Role of Glypican-6 and NG2 as metastasis promoting factors

Cell surface proteoglycans (PGs) are key molecules in the regulation of tumour progression and metastasis formation. Eleven primary surface PGs, including syndecans-1-4, glypicans-1-6 and NG2, are currently known to act as mediators of the cancer cell’s interaction with the host microenvironment, with the potential to function synergically or antagonistically in the promotion of tumour growth and spreading. Using soft-tissue sarcomas as a model system we have observed that a given tumour cell may constitutively and coincidently express on average 3-5 of the 11 surface PGs, suggesting that diverse combinations of surface PGs may dictate the behaviour of cancer cells in different manners. To start to investigate the PG surface profiles as pro- and anti-tumorigenic we delineated strategies to modify the PG expression of 143B osteosarcoma cells by stable gene transduction and by examining how these modifications affected the cells adhesive and migratory capabilities in response to selected ECM substrates, and endothelial monolayers. To date there are no notice about GPC6 implications either in cell-ECM interactions or in the behaviour motility of cells. For these reasons 143B cells were stable transfected for overexpressing GPC6 showed a modulation of relative expression of all surface PGs respect to vector control cells. GPC6 overexpression induces morphological modification in 143B cells which are seen as changes in the organization of actin filaments containing protrusions similar to fillopodia/lamellipodia and an increase of motility cells in monodimensional assay which could be in relationship with cytoskelatal reorganization. Moreover these cells showed a spreading ability and the lower invasion ability on different ECM molecules that could be correlated by surface PG profile modulation. Since NG2 as a cell surface ligand for collagen type VI has been postulated to be involved in tumor progression, we have also examinated the interaction of the NG2+ and NG2- sarcoma cells with collagen type VI and other ECM molecules. Sarcoma cells were NG2 abrogated by RNAi or cells immunosorted for NG2 expression were found to exhibit a rather elective, impaired ability to adhere and migrate on purified collagen type VI. These findings confirmed that the NG2 was capable of mediating tumour cell adhesion and migration through interaction with this specific collagen. The outcome of these investigations provide a first evidence that NG2 may represent a unique, malignancy promoting factor in several types of soft-tissue sarcomas and that defined surface PGs pattern differentially control tumour progression, with some profiles being specifically associated with an aggressive behaviour, whereas others with a more benign phenotype

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i. e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects

Fluorescence-based assays for in vitro analysis of cell adhesion and migration

Cell adhesion and cell migration are two primary cellular phenomena for which in vitro approaches may be exploited to effectively dissect the individual events and underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also afford an efficient way of conducting larger basic and applied research screenings on the factors affecting these processes and are potentially exploitable in the context of routine diagnostic, prognostic, and predictive tests in the biological and medical fields. Therefore, there is a longstanding continuum in the interest in devising more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescence cell tagging, the design of instruments capable of detecting fluorescent signals with high sensitivity, and informatic tools allowing sophisticated elaboration of data generated through these instruments. In this report, we describe three representative fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automated for high-to-medium throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup