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

Z. Li, J. Stanelle, C. Leurs, H. Hanenberg, Brigette M. Pützer

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 (∼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.

Original languageEnglish (US)
Pages (from-to)2813-2821
Number of pages9
JournalNucleic Acids Research
Volume33
Issue number9
DOIs
StatePublished - 2005
Externally publishedYes

Fingerprint

Gene Library
Apoptosis
Genes
Gene Silencing
E2F1 Transcription Factor
Cycloheximide
Cell Survival
Complementary DNA
Clone Cells
RNA
Neoplasms
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library. / Li, Z.; Stanelle, J.; Leurs, C.; Hanenberg, H.; Pützer, Brigette M.

In: Nucleic Acids Research, Vol. 33, No. 9, 2005, p. 2813-2821.

Research output: Contribution to journalArticle

Li, Z. ; Stanelle, J. ; Leurs, C. ; Hanenberg, H. ; Pützer, Brigette M. / Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library. In: Nucleic Acids Research. 2005 ; Vol. 33, No. 9. pp. 2813-2821.
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