Two studies have been initiated to elucidate biomarkers of stress, the first involving melanoma tumor progression and the second elucidating mechanisms cells use to protect against peroxide stress. Melanoma tumor progression is a dynamic biological process involving a wide spectrum of host and tumor proteins, and we hypothesized that many of these proteins contribute to tumor immune evasion. In wild type mice, melanoma has been treated successfully using immunotherapy three and four days post tumor challenge, but after one week of established tumor growth, immunotherapy generally fails suggesting that the tumor is somehow controling a host?s immune response against the tumor. Our approach was to first perform a comparative proteome analysis on the aggressive, B16-F10 mouse melanoma and the syngeneic, non-tumorigenic, melan-a cell lines. We then challenged C57Bl/6 mice with B16-F10, and resected the tumors on progressive days. The total proteins from the tumor and cell samples were separated using 2D gel electrophoresis, and spots were identified using mass spectrometry. Our data indicate significant alterations in the tumor proteome in vivo, as well as differences in the base proteomes of the cell lines. This work identifies proteins involved in melanoma tumor progression and potential proteins associated with tumor immune evasion. The second aspect of this study is to knock down the expression of key proteins using RNA interference approaches to determine mechanisms. Cells respond to oxidative challenge by changing gene expression, protein expression and post-translational modifications of proteins. The focus of this study is to define the mechanisms cells use for protection against hydrogen peroxide and lipid peroxides and to elucidate differences in the mechanisms, if any, that cells use against different peroxides. In this case cells were adapted to growth on high concentrations of peroxides and changes in the respective proteomes were determined. A proteomics approach was used to determine quantitative differences in protein expression in the presence and absence of each peroxide and to identify the proteins with altered expression. Our data indicate there are common mechanisms used with different peroxides but survival in the presence of lipid peroxides require additional responses.
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