Pathway In Jurkat Cells Research Articles

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2–3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

Indigofera suffruticosa aerial parts extract induce G2/M arrest and ATR/CHK1 pathway in Jurkat cells

BackgroundIndigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents.MethodsCytotoxicity of ISAE were determined by resazurin viability assay, multitox – Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach.ResultsWe showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine.ConclusionsISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.

Novel Ferrocene Derivatives Induce Apoptosis through Mitochondria-Dependent and Cell Cycle Arrest via PI3K/Akt/mTOR Signaling Pathway in T Cell Acute Lymphoblastic Leukemia.

Simple SummaryT cell acute lymphoblastic leukemia (T-ALL) is a malignant hematologic disease that urgently requires efficient therapeutic agents. The aim of this study is to explore the anti-T-ALL activity of novel ferrocene derivatives. It was found that ferrocene derivatives F1–F7 synthesized by our group inhibited the proliferation of several cancer cell lines in vitro. Among them, F1 and F3 displayed potent cytotoxicity against T-ALL cell lines, especially Jurkat cells, with low cytotoxicity for normal cells. Mechanistically, F1 and F3 could induce apoptosis through mitochondria-dependent pathway mediated by ROS, and cell cycle arrest at G0/G1 phase via the PI3K/Akt/mTOR signaling pathway in Jurkat cells. These results suggested that F1 and F3 could be potential candidates for future T-ALL therapy.T cell acute lymphoblastic leukemia (T-ALL) is one of the most common causes of death in pediatric malignancies. However, the clinical chemotherapy for T-ALL has been limited by numerous side effects, emphasizing that novel anti-T-ALL drugs are urgently needed. Herein, a series of 2-acyl-1-dimethylaminomethyl-ferrocenes for cancer therapy have been evaluated. Among them, F1 and F3 exhibited potent cytotoxicity against T-ALL cell lines, especially Jurkat cells, with low cytotoxicity for normal cells. Further mechanistic studies revealed that F1 and F3 could induce apoptosis in Jurkat cells by destructing mitochondrial membrane, enhancing reactive oxygen species (ROS) generation, decreasing the Bcl-2/Bax ratio, releasing Cytochrome c, and increasing the expression of Cleaved Caspase-9/-3 and Cleaved PARP. Additionally, F1 and F3 could suppress cell proliferation and arrest the cell cycle at G0/G1 phase through the PI3K/Akt/mTOR signaling pathway by down-regulating the expression of CDK6, Cyclin D1, p-Akt, p-GSK-3β, p-mTOR, p-p70 S6K, and up-regulating the expression of P21 and P27, which would also be a possible mechanism. Consequently, ferrocene derivatives F1 and F3 could induce apoptosis through a mitochondria-dependent pathway mediated by ROS, and cell cycle arrest at G0/G1 phase via the PI3K/Akt/mTOR signaling pathway in Jurkat cells. The present study provided fundamental insights into the clinical application of F1 and F3 for the treatment of T-ALL.

Cytotoxic Activity and Initiation of Apoptosis via Intrinsic Pathway in Jurkat Cells by Leaf Extract of Zanthoxylum rhetsa DC

Background Newer drugs are in demand for leukemia treatment that specifically targets tumor cells without affecting normal cells. Potent cytotoxic activities have been reported from various parts of Zanthoxylum rhetsa. Thus, the present study was conducted to evaluate antileukemic potential of leaf extract of Z. rhetsa along with probable mechanism of cytotoxicity. Materials and Methods: The antiproliferative activity of the extract on leukemic cell lines was evaluated using sulforhodamine B assay. The changes in cell death profile, cell cycle, and expression levels of pro-apoptotic markers (p53, Bax, cytochrome C, caspase 3, and MMP) and antiapoptic marker (Bcl2) on Jurkat cell lines were studied using flow cytometer. Comparison of oxidative stress induced by extract on Jurkat cells and normal mouse fibroblast cells was done. DNA fragmentation was studied using gel electrophoresis. Results: The leaf extract showed concentration-dependent cytotoxicity against Jurkat cell lines majorly via apoptotic mechanism. It arrested cells at G0/G1 and S phase of cell cycle. Apoptosis was associated with increase in the expression of pro-apoptotic markers and decrease of anti-apoptotic markers. The treatment with extract selectively increased the oxidative stress in Jurkat cells and showed DNA fragmentation. Conclusion: The methanol extract of leaves of Z. rhetsa show selective cytotoxic activity on Jurkat cell lines and induced apoptosis via intrinsic pathway.

20(S)-Ginsenoside Rh2 displays efficacy against T-cell acute lymphoblastic leukemia through the PI3K/Akt/mTOR signal pathway

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a kind of aggressive hematological cancer, and the PI3K/Akt/mTOR signaling pathway is activated in most patients with T-ALL and responsible for poor prognosis. 20(S)-Ginsenoside Rh2 (20(S)-GRh2) is a major active compound extracted from ginseng, which exhibits anti-cancer effects. However, the underlying anticancer mechanisms of 20(S)-GRh2 targeting the PI3K/Akt/mTOR pathway in T-ALL have not been explored. MethodsCell growth and cell cycle were determined to investigate the effect of 20(S)-GRh2 on ALL cells. PI3K/Akt/mTOR pathway–related proteins were detected in 20(S)-GRh2–treated Jurkat cells by immunoblotting. Antitumor effect of 20(S)-GRh2 against T-ALL was investigated in xenograft mice. The mechanisms of 20(S)-GRh2 against T-ALL were examined by cell proliferation, apoptosis, and autophagy. ResultsIn the present study, the results showed that 20(S)-GRh2 decreased cell growth and arrested cell cycle at the G1 phase in ALL cells. 20(S)-GRh2 induced apoptosis through enhancing reactive oxygen species generation and upregulating apoptosis-related proteins. 20(S)-GRh2 significantly elevated the levels of pEGFP-LC3 and autophagy-related proteins in Jurkat cells. Furthermore, the PI3K/Akt/mTOR signaling pathway was effectively blocked by 20(S)-GRh2. 20(S)-GRh2 suppressed cell proliferation and promoted apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in Jurkat cells. Finally, 20(S)-GRh2 alleviated symptoms of leukemia and reduced the number of white blood cells and CD3 staining in the spleen of xenograft mice, indicating antitumor effects against T-ALL invivo. ConclusionThese findings indicate that 20(S)-GRh2 exhibits beneficial effects against T-ALL through the PI3K/Akt/mTOR pathway and could be a natural product of novel target for T-ALL therapy.

Regulation of mPGES-1 composition and cell growth via the MAPK signaling pathway in jurkat cells.

Previous studies have suggested that microsomal prostaglandin E synthase-1 (mPGES-1) is highly expressed and closely associated with mitogen-activated protein kinase (MAPK) signaling pathways in various types of malignant cells. However, their expression patterns and function with respect to T-cell acute lymphoblastic leukemia (T-ALL) remain largely unknown. The present study investigated whether mPGES-1 served a crucial role in T-ALL and aimed to identify interactions between mPGES-1 and the MAPK signaling pathway in T-ALL. The results indicated that mPGES-1 overexpression in T-ALL jurkat cells was significantly decreased by RNA silencing. Decreasing mPGES-1 on a consistent basis may inhibit cell proliferation, induce apoptosis and arrest the cell cycle in T-ALL jurkat cells. Microarray and western blot analyses revealed that c-Jun N-terminal kinase served a role in the mPGES-1/prostaglandin E2/EP4/MAPK positive feedback loops. In addition, P38 and extracellular signal-regulated kinase 1/2 exhibited negative feedback effects on mPGES-1. In conclusion, the results suggested that cross-talk between mPGES-1 and the MAPK signaling pathway was very complex. Therefore, the combined regulation of mPGES-1 and the MAPK signaling pathway may be developed into a new candidate therapy for T-ALL in the future.

Proteogenomic analysis prioritises functional single nucleotide variants in cancer samples.

Massively parallel DNA sequencing enables the detection of thousands of germline and somatic single nucleotide variants (SNVs) in cancer samples. The functional analysis of these mutations is often carried out through in silico predictions, with further downstream experimental validation rarely performed. Here, we examine the potential of using mass spectrometry-based proteomics data to further annotate the function of SNVs in cancer samples. RNA-seq and whole genome sequencing (WGS) data from Jurkat cells were used to construct a custom database of single amino acid variant (SAAV) containing peptides and identified over 1,000 such peptides in two Jurkat proteomics datasets. The analysis enabled the detection of a truncated form of splicing regulator YTHDC1 at the protein level. To extend the functional annotation further, a Jurkat phosphoproteomics dataset was analysed, identifying 463 SAAV containing phosphopeptides. Of these phosphopeptides, 24 SAAVs were found to directly impact the phosphorylation event through the creation of either a phosphorylation site or a kinase recognition motif. We identified a novel phosphorylation site created by a SAAV in splicing factor SF3B1, a protein that is frequently mutated in leukaemia. To our knowledge, this is the first study to use phosphoproteomics data to directly identify novel phosphorylation events arising from the creation of phosphorylation sites by SAAVs. Our study reveals multiple functional mutations impacting the splicing pathway in Jurkat cells and demonstrates potential benefits of an integrative proteogenomics analysis for high-throughput functional annotation of SNVs in cancer.

Castanea sativa Mill. bark extract exhibits chemopreventive properties triggering extrinsic apoptotic pathway in Jurkat cells

BackgroundChemoprevention represents the possibility to prevent, stop or reverse the cancerogenetic process. In this context the interest towards natural extracts and botanical drugs has constantly grown due to their phytochemical content. Castanea sativa Mill. (CSM) extracts showed to exert positive effect in the prevention/counteraction of chronic/degenerative diseases, therefore, we evaluated the potential chemopreventive effect of CSM bark extract.MethodsFlow cytometry (FCM) analyses of Jurkat cells treated with CSM bark extract (0–500 μg·mL−1) for 24–72 h allowed evaluating its cytotoxicity and ability to induce apoptosis through the intrinsic or extrinsic pathways. Moreover, to evaluate CSM bark extract selectivity towards cancer cells, its cytotoxic and pro-apoptotic effect was also evaluated in human peripheral blood lymphocytes (PBL).ResultsCSM bark extract induced apoptosis in Jurkat cells in a dose- and time- dependent manner activating the extrinsic pathways as evidenced by the increase of activated caspase-8 positive cells. Moreover, IC50 calculated after 24 h treatment resulted 304 and 128 μg·mL−1 in PBL and Jurkat cells respectively.ConclusionsOur data suggest that CSM bark extract might be considered an interesting potential anti-cancer agent, since it induces apoptosis in cancer cells without appreciable cytotoxic effects on non-transformed cells.

Arsenic trioxide increases expression of secreted frizzled-related protein 1 gene and inhibits the WNT/β-catenin signaling pathway in Jurkat cells.

The aim of the present study was to investigate the demethylation effect of arsenic trioxide (As2O3) on the secreted frizzled-related protein 1 (SFRP1) gene and its ability to inhibit the Wingless-type MMTV integration site family (WNT) pathway in Jurkat cells. Methylation-specific polymerase chain reaction was used to examine the CpG island methylation status of the SFRP1 gene in leukemia cell lines. In addition, the effects on Jurkat cells of treatment with different concentrations of As2O3 for 48 h were investigated. Reverse transcription-quantitative polymerase chain reaction was employed to measure the expression of mRNAs, while western blot analysis was used to examine protein expression in cells. The SFRP1 gene was methylated in Jurkat cells. However, both methylated and unmethylated SFRP1 genes were detected in HL60 and K562 cells. In normal bone marrow mononuclear cells, the SFRP1 gene was unmethylated. Following treatment with As2O3 for 48 h, the SFRP1 gene was demethylated, and the mRNA and protein expression levels of the SFRP1 gene were increased. By contrast, the mRNA and protein expression levels of β-catenin and cyclin Dl were downregulated. The protein expression of c-myc was also downregulated, but As2O3 exhibited no significant effect on the mRNA expression of c-myc. Abnormal methylation of the SFRP1 gene was detected in Jurkat cells. These results suggest that As2O3 activates SFRP1 gene expression at the mRNA and protein levels in Jurkat cells by demethylation of the SFRP1 gene. Furthermore, they indicate that As2O3 regulates WNT target genes and controls the growth of Jurkat cells through the WNT/β-catenin signaling pathway.

Marsdenia tenacissimae extraction (MTE) inhibits the proliferation and induces the apoptosis of human acute T cell leukemia cells through inactivating PI3K/AKT/mTOR signaling pathway via PTEN enhancement.

Marsdenia tenacissimae extraction (MTE) as a traditional Chinese herb has long been used to treat some diseases such as tumors in China. However, the potential effectiveness of MTE in leukemia has not yet been fully understood, and the related molecular mechanism is still unknown. In the present study, we aimed to evaluate the effects of MTE on the proliferation and apoptosis of Jurkat cells (T-ALL lines) and lymphocytes from T-ALL (T-cell acute lymphoblastic leukemia) patients. Firstly, CCK8 assays and flow cytometry assays revealed that MTE dose-dependently reduced the proliferation of Jurkat cells by arresting cell cycle at S phase. Secondly, Annexin V-FITC/PI-stained flow cytometry and TUNEL staining assays showed that MTE promoted the apoptosis of Jurkat cells. Mechanistically, MTE enhanced PTEN (phosphatases and tensin homolog) level and inactivated PI3K/AKT/mTOR signaling pathway in Jurkat cells, which mediated the inhibition of cell proliferation by MTE and MTE-induced apoptosis. Finally, MTE significantly inhibited the proliferation and promoted the apoptosis of lymphocytes from T-ALL patients, compared with lymphocytes from healthy peoples. Taken together, these results reveal an unrecognized function of MTE in inhibiting the proliferation and inducing the apoptosis of T-ALL cells, and identify a pathway of PTEN/PI3K/AKT/mTOR for the effects of MTE on leukemia therapy.

Pyrvinium selectively induces apoptosis of lymphoma cells through impairing mitochondrial functions and JAK2/STAT5

Targeting mitochondrial respiration has emerged as an attractive therapeutic strategy in blood cancer due to their unique metabolic dependencies. In this study, we show that pyrvinium, a FDA-approved anthelmintic drug, selectively targets lymphoma T-cells though inhibition of mitochondrial functions and JAK2/STAT5. Pyrvinium induces apoptosis of malignant T-cell line Jurkat and primary T-cells from lymphoma patients while sparing T-cells from healthy donors. Increased level of active caspase-3 and decreased levels of Bcl-2 and Mcl-1 were also observed in Jurkat and lymphoma T-cells but not normal T-cells treated with pyrvinium. In addition, pyrvinium impairs mitochondrial functions by inhibit mitochondrial respiration, suppressing mitochondrial respiratory complex I activity, increasing ROS and decreasing ATP levels. However, the effects of pyrvinium were abolished in mitochondrial respiration-deficient Jurkat ρ0 cells, confirming that pyrvinium acts on lymphoma T-cells via targeting mitochondrial respiration. We further show that lymphoma T-cells derived from patients depend more on mitochondrial respiration than normal T-cells, and this explains the selective toxicity of pyrvinium in lymphoma versus normal T-cells. Finally, we demonstrate that pyrvinium also suppresses JAK2/STAT5 signaling pathway in Jurkat cells. Our study suggests that pyrvinium is a useful addition to T-cell lymphoma treatment, and emphasizes the potential therapeutic value of the differences in the mitochondrial characteristics between malignant and normal T-cells in blood cancer.

The Fas/Fas Ligand Apoptotic Pathway Is Involved in Abrin-Induced Apoptosis

Abrin is a plant glycoprotein toxin from the seeds of Abrus precatorius, sharing similarity in structure and properties with ricin. Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 µg/kg, causing death after accidental or intentional poisoning. It is a potent biological toxin warfare agent. There is no chemical antidote available against the abrin. The elucidation of molecular mechanism of abrin-induced cell death is important for development of therapy. Intrinsic pathway-mediated apoptosis has been well established in abrin-induced cell death. However, the detailed mechanism especially extrinsic receptor-mediated pathway remains uncharacterized. To assess whether some of the apoptosis known to occur after abrin exposure might be mediated by Fas/Fas ligand (Fas L) interactions, we analyzed effect of abrin on Fas pathway in Jurkat cells. Here, we report that activation of the Fas pathway is involved in abrin-induced apoptosis. Following treatment of abrin, Fas L was induced, which stimulated the Fas pathway by cross-linking Fas receptor (Fas R). Apoptosis was mediated by cleavage of the Fas R proximal caspase-8 and the downstream caspase-3, resulting in activation of the prototype caspase substrate poly-(ADP-ribose) polymerase and caspase-activated DNase. Blocking Fas L/Fas R interaction by using Fas inhibitor reduced abrin-induced apoptosis, further confirms involvement of Fas pathway. Activation of components of Fas pathway and caspases upon abrin treatment was also found in splenocytes in mice. Our findings offer new perspective for understanding the fundamental mechanism in abrin-induced apoptotic mechanism and may have implication in developing novel therapeutic strategies in the management for abrin-induced complications.

Insoluble substances produced from human cytomegalovirus-infected cells induce deaths of nearby cells through non-classical death signal pathway (P6116)

Abstract Human cytomegalovirus (HCMV) has many strategies to evade the host immune system, and eventually resides in some cells in latency. Although the involvement of Fas ligand in T cell death was reported in the pathogenesis of retinitis with HCMV infection to retinal pigment epithelial cells, the cell death near other lesions caused by HCMV infection was not studied in detail by now. The insoluble substances derived from HCMV-infected fibroblasts, induce the deaths of variety of cell lines including Jurkat, HL-60, K562, U937 and THP-1 cells, but not of fibroblasts, HEK293 and U373MG. They were resistant to treatment with 56°C for 30 min to lose death induceability to Jurkat cells. Cell death of Jurkat cells by these substances were not inhibited by the simultaneous treatment with neutralizing antibodies to Fas ligand or TRAIL, or caspase inhibitors. Insoluble substances induced cleavages of poly ADP-ribose polymerase-1 into 89 kDa fragment, but not through caspase 3, 7 or 9 pathway in Jurkat cells. AIF was detected in the cytoplasmic fraction, but not in the nuclear fraction of Jurkat cells from 6 hours following exposure to them. This result suggested AIF was not translocated from mitochondria to nucleus in Jurkat cells by the stimulation with insoluble substances. Collectively human cytomegalovirus infection would induce the death of myeloid and lymphoid cells near the infection site through non-classical death signal pathway.

Surface CD3 expression proceeds through both myosin regulatory light chain 9 (MYL9)-dependent and MYL9-independent pathways in Jurkat cells

CD3 is a complex of polypeptides which form part of the T cell receptor. Normal human peripheral pan T cells, express not only CD3, but the mRNA for myosin regulatory light chains MYL9, MYL12A, and MYL12B are also significantly expressed. In the Jurkat wild strain, an acute T cell leukemia cell line, CD3 on the surface and MYL9 mRNA are not expressed, while both MYL12A mRNA and MYL12B mRNA are expressed. Jurkat-I, a new clone was established by the transfection of the MYL9 gene into the Jurkat wild strain. As a result, the level of CD3 expressed on the surface of Jurkat-I cells was significantly higher than those in the Jurkat wild strain. Phorbol 12-myristate 13-acetate increased the surface CD3 levels in Jurkat wild strain cells without resulting in MYL9 gene expression, indicating that protein kinase C is partially involved in the expression of CD3 on the surface. These results suggest that surface CD3 expression proceeds through both MYL9-dependent and MYL9-independent pathways (i.e. the protein kinase C- dependent pathway) in Jurkat cells.

Vitamin D does not modulate NF-κB activity in Jurkat T cells

The active form of vitamin D, 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3], has been reported to influence the functioning of the immune system by targeting the activities of cellular signaling pathways, in addition to its direct genomic effects. One of the signaling pathways reported to be targeted by vitamin D is the NF-κB pathway, which is highly active in most immune cell types, including T cells. However, the effects of vitamin D on the NF-κB pathway in T cells are not fully understood. Therefore, we examined the effects of 1α,25(OH) 2D 3 on the NF-κB pathway in the Jurkat cell line, a human T cell line that constitutively expresses endogenous vitamin D receptor. We found that 1α,25(OH) 2D 3 does not inhibit the induction of IκBα degradation and the expression of an NF-κB-dependent reporter gene in Jurkat cells following treatment with PMA/ionomycin. Also, 1α,25(OH) 2D 3 did not suppress the activation of NF-κB by TNFα or PHA. Furthermore, we demonstrate that 1α,25(OH) 2D 3 does not block the induction of CD69, which is an NF-κB target gene and an early T cell activation marker. Therefore, we conclude that vitamin D does not modulate the activity of the NF-κB pathway in Jurkat cells.

Abstract B16: Expression of genes involved in PI3K/Akt signaling pathway in arsenic trioxide resistant clones derived from Jurkat cell line

