Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

BACKGROUND: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. RESULTS: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. CONCLUSION: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

Effect of materials for micro-electro-mechanical systems on PCR yield.

In this study we analyzed the surface properties of different silicon-based materials used for micro-electro-mechanical systems (MEMS) production, such as thermally grown silicon oxide, plasma-enhanced chemical vapor deposition (PECVD)-treated silicon oxide, reactive-ion etch (RIE)-treated silicon oxide, and Pyrex. Substrates were characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) to define the surface chemical and morphological properties, and by fluorescence microscopy to directly assess the absorption of the different polymerase chain reaction (PCR) components. By using microchips fabricated with the same materials we investigated their compatibility with PCR reactions, exploiting the use of different enzymes and reagents or proper surface treatments. We established the best conditions for DNA amplification in silicon/Pyrex microdevices depending on the type of device and fabrication method used and the quality of reagents, rather than on the passivation treatment or increment in standard Taq polymerase concentration.

Human plasma protein adsorption on carbon-based materials.

In this study the initial reactions of different carbon-based materials with human blood were investigated by short-time exposure to platelet poor plasma (PPP). Extent of protein adsorption and conformational changes of proteins adsorbed on material surfaces are known to be keys factors affecting further biological reactions. Plasma protein adsorption on multi-walled carbon nanotubes (MWCNTs), highly oriented pyrolytic graphite (HOPG) and nanocrystalline graphite (NG) were investigated and the results obtained on these materials were compared with those obtained studying pyrolytic carbon (PyC), a material showing good anti-trombogenic properties. The quantification of adsorbed plasma proteins on sample surfaces was obtained by Micro BCA Protein Assay, while immunofluorescence analysis was employed to monitor the surface density and distribution of two selected proteins, namely fibrinogen (Fg) and Hageman factor (FXII), proteins playing a leading role in mediating platelet adhesion. The dependence of the biological response on the surface chemical and morphological properties were also investigated and data obtained using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM) are presented. After PPP incubation PyC is characterized by the presence of low level of whole proteins and FXII adsorption, in contrast to a high adhesion of Fg. Compared to PyC the analysis of the other carbon-based materials result in a higher whole protein adsorption with an increasing trend moving from MWCNTs, NG and HOPG respectively. The Fg surface density on PyC, NG and MWCNTs is about four times higher than on HOPG while only HOPG show a detectable fluorescent signal of FXII. If AFM data indicate that surface morphology does not play a crucial role in protein adhesion, XPS analysis show chemical differences that can be correlated with this biological response.

Immunological and pathobiological roles of fibulin-1 in breast cancer.

Fibulin-1 (Fbln-1) is an immunogenic breast cancer-related glycoprotein identified by serological analysis of cDNA expression library (SEREX) strategy. Here, we show that dendritic cells from two breast cancer patients elicited a CD4(+)-mediated T-cell response to Fbln-1 presentation. In both patients, an antibody response to Fbln-1 was also found. By contrast, a Fbln-1-seronegative patient and a weakly seropositive patient demonstrated no such T-cell response. Analysis of human breast cancers for Fbln-1 RNA and protein expression revealed the presence of Fbln-1C and -1D variants. Fbln-1 was detected in the cytoplasm and at the cell surface of different human breast carcinoma cell lines. Immunohistochemical analysis of 528 archival primary breast carcinomas showed the expression of Fbln-1 in 35% of the cases. When the immunohistochemical findings were compared against pathobiological information associated with each specimen, an inverse relationship between Fbln-1 and cathepsin D expression was observed (P=0.04). Furthermore, even though long-term survival was similar between Fbln-1-positive and -negative cases, the survival of Fbln-1-positive cases improved when a lymphoid infiltrate was present at the tumour site. Taken together, our findings of an Fbln-1-specific immunity and the improved survival associated with Fbln-1 expression in the presence of lymphoid infiltration point to a role of Fbln-1 in tumour immunosurveillance.

New insights into the role of extracellular matrix during tumor onset and progression.

Recently, a view of the tumor as a functional tissue interconnected with the microenvironment has recently been described. For many years, the stroma has been studied in the context of the malignant lesion, and only rarely has its role been considered before carcinogenic lesions appear. Recent studies have provided evidence that stromal cells and their products can cause the transformation of adjacent cells through transient signaling that leads to the disruption of homeostatic regulation, including control of tissue architecture, adhesion, cell death, and proliferation. It is now well established that tumor progression requires a continually evolving network of interactions between neoplastic cells and extracellular matrix. A relevant step of this process is the remodeling of microenvironment which surrounds tumors leading to the release of ECM-associated growth factors which can then stimulate tumor and/or endothelial cells. Finally, tumor cells reorganizing the extracellular matrix to facilitate communications and escape the homeostatic control exerted by the microenvironment modify response to cytotoxic treatments.

Identification of breast cancer-restricted antigens by antibody screening of SKBR3 cDNA library using a preselected patient's serum.

Screening of a breast cancer cDNA library from SKBR3 human breast cancer cells by SEREX (serological analysis of cDNA expression library) using a preselected serum from a breast cancer patient revealed 13 genes, two of which, INT-MI-1 and INT-MI-2, encode novel gene products, while the remaining 11 genes and their products are identical with or highly homologous to known GenBank entries. Immunoscreening of the 13 clones using 20 allogeneic sera from breast cancer patients and 20 samples from age- and gender-matched healthy donors showed that lactate dehydrogenase-A (LDH-A), lactate dehydrogenase-B (LDH-B), fibulin-1, and thyroid hormone-binding protein (THBP) were recognized principally by the breast cancer patient sera, indicating the immunogenicity of these molecules in vivo. The other antigens were similarly recognized by normal and patients sera, and thus not tumor-restricted immunologically. RT-PCR analysis revealed strong expression of fibulin-1 in tumor cell lines and surgical specimen whereas in the same experimental conditions, normal tissues scored negative. Also THBP expression was found in various tumors whereas in normal tissues, its expression is restricted to the testis and, at lower levels, in ovary, liver, and spleen. In contrast, LDH-A and LDH-B were ubiquitously expressed in normal and tumor tissues, with LDH-B levels considerably lower and heterogeneous in normal samples compared to those expressed in tumor cell lines. The differential expression of fibulin-1 between the normal tissues and breast carcinoma cell lines (5/6) and surgical specimens (5/6) suggests the possible involvement of the overexpression of this extracellular matrix-associated glycoprotein in the pathogenesis of this neoplasm.