2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma.
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.
Department of Pediatrics, Division of Hematology-Oncology, University of Alabama at Birmingham, Birmingham, AL 35233, USA. jpressey@uab.edu
Joseph G
JG
Pressey
Christine S
CS
Pressey
Gloria
G
Robinson
Richie
R
Herring
Landon
L
Wilson
David R
DR
Kelly
Helen
H
Kim
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D017077:Culture Media, Conditioned; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008954:Models, Biological; D024510:Muscle Development; D009363:Neoplasm Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics; D015203:Reproducibility of Results; D018232:Rhabdomyosarcoma, Alveolar / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000952:Antigens, Polyomavirus Transforming; D014408:Biomarkers, Tumor; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D020543:Proteome