Transcriptome analysis of anaerobic glycolysis effects on Jurkat T cell proliferation.

Introduction: To explore the effects of anaerobic glycolysis on Jurkat T cell proliferation and clarify the possible mechanism via transcriptomic analysis. Material and methods: The monocarboxylate transporter 1 inhibitor AZD3965 was used to target and block the transmembrane transport of lactate, thereby inhibiting anaerobic glycolysis in Jurkat T cells. Then, genes with differential expression between treated and untreated cells were detected by transcriptomic analysis, and constructs were generated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses as well as protein-protein interaction (PPI) network analysis were performed to explore the potential mechanism. Results: Inhibition of anaerobic glycolysis reduced Jurkat T-cell proliferation. RNA sequencing identified 1723 transcripts that were differentially expressed, including 1460 upregulated genes and 263 downregulated genes. GO functional enrichment analysis showed that the differentially expressed genes were mainly involved in the biological processes of response to unfolded protein, response to topologically incorrect protein, and protein folding. KEGG pathway analysis of differentially expressed genes or hub genes from the PPI network analysis revealed enrichment in the estrogen signaling and PI3K-Akt pathways. Conclusions: Anaerobic glycolysis contributes to the regulation of Jurkat T-cell proliferation. The underlying mechanism may involve the estrogen signaling pathway or PI3K-Akt signaling pathway as well as protein metabolism.

Mechanics of leukemic T-cell.

Cell mechanics is a factor that determines cell growth, migration, proliferation, or differentiation, as well as trafficking inside the cytoplasm and organization of organelles. Knowledge about cell mechanics is critical to gaining insight into these biological processes. Here, we used atomic force microscopy to examine the elasticity, an important parameter of cell mechanics, of non-adherent Jurkat leukemic T-cells in both interphase and mitotic phases. We found that the elasticity of an individual cell does not significantly change at interphase. When a cell starts to divide, its elasticity increases in the transition from metaphase to telophase during normal division while the cell is stiffened right after it enters mitosis during abnormal division. At the end of the division, the cell elasticity gradually returned to the value of the mother cell. These changes may originate from the changes in cell surface tension during modulating actomyosin at the cleavage furrow, redistributing cell organelles, and constricting the contractile ring to sever mother cell to form daughters. The difference in elasticity patterns suggests that there is a discrepancy in the redistribution of the cell organelles during normal and abnormal division.

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

BACKGROUND: Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. METHODS AND RESULTS: In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. DISCUSSION: QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P < 0.001). These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

Jurkat T Cells are Immunophenotypically Distinct from T-Cell Acute Lymphoblastic Leukemia Cells Due to High-Level Surface Expression of CD5.

Human leukemic T cells show decreased surface CD5 (sCD5) and increased cytoplasmic CD5 (cCD5). When we examined their expressions in the Jurkat T cells, it showed increased sCD5 and decreased cCD5, which is in sharp contrast with the pattern of CD5 expression observed for human leukemic T cells. Furthermore, this opposite pattern was due to the absence of an exonal switch between E1A and E1B. This study suggests that Jurkat cell does not retain all characteristics of T-ALL cells; thus, we should carefully interpret the data obtained using Jurkat T cell as a model cell line of T-ALL.

Co-Application of Statin and Flavonoids as an Effective Strategy to Reduce the Activity of Voltage-Gated Potassium Channels Kv1.3 and Induce Apoptosis in Human Leukemic T Cell Line Jurkat.

