Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC

Targeted therapy against chemoresistant colorectal cancers: Inhibition of p38α modulates the effect of cisplatin in vitro and in vivo through the tumor suppressor FoxO3A

Chemoresistance is a major obstacle to effective therapy against colorectal cancer (CRC) and may lead to deadly consequences. The metabolism of CRC cells depends highly on the p38 MAPK pathway, whose involvement in maintaining a chemoresistant behavior is currently being investigated. Our previous studies revealed that p38a is the main p38 isoform in CRC cells. Here we show that p38a pharmacolog- ical inhibition combined with cisplatin administration decreases colony formation and viability of cancer cells and strongly increases Bax-dependent apoptotic cell death by activating the tumor suppressor pro- tein FoxO3A. Our results indicate that FoxO3A activation up-regulates transcription of its target genes (p21, PTEN, Bim and GADD45), which forces both chemosensitive and chemoresistant CRC cells to undergo apoptosis. Additionally, we found that FoxO3A is required for apoptotic cell death induction, as confirmed by RNA interference experiments. In animal models xenografted with chemoresistant HT29 cells, we further confirmed that the p38-targeted dual therapy strategy produced an increase in apoptosis in cancer tissue leading to tumor regression. Our study uncovers a major role for the p38- FoxO3A axis in chemoresistance, thereby suggesting a new therapeutic approach for CRC treatment; moreover, our results indicate that Bax status may be used as a predictive biomarker

po 393 notch3 and cxcr4 cross signalling sustains acute t cell leukaemia progression

Introduction Acute T-cell lymphoblastic leukaemia (T-ALL) is a childhood cancer, characterised by infiltration of immature T-cells in bone marrow. Notch hyperactivation is a major driver of T-ALL development where CXCL12/CXCR4 axis plays an important role in T-ALL maintenance. In thymus the lympho-stromal communication drives progressive maturation of T-cells. Notch receptors regulate T-cell fate choices, dominating early steps of thymocyte maturation. In T-cell differentiation, Notch3, in association with pre-TCR and chemochine receptor CXCR4, govern the transition from double negative (DN) to double positive (DP) thymocytes. Previously, our laboratory demonstrated the lymphomagenic potential of Notch3 by creating a transgenic mouse model (N3-ICtg), characterised by the constitutive activation of the intracellular domain (IC) of Notch3 receptor (N3-IC) in immature thymocytes. In order to investigate the oncogenic cross-talk between Notch3 and CXCR4 in T-ALL progression, we analysed DP T-cells in different lymphoid compartments of N3-ICtg mice. Material and methods Freshly isolated cells from thymus, blood and bone marrow of N3-ICtg and WT mice were analysed by flow cytometry in order to verify the presence of DP T-cells and their cell-surface expression of CXCR4 and Notch3 receptors. Experiments in TALL1, a human T-ALL leukemic CD3 + /CD4 + /CD8 + cell line characterised by the activation of Notch3 and high expression of CXCR4, were also performed. TALL1 cells were treated with γ-secretase inhibitor (GSI) or their gene expression of Notch3 was silenced and then analysed by flow cytometry, RT-PCR and western blot. Statistical interpretation of the results was performed. Results and discussions DP-gated thymocytes obtained by N3-ICtg mice have shown a high co-expression of Notch3 and CXCR4 and a high migratory ability induced by SDF-1. An anomalous percentage representation of these DP T-cells at different ages in circulating blood, spleen and bone morrow may suggest an interaction between CXCR4 and Notch3 in T-ALL cell propagation. Experiments in human TALL1 cell line with Notch3 targeted inhibition suggest a modulated expression of CXCR4 through a β-arrestin1-mediated mechanism. CXCR4-antagonists treatment will further elucidate the molecular crosstalk between the two receptors. Conclusion Notch3 abnormal pathway, through boosting the expression of CXCR4 on cell-surface, may play a role in DP T-cells egress from thymus, and define a possible mechanism of 'pre-leukemic-cells' dissemination

HTLV-1 propels thymic human T cell development in “human immune system” Rag2-/- IL-2R γc-/- Mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that TaxHTLV-1 interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a “Human Immune System” (HIS) Rag2-/-γc-/- mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2-/-γc-/- mice, mature single-positive (SP) CD4+ and CD8+ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2-/-γc-/- animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Electrochemical and mechanical properties of hydrogels based on conductive poly (3,4-ethylene dioxythiophene)/poly (styrenesulfonate) and PAAm

This paper reports on the effects of poly(3,4-ethylene dioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) entangled in the polyacrylamide (PAAm) network by the formation of a semi-IPN on hydrogel performance. Hydrogel properties were evaluated by scanning electron microscopy, water uptake, compressive load tests, ionic conductance and capacitance measurements. It has been found that the introduction of PEDOT/PSS leads to changes in the hydrogel morphology as compared to that of PAAm hydrogels. In addition, PAAm networks with good mechanical properties have been obtained. The presence of PEDOT/PSS increased the ionic conductance of swollen semi-IPN hydrogels substantially. Electrochemical experiments demonstrated that PAAm-PEDOT/PSS hydrogel is electrochemically stable and presents reversible responses to electrochemical stimuli. (c) 2005 Elsevier Ltd. All rights reserved.25215816

Thermo-responsive sandwiched-like membranes of IPN-PNIPAAm/PAAm hydrogels

The synthesis of sandwiched-like IPN hydrogels having cross-linked PNIPAAm interpenetrated into PAAm networks was carried out by UV-induced polymerization using potassium periodate as a sensitizer. NIPAAm monomers and MBAAm cross-linker were co-polymerized inside previously synthesized cross-linked PAAm. Through SEM images, we observed that the hydrogel membranes were formed by three layers; the internal layer was fully involved by the two others. Significant morphological difference between the internal and external layers was also observed. Thus, sandwiched-like interpenetrating polymer networks were obtained. The internal layer shrank significantly after warming the swollen hydrogel above LCST of PNIPAAm in water, 30-35 degrees C while the external layers remained swollen and highly porous due to the hydrophilicity of PAAm. The experiments performed at 40 degrees C revealed that the hydrogels shrank considerably. Collapsed PNIPAAm chains induced a substantial contraction of the internal hydrogel layer. The hydrogel contraction was accompanied by an increase in gel strength and elasticity modulus. The presence of the PNIPAAm network in the internal layer reinforced the hydrogel and this effect was more pronounced above LCST. The permeability of sandwich-like membrane significantly rather decreases as the temperature increases. There was a decrease in the permeability of 52% as the temperature was increased from 25 to 40 degrees C. It was suggested that the structural changes in the sandwich membrane induce to significant flux control. (c) 2005 Elsevier B.V. All rights reserved.2754167118719

Porous alginate-Ca2+ hydrogels interpenetrated with PNIPAAm networks: Interrelationship between compressive stress and pore morphology

Thermo-sensitive porous hydrogels composed of interpenetrated networks (IPN) of alginate-Ca2+ and PNIPAAm have been obtained. The hydrogels were prepared by cross-linking alginate-Na+ with Ca2+ ions inside PNIPAAm networks. Compressive tests and scanning electron microscopy were used to evaluate gel strength and pore morphology, respectively. IPN hydrogels displayed two distinct pore morphologies under thermal stimuli. Below 3035 degrees C, the LCST of PNIPAAm in water, IPN hydrogels were highly porous. The pore size of hydrogel heated above LCST became progressively smaller. Alginate-Ca2+ and PNIPAAm hydrogels, used as references, did not present such behaviour, indicating that the porous effect is due to IPN hydrogel. It was verified that higher strength is achieved when the hydrogel presents small pore size and the temperature is increased. It is suggested that at temperatures above LCST, the PNIPAAm chains shrink and pull the alginate-Ca2+ networks back. During shrinking, the polymer chains occupy the open spaces (pores from which water is expelled), and therefore, the hydrogel becomes less deformable when subjected to compressive stress. The results presented in this work indicate that the mechanical properties as well as the pore morphologies of these IPN hydrogels can be tailored by thermal stimulus. (c) 2005 Elsevier Ltd. All rights reserved.41122845285