Proteomic analysis in melanoma cells with regulated SPARC expression reveals candidates for SPARC activity

MARIA ROMINA GIROTTI; JUAN PABLO ALBAR; OSVALDO PODHAJCER; ANDREA LLERA

Heidelberg, Alemania

Simposio; 7th EMBL International PhD Student Symposium "Biology at Work"; 2005

EMBL

Proteomic analysis in melanoma cells with regulated Sparc expression reveals candidates for Sparc activity SPARC is a secreted glycoprotein related to tumor progression and metastasis and overexpressed in different tumors. Previous studies of our laboratory showed that stable transfection of tumor cells with antisense SPARC DNA abolished tumorigenicity in an in vivo melanoma murine model through still unclear molecular mechanisms. In order to identify putative secreted proteins that may mediate SPARC biological function, we performed a proteomic analysis of conditioned media of human melanoma MEL-LES cells with antisense-mediated downregulation of SPARC expression (clone L-1D) and its control cell line L-CMV. By using two-dimensional electrophoresis we identified 13 differentially expressed proteins, from which three were chosen for validation by different techniques. Differences in levels of expression of these three 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 expression 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. SPARC is a secreted glycoprotein related to tumor progression and metastasis and overexpressed in different tumors. Previous studies of our laboratory showed that stable transfection of tumor cells with antisense SPARC DNA abolished tumorigenicity in an in vivo melanoma murine model through still unclear molecular mechanisms. In order to identify putative secreted proteins that may mediate SPARC biological function, we performed a proteomic analysis of conditioned media of human melanoma MEL-LES cells with antisense-mediated downregulation of SPARC expression (clone L-1D) and its control cell line L-CMV. By using two-dimensional electrophoresis we identified 13 differentially expressed proteins, from which three were chosen for validation by different techniques. Differences in levels of expression of these three 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 expression 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.