HIV-1 Nef Induces Hck/Lyn-Dependent Expansion of Myeloid-Derived Suppressor Cells Associated with Elevated Interleukin-17/G-CSF Levels.

Human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection causes myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+) through largely unknown cellular and molecular pathways. The mouse cells thought to be equivalent to human CD14+ CD16+ cells are CD11b+ Gr1+ myeloid-derived suppressor cells (MDSC). We used HIV transgenic (Tg) mouse models to study MDSC, namely, CD4C/Nef Tg mice expressing nef in dendritic cells (DC), pDC, CD4+ T, and other mature and immature myeloid cells and CD11c/Nef Tg mice with a more restricted expression, mainly in DC and pDC. Both Tg strains showed expansion of granulocytic and CD11b+ Gr1low/int cells with MDSC characteristics. Fetal liver cell transplantation revealed that this expansion was stroma-independent and abrogated in mixed Tg/non-Tg 50% chimera. Tg bone marrow (BM) erythroid progenitors were decreased and myeloid precursors increased, suggesting an aberrant differentiation likely driving CD11b+ Gr1+ cell expansion, apparently cell autonomously in CD4C/Nef Tg mice and likely through a bystander effect in CD11c/Nef Tg mice. Hck was activated in Tg spleen, and Nef-mediated CD11b+ Gr1+ cell expansion was abrogated in Hck/Lyn-deficient Nef Tg mice, indicating a requirement of Hck/Lyn for this Nef function. IL-17 and granulocyte colony-stimulating factor (G-CSF) were elevated in Nef Tg mice. Increased G-CSF levels were normalized in Tg mice treated with anti-IL-17 antibodies. Therefore, Nef expression in myeloid precursors causes severe BM failure, apparently cell autonomously. More cell-restricted expression of Nef in DC and pDC appears sufficient to induce BM differentiation impairment, granulopoiesis, and expansion of MDSC at the expense of erythroid maturation, with IL-17→G-CSF as one likely bystander contributor. IMPORTANCE HIV-1 and SIV infection often lead to myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+), with the latter likely involved in neuroAIDS. We found that some transgenic (Tg) mouse models of AIDS also develop accumulation of mature and immature cells of the granulocytic lineage, decreased erythroid precursors, and expansion of MDSC (equivalent to human CD14+ CD16+ cells). We identified Nef as being responsible for these phenotypes, and its expression in mouse DC appears sufficient for their development through a bystander mechanism. Nef expression in myeloid progenitors may also favor myeloid cell expansion, likely in a cell-autonomous way. Hck/Lyn is required for the Nef-mediated accumulation of myeloid cells. Finally, we identified G-CSF under the control of IL-17 as one bystander mediator of MDSC expansion. Our findings provide a framework to determine whether the Nef>Hck/Lyn>IL-17>G-CSF pathway is involved in human AIDS and whether it represents a valid therapeutic target.

Inhibition of the Na+/H+ antiporter induces cell death in TF-1 erythroleukemia cells stimulated by the stem cell factor.

Leukemia cells produce acidic metabolites due to their high metabolic condition. An alkaline pHi (intracellular pH) shift, caused by activation of the Na+/H+ exchange, is an important event in the mechanism of growth factor activity. However, the role of the Na(+)/H(+) exchanger in the survival of erythroleukemia TF-1 cells has not yet been studied in detail. The aim of this study was to identify the effects of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a highly specific blocker of the Na(+)/H(+) exchanger, on the survival of SCF-dependent TF-1 cells. The effects of EIPA on survival and mitochondrial membrane potential were studied when exposing wild type TF-1 cells and TF-1 cells expressing bcl-2 to EIPA for 48h. Ectopic expression of the bcl-2 gene maintained a mildly alkaline pH and prevented the simultaneous appearance of apoptosis and autophagy (typically displayed by TF-1 cells) in the presence of EIPA. Consistent with Stem Cell Factor (SCF) function, we found that this molecule rescued TF-1 cells during autophagy but not apoptosis, allowing these cells to subsequently respond to GM-CSF. Serum deprivation or SCF withdrawal induced cell death at 36h in TF-1 and TF-1 neo cells, whereas TF-1/bcl-2 cells tended to undergo apoptosis and show acidic vacuoles after 96h, pointing to a transient anti-apoptotic effect. The present study shows the suppressive effect of EIPA on the proliferation of leukemia cell line stimulated with SCF, apparently by decreasing the mitochondria membrane potential and averting alkalinization. Through the constitutive expression of bcl-2, TF-1 cells were survival factor independent. Proliferation in these cells was not affected by EIPA at the concentrations used against parental TF-1 cells, indicating that the inhibitory effect in SCF-stimulated cells can be attributed to specific blocking of the Na(+)/H(+) exchanger.

