Proteomics Analysis of Melanoma Cell Lines

K.A.Resing, K.Bernard, L.Aveline, K.Jonscher, N.G.Ahn

Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA

Biochemical, molecular biological, and genetic studies have led to a model for carcinogenesis wherein tumor progression involves increasing mutational burden resulting in progressive release from growth regulation and apoptosis surveillance, and increasing likelihood of metastasis. In melanoma, this progression is reflected in morphological "stages": atypical naevus, radial growth phase (RGP), vertical growth phase (VGP), and metastasis. When melanoma cells are cultured from tumors, these stages are correlated with ability to produce tumors or metastases in nude mice, or with differences in motility, adhesion, or sensitivity to apoptosis signals. Although changes in these properties are driven by changes in signaling pathways, the complexity of these pathways makes it impossible to directly or quantitatively connect these functional assays to specific signaling events.

We are using immobiline dry strip method for isoelectric focusing and two-dimensional gel electrophoresis to provide a more precise method of delineating the phenotype of the cell in a quantifiable way that can be directly related to the underlying signaling status. Using pI range 4.5 to 6.5 gels, approximately 3500 protein spots can be identified. Initial analyses of 15 cell lines (5 RGP, 4 VGP, 6 Metastatic) in the size range 10-55 kDa, shows most of the variability in this pI and size range is related to the growth rate and cell morphology in culture. Eight proteins can be related to the functional stages; but in each case, there are some cell lines that do not fit the pattern. Identification of these proteins reveals that they include known carcinogenesis markers, as well as potential new markers. Using pharmacological agents and transient transfections we are testing the role of specific signaling pathways in the expression of the variable proteins that vary in comparisons between these cell lines and with primary melanocytes. For example, we have identified a group of the variable proteins as those that are turned on by TPA, but not growth factors, suggesting that they are regulated by a PKC dependent mechanism.