Jurkat E6 Research Articles

NK Cell Mediated Cytotoxicity Against Malignant Promyelocytes Enhanced By Arsenic Trioxide: Potential Clinical Relevance

Abstract NK cells are primary effectors of the innate immune response against cells that have undergone malignant transformation. Several lines of evidence indicate that the expression level of NK ligands on leukemic cells affects the sensitivity of the leukemic cells to cytolytic activity by NK cells. Various agents have been evaluated for their ability to induce these ligands on leukemic cells to augment the NK cell mediated anti-leukemia effect. There is substantial evidence that has established the importance of the adaptive immune system in the treatment of acute promyelocytic leukemia (APL) (Rose Ann Padua et al. Nat Med 2003). While there is significant data which address the mechanisms of arsenic trioxide (ATO) on malignant promyelocytes, limited data is available of its effect on the innate and adaptive immune system. We undertook a series of experiments to address the impact of ATO on NK cell receptor and malignant promyelocyte ligand expression and its effect on NK cell mediated cytotoxicity. We also evaluated NK cell reconstitution in patients treated with ATO and the impact of KIR genotypes on relapse. We first evaluated the cytotoxic activity of NK92MI (NK cell line) against 5 different myeloid (K562, U937, HL60, UF1, NB4) and 2 lymphoid cell lines (Jurkat E6.1, SUP-B15) by CFSE/ 7AAD cytotoxicity assay. Target (T) cells (1x 105/100 µL/well) pre-treated with CFSE were co-cultured with effector NK cells (E) at a E:T ratio of 1:1, 2:1 and 5:1 for 5 hours at 37°C in 96 well plates. The percentage cytolytic activity of the NK cells was then calculated after adding 7AAD and acquired in FACS Calibur (Becton Dickinson, San Jose, CA, USA). Significant cytolytic activity was noted against K562 and NB4 cell lines. At the highest E:T ratio there was a median 22% cytolytic activity against NB4 (N=5). We observed that NB4 when treated overnight with 1µM ATO (>99% viability retained after this exposure) significantly increased the cytotoxic effect of NK92MI cell line at all the E:T ratios as shown in figure 1A (n=5; P=0.0023). No other cell line showed a similar increase in cytotoxic effect following exposure to ATO at these concentrations (data not shown). We next evaluated the effect of exposure of NB4 cells to ATO at 1µM for 6 hours on NK ligand expression by flowcytometry. As shown in figure 1B there was a significant increase in activating ligand MICA/B in NB4 cell lines (n=3; P=0.016) which was not seen in any of the other cell lines. Similar significant increased expression of Nectin-2 (DNAM-1 ligand) and HLA Class I was seen. Exposure of NK92MI to ATO for 6 hours at 1uM (non cytotoxic dose:IC50-3.8uM) resulted in increased expression of activating receptors NKG2D, NKP30 and KIR2DS4 (figure 1C) and inhibitory receptor NKG2A and decrease in inhibitory receptors KIR3DL1/DL2. There were no changes in the expression of NKP46, KIR2DL1, KIR2DL2 and DNAM1 receptors. We undertook a prospective study to evaluate the pattern of NK (CD56+CD3-) reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen (Mathews et al. JCO 2011). The mean NK cell counts in patients were below the 2SD deviation level of the normal range even after completion of therapy (approximately a year)(figure 1D). All other subsets evaluated (CD4, CD8, CD3, CD19, CD56+CD3+, CD4CD45RO) had returned to levels within the normal range by the end of consolidation therapy (approximately 3 months from diagnosis). KIR genotyping was done on 55 patients with APL who received treatment with single agent ATO based regimen. The median follow up of this cohort was 20 months and 14 cases relapsed following initial therapy. The presence or absence of 17 KIR genes was done by PCR-SSP method (KIR Typing kit, Miltenyi Biotech Inc, CA). There was no association with any specific genotype or haplotype with risk of relapse. In summary we have noted that there is up regulation of receptors on NK cells and ligands on malignant promyelocytes following exposure to ATO that favors NK cell mediated cytotoxicity. In-vitro we have demonstrated a significant increase in NK cell mediated cytolytic activity against malignant promyelocytes exposed to ATO even at relatively low E:T ratios. This could be an important mechanism by which ATO induces durable remissions in patients with APL. The delayed NK cell recovery following treatment with ATO raises the possibility of using NK cell therapy to augment the effect of ATO in the treatment of patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

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

Epigonal Conditioned Media from Bonnethead Shark, Sphyrna tiburo, Induces Apoptosis in a T-Cell Leukemia Cell Line, Jurkat E6-1

Representatives of Subclass Elasmobranchii are cartilaginous fish whose members include sharks, skates, and rays. Because of their unique phylogenetic position of being the most primitive group of vertebrates to possess all the components necessary for an adaptive immune system, the immune regulatory compounds they possess may represent the earliest evolutionary forms of novel compounds with the potential for innovative therapeutic applications. Conditioned medium, generated from short term culture of cells from the epigonal organ of bonnethead sharks (Sphyrna tiburo), has been shown to have potent reproducible cytotoxic activity against a variety of human tumor cell lines in vitro. Existing data suggest that epigonal conditioned medium (ECM) exerts this cytotoxic activity through induction of apoptosis in target cells. This manuscript describes apoptosis induction in a representative tumor cell line, Jurkat E6-1, in response to treatment with ECM at concentrations of 1 and 2 mg/mL. Data indicate that ECM exposure initiates the mitochondrial pathway of apoptosis through activation of caspase enzymes. Future purification of ECM components may result in the isolation of an immune-regulatory compound with potential therapeutic benefit for treatment of human cancer.

Mechanisms of CD154 release from T cells and subsequent biological activities (P3375)

Abstract CD154 is a type II transmembrane protein critically involved in humoral immune responses. The soluble form of CD154 (sCD154) has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is release from the cell surface is of primordial importance. Using co-culture experiments, we show that CD154 is predominantly shed upon its engagement with CD40. Indeed, only CD40 and not α5β1 or αMβ2 is implicated in the cleavage of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers, a process previously shown to be involved in specific elements of CD154-induced signaling. We previously reported that the translocation of CD154 into lipid rafts is required for critical elements of CD154 signaling. Here, we show that CD154 is shed from the cell surface independently of this phenomenon. In contrast, we found that the protein kinase C signaling family and the metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. Moreover, released sCD154 remains biologically active and can regulate critical biological responses such as activation of key survival signaling pathways and inhibition of cell death in α5β1 positive T-cells. In conclusion, our data add significant insights into the mechanisms by which CD154 is released and may explain the link between enhanced levels of sCD154 and T-cell survival and persistence during chronic inflammatory diseases.

Design, synthesis, in vitro cytotoxicity evaluation and structure–activity relationship of Goniothalamin analogs

A series of six/five member (E/Z)-Goniothalamin analogs were synthesized from commercially available (3,4-dihydro-2H-pyran-2-yl)methanol/5-(hydroxymethyl)dihydrofuran-2(3H)-one in three steps with good to moderate overall yields and their cytotoxicity against lymphoblastic leukemic T cell line (Jurkat E6.1) have been evaluated. Among the synthesized analogs, (Z)-Goniothalamin appeared to be the most active in cytotoxicity (IC50 = 12 μM). Structure-activity relationship study indicates that introducing substituent in phenyl ring or replacing phenyl ring by pyridine/naphthalene, or decreasing the ring size of lactones (from six to five member) do not increase the cytotoxicity.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits IL-13 production in T cells via an NF-κB-dependent mechanism

Interleukin (IL)-13 is a cytokine produced by activated CD4+ T cells that plays a critical role in promoting allergic responses and tumor cell growth. The 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a natural ligand for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), a known regulator of anti-inflammatory activities. We determined the effects of 15d-PGJ2 on IL-13 expression in the Jurkat E6.1 T-cell line and in peripheral blood mononuclear cells. Semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay revealed that treatment of activated T cells with 15d-PGJ2 significantly decreased IL-13 mRNA transcription and secretion, respectively. This inhibition by 15d-PGJ2 was independent of PPAR-γ since treatment with GW9662, an irreversible antagonist of the nuclear receptor, produced no effect. Our data also revealed the involvement of nuclear factor-κB in mediating 15d-PGJ2-dependent down regulation of IL-13 expression. Collectively, these results demonstrate the potential of 15d-PGJ2 in attenuating expression and production of IL-13 in activated T cells.

Static magnetic field of 6 mT induces apoptosis and alters cell cycle in p53 mutant Jurkat cells

This study aimed to investigate the effect of 6 milliTesla (mT) static magnetic field (SMF) on apoptosis induction and cell cycle alteration in T-lymphoblastoid Jurkat E6.1 cells. Exposure of human p53 mutant Jurkat cells to 6 mT SMF resulted in apoptosis, which was detected by luminometric and flow cytometric analysis also, phosphorylated ATM and E2F1 proteins were detected by western blot analysis.Based on luminescence detection data, apoptosis initiated 36 h after exposure to 6 mT SMF. Apoptosis also reached its maximum rate 48 h after treatment. Flow cytometric analysis revealed a temporary G2 arrest after exposure to 6 mT SMF. Indeed, cellular population of S and G2 phases was increased. Based on reports of other investigations on the effect of magnetic fields on Ca2+flux changes in cell membranes and the effect of MFs on free radical formation, it can be suggested that the magnetic fields may induce the apoptosis and alter the cell population in different cell cycle phases of Jurkat cells via changing the Ca2+fluxes through cell membranes and playing a role in free radical formation. Western blot analysis showed that the amount of phosphorylated ATM and E2F1 proteins were increased in treated cells. The results of luminometric and flow cytometric detection did not show a significant difference in the apoptosis rate between 6 h-treated and 24 h-treated cells by 6 mT SMF. Thus, 6 mT SMF can induce apoptosis and alter cell cycle in Jurkat cells via a p53-independent pathway.

Synthesis, Characterization and Crystal Structure of Organotin(IV) N-Butyl-N-Phenyldithiocarbamate Compounds and their Cytotoxicity in Human Leukemia Cell Lines

Organotin complexes are recognized as the biologically active compounds in inducing cancerous cells death at very low doses. To date, organotin compounds currently appear among the most potent candidates in research related to the new anticancer drugs. In this study, new organotin(IV) N-butyl-N-phenyldithiocarbamate compounds have been successfully synthesized between the reaction of N-butylaniline amine with organotin(IV) chloride in 1:2/1:1 molar ratio. All compounds were characterized using the elemental analysis, FT-IR and NMR spectroscopy. The single crystal structure was determined by X-ray single crystal analysis. The elemental analysis showed good agreement with the suggested formula (C4H9)2Sn[S2CN(C4H9)(C6H5)]2 (Compound 1 and 2), (C6H5)2Sn[S2CN(C4H9)(C6H5)]2 (Compound 3) and (C6H5)3Sn[S2CN(C4H9)(C6H5)] (Compound 4). The important infrared absorbance peaks, v (C = N) and v(C = S) were detected in range between 1457-1489 cm(-1) and 951-996 cm(-1), respectively. The chemical shift of carbon in NCS2 group obtained from 13C NMR was found in range 198.86-203.53 ppm. The crystal structure of compound 4 showed that the dithiocarbamate ligand coordinates in a monodentate fashion. It crystallized in monoclinic P2(1)/n space group with the crystal cell parameter: a = 10.0488(1) angstroms, b = 18.0008(2) angstroms, c = 15.2054(2) angstroms, beta = 102.442(1) degrees and R = 0.044. The cytotoxicity (IC50) of these compounds against Jurkat E6.1 and K-562 leukemia cells were in the range between 0.4-0.8 and 1.8-5.3 microM, respectively as assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazholium bromide (MTT) assay. In conclusion, our study demonstrate that all compounds showed potent cytotoxicity towards both cell lines tested with the triphenyltin(IV) compound displayed the greatest effect.

30-Normedicagenic Acid Glycosides from Chenopodium Foliosum

Two new glycosides of 30-normedicagenic acid, namely 3-O-[beta-D-glucuronopyranosyl methyl ester]-2beta,3beta-dihydroxy-30-noroleane-12,20(29)-diene-23,28-dioic acid 28-O-beta-D-glucopyranosyl ester, and 3-O-beta-D-glucopyranosyl-2beta,3beta-dihydroxy-30-noroleane- 12,20(29)-diene-23,28-dioic acid, together with the known 3-O-beta-glucopyranosyl-2beta,3beta-dihydroxy-30-noroleane-12,20(29)-diene-23 ,28-dioic acid 28-O-beta-glucopyranosyl ester, and 3-O-beta-glucuronopyranosyl-2beta,3beta-dihydroxy-30-noroleane-12,20(29)-diene-23,28-dioic acid 28-O-beta-glucopyranosyl ester were isolated from the aerial parts of Chenopodium foliosum Asch. The structures of the compounds were determined by means of spectroscopic methods (1D and 2D NMR, UV, IR) and HRMS-ESI. The compounds were tested for cytotoxicity on three leukemic cell lines (BV-173, SKW-3, HL-60). In addition, the saponins showed moderate stimulatory effects on interleukin-2 production in PHA/PMA stimulated Jurkat E6.1 cells.

