INTERPLAY BETWEEN NUCLEOSOMES AND TELOMERIC PROTEINS. DO THEY INTERACT OR COMPETE FOR TELOMERIC SEQUENCES?

In higher eukaryotes, both specific proteins and the histone octamer bind to telomeric repeats. Whether histones and specific telomeric proteins compete for telomeric DNA binding (and hence occupy different telomere domains) or whether they cooperate in the formation of the telomeric complex is a relevant issue in order to understand telomeric structure and its dynamics. In humans, TRF1 and TRF2 bind as preformed homodimers recognizing two telomeric TTAGGG repeats. Since most of telomeric DNA is packed in nucleosomes, a possible hypothesis is that TRF proteins, after saturating the short DNA linkers between nucleosomes, could interact with nucleosomal binding sites. We have previously shown that hTRF1 forms stable ternary complexes with telomeric nucleosomes. By means of in vitro model systems we show that also hTRF2 recognize nucleosomal binding sites, but with a lower affinity with respect to hTRF1. This difference does not depend on DNA binding domains (DBDs), since the DBDs of hTRF1 and hTRF2 have the same affinity for nucleosomal binding sites. Binding of hTRF1 to naked DNA, but not of hTRF2, is favored by the presence of an adjacent nucleosome. Importantly, hTRF1 binding induces nucleosome sliding, enhancing the intrinsic mobility of telomeric nucleosomes, whereas hTRF2 does not apparently influence nucleosome mobility. These results suggest that the different interactions of hTRF1 and hTRF2 with nucleosomes may play a major role in the architecture of telomeric chromatin

Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis

Alpha-tocopheryl succinate (\u3b1TOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although \u3b1TOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 \u3bcM \u3b1TOS/20 \u3bcM AA/0.2 \u3bcM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention

Human CAR NK cells: A new non-viral method allowing high efficient transfection and strong tumor cell killing

CAR-NK cells may represent a valuable tool, complementary to CART cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer

SMN affects membrane remodelling and anchoring of the protein synthesis machinery

Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis