  
Georgina Natalia Díaz Napal ◽  
Sara María Palacios ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cytotoxic Activity ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Multidrug Resistant ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Cycle Arrest ◽  
M Phase ◽  
Lower Effect

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract ofFlourensia oolepiswere evaluated. Bioguided fractionation led to five flavonoids, identified as 2′,4′-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4′-dihydroxy-3′-methoxyflavanone (5). Compound1showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds1–5was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound1induced a remarkable cytotoxic activity toward ALL cells (IC50= 6.6–9.9 μM) and a lower effect against CML cells (IC50= 27.5–30.0 μM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment,1induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites ofF. oolepisextract, with1being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

Bone Marrow Mesenchymal Stromal Cells Regulate the Metabolism of H2O2 In Human Leukemic Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1058-1058
Author(s):  
Christine Vignon ◽  
Marie-Thérèse Georget ◽  
Yves Levern ◽  
Elfi Ducrocq ◽  
Marie-Christine Bernard ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Leukemic Cells ◽  
Penicillin G ◽  
Adherent Cells ◽  
Alexa Fluor ◽  
Human Leukemic Cells ◽  
H2o2 Level

Abstract Abstract 1058 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic cells and it has been recently established by the Guy Sauvageau's group (Institute for Research in Immunology and Cancer, Montréal, QC) that glutathione peroxydase 3 (GPx-3) promotes competitiveness of Hoxa9-Meis1 induced leukemic stem cells in which Gpx3 overexpression is concomitant of a decrease in H2O2 level and inactivation of p38 MAPK (Herault O et al, ASH annual meeting, 2010 - submitted). Leukemic cells located in the bone marrow (BM) are interacting with a microenvironment which plays a crucial role in the development and progression of leukemia. Mesenchymal stromal cells (MSCs) constitute a population of multipotential cells giving rise to the different hematopoietic microenvironmental cells (adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells). The aim of our study was to evaluate the effects of MSC-contact on the GPx-3/H2O2/p38 MAPK axis in human leukemic cells and to assess the cell cycle changes associated with the modification of H2O2 metabolism. BM MSCs were obtained from informed and consenting patients undergoing orthopedic surgery, following a procedure approved by the local ethical committee. Nucleated cells harvested from the iliac crest were seeded (5.104 cells per cm2) in α-MEM supplemented with 10% fetal calf serum (FCS), 20 mmol/l L-glutamine, and 100 units/mL penicillin G. Cells were incubated at 37°C in 95% humidified air and 5% CO2. When fully confluent, the layer of adherent cells was trypsinized (0.25% trypsin-EDTA), and the cells were resuspended in culture medium and seeded at 103 cells per cm2 (passage 1 - P1). BM MSCs were used at P2 in all experiments. The three-lineage mesenchymal differentiation of the BM MSCs used was systematically checked by culturing cells in adipogenic, chondrogenic, and osteogenic induction media as previously described (Delorme et al, Blood 2008, 111:2631). The human KG1a leukemic cell line (FAB M0/M1 CD34+ leukemic cells) was cultured in RPMI 1640 with 20 mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100 mg/mL streptomycin. KG1a cells were seeded at 1.5 105 cells/cm2 and cocultured with MSCs for 72 h. We have defined two distinct localizations of leukemic cells relative to MSC layer: those in supernatant (non-adherent cells) and cells adhering on the surface of MSCs. The expression of antioxydative enzymes, H2O2 level, p38 MAPK activation (T180/Y182), cell cycle, proliferation and immunophenotype of these two cell fractions were evaluated at day 0 and day 3. The expression of SOD1, SOD2, SOD3, CAT, TXN, TXN2, GLRX, GLRX2, GLRX3, GLRX5, PRDX, PRDX2, PDRX3, PRDX4, PRDX5, PRDX6, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 and GSR antioxydative genes and CDKN1A (p21CIP1) gene was quantified by qRT-PCR (Universal ProbeLibrary, Roche). SDS-PAGE and western-blot experiments were realized to quantify GPx-3 expression and p38 MAPK activation. Flow cytometry studies were performed: (a) to quantify H2O2 level by dichlorodihydrofluorescein diacetate (DCF-DA) staining; (b) to analyze the cell cycle by staining with 7-aminoactinomycin D (7AAD), Alexa Fluor®488-conjugated anti-human Ki67 and Alexa Fluor®488-conjugated anti-phospho-histone H3 (ser10) antibodies; (c) to track the cell divisions with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Supernatant of MSCs did not modify the GPx-3/H2O2/p38 MAPK axis in KG1a cells. Conversely, when compared with cells in the supernatant of MSCs, adhering KG1a cells were characterized by the exclusive overexpression of GPX3 antioxydative gene, the induction of GPx-3 production, a major decrease in H2O2 concentration and the inactivation of p38 MAPK. These effects were concomitant of cell cycle inhibition: increase in G0 phase, decrease in S and M phase, overexpression of CDKN1A and reduced mitotic activity (CFSE). Altogether these findings suggest that the bone marrow microenvironment plays a key role in the regulation of the oxidative metabolism of leukemic cells by promoting the inhibition of the H2O2/p38 MAPK axis via the induction of GPx-3. Modulation of the GPx-3/H2O2/p38 MAPK pathway by targeting of microenvironmental interactions in leukemia may have clinical relevance and it will be important to verify if these results can be extended in vivo to other models of human leukemias. Disclosures: No relevant conflicts of interest to declare.

