Using proteomics to study the molecular events associated to the role of SPARC in tumor progression.

ANDREA S. LLERA; MARÍA ROMINA GIROTTI; MARÍA SOLEDAD SOSA; OSVALDO PODHAJCER

Pinamar, Provincia de Buenos Aires

Congreso; X Congress of the Panamerican Association for Biochemistry and Molecular Biology (PABMB), XLI Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology (SAIB) and XX Annual Meeting of the Argentine Society for Neurochemistry (SAN); 2005

Sociedad Argentina de Investigaciones Bioquímicas

SPARC is a glycoprotein from the extracellular matrix that elicits changes in cell shape and proliferation. SPARC is overexpressed in different tumors, in association with tumor progression. Our previous results showed that stable transfection of tumor cells with antisense SPARC DNA abolished tumorigenicity in an in vivo melanoma murine model, through still not clear molecular mechanisms. We have started a proteomic analysis of proteins expressed by human melanoma cells with modulated SPARC expression, in order to identify putative mediators of SPARC activity. We have used 2D-electrophoresis to compare conditioned media from human melanoma MEL-LES cells with antisense-mediated downregulation of SPARC expression (clone L-1D) and its control cell line L-CMV. We found 25 differential spots, 17 of which were identified by peptide fingerprinting analysis. A selected group of three of these proteins was chosen for validation by immunoblotting, real time PCR and other techniques. Differences in those proteins were confirmed not only in the aforementioned cells but also using other transient (i.e. adenoviral) and stable (i.e.shRNAi) methods of SPARC down-regulation on MEL-LES cells. Furthermore, L-1D cells transduced with SPARC-expressing adenovirus reverted levels of differential proteins to those in L-CMV. This is the first evidence that SPARC and these three proteins may participate in a single molecular network that leads to tumor progression