Influence of Ki-ras-driven oncogenic transformation on the protein network of murine fibroblasts

Ki-ras gene mutations that specifically occur in codons 12, 13 and 61 are involved in the carcinogenesis of acute myeloid leukemia, melanoma and different carcinomas. In order to define potential mutation-specific therapeutic targets, stable transfectants of NIH3T3 cells carrying different Ki-ras4B gene mutations were generated. Wild type Ki-ras transformants, mock transfectants and parental cells served as controls. These in vitro model systems were systematically analyzed for their protein expression pattern using two-dimensional gel electrophoresis followed by mass spectrometry and/or protein sequencing. Using this approach, a number of target molecules that are differentially but coordinately expressed in the ras transfectants were identified next to other proteins that exhibit a distinct regulation pattern in the different cell lines analyzed. The differentially expressed proteins predominantly belong to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Their validation was assessed by real-time quantitative RT-PCR and/or Western blot analysis. Our results suggest that the Ki-ras-transformed cells represent a powerful tool to study Ki-ras gene mutation-driven protein expression profiles. In addition, this approach allows the discovery of ras-associated cellular mechanisms, which might lead to the identification of physiological targets for pharmacological interventions of the treatment of Ki-ras-associated human tumors.