Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library
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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
1Department 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.
Insights
Researchers identified novel E2F1 targets crucial for apoptosis. Inactivating these genes significantly enhanced cell survival, revealing new insights into E2F1-mediated anti-tumor mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The E2F1 transcription factor plays a critical role in apoptosis, acting as an anti-tumor mechanism.
- Understanding E2F1's targets is key to elucidating its role in both p53-dependent and p53-independent cell death pathways.
Purpose of the Study:
- To identify novel p53-independent target genes of the E2F1 transcription factor.
- To investigate the role of these identified genes in E2F1-induced apoptosis.
Main Methods:
- A functional technical knockout (TKO) approach using Saos-2ERE2F1 cells with conditional E2F1 activation.
- Retroviral transfer of an antisense cDNA library enriched with E2F1-induced genes.
- Selection of surviving cells under apoptotic stimulus and subsequent gene identification.
Main Results:
- Thirteen novel E2F1 target genes involved in apoptosis and RNA binding were identified.
- Inactivation of these genes significantly attenuated E2F1-induced apoptosis, increasing cell viability by over 80%.
- Four genes demonstrated direct transcriptional regulation by E2F1, while one showed indirect regulation.
Conclusions:
- The study identified novel genes that mediate E2F1-induced apoptosis, highlighting their importance in the anti-tumor function of E2F1.
- These findings offer new insights into the regulatory network controlling E2F1-mediated cell death pathways.
