Imatinib Treatment Induces CD5+ B Lymphocytes and IgM Natural Antibodies with Anti-Leukemic Reactivity in Patients with Chronic Myelogenous Leukemia

Imatinib mesylate is a first line treatment of Chronic Myelogenous Leukemia and of a rare form of gastrointestinal stromal cancer, where the response to the drug is also linked to the immune system activation with production of antineoplastic cytokines. In this study, forty patients in the chronic phase of disease, treated with imatinib mesylate, were analyzed. Bone marrow aspirates were drawn at diagnosis, after 3, 6, 12, 18 months for haematological, cytofluorimetric, cytogenetic, biomolecular evaluation and cytokine measurement. Responder and non responder patients were defined according to the European LeukemiaNet recommendations. In responder patients (n = 32), the percentage of bone marrow CD20+CD5+sIgM+ lymphocytes, and the plasma levels of IgM, were significantly higher, at 3 months and up to 9 months, than in non responders. These IgM reacted with O-linked sugars expressed by leukemic cells and could induce tumor cell apoptosis. In responeìder patients the stromal-derived factor-1 and the B-lymphocyte-activating factor of the tumor necrosis factor family significantly raised in the bone marrow after imatinib administration, together with the bone morphogenetic proteins-2 and −7. All patients with high number of CD20+CD5+sIgM+ cells and high stromal-derived factor-1 and B lymphocyte activating factor levels, underwent complete cytogenetic and/or molecular remission by 12 months. We propose that CD20+CD5+sIgM+ lymphocytes producing anti-carbohydrate antibodies with anti-tumor activity, might contribute to the response to imatinib treatment. As in multivariate analysis bone marrow CD20+CD5+sIgM+ cells and stromal-derived factor-1 and B-lymphocyte-activating factor levels were significantly related to cytogenetical and molecular changes, they might contribute to the definition of the pharmacological response

Modulation of the CD95-Induced Apoptosis: The Role of CD95 N-Glycosylation

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems

17β-Estradiol Enhances Breast Cancer Cell Motility and Invasion via Extra-Nuclear Activation of Actin-Binding Protein Ezrin

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr567 in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers

Heparin modification of a biomimetic bone matrix modulates osteogenic and angiogenic cell response in vitro

In this study, the effect of heparin-modified collagen type I/hydroxyapatite (HA) nanocomposites on key processes of bone regeneration - osteogenesis and angiogenesis was characterised in vitro. Two approaches were applied for heparin modification: it was either integrated during material synthesis (in situ) or added to the porous scaffolds after their fabrication (post). Cultivation of human bone marrow-derived stromal cells (hBMSC), in heparin-modified versus heparin-free scaffolds, revealed a positive effect of the heparin modification on their proliferation and osteogenic differentiation. The amount of heparin rather than the method used for modification influenced the cell response favouring proliferation at smaller amount (30 mg/g collagen) and differentiation at larger amount (150 mg/g collagen). A co-culture of human umbilical vein endothelial cells (HUVEC) and osteogenically induced hBMSC was applied for in vitro angiogenesis studies. Pre-vascular networks have formed in the porous structure of scaffolds which were not modified with heparin or modified with a low amount of heparin (30 mg/g collagen). The modification with higher heparin quantities seemed to inhibit tubule formation. Pre-loading of the scaffolds with VEGF influenced formation and stability of the prevascular structures depending on the presence of heparin: In heparin-free scaffolds, induction of tubule formation and sprouting was more pronounced whereas heparin-modified scaffolds seemed to promote stabilisation of the pre-vascular structures. In conclusion, the modification of mineralised collagen with heparin by using both approaches was found to modulate cellular processes essential for bone regeneration; the amount of heparin has been identified to be crucial to direct cell responses

HEPARIN MODIFICATION OF A BIOMIMETIC BONE MATRIX MODULATES OSTEOGENIC AND ANGIOGENIC CELL RESPONSE IN VITRO

In this study, the effect of heparin-modified collagen type I/hydroxyapatite (HA) nanocomposites on key processes of bone regeneration - osteogenesis and angiogenesis was characterised in vitro. Two approaches were applied for heparin modification: it was either integrated during material synthesis (in situ) or added to the porous scaffolds after their fabrication (post). Cultivation of human bone marrow-derived stromal cells (hBMSC), in heparin-modified versus heparin-free scaffolds, revealed a positive effect of the heparin modification on their proliferation and osteogenic differentiation. The amount of heparin rather than the method used for modification influenced the cell response favouring proliferation at smaller amount (30 mg/g collagen) and differentiation at larger amount (150 mg/g collagen). A co-culture of human umbilical vein endothelial cells (HUVEC) and osteogenically induced hBMSC was applied for in vitro angiogenesis studies. Pre-vascular networks have formed in the porous structure of scaffolds which were not modified with heparin or modified with a low amount of heparin (30 mg/g collagen). The modification with higher heparin quantities seemed to inhibit tubule formation. Pre-loading of the scaffolds with VEGF influenced formation and stability of the prevascular structures depending on the presence of heparin: In heparin-free scaffolds, induction of tubule formation and sprouting was more pronounced whereas heparin-modified scaffolds seemed to promote stabilisation of the pre-vascular structures. In conclusion, the modification of mineralised collagen with heparin by using both approaches was found to modulate cellular processes essential for bone regeneration; the amount of heparin has been identified to be crucial to direct cell responses

Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes

The ezrin/radixin/moesin (ERM) family of actin-binding proteins act both as linkers between the actin cytoskeleton and plasma membrane proteins and as signal transducers in responses involving cytoskeletal remodelling. The Rho family of GTPases also regulate cytoskeletal organisation, and several molecular pathways linking ERM proteins and Rho GTPases have been described. This review discusses recent findings on ERM protein function in leucocytes and how these may be integrated with Rho GTPase signalling