RESUMEN
To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.
Asunto(s)
Electroforesis en Gel Bidimensional , Proteínas de Neoplasias/metabolismo , Proteoma/análisis , Proteómica , Rabdomiosarcoma Alveolar/metabolismo , Animales , Antígenos Transformadores de Poliomavirus , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Medios de Cultivo Condicionados , Fibroblastos , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Desarrollo de Músculos , Mapeo Peptídico , Proteoma/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Rat mammary gland proteomes at day 21 (prepubertal) and day 50 (late puberty) were compared by 2D difference gel electrophoresis. Two-hundred fifty-one spots were significantly different ( p < 0.05) in abundance. Peptide mass fingerprint analysis of a subset of these proteins identified two significantly over-represented classes including structural and blood proteins (increased), and metabolism-relevant proteins (reduced) in day 50 relative to day 21 glands. This is a first report of mammary gland proteome differences at these important breast cancer-relevant time-points.