T-cell Leukemia Jurkat Research Articles

Study of molecular mechanisms of proapoptotic action of novel heterocyclic 4-thiazolidone derivatives

Aim. Mechanisms of induction of apoptosis signaling pathways in mammalian tumor cells treated by novel heterocyclic 4-thiazolidones with different side groups were studied. Methods. Annexin V/propidium iodide and DAPI (4',6-diamidino-2-phenylindole) staining of cells, Western-blot analysis of specific proteins. Results. 4-Thiazolidone derivatives of various structure possess similar cytotoxic activity in vitro (²N50 = 5 µM), and induce apoptosis in both leukemia (Jurkat, CCRF-CEM) and carcinoma (MCF-7, MDA-MD-231) cells. Western-blot analysis of the expression of several proteins of apoptosis signaling showed that the structure of lateral groups of 4thiazolidones may directly affect biological activity of these proteins in leukemia cells. In particular, compounds Les-3120 (pyrazoline-substituted thiazolidinone) and Les-3166 (thiazolidinone-benzothiazole conjugate) induced receptor-mediated apoptosis in Jurkat T-leukemia cells. 4-Iminothiazolidinone Les-3372 caused mitochondrial type apoptosis, mediated by AIF protein. Conclusions. Structure-functional relationships between the presence of specific side groups in novel 4-thiazolidones and the signaling apoptotic pathways induced by these compounds have been established. The obtained results allow designing new, «hybrid» compounds which can simultaneously induce more than one apoptotic pathway in tumor cells.

Probing nanoparticle interactions in cell culture media

Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco's Modified Eagle's Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100nm, regardless of their nominal sizes. The DLS and UV–vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50nm particles enter cells at faster rates than the 25nm particles.

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.

Abstract C29: Elevated CDKN1A (p21) expression in human cancer cell variants selected for resistance to the novel microtubule depolymerizing agent BAL27862 (active moiety of BAL101553).

Abstract BAL27862 is a synthetic small molecule that induces tumor cell apoptosis due to inhibition of tubulin polymerization via a potentially new mechanism of action. Administered as the prodrug BAL101553, BAL27862 is currently undergoing Phase I evaluation in advanced cancer patients. We have previously demonstrated that BAL27862 retains activity in a broad panel of MDR1(+) and MDR1(−) human breast and ovarian cancer cell variants selected for resistance to taxanes (Abstract 4412, AACR 2010). In this current study, we established five variants selected for stable drug resistance in cell lines from human lung cancer (H460 and A549), acute T-cell leukemia (Jurkat), ovarian cancer (SK-OV-3) and cervical cancer (HeLa). These variants are approximately 3- to 24-fold resistant to BAL27862. The resistance is not transporter mediated, with no activation of ABCB1 (MDR1/P-glycoprotein) following drug selection, no change in activity in cells co-incubated with known P-glycoprotein inhibitors (PSC-833, 2 μM) and no difference in either accumulation or retention of [14C]-BAL27862 relative to parental controls. Tubulin polymerization assays indicated a higher proportion of tubulin in the polymerized fraction from resistant lines, indicating microtubule stabilization. Consistently, most lines exhibited increased sensitivity to microtubule stabilizers (e.g. resistance factors [RF = antiproliferative IC50 resistant line/parental line] for paclitaxel: H460=0.4; Jurkat=0.7). Strikingly, differential responses to standard microtubule-destabilizing agents were evident. For example, RFs for Jurkat cells were lower for both vincristine (2.5) and colchicine (10) as compared to BAL27862 (24); whereas, similar RFs were measured in SK-OV-3 (3, 5 and 4, respectively). Finally, relative to parental controls, altered tubulin isoform content was observed in some BAL27862-selected lines, but elevated CDKN1A (p21) protein expression was observed in 4 out of 5 of the resistant variants (SK-OV-3, H460, A549 and HeLa). Q-PCR analysis indicated an associated elevation in p21 mRNA levels only in lines with enhanced protein expression. As aberrant p21 expression has been previously related to poor prognosis and chemotherapy resistance in cancer patients, the potential of using this biomarker to support patient stratification efforts in future trials is worthy of further study. The functional significance of p21 to the resistance phenotype, and other key alterations identified through more detailed genomic profiling (Stanford's HEEBO microarrays), will be studied by gene transfer and gene silencing experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C29.

