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Nucleic Acids Res. 2005 May 16;33(9):2813-21. Print 2005.

Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library.

Author information

1
Department of Vectorology and Experimental Gene Therapy, University of Rostock Medical School Institute Building, Schillingallee 70, 18057 Rostock, Germany.

Abstract

The E2F1 transcription factor is an essential mediator of p53-dependent and p53-independent apoptosis as part of an anti-tumour safeguard mechanism. In this study, a functional so-called technical knockout (TKO) approach was applied to Saos-2ERE2F1 cells that conditionally activate E2F1 by the addition of 4-hydroxytamoxifen to search for p53-independent pro-apoptotic E2F1 targets. The approach was based on random inactivation of genes after retroviral transfer of an antisense cDNA library enriched of E2F1-induced genes, followed by the selection of Saos-2ERE2F1 cells that survive in the presence of the apoptotic stimulus. We identified 13 novel E2F1 target genes encoding proteins of known cellular function, including apoptosis and RNA binding. FACS analysis revealed that E2F1-induced apoptosis was significantly attenuated in cell clones containing the antisense cDNA fragments of these genes, demonstrating their participation in E2F1 death pathways. Moreover, inactivation of the target genes resulted in a clear increase of cell viability (>80%) in response to E2F1 activation compared with controls (approximately 30%). Four genes showed an increase in expression intensity in the presence of cycloheximide, suggesting a direct effect of E2F1 on gene transcription, whereas one gene was identified as an indirect target. Our data provide new insight in the regulation of E2F1-induced apoptosis.

PMID:
15897325
PMCID:
PMC1129028
DOI:
10.1093/nar/gki581
[Indexed for MEDLINE]
Free PMC Article

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