Human T-cell Leukemia Cell Line Research Articles

Rhizoctonia Bataticola Lectin (RBL) Induces Caspase-8-Mediated Apoptosis in Human T-Cell Leukemia Cell Lines but Not in Normal CD3 and CD34 Positive Cells

We have previously demonstrated immunostimulatory activity of a fungal lectin, Rhizoctonia bataticola lectin (RBL), towards normal human peripheral blood mononuclear cells. The present study aimed to explore the anticancer activities of RBL using human leukemic T-cell lines, Molt-4, Jurkat and HuT-78. RBL exhibited significant binding (>90%) to the cell membrane that was effectively inhibited by complex glycoproteins such as mucin (97% inhibition) and asialofetuin (94% inhibition) but not simple sugars such as N-acetyl-D-galactosamine, glucose and sucrose. RBL induced a dose and time dependent inhibition of proliferation and induced cytotoxicity in the cell lines. The percentage of apoptotic cells, as determined by hypodiploidy, was 33% and 42% in Molt-4 and Jurkat cells, respectively, compared to 3.11% and 2.92% in controls. This effect was associated with a concomitant decrease in the G0/G1 population. Though initiator caspase-8 and -9 were activated upon exposure to RBL, inhibition of caspase-8 but not caspase-9 rescued cells from RBL-induced apoptosis. Mechanistic studies revealed that RBL induced cleavage of Bid, loss of mitochondrial membrane potential and activation of caspase-3. The expression of the anti-apoptotic proteins Bcl-2 and Bcl-X was down regulated without altering the expression of pro-apoptotic proteins- Bad and Bax. In contrast to leukemic cells, RBL did not induce apoptosis in normal PBMC, isolated CD3+ve cells and undifferentiated CD34+ve hematopoietic stem and progenitor cells (HSPCs). The findings highlight the differential effects of RBL on transformed and normal hematopoietic cells and suggest that RBL may be explored for therapeutic applications in leukemia.

The effect of Z36 on Human T-cell Leukemia

Event Abstract Back to Event The effect of Z36 on Human T-cell Leukemia Tsukasa Nakanishi1* 1 Department of Immunology and Parasitology, School of Medicine, Japan Human T-cell Leukemia is aggressive and malignant blood disease. Above all, Adult T-cell Leukemia (ATL) has a very poor prognosis even with intensive chemotherapy, indicating the need for development for new drugs to treat the disease. Z36 which is BCL-XL inhibitor have been known induces autophagy. But the effect in Human T-cell Leukemia of Z36 is not reported. In this study, we demonstrate that the effect of Z36 on cell viability of Human T-cell Leukemia cell lines such as Hut 78, Hut 102, jurkat, MT-1, MT-2 and MT-4 in vitro. Z36 inhibited proliferation of all Human T-cell Leukemia cell lines with different sensitivity at 24 hr after the treatment. The effect of Z36 on Human T-cell Leukemia was restored by bafilomycin-A but not by 3-MA and choloroqine, which are known to be autophagy inhibitors. Western blot showed that Z36 induced LC3-II, which is known as a marker for autophagy. Proliferation of SiATG5-treated cells did not inhibit by Z36. These results suggest that Z36 induced autophagic cell death through ATG5 in human T-cell leukemia. Keywords: T-cell leukemia, cell-death, Apoptosis, Autophagy, z36, cucurbitacin D Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Nakanishi T (2013). The effect of Z36 on Human T-cell Leukemia. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00870 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Tsukasa Nakanishi, Department of Immunology and Parasitology, School of Medicine, Kitakyushu, Japan, naka-t1@med.uoeh-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tsukasa Nakanishi Google Tsukasa Nakanishi Google Scholar Tsukasa Nakanishi PubMed Tsukasa Nakanishi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells

Consumers are exposed daily to several pesticide residues in food, which can be of potential concern for human health. Based on a previous study dealing with exposure of the French population to pesticide residues via the food, we selected 14 pesticides frequently found in foodstuffs, on the basis of their persistence in the environment or their bioaccumulation in the food chain. In a first step, the objective of this study was to investigate if the 14 selected pesticides were potentially cytotoxic and genotoxic. For this purpose, we used a new and sensitive genotoxicity assay (the γH2AX test, involving phosphorylation of histone H2AX) with four human cell lines (ACHN, SH-SY5Y, LS-174T and HepG2), each originating from a potential target tissue of food contaminants (kidney, nervous system, colon, and liver, respectively). Tebufenpyrad was the only compound identified as genotoxic and the effect was only observed in the SH-SY5Y neuroblastoma cell-line. A time-course study showed that DNA damage appeared early after treatment (1h), suggesting that oxidative stress could be responsible for the induction of γH2AX. In a second step, three other pesticides were studied, i.e. bixafen, fenpyroximate and tolfenpyrad, which – like tebufenpad – also had a methyl-pyrazole structure. All these compounds demonstrated genotoxic activity in SH-SY5Y cells at low concentration (nanomolar range). Complementary experiments demonstrated that the same compounds show genotoxicity in a human T-cell leukemia cell line (Jurkat). Moreover, we observed an increased production of reactive oxygen species in Jurkat cells in the presence of the four methyl-pyrazoles. These results demonstrate that tebufenpyrad, bixafen, fenpyroximat and tolfenpyrad induce DNA damage in human cell lines, very likely by a mode of action that involves oxidative stress. Nonetheless, additional in vivo data are required before a definitive conclusion can be drawn regarding hazard prediction to humans.

Abstract 796: Novel mechanisms of drug resistance in leukemia cells associated with changes in HoxA9 and microRNA expression

Abstract The acquisition of resistance to anticancer drugs is a key obstacle to successful cancer therapy. An increasing number of studies have investigated the roles of microRNAs in drug resistance. We previously reported that the upregulation of miR-135b and miR-196b correlated positively with acquired drug resistance in the human T-cell leukemia cell line, CCRF-CEM (CEM), and that the elevation of expression of these two microRNAs in response to the DNA damaging agent etoposide appears to be histiotype-specific (T-T Ho et al, Proceedings of the 102nd Annual Meeting of the AACR 2011). To develop a mechanistic understanding of why these microRNAs are differentially expressed, we have studied miR-196b, which maps between the HoxA9 and HoxA10 genes on chromosome 7p15.2, suggesting that miR-196b and HoxA genes might be co-activated. Indeed, we found upregulation of HoxA9 mRNA after short-term (48 h) exposure of CEM cells to the topoisomerase II inhibitors etoposide and doxorubicin. Of note, we also found that HoxA9 is constitutively overexpressed in multidrug-resistant CEM/VM-1-5 cells. Of interest, we found that HoxA9 is clearly associated with drug resistance, as knockdown of HoxA9 using esiRNA (a heterogeneous mixture of siRNAs that all target the same mRNA) partially sensitized multidrug-resistant CEM/VM-1-5 cells to etoposide. This indicates that the expression of miR-196b is linked to HoxA gene transcription and to drug responsiveness. We subsequently confirmed that the expression of HoxA9 mRNA follows the same pattern in other leukemia cells. For example, both miR-196b and HoxA9 are upregulated in response to short-term etoposide exposure in HL-60 (acute promyelocytic leukemia), but not in solid tumor-derived MCF7 (breast cancer cell line) and its etoposide-resistant subline MCF7/VP, or Rh30 (rhabdomyosarcoma cell line) and its etoposide-resistant subline, Rh30/V1. We are presently assessing the role of HoxA9 in drug resistance in other leukemia cell lines and investigating the mechanistic basis for this effect. In summary, we report here a novel mechanism of anticancer drug resistance in leukemia cells that is associated with HoxA9 and specific changes in microRNA expression. This is the first report that HoxA9 may impact on anticancer drug resistance. (Supported in part by R01 CA40570 from NCI to WTB, and in part by UIC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 796. doi:1538-7445.AM2012-796

IN-VITRO CYTOTOXICITY ANALYSIS OF TAMOXIFEN CITRATE LOADED CROSS-LINKED GUAR GUM NANOPARTICLES ON JURKAT (HUMAN T-CELL LEUKEMIA) CELL LINE

The present investigation was aimed to study the antiproliferative action of tamoxifen citrate (TMX), a non steroidal antiestrogen, on a human T-cell leukemia cell line, Jurkat, as free drug and TMX loaded guar gum nanoparticles. For this we developed a new formulation containing chemically cross-linked guar gum nanoparticles (GG NPs) loaded with tamoxifen citrate (TMX). Single step (oil in water) emulsion and in-situ polymer cross-linking technique was employed to prepare spherical and smooth surfaced nanoparticles in the size range of 200-300nm. Nanoparticle size and shape was confirmed from observation in transmission electron microscope (TEM) analysis. Cytotoxicity on Jurkat (human T-cell leukemia) cell lines as determined by cell growth inhibition after 48 hrs of incubation indicated that tamoxifen citrate loaded guar gum nanoparticles were as efficient as the free drug when applied to the cancer line. However, the crosslinked guar gum nanoparticles loaded with tamoxifen citrate exhibited sustained release of the drug and delayed apoptosis over a long period of time making it suitable for cancer treatment. Keywords: Nanoparticles, guar gum, tamoxifen citrate, Jurkat (human T-cell leukemia) cell line, cytotoxicity, cell death, WST-1 assay.

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

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

Introduction of pro-interleukin-16 inhibits T-lymphoblastic leukemia growth in mice

Pro-interleukin-16 (pro-IL-16) is the precursor to mature interleukin-16 (IL-16) protein. Previous studies have demonstrated that pro-IL-16 can function as a regulator of cell cycle. A number of human T-cell leukemia and lymphoma cell lines are pro-IL-16 deficient. Intracellular expression of pro-IL-16 causes these cell lines to become quiescent, implicating loss of pro-IL-16 as a contributory step in T-cell malignancy. Therefore, we tested whether or not reintroduction of pro-IL-16 into solid tumors in mice could halt tumor growth. MOLT-4 lymphoblastic leukemia cells were stably transfected with a dsRed-tomato virus and were injected subcutaneously into NOD/SCID/γ chain-knockout mice. Tumor growth was monitored with an in vivo imaging system. A pro-IL-16-GFP fusion virus or control GFP only virus was injected into the tumors, and mice were monitored for 1week. Injection of the pro-IL-16-containing lentivirus inhibited growth of established MOLT-4 tumors in mice. Tumor explants exhibited diminished proliferative capacity. Our data support the concept that restoration of pro-IL-16 expression in malignant T cells may have therapeutic value.

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

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

Pseudoephedrine inhibits T-cell activation by targeting NF-κB, NFAT and AP-1 signaling pathways

Pseudoephedrine (PSE) is a stereoisomer of ephedrine that is commonly used as a nasal decongestant in combination with other anti-inflammatory drugs for the symptomatic treatment of some common pathologies such as common cold. Herein, we describe for the first time the effects of PSE on T-cell activation events. We found that PSE inhibits interleukin-2 (IL-2) and tumor necrosis factor (TNF) alpha-gene transcription in stimulated Jurkat cells, a human T-cell leukemia cell line. To further characterize the inhibitory mechanisms of PSE at the transcriptional level, we examined the transcriptional activities of nuclear factor kappa B (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1) transcription factors and found that PSE inhibited NF-κB-dependent transcriptional activity without affecting either the phosphorylation, the degradation of the cytoplasmic NF-κB inhibitory protein, IκBα or the DNA-binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by PSE in stimulated cells. In addition, PSE inhibited the transcriptional activity of NFAT without interfering with the calcium-induced NFAT dephosphorylation event, which represents the major signaling pathway for its activation. NFAT cooperates with c-Jun, a compound of the AP-1 complex, to activate target genes, and we also found that PSE inhibited both JNK activation and AP-1 transcriptional activity. These findings provide new mechanistic insights into the potential immunomodulatory activities of PSE and highlight their potential in designing novel therapeutic strategies to manage inflammatory diseases.

Cycloheximide Suppresses Radiation-induced Apoptosis in MOLT-4 Cells with Arg72 Variant of p53 through Translational Inhibition of p53 Accumulation

The human T-cell leukemia cell line MOLT-4 is highly radiosensitive, and thus it is often used as a model of p53-dependent radiation-induced apoptosis. Two branches of the p53-mediated apoptotic pathway are reported: "transcription-dependent" and "transcription-independent." However, the relative contribution of each in different types of cells is not yet clearly defined. Moreover, recent studies have shown that the codon 72 polymorphic variants of p53 show different sensitivities to apoptosis signals. The Arg72 variant has a more potent apoptosis-inducing activity in mitochondria than the Pro72 variant. Here, we initially investigated the codon 72 polymorphism of p53 in MOLT-4 cells. Analysis of the p53 exon 4 genomic DNA sequence, which includes codon 72, revealed that MOLT-4 cells are homozygous for the allele encoding Arg72. We next investigated the involvement of the transcription-independent function of p53 using an RNA synthesis inhibitor, actinomycin D (ActD), and a protein synthesis inhibitor, cycloheximide (CHX), and found that the apoptosis was suppressed by CHX but not by ActD. We also revealed that the suppressive effect of CHX on apoptosis was specifically mediated by p53, using a p53-knockdown MOLT-4 transfectant. Furthermore, the suppressive effect of CHX on apoptosis was highly correlated with the suppression of p53 protein accumulation, and less correlated with the suppression of p53 target genes expression. These results indicated that p53 transactivation is not necessary to induce apoptosis, and that p53 protein accumulation itself is both necessary and sufficient to do so.

Abstract 2073: Changes in microRNA expression in response to short-term chemotherapeutic exposure is associated with transient anticancer drug resistance

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that act as posttranscriptional repressors of gene expression by binding the 3’ untranslated region of target genes. There is emerging evidence showing that miRNAs regulate diverse biologic processes including multidrug resistance (MDR). Our previous profiling study revealed that certain miRNAs are differentially expressed in multidrug resistant human T-cell leukemia cell line CEM/VM-1-5 compared to drug-sensitive parental cell line CCRF-CEM. To investigate whether these expression-altered miRNAs have a role in MDR, we ectopically expressed three miRNAs that are highly upregulated in CEM/VM-1-5 cells (hsa-miR-135b, −146b, and −196b) in CCRF-CEM cells with the use of lentiviral vectors. We found that overexpression of these miRNAs, individually or in combination, resulted in only slight increases in resistance of CCRF-CEM cells to teniposide, the agent used to select CEM/VM-1-5 cells. Moreover, we were unable to sensitize CEM/VM-1-5 cells to etoposide by suppression of hsa-miR-135b expression using LNA antisense oligonucleotide. Our data implied that these miRNAs do not confer drug resistance directly. However, it is still not clear whether these miRNAs play any role in the development of MDR. Therefore, we asked whether the expression of these miRNAs changed in CCRF-CEM cells after short exposure (3 to 48 hours) to various chemotherapeutic drugs at concentrations below their IC50-values. We observed substantial increases in hsa-miR-135b and −196b expression in cells treated with etoposide, doxorubicin and topotecan, but not in cells treated with vinblastine. These results suggested that the upregulation of these two miRNAs might be the consequence of DNA damage. Time-course and dose-response studies showed that the elevation of hsa-miR-135b and −196b correlated positively with the drug exposure time and drug concentration. Furthermore, the levels of miRNAs gradually decreased after the withdrawal of etoposide from the media following 48 h exposure to etoposide. Strikingly, the elevated expression of the miRNAs accompanied increased drug resistance. CCRF-CEM cells exposed to etoposide for 48 h were over 50-fold more resistant than control cells. The acquired drug resistance, like elevated expression of miRNAs, did not appear to be stable and became attenuated with increasing duration of the drug-free incubation. Together, our results suggest that miRNAs might play a significant role in cellular response to genotoxic agents and could be involved in the development of cancer drug resistance, but their roles in conferring resistance remain to be determined. That said, targeting miRNA expression could be a potential approach to prevent multidrug resistance. (Supported in part by 5RO1CA040570 from NCI to WTB, and in part by UIC) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2073.

Establishment of a high-throughput screening system for universal anti-HIV targets

The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research. Virus-cell-based screening approaches offer some advantages in the quest for novel inhibitors because they include multiple targets in a single screen and in some cases reveal targets not captured in biochemical assays. In this study, a high-throughput screening (HTS) system for HIV-1 inhibitors was developed, which allows the simultaneous screening of all the HIV-1 targets required for replication in the cell culture. HeLa/cluster of differentiation 4 (CD4)/long terminal repeat (LTR) indicator cells, which stably expressed high levels of HIV receptor CD4 and contained the firefly luciferase reporter gene under the control of the HIV-1 LTR promoter, were generated. The expression of CD4 and LTR function in this cell line was validated by Western blot and luciferase assay. MT2 cells, a human T-cell leukemia cell line that support high levels of HIV-1 replication, were infected with HIV-1, and then the infected MT2 cells were co-cultured with HeLa/CD4/LTR cells. In the optimized assay conditions, HIV-1 replication occurs rapidly in the MT2 cells, resulting in the infection of the HeLa/CD4/LTR cells and a significant induction of luciferase signals through LTR, which is activated by the expression of HIV-1 tat gene. The luciferase signals of HeLa/CD4/LTR cells co-cultured with HIV-1 infected MT2 cells were significantly stronger than the signals of noninfected HeLa/CD4/LTR cells (P < 0.001). The inhibitory effects of HIV-1 inhibitors (3′-Azido-3′-deoxythymidine [AZT], efavirenz [EFV], and nevirapine [NVP]) were evaluated with this assay, and the inhibitory concentration 50% (IC50) values of the above three inhibitors were 58, 1.4, and 85 nmol/L, respectively, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. The Z′ factor had a value of 0.563, indicating this is a very robust assay. These results suggested that HIV-1 infection assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of HTS campaigns.

Dispersion state and toxicity of mwCNTs in cell culture medium with different T80 concentrations

In this study, size distribution, zeta potential, shape, and toxicity of multi-walled carbon nanotubes (mwCNTs), and effect of non-ionic detergent Tween 80 (T80) concentrations (0%, 0.2%, and 1%) on the dispersion quality and cell viability are investigated. Nanotubes are suspended in biological solutions (DMEM, RPMI) with three different concentrations (10, 50, and 100 μg/ml) and toxicological investigations are carried on human T-cell leukemia (Jurkat) and human pancreatic carcinoma (PANC1) cell lines. According to light and transmission electron microscopy results, mwCNTs form well-defined and regular bundles in the presence of 1% T80 surfactant; whereas more irregular structures are present in absence of T80. Dispersion quality is represented in terms of the size distribution from dynamic light scattering (DLS) experiments and its second moment. Dispersion quality of the mwCNTs decreases with decreasing T80 concentration, while the constituents of RPMI and DMEM increase the dispersion quality. No significant differences between the dispersive effects of RPMI and DMEM suspensions are observed. Zeta potential of the mwCNTs is measured using electrophoretic light scattering. Variations in the nanotube and T80 concentrations do not change the zeta potential significantly. T80 concentrations above 0.2% are found to be toxic for Jurkat andPANC1 cells.

Apoptosis in immunocytes induced by several types of pesticides

Several types of pesticides, such as organophosphates and organochlorines, can induce thymocyte apoptosis, resulting in thymic atrophy and predisposing the highly sensitive fetal immune system to loss of tolerance to self-antigens and subsequent increased risk for autoimmune disease and allergies. In the studies here, mouse primary thymocytes and a human acute T-cell leukemia cell line (J45.01) were employed to examine potential thymocyte apoptosis induced by several types of chemicals, including several commonly-used pesticides. Thymocytes and J45.01 cells were treated for 4 or 8 hr with varying doses of metamidophos, parathion, PNMC, or methoxychlor; dexamethasone was used as a positive control. Apoptosis, cell viability, the proportion of Annexin-V+ cells, the activities of caspases 3/7, 8, and 9, and the levels of DNA fragmentation in both the J45.01 cells and thymocytes were then examined. The results here show that with both cell types, there was an increase in the proportion of annexin-V+ cells and levels of DNA fragmentation following exposure to parathion, PNMC, methoxychlor, or dexamethasone (positive control); however, the levels of sensitivity appeared to differ between the cell types. Furthermore, caspase-7 and -8 activities also differed between the J45.01 cells and thymocytes when treated with PNMC, methoxychlor, or dexamethasone. A more precise characterization of these inter-cellular differences is the logical next step in our studies of the effects of these (and other) pesticides on immune cell integrity. These specific types of follow-on mechanistic experiments are currently underway in our laboratories.

Effects of (15S)‐hydroperoxyeicosatetraenoic acid and (15S)‐hydroxyeicosatetraenoic acid on the acute‐ lymphoblastic‐leukaemia cell line Jurkat: activation of the Fas‐mediated death pathway

The antiproliferative effects of 15-LOX (15-lipoxygenase) metabolites of arachidonic acid {(15S)-HPETE [(15S)-hydroperoxyeicosatetraenoic acid] and (15S)-HETE [(15S)-hydroxyeicosatetraenoic acid]} and the mechanism(s) involved were studied in the human T-cell leukaemia cell line Jurkat. (15S)-HPETE, the hydroperoxy metabolite of 15-LOX, inhibited the growth of Jurkat cells 3 h after exposure and with an IC(50) value of 10 microM. The hydroxy metabolite of 15-LOX, (15S)-HETE, on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC(50) value of 40 microM. The cells exposed to 10 microM (15S)-HPETE for 3 h or to 40 microM (15S)-HETE for 6 h showed increased expression of Fas ligand and FADD (Fas-associated death domain), caspase 8 activation, Bid (BH3-interacting domain death agonist) cleavage, decrease in mitochondrial membrane potential, cytochrome c release, caspase 3 activation, PARP-1 [poly(ADP-ribose) polymerase-1] cleavage and DNA fragmentation, suggesting the involvement of both extrinsic and intrinsic death pathways. Further studies on ROS (reactive oxygen species) generation revealed the involvement of NADPH oxidase. In conclusion, the present study indicates that NADPH oxidase-induced ROS generation activates the Fas-mediated death pathway.

NTAL Potentiates Glucocorticosteroid-Induced Apoptosis in T-ALL – in Vivo Observations and Experimental Model

PAG, LAT, NTAL and LIME belong to category of transmembrane adaptor proteins (TRAPs). They do not possess an enzymatic or kinase function, but they are involved in mediation of signal transmission from surface receptors to cell nucleus. We propose that some of them are engaged in development or maintenance of leukaemia.We have previously demonstrated that expression status of TRAPs at mRNA level is specific in some subgroups of childhood ALL, particularly in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) with the TEL/AML1 fusion gene. Furthermore, we have described that variable expression of NTAL mRNA is related to the response to initial glucocorticosteroid pre-phase in the treatment of childhood T-ALL; patients with high NTAL levels show better treatment response compared to the low-NTAL cases.In the current study, we aimed to prove experimentally that different levels of NTAL protein influence response of leukaemic T cells to glucocorticosteroids.In the wild type Jurkat cells (human T-cell leukaemia cell line) the NTAL protein is undetectable. For the in-vitro experiments we created a derivative Jurkat cell line transfected with the NTAL construct. In the derivative cell line, the NTAL positivity at both mRNA and protein level was verified using RT-PCR and Western blotting. The derivative cell line in a cell culture behaves similar as wild type Jurkat cell line. Transfectants and wild type Jurkat cells were incubated with methylprednisolone, dexamethasone or with solvent alone (H2O) as a negative control. Using flow cytometry we determined a percentage of surviving cells after 24, 48 and 72 hours of treatment. Cells were stained with Annexin V and DAPI and living cells were defined as Annexin V, DAPI negative. At each time point, the number of living cells in the negative control was set to 100%. After 48 hours of dexamethasone treatment the number of surviving cells in the Jurkat wild-type was higher by 12% compared to the Jurkat cells expressing NTAL. After 72 hours this difference was even more prominent reaching 46%. The same effect of methylprednisolone treatment was less pronounced (non-significant difference at 48 hours and 5% difference at 72 hours). Based on our experimental data we propose that NTAL acts in T-cells as a putative tumour suppressor. A plausible mechanism of its function is that NTAL competitively inhibits another adaptor LAT, which is required for signal propagation to the nucleus in T-cells. NTAL and LAT can compete for its localisation in proper “lipid rafts” or for palmitoylation. Our data can be used for subsequent functional analysis of signaling pathways in leukaemic blasts as well as in physiological lymphoid cells.

