IGFBP-3 inhibits Wnt signaling in metastatic melanoma cells.

In previous works, we have shown that insulin-like growth factor-binding protein-3 (IGFBP-3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP-3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti-tumoral effects of IGFBP-3. We show that the anti-tumoral effect of IGFBP-3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP-3 binds the Wnt signalosome interacting specifically with its component GSK-3β. As a consequence, the β-catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK-3β is activated through dephosphorylation, becoming free to target cytoplasmic β-catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP-3 is a novel and effective inhibitor of Wnt signaling. As IGFBP-3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling

Il trattamento chirurgico dei tumori maligni del labbro. Esperienza personale

I tumori maligni del labbro rappresentano circa l’1-2% delle neoplasie cervico-facciali. Tali lesioni sono più frequentemente costi - tuite da carcinomi spinocellulari e basocellulari che nel complesso rappresentano il 25% di tutti i tumori orali. Mentre il carcinoma spinocellulare si localizza soprattutto sul labbro inferiore, il carcino - ma basocellulare si riscontra maggiormente sul labbro superiore .Il sesso maschile risulta essere sensibilmente il più colpito. L’etiopatoge - nesi di queste neoplasie è legata all’esposizione solare, al fumo, alla predisposizione genetica (mutazione del gene soppressore p53), all’e - voluzione di forme di precancerosi, quali radiodermiti, cheiliti croni - che, xeroderma pigmentosum. Inoltre, alcuni Autori enfatizzano il ruolo svolto da diversi agenti virali quali HPV16, HPV24, HSV1 e HSV2. Il trattamento dei carcinomi del labbro è prevalentemente chirurgico ed è fondamentalmente rappresentato dall’escissione della lesione e successiva ricostruzione del labbro. Le tecniche riparative sono numerose e per la maggior parte basate sull’utilizzo di lembi cutanei locali di scorrimento e di rotazione. La ricostruzione del lab - bro comporta un notevole impegno al fine di preservare quanto più possibile forma e funzioni essendo il labbro fondamentale nell’ali - mentazione, nella fonazione e nella mimica espressiva. Gli Autori riportano la loro esperienza riguardo il trattamento chi - rurgico di 19 pazienti, 17 di sesso maschile, di età compresa tra 58 e 84 anni (età media 72 anni), affetti da carcinoma del labbro (16 spi - nocellulari, 3 basocellulari) con prevalenza di forme ben differenziate. Vengono inoltre discusse le principali problematiche ricostruttive ine - renti la preservazione degli aspetti estetici e funzionali delle labbra

Novel strategies for the treatment of myelofibrosis driven by recent advances in understanding the role of the microenvironment in its etiology

Myelofibrosis is the advanced stage of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by systemic inflammation, hematopoietic failure in the bone marrow, and development of extramedullary hematopoiesis, mainly in the spleen. The only potentially curative therapy for this disease is hematopoietic stem cell transplantation, an option that may be offered only to those patients with a compatible donor and with an age and functional status that may face its toxicity. By contrast, with the Philadelphia-positive MPNs that can be dramatically modified by inhibitors of the novel BCR-ABL fusion-protein generated by its genetic lesion, the identification of the molecular lesions that lead to the development of myelofibrosis has not yet translated into a treatment that can modify the natural history of the disease. Therefore, the cure of myelofibrosis remains an unmet clinical need. However, the excitement raised by the discovery of the genetic lesions has inspired additional studies aimed at elucidating the mechanisms driving these neoplasms towards their final stage. These studies have generated the feeling that the cure of myelofibrosis will require targeting both the malignant stem cell clone and its supportive microenvironment. We will summarize here some of the biochemical alterations recently identified in MPNs and the novel therapeutic approaches currently under investigation inspired by these discoveries

p53 and telomerase control rat myocardial tissue response to hypoxia and ageing

Cellular senescence implies loss of proliferative and tissue regenerative capability. Also hypoxia, producing Reactive Oxygen Species (ROS), can damage cellular components through the oxidation of DNA, proteins and lipids, thus influencing the shortening of telomeres

Shared and Distinctive Ultrastructural Abnormalities Expressed by Megakaryocytes in Bone Marrow and Spleen From Patients With Myelofibrosis

Numerous studies have documented ultrastructural abnormalities in malignant megakaryocytes from bone marrow (BM) of myelofibrosis patients but the morphology of these cells in spleen, an important extramedullary site in this disease, was not investigated as yet. By transmission-electron microscopy, we compared the ultrastructural features of megakaryocytes from BM and spleen of myelofibrosis patients and healthy controls. The number of megakaryocytes was markedly increased in both BM and spleen. However, while most of BM megakaryocytes are immature, those from spleen appear mature with well-developed demarcation membrane systems (DMS) and platelet territories and are surrounded by platelets. In BM megakaryocytes, paucity of DMS is associated with plasma (thick with protrusions) and nuclear (dilated with large pores) membrane abnormalities and presence of numerous glycosomes, suggesting a skewed metabolism toward insoluble polyglucosan accumulation. By contrast, the membranes of the megakaryocytes from the spleen were normal but these cells show mitochondria with reduced crests, suggesting deficient aerobic energy-metabolism. These distinctive morphological features suggest that malignant megakaryocytes from BM and spleen express distinctive metabolic impairments that may play different roles in the pathogenesis of myelofibrosis

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils of polycythemia vera.