Toxic effects induced by curcumin in human astrocytoma cell lines.

OBJECTIVE: The objective of this study was to describe the toxicity induced by curcumin in human astrocytoma cell lines. METHODS: The effects induced by curcumin, at 100 µM for 24 h, were evaluated in four astrocytoma cell lines using crystal violet assay and through the evaluation of morphological and ultrastructural changes by electron microscopy. Also, the results of vital staining with acridine orange and propidium iodide for acidic vesicles and apoptotic bodies were analyzed and the expression of the Beclin1 gene was assessed by RT-PCR. RESULTS: The cells treated with curcumin at 100 µM induced an inhibitory concentration50 of viability with morphological changes characterized by a progressive increase in large, non-acidic vesicles devoid of cytoplasmic components and organelles, but that conserved the cell nuclei. No DNA breakage was observed. The astrocytoma cells showed no apoptosis, necrosis or autophagy. Expression of BECLIN1 was not induced (p < 0.05) by curcumin in the astrocytoma cells. CONCLUSIONS: Curcumin at 100 µm induced a new type of death cell in astrocytoma cell lines.

Low expression of stem cell antigen-1 on mouse haematopoietic precursors is associated with erythroid differentiation.

Sca1 is a surface marker of haematopoietic stem cell but its role in erythropoiesis is still largely unknown. In this work we evaluated the ability of Sca1⁺ cells to differentiate into cells of the erythrocytic lineage. We performed FACS analysis of complete and purified Sca1⁺ bone marrow cells from C3H/HeNHsd mice and measured the expression of CD71 and Terr119 to evaluate the stages in erythroid development. Definitive erythropoiesis was evident within the complete bone marrow, while only proerythroblasts were found in Sca1⁺ cells, suggesting that Sca1 is a negative regulator of erythropoiesis. We also used FDCP-mix cells and their PU.1 and SCL transfectants. The PU.1 transfectant showed significantly increased expression of Sca1 and was not induced to differentiate into red blood cells, while the SCL transfectant showed significantly lower expression of Sca1 and produced red blood cells. The results of this study suggest that increased Sca1 expression on erythropoietic precursors inhibits erythroid differentiation.

[Osteogenesis of human vascular endothelial cells in culture]. / Osteogénesis en cultivo de endotelio vascular humano.

INTRODUCTION: Mesenchymal stem cells have the potential to differentiate into several types of cells including osteoblasts. These stem cells have cell surface markers found on cells of endothelial and subendothelial origin of the umbilical cord vein. Taking this into consideration we have postulated that human umbilical vein endothelial cells (HUVEC) could present osteogenic differentiation as well. Gene activation that could drive osteogenic differentiation is regulated by exogenous and endogenous factors. OBJECTIVE: The induction osteogenesis in HUVEC. MATERIAL AND METHODS: We used: a) an osteogenic medium containing 0.1 microM dexamethasone, 10 microM beta-glycerophosphate, 50 microM L-ascorbic-acid 2-phosphate, 20% MCGS serum; and b) a treatment with DNA demethylating agents hydralazine and 5'-aza-2'-deoxycytidine (0.39-200 microM). Phenotypic characteristics of HUVEC were their spindle and stellate shapes with fine homogenous cytoplasm, typically associated with fibroblast-like cells. RESULTS: The control cells (without osteogenic treatment) exhibited little extracellular matrix, whereas the osteogenically treated cells appeared shortened and flattened, and they were surrounded by extracellular matrix that subsequently became mineralized in vitro. After 28 days in culture, morphologic and histochemical studies confirmed that osteogenic medium had a strong stimulatory effect on the alkaline phosphatase activity of endothelial cells, a very early marker of cell differentiation into the osteogenic lineage. Hydralazine and 5'-aza-2'-deoxycytidine, two drugs utilized in chromatin remodeling leading to gene re-expression of inactivated DNA hypermethylated islands, did not favor osteoblast differentiation. CONCLUSION: Our study shows that HUVEC can differentiate along an osteogenic lineage and thus provide an alternative source for cell-based therapies and tissue engineering strategies.