The potential of a mistletoe ( Viscum album L.) extract to alleviate adverse effects of cancer chemotherapy: An in vitro study

Objectives: Mistletoe extracts have been shown to provide deoxyribonucleic acid (DNA)-stabilizing effects in human peripheral blood mononuclear cells (PBMC) in vitro. We investigated the effect of a mistletoe extract on PBMC with and without concomitant treatment with cyclophosphamide and compared mitochondrial activity and replication of normal PBMC with that of a T-cell leukemia cell line. Design: The experiments were performed with PBMC of healthy blood donors and the T-cell leukemia Jurkat cell line. Cells were pre-incubated with mistletoe extract for 60–65 h. 4-Hydroperoxycyclophosphamide (4-hpc, precursor of 4-hydroxycyclophosphamide) was added for 2 h, after which mitochondrial activity and replication were measured. All experiments were randomized and blinded. Main outcome measures: Cell mitochondrial activity and replication were assessed with spectrophotometric analysis of WsT-1 reduction and BrdU incorporation. Results: The application of 4-hpc consistently reduced mitochondrial activity and replication of PBMC and Jurkat cells. Mistletoe extract strongly enhanced PBMC mitochondrial activity and replication (with or without 4-hpc) and partially inhibited Jurkat cell replication (with 4-hpc only). Compared to mistletoe untreated cells, enhancement of PBMC mitochondrial activity by mistletoe extract was independent of treatment with 4-hpc, but enhancement of PBMC replication by mistletoe extract was stronger when treated with 4-hpc. Conclusions: Mistletoe extract strongly stimulated healthy PBMC but not malignant Jurkat cells. In addition, mistletoe extract seemed to partially protect healthy PBMC – but not malignant Jurkat cells – from the cytostatic effect of 4-hpc. The results motivate further preclinical and clinical investigations of mistletoe extracts as an adjuvant medication in cancer therapy to alleviate side effects of conventional therapy.

Aqueous mistletoe extracts versus purified mistletoe lectin-I (pML-I): Effects on melanoma growth and spread in a human melanoma xenograft scid mouse model

Objectives: Mistletoe extracts have been shown to provide eoxyribonucleic acid (DNA)-stabilizing effects in human periphral blood mononuclear cells (PBMC) in vitro. We investigated the ffect of amistletoe extract onPBMCwith andwithout concomitant reatment with cyclophosphamide and compared mitochondrial ctivity and replication of normal PBMC with that of a T-cell eukemia cell line. Design: The experiments were performed with PBMC of ealthy blood donors and the T-cell leukemia Jurkat cell ine. Cells were pre-incubated with mistletoe extract for 0–65h. 4-Hydroperoxycyclophosphamide (4-hpc, precursor of -hydroxycyclophosphamide)was added for 2h, after whichmitohondrial activity and replication were measured. All experiments ere randomized and blinded. Main outcomemeasures: Cell mitochondrial activity and repliation were assessed with spectrophotometric analysis of WsT-1 eduction and BrdU incorporation. Results: The application of 4-hpc consistently reduced mitohondrial activity and replication of PBMC and Jurkat cells. istletoe extract strongly enhanced PBMC mitochondrial activty and replication (with or without 4-hpc) and partially inhibited urkat cell replication (with 4-hpc only). Compared to mistleoe untreated cells, enhancement of PBMC mitochondrial activity y mistletoe extract was independent of treatment with 4-hpc, ut enhancement of PBMC replication by mistletoe extract was tronger when treated with 4-hpc. Conclusions: Mistletoe extract strongly stimulated healthy BMC but not malignant Jurkat cells. In addition, mistletoe extract eemed to partially protect healthy PBMC – but not malignant urkat cells – from the cytostatic effect of 4-hpc. The results motiate further preclinical and clinical investigations of mistletoe xtracts as an adjuvant medication in cancer therapy to alleviate ide effects of conventional therapy.

Synthesis and antiproliferative evaluation of 3,5-disubstituted 1,2,4-triazoles containing flurophenyl and trifluoromethanephenyl moieties

An efficient 1,3-dipolar cycloaddition method was performed for the synthesis of a series of monofluoro- and trifluoromethane-3,5-disubstituted 1,2,4-triazoles. This efficient cycloaddition method was to react hydrazonoyl hydrochlorides with a series of aldehydes in the presence of NEt(3) as catalytic basic agent to provide the corresponding product in 28-94%. Their growth inhibitory results against cancer cells indicated that some of the fluorine- and trifluoromethane-containing compounds could effectively inhibit the growth of NCI-H226 and T-cell leukemia (Jurkat) cells. Among the compounds, trifluoromethane-containing 1,2,4-triazoles possessed the five-membered ring groups on the C-5 position of the triazolic ring, including cyclopentyl, 3-furyl, 3-thienyl, and 2-pyrrolyl, possessed the significant inhibitory activity for NCI-H226 cancer cells.

Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.

Continuous cell from cell separation by traveling wave dielectrophoresis

AbstractIn this contribution we present a label-free continuous separation device for suspended-grown biological cells based on traveling wave dielectrophoresis (twDEP). Suspended-grown cells could otherwise only be separated by flow-cytometry which requires elaborate labeling steps. The chip consists of a structured PDMS layer on glass in which the channels have been defined. Parallel electrodes positioned along the microfluidic channel were used to expose selected cells to a twDEP force perpendicular to a pressure driven flow. With this device we have successfully separated viable Saccharomyces cerevisiae and Jurkat (T-cell leukemia) cells from a mix also containing cell culture debris, non-viable cells, and Lactobacillus casei bacteria.

Cyclin-Dependent Kinase Inhibitors Enhance Sensitivity to Methotrexate In Human T-Cell Leukemia Jurkat Cells

AbstractAbstract 3976Methotrexate (MTX), a classical anti-folate drug, has been used in the treatment of various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. In fact, it is well known that elevated expression of DHFR is a direct factor in clinical therapeutic resistance to MTX. Therefore, small compounds that repress DHFR expression may be useful as chemosensitizers in combination with MTX.First of all, we demonstrated that the sensitivity to MTX was enhanced by the knockdown of the DHFR gene in Jurkat cells. Concretely, the expression of DHFR was efficiently knocked down by siRNA transiently transfected using electroporation. We subsequently confirmed that pretreatment with DHFR siRNA significantly enhanced sensitivity of Jurkat cells to MTX in vitro with colony formation assays. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX.On the other hand, the expression of DHFR has been assumed to be controlled by the transcription factor E2F. Unphosphorylated retinoblastoma (RB) protein is known to bind and inhibit the E2F family. Actually, it has been reported RB protein, which acts as a negative regulator of cell cycle transition, suppresses the expression of DHFR. When RB is phosphorylated by cyclin-dependent kinase (CDK)-cyclins, E2F is released from the complex and binds to a cis-element in the promoter region of various genes involved in cell proliferation and DNA synthesis. In other words, CDK inhibitors activate RB function and inhibit E2F function. Then we investigated whether CDK inhibitor acted as a suppressor of DHFR through the E2F site of the promoter, and whether it could be used to enhance sensitivity to MTX.We found that SU9516, a small chemical CDK inhibitor, showed the inhibition of cell growth without the significant increase of dead cells. Furthermore we also found that SU9516 decreased phospho-RB (Ser780) and reduced the expression of both DHFR mRNA and protein, suggesting that SU9516 activates the RB pathway. The DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. The results suggested that SU9516 inhibited DHFR expression at the promoter level. Furthermore, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay.To expand these results in Jurkat cells, we have also examined other leukemic cell lines (human T cell leukemia CCRF-CEM cells and human erythroleukemia K562 cells) and confirmed that SU9516 also repressed the levels of DHFR protein and phopho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in both of cell lines. These results might indicate that pretreatment of SU9516 is effective to enhance sensitivity to MTX in not only the T-cell leukemia cell lines but also erythroleukemia cell lines such as K562 cells.Finally, to expand these results by SU9516, we have examined other CDK inhibitors (Purvalanol A and Cdk4/6 Inhibitor IV) and confirmed that these agents also decreased DHFR protein and phospho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in Jurkat cells as similar as SU9516.In conclusion, we showed for the first time that SU9516 significantly enhanced sensitivity to MTX through the inhibition of DHFR expression in human T-cell leukemia Jurkat cells and CCRF-CEM cells, and human erythroleukemia K562 cells. Although further study is needed, these results raise the possibility that a combination of CDK inhibitors and MTX may be a suitable chemotherapeutic option for enhancing sensitivity to MTX when treating hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

The Control of Human Immunosystem by Using Paeony Root Drug

Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1200 years in China, Korea and Japan for its anti-allergic, anti-inflamatory and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T-lineage cells and human T-cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N-acetyl cysteine (NAC), successfully attenuated the PF-induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen-activated protein (MAP) family kinases, extracellular signal-regulated kinase, c-Jun amino-terminal kinase (JNK) and p38 MAP kinase. Curcumin, an anti-oxidant and JNK inhibitor, inhibited PF-induced apoptosis, suggesting the possible involvement of curcumin-sensitive JNK or other redox-sensitive elements in PF-induced apoptosis. These results partially explain the action mechanism of PF-containing paeony root as a herbal medicine.Keywords: paeoniflorin; paeony root; apoptosis; reactive oxygen species (ROS); redox; CD8+ CD122+ regulatory T cells

Guided Dielectrophoresis: A Robust Method for Continuous Particle and Cell Separation

In this paper, we report on an on-chip separation device that is based on guided dielectrophoresis (DEP). The simple design comprises a pair of electrodes and a microfluidic channel for sample delivery. Continuous separation into two distinct populations (corresponding to positive and negative DEP, respectively) in the microchannel was achieved without the need of accurate hydrodynamic focusing or multiple excitation frequencies. In this device, the sample position after separation is only determined by the geometry of the channel and the electrodes, which results in a separation process that is robust against fluctuations in separation voltage and flow caused by vibrations in the setup. The separation principle has been simulated and proven by measurements comprising polystyrene beads and two human cell lines: Jurkat (T-cell leukemia) and HeLa (cervical cancer). Automatic image processing was used to detect the particle positions at the separation channel outlet.

Effect of bovine lactoferricin on DNA methyltransferase 1 levels in Jurkat T-leukemia cells

Abnormal methylation of the promoter of several genes is common in patients with acute lymphoblastic leukemia. Methylation of DNA is brought about by DNA methyltransferases (DNMT). Bovine lactoferricin (Lfcin B) is a cationic peptide that possesses potent in vitro and in vivo anticancer activity and might affect the expression of DNMT1. In the current study, we determined the mRNA and protein expression of DNMT1 in Jurkat T-leukemia cells, after incubation with Lfcin B, by real-time quantitative reverse transcription PCR and Western blot analysis. The results of real-time quantitative reverse transcription PCR showed that DNMT1 expression in Jurkat T-leukemia cells was reduced after treatment with Lfcin B, and Lfcin B reduced the half-life of DNMT1 mRNA from approximately 8 to 2h. The results of Western blot analysis showed that the expression of DNMT1 protein was down-modulated by Lfcin B in Jurkat T-leukemia cells. Moreover, we found that protein biosynthesis in Jurkat T-leukemia cells was essential for Lfcin B to down-modulate the expression of DNMT1.

Proteomic analysis revealed association of aberrant ROS signaling with suberoylanilide hydroxamic acid-induced autophagy in Jurkat T-leukemia cells

Suberoylanilide hydroxamic acid (SAHA) is a newly emerging histone deacetylase inhibitor (HDACi) and has been approved in phase II clinical trials for treating patients with cutaneous T-cell lymphoma. Autophagy is a conserved self-digestion process that degrades cytoplasmic materials and recycles long-lived proteins and organelles within cells. In this study, we demonstrate that SAHA stimulates autophagy in Jurkat T-leukemia cells, which was evidenced by the appearance of autophagic vacuoles, formation of acidic vesicular organelles, recruitment of LC3-II to the autophagosomes and conversion of LC3-I to LC3-II. Moreover, SAHA treatment upregulated expression of Beclin 1 and Atg7 and promoted formation of the Atg12-Atg5 conjugate. Furthermore, inhibition of autophagy by chloroquine (CQ) enhanced SAHA-induced apoptosis. To determine the underlying mechanism of SAHA-induced autophagy, two complementary proteomic approaches (2-DE and SILAC), coupled with ESI-Q-TOF MS/MS analysis are utilized to profile differentially expressed proteins between control and SAHA-treated Jurkat T-leukemia cells. In total, 72 proteins were identified with significant alterations. Cluster analysis of the changed proteins reveal several groups of enzymes associated with energy metabolism, anti-oxidative stress and cellular redox control, which suggested an abnormal reactive oxygen species (ROS) production in SAHA-treated Jurkat T-leukemia cells. These observations were further confirmed by ROS chemiluminescence assay. Mechanistic studies revealed that SAHA-triggered autophagy was mediated by ROS production, which could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we illustrated that Akt-mTOR signaling, a major suppressive cascade of autophagy, was inactivated by SAHA treatment. Taken together, our study identifies autophagy as a reaction to counter increased ROS and is thus involved as a cellular prosurvival mechanism in response to SAHA treatment.

