Use of DIGE for the discovery of molecular pathways affected by the tumorigenic protein SPARC

MARÍA ROMINA GIROTTI; MARISOL FERNÁNDEZ; ELMER FERNÁNDEZ; EDGARDO SALVATIERRA; JUAN PABLO ALBAR; OSVALDO PODHAJCER; ANDREA LLERA

rosario

Congreso; SAIB; 2006

SPARC is a secreted glycoprotein related to tumor progression and metastasis and overexpressed in different tumors. However, little is known about the molecular mechanisms affected by SPARC during tumor growth. We have developed stable cell clones of human melanoma cells (L3B9 and L2F6) in which SPARC expression was downregulated by the use of 2 different RNAi. SPARC downregulation diminished (L3B9) or abolished (L2F6) tumor growth in a murine in vivo model. In order to identify putative secreted proteins that may mediate SPARC biological function, we performed a proteomic analysis of conditioned media of clones L3B9 and L2F6 and the control cell line LBLAST. For this purpose we applied the novel technology of DIGE (DIfferential Gel Electrophoresis) that proved to be superior to traditional silver-stained 2D electrophoresis in many aspects. Along with a thorough description of processing and normalization steps applied to our DIGE data, we will show that unsupervised multivariate analysis was able to identify a set of spots that could distinguish the three different treatments used in the study. These results define a set of proteins potentially related to SPARC role in tumor progression. Further work is in progress to validate our candidates both technically and biologically.