Jurkat E6-1 Cells Research Articles

Preliminary exploration of the expression of acetylcholinesterase in normal human T lymphocytes and leukemic Jurkat T cells.

The classic enzymatic function of acetylcholinesterase (AChE) is the hydrolysis of acetylcholine (ACh) in the neuronal synapse. However, AChE is also present in nonneuronal cells such as lymphocytes. Various studies have proposed the participation of AChE in the development of cancer. The ACHE gene produces three mRNAs (T, H and R). AChE-T encodes amphiphilic monomers, dimers, tetramers (G1 A, G2 A and G4 A) and hydrophilic tetramers (G4 H). AChE-H encodes amphiphilic monomers and dimers (G1 A and G2 A). AChE-R encodes a hydrophilic monomer (G1 H). The present study considered the differences in the mRNA expression (T, H and R) and protein levels of AChE, as well as the molecular forms of AChE, the glycosylation pattern and the enzymatic activity of AChE present in normal T lymphocytes and leukemic Jurkat E6-1 cells. The results revealed that AChE enzymatic activity was higher in normal T lymphocytes than in Jurkat cells. Normal T cells expressed AChE-H transcripts, whereas Jurkat cells expressed AChE-H and AChE-T. The molecular forms identified in normal T cells were G2 A (5.2 S) and G1 A (3.5 S), whereas those in Jurkat cells were G2 A (5.2 S), G1 A (3.5 S) and G4 H (10.6S). AChE in Jurkat cells showed altered posttranslational maturation since a decrease in the incorporation of galactose and sialic acid into its structure was observed. In conclusion, the content and composition of AChE were altered in Jurkat cells compared with those in normal T lymphocytes. The present study opened new avenues for exploring the development of novel therapeutic strategies against T-cell leukemia and for identifying potential molecular targets for the early detection of this type of cancer.

FIP-fve Stimulates Cell Proliferation and Enhances IL-2 Release by Activating MAP2K3/p38α (MAPK14) Signaling Pathway in Jurkat E6-1 Cells.

FIP-fve, a fungal fruiting body protein from Flammulina velutipes, has potential immunomodulatory properties. Here, we investigated the immunomodulation mechanism of FIP-fve in Jurkat E6-1 cells by conducting a cell viability assay and IL-2 release assay. Kinase inhibitors experiment and proteomics analysis were also involved in the mechanism study. It was found that FIP-fve stimulated cell proliferation and enhanced IL-2 secretion in a dose-dependent manner in Jurkat E6-1 cells. Unbiased high-throughput proteomics analysis showed that 4 T cell immune activation markers, including ZAP-70, CD69, CD82, and KIF23, were upregulated in response to FIP-fve treatment. Further pathway analysis indicated that MAP2K3/p38 pathway-related proteins, including MAP2K, p38, ELK, AATF, FOS, and JUN-B, were unregulated. In addition, losmapimod (p38 inhibitor) and gossypetin (MAP2K3 inhibitor) inhibited FIP-fve enhanced cell proliferation and IL-2 release in Jurkat E6-1 cells. Our results demonstrate that FIP-fve stimulates cell proliferation and enhances IL-2 secretion through MAP2K3/p38α activation.

Mechanism of Mycobacterium tuberculosis on interleukin-6 receptor 3'-untranslated region methylation in CD4+T cells

Objective: To investigate the role and mechanism of DNA methylation in Mycobacterium tuberculosis (MTB lysate) -induced downregulation of interleukin-6 receptor(IL-6R) expression in CD4+T cells. Methods: A prospective study was conducted. Bisulfite sequencing (BSP) was applied to determine the methylation levels of CpG island in IL-6R promoter region and 3'untranslated region (3'UTR) region in CD4+T cells from peripheral blood mononuclear cells (PBMC) of control group (healthy person, n=10) and TB group (tuberculosis patients, n=10) in Shenzhen Third People's Hospital between 2019 and 2020. Quantitative reverse transcription-PCR (RT-qPCR) and Western blotting were used to detect the expression of IL-6R, DNMT1, DNMT3A and DNMT3B in MTB lysate-stimulated CD4+T cells and Jurkat E6-1 cells. Furthermore, PBMC in control group and Jurkat E6-1 cells activated by anti-CD3/CD28 antibody were stimulated by MTB lysates to detect the methylation levels of CpG island and IL-6R and DNMT expression. Transcriptional activity of differently methylation regions of IL-6R 3'UTR was detected by using luciferase reporter gene system. Results: IL-6R expression in TB group was lower than that in control group, but DNMT1 and DNMT3B expressions were higher than those in control group in CD4+T cells isolated from PBMC. There was no significant difference in the methylation rate of IL-6R promoter CpG island of CD4+T cells between control and TB group. However, the methylation rates of CpG island in 3'UTR region were significantly higher (P<0.001) in TB (69.5%±3.4%), compared with control (54.3%±4.7%). Besides, IL-6R expression was lower than unstimulated, while DNMT1 and DNMT3B expression was higher than unstimulated after MTB lysate-stimulation of activated control PBMC in vitro. The methylation rate of CpG island in IL-6R 3'UTR region of CD4+T cells increased from 58.8%±11.6% to 79.4%±10.9% (P<0.001) after MTB lysate-stimulated PBMC of the control. The same results were observed in the MTB lysate-stimulated CD4+T cells isolated from PBMC in control and Jurkat E6-1 cell line. Furthermore, IL-6R expression after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysate was higher than that stimulated by MTB lysate alone. In addition, the methylation levels of CpG islands in the 3' UTR region of IL-6R were lower than those stimulated by MTB lysates alone after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysates. The transcriptional activity of the fully unmethylated IL-6R 3'UTR CpG island reporter gene was higher than that of the fully methylated IL-6R 3'UTR CpG island. Conclusions: MTB lysates stimulation inhibited IL-6R expression transcriptionalely as well as on the protein level by inducing hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells. The hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells induced by MTB may be related to the increased expression of DNMT1 and DNMT3B.

Increased Expression of Cytoskeleton Coordinator Protein MACF1 at the Immune Synapse during Jurkat T Cell Activation

