Article

C/EBPbeta cooperates with RB:E2F to implement Ras(V12)-induced cellular senescence.

Laboratory of Protein Dynamics and Signaling, NCI-Frederick, Frederick, MD 21702-1201, USA.
The EMBO Journal (Impact Factor: 10.75). 10/2005; 24(18):3301-12. DOI: 10.1038/sj.emboj.7600789
Source: PubMed

ABSTRACT In primary cells, overexpression of oncogenes such as Ras(V12) induces premature senescence rather than transformation. Senescence is an irreversible form of G1 arrest that requires the p19ARF/p53 and p16INK4a/pRB pathways and may suppress tumorigenesis in vivo. Here we show that the transcription factor C/EBPbeta is required for Ras(V12)-induced senescence. C/EBPbeta-/- mouse embryo fibroblasts (MEFs) expressing Ras(V12) continued to proliferate despite unimpaired induction of p19ARF and p53, and lacked morphological features of senescent fibroblasts. Enforced C/EBPbeta expression inhibited proliferation of wild-type MEFs and also slowed proliferation of p19Arf-/- and p53-/- cells, indicating that C/EBPbeta acts downstream or independently of p19ARF/p53 to suppress growth. C/EBPbeta was unable to inhibit proliferation of MEFs lacking all three RB family proteins or wild-type cells expressing dominant negative E2F-1 and, instead, stimulated their growth. C/EBPbeta decreased expression of several E2F target genes and was associated with their promoters in chromatin immunoprecipitation assays, suggesting that C/EBPbeta functions by repressing genes required for cell cycle progression. C/EBPbeta is therefore a novel component of the RB:E2F-dependent senescence program activated by oncogenic stress in primary cells.

0 Followers
 · 
106 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.
    PLoS ONE 10/2013; 8(10):e76279. DOI:10.1371/journal.pone.0076279 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Expression of the BRAFV600E oncoprotein is known to cause benign lesions, for example melanocytic nevi (moles). In spite of the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called Oncogene-Induced Senescence (OIS). Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor suppression mechanism, we performed a mass spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells as well as cells that have abrogated senescence. Proteome analysis of senescent cells revealed the upregulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti4+-IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK and CDK-retinoblastoma (Rb) pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAFV600E-expressing senescent cells provides molecular clues as to how OIS is initiated, maintained or evaded, serving as a comprehensive proteomic basis for functional validation.
    Molecular &amp Cellular Proteomics 06/2014; 13(8). DOI:10.1074/mcp.M113.035436 · 7.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Oncogene-induced senescence (OIS) protects normal cells from transformation by Ras, whereas cells lacking p14/p19(Arf) or other tumor suppressors can be transformed. The transcription factor C/EBPβ is required for OIS in primary fibroblasts but is down-regulated by H-Ras(V12) in immortalized NIH 3T3 cells through a mechanism involving p19(Arf) loss. Here we report that members of the serum-induced early growth response (Egr) protein family are also down-regulated in 3T3(Ras) cells and directly and redundantly control Cebpb gene transcription. Egr1-3 recognize three sites in the Cebpb promoter and associate transiently with this region after serum stimulation, coincident with Cebpb induction. Co-depletion of all three Egrs prevented Cebpb expression, and serum induction of Egrs was significantly blunted in 3T3(Ras) cells. Egr2-3 levels were also reduced in Ras(V12)-expressing p19(Arf) null MEFs, and overall Egr DNA-binding activity was suppressed in Arf-deficient but not WT MEFs, leading to Cebpb down-regulation. Analysis of human cancers revealed a strong correlation between EGR levels and CEBPB expression, regardless of whether CEBPB was increased or decreased in tumors. Moreover, over-expression of Egrs in tumor cell lines induced CEBPB and inhibited proliferation. Thus, our findings identify the Arf-Egr-C/EBPβ axis as an important determinant of cellular responses (senescence or transformation) to oncogenic Ras signaling. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Molecular and Cellular Biology 12/2014; 35(5). DOI:10.1128/MCB.01489-14 · 5.04 Impact Factor

Full-text (2 Sources)

Download
60 Downloads
Available from
May 22, 2014