In vivo effects of high AA on progression of leukemia.

<p>A) High AA or the vehicle was injected intravenously for 6 days with a rest period of 2 days between 3 daily injections of mice transplanted with HL60 cells. Compared with vehicle (blue line), high AA (red line) significantly inhibited tumor growth (*<i>P</i><0.01). The values represent the mean ± SD values of 5 mice. B) Appearance of mice treated with vehicle (left) and high AA (right), 4 days after the final injection. C) Representative macroscopic appearance of tumors of mice treated with the vehicle (left) and high AA (right). Note that the tumors of high AA-treated mice were smaller and less erythematous than those of vehicle-treated mice. D) Immunohistochemical analysis of tumor neoangiogenesis in mice treated with the vehicle (left) and high AA (right). The green and blue signals represent CD31 and 4′,6-diamidino-2-phenylindole (DAPI), respectively. The bars indicate 100 μm.</p

Effects of high AA on tumor growth in the presence or absence of overexpression of <b><i>HIF-1α</i></b><b>.</b>

<p>A) Immunohistochemical analysis of tumor neoangiogenesis in vehicle-treated (left) and high AA-treated (right) mice transplanted with K562 (upper column) or K562-HIF1α cells (lower column). The green and blue signals represent CD31 and DAPI, respectively. The bars indicate 100 μm. Note that administration of high AA suppressed tumor neoangiogenesis in mice transplanted with K562 cells, but not in mice transplanted with K562-HIF1α cells. B) In the xenogeneic transplant model, high AA or vehicle was injected for 5 days. Administration of high AA significantly inhibited tumor growth of K562 cells (*<i>P</i><0.05) but not of K562-HIF1α cells (<i>P</i>>0.05). Tumor growth rate was estimated using the following equation: tumor volume on day 4 after high AA treatment/tumor volume just before high AA treatment. The values represent the mean ± SD values for 4 mice.</p

In vitro effects of AA on human leukemic and CB-CD34⁺ cells, relative to catalase activity.

<p>A) Cell viability assay of various leukemic cell lines and 2 independent isolates of CB-CD34⁺ cells. Cells were treated with different concentrations of AA for 1 h, and then washed, cultured, and analyzed after 72 h. The viability of all cell lines reduced significantly in the presence of 280 and 2800 µM AA (*<i>P</i><0.0001, as compared with vehicle), but this finding was not obtained for CB-CD34⁺ cells (<i>P</i>>0.05). The values represent the mean ± SD values of quadruplicate samples. B) Flow cytometric measurement of apoptosis of HL60 cells. Cells were treated with vehicle or AA for 1 h, and then washed, cultured, and analyzed after 18 h. Representative profiles are shown. The annexin V⁺ propidium iodide (PI)⁺ cell fraction indicates apoptotic cells. Note that AA-induced apoptosis was almost completely abrogated by the addition of catalase. C) Intracellular catalase activity. Leukemic cells generally expressed lower catalase activities than did CB-CD34⁺ isolates (*<i>P</i><0.001, as compared with each cell line). The values represent the mean ± SD values of quadruplicate samples. D) Histochemical analysis demonstrated lower catalase activity in HL60 cells than in CB-CD34⁺ cells. The bars indicate 50 μm.</p

Relationship between antileukemic effects of high AA and HIF-1α expression.

<p>A) Quantitative real-time PCR analysis of <i>HIF-1α</i> mRNA expression in K562 and K562-HIF1α cells. Cells were treated with the vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. After high AA exposure, <i>HIF-1α</i> mRNA expression significantly reduced in K562 (*<i>P</i><0.01), but not in K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. High AA exposure significantly reduced the HIF-1α protein level in both types of cells. However, the HIF-1α protein level in K562-HIF1α cells was significantly higher than that in K562 cells after vehicle or high AA exposure. *<i>P</i><0.01, **<i>P</i><0.0001, ***<i>P</i><0.00001. The values represent the mean ± SD values of triplicate samples. C) Flow cytometric measurement of apoptosis of K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 18 h. There was a significant difference in the number of apoptotic (annexin V⁺ propidium iodide (PI)⁺) cells between high AA-treated K562 and K562-HIF1α cells (*<i>P</i><0. 001). The values represent the mean ± SD values of triplicate samples. D) Flow cytometric measurement of cleaved caspase-3 expressed by K562 and K562-HIF1α cells. Cells were treated with vehicle (gray lines) or high AA (black lines) for 1 h, washed, cultured, and analyzed after 24 h. Activation of caspase-3 by high AA was lower in K562-HIF1α than in K562 cells. E) Western blotting analysis of Mcl-1, Bcl-x_L, and Bcl-2 in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.05) and between the vehicle-treated and high AA-treated K562 cells (**<i>P</i><0.0001). There was no significant difference between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. F) Western blotting analysis of Sp1, Sp3, Sp4, and VEGF. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels of these molecules between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.01, ** <i>P</i><0.0001). There were significant differences in the expression levels of Sp1, Sp3, and Sp4 between the vehicle-treated and high AA-treated K562 or K562-HIF1α cells (^†<i>P</i><0.01, ^††<i>P</i><0.001, ^†††<i>P</i><0.0001). There was a significant difference in the expression level of VEGF between the vehicle-treated and high AA-treated K562 (^†††<i>P</i><0.0001), but not between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05).</p

Expression of angiogenesis-related molecules in human leukemic and CB-CD34⁺ cells exposed to the vehicle or to high AA.

<p>A) Quantitative real-time PCR (qRT-PCR) analysis of <i>HIF-1α</i> mRNA in CB-CD34⁺ and HL60 cells. The cells were treated with vehicle or high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There were no significant differences in the expression levels for the 2 conditions (<i>P</i>>0.05) in CB-CD34⁺ cells. In contrast, there were significant differences in the expression levels between the 2 conditions (*<i>P</i><0.0001) in HL60 cells. The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in CB-CD34⁺ and HL60 cells. The cells were treated with vehicle or high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels (*<i>P</i><0.01, **<i>P</i><0.0005). The values are mean ± SD values of triplicate samples. C) Sequential analysis of qRT-PCR results of <i>HIF-1α</i> and <i>VEGF</i> mRNA in HL60 cells. The cells were treated with high AA for 1 h, and then washed, cultured, and analyzed after 1, 3, 22, and 26 h. The expression of <i>VEGF</i> mRNA reduced along with that of <i>HIF-1α</i> over time. Compared with the expression levels at 0 h, there were significant differences in the expression levels (*<i>P</i><0.01, **<i>P</i><0.001, ***<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples.</p

Differences in NF-κB activation and intracellular AA content between human leukemic and CB-CD34⁺ cells in the presence of high AA.

<p>A) Western blotting analysis of p-IκB in HL60 cells. Cells were treated with the vehicle or with high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There was a significant difference in the expression levels (*<i>P</i><0.001). Values represent the mean ± SD of triplicate samples. B) Immunocytochemical (left) and Western blotting (right) analyses of NF-κB in CB-CD34⁺ and HL60 cells. Cells were treated with vehicle or high AA for 1 h, then washed, cultured, and analyzed after 24 h. Note that translocation of NF-κB into the nucleus was markedly decreased in high AA-treated HL60 cells. Green and blue signals represent NF-κB and DAPI, respectively. Bars indicate 20 μm. There were significant differences in the expression levels (*<i>P</i><0.001, **<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples. C) Intracellular AA content of human leukemic cells and 2 different isolates of CB-CD34⁺ cells. Cells were treated with high AA for 1 h, washed in PBS, and analyzed immediately. There were significant differences in the content between leukemic and CB-CD34⁺ cells. *<i>P</i><0.001, as compared with CB-CD34⁺ cells (1) or (2). The values are mean ± SD values of triplicate samples.</p

Dimorfismo, uso de hábitat y dieta de Anolis Maculiventris (Lacertilia: Dactyloidae), en bosque pluvial tropical del chocó, Colombia

Se estudiaron algunos aspectos de la biología poblacional de Anolis maculiventris, en zonas de bosque pluvial tropical de la región natural chocoana. Los machos y hembras presentaron una coloración café claro con un pequeño lunar en la parte posterior de la cabeza, las proporciones de sexo en los individuos capturados mostraron diferencias estadísticas significativas (X2 : 4,81, gl: 1, p: 0,028). El grado de dimorfismo sexual (GDS) e índice de dimorfismo sexual fue de 1,08 mm y un de 8,5 mm respectivamente. Las hembras difieren significativamente de los machos en todas las regiones corporales medidas. Se disectaron 30 individuos a los cuales se les determino su estado reproductivo, en donde la talla mínima de madures en hembras (LHA) fue de 30,7 mm, con un máximo de 39,9 mm y una media de 36,5 mm. En machos la talla mínima de madurez fue de 30,4 mm, y máxima de 36,7 mm, con una media de 33,8 mm; lo cual indica que las hembras fueron más grandes que los machos. Se evaluó el uso de hábitat, mostrando diferencias estadísticas significativas entre los sustrato utilizados (X2 =16,89, gl: 3, p: 0,001); y para las posiciones verticales mostraron el mismo comportamiento (X2 =3,77, gl: 3, p: 0,207). Para el análisis dietario de la especie se disectaron 30 estómagos, de los cuales 15 pertenecieron a machos y 15 a hembras, se registróun total 46 presas gastrointestinales que componen su dieta mostrando a los órdenes Himenóptera (Formicidae) y Coleóptera como los de mayor importancia relativa en la dieta.We studied some aspects of the population biology of A. maculiventris, in areas of tropical rainforest in the natural region of Choco. The males and females brown coloration with a small mole on the back of the head, the proportions and gender in the captured individuals showed significant statistical differences (X2 : 4,81, gl: 1, p: 0,028). The degree of sexual dimorphism (GDS) and sexual dimorphism index was 1.08, mm and 8.5 mm respectively. Females differ significantly from males in all the parts of the body measures. 30 Individuals have spread, which was determined to their reproductive status and failed to register a size minimum of maturity (LHA) in females is 30.7 mm, with a maximum of 39.9 mm and an average of 36.5 mm. In the male the minimum maturity sexual 30.4 mm, and a maximum of 36.7 mm, with an average of 33.8 mm; which indicates that females were more large than males. Assessed habitat use, showing significant statistical differences between the used substrate (X2 =16,89, gl: 3, p: 0,001); and for vertical positions, showed the same behavior (X2 =3,77, gl: 3, p: 0,207). For analyzing dietary species spread 30 stomach, of which 15 belonged to males and 15 females, will register a total 46 gastrointestinal dams that compose its diet showing the orders Coleoptera and Hymenoptera (Formicidae) as the of greater relative importance in the diet