Cytotoxic effects of Fisturalin-3 and 11-Deoxyfisturalin-3 on Jurkat and U937 cell lines

Fisturalines are bromotyrosine compounds isolated from marine sponges. Previous studies have shown antineoplasic, antiviral and antibacterial effects in Vitro; however, the possible effects of these compounds in hematologic malignancies have not been assessed. In the present study, the antiproliferative and pro apoptotic effects of Fistularin-3 (F) and 11-Deoxyfistularin-3 (DF) were assessed using the MTT method and annexin V/propidium iodide by flow cytometry using the cell lines: Jurkat E6.1 and U937. In addition, the cell cycle was assessed by flow cytometry. Inhibition of the proliferative response was concentration and time dependent. The IC50 of F was 7.39 and 8.10 µM for Jurkat E6.1 and U937 respectively. At 24 and 48 h, in the U937 cell line, but not in the Jurkat cell line, both compounds induced up to 35% annexin V increase. Necrosis was not observed in any case. Compound F induced, in both cell lines, a decrease in the number of cells in the S phase and increase in the G0/G1 phase. In the Jurkat cell line only, there was an increase in the number of cells in the G2/M phase. Compound DF was not as effective as F. F is more active than DF in repressing the cell cycle and inducing apoptosis. Both compounds are potentially useful in the development of new drugs to treat hematologic malignancies.

An investigation of antileukemia activity of violacein-loaded dendrimer in Jurkat cell lines

Violacein, a bacterial pigment, has been reported to have in vitro antitumor effects. The objective of the current research was investigation of antileukemia activity of violacein – loaded dendrimer with ascorbic acid in Jurkat E6.1 cell lines as human acute lymphoblastic leukemia in a nanocomplex delivery system. Violacein was extracted and purified from Janthinobacterium lividum DSM1522T. Generation2 (PAMAM) dendrimer with extracted pigment and L-ascorbic acid were used as a nanocomplex delivery system.Chemical bonds between violacein and dendrimer were identified by fourier transform infrared (FTIR). Nanoparticles sizes were determined by scanning electron microscopy.The in vitro antileukemia activity of nanocomplex system was evaluated using MTT assay and caspase activity assay. FTIR assay results showed bands characteristic of the PAMAM dendrimer with violacein. Nanoparticles sizes were demonstrated almost 200 nm.The results showed dendrimer containing voilacein (IC50=4 μM) was almost 2 fold more efficient compared with free violacein. Flow cytometry studies of caspases activitiesdemonstrated that caspase -3, -7 and -8 caused apoptosis in 29, 34 and 41% of Jurkat E6.1 cells respectively. Cancer cells can uptake ascorbic acid higher than normal cells and it can operate specific drug delivery systems. This nanocomplex system can lead to pharmaceutical delivery at specific sites and reduce side effects. Key words: Violacein, dendrimer, antileukemia, jurkat cell line, Janthinobacterium lividum.

Aggregation behavior and in vitro biocompatibility study of octopus-shaped macromolecules based on tert-butylcalix[4]arenes

A series of products based on tert-butylcalix[4]arene have been synthesized by anionic polymerization of ethylene oxide. The resulting products are amphiphilic octopus-shaped macromolecules, consisting of a hydrophobic calix[4]arene core and four arms of hydrophilic poly(ethylene oxide) chains. In aqueous solutions the polyoxyethylated tert-butylcalix[4]arenes were found to self-associate above certain CMC determined by dye solubilization technique. The light scattering study reveals that the polyoxyethylated tert-butylcalix[4]arenes form aggregates of narrow size distribution and hydrodynamic diameters ranging from about 155 to 245 nm and aggregation numbers from tens to hundreds macromolecules per particle depending on the degree of polymerization of the PEO chains. An in vitro biocompatibility study showed that the tested compounds are practically devoid of intrinsic cytotoxic and hemolytic effects and moreover they failed to modulate the mitogen-induced interleukin-2 release from the human T-lymphocyte cell line Jurkat E6-1. Taken together the excellent in vitro biocompatibility profile and the favorable physicochemical characteristics of the tested polyoxyethylated calix[4]arenes give us reason to consider them as promising for further evaluation as drug delivery platforms.

Jurkat cell proliferation is suppressed by Chlamydia (Chlamydophila) pneumoniae infection accompanied with attenuation of phosphorylation at Thr389 of host cellular p70S6K

Chlamydia (Chlamydophila) pneumoniae infects T lymphocytes and multiplies within them. Our previous studies have indicated that C. pneumoniae infection suppresses proliferation of peripheral blood mononuclear cells stimulated with Staphylococcus-enterotoxin B; however, the mechanism of suppression was unclear. In this study, we explored the molecular mechanism involved in C. pneumoniae infection by using human acute T cell leukemia cell line, Jurkat E6-1. Proliferation of Jurkat cells was suppressed in an m.o.i.-dependent manner by C. pneumoniae infection. The suppression by the infection was particularly evident during the initial 24h of the infection, and down modulation of cyclin D3 protein levels were observed at the same time period by immunoblot analysis. The suppression of the Jurkat cell proliferation and the down modulation of cyclin D3 protein level were only induced by viable C. pneumoniae infection, not by exposure to UV-killed or heat-killed C. pneumoniae. Phosphorylations at Thr308 and Ser473 of AKT were induced by C. pneumoniae infection; however, phosphorylation at Thr389 of the downstream kinase, p70S6K was inhibited by unidentified mechanism associated with C. pneumoniae infection. Taking into account that G1 arrest of the C. pneumoniae infected Jurkat cells were not observed and that p70S6K is one of the most important regulators of protein synthesis, it was suggested that the suppression of Jurkat cell proliferation by C. pneumoniae was at least in part mediated by down modulation of protein synthesis through attenuation of Thr389 phosphorylation of p70S6K.

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.

NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation

NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-κB activation. However, the exact mechanism of its function has not been fully understood. Here, we report that knockdown of NEMO by RNA interference in Jurkat E6.1 cells enhanced TCR-induced NF-κB report gene activity and IL-2 production by promotion of IκBα degradation and p65 nuclear translocation, whereas inhibited TNF-α and LPS-induced IκBα degradation without influencing the phosphorylation of MAPKs. In human primary T and Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-κB activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-κB report gene activity was increased along with ascending doses of transfected siNEMO and reached the highest activity when knockdown about 70% of NEMO, then turned to decline and gradually be blocked once almost thoroughly knockdown of NEMO. Meanwhile, TNF-α induced NF-κB was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKKβ may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKKβ continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKKβ. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-κB activation and differentially regulates TNF-α and TCR-induced NF-κB pathways.

The activity of a new 2-amino-1,3,4-thiadiazole derivative 4ClABT in cancer and normal cells

The 2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole set are well known compounds with interesting in vitro and in vivo anti-cancer profiles. The aim of this study was an in vitro evaluation of the anti-cancer activity of a new synthesized aminothiadiazole derivative 2-(3-chlorophenyloamino)-5-(2,4-dihydroxyphenyl)- -1,3,4-thiadiazole 4ClABT. The effect on tumor cell proliferation, motility and morphology, DNA synthesis as well as the influence on normal cells was assessed. The antiproliferative activity of 4ClABT in tumor cells derived from peripheral cancers including breast carcinoma (T47D), colon carcinoma (HT-29), thyroid carcinoma (FTC-238), teratoma (P19), and T-cell leukemia (Jurkat E6.1), as well as cancers of the nervous system including rhabdomyosarcoma/medulloblastoma (TE671), brain astrocytoma (MOGGCCM) and glioma (C6) was studied by means of MTT assay. DNA synthesis level was determined in BrdU ELISA test. Wound assay model was applied for tumor cell motility assessment. Morphological changes induced by 4ClABT in cancer and normal cells were analyzed in HE staining specimens. Moreover, the influence of 4ClABT on normal cells including skin fibroblasts (HSF), hepatocytes (Fao), astroglia and neurons was studied by means of LDH assay. The tested compound inhibited the proliferation of tumor cells in dose-dependent fashion. The anti-cancer effect was attributed to decreased DNA synthesis, prominent changes in tumor cell morphology as well as reduced cell motility. In antiproliferative concentrations, 4ClABT was not toxic to normal cells. Our study showed prominent anti-cancer effects of the tested aminothiadiazole derivative in the absence of toxicity in normal cells. The obtained results confirmed the promising anti-cancer profile of previously tested 2-(monohalogenphenylamino)- -5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole derivatives (ClABT - chlorophenyl derivative, FABT and 3FABT - fluorophenyl derivatives and 4BrABT - bromophenyl derivative). The molecular mechanisms and the in vivo activity of aminothiadiazole derivatives will be the subject of further studies.

Use of RNA interference to discover pathways involved in HIV infection and replication: cell lines tell many stories, primary cells might tell the truth

Infection by Human Immunodeficiency Virus is difficult to treat thanks to its persistent viral reservoir and to its high rate of mutation that allows appearance of resistance to the available treatment. An approach to discover drugable targets is the identification of cellular partner proteins interacting with the virus during its life cycle. We used RNAi technology to identify new HIV partners in the host cytoskeleton since it has been shown that cytoskeleton components and the irregulators have a role during several steps of HIV life cycle. By transducingseveral Tcell lines such as Jurkat CD4 CCR5, Jurkat E6-1 and SupT1 with lentiviral vectors expressings hRNA sequences, we silenced different target genes, members of pathways involved inactinrearrangement. By infection with HIV-NL4.3-eGFP reporter virus we evaluated HIV replication ratesin transducedcells. Surprisingly, the infection rate affected by the specific knock-down was dependent on the cell line used. lndeed, ashRNAtransduced in one cell line affected infection differently to what it did in another. Moreover, we observed that transduction on itself with a control vector expressinga scrambled shRNA sequence affected HIV infection rate in some but not all cell lines. Therefore, to obtain relevant results in screening co-factors for HIV infection, we turned toprimary cells, the naturaltargets of the virus in vivo. We optimized combinedlentiviral transduction and HIV infection in cultured peripheral blood CD4+ lymphocytes. In this setting, transduction with scrambled shRNA expressing lentivirus did not affect HIV replication, providing us a platform to assay gene-knockdown likely to generate the most relevant information for natural HIV infection invivo.

Synthesis and biological evaluation of α,β-unsaturated lactones as potent immunosuppressive agents

Compounds having α,β-unsaturated lactones display a variety of biological activities. Many research groups have tested both natural and unnatural α,β-unsaturated lactones for as-yet undiscovered biological properties. We synthesized α,β-unsaturated lactones with various substituents at the δ-position and studied their immunosuppressive effects, that is, the inhibition of Interleukin-2 (IL-2) production. Among the compounds synthesized, the benzofuran-substituted α,β-unsaturated lactone 4h showed the best inhibitory activity toward IL-2 production in Jurkat e6-1 T lymphocytes (IC(50)=66.9 nM) without cytotoxicity at 10 μM. The results indicated that 4h may be useful as a potent immunosuppressive agent, as well as in IL-2-related studies.

An analysis of the phosphoproteome of immune cell lines exposed to the immunomodulatory mycotoxin deoxynivalenol

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500 ng/mL) for 24 h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71 kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanism-based phosphoprotein biomarkers for DON exposure.

Viral envelope protein folding and membrane hemifusion are enhanced by the conserved loop region of HIV-1 gp41.

Fusion of human immunodeficiency virus (HIV-1) with target cells is mediated by the gp41 transmembrane envelope protein. The loop region within gp41 contains 2 crucial cysteines that play an unknown role in HIV-cell fusion. On the basis of cell-cell fusion assay, using human T-cell lines [Jurkat E6-1 and Jurkat HXBc2(4)], and virus-cell fusion assay, using fully infectious HIV-1 HXBc2 virus and TZM-bl human cell line, we provide evidence that the oxidation state of the disulfide bond within a loop domain peptide determines its activity. The oxidized (closed) form inhibits fusion, while the reduced (opened) form enhances hemifusion. These opposite activities reach 60% difference in viral fusion. Both forms of the loop domain interact with gp41: the opened form enhances gp41 folding into a bundle, whereas the closed form inhibits this folding. Therefore, the transformation of the cysteines from a reduced to an oxidized state enables the loop to convert from opened to closed conformations, which assists gp41 to fold and induces hemifusion. The significant conservation of the loop region within many envelope proteins suggests a general mechanism, which is exploited by viruses to enhance entry into their host cells.