Voltage-gated potassium channels of the Kv1.3 type are considered a potential new molecular target in several pathologies, including some cancer disorders and COVID-19. Lipophilic non-toxic organic inhibitors of Kv1.3 channels, such as statins and flavonoids, may have clinical applications in supporting the therapy of some cancer diseases, such as breast, pancreas, and lung cancer; melanoma; or chronic lymphocytic leukemia. This study focuses on the influence of the co-application of statins-simvastatin (SIM) or mevastatin (MEV)-with flavonoids 8-prenylnaringenin (8-PN), 6-prenylnarigenin (6-PN), xanthohumol (XANT), acacetin (ACAC), or chrysin on the activity of Kv1.3 channels, viability, and the apoptosis of cancer cells in the human T cell line Jurkat. We showed that the inhibitory effect of co-application of the statins with flavonoids was significantly more potent than the effects exerted by each compound applied alone. Combinations of simvastatin with chrysin, as well as mevastatin with 8-prenylnaringenin, seem to be the most promising. We also found that these results correlate with an increased ability of the statin-flavonoid combination to reduce viability and induce apoptosis in cancer cells compared to single compounds. Our findings suggest that the co-application of statins and flavonoids at low concentrations may increase the effectiveness and safety of cancer therapy. Thus, the simultaneous application of statins and flavonoids may be a new and promising anticancer strategy.

Statins as inhibitors of voltage-gated potassium channels Kv1.3 in cancer cells.

Voltage-gated potassium channels are integral membrane proteins selectively permeable for potassium ions and activated upon change of membrane potential. Voltage-gated potassium channels of the Kv1.3 type were discovered both in plasma membrane and in inner mitochondrial membrane (mito Kv1.3 channels). For some time Kv1.3 channels located both in plasma membrane and in mitochondria are considered as a potentially new molecular target in several pathologies including some cancer disorders. Lipophilic nontoxic organic inhibitors of Kv1.3 channels may potentially find a clinical application to support therapy of some cancer diseases such as breast, pancreas and lung cancer, melanoma or chronic lymphocytic leukaemia (B-CLL). Inhibition of T lymphocyte Kv1.3 channels may be also important in treatment of chronic and acute respiratory diseases including severe pulmonary complication in corona virus disease Covid 19, however further studies are needed to confirm this supposition. Statins are small-molecule organic compounds, which are lipophilic and are widely used in treatment of hypercholesterolemia and atherosclerosis. Electrophysiological studies performed in our laboratory showed that statins: pravastatin, mevastatin and simvastatin are effective inhibitors of Kv1.3 channels in cancer cells of human T cell line Jurkat. We showed that application of the statins in the concentration range from 1.5 µM to 50 µM inhibited the channels in a concentration-dependent manner. The inhibitory effect was the most potent in case of simvastatin and the least potent in case of pravastatin. The inhibition was partially irreversible in case of simvastatin and fully reversible in case of pravastatin and mevastatin. It was accompanied by a significant acceleration of the current inactivation rate without any significant change of the activation rate. Mechanism of the inhibition is probably complex, including a direct interaction with the channel protein and perturbation of lipid bilayer structure, leading to stabilization of the inactivated state of the channels.

Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.

Integration of conventional quantitative and phospho-proteomics reveals new elements in activated Jurkat T-cell receptor pathway maintenance.

Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T-cell receptor (TCR) pathway in a T-cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T-cells and phosphopeptides enriched via a TiO2-based chromatographic step. Both phosphopeptide-enriched and flow-through fractions were analyzed by LC-MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow-through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR-related function were detected. A kinase-substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR-related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T-cells.

Individual cytotoxicity of three major type A trichothecene, T-2, HT-2, and diacetoxyscirpenol in human Jurkat T cells.

Mycotoxins are toxic, fungal secondary metabolites that contaminate agricultural commodities, food, and feed. Among them, T-2, HT-2, and diacetoxyscirpenol (DAS; the major type A trichothecene) are primarily produced from Fusarium species. These mycotoxins exert numerous toxicological effects in animals and humans, such as dermatotoxicity, haematotoxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, and immunotoxicity. In the present study, human Jurkat T cells were used as a model to investigate apoptotic cell death induced by T-2, HT-2, and DAS. The results showed that T-2, HT-2, and DAS decreased cell viability and increased production of Reactive Oxygen Species in a time- and dose-dependency. Based on their IC50 values, they could be ranked in decreasing order of cytotoxicity as T-2 > HT-2 > DAS. All tested mycotoxins caused DNA fragmentation, up-regulated cytochrome C, caspase 3, and caspase 9 mRNA levels, and down-regulated the relative expression of Bcl-2 and caspase 8. The effects of these trichothecenes on apoptosis were determined based on flow cytometry. At the IC50 concentrations, the percentages of apoptotic cells were significantly higher than for the controls. Taken together, these data suggested that T-2, HT-2, and DAS could induce apoptosis through the mitochondrial apoptotic pathway.

