Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages.

The intracellular iron level exerts a negative feedback on transferrin receptor (TfR) expression in cells requiring iron for their proliferation, in contrast to the positive feedback observed in monocytes-macrophages. It has been suggested recently that modulation of TfR and ferritin synthesis by iron is mediated through a cytoplasmic protein(s) (iron regulatory element-binding protein(s) (IRE-BP)), which interacts with ferritin and TfR mRNA at the level of hairpin structures (IRE), thus leading to inhibition of transferrin mRNA degradation and repression of ferritin mRNA translation. In the present study we have evaluated in parallel the level of TfR expression, ferritin, and IRE-BP in cultures of: (i) circulating human lymphocytes stimulated to proliferate by phytohemagglutinin (PHA) and (ii) circulating human monocytes maturing in vitro to macrophages. The cells were grown in either standard or iron-supplemented culture. TfR and ferritin expression was evaluated at both the protein and mRNA level. IRE-BP activity was measured by gel retardation assay in the absence or presence of beta-mercaptoethanol (spontaneous or total IRE-BP activity, respectively). Spontaneous IRE-BP activity, already present at low level in quiescent T lymphocytes, shows a gradual and marked increase in PHA-stimulated T cells from day 1 of culture onward. This increase is directly and strictly correlated with the initiation and gradual rise of TfR expression, which is in turn associated with a decrease of ferritin content. Both the rise of TfR and spontaneous IRE-BP activity are completely inhibited in iron-supplemented T cell cultures. In contrast, the total IRE-BP level is similar in both quiescent and PHA-stimulated lymphocytes, grown in cultures supplemented or not with iron salts. Monocytes maturing in vitro to macrophages show a sharp increase of spontaneous and, to a lesser extent, total IRE-BP; the addition of iron moderately stimulates the spontaneous IRE-BP activity but not the total one. Here again, the rise of spontaneous IRE-BP from very low to high activity is strictly related to the parallel increase of TfR expression and, suprisingly, also with a very pronounced rise of ferritin expression observed at both the mRNA and protein level. It is noteworthy the effect of beta-mercaptoethanol is cell specific, i.e. the ratio of total versus spontaneous IRE-BP activity is different in activated lymphocytes and maturing monocytes.(ABSTRACT TRUNCATED AT 400 WORDS

HOXB7: a key factor for tumo-associated angiogenic switch.

We had demonstrated previously a functional bridge between altered homebox (HOX) gene expression and tumor progression through HOXB7 transactivation of basic fibroblast growth factor. Here, we have studied whether HOXB7, in addition to basic fibroblast growth factor, may induce other genes directly or indirectly related to neoangiogenesis and tumor invasion. Parental, beta-galactosidase-transduced, and HOXB7-transduced SkBr3 cell lines were examined for the expression of several growth factors and growth factor receptors involved in the proliferative and angiogenic processes. Vascular endothelial growth factor, melanoma growth-stimulatory activity/growth-related oncogenene alpha, interleukin-8, and angiopoietin-2 were up-regulated by HOXB7 transduction. The exception was angiopoietin-1 expression that was abrogated. Additional analyses included the expression levels of enzymes such as matrix metalloprotease (MMP)-2 and MMP-9 and heparanase, capable of proteolytic degradation of extracellular matrix and basement membranes. Results showed an induction of only MMP-9. The functional implication of such a finding was tested using an in vitro coculture assay in a three-dimensional matrix. A delay of differentiation with persistent nests of proliferating cells was found in endothelial cells cocultured with HOXB7-transduced SkBr3 cells. Tumorigenicity of these cells has been evaluated in vivo. Xenograft into athymic nude mice showed that SkBr3/HOXB7 cells developed tumors in mice, either irradiated or not, whereas parental SkBr3 cells did not show any tumor take unless mice were sublethally irradiated. Comparison of tumor nodules for vascularization by CD-31 and CD-34 immunostaining revealed an increased number of blood vessels in tumors expressing HOXB7. Together, the results indicate HOXB7 as a key factor up-regulating a variety of proangiogenic stimuli. Thus, HOXB7 gene or protein is a target to aim at to inhibit tumor-associated neoangiogenesis, considering the number and the redundancy of proangiogenic molecules that should be targeted one by one to theoretically achieve the same effect

Isolation and characterization of CD146+ multipotent mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) represent a bone marrow (BM) population, classically defined by five functional properties: extensive proliferation, ability to differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells−supporting hematopoiesis. However, research progress in this area has been hampered by lack of suitable markers and standardized procedures for MSC isolation. We have isolated a CD146+ multipotent MSC population from 20 human BM donors displaying the phenotype of self-renewing osteoprogenitors; an extensive 12-week proliferation; and the ability to differentiate in osteoblasts, chondrocytes, adipocytes, and stromal cells supporting hematopoiesis. Furthermore, the CD146+ MSCs secrete a complex combination of growth factors (GFs) controlling hematopoietic stem cells (HSCs) function, while providing a >2-log increase in the long-term culture (LTC) colony output in 8-week LTC over conventional assays. The hematopoietic stromal function exhibited by the MSCs was further characterized by manipulating LTCs with the chemical inhibitors Imatinib or SU-5416, targeting two GF receptors (GFRs), KIT or VEGFR2/1, respectively. Both treatments similarly impaired LTC colony output, indicating key roles for these two GF/GFR interactions to support LTC-initiating cell activity. CD146+ MSCs may thus represent a tool to explore the MSC-HSC cross-talk in an in vitro surrogate model for HSC “niches,” and for regenerative therapy studies. In addition, the MSC microRNA (miRNA) expression profile was analyzed by microarrays in both basic conditions and chondrogenic differentiation. Our analysis revealed that several miRNAs are modulated during chondrogenesis, and many of their putative targets are genes involved in chondrogenic differentiation

KDR receptor: A key marker defining hematopoietic stem cells

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34+cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR-subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors

Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma