WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.     

Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of Dasypyrum villosum L. (H. villosa) by a Single DNA Probe

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.     

Properties of the endogenous components of the thioredoxin system in the psychrophilic eubacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC 125

The endogenous components of the thioredoxin system in the Antarctic eubacterium Pseudoalteromonas haloplanktis have been purified and characterised. The temperature dependence of the activities sustained by thioredoxin (PhTrx) and thioredoxin reductase (PhTrxR) pointed to their adaptation in the cold growth environment. PhTrxR was purified as a flavoenzyme and its activity was significantly enhanced in the presence of molar concentration of monovalent cations. The energetics of the partial reactions leading to the whole electron transfer from NADPH to the target protein substrate in the reconstituted thioredoxin system was also investigated. While the initial electron transfer from NADPH to PhTrxR was energetically favoured, the final passage to the heterologous protein substrate enhanced the energetic barrier of the whole process. The energy of activation of the heat inactivation process essentially reflected the psychrophilic origin of PhTrxR. Vice versa, PhTrx possessed an exceptional heat resistance (half-life, 4.4 h at 95 °C), ranking this protein among the most thermostable enzymes reported so far in psychrophiles. PhTrxR was covalently modified by glutathione, mainly by its oxidised or nitrosylated forms. A mutagenic analysis realised on three non catalytic cysteines of the flavoenzyme allowed the identification of C₃₀₃ as the target for the S-glutathionylation reaction.     

Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres

Increasing evidence indicates that invasive properties of breast cancers rely on gain of mesenchymal and stem features, which has suggested that the dual targeting of these phenotypes may represent an appealing therapeutic strategy. It is known that the fraction of stem cells can be enriched by culturing breast cancer cells as mammospheres (MS), but whether these pro-stem conditions favor also the expansion of cells provided of mesenchymal features is still undefined. In the attempt to shed light on this issue, we compared the phenotypes of a panel of 10 breast cancer cell lines representative of distinct subtypes (luminal, HER2-positive, basal-like and claudin-low), grown in adherent conditions and as mammospheres. Under MS-proficient conditions, the increment in the fraction of stem-like cells was associated to upregulation of the mesenchymal marker Vimentin and downregulation of the epithelial markers expressed by luminal cells (E-cadherin, KRT18, KRT19, ESR1). Luminal cells tended also to upregulate the myoepithelial marker CD10. Taken together, our data indicate that MS-proficient conditions do favor mesenchymal/myoepithelial features, and indicate that the use of mammospheres as an in vitro tumor model may efficiently allow the exploitation of therapeutic approaches aimed at targeting aggressive tumors that have undergone epithelial-to-mesenchymal transition.     

Cutting edge: a naturally occurring mutation in CD1e impairs lipid antigen presentation

The human CD1a-d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM(6). CD1e is formed by the association of beta(2)-microglobulin with an alpha-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM(6) presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.     

Synthesis and proteomic activity evaluation of a new isotope-coded affinity tagging (ICAT) reagent

During recent years, quantitative proteome profiling has taken advantage of incorporating the traditional stable isotope dilution analysis into global scale or discovery-based proteomic experiments that use mass spectrometers as detectors to allow the pairwise study of differently expressed proteins. Quantitative protein analysis by means of the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pairwise comparison of protein expression levels in biological samples. Herein, a modified ICAT reagent, named BAA-ICAT (beta-alanine-arm-ICAT) in which the polyether linker is replaced by a more water-soluble polyamide one, was investigated.