T-cell immunoglobulin and mucin 1 (TIM-1) mediates infection of Hantaan virus in Jurkat T cells.

Hantaan virus (HTNV) is a major public health concern due to its ability to cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia. Symptoms of HFRS include fever, hemorrhage, immune dysfunction and renal impairment, and severe cases can be fatal. T cell-mediated adaptive immune responses play a pivotal role in countering HTNV infection. However, our understanding of HTNV and T cell interactions in the disease progression is limited. In this study, we found that human CD4+ T cells can be directly infected with HTNV, thereby facilitating viral replication and production. Additionally, T-cell immunoglobulin and mucin 1 (TIM-1) participated in the process of HTNV infection of Jurkat T cells, and further observed that HTNV enters Jurkat T cells via the clathrin-dependent endocytosis pathway. These findings not only affirm the susceptibility of human CD4+ T lymphocytes to HTNV but also shed light on the viral tropism. Our research elucidates a mode of the interaction between the virus infection process and the immune system. Critically, this study provides new insights into the pathogenesis of HTNV and the implications for antiviral research.

Use of tetraethylammonium (TEA) and Tris loading for blocking TRPM7 channels in intact cells.

Tetraethylammonium (TEA), a quaternary ammonium compound, is a well-known blocker of potassium channels belonging to various subfamilies, such as KV1-3, KCa1, 2 and prokaryotic KcsA. In many cases, TEA acts from the extracellular side by open pore blockade. TEA can also block transient receptor potential (TRP) cation channels, such as TRPM7, in a voltage-dependent manner. In human T lymphocytes, intracellular (cytosolic) TEA and its analog TMA (tetramethylammonium) inhibit TRPM7 channel currents in the outward but not inward direction. By contrast, intracellular Mg2+, protons and polyamines inhibit both outward and inward current components equally. Likewise, the majority of available pharmacological tools inhibit TRPM7 channels in a voltage-independent manner. Since TRPM7 is a steeply outwardly rectifying conductance, voltage-dependent blockers can be useful for studying the cellular functions of this channel. TRPM7 protein is endogenously expressed in diverse cell lines, including HEK, HeLa, CHO, RBL and Jurkat. Using patch-clamp electrophysiology, we found that incubating HEK293 and Jurkat T cells overnight in the presence of 20 mM TEA-Cl, resulted in the nearly complete blockade of whole-cell TRPM7 outward current, measured at break-in. By contrast, the inward current was unchanged in TEA-loaded cells. The blockade was fully reversible after washout of intracellular solution in whole-cell but not in perforated-patch recording configurations. Overnight incubation with 20 mM TMA-Cl resulted in a more modest blockade of the outward TRPM7 current. Internal 129 mM TMA and TEA eliminated most of the outward current. TEA uptake in transfected HEK293 cells led to blockade of recombinant murine TRPM7 and the Mg2+ and pH insensitive Ser1107Arg variant. Unexpectedly, Tris-HCl, a widely used pH buffer, could similarly be loaded into Jurkat and HEK cells, and preferentially blocked outward TRPM7 currents. 20 mM and 129 mM Tris in the internal solution blocked TRPM7 current in outward but not inward direction. Voltage-dependent channel blockade by TEA, TMA and Tris loading will be useful for studying the properties and functions of TRPM7-mediated ion transport in intact cells.

Loureirin B Exerts its Immunosuppressive Effects by Inhibiting STIM1/Orai1 and KV1.3 Channels.

Loureirin B (LrB) is a constituent extracted from traditional Chinese medicine Resina Draconis. It has broad biological functions and an impressive immunosuppressive effect that has been supported by numerous studies. However, the molecular mechanisms underlying Loureirin B-induced immune suppression are not fully understood. We previously reported that Loureirin B inhibited KV1.3 channel, calcium ion (Ca2+) influx, and interleukin-2 (IL-2) secretion in Jurkat T cells. In this study, we applied CRISPR/Cas9 to edit KV1.3 coding gene KCNA3 and successfully generated a KV1.3 knockout (KO) cell model to determine whether KV1.3 KO was sufficient to block the Loureirin B-induced immunosuppressive effect. Surprisingly, we showed that Loureirin B could still inhibit Ca2+ influx and IL-2 secretion in the Jurkat T cells in the absence of KV1.3 although KO KV1.3 reduced about 50% of Ca2+ influx and 90% IL-2 secretion compared with that in the wild type cells. Further experiments showed that Loureirin B directly inhibited STIM1/Orai1 channel in a dose-dependent manner. Our results suggest that Loureirin B inhibits Ca2+ influx and IL-2 secretion in Jurkat T cells by inhibiting both KV1.3 and STIM1/Orai1 channels. These studies also revealed an additional molecular target for Loureirin B-induced immunosuppressive effect, which makes it a promising leading compound for treating autoimmune diseases.

Amorphous-Crystalline Calcium Phosphate Coating Promotes In Vitro Growth of Tumor-Derived Jurkat T Cells Activated by Anti-CD2/CD3/CD28 Antibodies.

A modern trend in traumatology, orthopedics, and implantology is the development of materials and coatings with an amorphous-crystalline structure that exhibits excellent biocopatibility. The structure and physico-chemical and biological properties of calcium phosphate (CaP) coatings deposited on Ti plates using the micro-arc oxidation (MAO) method under different voltages (200, 250, and 300 V) were studied. Amorphous, nanocrystalline, and microcrystalline statesof CaHPO4 and ß-Ca2P2O7 were observed in the coatings using TEM and XRD. The increase in MAO voltage resulted in augmentation of the surface roughness Ra from 2.5 to 6.5 µm, mass from 10 to 25 mg, thickness from 50 to 105 µm, and Ca/P ratio from 0.3 to 0.6. The electrical potential (EP) of the CaP coatings changed from -456 to -535 mV, while the zeta potential (ZP) decreased from -53 to -40 mV following an increase in the values of the MAO voltage. Numerous correlations of physical and chemical indices of CaP coatings were estimated. A decrease in the ZP magnitudes of CaP coatings deposited at 200-250 V was strongly associated with elevated hTERT expression in tumor-derived Jurkat T cells preliminarily activated with anti-CD2/CD3/CD28 antibodies and then contacted in vitro with CaP-coated samples for 14 days. In turn, in vitro survival of CD4+ subsets was enhanced, with proinflammatory cytokine secretion of activated Jurkat T cells. Thus, the applied MAO voltage allowed the regulation of the physicochemical properties of amorphous-crystalline CaP-coatings on Ti substrates to a certain extent. This method may be used as a technological mechanism to trigger the behavior of cells through contact with micro-arc CaP coatings. The possible role of negative ZP and Ca2+ as effectors of the biological effects of amorphous-crystalline CaP coatings is discussed. Micro-arc CaP coatings should be carefully tested to determine their suitability for use in patients with chronic lymphoid malignancies.

Anti-leukemic activity of Satureja bachtiarica occurs by apoptosis in human cells.

Failure of apoptosis contributes to leukemia progression. We investigated extracts of a native Iranian plant, Satureja bachtiarica, for possible anti-leukemia activity by induction of apoptosis and changes to the cell cycle. Growth inhibition caused by aqueous, butanol, dichloromethane and hexane extracts of S. bachtiarica on K562 and Jurkat leukemia cells was assessed using a colorimetric assay. Extracts were analyzed for induction of apoptosis and cell cycle arrest using flow cytometry and measurement of caspase-3 activity. Dichloromethane and hexane extracts inhibited leukemia cell proliferation in a dose-dependent manner. The IC50 values of these extracts were 22-33 µg/ml. Flow cytometric determination of annexinV/propidium iodide positive cells verified a significantly increased percentage of apoptotic cells compared to negative controls. Both 50 µg/ml dichloromethane and hexane extracts induced apoptosis in 89-97% of K562 and 94-97% of Jurkat cells 48 h after treatment. The effects of extracts on the cell cycle included significantly increased numbers of K562 and Jurkat cells in the subG1 phase and decreased numbers of cells in the G1, S and G2/M phases. After 24 h, we found increased levels of caspase-3 activation in cells treated with 25 µg/ml dichloromethane and hexane extracts compared to untreated cells. Our findings indicate the anti-leukemic effects of dichloromethane and hexane extracts of S. bachtiarica due to induction of apoptosis and inhibition of cell cycle progression.

Quantification of T Cell Binding Polyclonal Rabbit Anti-thymocyte Globulin in Human Plasma with Liquid Chromatography Tandem-Mass Spectrometry.

The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft-versus-host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and - 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.

Individual and combined cytotoxicity of major trichothecenes type B, deoxynivalenol, nivalenol, and fusarenon-X on Jurkat human T cells.

Food and feed commodities are often contaminated by more than one mycotoxin. Among the several combinations that frequently occur, fusariotoxins are often mentioned. The multi-mycotoxins contamination is a serious threat for health. In the present study we investigated the toxic interactions between deoxynivalenol (DON), nivalenol (NIV), and fusarenon-X (FX) in Jurkat T cells by using the MTT assay. The tested mycotoxins alone or in combination had a dose dependent effect on proliferating lymphocytes. According to IC50, it could be classified in decreasing order of toxicity: FX > NIV > DON > DON + FX > NIV + FX > DON + NIV > DON + NIV + FX. The type of mycotoxin interactions was assessed by calculating the combination index (CI) and the dose reduction index (DRI). Our data indicate that an antagonistic effect was strongly observed in binary combination between DON and NIV at higher concentrations and ternary mixtures. Meanwhile, the DON and FX mixtures generated moderate antagonism, while NIV and FX provoked different interactions. The effects of tested mycotoxins on apoptosis were also determined using a FACScan flow cytometer. At IC75, the percentages of apoptotic cells in all treatment groups were significantly different when compared to control group but no significant differences among treatment groups was observed. Taken together, our data suggest that immunotoxicity among multi-mycotoxins contamination cannot be predicted based on individual effects but depends on the mixtures, ratio and/or concentrations in the product, as well as type of target cells.

Tetrandrine and cepharanthine induce apoptosis through caspase cascade regulation, cell cycle arrest, MAPK activation and PI3K/Akt/mTOR signal modification in glucocorticoid resistant human leukemia Jurkat T cells.

Tetrandrine (TET) and cepharanthine (CEP) are two bisbenzylisoquinoline alkaloids isolated from the traditional herbs. Recent molecular investigations firmly supported that TET or CEP would be a potential candidate for cancer chemotherapy. Prognosis of patients with glucocorticoid resistant T cell acute lymphoblastic leukemia (T-ALL) remains poor; here we examined the anti-T-ALL effects of TET and CEP and the underlying mechanism by using the glucocorticoid resistant human leukemia Jurkat T cell line in vitro. TET and CEP significantly inhibited cell viabilities and induced apoptosis in dose- and time-dependent manner. Further investigations showed that TET or CEP not only upregulated the expression of initiator caspases such as caspase-8 and 9, but also increased the expression of effector caspases such as caspase-3 and 6. As the important markers of apoptosis, p53 and Bax were both upregulated by the treatment of TET and CEP. However, TET and CEP paradoxically increased the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, and activated the survival protein NF-κB, leading to high expression of p-NF-κB. Cell cycle arrest at S phase accompanied by increase in the amounts of cyclin A2 and cyclin B1, and decrease in cylcin D1 amount in cells treated with TET or CEP will be another possible mechanism. During the process of apoptosis in Jurkat T cells, treatment with TET or CEP also increased the phosphorylation of JNK and p38. The PI3K/Akt/mTOR signaling pathway modification appears to play significant role in the Jurkat T cell apoptosis induced by TET or CEP. Moreover, TET and CEP seemed to downregulate the expressions of p-PI3K and mTOR in an independent way from Akt, since these two drugs strongly stimulated the p-Akt expression. These results provide fundamental insights into the clinical application of TET or CEP for the treatment of patients with relapsed T-ALL.

6-mercaptopurine promotes energetic failure in proliferating T cells.

The anticancer drug 6-mercaptopurine (6-MP) inhibits de novo purine synthesis and acts as an antiproliferative agent by interfering with protein, DNA and RNA synthesis and promoting apoptosis. Metabolic reprogramming is crucial for tumor progression to foster cancer cells growth and proliferation, and is regulated by mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) as well as the oncogenes Myc and hypoxia inducible factor 1α (HIF-1α). We hypothesized that 6-MP impacts metabolic remodeling through its action on nucleotide synthesis. The aim of our study is to provide a comprehensive characterization of the metabolic changes induced by 6-MP in leukemic T cells. Our results indicate that exposition to 6-MP rapidly reduces intracellular ATP concentration, leading to the activation of AMPK. In turn, mTOR, an AMPK target, was inhibited, and the expression of HIF-1α and Myc was reduced upon 6-MP incubation. As a consequence of these inhibitions, glucose and glutamine fluxes were strongly decreased. Notably, no difference was observed on glucose uptake upon exposition to 6-MP. In conclusion, our findings provide new insights into how 6-MP profoundly impacts cellular energetic metabolism by reducing ATP production and decreasing glycolytic and glutaminolytic fluxes, and how 6-MP modifies human leukemic T cells metabolism with potential antiproliferative effects.

Apoptosis and gene expression in Jurkat human T cells and lymphoid tissues of fusarenon-X-treated mice.

Fusarenon X, a member of the type B trichothecene mycotoxin group, has been frequently observed, along with deoxynivalenol (DON) and nivalenol (NIV) as a contaminant in cereals. Our previous study demonstrated that a 14-day FX exposure caused apoptosis in the lymphoid tissues of mice, especially at 0.5 mg/kg bodyweight. However, the relationship between low concentrations of FX and apoptotic molecular machinery remains unclear. In the present study, we investigated the genetic regulatory mechanisms in the thymus and Peyer's patches of mice after 14 days oral administration of FX at 0.5 mg/kg bodyweight. FX caused the up-regulation of Bax, Bid, Trp53, and Caspase-9 mRNA but the relative expression of Fas, TNF, and Caspase-8 remained unchanged. Furthermore, we also determined the toxicity of FX in Jurkat T-cells. FX exhibited a concentration- and time-dependent inhibition of cell viability. Thus, incubation time and FX concentration influence the percentage of apoptotic cells. These data suggested that treatment with low dosage of FX can induce apoptosis in lymphocytes through an effect on Bax, Bid, Trp53, and Caspase-9 and therefore the mitochondrial apoptotic pathway.

PKC-θ is a negative regulator of TRAIL-induced and FADD-mediated apoptotic spectrin aggregation.

INTRODUCTION: During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. MATERIAL AND METHODS: For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging. RESULTS: TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down ofPRKCQin Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of theß-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene,FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane. CONCLUSIONS: In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ.