RESUMO
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.
Assuntos
Apoptose/genética , Proteínas de Ciclo Celular/metabolismo , DNA Antissenso/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Retroviridae/genética , Tamoxifeno/análogos & derivados , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Antissenso/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Tamoxifeno/farmacologia , Fatores de Transcrição/genéticaRESUMO
The transcription factor E2F1 does not only induce cell proliferation but also shows the strongest proapoptotic effect of all E2F family members as part of an antitumor safeguard mechanism. We have recently identified KIAA0767 as a novel p53-independent target of E2F1. Here, we investigated the biological function of interaction. Overexpression studies of KIAA0767, termed D(eath)-I(nducing)-P(rotein), revealed its strong proapoptotic effect. DIP greatly reduced cell viability in several in vitro systems accompanied by typical apoptotic features such as caspase-3 activation and cleavage of poly(ADP-ribose)-polymerase. Endogenous DIP levels increased following E2F1 activation. Yet, inhibition of endogenous DIP function by small interfering RNA rescued p53-negative cells from E2F1-induced apoptosis, indicating that DIP is an essential mediator of the p53-independent E2F1 death pathway. Localization studies showed that DIP localizes to the mitochondria, where endogenous DIP is upregulated following E2F1 induction. These results provide new insights to the incompletely understood regulatory mechanisms of E2F1-induced apoptosis.