T-cell Leukemia Cell Line Jurkat Research Articles

Nicotinamidephosphoribosyltransferase (NAMPT) Induces Ubiquitination of LEF-1 Transcription Factor By Deacetylation

We and others demonstrated, that transcription factor lymphoid enhancer-binding factor 1 (LEF-1) is crucial for neutrophil granulocytopoiesis and lymphopoiesis. LEF-1 expression is severely diminished in myeloid progenitors of severe congenital neutropenia (CN) patients, leading to "maturation arrest" of granulopoiesis.In the present study we evaluated the mechanisms of LEF-1 regulation. Previously we identified elevated levels of the enzyme Nicotinamide phosphoribosyltransferase (NAMPT) and its target Sirtuin 1 (SIRT1) in myeloid cells of CN patients. SIRT1 is NAD+-dependent protein deacetylase and in this study we analyzed whether LEF-1 could be regulated by NAMPT/SIRT1-triggered deacetylation. We identified lysine residue in LEF-1 protein, which could be de-/acetylated, generated specific rabbit polyclonal antibody recognizing this acetyl-Lys and confirmed NAMPT/SIRT1-triggered deacetylation of LEF-1 protein on this lysine in T-cell leukemia cell line Jurkat and in AML cell line LW/SO.We first studied the effects of LEF-1 deacetylation by NAMPT/SIRT1 on its expression and functions. We found markedly diminished levels of total LEF-1 protein as well as its nuclear translocation in Jurkat and LW/SO cells treated with NAMPT. At the same time, inhibition of NAMPT using specific NAMPT inhibitor FK866 resulted in elevated mRNA and protein levels of LEF-1 and its target genes survivin, c-myc and cyclin D1.We also performed reporter gene assay using WT or acetyl-Lys LEF-1 mutant and TOP promoter construct, containing six LEF-1/TCFs binding sites. Acetyl-Lys LEF-1 mutant was not able to activate TOP promoter, as compared to its strong activation by WT LEF-1. Moreover, co-transfection with NAMPT- or SIRT1-expression constructs also showed inhibitory effects on LEF-1-triggered activation of TOP promoter. These experiments clearly demonstrated that NAMPT/SIRT1-triggered deacetylation of the LEF-1 protein inhibited its expression and functions.We further evaluated the mechanism of NAMPT-triggered reduction of LEF-1 expression. In silico analysis of LEF-1 protein using CPLM software revealed that lysine residue in LEF-1 protein, which is de-/acetylated, could be also ubiquitinated. We therefore studied the effects of deacetylation on the ubiquitination of LEF-1 protein in Jurkat cells. We evaluated the effects of 26S proteasome inhibitor, bortezomib, on LEF-1 protein levels after treatment with NAMPT. Indeed, upon treatment with bortezomib the level of LEF-1 protein was restored. This confirmed, that deacetylation of LEF-1 by NAMPT led to degradation of LEF-1 by ubiquitination. We assumed, that in CN patients, hyperactivation of NAMPT/SIRT1 pathway might lead to downregulation of LEF-1 by deacetylation and ubiquitination. Indeed, we observed decreased G-CSF-triggered granulocytic differentiation and proliferation of CD34+ cells from healthy individuals transduced with LEF-1 acetyl-lysine mutant, as compared to cells transduced with WT LEF-1. Moreover, treatment of CD34+ cells of CN patients with bortezomib resulted in restoration of defective LEF-1 expression ultimately leading to normalization of granulocytic differentiation.In conclusion, NAMPT/SIRT1-triggered deacetylation of LEF-1 resulted in its degradation by ubiquitination. DisclosuresNo relevant conflicts of interest to declare.

Characterization of the apoptotic response induced by the cyanine dye D112: a potentially selective anti-cancer compound.

Chemotherapeutic drugs that are used in anti-cancer treatments often cause the death of both cancerous and noncancerous cells. This non-selective toxicity is the root cause of untoward side effects that limits the effectiveness of therapy. In order to improve chemotherapeutic options for cancer patients, there is a need to identify novel compounds with higher discrimination for cancer cells. In the past, methine dyes that increase the sensitivity of photographic emulsions have been investigated for anti-cancer properties. In the 1970's, Kodak Laboratories initiated a screen of approximately 7000 dye structural variants for selective toxicity. Among these, D112 was identified as a promising compound with elevated toxicity against a colon cancer cell line in comparison to a non-transformed cell line. Despite these results changing industry priorities led to a halt in further studies on D112. We decided to revive investigations on D112 and have further characterized D112-induced cellular toxicity. We identified that in response to D112 treatment, the T-cell leukemia cell line Jurkat showed caspase activation, mitochondrial depolarization, and phosphatidylserine externalization, all of which are hallmarks of apoptosis. Chemical inhibition of caspase enzymatic activity and blockade of the mitochondrial pathway through Bcl-2 expression inhibited D112-induced apoptosis. At lower concentrations, D112 induced growth arrest. To gain insight into the molecular mechanism of D112 induced mitochondrial dysfunction, we analyzed the intracellular localization of D112, and found that D112 associated with mitochondria. Interestingly, in the cell lines that we tested, D112 showed increased toxicity toward transformed versus non-transformed cells. Results from this work identify D112 as a potentially interesting molecule warranting further investigation.

Microfluidic impedance spectroscopy as a tool for quantitative biology and biotechnology.

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.

Combined treatment with fenretinide and indomethacin induces AIF-mediated, non-classical cell death in human acute T-cell leukemia Jurkat cells

Currently used cytotoxic drugs in cancer therapy have a similar mechanism of action and low specificity. Applied simultaneously, they show an additive effect with strong side effects. Clinical trials with the use of different agents in cancer therapy show that the use of these compounds alone is not very effective in fighting cancer. An alternative solution could be to apply a combination of these agents, because their combination has a synergistic effect on some cancer cells. Therefore, in our investigations we examined the effects of a synthetic retinoid-fenretinide when combined with a non-steroidal anti-inflammatory drug–indomethacin on the process of apoptosis in the acute human T-cell leukemia cell line Jurkat. We demonstrate that treatment with the combination of the tested compounds induces the death of cells, that is peculiar and combines features of apoptosis as well as non-apoptotic cell death. In detail we observed, cell membrane permeabilization, phosphatydylserine exposure, no oligonucleosomal DNA fragmentation, no caspase-3 activation, but apoptosis inducing factor (AIF) nuclear translocation. Taken together these results indicate, that Jurkat cells after treatment with a combination of fenretinide and indomethacin undergo AIF-mediated programmed cell death.

Effects of NVP-BSK805, a Novel JAK2 Inhibitor, on BCR-ABL and JAK2V617F Positive Cell Lines

Abstract 5002 Background:Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. The V617F mutation in JAK2 occurs in 95% of patients with polycythemia vera, 50% of those with essential thrombocythemia and 50% of primary myelofibrosis patients. Preclinical results strongly support that JAK2 inhibitors could be effectively used in these three indications. Replacement of valine 617 with phenylalanine upregulates the tyrosine kinase activity of JAK2, causing constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapy strategies for CMPNs. In this study the role of the novel JAK2 inhibitor, NVP-BSK805 (Novartis Pharmaceuticals), has been investigated in cells expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed. Methods:The in vitro activity of NVP-BSK805 was analyzed in 12 hematopoietic cell lines, including 7 BCR-ABL positive (K562, KCL22, KU812, Lama87, BV173, EM3, SUP-B15), 4 JAK2 mutated (CHRF288, SET2, UKE1, HEL), the T-cell leukemia cell line Jurkat, and the neuroendocrine colonic tumour line LCC-18. Concentration kinetics from 0 up to 25 μM were established using XTT proliferation assays and flow cytometry for measuring apoptosis. Protein levels of JAK2, phospho-JAK2, STAT5, phospho-STAT5 and BCR-ABL were analyzed using Western blotting. NVP-BSK805 was also tested in combination with imatinib and nilotinib. JAK2 was sequenced in all cell lines in order to detect possible mutations in the gene. Results:Of the JAK2 mutated cell lines tested, 3 of 4 (CHRF288, SET2, UKE1) showed a significant reduction of proliferation, as well as viability, compared to the other cell lines. CHRF288 responded best to NVP-BSK805 with an IC50 value of 0.22 ± 0.04 μM. UKE1 and SET2 had similar values of 0.35 ± 0.03 μM and 0.37 ± 0.05 μM. Interestingly, HEL (V617F positive) cells showed only an IC50 value (1.8 ± 0.17 μM) for NVP-BSK805, comparable with that of the non-mutated BCR-ABL positive cell lines (1.5 to 2.7 μM). LCC-18 showed the weakest response of all cell lines tested, with an IC50 value of 9.93 ± 0.202 μM. Each cell line responded to concentrations higher than 5 μM with a strong reduction of proliferation due to inhibition of various kinases. Combination of the JAK2 inhibitor with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Western blotting of proteins of the JAK-STAT pathway confirmed the results of the proliferation and apoptosis tests showing a strong reduction of phoshorylated STAT5 in CHRF288 cells after a 30 min incubation even with NVP-BSK805 concentrations as low as 0.01 μM. UKE-1 and SET-2 showed reduction of pSTAT5 from 0.1 μM. Levels of total STAT5 were not affected. In all the other cell lines no changes were detected in any of the proteins tested. Conclusions:Here, we tested a novel JAK2 inhibitor in cells carrying the V617F mutation. Interestingly, not every cell line with the JAK2 V617F mutation showed a good response upon JAK2 inhibition, indicating that there are additional factors determining response. On the other hand, clinical trials with JAK inhibitors in myelofibrosis have shown responses in V617F-mutated and non-mutated patients, warranting further research to identify predictors of response. In BCR-ABL mutant cells not harbouring JAK2 mutations no significant inhibition of proliferation or apoptosis was detected following JAK2 inhibition, indicating that there are JAK2 independent signal transduction pathways of BCR-ABL to avoid apoptosis. Disclosures:le Coutre:Novartis Pharmaceuticals: Honoraria, Research Funding.

The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 μM caused accumulation of Jurkat cells in the G 1 cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

Oridonin, An Ent-Kaurane Diterpenoid, Shows Antileukemic Effect and Enhances the Effect of Indarubicin on Human T-Cell Leukemia Cell Line

AbstractAbstract 4933 IntroductionAcute lymphoblastic leukemia (ALL) is the most common type of leukemia in young children, and also affects adults, especially those aged 65 years or older. Although the outcome of children with ALL has been improved dramatically with current chemotherapy strategy, adult patients still have a worse outcome and the incidence of relapse remains high either in children or adult patients. Furthermore, there has been very little progress in improving the outcome of patients who experience relapse. Oridonin, an ent-kaurane diterpenoid derived from the herbal Rabdosia rubescens, has been proved to have significant antitumor effect on a large variety of cancer cells. In this study, we aimed to assess whether oridonin had antileukemic effection and the possibility of combination effect with indarubicin (IDA), an important component commonly used in T-ALL induction regimen. MethodsHuman T-cell leukemia cell line Jurkat cells in culture medium were treated with different concentrations of oridonin (1umol/L~ 20umol/L) for 24h and 48h. Cell Counting Kit-8 (CCK8) assay was used to detect the cell growth inhibitory rate. Apoptosis was measured with flow cytometric (FCM) by staining cells with Annexin V-fluorescein-isothiocyanate(FITC) and propidium iodide (PI). Hoechst 33258 staining was used to observe the apoptotic morphology. The expression of caspase-3, caspase-8 and other apoptosis modulators, including Bcl-2 family members, was analyzed by western blotting. When the combination effect of oridonin and indarubicin was investigated, oridonin was given one hour before indarubicin. The calculation of combined effect was used Isobologram Analysis. ResultsCCK8 assay revealed that oridonin could significantly inhibit the growth of Jurkat cells in vitro. The suppression was both time- and dose-dependent. The concentration of oridonin lower than 5 umol/L showed little inhibitory effect, but it presented significant inhibition of the proliferation at a higher concentration, especially when the concentration was higher than 10 umol/L. The 50% inhibitory concentration (IC50) values for 24h and 48h were 9.44 and 9.06 umol/L respectively. After Jurkat cells were treated with higher than 5 umol/L of oridonin for 24–48h., the percentage of Annexin VFITC positive cells was gradually increased in a time and dose-dependent manner and the percentage of Annexin VFITC positive cells could reach 40% to 50% after 10 umol/L of oridonin treatment for 48h. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed using Hoechst 33258 stain after 48h. Western blotting analysis showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 20-kDa subunit when apoptosis occurred; Expression of Bcl-2 was downregulated significantly after the cells were treated with oridonin for 24 h. The expression of caspase-8 remained constant before and after apoptosis occurred. We therefore concluded that oridonin has significant antiproliferation effects on Jurkat cells by activation of caspase-3 as well as down-regulation of anti-apoptotic protein Bcl-2. The 24h IC50 of IDA for Jurkat cells was 25.85nmol/L. When combined with 1umol/L of oridonin, the IC50 of IDA decreased to 4.93nmol/L. The combine index was < 1, indicating the synergistic antitumor effect of IDA and oridonin. The data from FCM showed that 2.5 umol/L of oridonin could augment the apoptosis effect of 2.5nmol/L of IDA from 5.1% to 12.7%. During the experiment, we found that oridonin given one hour before IDA could make a better combination effect than given simultaneously. We also discovered that low dose oridonin (lower than 5 umol/L) had little inhibitory effect on normal bone marrow mononuclear cells. ConclusionsThe results showed oridonin could effectively induce ALL cells apoptosis, and in low dose showed a significant synergistic effect with IDA, indicating oridonin may serve as a potential and secure antileukemia reagent for ALL, especially in the combination chemotherapy. The underlying machanisms of the combination effect with anthracycline should be explored. Disclosures:No relevant conflicts of interest to declare.

Antiproliferative effect of rapamycin on human T-cell leukemia cell line Jurkat by cell cycle arrest and telomerase inhibition

To examine the ability of rapamycin to suppress growth and regulate telomerase activity in the human T-cell leukemia cell line Jurkat. Cell proliferation was assessed after exposure to rapamycin by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were determined by flow cytometry. The proteins important for cell cycle progression and Akt/mammalian target of rapamycin signaling cascade were assessed by Western blotting. Telomerase activity was quantified by telomeric repeat amplication protocol assay. The human telomerase reverse transcriptase (hTERT) mRNA levels were determined by semi-quantitative RT-PCR. Rapamycin inhibited the proliferation of Jurkat, induced G1 phase arrest, unregulated the protein level of p21 as well as p27, and downregulated cyclinD3, phospho-p70s6k, and phospho-s6, but had no effect on apoptosis. Treatment with rapamycin reduced telomerase activity, and reduced hTERT mRNA and protein expression. Rapamycin displayed a potent antileukemic effect in the human Tcell leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through the suppression of telomerase activity, suggesting that rapamycin may have potential clinical implications in the treatment of some leukemias.

Cytoskeleton-mediated Death Receptor and Ligand Concentration in Lipid Rafts Forms Apoptosis-promoting Clusters in Cancer Chemotherapy

While investigating the mechanism of action of the novel antitumor drug Aplidin, we have discovered a potent and novel cell-killing mechanism that involves the formation of Fas/CD95-driven scaffolds in membrane raft clusters housing death receptors and apoptosis-related molecules. Fas, tumor necrosis factor-receptor 1, and tumor necrosis factor-related apoptosis-inducing ligand receptor 2/death receptor 5 were clustered into lipid rafts in leukemic Jurkat cells following Aplidin treatment, the presence of Fas being essential for apoptosis. Preformed membrane-bound Fas ligand (FasL) as well as downstream signaling molecules, including Fas-associated death domain-containing protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid, were also translocated into lipid rafts, connecting death receptor extrinsic and mitochondrial intrinsic apoptotic pathways. Blocking Fas/FasL interaction partially inhibited Aplidin-induced apoptosis. Aplidin was rapidly incorporated into membrane rafts, and drug uptake was inhibited by lipid raft disruption. Actin-linking proteins ezrin, moesin, RhoA, and RhoGDI were conveyed into Fas-enriched rafts in drug-treated leukemic cells. Disruption of lipid rafts and interference with actin cytoskeleton prevented Fas clustering and apoptosis. Thus, Aplidin-induced apoptosis involves Fas activation in both a FasL-independent way and, following Fas/FasL interaction, an autocrine way through the concentration of Fas, membrane-bound FasL, and signaling molecules in membrane rafts. These data indicate a major role of actin cytoskeleton in the formation of Fas caps and highlight the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in apoptosis, acting as concentrators of death receptors and downstream signaling molecules and as the linchpin from which a potent death signal is launched.

An Automated Method for the Analysis of T-Cell Receptor Repertoires: Rapid RT-PCR Fragment Length Analysis of the T-Cell Receptor β Chain Complementarity-Determining Region 3

The examination of T-cell receptor (TCR) repertoires has an important role in the study of lymphoproliferative disorders and autoimmune diseases. Analysis of the complementarity-determining region 3 (CDR3) of the TCR beta chain is used to assess the clonality of T-cell populations. We developed a rapid fluorescence-based method for CDR3 length analysis of expressed TCR gene families. TCR beta chain complementary DNA is amplified by a nested polymerase chain reaction with V beta family-specific oligonucleotide primers and a fluorochrome-labeled C beta primer. The polymerase chain reaction products were analyzed on a compact automated DNA sequencing system (OpenGene system, Visible Genetics, Toronto, Ontario). To demonstrate the usefulness of our technique, we examined the CDR3 length distribution of peripheral blood T cells from a healthy subject, intestinal T cells from a patient with ulcerative colitis, and the T-cell leukemia cell line Jurkat. The analysis revealed polyclonal, oligoclonal, and monoclonal CDR3 distributions, respectively, for the 3 T-cell populations. Our new method shows virtually identical CDR3 length patterns compared with the traditional radioisotope-based method. The new technique offers the convenience of rapid throughput, nonradioactive labeling, and quality data analysis.

CD7 false‐positive acute myelogenous leukemia and promyelocytic leukemia cell line HL‐60: Characterization of CD7 epitopes by four monoclonal antibodies

Phenotypes of cells from 12 patients with acute myelogenous leukemia (AML) were analysed by means of a fluorescence-activated cell sorter utilizing a panel of monoclonal antibodies (MAbs). A majority of the cells from peripheral blood coexpressed the antigens against MAbs CD11, CD13, and CD33 but did not express the antigens against CD1, CD3, CD4, CD5, CD8, CD19, CD20, CD21, CD41 and 42, and glycophorin A. Three out of the 12 cases expressed CD7 antigen. However, one of them showed no reaction with Tp40 MAb, whereas the others showed reaction with Leu9 and T55. The discrepancy of reactivities between Leu9 and Tp40 MAbs prompted us to study the promyelocytic leukemia cell line HL-60, which showed similar reactions against Leu9 and Tp40 MAbs. Leu9, OKT16, and T55 MAbs reacted strongly with HL60 cells, whereas Tp40 MAb, which reacted strongly with T-cell leukemia cell line Jurkat, showed no reaction. The reactivity of Leu9, OKT16, and T55 MAbs with HL-60 cells was completely inhibited after preincubation with aggregated human immunoglobulin G (AHIG), which clearly shows the existence of nonspecific binding between these 3 MAbs and HL-60 cells via Fc gamma R. On the basis of our experiments, we conclude that HL-60 cells bind nonspecifically with Leu9, OKT16, and T55 MAbs via FcRI, and this is suggestive that de novo AML cells probably behave in the same fashion. Hence, we recommend that the utilization of murine IgG2a and IgG3 MAbs should be avoided especially in cell surface analysis of myeloid leukemic cells.

Activation of protein kinase C inhibits prostaglandin- and potentiates adenosine receptor-stimulated accumulation of cyclic AMP in a human T-cell leukemia line

Accumulation of cAMP in the human T-cell leukemia cell line Jurkat was stimulated by the adenosine analogue 5′- N-ethylcarboxamidoadenosine (NECA) and by prostaglandin E 2, (PGE 2). Addition of two phorbol esters, PDiBu and TPA, markedly enhanced the NECA-stimulated accumulation of cAMP whereas the PGE 2-stimulated cAMP accumulation was substantially reduced. The non-tumor-promoting phorbol ester, 4α-PDD, had no effect on either NECA- or PGE 2-stimulated cAMP accumulation. The ability of PDiBu to inhibit the effect of PGE 2 and to stimulate the effect of NECA remained in the presence a low concentration of forskolin (0.3 μM), which per se increased both NECA- and PGE 2-stimulated cAMP accumulation. Our results suggest that the effect of PK-C-activating drugs on receptor-mediated cAMP accumulation is entirely dependent on which receptor is being stimulated