An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours.

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.

Genomic profiling of malignant melanoma using tiling-resolution arrayCGH.

Malignant melanoma is an aggressive, heterogeneous disease where new biomarkers for diagnosis and clinical outcome are needed. We searched for chromosomal aberrations that characterize its pathogenesis using 47 different melanoma cell lines and tiling-resolution bacterial artificial chromosome-arrays for comparative genomic hybridization. Major melanoma genes, including BRAF, NRAS, CDKN2A, TP53, CTNNB1, CDK4 and PTEN, were examined for mutations. Distinct copy number alterations were detected, including loss or gain of whole chromosomes but also minute amplifications and homozygous deletions. Most common overlapping regions with losses were mapped to 9p24.3-q13, 10 and 11q14.1-qter, whereas copy number gains were most frequent on chromosomes 1q, 7, 17q and 20q. Amplifications were delineated to oncogenes such as MITF (3p14), CCND1 (11q13), MDM2 (12q15), CCNE1 (19q12) and NOTCH2 (1p12). Frequent findings of homozygous deletions on 9p21 and 10q23 confirmed the importance of CDKN2A and PTEN. Pair-wise comparisons revealed distinct sets of alterations, for example, mutually exclusive mutations in BRAF and NRAS, mutual mutations in BRAF and PTEN, concomitant chromosome 7 gain and 10 loss and concomitant chromosome 15q22.2-q26.3 gain and 20 gain. Moreover, alterations of the various melanoma genes were associated with distinct chromosomal imbalances suggestive of specific genomic programs in melanoma development.

Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile.

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the digestive tract, are believed to arise from the interstitial cells of Cajal. GISTs are characterized by mutations in the proto-oncogene KIT that lead to constitutive activation of its tyrosine kinase activity. The tyrosine kinase inhibitor STI571, active against the BCR-ABL fusion protein in chronic myeloid leukemia, was recently shown to be highly effective in GISTs. We used 13,826-element cDNA microarrays to define the expression patterns of 13 KIT mutation-positive GISTs and compared them with the expression profiles of a group of spindle cell tumors from locations outside the gastrointestinal tract. Our results showed a remarkably distinct and uniform expression profile for all of the GISTs. In particular, hierarchical clustering of a subset of 113 cDNAs placed all of the GIST samples into one branch, with a Pearson correlation >0.91. This homogeneity suggests that the molecular pathogenesis of a GIST results from expansion of a clone that has acquired an activating mutation in KIT without the extreme genetic instability found in the common epithelial cancers. The results provide insight into the histogenesis of GIST and the clinical behavior of this therapeutically responsive tumor.

Estrogen receptor status in breast cancer is associated with remarkably distinct gene expression patterns.

To investigate the phenotype associated with estrogen receptor alpha (ER) expression in breast carcinoma, gene expression profiles of 58 node-negative breast carcinomas discordant for ER status were determined using DNA microarray technology. Using artificial neural networks as well as standard hierarchical clustering techniques, the tumors could be classified according to ER status, and a list of genes which discriminate tumors according to ER status was generated. The artificial neural networks could accurately predict ER status even when excluding top discriminator genes, including ER itself. By reference to the serial analysis of gene expression database, we found that only a small proportion of the 100 most important ER discriminator genes were also regulated by estradiol in MCF-7 cells. The results provide evidence that ER+ and ER- tumors display remarkably different gene-expression phenotypes not solely explained by differences in estrogen responsiveness.

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

Binding of human plasminogen and lactoferrin by Helicobacter pylori coccoid forms.

The interactions between Helicobacter pylori spiral and coccoid forms, extracellular matrix (ECM) and plasma proteins were studied in an 125I-labelled protein assay. The range of binding of collagen V, plasminogen, human lactoferrin (HLf) and vitronectin to coccoid forms of H. pylori NCTC 11637 was 26-48%. In contrast, binding of radiolabelled fibronectin and collagen types I and III was low (3-8%). The coccoid forms of 14 strains of H. pylori showed significant HLf binding (median 26%). With plasminogen, no significant difference was found between binding to the coccoid (median = 13%) and spiral (median = 12%) forms, of 13 of the 14 strains of H. pylori tested; the exception was strain NCTC 11637. 125I-plasminogen showed a dose-dependent binding to both the coccoid and spiral forms. Plasminogen binding to both forms was specific; the binding was inhibited by non-labelled plasminogen, plasmin, lysine, EACA (epsilon-aminocaproic acid) but not by fetuin or various carbohydrates. Similarly, HLf binding was found to be specific and was inhibited by non-labelled HLf and BLf. The coccoid forms showed either similar or enhanced ECM binding capabilities compared with the spiral forms. As the binding of ECM proteins may be an important mechanism of tissue adhesion for various pathogenic bacteria, the coccoid differentiated form of H. pylori can be considered as an infective form in the pathogenesis of helicobacter infection and type B gastritis.

Inhibition of heparan sulphate and other glycosaminoglycans binding to Helicobacter pylori by various polysulphated carbohydrates.

Heparan sulphate binding to Helicobacter pylori at pH 4 to 5 was inhibited with various sulphated polysaccharides (heparin and chondroitin sulphates, fucoidan, carrageenans and some others), but not by carboxylated or nonsulphated compounds. Heparin binding proteins are exposed on the cell surface.

Binding of vitronectin and plasminogen to Helicobacter pylori.

We have studied how some extracellular matrix proteins, fibronectin, fibrinogen, collagen type I and type IV, plasminogen and vitronectin bind to Helicobacter pylori. Radiolabelled vitronectin and plasminogen bound to the haemagglutinating H. pylori strain 17874 at a high level (53% and 32%, respectively), type IV collagen showed an intermediate level of binding (16%), while binding by 125I-labelled fibrinogen, fibronectin and collagen type I remained at a low level (5-7%). Both 125I-vitronectin and plasminogen showed a dose-dependent binding to cells of H. pylori 17874. Plasminogen binding by this strain was specific since the binding was inhibited by nonlabelled plasminogen, but not by highly glycosylated glycoproteins such as fetuin and orosomucoid or by a variety of monosaccharides. We have previously shown that 125I-vitronectin shows a specific and saturable binding to H. pylori 17874, and that sialic acid-rich glycoproteins such as fetuin and orosomucoid drastically reduced binding. We now report that a simultaneous incubation of 125I-vitronectin and 125I-plasminogen with cells of H. pylori 17874 showed a total binding approximately similar to the level of binding when either 125I-plasminogen, or 125I-vitronectin only were incubated with the bacterial cells. Nonlabelled vitronectin inhibited the binding of 125I-plasminogen by H. pylori, but nonlabelled plasminogen had no effect on the binding of 125I-vitronectin. Our findings suggest that there are different but probably closely localized binding sites for vitronectin and plasminogen on H. pylori 17874.

Adherence of haemagglutinating Helicobacter pylori to five cell lines.

Helicobacter pylori causes gastritis and is an important factor for the development of peptic ulcer disease in man. We used two different methods to examine the adhesion of nine H. pylori strains, with different haemagglutinating properties, to five cell lines, HeLa S3, HFI, Vero, SW1222 and WEHI cells. The adhesion studies were performed a) as bacterial adhesion to a monolayer of tissue culture cells, visualizing bacteria with fluorescein-isothiocyanate-labelled antibodies, b) as cell agglutination with bacteria and eucaryotic cells mixed in a suspension. The H. pylori strains were divided into three groups according to their cell adhesion properties. In general, H. pylori strains which showed the best adhesion to the five cell lines were strains which showed the best capability of agglutinating erythrocytes of several animal species. It is likely that the same adhesins are involved in cell adhesion and in haemagglutination. The two methods gave similar results.

High-affinity binding of laminin by Helicobacter pylori: evidence for a lectin-like interaction.

Laminin, the major glycoprotein of basement membranes, was shown to be bound by the human gastric pathogen Helicobacter pylori. Binding of 125I-laminin by strain 17874 was time-dependent, specific and saturable. Scatchard analysis of specific binding indicated about 2000 binding sites per cell with a dissociation constant of 8.5 pM. Treatment of the cells by heat (80 degrees) and with proteolytic enzymes drastically reduced laminin binding, suggesting that the laminin receptors are surface proteins. Some highly glycosylated glycoproteins inhibited laminin binding by 50%. Furthermore, N-acetylneuraminyllactose decreased laminin binding by 70% and neuraminidase treatment of laminin by 50%, while a recombinant B1 chain of laminin, containing high-mannose type oligosaccharides, inhibited binding by only 25%. This suggests that terminal sialic acids on laminin compete for a specific sugar binding protein(s) on H. pylori cells.

Vitronectin binding by Helicobacter pylori.

Vitronectin, a serum and extracellular matrix protein involved in immunological reactions, interacts with Helicobacter pylori strains. Of the 20 H. pylori strains tested three strains bound more than 50% of the vitronectin added, five strains bound 25-40%, nine strains bound 10-20% and three strains bound 5-8% vitronectin. Two strains, one with high- and one with low-binding properties, were selected for further characterization of 125I-vitronectin binding. Binding to the urea-activated 125I-labelled vitronectin was fast, saturable and reversible when an excess of unlabelled vitronectin was added to the bacteria with bound 125I-vitronectin. The binding was heat- and protease-sensitive, suggesting that the binding was mediated by bacterial cell-surface proteins. Since components such as fetuin and orosomucoid but not asialofetuin inhibited the binding, sialic-acid specific proteins, related to H. pylori sialic-acid specific haemagglutinins, were probably involved.