Abstract Arsenic trioxide (ATO) is a well-known carcinogen but also an effective cancer therapeutic drug for acute promyelocytic leukemia. Despite intensive research efforts, the molecular mechanisms underlying antileukemic effect of ATO as well as resistance to arsenic therapy appearing during treatment are not well understood. There are data showing that dysregulation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, a central integrating point of survival signals, may be the reason of apoptosis resistance in leukemic cells. The aim of the present study was to assess expression of 84 genes involved in PI3K/Akt signaling pathway in Jurkat cells exposed to ATO at 1, 2.5 and 5 μM for 16 hrs as well as in ATO resistant clones derived from the cells. First screening of genes was performed using the Human PI3K-AKT Signaling Pathway RT2 ProfilerTM PCR Array (Superarray) and then selected genes were verified in a standard real-time PCR. Results of our study showed that ATO induced more intensive changes in expression of PI3K/Akt pathway genes in ATO resistant clones than in Jurkat cells exposed to arsenic. Among genes differently regulated in both cell types were these playing a key role in regulating cell growth e.g. GRB10 (growth factor receptor-bound protein 10), FASLG (Fas ligand), CCND1 (cyclin D1). Interestingly, the level of FASLG mRNA was strongly down-regulated in ATO treated Jurkat cells and up-regulated in ATO resistant clones. Expression of CCND1 and GRB10 varied between resistant clones in a concentration-dependent manner. Moreover, concentration-dependent up-regulation of JUN (jun oncogene) was the most characteristic effect induced by ATO in Jurkat cells. Comparing to untreated Jurkat cells, we identified several genes differently regulated in ATO resistant clones (but not in Jurkat ATO exposed cells) that were mainly involved in anti-apoptotic pathways (e.g. IRS1 (Insulin receptor substrate 1), MAP2K1 (Mitogen-activated protein kinase kinase 1), MAPK3 (Mitogen-activated protein kinase 3), RPS6KA1 (Ribosomal protein S6 kinase, 90kDa, polypeptide 1), PRKCB (Protein kinase C, beta 1), PRKCZ (Protein kinase C, zeta) or Akt activation and growth regulation (e.g. PDK1 (Pyruvate dehydrogenase kinase, isozyme 1), MTOR (Mechanistic target of rapamycin), MTCP1 (Mature T-cell proliferation 1)). In conclusion, our in vitro study identified several targets of ATO that may mediate cytotoxic effects in Jurkat cells. Moreover, the data suggest that the PI3K/Akt signaling pathway plays an important role in the ATO resistance in Jurkat leukemic cells. The work was supported by the Polish State Committee for Scientific Research (grant No PB 2806/B/PO1/2007/33). Citation Information: Clin Cancer Res 2010;16(7 Suppl):B16

Noxa is necessary for hydrogen peroxide-induced caspase-dependent cell death

Oxidative stress induces apoptosis or necrosis of many cell types, which can cause tissue injury. Hydrogen peroxide (H2O2) induced apoptotic death of Jurkat cells. This effect was inhibited by overexpression of human Bcl-2, by silencing of cytochrome c, and by ablation of Bax/Bak, indicating that H2O2-induced apoptosis was mediated by the mitochondrial pathway in Jurkat cells. Treatment with H2O2 caused an increase of Noxa protein, via activating transcription factor 4-dependent accumulation of Noxa mRNA and inhibition of Noxa protein degradation. H2O2-induced apoptosis was strongly suppressed by silencing of Noxa, indicating that Noxa plays a crucial role in this form of apoptosis. Structured summaryMINT-7543162: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0914) with Bim EL (uniprotkb:O43521), Bim L (uniprotkb:O43521) and NOXA (uniprotkb:Q13794) by anti bait coimmunoprecipitation (MI:0006)

IκB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-κB activity by glycine chloramine through methionine oxidation

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 μM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.

TCR pathway involves ICBP90 gene down-regulation via E2F binding sites

Antigen-induced cell death is essential for function, growth and differentiation of T-lymphocytes through legation of the T cell receptor. Since TCR-induced cell death occurs at late G1 checkpoint of the cell cycle and considering that ICBP90 is critical for G1/S transition, we studied the ICBP90 regulation through the TCR pathway in Jurkat cells. ICBP90 expression was strongly decreased after TCR triggering concomitantly to cyclin D3 and topoisomerase IIα expression decreases. Cell stimulation with PMA and/or calcium ionophore A23187 down-regulated ICBP90 expression. The decrease of ICBP90 protein and mRNA expressions was accompanied with cell growth arrest. A luciferase reporter assay demonstrated that activation of TCR pathways inhibit ICBP90 gene promoter activity. Three consensus E2F binding sites (called from E2F-a to E2F-c) were identified in the ICBP90 gene promoter and were subjected to mutations. The E2F-a, located in a highly active promoter fragment, shows a strong positive functional activity in proliferating cells. E2F-a and E2F-c binding sites are involved in the TCR-induced down-regulation of ICBP90 gene transcription. Altogether, our data demonstrate that TCR signaling pathways regulate ICBP90 gene expression through pRb/E2F complex. We propose that ICBP90 down-regulation is a key event in G1 arrest preceding T cell death.