Arsenic trioxide concentration determines the fate of Ewing’s sarcoma family tumors and neuroblastoma cells in vitro

AbstractArsenic trioxide (As2O3) induces both the differentiation and apoptosis of acute promyelocytic leukemia cells in a concentration dependent manner. We assessed the effects of As2O3 in CADO-ES Ewing’s sarcoma (ES), JK-GMS peripheral primitive neuroectodermal tumor (PNET), and SH-SY5Y neuroblastoma cells, as they share common histogenetic backgrounds. As2O3 at low concentrations (0.1–1μM) induced SH-SY5Y differentiation, and whereas PNET cells acquired a slightly differentiated phenotype, change was minimal in ES cells. Extracellular signal-regulated kinase 2 (ERK2) was activated at low As2O3 concentrations, and PD98059, an inhibitor of MEK-1, blocked SH-SY5Y cell differentiation by As2O3. High concentrations (2–10μM) of As2O3 induced the apoptosis in all three cell lines, and this was accompanied by the activation of c-jun N-terminal kinase. The generation of H2O2 and activation of caspase 3 were identified as critical components of As2O3-induced apoptosis in all of the above cell lines. Fibroblast growth factor 2 enhanced As2O3-induced apoptosis in JK-GMS cells. The overall effects of As2O3 strongly suggest that it has therapeutic potential for the treatment of ES/PNET

Functional Modulation of Regulatory T Cells by IL-2

<div><p>The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis <i>in vivo</i> and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4⁺CD25⁺ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4⁺CD25⁺ fraction is still frequently used for functional assays of Tregs. Although, the CD4⁺CD25⁺ fraction substantially overlaps with the FoxP3⁺ fraction, the minor mismatch between CD4⁺CD25⁺ and FoxP3⁺ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4⁺FoxP3⁺ as well as CD4⁺CD25⁺ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4⁺CD25⁺ fraction the FoxP3⁺ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions were more sensitive than the CD4⁺CD25^- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells.</p></div

Low dose IL-2 enhanced, while high dose abrogated the suppressive activity of Tregs.

<p>CFSE-labeled CD4⁺CD25^- responder cells were cultured in the absence or presence of Tregs (CD4⁺CD25⁺ or CD4⁺FoxP3⁺ cells) to study the suppressive activity of Tregs. Responder cells, Tregs and CD11c⁺ dendritic cells were cultured together at a ratio of 1:0.5:0.2, respectively, with stimulation by soluble anti-CD3e (333 ng/mL). After three days of co-culture, the cells were harvested and stained with CD4-Pacific blue, and whole cells were acquired for analysis. Live CD4⁺CFSE⁺ cells were gated for analysis of the proliferative response of CFSE-labeled responder cells. A representative series of histograms from three separate experiments with the same pattern of results are shown (A). The numbers are precursor frequency (Pf) (%). Data are mean ± SE of three separate experiments with 4 replicate wells per dilution (B and C). P<0.05, compared with the control values (in the absence of IL-2) of CD4⁺CD25⁺ cells (*) or CD4⁺FoxP3⁺ cells (¶).</p

<i>In vitro</i>, CD4⁺CD25⁺ and CD4⁺FoxP3⁺ cells were hypo-proliferative compared with CD4⁺CD25^- responder cells in separate cultures, but hyperproliferative in the co-cultures with the responder cells.

<p>Proliferative responses of CD4⁺CD25⁺, CD4⁺FoxP3⁺ and CD4⁺CD25^- cells in separate (left column) or co-cultures (middle and right columns). The cells were stimulated with varying concentrations of soluble anti-CD3e alone (upper) or in combination with 100 ng/mL of soluble anti-CD28 (lower) in the presence of CD11c⁺ dendritic cells. Data are mean ± SE of three separate experiments with 4 replicate wells per dilution. *, P<0.05, between CD4⁺CD25⁺ or CD4⁺FoxP3⁺ fraction and CD4⁺CD25^- responder cells. ¶, P<0.05, between CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions.</p

In presence of exogenous IL-2, CD4⁺CD25⁺ and CD4⁺FoxP3⁺ cells were more sensitive than CD4⁺CD25^- responder cells in a proliferative response <i>in vitro</i>.

<p>CFSE-labeled CD4⁺CD25⁺, CD4⁺FoxP3⁺ or CD4⁺CD25^- responder cells were stimulated with varying amounts of immobilized anti-CD3e (numbers in ng/well) plus soluble anti-CD28 (100 ng/mL) in the presence of varying concentrations of exogenous IL-2 in an APC-free system. Data are mean ± SE of three separate experiments with 4 replicate wells per dilution. *, P<0.05, between CD4⁺CD25⁺ or CD4⁺FoxP3⁺ fraction and CD4⁺CD25^- responder cells.</p