Blastic leukaemias (AML): a biologist's view.

Acute myeloblastic leukaemia is characterised by the extreme clonal proliferation of haematopoietic precursor cells with abnormal or arrested differentiation. Chemotherapy of acute leukaemia is channelled towards the reduction and eradication of leukaemic cells. However, relapse is generally assumed to occur in residual host cells, which are refractory to or elude therapy. The cancer stem cell hypothesis has gained considerable importance in recent years and could interpret this behaviour. This persuasive theory states that cells within a tumour are organised in a hierarchy similar to that of normal tissues and are maintained by a small subset of cells responsible for tumour dormancy. These cells, defined as 'tumour initiating cells' (TICs), possess several properties of normal tissue stem cells. Recently, the TICs associated with AML have been shown to comprise distinct, hierarchically arranged classes similar to those observed for haematopoietic stem cells. We know now that the growth and survival of blasts in AML are driven by the same growth factors that stimulate normal cells. Furthermore, direct evidence of the role of membrane stem cell factor and its receptor c-Kit in cell-cell interactions and cell survival in primary AML blasts have been provided, defining the importance of juxtacrine stimulation. Inhibition of c-Kit signalling induces combinations of cell death: autophagy (compensatory mechanism towards survival) and apoptosis. While recent work confirmed that c-Kit inhibitors reduce cancer cell proliferation, it also demonstrated that future inappropriate prescriptions could cause normal tissue deterioration. The purpose of this paper was to review some of the salient features of leukaemic blasts in support of the proposal that research into neoplasia be increased. Rather than presenting the details of various studies, I have attempted to indicate general areas in which work has been done or is in progress. It is hoped that this survey of the subject will demonstrate a variety of opportunities for additional research in human neoplasia.

On the nature of the tumor-initiating cell.

Certain aspects of tumors that may influence areas of basic biology and medicine are reviewed. The hypothesis that malignant stem cells evolve from normal stem cells, is considered. Information is being accumulated on the possibility that certain cell populations that can be propagated as cell lines in vitro can produce cells with features of differentiated cells in addition to others that maintain the line and, in some cases may also initiate tumor formation in vivo. Up to the present time, there is evidence to show that cancer stem cells persist in many cell lines. Tyrosine kinase inhibition produces combinations of autophagy and apoptosis in the human erythroleukemia cell line TF-1 hinting at a heterotypic aggregation of cells containing cancer stem cells. Finally, the mechanisms of cancer development, invasion and metastasis are operatively defined. The purpose of this paper is to review some of the salient features of cancer stem cells in support of the proposal that research in neoplasia be increased. Rather than presenting details of various studies, we have attempted to indicate general areas in which work has been done or is in progress. It is hoped that this survey of the subject will demonstrate a variety of opportunities for additional research in human neoplasia.

Cationic liposomes bearing IL-2 on their external surface induced mice leukocytes to kill human cervical cancer cells in vitro, and significantly reduced tumor burden in immunodepressed mice.

Tumor cells are known to modify their surroundings in order to escape immunologic detection, and IL-2, a killer cell activator, is one of the factors known to overcome this escape mechanism. In this regard, when we cocultured cells from the human cervical cancer cell line INBL with mice blood leukocytes, no inhibition of tumor cell growth was observed, but when a similar coculture was done in the presence of cationic liposomes bearing IL-2 on their external surface (CL-IL-2), all the INBL cells were killed. In order to evaluate whether this in vitro property of CL-IL-2 to overcome tumor cell detection by lymphocytes could also be reproduced in vivo, INBL cells were intraperitoneally (i.p.) inoculated into immunodepressed mice to produce solid tumors. We observed that the subsequent i.p. delivery of CL-IL-2 rendered the tumor masses significantly smaller. The presence of a large number of infiltrating lymphocytes on those tumors, and the fact that many had a cytotoxic CD8(+) phenotype suggests that these lymphocytes were responsible for the observed antitumor effect. Finally, the possible formation of a bridge between the IL-2R receptors on both, the lymphocytes and the INBL cells, mediated by the IL-2-bearing liposomes, and its possible effect on the activation of antitumor cytotoxic lymphocytes is discussed.