BACKGROUND: The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. DESIGN AND METHODS: We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63(+) basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. RESULTS: We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63(+) basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63(+) basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. CONCLUSIONS: These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus

Resident Self-Tissue of Proinflammatory Cytokines Rather than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-β1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakar-yocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice

Inhibition of TGF-β1 signaling restores both microenvironmental and stem cells abnormalities in the Gata-1low Mouse Model of Myelofibrosis

Primary myelofibrosis (PMF) is characterized by abnormal megakaryocyte (Mk) development, fibrosis and ineffective hematopoiesis in the marrow and hematopoiesis in extramedullary sites [1]. Studies in animal models have suggested that fibrosis is established by fibroblasts activated by TGF-β1 released by the abnormal Mk. Increased levels of TGF-β1 expression in Mk have been implicated in the development of PMF. To clarify whether TGF-β1 alterations are involved in the development of PMF in Gata1low mice, the TGF-β1 content of Mk from the marrow and spleen from PMF patients and Gata1low mice was compared, the TGF-β1 pathway of the marrow and spleen of the Gata1low mouse PMF model was profiled and the consequences of pharmacological inhibition of TGF-β1 signaling, obtained through treatment with SB431542, was determined. Bone marrow (BM) sections from PMF patients contain 4-times more Mk than those from normal donors and great numbers of Mk are also detectable in their spleen. In addition, Mk from both BM and spleen of PMF patients reacted 34-times more intensely than normal Mk with the TGF-β1 antibody. Similarly the number of Mk in BM and spleen of Gata-1low mice was 2-3- fold greater than normal and these cells reacted 3-8-times more intensely with the TGF-β1 antibody than wild-type(wt)Mk. These results were confirmed by immunoelectron- microscopy. On average, one Mk from wild-type and Gata1low mice contained 10.3±2.2 and 54.3±6.5 immunogold-particles per area (p<0.01). SB431542-treatment reduced the intensity of TGF-β1 staining of Gata1low Mk both in BM (5.3±1.1) and spleen (9.2±0.7) compared to Mk both in BM (18.9±0.8) and spleen (24.3±0.9) of Gata- 1low vehicle-treated mice, while had modest effects on the expression of VEGF and CXCL12. Inhibition of TGF-β1 signaling activates hematopoiesis in BM while reducing extramedullary hematopoiesis in spleen of Gata-1low mice. In addition, it reduced fibrosis, vessel microdensity, increases Ptl counts and decreases WBC and poikilocytes in the blood of Gata1low mice suggesting a potential benefit for treatments targeting microenvironment abnormalities in PMF

Thrombotic events in models GATA-1 low myelofibrosis characterized by altered localization of P-selectin during megakaryocyte development

Patients with primary myelofibrosis (PMF) have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions.This interactions are mediated by P-selectin and even though the megakaryocytes(Mk)of MF patients express normal levels of P-selectin,it remains abnormally localized to the DMS rather than being assembled into the a-granules in platelets.Mice carrying the hypomorphic Gata1low mutation express the same Mk abnormalities presented by PMF patients,including abnormal P-selectin localization to the DMS and develop with age myelofibrosis,that closely resembles human PMF.The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and,in this case,the role played by P-selectin in the development of the trait.To this aim,Gata1low mice were crossed with P-selnull mice according to standard genetic protocols and Gata1lowP-selWT,Gata1lowP-selnull and Gata1WTP-selnull or Gata1WTP-selWT littermates obtained.Platelet count,hematocrit as well as platelet microparticle levels were determined on all the different mutants.It was shown that platelet counts are reduced in Gata1low mice.Moreover,platelet microparticles are reduced in Gata1low mice and P-selectin positive platelet microparticles were not found.The presence of thrombosis was determined by immunohistological staining of organs.Gata1low mice with or without the P-selectin null trait had a prolonged bleeding time and thrombosis was seen adult and old Gata1low mice,but the Gata1low/P-selnull mice were rescued.Thus,presence of the P-selectin null trait rescued Gata1low mice from the thrombotic phenotype,but did not change level of platelet microparticles.All these data indicate that abnormal localization of P-selectin,induced by the Gata1low mutation,and thus, increased pathological interactions with leucocytes,is responsible for the increased presence of thrombosis seen in these mice

Repercussion of megakaryocyte-specific Gata1 Loss on megakaryopoiesis and the hematopoietic precursor compartment

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cK-OMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches. © 2016 Meinders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited