Tumor cell viability in clear cell sarcoma requires DNA binding activity of the EWS/ATF1 fusion protein.
Chimeric proteins resulting from characteristic chromosomal translocations are believed to play a key role in the development of neoplasia. The consistent chromosomal translocation t(12;22) found in Clear Cell sarcoma (CCS) fuses the genes for Ewing's sarcoma protein (EWS) and activating transcription factor 1 (ATF1). Contribution of the chimeric EWS/ATF1 protein to maintenance of the tumor phenotype was investigated using intracellular expression of an inhibitory anti-ATF1 single chain antibody fragment (scFv4). Transfection of scFv4 into a cell line (SU-CCS-1) derived from CCS resulted in a 90% reduction in cyclic AMP response element-driven reporter activity. The delivery of scFv4 into SU-CCS-1 cells by a Moloney sarcoma retroviral vector (SRalpha-Fv4) significantly reduced viability and induced apoptosis as measured by terminal deoxynucleotidetransferase-mediated dUTP-biotin nick end labeling and flow cytometry. Conversely, scFv4 had no effect on viability of HeLa cells. The level of EWS/ATF1 expression was found to be significantly higher in primary tumor tissue than in SU-CCS-1 cells or in 293T cells following introduction of an EWS/ATF1 expression vector. These studies demonstrate a direct role for the EWS/ATF1 fusion protein in maintaining tumor cell viability of Clear Cell sarcoma and indicate that intracellular antibodies may be used to achieve a phenotypic knockout of tumor-related proteins as a method to explore their function.
Bosilevac
J M
JM
Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Olsen
R J
RJ
Bridge
J A
JA
Hinrichs
S H
SH
D017209:Apoptosis; D002470:Cell Survival; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes; D005434:Flow Cytometry; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D015514:Oncogene Proteins, Fusion / Q000276:immunology / Q000378:metabolism*; D012190:Retroviridae; D018227:Sarcoma, Clear Cell / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C111598:EWS-ATF1 fusion protein, human; D000939:Epitopes; D007128:Immunoglobulin Fragments; D015514:Oncogene Proteins, Fusion; D014157:Transcription Factors