Silencing of TEL/AML1 In Definitive Leukemic Cells Does Not Impair Cell Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3229-3229
Author(s):  
Marketa Zaliova ◽  
Jozef Madzo ◽  
Gunnar Cario ◽  
Jan Trka
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Fusion Gene ◽  
Global Gene Expression ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Wild Type ◽  
Egfp Reporter

Abstract Abstract 3229 The most frequent structural chromosomal aberration in childhood acute lymphoblastic leukemia t(12;21) generates TEL/AML1 fusion gene. Resulting TEL/AML1 protein probably acts as an aberrant transcription factor that deregulates AML1-dependent transcription but its target genes and thus also the exact role in leukemic cells remain unknown. In vivo studies showed that TEL/AML1 itself is not sufficient to cause leukemia but may induce a preleukemic state characterized by the increased numbers of multipotent or B-cell progenitors with an incomplete block of differentiation. Despite its role for leukemia establishment the relevance of TEL/AML1 fusion gene for leukemia persistance has not been studied enough.To address this question and to explore the possibility of TEL/AML1-targeted therapy, we studied the effects of RNAi-mediated TEL/AML1 silencing on leukemic cells. As the only siRNA used for TEL/AML1 silencing published so far (Diakos et al, Blood, 2007) targets also the wild type AML1 (46% transcript reduction, our data), our first goal was to identify efficient and TEL/AML1-specific siRNA. We designed eleven different siRNAs spanning the fusion point of TEL/AML1 lacking the total sequence homology to wild type TEL and AML1 alleles to avoid their silencing. These 11 siRNAs were tested in HeLa cells transgenic for TEL/AML1-ires2-EGFP reporter. After lipofection into HeLa cells the efficiency of individual siRNAs was measured as a decrease of EGFP reporter fluorescence by flow cytometry. The best five siRNAs, that induced 50–58% silencing of the EGFP reporter, were tested at the mRNA level in TEL/AML-positive leukemic cell line. 24h after electroporation of siRNAs, when the silencing reached its maximum, two most efficient siRNAs induced 58% and 57% TEL/AML1 transcript reduction, respectively. We achieved 61% TEL/AML1 transcript reduction with the pool of both siRNAs while there was only slight reduction (14%) of wild type AML1 transcript. We used this efficient and specific siRNA pool to silence TEL/AML1 in REH and UOC-B6 TEL-AML1 positive cell lines and studied its effect on cell viability, proliferation and global gene expression. Applying two rounds of siRNA electroporation within 48 hours interval we achieved 74% and 86% TEL/AML1 protein knockdown in REH and UOC-B6 cells, respectively. We used trypan blue staining followed by optical microscopy to monitor cell viability and staining with annexin V and propidium idode to assess apoptosis rate by flow cytometry. Analysis of DNA content using staining with propidium iodide was performed to assess cell-cycle distribution. Incorporation of nucleoside analog was measured by flow cytometry to analyse de novo DNA synthesis as an indicator of proliferation rate. Despite the common expectation derived from studies on other fusion oncogenes (BCR/ABL, AML1/ETO, E2A/PBX), TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, TEL/AML1 depletion was accompanied by the slight but significant increase in the fraction of S-phase cells and corresponding rise in proliferation rate. Opposite effects on cell cycle distribution and proliferation were induced when we silenced wild type AML1. These findings support our hypothesis that TEL/AML1 may block previously established AML1 function in G1/S progression through the cell cycle. In line with the lack of effect on cell viability and discreet effect on cell-cycle distribution and proliferation we found no significant changes in global gene expression pattern upon TEL/AML1 depletion. Our data indicate, that TEL/AML1 is dispensable for the survival of definitive leukemic cells. This work was supported by grants MSM0021620813 and MZOFNM2005. Disclosures: No relevant conflicts of interest to declare.

