Tumor cell viability in clear cell sarcoma requires DNA binding activity of the EWS/ATF1 fusion protein.

Abstract

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.

• Authors: J M Bosilevac, R J Olsen, J A Bridge, S H Hinrichs
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 274 Issue 49 Pg. 34811-8 (Dec 03 1999) ISSN: 0021-9258 [Print] United States
• PMID: 10574952 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA-Binding Proteins
• EWS-ATF1 fusion protein, human
• Epitopes
• Immunoglobulin Fragments
• Oncogene Proteins, Fusion
• Transcription Factors

Topics

• Apoptosis
• Cell Survival
• DNA-Binding Proteins (immunology, metabolism)
• Epitopes
• Flow Cytometry
• Genes, Reporter
• HeLa Cells
• Humans
• Immunoglobulin Fragments (metabolism)
• In Situ Nick-End Labeling
• Oncogene Proteins, Fusion (immunology, metabolism)
• Retroviridae
• Sarcoma, Clear Cell (metabolism, pathology)
• Time Factors
• Transcription Factors (metabolism)
• Transfection
• Tumor Cells, Cultured