The induction of myeloid differentiation in the APL cells induced by ATRA treatment <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Cytospin slides of the induced APL cells cultured with or without 1 µM ATRA for 6 days. The mature neutrophils with Auer rods are seen in the ATRA-treated group. The images were captured with a BX41 microscope (Olympus). The bar indicates 10 µm. (<b>B</b>) The differential cellular counts of the induced APL cells cultured with or without ATRA. The average leukocyte differentiation in three independent experiments is shown. (<b>C</b>) The results of an immunofluorescent analysis of the PML distribution in the induced APL cells before and after treatment with ATRA. The images were captured with an LSM510 META confocal microscope (Carl Zeiss). All the bars indicate 10 µm. (<b>D, E</b>) The induction of myeloid differentiation in the induced APL cells by ATRA <i>in vivo</i>. The secondary recipients transplanted with the induced APL cells were then intraperitoneally treated with ATRA for 21 days. Cytospin slides of EGFP⁺/hCD45⁺/hCD33⁺ cells from the secondary recipients transplanted with the induced APL cells are shown (<b>D</b>). The images were captured with a BX41 microscope (Olympus). The bar indicates 10 µm. Their differentiated cellular counts were evaluated, and the representative series data are indicated (<b>E</b>).</p

Ribosomal Biogenesis and Translational Flux Inhibition by the Selective Inhibitor of Nuclear Export (SINE) XPO1 Antagonist KPT-185

<div><p>Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators (e.g., XPO1, cyclin D1, c-Myc, PIM1, and Bcl-2 family members), repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins (e.g., PIM2, EEF1A1, EEF2, and HSP70) that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1.</p></div

Establishment of humanized APL <i>in vivo</i>.

<p>(<b>A</b>) The proportion of EGFP⁺/human CD45⁺ cells in the bone marrow of leukemic NOG mice. Each dot represents a single mouse. The horizontal line represents the median value. (<b>B</b>) The proportion of CD33⁺ cells among the EGFP⁺/human CD45⁺ cells in the bone marrow of the leukemic NOG mice. Each dot represents a single mouse. The horizontal line represents the median value. (<b>C</b>) The expression of <i>PML-RARA</i> in RT-PCR was detected only in the EGFP⁺ fraction obtained from the engrafted human CD45⁺/CD33⁺ cells. The cells were obtained from bone marrow 16 weeks after transplantation. <i>B2M, beta 2 microglobulin</i>. The <i>PML-RARA</i> expression vector and human CD34⁺ cells were used as a positive control for the <i>PML-RARA</i> and <i>B2M</i> analysis, respectively. (<b>D</b>) The differential counts of the engrafted CD45⁺/CD33⁺/EGFP⁺ and EGFP⁻ cells from the mice transplanted with <i>PML-RARA</i>-induced human CD34⁺ cells. They were obtained from bone marrow 16 to 20 weeks after transplantation. The data represent the means. (<b>E</b>) A representative photograph of the resulting leukemic cells which morphologically recapitulated APL. Faggot cells were recognized, as seen in the right top corner. The images were captured with a BX41 microscope (Olympus). The bar indicates 10 µm. (<b>F</b>) The results of a Southern blot analysis of the genomic DNA from induced APL cells with an EGFP probe. Clonal bands are shown by arrow heads: white, seen in both BM and SP; orange, seen only in BM or SP. BM, bone marrow; SP, spleen. (<b>G</b>) The leukemia-free duration in NOG mice transplanted with <i>PML-RARA-</i>transduced CD34⁺ cells.</p