Survivin is a member of the inhibitor of apoptosis family. This apoptosis inhibitor also has an evolutionarily conserved role as a mitotic spindle checkpoint protein. Previous studies on p53-repressed genes have implicated several genes involved in the G 2 /M transition of the cell cycle as targets of negative regulation by p53. However, few targets of p53 repression that are antiapoptotic have been identified. This study identifies the anti-apoptotic survivin gene as a p53-repressed gene. Notably, Survivin repression by p53 is shown to be distinct from p53-dependent growth arrest. Chromatin immunoprecipitations indicate that p53 binds the survivin promoter in vivo; immunobinding studies indicate that this site overlaps with a binding site for E2F transcription factors and is subtly distinct from a canonical p53-transactivating element. The survivin-binding site contains a 3-nucleotide spacer between the two decamer "half-sites" of the p53 consensus element; deletion of this spacer is sufficient to convert the survivin site into a transactivating element. Finally, we show that overexpression of Survivin in cells sensitive to p53-dependent cell death markedly inhibits apoptosis induced by ultraviolet light. The identification of survivin as a p53 repressed gene should aid in the elucidation of the contribution of transcriptional repression to p53-dependent apoptosis.
RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.
The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.
The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G 1 /S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.
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