Enforced Expression Of MiR-24, MiR-126 and MiR-365 Does Not Alter Cellular Viability In TCF3-Rearranged Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2630-2630
Author(s):  
Farhad Akbari Moqadam ◽  
Judith M. Boer ◽  
Ellen Lange-Turenhout ◽  
Rob Pieters ◽  
Monique L. Den Boer
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Leukemic Cell ◽  
Expression Level ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Expression Levels ◽  
Leukemic Cell Lines ◽  
Candidate Target

Abstract MicroRNAs (miRNAs) are involved in fine-tuning of the several cellular processes in human healthy and malignant tissues. In various types of tumors, miR-24, miR-126 and miR-365 were shown to regulate cell cycle progression and apoptosis. Interestingly, these three miRNAs were downregulated in pediatric TCF3-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) (Schotte et al. Haematologica 2011). Here, we investigated whether these three miRNAs, individually or in combination, may alter cell cycle distribution and amount of apoptosis in TCF3-rearranged leukemia. In addition, we used a new miRNA target identification method (van Iterson et al. Nucleic Acid Res 2013) which integrates the mRNA and miRNA expression profiles of 37 cases with childhood BCP-ALL (4 BCR-ABL1-positive, 3 TCF3-rearranged, 7 ETV6-RUNX1-positive, 13 hyperdiploid (>50 chromosomes) and 10 unclassified B-others negative for the above mentioned genetic aberrations). Following overexpression of miRNAs in TCF3-rearranged leukemic cells, changes in the expression levels of predicted candidate target genes in our miRNA-mRNA integration cohort were analyzed to discover functional relationships between these miRNAs and their predicted candidate target genes in BCP-ALL. MiRNAs were over-expressed in three TCF3-rearranged leukemic cells; i.e. 697, KASUMI-2 and MHH-CALL-3 cells, using miRNA precursors transfection. Expression level of mature miRNAs and candidate target genes were analyzed by qRT-PCR. The cell cycle distribution and the amount of leukemic cells in apoptosis were determined by flow cytometry of propidium iodide stained nuclei and Annexin V-propidium iodide stained cells, respectively. Our data revealed that the expression level of mature miR-24, miR-126 and miR-365 was raised >100-fold upon precursor miRNA transduction compared to basal expression levels of Three TCF3-rearranged leukemic cell lines. Individual or combined overexpression of miR-24, miR-126 and miR-365 did not alter cell cycle progression and amount of apoptosis in these three different leukemic cell lines. Integration of miRNA and mRNA expression levels of 37 newly diagnosed children with BCP-ALL revealed significant association between the expression of 29 miRNAs and their predicted target genes (FDR<0.05, p<0.005), including miR-24 (ranked at position #5), miR-126 (ranked at position #17) and miR-365 (ranked at position #20). Candidate target genes were selected for further analysis: ELL, EBF3 and IRF4 for miR-24 (Pearson r <-0.6, p<0.005), PITPNC1 for miR-126 (Pearson r <-0.5, p<0.01) and ZAP-70 for miR-365 (Pearson r <-0.4, p<0.01). However, miRNAs overexpression in MHH-CALL-3 cells did not reduce the expression levels of these selected candidate target genes. Considering the presence of only 3 patients with TCF3-rearranged ALL in our miRNA-mRNA expression data integration cohort, the observed significant inverse correlation between miRNA-mRNA pairs might mainly originate from other subtypes with higher number of cases in this cohort, such as hyperdiploid (13 cases) or unclassified B-others BCP-ALL (10 cases). Interestingly, expression level of AURKB – a validated target for miR-24 – was reduced by ∼4-fold upon miR-24 overexpression in hepatocarcinoma HEP-G2 cells, while overexpression of similar amount of miR-24 cannot alter AURKB expression levels in MHH-CALL-3 leukemic cells. Together, our findings indicate that individual and combined expression of miR-24, miR-126 and/or miR-365 does not affect the amount of apoptosis and cell cycle distribution in TCF3-rearranged leukemia. Moreover, our data suggest that miRNAs function is highly cell type-dependent and a defined biological target gene or function of one miRNA in a specific cellular context cannot be generalized for all types of cells/tissues. Disclosures: No relevant conflicts of interest to declare.

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