El factor Steel / The steel factor

Los ratones mutantes dominant white spotting (W) y Steel (SI) presentan defectos en el desarrollo de tres linajes celulares: melanocitos, células primordiales y hematopoyéticas. La genética ha revelado que el locuss SI codifica para el factor Steel (SF), el cual es el ligando del receptor de tipo tirosina-cinasa cinasa c-kit que es producto del locus W. El SF es sintetizado principalmente por las células estromales en su forma membranal y puede ser procesado para producir una forma soluble. Las interacciones célula-célula son importantes en la producción de células hematopoyéticas normales in vitro e in vivo y en la expansión de las células leucémicas. Aquí se discute cómo el SF soluble disminuye el requerimiento de la interacción celular en la formación de colonias blásticas en la leucemia mieloblástica aguda (LMA). Se discute cómo la presencia de SF membranal sobre los blastos de la LMA contribuye al crecimiento poblacional dependiente de la densidad celular. Se explica auquí cómo el SF es un factor principalmente de supervivencia para las células hematopoyéticas de poco efecto proliferativo y que mantiene a las células hamatopoyéticas CD34+ en un estado no diferenciado. Estas propiedades potencialmente permitirán mantener las células hematopoyéticas en cultivo, permitiendo purgar a la médula ósea de células malignas, por ejemplo, previo a un trasplante de médula ósea y en terapia génica. Igualmente, la activación de la vía de transducción de señales de c-kit puede jugar un papel en el desarrollo de muchas malignidades no hematológicas al romper la interacción celular y permitir el crecimiento de cánceres. En resumen, el SF juega un papel en la supervivencia celular durante el proceso normal de diferenciación, en la proliferación de la LMA, y en el mantenimiento de muchos tumores c-kit positivo

La transfección de la línea celular TF-1 con el proto-oncogen bcl-2 humano proporciona supervivencia a corto plazo en ausencia de GM-CSF sin cambiar el fenotipo / Transfection of the tf-1 cell line with the human protooncogen bcl-2 favors short term cell survival and does not change phenotype

La muerte celular programada (apoptosis) es un proceso controlado genéticamente. El producto del gen bcl-2 (Bcl-2) es una proteína anti apoptótica integrada a la membrana externa mitocondrial. Objetivo. El objetivo del presente trabajo fue valorar los efectos de la transfección de bcl-2 sobre la supervivencia, proliferación, fenotipo y morfología en las líneas celulares TF-1, TF-1 neo (control negativo) y TB-1 (TF-1 transfectada con bcl-2). Metodología. Para ello utilizamos el método siguiente: examen del nivel de expresión de los antígenos de superficie CD13, CD34 y c-Kit, respuesta celular al GM-CSF y capacidad de supervivencia luego del retiro del factor de viabilidad. La apoptosis fue evaluada mediante la observación del blebbing membranal a diferentes tiempos luego del retiro del suero, el factor de viabilidad GM-CSF y la tolerancia a toxinas presentes en Justicia spicigera. Resultados. La expresión ectópica de bcl-2 en las células TB-1 previno la muerte celular por apoptosis sin inducir un cambio importante en el fenotipo y morfología en ausencia de GM-CSF, suero o en presencia del extracto de Justicia spicigera. Consistente con la función de Bcl-2, encontramos que la proliferación de la línea celular TB-1 fue muy similar a la línea parental TF-1 en respuesta al GM-CSF y por lo tanto Bcl-2 no altera dicha función. El retiro de suero y GM-CSF al medio de cultivo de las líneas TF-1 y TF-1 neo, induce apoptosis en 36 h. Por el contrario, la línea TB-1 entra en apoptosis a 96 h bajo las mismas condiciones. Conclusiones. Reuniendo los resultados, éstos sugieren que Bcl-2 es un inhibidor de la apoptosis a corto plazo en la línea celular TB-1, no cambia la respuesta al GM-CSF y no afecta el nivel de expresión de otros antígenos de superficie como CD13, CD34 y c-Kit.

Biología de las células totipotenciales hematopoyéticas / Biology of hemopoietic stem cells

Todas las células que comprenden el sistema hematopoyético son derivadas de un precursor común, la célula totipotencial hematopoyética (CTH), la cual por procesos de proliferación y diferenciación da lugar a las células maduras que se localiza en la sangre y órganos linfohematopoyéticos. Para que se lleven a cabo los procesos de proliferación, supervivencia, apoptosis,inhibición y diferenciación desde las CTH hasta las células maduras funcionales, se requiere de la participación de proteínas denominadas colectivamente citocinas, las cuales promueven y regulan una o varias funciones celulares, e intervienen en una o varías etapas de diferenciación de las CTH. Por el empleo de diferentes técnicas de cultivo, se concluyó que el estroma de la médula ósea: fibroblastos, células endoteliales y adipocitos, tiene un papel importante. La participación de esta células radica en la producción de citocinas, así como en proveer de un soporte sobre el cual se asientan las CTH. Aunque podría parecer que las citocinas son los factores fundamentales para regular los procesos mencionados de las células hematopoyéticas, se han propuesto dos hipótesis principales para explicar cómo se lleva a cabo la hematopoyesis: el modelo determinístico y el modelo estocástico. Ambos modelos aportan pruebas para apoyar sus postulados; sin embargo, a la fecha decidir cuál modelo es el acertado es todavía materia de controversia. Aunque el estudio de las CTH plantea muchas preguntas de orden básico, en muchos laboratorios en el mundo se han comenzado a indagar sobre la aplicación de las CTH en terapia humana: su uso en transplantes, sustituyendo el trasplante de médula ósea completa y, aún en etapas experimentales, el empleado de las CTH para insertar genes que pudieran ser de importancia terapéutica en enfermedades deficitarias o incluso en el tratamiento contra el cáncer

El factor de células totipotenciales (stem cell factor; SCF) sostiene la supervivencia de progenitores de granulocitos en cultivos de médula ósea de ratón / Stem cell factor supports survival of granulocyte progenitors in mouse bone marrow cultures

La regulación de la proliferación y la muerte celular es crucial para la generación de células hematopoyéticas tanto in vitro como in vivo. El papel biológico del factor de células totipotenciales (Stem Cell factor; SCF) en el desarrollo del sistemahematopoyético no está bien definido. El objetivo del presente trabajo fue estudiar la supervivencia, proliferación y diferenciación de células de médula ósea de ratón en cultivo en presencia de SCF. Para ello utilizamos la siguiente metodología: examen de la formación de colonias, MTT y el suicidio por timidina tritiada de las células de médula ósea los cuales revelaron que el SCF es un factor de supervivencia principalmente. Nuestros resultados muestran que el SCF mantiene a las células en un estado indiferenciado. Los progenitores de macrófagos y granulocitos (CFU-GM) sobreviven por siete días en presencia de SCF, y no se obtienen granulocitos ni macrófagos. La exposición de estas células a cultivos que contienen el factor estimulante de la formación de colonias de macrófagos y granulocitos (GM-CSF), que favorece la emergencia de macrófagos y granulocitos, provoca la proliferación y diferenciación terminal de ellas. Nuestros datos indican que el SCF induce la supervivencia de progenitores hematopoyéticos y en especial favorece la de progenitores granulocíticos sobre la de progenitores de monocitos. En ausencia de factores tardíos, tales como el GM-CSF, las células que progresan más allá del estadio CFU-GM pierden la expresión de c-Kit y mueren. De ahí que la ausencia de expansión celular en presencia de SCF sea debida a la supervivencia, y un balance entre una reducida proliferación y muerte celular. En contraste, la presencia de SCF y GM-CSF permite la continua supervivencia y expanción de los progenitores hematopoyéticos en cultivo y su posterior diferenciación en macrófagos y granulocitos. La producción de factor estimulante de colonia de granulocitos (G-CSF) e (interleucina-6 (IL-6), dos citocinas involucradas en la producción de granulocitos, en macrófagos generados con GM-CSF sugiere una inducción indirecta del GM-CSF durante la granulopoyesis. Finalmente, nuestros datos sugieren que las señales de supervivencia del CSF son cruciales durante el proceso de diferenciación de los granulocitos, apoyando el modelo determinístico