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24 h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1 alpha was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl(4)-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1 alpha, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl(4) damaged liver. (C) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.Hu XY, 2008, J THORAC CARDIOV SUR, V135, P799, DOI 10.1016/j.jtcvs.2007.07.071Jung YJ, 2006, STEM CELLS DEV, V15, P687Uemura R, 2006, CIRC RES, V98, P1414, DOI 10.1161/01.RES.0000225952.61196.39Guo Y, 2005, STEM CELLS, V23, P1324, DOI 10.1634/stemcells.2005-0085Kucia M, 2005, STEM CELLS, V23, P879, DOI 10.1634/stemcells.2004-0342Togel F, 2005, KIDNEY INT, V67, P1772Zernecke A, 2005, CIRC RES, V96, P784, DOI 10.1161/01.RES.0000162100.52009.38Terai S, 2005, J HEPATO-BILIARY-PAN, V12, P203, DOI 10.1007/s00534-005-0977-0Kucia M, 2004, CIRC RES, V95, P1191, DOI 10.1161/01.RES.0000150856.47324.5bAcs G, 2004, CANCER LETT, V214, P243, DOI 10.1016/j.canlet.2004.04.027Ceradini DJ, 2004, NAT MED, V10, P858, DOI 10.1038/nm1075Weinmann M, 2004, INT J RADIAT ONCOL, V58, P386Schioppa T, 2003, J EXP MED, V198, P1391, DOI 10.1084/jem.20030267Staller P, 2003, NATURE, V425, P307, DOI 10.1038/nature01874Kollet O, 2003, J CLIN INVEST, V112, P160, DOI 10.1172/JCI200317902Helbig G, 2003, J BIOL CHEM, V278, P21631, DOI 10.1074/jbc.M300609200Yamaguchi J, 2003, CIRCULATION, V107, P1322, DOI 10.1161/01.CIR.0000055313.77510.22Ratajczak MZ, 2003, STEM CELLS, V21, P363Bagri A, 2002, DEVELOPMENT, V129, P4249HATCH HM, 2002, CLONING STEM CELLS, V4, P339Ponomaryov T, 2000, J CLIN INVEST, V106, P1331FRESHNEY RI, 2000, CULTURE ANIMAL CELLS, P357Paz-Miguel JE, 1999, J IMMUNOL, V163, P5399Okabe M, 1997, FEBS LETT, V407, P313TSUJIMOTO Y, 1997, LEUKEMIA S3, V11, P380Briehl MM, 1997, ONCOL RES, V9, P281Nagasawa T, 1996, NATURE, V382, P635SHIMIZU S, 1995, NATURE, V374, P811

Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow

We have previously reported that bone marrow cells (BMCs) participate in the regeneration after liver injury. However, it is not established that this is the result of differentiation of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or the combination of both. We investigated the contribution of each cell fraction to the regenerative process. First, we confirmed that transplanted stem cells migrate directly to injured liver tissue without dispersing to other organs. Next, we divided green fluorescent protein (GFP)-expressing BMCs into three populations as mononuclear cells, MSCs and HSCs. We then compared the engraftment capacity after transplantation of each fraction of cells into liver-injured mice. Of these, the MSCs transplanted group showed the highest GFP fluorescence intensities in liver tissue by flow cytometry analysis and confocal microscopic observation. Furthermore, MSCs showed differentiation potential into hepatocytes when co-cultured with injured liver cells, which suggests that MSCs showed highest potential for the regeneration of injured liver tissue compared with those of the other two cell refractions. (c) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.Chen Y, 2007, J CELL BIOCHEM, V102, P52, DOI 10.1002/jcb.21275Ong SY, 2006, TISSUE ENG, V12, P3477Jung YJ, 2006, STEM CELLS DEV, V15, P687Terai S, 2006, STEM CELLS, V24, P2292, DOI 10.1634/stemcells.2005-0542Talens-Visconti R, 2006, WORLD J GASTROENTERO, V12, P5834Chen Y, 2006, CYTOTHERAPY, V8, P381, DOI 10.1080/14653240600735800Russo FP, 2006, GASTROENTEROLOGY, V130, P1807, DOI 10.1053/j.gastro.2006.01.036Lange C, 2006, WORLD J GASTROENTERO, V12, P2394Hristov M, 2006, MED KLIN, V101, P186Okumoto K, 2006, J GASTROENTEROL, V41, P62, DOI 10.1007/s00535-005-1723-8CAO BQ, 2006, WORLD J GASTROENTERO, V13, P1851CAO BQ, 2006, WORLD J GASTROENTERO, V13, P1854Terai S, 2005, J HEPATO-BILIARY-PAN, V12, P203, DOI 10.1007/s00534-005-0977-0Sato Y, 2005, TRANSPLANT P, V37, P273, DOI 10.1016/j.transproceed.2005.01.035Okumoto K, 2003, BIOCHEM BIOPH RES CO, V304, P691, DOI 10.1016/S0006-291X(03)00637-5Strauer BE, 2002, CIRCULATION, V106, P1913, DOI 10.1161/01.CIR.0000034046.87607.1CMorio LA, 2001, TOXICOL APPL PHARM, V172, P44, DOI 10.1006/taap.91331