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

Joseph M. Bosilevac, Randall J. Olsen, Julia A. Bridge, Steven Heye Hinrichs

Research output: Contribution to journalArticle

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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 (SRα-Fv4) significantly reduced viability and induced apoptosis as measured by terminal deoxynucleotide-transferase- 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.

Original languageEnglish (US)
Pages (from-to)34811-34818
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number49
DOIs
StatePublished - Dec 3 1999

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Activating Transcription Factor 1
Clear Cell Sarcoma
Ewing's Sarcoma
Tumors
Cell Survival
Fusion reactions
Cells
DNA
Neoplasms
Proteins
Genetic Translocation
Single-Chain Antibodies
Immunoglobulin Fragments
Flow cytometry
HEK293 Cells
Response Elements
Electric fuses
Biotin
Transferases
HeLa Cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tumor cell viability in clear cell sarcoma requires DNA binding activity of the EWS/ATF1 fusion protein. / Bosilevac, Joseph M.; Olsen, Randall J.; Bridge, Julia A.; Hinrichs, Steven Heye.

In: Journal of Biological Chemistry, Vol. 274, No. 49, 03.12.1999, p. 34811-34818.

Research output: Contribution to journalArticle

Bosilevac, Joseph M. ; Olsen, Randall J. ; Bridge, Julia A. ; Hinrichs, Steven Heye. / Tumor cell viability in clear cell sarcoma requires DNA binding activity of the EWS/ATF1 fusion protein. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 49. pp. 34811-34818.
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