Transcription of HERV-E and HERV-E-related sequences in malignant and non-malignant human haematopoietic cells

Infection of a human T-cell leukaemia cell line (HSB-2) with HHV-6 led to the induction of exosome-like-particles attached to newly formed HHV-6 enveloped particles and to amplification of a 1642 bp molecule consisting of a partial human endogenous retrovirus (HERV)-E polymerase gene and repetitive sequences. We initiated an analysis of transcriptional patterns of predicted genes from HERV-E sequences in normal and malignant haematopoietic cells. Transcription patterns of regions corresponding to gag, pol and env genes at different chromosomal loci varied among cell types tested. Several specific transcripts were only observed in malignant haematopoietic cells and transcriptional activity varied among different malignant cell types. A transcript of 7.1 kb spanning the complete gag, pol and env gene region, originating from chromosome 8p23, was identified in normal peripheral blood cells and cells of the chronic myeloid leukaemia cell line K562. Our study describes new active HERV-E sequences and new loci throughout the human genome.

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

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

GaAlAs (830 nm) low‐level laser enhances peripheral endogenous opioid analgesia in rats

Low-level laser therapy (LLLT) has been reported to relieve pain with minimal side effects. Recent studies have demonstrated that opioid-containing immune cells migrate to inflamed sites and release beta-endorphins to inhibit pain as a mode of peripheral endogenous opioid analgesia. The present study investigates whether LLLT may enhance peripheral endogenous opioid analgesia. The effect of LLLT on opioid analgesia and production was evaluated in vivo in a rat model of inflammation as well as in vitro in Jurkat cells, a human T-cell leukemia cell line. mRNA expression of the beta-endorphin precursors proopiomelanocortin and corticotrophin releasing factor was assessed by reverse transcription polymerase chain reaction. LLLT produced an analgesic effect in inflamed peripheral tissue which was transiently antagonized by naloxone. Beta-endorphin precursor mRNA expression increased with LLLT, both in vivo and in vitro. This study demonstrates that LLLT produces analgesic effects in a rat model of peripheral inflammation. We further revealed an additional mechanism of LLLT-mediated analgesia via enhancement of peripheral endogenous opioids. These findings suggest that LLLT induces analgesia in rats by enhancing peripheral endogenous opioid production in addition to previously reported mechanisms.

Telomere attrition, and chromosome instability via downregulation of TRF2 contributes to arsenic trioxide-induced apoptosis of human T-cell leukemia cell line molt-4 cells

Overexpression of human telomere repeat binding factor 2 (TRF2), which may play an important role in the fate of cancer cells, has been observed in adult T-cell leukemia. Previous reports have shown that the inhibition of TRF2 results in the apoptosis of cancer cells. In this study, we demonstrated that arsenic trioxide (As2O3) induced in vitro growth inhibition and/or apoptosis of human T-cell leukemia cell line Molt-4 in a caspase-independent manner. Telomerase activity was not inhibited, although the level of the reverse transcriptase subunit of the human telomerase gene (hTERT) mRNA expression was down regulated during the early times and then recovered to the level found in untreated controls about 48 hours after treatment with As2O3. Furthermore, a remarkable telomere shortening related to exposure of As2O3 was observed in 50 population doubling. In contrast, the alteration of telomere length did not occur after exposure to higher concentration of As2O3 (10 µM) for 24 hours and 48 hours, respectively, suggesting that the shortening of telomeres induced by As2O3 is dependent of a series of cell division cycles. Chromosomal analysis showed that As2O3 exposure caused chromosomal end-to-end fusion in human T-cell leukemia cells while downregulation of TRF2 was observed. Finally, the inhibition of TRF2 protein expression and the sensitivity to As2O3 in a panel of leukemia cell lines were checked. The data revealed that inhibition of TRF2 rendered leukemia cells more susceptible to As2O3. In conclusion, the downregulation of TRF2 by As2O3 contribute to chromosomal end-to-end fusion, and apoptosis in leukemia cells, suggesting that TRF2 could be an attractive target for new therapies of leukemia.