Introduction: Activation of the T cell Receptor (TCR) by antigen on the surface of an Antigen Presenting Cell (APC) leads to a dramatic change in T cell morphology and formation of a specialized signaling interface with the APC known as the Immune Synapse (IS). The IS allows for controlled cytokine secretion and is an important regulator of T cell activation. IS formation has been found to be dysfunctional in many immune disorders including T cell acute lymphoblastic leukemia (ALL).The actin and microtubule (MT) cytoskeletons have individually been implicated in regulating both IS formation and TCR signaling, but the crosstalk between actin and MTs in T cells has not been well explored. We hypothesized that MACF1 (Microtubule Actin Crosslinking Factor 1), a giant cytoskeleton crosslinker from the spectraplakin family, is present at the IS following Jurkat T cell activation and coordinates actin and MTs. To investigate this, we used TIRFM (Total Internal Reflection Fluorescence Microscopy) and confocal microscopy to image MACF1 organization at the IS. Methods: Jurkat E6-1 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Glass coverslips were coated with Poly-L-Lysine 0.01% and then with 10 µg/mL anti-CD3 for 2 hours at 37 °C to create an activating surface. Cells were dropped carefully and allowed to spread on these coverslips at 37 °C for the indicated time. In the inhibition experiments,5 µM SB415286 or 5 µM PP2 was added to cells at 8 minutes following the initiation of activation and allowed to incubate for an additional 8 minutes. Cells were fixed with 3.7% paraformaldehyde following activation and then permeabilized with 0.1% Triton X-100. Cells were stained for anti-MACF1, anti-beta tubulin clone KMX-1, respective secondary antibodies, actin-stain 488 phalloidin, and DAPI (4',6-diamidino-2-phenylindole). Fluorescence of labeled cells was imaged using TIRFM with a Nikon Ti microscope and Andor Zyla CMOS camera with a 100x magnification objective (1.49 NA). TIRFM images were analyzed for Corrected Total Cell Fluorescence, which is the total fluorescence of the cell corrected for the background. Mann-Whitney U tests were used for all statistical comparisons. Cells were imaged on Leica SP5X laser confocal microscope with 63x objective for localization of MACF1 with actin and tubulin. Confocal images were deconvolved using the deconvolution lab2 plugin in FIJI (FIJI Is Just ImageJ). Results: Corrected Total Cell Fluorescence (CTCF) of MACF1 was higher at 4, 8 and 16 minutes following activation compared to the PLL control (Figures 1A and 1B). There was no significant difference in CTCF across the different activation times. To test whether MACF1 is dis-inhibited in Jurkat T cells during activation, we used SB415286 to inhibit Glycogen Synthase Kinase 3-Beta (GSK3B), a protein that inhibits MACF1 function, on PLL-only coverslips. We found MACF1 CTCF to be higher following SB415286 treatment compared to the control, indicating that MACF1 levels increase with GSK3B inhibition (Figure 1C). Thus, SB415286 inhibition of GSK3B mimics T cell activation where Akt upregulation downstream of TCR signaling inhibits GSK3B. To further test whether CD3 signaling activates MACF1, we stimulated Jurkat cells on anti-CD3 coated glass and then removed the activation signal with the src-kinase inhibitor PP2, which inhibits lymphocyte-specific protein tyrosine kinase (Lck) - an important regulator of early T cell signaling. MACF1 CTCF was lower after PP2 inhibition compared with the control, suggesting that continued CD3 stimulation is necessary for MACF1 accumulation at the IS (Figure 1D). We then used confocal microscopy to image the spatial distribution of MACF1 and assess whether it localizes with actin and MTs at the IS. We found that MACF1 puncta are present at the F-actin rich lamellipodial region of the IS and appear to localize with MT filaments, suggesting that MACF1 coordinates the two cytoskeleton components at the IS (Figure 2). Conclusion: Using high resolution microscopy, we show that MACF1 is upregulated at the IS during Jurkat T cell activation downstream of CD3 signaling through a GSK3B dependent pathway. At the IS, we found the novel localization of MACF1 with actin and MTs. This upregulation and expression of MACF1 during activation warrants more studies of the role MACF1 plays in T cell activation, cytoskeletal dynamics and IS function. Disclosures No relevant conflicts of interest to declare.

Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and HA-1 negative donors were expanded in vitro using autologous dendritic cells pulsed with HA-1 peptide. Antigen-specific cells were enriched by CD137 marker expression or by HLA-tetramer staining, RNA from positive and negative fractions was isolated for cDNA library preparation. The α and β TCR-repertoires were sequenced using the Illumina MiSeq system. The representative enrichment plot is shown in Figure 1 (A - α chains, B - β chains). Each circle represents a unique TCR. The vertical axis shows the normalized frequency in enriched fraction, the horizontal axis shows the normalized frequency in tetramer or CD137 negative flowthrough. HA-1-specific TCRs are denoted by green filled circles.TCRs were considered to be HA-1-specific if they were significantly enriched in CD137+ or tetramer+ fraction (exact Fisher test, p=0.05). In total, 49 α and 80 β chains were described. To determine the degree of similarity between HA-1-specific TCRs Levenstein distance was calculated between amino acid sequences of complementarity-determining region 3 for both chains. Sequences of previously published HA-1-specific TCRs were also included in the analysis (Verdijk et.al., Haematologica, 2002; Bleakley et.al., 2017, WO2018058002A1). α chains demonstrated low degree of mutual similarity, the majority of sequences did not belong to any cluster (Figure 2A, sequences with the Levenstein distance &lt;3 are connected). In contrast, a significant proportion of β chains were organized in a few clusters containing sequences from all 3 donors and previously published data (Figure 2B). We selected 14 α and 12 β HA-1-specific TCR chains (marked by the black dots in Figure 2). Clones were picked to represent separate clusters of similarity to Levenstein metrics, and unique sequences. Selected α and β-chains were cloned for subsequent functional screening in different combinations. Besides, we developed the modular lentiviral backbone for manufacturing HA-1 specific transgenic CD8+ T-cells. Our approach utilizes Golden Gate Cloning, which allows rapid assembly of lentiviral backbone carrying any combination of TCR α and β chains fused with the selective marker for sorting via p2A peptides. We used truncated CD34 as a transduced cell surface marker for the rapid separation of transduced cells by clinical-grade antibodies and subsequent expansion. In order to prevent the mispairing of transgenic TCR with endogenous one, CRISPR/Cas9 knockout strategy of endogenous TCR chains was developed. We used guide RNAs specific to TRAC,TRBC1 and TRBC2 genes and recombinant Cas9. The efficiency was demonstrated on Jurkat E6-1 cell line, the knockout was confirmed both by flow cytometry and genotyping of the modified cells using fragment analysis. Constant regions of the transgenic TCRs were modified to prevent cleavage by Cas9, the resistance was confirmed by in vitro Cas9 digestion assay. Moreover additional cysteines were introduced in the constant regions of transgenic TCRs for increased transgenic TCR stability. Cytotoxic activity of modified cells will be confirmed on lymphoblastoid cell lines and patient leukemia samples, cytokine secretion of modified cells will be detected using ELISPOT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. Disclosures No relevant conflicts of interest to declare.

Liraglutide Exerts Potential Anti-inflammatory Effect in Type 1 Diabetes by Inhibiting IFN-γ Production via Suppressing JAK-STAT Pathway

Type 1 diabetes is a T cell-mediated autoimmune disease. Interferon γ plays a critical role in the pathogenesis of type 1 diabetes. Signal transducer and activator of transcription transduces type I interferon cytokines in T cells, leading to Th1 cell differentiation and production of interferon γ. Recent studies suggest that liraglutide reduces the plasma concentration of C-reative protein in patients with type 1 diabetes and protects β cell function in the non-obese diabetic mouse. The study aimed to explore the effect of glucagon-like peptide-1 analogue on interferon γ production and the underlying signaling pathway in vitro. Jurkat E6-1 cells were intervened with different concentrations of glucose and liraglutide during different time periods. Protein was extracted from Jurkat E6-1 cells. The target proteins (total and activated Janus kinase 2, signal transducers and activators of transcription 4 and interferon γ) were detected by Western blot. Glucose stimulates interferon γ expression and activates Janus kinase 2/signal transducers and activators of transcription 4 signaling pathway in Jurkat E6-1 cells in a concentration and timedependent manner. Under high glucose condition, liraglutide inhibits interferon γ expression and Janus kinase 2/signal transducers and activators of transcription 4 signaling pathway in Jurkat E6-1 cells in a concentration and time-dependent manner. The Janus kinase responsible for liraglutide-inhibited signal transducers and activators of transcription 4 phosphorylation is Janus kinase 2, which is also required for the interferon γ induction. Finally, we demonstrated that under high glucose condition, liraglutide inhibits interferon γ expression via Janus kinase 2/signal transducers and activators of transcription 4 signaling pathway in Jurkat E6-1 cells. Liraglutide inhibits Jurkat E6-1 cells to produce interferon γ via the Janus kinase/signal transducers and activators of transcription signaling pathway under high glucose condition, which implies its potential in the immunoregulatory effect of type 1 diabetes.

Induction of apoptosis in leukemic cells by the alkaloid extract of garden cress (Lepidium sativum L.)

ObjectiveGarden cress (Lepidium sativum L.) is an important herb in traditional medicine used to improve production of breast milk in women and semen in men. In the present research the authors evaluated its ability to destroy leukemic cancer (Jurkat E6-1) cells, using the alkaloid extract of this plant. MethodsConstituents of the alkaloid extract were analyzed by gas chromatography–mass spectrometry (GC–MS) and their cytotoxicity in leukemic cancer cells and healthy peripheral blood mononuclear cells (PBMCs) was assessed. Cell death via apoptosis was confirmed by DNA laddering, caspase-3 activity, annexin V-fluorescein isothiocyanate and mitochondrial toxicity assays. The specific course of gene activation in treated cells was determined through quantitative polymerase chain reaction (qPCR). ResultsGC–MS analysis identified six alkaloids and proto-alkaloids, namely, benzyl isothiocyanate (1), 2-ethoxy-4H-3,1-benzoxazin-4-one (2), (4R)-2-(2-aminophenyl)-4-phenyloxazoline (3), 5-acetyl-1,2-dihydro-6-methyl-2-oxo-4-phenyl-3-pyridinecarbonitrile (4), benzo[b][1,8]-naphthyridin-5(10H)-one,2,4,7-trimethyl (5) and 1,4-diaminoanthraquinone (6), in the alkaloid extract of L. sativum. Of these, compound 1 was previously identified in the seeds of L. sativum. Exposure to the alkaloid extract caused death of Jurkat E6-1 cells, with median lethal concentration (LC50) of 75.25 µg/mL. However, the alkaloid extract also showed a nontoxic and proliferative (1.6-fold) effect in healthy PBMCs. Further experiments performed with Jurkat cells at LC50 and sub-LC50 doses demonstrated DNA fragmentation, activation of caspase-3 and time-dependant phosphatidylserine translocation (apoptosis) from inner to outer cell membranes. Cell toxicity and assessment of adenosine triphosphate level, together with using qPCR to evaluate expression profile of major apoptosis genes, revealed that apoptosis may be induced by disruption in the mitochondrial outer membrane potential, through activation of extrinsic and intrinsic apoptosis pathways in Jurkat cells. ConclusionThe ability of the alkaloid extract of L. sativum seeds to induce apoptosis indicates a potential pharmacological use in cancer chemotherapy. The separation of individual active compounds and further in-depth exploration of the molecular mechanism of apoptosis may lead to novel chemotherapeutic compounds in our future antineoplastic research.

Saponins from the roots of Chenopodium bonus-henricus L.

Two new glycosides of phytolaccagenin and 2β-hydroxyoleanoic acid, namely bonushenricoside A (3) and bonushenricoside B (5) together with four known saponins, respectively compounds 3-O-L-α-arabinopyranosyl-bayogenin-28-O-β-glucopyranosyl ester (1), 3-O-β-glucuronopyranosyl-2β-hydroxygypsogenin-28-O-β-glucopyranosyl ester (2), 3-O-β-glucuronopyranosyl-bayogenin-28-O-β-glucopyranosyl ester (4) and 3-O-β-glucuronopyranosyl-medicagenic acid-28-β-xylopyranosyl(1→4)-α-rhamnopyranosyl(1→2)-α-arabinopyranosyl ester (6) were isolated from the roots of Chenopodium bonus-henricus L. The structures of the compounds were determined by means of spectroscopic methods (1D and 2D NMR, IR and HRMS). The MeOH extract and compounds were tested for cytotoxic activity on five leukemic cell lines (HL-60, SKW-3, Jurkat E6-1, BV-173 and K-562). In addition, the ability of metanolic extract and saponins to modulate the interleukin-2 production in PHA/PMA stimulated Jurkat E6-1 cells was investigated as well.

Indirect Measurement of CRAC Channel Activity Using NFAT Nuclear Translocation by Flow Cytometry in Jurkat Cells.

Flow cytometry is a powerful technology to assess the presence of NFAT in the nuclei after CRAC channel activation. Here we described a simplified procedure for the analysis of CRAC channel activity using NFAT nuclear translocation by flow cytometry, based on the isolation of Jurkat E6-1 cell nuclei.

Molecular mechanism of apoptosis induction in Jurkat E6-1 cells by Tribulus terrestris alkaloids extract

The present study demonstrates apoptosis-inducing potential and mechanism of action of Tribulus terristris alkaloid extract in Jurkat E6-1 cancer cell line. Liquid Chromatography-Mass Spectrometry and High Resolution-Mass Spectrometry analysis identified the presence of four N-feruloyltyramine derivatives, namely trans-N-feruloyl-3-hydroxytyramine (1), trans-N-coumaroyltyramine (2), trans-N-feruloyltyramine (3) and trans-N-feruloyl-3-ethoxytyramine (4) in the alkaloid extract. Compounds 2 and 3 have not been yet reported in the alkaloid extract of T. terristris. In silico analysis revealed therapeutic potential of N-feruloyltyramine derivatives and strong binding efficiency to both chains of Tumor Necrosis Factor Receptor 1. Treatment of alkaloids extract to Jurkat E6-1 clone induced dose-dependent cytotoxicity (LC50 140.4 μg mL−1). Jurkat cells treated with alkaloids extract at sub-lethal concentration showed DNA fragmentation, enhancement in caspase-3 activity and phosphatidylserine translocation (apoptosis indicator) compared to control cells. Gene expression analysis using Human Apoptosis RT2 Profiler PCR Array analysis upon alkaloid treatment was found to significantly alter expression of critical genes such as TNFR1, FADD, AIFM, CASP8, TP53, DFFA and NFKB1. These genes are predicted to mediate apoptotic cell death via both intrinsic and extrinsic apoptosis pathway. In summary, we report the identification of new N-feruloyltyramine derivatives from alkaloid extract of T. terristris fruit with probable anti-leukemic and pharmacological potential.

Germline variation in the 3'-untranslated region of the POU2AF1 gene is associated with susceptibility to lymphoma.

Genetic variations in certain genes may alter the susceptibility to lymphoma. We searched electronic databases and selected candidate single-nucleotide polymorphisms (SNPs) located within 3'-untranslated regions (3'-UTRs) that might affect miRNA-binding ability in the 50 most dysregulated genes in lymphoma for further study. We found that rs1042752-located in the 3'-UTR of POU2AF1, which plays a vital role in lymphomagenesis-was significantly associated with lymphoma risk in a case-control study with 793 patients and 939 controls. Compared with individuals with the rs1042752TT genotype, those with the rs1042752CC genotype had a higher risk of lymphoma (OR = 2.14, 95% CI: 1.55-2.95, P < 0.001), even in stratified analysis for non-Hodgkin lymphoma (OR = 4.58, 95% CI: 2.38-8.81, P < 0.001), B-cell lymphoma (OR = 4.89, 95% CI: 2.46-9.73, P < 0.001), T-cell lymphoma (OR = 4.20, 95% CI: 1.76-10.05, P = 0.001), and Hodgkin lymphoma (OR = 3.62, 95% CI: 1.25-10.46, P = 0.018). Similar results were also observed in a recessive genetic model. Database findings suggested that rs1042752 might affect the interaction of POU2AF1 mRNA with hsa-miR-633. Functional assays confirmed that rs1042752C altered the binding site of hsa-miR-633 and increased POU2AF1 expression in Ramos, HuT 102, and Jurkat E6-1 cell lines. These findings demonstrate for the first time that functional polymorphism in the 3'-UTR of POU2AF1 is associated with susceptibility, and that SNP interaction with hsa-miR-633 affects gene expression and increases the risk of lymphoma.

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

BackgroundWe have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1.MethodsAfter treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA.ResultsThe initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15–20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL.ConclusionNano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P2 in coupling glycolysis to cell proliferation and protein synthesis in this model.

Comprehensive analysis of venom from the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10–100 μg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 μg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 μg/mL.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific.

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments.

Epigenetic regulatory effects of the methylation of CpG island shore on the expression of lag3 gene in various human T cell lines

Objective To investigate the effects of DNA methylation on the expression of lymphocyte activation gene 3 (lag3) in different human T cell lines. Methods A quantitative PCR and a flow cytometry analysis were performed to measure the expression of lag3 gene in various T cell lines at mRNA and protein levels. The distribution of CpG sites within the promoter and body of lag3 gene was detected to locate the potential regulatory region(s) (CpG island and CpG island shore). The levels of DNA methylation in each cell line were analyzed. The T cell lines were demethylated with 5-Aza-2'-deoxycytidine (5-Aza-2'-dc) for further investigation on the changes of lag3 gene expression and DNA methylation. Results Jurkat E6-1 cells showed the highest expression level of lag3 gene as compared with J. CaM1.6 and CEM cells. Hypermethylated CpG islands were detected in cells of each cell line. The methylation levels of CpG island shore in J. CaM1.6 and CEM cells were higher than that in Jurkat E6-1 cells. Treatment of J. CaM1.6 and CEM cells with 5-Aza-2'-dc significantly promoted the expression of lag3 gene at mRNA and protein levels as well as the demethylation of CpG island shore. No significant differences with the expression of lag3 gene and the methylation of CpG island were observed in Jurkat E6-1 cells with or without 5-Aza-2'-dc stimulation. Conclusion Methylation and demethylation of CpG island shore played important roles in regulating the transcription of lag3 gene. Key words: DNA methylation; CpG island shore; lag3; Human T cell line

Immune suppressive effect of cinnamaldehyde due to inhibition of proliferation and induction of apoptosis in immune cells: implications in cancer.

BackgroundBesides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells.MethodsActivation of nuclear factor-κB by cinnamaldehyde (0–10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV.ResultsLow concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde.ConclusionWe attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation.

Identification and function analysis of a novel transcript 630 in acute T-cell lymphoblastic leukemia Jurkat cell lines

Objective To identify and analyze a novel transcript 630 in acute T-cell lymphoblastic leukemia cell (T-ALL) Jurkat cell lines. Methods RNA-sequencing was applied to transcriptome profiling. The novel transcript 630 was identified by reverse transcription polymerase chain reaction (RT-PCR) combined with sequencing. The expression level of the novel transcript was detected in some tumor cell lines and normal cells. The sequences for small interfering RNA (siRNA) against 630 were designed and synthesized. Then, the effects of siRNA were determined by real-time PCR. Apoptotic cells and cell cycle analysis were investigated with the flow cytometry after siRNA-mediated knockdown of 630. Results The novel transcript 630 was identified. High expression level of 630 was detected in Jurkat E6-1, HL60 and human umbilical vein endothelial(HUVE) cells. The knockdown of transcript 630 induced a decrease of cellular proliferation. Conclusions The novel transcript 630 is one of the novel transcripts in Jurkat cells. The decreased expression level of transcript 630 can suppress the proliferation of leukemia cells. The novel transcript 630 might contribute to the treatment of T-ALL. Key words: Leukemia; Jurkat cell; Transcriptome

New modes of ERK feedback regulation in T cell receptor signaling revealed by quantitative phosphoproteomic analysis (121.29)

Abstract Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor (TCR) activation by selectively enhancing responses to foreign ligands while suppressing signals from self-peptides. In response to TCR agonist engagement, ERK is activated to positively regulate TCR signaling through phosphorylation of Ser59 on Lck. To obtain a wide-scale view of the role of ERK in propagating TCR signaling, a quantitative phosphoproteomic analysis of 417 tyrosine phosphorylation sites by mass spectrometry was performed on Jurkat E6-1 cells in the presence of U0126, a MEK1/2 specific inhibitor of ERK activation. Relative to untreated cells, ERK-inhibited cells showed constitutive decreases in phosphorylation through a TCR stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (LAT, SLP-76, PLCγ1, Itk), consistent with ERK’s proposed role in TCR positive feedback signaling. Interestingly, ZAP-70 Tyr598 and CD3 ζ Tyr123 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of these sites via ERK feedback. Furthermore, elevated phosphorylation on the activation site of Fyn was observed in response to ERK inhibition. These findings shed light on novel signaling events that are regulated by this kinase which may fine tune TCR activation.

Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.