Synthesis, Characterization, Mechanism of Decomposition, and Antiproliferative Activity of a Class of PEGylated Benzopolysulfanes Structurally Similar to the Natural Product Varacin

Benzopolysulfanes, 4-CH(3)(OCH(2)CH(2))(3)NHC(O)-C(6)H(4)-1,2-S(x) (x = 3-7 and 9) were synthesized with a PEG group attached through an amide bond and examined for water solubility, antitumor activity, and propensity to equilibrate and desulfurate. LCMS and HPLC data show the PEG pentasulfane ring structure predominates, and the tri-, tetra-, hexa-, hepta-, and nonasulfanes were present at very low concentrations. The presence of the PEG group improved water solubility by 50-fold compared to the unsubstituted benzopolysulfanes, C(6)H(4)S(x) (x = 3, 5, and 7), based on intrinsic solubility measurements. Polysulfur linkages in the PEG compounds decomposed in the presence of ethanethiol and hydroxide ion. The PEG pentathiepin desulfurated rapidly, and an S(3) transfer reaction was observed in the presence of norbornene; no S(2) transfer reaction was observed with 2,3-dimethylbutadiene. The antitumor activities of the PEG-substituted benzopolysulfane mixtures were analyzed against four human tumor cell lines PC3 (prostate), DU145 (prostate), MDA-MB-231 (breast), and Jurkat (T-cell leukemia). The PEG-conjugated polysulfanes had IC(50) values 1.2-5.8 times lower than the parent "unsubstituted" benzopolysulfanes. Complete cell killing was observed for the PEG polysulfanes at 4 microM for PC3 and DU145 cells and at 12 muM for MDA-MB-231 cells. The results suggest that solubilization of the polysulfur linkage is a key parameter to the success of these compounds as drug leads.

CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

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.

The Ca2+ channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells

Flunarizine is a Ca(2+) channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.

Continuous separation of viable cells by travelling wave dielectrophoresis

In this contribution we present a label-free continuous separation device for suspended-grown biological cells based on travelling wave dielectrophoresis (twDEP). Suspended-grown cells could otherwise only be separated by flow-cytometry which requires elaborate labelling steps. The biochip consists of a structured PDMS layer on glass in which the channels have been defined. Parallel electrodes positioned along the microfluidic channel were used to expose selected cells to a twDEP force perpendicular to a pressure driven flow. With this device we have successfully separated suspended-grown viable Jurkat (T-cell leukaemia) cells from a mix also containing non-viable cells and L. casei bacteria.

Role of microRNA-143 in Fas-mediated apoptosis in human T-cell leukemia Jurkat cells

Treatment of Jurkat T cells with Fas-activating antibody (CH-11) facilitated rapid cell death that was shown to be caspase-dependent apoptosis. The expression of miR-143 was up-regulated during the apoptosis with time. The increased expression of miR-143 emerged from 1 to 2h after the treatment, at which time the caspases-8 and -3 were also activated; and this increase was almost canceled by the pretreatment with an inhibitor of caspase-3 or -8. Furthermore, the transfection of Jurkat cells with mature miR-143 induced a significant growth suppression and enhancement of CH-11-induced apoptosis. On the contrary, an extracellular signal-regulated protein kinase 5 (ERK5), which was determined to be a target of miR-143 in colon cancer DLD-1 cells, was time-dependently down-regulated at the translational level after the treatment. During the apoptosis, the expression level of FasL was maintained and the level of nuclear-Foxo3a was increased in the early phase. These data suggest that the up-regulation of miR-143 could be related to the apoptosis in part by targeting ERK5, which leads to promotion of Foxo3a/FasL positive feedback loop.